METHODS OF TREATING DISEASE WITH PFKFB3 INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/343,377, filed May 31, 2016, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under DK059579, awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION

I. FIELD OF THE INVENTION

[0003] Embodiments are directed generally to biology and medicine. In certain aspects there are methods and compositions for treating protein misfolding disorders.

II. BACKGROUND

[0004] In medicine, a protein misfolding disease refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function) or they can lose their normal function. The diseases (also known as proteinopathies, protein conformational disorders, or proteopathies) include such diseases as Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, prion disease, amyloidosis, and a wide range of other disorders.

[0005] Protein aggregation diseases are not exclusive to the central nervous system; they can also appear in peripheral tissues. In general, the genes and protein products involved in these kinds of diseases are called amyloidogenic. Such diseases include type 2 diabetes, inherited cataracts, some forms of atherosclerosis, hemodialysis-related disorders, and short- chain amyloidosis, among many others. All these diseases have in common the expression of a protein outside its normal context, leading to an irreversible change into a sticky conformation rich in beta sheets that make the protein molecules interact with each other. [0006] The general pattern that emerges in all these diseases is an abnormal tendency of proteins to aggregate as a result of misfolding. The aggregation can be caused by chance; by protein hyperphosphorylation (a condition where multiple phosphate groups are added to the protein), by prion self-catalytic conformational conversion, or by mutations that make the protein unstable. Aggregation can also be caused by an unregulated or pathological increase in the intracellular concentration of some of these proteins. Such imbalances in protein concentration can be a consequence of mutations such as duplications of the amyloidogenic gene or changes in the protein's amino acid sequence. Imbalances can also be caused by deficiencies in the proteasome, the cellular machinery involved in the degradation of aging proteins. Inhibition of autophagy (a process by which cells engulf themselves) also promotes amyloid aggregation. In addition, some evidence suggests that the severity of these diseases correlates with an increase in oxidative stress, mitochondrial dysfunction, alteration of cytoplasmic membrane permeability, and abnormal calcium concentration.

[0007] Protein misfolding can cause an acute stress response that may have beneficial survival-promoting effects in the short-term, but can be detrimental to cells experiencing chronic stresses, such as those involved in protein misfolding deases. While traditional therapeutic design has been to target the misfolded protein, there is a need in the art for therapies that attenuate the acute stress response resulting from the misfolded proteins, since this response can contribute to the pathology of the disease.

SUMMARY OF THE INVENTION

[0008] The current disclosure fulfills the aforementioned need in the art by providing therapeutics that treat protein misfolding disease by inhibiting PFKFB3. Accordingly, aspects of the disclosure relate to a method of treating a protein misfolding disease in a subject comprising administering a PFKFB3 inhibitor to the subject. Further aspects of the disclosure relate to a method of treating a protein misfolding disease in a subject comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the subject.

[0009] Further aspects relate to a method for treating or preventing protein misfolding- induced cell death in a cell, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the cell. In some embodiments, the cell is in a subject.

[0010] Further aspects relate to a method for inhibiting or reducing β-cell death in a subject with Type 2 diabetes, the method comprising administering a PFKFB3 inhibitor to the subject. Further aspects relate to a method for inhibiting or reducing β-cell death in a subject with Type 2 diabetes, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the subject.

[0011] Yet further aspects relate to a method for treating or preventing an acute stress response in a cell or subject in need thereof, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the cell or subject. In some embodiments, the cell is in vitro. In some embodiments, the cell is transplanted into the subject. In some embodiments, the subject is one that has or will receive transplanted cells. In some embodiments, the cell or transplanted cells are pancreatic islet cells.

[0012] In some embodiments, the subject has a protein misfolding disease or the cells has pathogenic misfolded protein. In some embodiments, the misfolded protein is one described herein. In some embodiments, the protein misfolding disease is one known in the art or described herein. In some embodiments, the protein misfolding disease is Alzheimer's, Parkinsin's, Type 2 Diabetes, or prediabetes. In some embodiments, the protein misfolding disease is Type 2 diabetes. In some embodiments, the subject does not have cancer or is not diagnosed as having cancer. In some embodiments, the subject has been diagnosed with a protein misfolding disease. In some embodiments, the subject has previously been treated for a protein misfolding disease.

[0013] The PFKFB3 inhibitor or K(ATP) channel opener may be a protein inhibitor, a nucleic acid inhibitor, or a small molecule inhibitor. In some embodiments, the the protein inhibitor is an antibody. In some embodiments, the antibody binds to a PFKFB3 protein and inhibits the activity of PFKFB3. In some embodiments, the inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is 3-(3-Pyridinyl)-l-(4- pyridinyl)-2-propen-l-one (3-PO) or an analog thereof. In some embodiments, the analog is is 1- (4-pyridinyl)-3-(2-quinolinyl)-2-propen-l-one (PFK15). In some embodiments, the small molecule inhibitor is one known in the art, described herein, or incorporated by reference. In some embodiments, the PFKFB3 inhibitor is an antisense nucleic acid. In some embodiments, the antisense nucleic acid is an siRNA, a double stranded RNA, a short hairpin RNA.

[0014] K(ATP) channel openers include, for example, diazoxide, NN 414 - Tifenazoxide, nicorandil, iptakalim, aprikalim, cromakalim, pinacidil, BMS- 180448, BPDZ 44, BPDZ 49, BPDZ 62, BPDZ 73, BPDZ 79, BPDZ 83, BPDZ 109, BPDZ 154, BPDZ 216 (=NNC 55- 9216), NNC 55-0118 (see e.g. T. M. Tagmose et al., J. Med. Chem. 47 (2004) 3202-3211); NNC 55-0462, MCC-134 (see e.g. M. J. Coghlan et al., J. Med. Chem. 44 (2001) 1627- 1653); losimendan, SR 47063, and WAY 135201.

[0015] In some embodiments, the inhibitor or K(ATP) channel opener is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, topically, through inhalation, or through a combination of two or more routes of administration. In some embodiments, the inhibitor is administered by a method described herein.

[0016] In some embodiments, the inhibitor is administered to a specific cell type, such as a pancreatic cell, a bone cell, a neural cell, a blood cell, a breast cell, an epithelial cell, an adipocyte, a kidney cell, a muscle cell, a pancreatic islet, an alpha cell, a beta cell, a delta cell, a pancreatic polypeptide secreting cell, or an epsilon cell.

[0017] The terms "ameliorating," "inhibiting," or "reducing," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. [0018] A "subject," "individual" or "patient" is used interchangeably herein and refers to a vertebrate, for example a primate, a mammal or preferably a human. Mammals include, but are not limited to equines, canines, bovines, ovines, murines, rats, simians, humans, farm animals, sport animals and pets. In some embodiments, the subject is a human subject.

[0019] Use of the one or more compositions may be employed based on methods described herein. Use of one or more compositions may be employed in the preparation of medicaments for treatments according to the methods described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Example section are understood to be embodiments that are applicable to all aspects of the technology described herein.

[0020] As used herein, "treatment" or "therapy" is an approach for obtaining beneficial or desired clinical results. This includes: reduce the alleviation of symptoms, the reduction of inflammation, the inhibition of cell death, and/or the restoration of cell function. In some embodiments, the term treatment refers to the inhibition or reduction of cell death, such as the rejction of β-cell death.

[0021] The term "therapeutically effective amount" refers to an amount of the drug that treats or inhibits disease in a subject. In some embodiments, the therapeutically effective amount inhibits at least or at most or exactly 100, 99, 98, 96, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 30, 20, or 10%, or any derivable range therein, of PFKFB3 activity.

[0022] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0023] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0024] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." It is also contemplated that anything listed using the term "or" may also be specifically excluded.

[0025] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026] As used in this specification and the claim(s), when referring to a particular therapeutic drug regimen, the words "consisting essentially of includes therapeutic drug remiments including, as active ingredients, only the recited active ingredients and excludes any active ingredients not recited.

[0027] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIG. 1A-D. hAPP and autoreactive cytokines induce β-cell mitochondrial fragmentation, a) scheme of β-cell synchronization by serum deprivation in combination with Aphidicolin treatment (5 μg/ml) for enrichment of cells in Gl/S. b) confocal images of Gl/S synchronized control (CTRL), hIAPP-expressing and cytokine mix treated INS 823/13 β-cells stained with Mitotracker Red (Mito Red), c) Islet-derived primary β-cells from wild- type (WT) or hIAPP (hTG) transgenic mouse show mitochondrial fragmentation after staining with Mitotracker Red (Mito Red) and insulin, d) FACS diagrams overlay showing the mitochondrial membrane potential of control (CTRL) and hIAPP expressing β-cells at Oh and 4h post-release from the synchronization block measured using TMRE in presence or absence of oligomycin (Oligo) or 2-deoxy glucose (DOG).

[0030] FIG. 2A-G. Mitochondrial dysfunction leads to compensatory enhancement of aerobic glycolysis and PFKFB3 expression, a) Lactate rate in isolated islets from wild type (WT) and hIAPP transgenic (HIP) rats, b) Protein expression of PFKFB3 in islet whole cell extracts from 2 WT and 2 HIP rats at 2, 4 and 6 months (mo) of age as measured by immunoblotting. c) time course of the lactate rate in synchronized CTRL and hIAPP expressing cells, d) Protein expression of PFKFB3 in GO cells after treatment with either Ad- hlAPP or cytokine mix before and after silencing of PFKFB3 and in islet whole cell extracts from WT, human IAPP (hIAPP) and rodent IAPP (rIAPP) transgenic mice e) lactate levels in hIAPP expressing INS 832/13 cells relative to control (CTRL) or hIAPP expressing cells after silencing of PFKFB3 (PFKFB3 si) as measured by mass spectroscopy f) PFKFB3 protein expression in synchronized INS 823/13 after 0-, 4- and 12h release from Ahidicolin block, g) PFKFB3 staining (white) of wild type- (WT), rodent IAPP- (rTG) and human IAPP (hTG) transgenic mouse islets.

[0031] FIG. 3A-C. PFKFB3 expression is enhanced in islets from prediabetic HIP rats and in islets from human obese (obese T2D) and lean (lean T2D) diabetes. Confocal images (upper panel) showing PFKFB3 expression in a) WT compared to prediabetic HIP rat at 7 months of age. b) (middle panel) showing PFKFB3 cytoplasmic enrichment in islets from obese type 2 (T2D) diabetes and c) (lower panel) PFKFB3 nuclear enrichment in lean type 2 (T2D) diabetes. [0032] FIG. 4A-C. β-cell rest by suppressed glycolysis permits the mitochondrial network to withstand stress and enhances β-cell survival, a) Confocal images (upper panel) of GO- hIAPP expressing- (36h) versus control (CTRL) INS 823/13 β-cells and (lower panel) primary β-cells isolated from HIP rats showing reduction of γΗ2Α.Χ foci and restoration of mitochondrial morphology after knocking down of PFKFB3. b) cell cycle distribution map. c) FACS histograms of GO INS 823/13 β-cells with indicated treatments to demonstrate rescue of hIAPP-induced cell death by PFKFB3 knockdown or K ⁺ _ATP channel opener NN-414 and reversal to cell death after addition of Glibenclamide (Gli) (hi APP+PFKFB 3 si+Gli). [0033] FIG. 5A-C. β-cell rest by suppressed glycolysis through inhibition of PFKFB3 with small molecule inhibitor 3PO enhances β-cell survival in dose-dependent fashion and stimulates insulin secretion after high glucose challenge in prediabetic HIP rat islets, a) FACS histograms of GO INS 823/13 β-cells with indicated treatments to demonstrate partial rescue of hIAPP-induced cell death by inhibition of PFKFB3 with 10 μΜ 3PO and prevention of cell death with 30 and 50 μΜ 3PO. DMSO and Ad-LacZ with DMSO in presence or absence of either 10 μΜ or 50 μΜ 3PO were used as controls, b) Confocal images of islets isolated from WT or HIP rats at 6 months of age showing enhanced nuclear staining for PFKFB3 in islets of HIP rats, c) Dynamic insulin secretion presented as concentration of measured insulin by spectrophotometry (AU* 1000) in perifused WT versus HIP islets at 6 months of age in presence or absence of PFKFB3 inhibitor - 3PO.

[0034] FIG. 6A-E. β-cell mitochondrial fragmentation in T2D is reproduced in hIAPP model. Images of mitochondria in (A) human pancreatic sections from non-diabetic (ND) and (B) T2D subjects stained with Tom20 (mitochondria), insulin, and DAPI (nuclei) (C) Quantification of mitochondrial area per β-cell in ND and T2D subjects. (D) Images of mitochondria in control (CTRL, LacZ-AdV) and hIAPP-expressing (hIAPP-AdV) INS 832/13 cells synchronized at the Gl/S cell cycle stage, stained with the mitochondrial probe Mitotracker Red (MTR). (E) Quantification of mitochondrial morphology in Gl/S enriched INS 832/13 cells after indicated treatments to overt fragmented- (illustrated in D, hIAPP) or overt intermediate-to-fused- (illustrated in D, CTRL) mitochondria. Data are presented as mean ± SEM, n=3, ***p<0.005 relative to CTRL, and ft ft ftp<0.005 relative to rIAPP.

[0035] FIG. 7A-7D. Mitochondrial respiration but not mitochondrial membrane potential is decreased by hIAPP. (A) Oxygen consumption rate (OCR) by Seahorse, in isolated islets from 4-6 months old HIP and WT rats after stimulation with high glucose (20 mM). (B) Quantification of OCR presented as a fold change of stimulated respiration versus WT as measured by Seahorse in FIG. 6A. Mitochondrial membrane potential measured by FACS after labelling with TMRE (C) CTRL and (D) hIAPP overexpressing Gl/S synchronized cells in the presence or absence of oligomycin or 2-deoxy-glucose (DOG). Data are presented as mean ± SEM, n=3, **p<0.01.

[0036] FIG. 8A-8E. hIAPP leads to upregulation of HIF 1 a-PFKFB3 stress pathway and increases aerobic glycolysis. (A) Summary of the differentially expressed genes of interest after microarray analysis performed on RNA isolated from rat WT and HIP islets (4-6 months) presented as a -fold change over WT. * represents genes that were differentially expressed in one set (B) LDHA and MCT1 mRNA levels in HIP vs. WT as measured by qRT-PCR (C) Lactate production rate (-fold change) measured in isolated islets from HIP relative to WT. (D) PFKFB3 protein expression assessed by immunoblotting in whole cell extracts from islets from 2 WT and 2 HIP rats from 2-6 months of age. (E) γΗ2Α.Χ immunostaining of: upper panel - hIAPP or LacZ (CTRL) transfected INS 832/13 cells (mitochondria visualized by MTR and nuclei by DAPI) and lower panel - pancreatic sections from 6 months old HIP and WT rats. Data are presented as mean ± SEM, n=3, *p<0.05.

[0037] FIG. 9A-9D. HIF 1 a PFKFB3 stress pathway is upregulated in β-cells from transgenic hIAPP rats and humans with T2D. Images of PFKFB3 immunostaining in (A) islets from HIP and WT rats and in (B) islets from non-diabetic (ND) and T2D subjects. (C) Quantification of frequency of PFKFB3 positive β-cells in HIP vs. WT rats, left, and T2D vs. ND subjects, right. (D) Immunoblotting of PFKFB3 and HIF la in nuclear enriched- (RIPA post NP-40) and whole cell extracts (NP-40) from human non-diabetic (ND) and T2D donor islets. Data are presented as mean ± SEM, n=3, *p<0.05, ***p<0.001.

[0038] FIG. 10A-10B. Silencing of PFKFB3 restores β-cell metabolome (A) Heatmap of relative metabolite composition in CTRL and hIAPP overexpressing INS 832/13 cells in presence or absence of PFKFB3 siRNA. (B) Mass isotopomer distribution (MID) of the TCA metabolic intermediates from cultured INS 832/13 cells with [U-13C6] glucose. Citrate - Cit; Fumarate- Fum; a -ketoglutarate - a-KG; Aspartate - Asp. Data are presented as mean ± SEM, n=3, *p<0.05, **p<0.01, ***p<0.005. [0039] FIG. 11A-D. PFKFB3 silencing restores Ca ²⁺ homeostasis, mitochondrial networks and reduces β-cell death. (A) Images illustrating restoration of mitochondrial networks as visualized by Mitotracker red (MTR) and reduction of genotoxic stress illustrated by γΗ2Α.Χ staining in INS 832/13 b-cells treated with hIAPP and PFKFB3 siRNA and quantification of mitochondrial morphology. (B) FACS histograms of GO INS 823/13 b-cells with indicated treatments demonstrate rescue from hIAPP induced cell death after PFKFB3 silencing and quantification of the percentage (%) of the subGl (apoptotic) cells as measured by FACS. (C) Immunoblotting analysis of whole cell extracts from INS 832/13 cells demonstrating reduction of γΗ2Α.Χ, cleaved caspase 3 and truncated (p89) PARP-1 upon PFKFB3 silencing and quantification of WB signals using ImageJ analysis. LacZ-AdV represents CTRL. (D) Comparison of cytosolic, ER and mitochondrial calcium in GO INS 832/13 cells overexpressing hIAPP or LacZ (CTRL) in the presence or absence of PFKFB3. Data are presented as mean ± SEM, n=3, **p<0.01 and ***p<0.005 relative to CTRL and ft ftp<0.01 and ft ftftp<0.005 relative to hIAPP, respectively.

[0040] FIG. 12A-B Scheme and working model. (A) Schematic presentation of the isotopologue distribution depicts a change in the metabolic pathway contribution after silencing of PFKFB3 in presence of hIAPP. (B) Working model depicting hIAPP toxicity inducing mitochondrial adaptation to perturbed Ca ²⁺ homeostasis leading to increase in the aerobic glycolysis and PFKFB3 upregulation, thus resulting in loss of a metabolic-insulin secretion coupling and β-cell loss. This phenotype is partly reversed via inhibition of PFKFB3 by restoration of Ca ²⁺ homeostasis and mitochondrial networks.

[0041] FIG. 13A-B. hIAPP induces cell death in synchronized INS 832/13 cells. FlowJo overlay of FACS diagrams from INS 832/13 cells at (A) Oh and (B) 12h post-release from aphidicolin block showing subGl accumulation at 12h (36h treatment with hlAPP- AdV).

[0042] FIG. 14A-B. hIAPP affects fusion by reducing MFN-2 levels but not mitochondrial fission. (A) Immunoblotting of indicated dynamin-related proteins in untreated (UT), control (CTRL, LacZ) and hIAPP overexpressing (hIAPP) INS 832/13 cells. (B) Images of CTRL-LacZ and hIAPP-AdV transduced INS 823/13 β-cells in presence or absence of the dominant negative DrplK48A mutant illustrate non-sustained fusion after hIAPP overexpression.

[0043] FIG. 15A-E. hIAPP reduces the flux through TCA cycle (A-B) and hIAPP increases the flux through de novo purine synthesis and oxidized glutathione (C-E). (A) Relative metabolite composition of hIAPP (30h) overexpressing INS 832/13 cells presented as a -fold change of control rIAPP overexpressing cells. (B) Mass isotopomer distribution (MID) of the TCA intermediates derived from culturing INS 832/13 with [U- ¹³ C ₆ ] glucose; Glucose - Glc; Aspartate - Asp; Citrate - Cit; a— ketoglutarate - a— KG; Succinate - Sue; Fumarate - Fum; Malate -Mai; Glutamate - Glu. Data are presented as mean ±SEM, n=3. *p<0.05, **p<0.01, ***p<0.005. (C) Mass isotopomer distribution (MID) of the TCA intermediates derived from culturing INS 832/13 with [U- ¹³ C ₆ ] glucose. Inositol monophosphat - IMP; Adenosin diphosphate - ADP; Adenosin triphosphate - ATP; Cytidin diphosphate -CDP; Cytidin triphosphate - CTP; Uridin diphosphate - UDP; Uridin triphosphate - UTP (D) reduced Glutathione -GSH; oxidized Glutathione - GSSG. (E) Schematic presentation of the isotopologue distribution depicts metabolic detachment of aerobic glycolysis from TCA in presence of hIAPP. Data are presented as mean ± SEM, n=3. *p<0.05, **p<0.01, ***p<0.005.

[0044] FIG. 16A-F. hIAPP induced apoptosis is linked to PFKFB3. (A) PFKFB3 protein levels in GO synchronized INS 832/13 after transduction with hIAPP-AdV for 36h (upper panel) with or without PFKFB3 siRNA and after transduction with rIAPP- or hlAPP- AdV for 30h (lower panel) as assessed by immunoblotting of whole cell extracts. (B) Relative PFKFB3 mRNA levels in indicated treatments of INS 832/13 cells as measured by qRT-PCR and presented as -fold change to CTRL. (C) PFKFB3 immunostaining of INS 832/13 cells transfected with hIAPP or LacZ (CTRL) adenoviruses. (D) Quantification of PFKFB3 immunopositive cells in different treatments, shown as % of all cells visualized by DAPI. Quantification of γΗ2Α.Χ (E) or propidium iodide (F) positive INS 832/13 cells in the indicated treatments, expressed as % over all cells. Data are presented as mean ±SEM, n=3. *p<0.05, **p<0.01, ***p<0.005.

[0045] FIG. 17A-B. Silencing of PFKFB3 stimulates the PPP flux. (A) Mass isotopomer distribution (MID) of the TCA intermediates derived from culturing INS 832/13 with [U- ¹³ C ₆ ] glucose. Glucose - Glc; Malate - Mai; Succinate -Sue; Glutamate - Glu; and the (B) pentose phosphate pathway (PPP) metabolites G6P--F6P - Glucose— 6— phosphae- Fructose~6~phosphate; R5P--Ribose--5— phosphate; IMP-Inositol monophosphate; S7P- -sedoheptulose-7-phosphate. Data are presented as mean ±SEM, n=3. *p<0.05, **p<0.01, ***p<0.005. [0046] FIG. 18A-D. NN-414 K(ATP) opener prevents β-cell death by restoring the cell cycle distribution and mitochondrial networks similar to PFKFB3 silencing. (A)

FACS analysis of DNA content distribution in INS 832/13 cells overexpressing hIAPP in presence or absence of K(ATP) channel opener, NN-414, or K(ATP) inhibitor, Glybenclamide (Gly). (B) Quantification of apoptotic cells, expressed as % of cells in subGl after treatments described in (A) and assessed by FACS. (C) Images of INS 832/13 cells overexpressing hIAPP in presence or absence of the K(ATP) channel opener, NN-414, or the K(ATP) inhibitor, Glybenclamide, and stained with Mitotracker red (MTR), γΗ2Α.Χ and DAPI. (D) Quantification of mitochondrial morphology in either fragmented or intermediate- -to-fused in GO synchronized INS 832/13 cells after indicated treatments. Data are presented as mean ± SEM, n=3, *p<0.05, **p<0.01, ***p<0.005 relative to CTRL and ft ft ft p<0.005 relative to hIAPP.

DETAILED DESCRIPTION OF THE INVENTION

[0047] In health, β-cells (collectively referred to as β-cell mass) regulate glucose metabolism by secretion of insulin in a glucose dependent manner that constrains hepatic glucose release. The mitochondrial network is critical for β-cell function and viability. In healthy β-cells, the prevailing glucose concentration is sensed by the rate of flux through glycolysis regulated by glucokinase, with tight linkage of acetyl CoA generated by glycolysis to ATP synthesis by mitochondria. The ATP acts to close K ⁺⁽ ATP) sensitive channels and thus opening of voltage gated Ca ²⁺ channels to elicit insulin secretion in response to glucose. In order to ensure the tight linkage between glucose sensing and insulin secretion by β-cells, the mitochondrial network is highly fused and interconnected, a form that favors oxidative phosphorylation. [0048] The inventors of the application have now, for the first time, investigated the mitochondrial network in replicating β-cells and confirmed its departing from highly fused to a fragmented form prior to mitosis, switching from aerobic to a high rate of aerobic glycolysis during Gl-to-S phase transition, an adaptation synchronized by signaling cross talk between regulators of cell cycle and the mitochondrial network. These changes permit synthesis of nucleotides required for new DNA and elimination of compromised mitochondria (mitophagy) prior to sorting the remainder to daughter cells. Mitochondrial morphology is highly disrupted in islet amyloid pancreatic peptide (IAPP)-expressing-cells and in β-cells isolated from the islets of IAPP -transgenic rodents, being represented by shortened spheroid mitochondria with reduced motility and reflecting mitochondrial dysfunction in type 2 diabetes (T2D) in vivo. Mitochondrial fragmentation, and a switch to aerobic glycolysis, is also an adaptive defense by cells in response to hypoxia and related stress. Of interest, this metabolic pattern is also present in β-cells in infancy that are characterized by high rates of aerobic glycolysis, and therefore minimal glucose responsiveness. This adaptation likely is to permit the high rate of β-cell replication in infancy.

[0049] The inventors found that the flux of aerobic glycolysis is mainly enhanced by upregulation of phospho-fructokinase 2,6-biphosphatase (PFKFB3/PFK-2) in IAPP expressing cultured and primary β-cells from either islets from IAPP transgenic rodents or human pancreas. It was found that metabolic switch to aerobic glycolysis, PFKFB3 expression and mitochondrial network disruption were mechanistically linked to closure of K ⁺ ATP channels and sustained Ca ²⁺ flux leading to enhanced β-cell demise associated with a progressive decline in β-cell mass and function in T2D. Once diabetes occurs, glucose toxicity is superimposed on these initial stressors. Therefore, the inventors have demonstrated that initial β-cell stressors converge on the mitochondrial network, which by adapting a defensive fragmented form, disrupts the coupling of glycolysis to ATP production, and thus insulin secretion. It was further found that the fragmented mitochondrial network leads to aberrant signal cross talk between the mitochondria and cell cycle regulatory network, recruits β-cells into cell cycle but that under stress conditions, this leads to β-cell loss rather than β-cell regeneration.

[0050] The inventors identified a strategy based on targeting PFKFB3 from the aerobic glycolysis that maintains a fused mitochondrial network, during entry into cell cycle, and in the face of these initial stressors sustains β-cell survival. The studies described in the examples support a concept of benefit by β-cell rest, as achieving protection by attenuating sustained high glycolytic flux induced by activation of PFKFB3 and its action to fragment the mitochondrial network, at least in part by sustained activation of K ⁺ ATP channels and Ca ²⁺ activation of calcineurin. This strategy confers prevention of β-cell death and promotion of β- cell survival. [0051] This is also a novel and original approach which attempts to support β-cell survival and stimulate β-cell renewal after restoring the mitochondrial architecture, function and related physiological metabolic profile which then secures restoration of β-cell function by orderly insulin secretion.

I. PFKFB3 [0052] PFKFB3 is also referred to as 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3, 6PF-2-K/Fru-2,6-P2ase Brain/Placenta- Type Isozyme, renal carcinoma antigen NY-REN-56, 6PF-2-K/Fru-2,6-P2ase 3, PFK/FBPase 3, IPFK-2, Inducible 6- Phosphofoicto-2-Kinase/Fructose-2,6-Bisphosphatase 3, Fructose-6-Phosphate,2- Kinase/Fructose-2, 6-Bisphosphatase 3, 6-Phosphofructo-2-Kinase/ Fructose-2,6- Bisphosphatase 3, IPFK2, and PFK2.

[0053] The protein encoded by this gene belongs to a family of bifunctional proteins that are involved in both the synthesis and degradation of fructose-2,6-bisphosphate, a regulatory molecule that controls glycolysis in eukaryotes. The encoded protein has a 6-phosphofructo- 2-kinase activity that catalyzes the synthesis of fructose-2,6-bisphosphate (F2,6BP), and a fructose-2,6-biphosphatase activity that catalyzes the degradation of F2,6BP. This protein is required for cell cycle progression and prevention of apoptosis. It functions as a regulator of cyclin-dependent kinase 1, linking glucose metabolism to cell proliferation and survival in tumor cells. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.

II. PFKFB3 INHIBITORS

[0054] A PFKFB3 inhibitor may refer to any member of the class of compound or agents having an IC50 of 100 μΜ or lower concentration for a PFKFB3 activity, for example, at least or at most or about 200, 100, 80, 50, 40, 20, 10, 5, 1 μΜ, 100, 10, 1 nM or lower concentration (or any range or value derivable therefrom) or any compound or agent that inhibits the expression of PFKFB3. Examples of PFKFB3 activity or function may include, but not be limited to, regulation of glycolysis, kinase activity, regulation of CDK1, 6- phosphofructo-2-kinase activity, fructose-2,6-bisphosphate 2-phosphatase activity, ATP binding activity, and enzyme catalysis activity. In some embodiments, the inhibition can be a decrease as compared with a control level or sample. In further embodiments, functional assay such as MTT assay, cell proliferation assay, Ki67 immunofluoresence, apoptosis assay, or glycolysis assay may be used to test the PFKFB3 inhibitors. [0055] The following sequences exemplifies the PFKFB3 mRNA in humans (SEQ ID NO: l):

ccctttcccc tccctcgccc gccccgccgc ccgcaggcgc cccgagtcgc ggggctgccg cttggacgtc gtcctgtctg ggtgtcgcgg gccggccccg cggggagcgc ccccggcgcg atgcccttca ggaaagcctg tgggccaaag ctgaccaact cccccaccgt catcgtcatg gtgggcctcc ccgcccgggg caagacctac atctccaaga agctgactcg ctacctcaac tggattggcg tccccacaaa agtgttcaac gtcggggagt atcgccggga ggctgtgaag cagtacagct cctacaactt cttccgcccc gacaatgagg aagccatgaa agtccggaag caatgtgcct tagctgcctt gagagatgtc aaaagctacc tggcgaaaga agggggacaa attgcggttt tcgatgccac caatactact agagagagga gacacatgat ccttcatttt gccaaagaaa atgactttaa ggcgtttttc atcgagtcgg tgtgcgacga ccctacagtt gtggcctcca atatcatgga agttaaaatc tccagcccgg attacaaaga ctgcaactcg gcagaagcca tggacgactt catgaagagg atcagttgct atgaagccag ctaccagccc ctcgaccccg acaaatgcga cagggacttg tcgctgatca aggtgattga cgtgggccgg aggttcctgg tgaaccgggt gcaggaccac atccagagcc gcatcgtgta ctacctgatg aacatccacg tgcagccgcg taccatctac ctgtgccggc acggcgagaa cgagcacaac ctccagggcc gcatcggggg cgactcaggc ctgtccagcc ggggcaagaa gtttgccagt gctctgagca agttcgtgga ggagcagaac ctgaaggacc tgcgcgtgtg gaccagccag ctgaagagca ccatccagac ggccgaggcg ctgcggctgc cctacgagca gtggaaggcg ctcaatgaga tcgacgcggg cgtctgtgag gagctgacct acgaggagat cagggacacc taccctgagg agtatgcgct gcgggagcag gacaagtact attaccgcta ccccaccggg gagtcctacc aggacctggt ccagcgcttg gagccagtga tcatggagct ggagcggcag gagaatgtgc tggtcatctg ccaccaggcc gtcctgcgct gcctgcttgc ctacttcctg gataagagtg cagaggagat gccctacctg aaatgccctc ttcacaccgt cctgaaactg acgcctgtcg cttatggctg ccgtgtggaa tccatctacc tgaacgtgga gtccgtctgc acacaccggg agaggtcaga ggatgcaaag aagggaccta acccgctcat gagacgcaat agtgtcaccc cgctagccag ccccgaaccc accaaaaagc ctcgcatcaa cagctttgag gagcatgtgg cctccacctc ggccgccctg cccagctgcc tgcccccgga ggtgcccacg cagctgcctg gacaaaacat gaaaggctcc cggagcagcg ctgactcctc caggaaacac tgaggcagac gtgtcggttc cattccattt ccatttctgc agcttagctt gtgtcctgcc ctccgcccga ggcaaaacgt atcctgagga cttcttccgg agagggtggg gtggagcagc gggggagcct tggccgaaga gaaccatgct tggcaccgtc tgtgtcccct cggccgctgg acaccagaaa gccacgtggg tccctggcgc cctgccttta gccgtggggc ccccacctcc actctctggg tttcctagga atgtccagcc tcggagacct tcacaaagcc ttgggagggt gatgagtgct ggtcctgaca ggaggccgct ggggacactg tgctgttttg tttcgtttct gtgatctccc ggcacgtttg gagctgggaa gaccacactg gtggcagaat CCt3333tt3 aaggaggcag gctcctagtt gctgaaagtt aaggaatgtg taaaacctcc acgtgactgt ttggtgcatc ttgacctggg aagacgcctc atgggaacga acttggacag gtgttgggtt gaggcctctt ctgcaggaag tccctgagct gagacgcaag ttggctgggt ggtccgcacc ctggctctcc tgcaggtcca cacaccttcc aggcctgtgg cctgcctcca aagatgtgca agggcaggct ggctgcacgg ggagagggaa gtattttgcc gaaatatgag aactggggcc tcctgctccc agggagctcc agggcccctc tctcctccca cctggacttg gggggaactg agaaacactt tcctggagct gctggctttt gcactttttt gatggcagaa gtgtgacctg agagtcccac cttctcttca ggaacgtaga tgttggggtg tcttgccctg gggggcttgg aacctctgaa ggtggggagc ggaacacctg gcatccttcc ccagcacttg cattaccgtc cctgctcttc ccaggtgggg acagtggccc aagcaaggcc tcactcgcag ccacttcttc aagagctgcc tgcacactgt cttggagcat ctgccttgtg cctggcactc tgccggtgcc ttgggaaggt cggaagagtg gactttgtcc tggccttccc ttcatggcgt ctatgacact tttgtggtga tggaaagcat gggacctgtc gtctcagcct gttggtttct cctcattgcc tcaaaccctg gggtaggtgg gacggggggt ctcgtgccca gatgaaacca tttggaaact cggcagcaga gtttgtccaa atgacccttt tcaggatgtc tcaaagcttg tgccaaaggt cacttttctt tcctgccttc tgctgtgagc cctgagatcc tcctcccagc tcaagggaca ggtcctgggt gagggtggga gatttagaca cctgaaactg ggcgtggaga gaagagccgt tgctgtttgt tttttgggaa gagcttttaa agaatgcatg tttttttcct ggttggaatt gagtaggaac tgaggctgtg cttcaggtat ggtacaatca agtgggggat tttcatgctg aaccattcaa gccctccccg cccgttgcac ccactttggc tggcgtctgc tggagaggat gtctctgtcc gcattcccgt gcagctccag gctcgcgcag ttttctctct ctccctggat gttgagtctc atcagaatat gtgggtaggg ggtggacgtg cacgggtgca tgattgtgct taacttggtt gtatttttcg atttgacatg gaaggcctgt tgctttgctc ttgagaatag tttctcgtgt ccccctcgca ggcctcattc tttgaacatc gactctgaag tttgatacag ataggggctt gatagctgtg gtcccctctc ccctctgact acctaaaatc aatacctaaa tacagaagcc ttggtctaac acgggacttt tagtttgcga agggcctaga tagggagaga ggtaacatga atctggacag ggagggagat actatagaaa ggagaacact gcctactttg caagccagtg acctgccttt tgaggggaca ttggacgggg gccgggggcg ggggttgggt ttgagctaca gtcatgaact tttggcgtct actgattcct ccaactctcc accccacaaa ataacgggga ccaatatttt taactttgcc tatttgtttt tgggtgagtt tcccccctcc ttattctgtc ctgagaccac gggcaaagct cttcattttg agagagaaga aaaactgttt ggaaccacac caatgatatt tttctttgta atacttgaaa tttatttttt tattattttg atagcagatg tgctatttat ttatttaata tgtataagga gcctaaacaa tagaaagctg tagagattgg gtttcattgt taattggttt gggagcctcc tatgtgtgac ttatgacttc tctgtgttct gtgtatttgt ctgaattaat gacctgggat ataaagctat gctagctttc aaacaggaga tgcctttcag aaatttgtat attttgcagt tgccagacca

tggttgaaat acatggacga agtaaa .

[0056] The protein sequence is exemplified by the following (SEQ ID NO:2):

MPFRKACGPKLTNSPTVIVMVGLPARGKTYISKKLTRYLNWIGVPTKVFNVGEYRRE AVKQYSSYNFFRPDNEEAMKVRKQCALAALRDVKSYLAKEGGQIAVFDATNTTRE RRHMILHF AKENDFKAFFIES VCDDPTVVASNIMEVKIS SPD YKDCNS AEAMDDFMK RISCYEASYQPLDPDKCDRDLSLIKVIDVGRRFLVNRVQDHIQSRIVYYLMNIHVQPR TIYLCRHGENEHNLQGRIGGDSGLSSRGKKFASALSKFVEEQNLKDLRVWTSQLKSTI QTAEALRLPYEQWKALNEIDAGVCEELTYEEIRDTYPEEYALREQDKYYYRYPTGES YQDLVQRLEPVIMELERQENVLVICHQAVLRCLLAYFLDKSAEEMPYLKCPLHTVLK LTPVAYGCRVESIYLNVESVCTHRERSEDAKKGPNPLMRRNSVTPLASPEPTKKPRIN SFEEHVASTSAALPSCLPPEVPTQLPGQNMKGSRSSADSSRKH.

[0057] The above protein and mRNA sequence represents one isoform (isoform 2) of the gene, but other isoforms are known in the art. For example, the Genbank numbers below represent additional isoforms. The sequences associated with these Genbank numbers are incorporated by reference for all purposes.

Isoform GenBank mRNA GenBank protein

6-phosphofructo-2-kinase/fructose-2,6- NM_001145443.2 NP_001138915.1 bisphosphatase 3 isoform 2

6-phosphofructo-2-kinase/fructose-2,6- NM_001282630.2 NP_001269559.1 bisphosphatase 3 isoform 3

A. PFKFB3 inhibitory nucleic acids

[0058] Inhibitory nucleic acids or any ways of inhibiting gene expression of PFKFB3 known in the art are contemplated in certain embodiments. Examples of an inhibitory nucleic acid include but are not limited to antisense nucleic acids such as: siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an any other antisense oligonucleotide. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long. The nucleic acid may have nucleotides of at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 or any range derivable therefrom.

[0059] As used herein, "isolated" means altered or removed from the natural state through human intervention. For example, an siRNA naturally present in a living animal is not "isolated," but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered. [0060] Inhibitory nucleic acids are well known in the art. For example, siRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.

[0061] Particularly, an inhibitory nucleic acid may be capable of decreasing the expression of PFKFB3 by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95% or more or any range or value in between the foregoing.

[0062] In further embodiments, there are synthetic nucleic acids that are PFKFB3 inhibitors. An inhibitor may be between 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature PFKFB3 mRNA. In certain embodiments, an inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, an inhibitor molecule has a sequence (from 5' to 3') that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature PFKFB3 mRNA, particularly a mature, naturally occurring mRNA. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature mRNA as the sequence for an mRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature mRNA.

[0063] Inhibitor nucleic acids for PFKFB3 are also commercially available. For example, the following miRNAs may inhibit PFKFB3 : hsa-mir-26b-5p (MIRT028775), hsa-mir-330- 3p (MIRT043840), hsa-mir-6779-5p (MIRT454747), hsa-mir-6780a-5p (MIRT454748), hsa- mir-3689c (MIRT454749), hsa-mir-3689b-3p (MIRT454750), hsa-mir-3689a-3p (MIRT454751), hsa-mir-30b-3p (MIRT454752), hsa-mir-1273h-5p (MIRT454753), hsa-mir- 6778-5p (MIRT454754), hsa-mir-1233-5p (MIRT454755), hsa-mir-6799-5p (MIRT454756), hsa-mir-7106-5p (MIRT454757), hsa-mir-6775-3p (MIRT454758), hsa-mir-1291 (MIRT454759), hsa-mir-765 (MIRT454760), hsa-mir-423-5p (MIRT454761), hsa-mir-3184- 5p (MIRT454762), hsa-mir-6856-5p (MIRT454763), hsa-mir-6758-5p (MIRT454764), hsa- mir-3185 (MIRT527973), hsa-mir-6892-3p (MIRT527974), hsa-mir-6840-5p (MIRT527975), and hsa-mir-6865-3p (MIRT527976). [0064] siRNAs and shRNAs are also commercially available from, for example, Santa Cruz biotechnology (sc-44011 and sc-44011-SH, respectively). B. PFKFB3 inhibitory polypeptides

[0065] In certain embodiments, an antibody or a fragment thereof that binds to at least a portion of PFKFB3protein and inhibits PFKFB 3 activity and/or function is used in the methods and compositions described herein. [0066] In some embodiments, the PFKFB3 inhibitor polypeptide is a PFKFB3 antibody. In some embodiments, the anti- PFKFB3 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a chimeric antibody, an affinity matured antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment comprises a Fab, Fab', Fab'-SH, F(ab')2, or scFv. In one embodiment, the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non-human, human or humanized sequence (e.g., framework and/or constant domain sequences). In one embodiment, the non-human donor is a mouse. In one embodiment, an antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening, etc.). In one embodiment, a chimeric antibody has murine V regions and human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain or a human IgGl C region.

[0067] Examples of antibody fragments include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL and CHI domains; (ii) the "Fd" fragment consisting of the VH and CHI domains; (iii) the "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) the "dAb" fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv"), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers (see U.S. Pat. No. 5,091,513) and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Pub. 2005/0214860). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, 1996). [0068] A monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B-lymphocyte cell line. Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a PFKFB3 antigen with an immortal myeloma cell (usually mouse myeloma). This technology provides a method to propagate a single antibody-producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced. However, in therapeutic applications a goal of hybridoma technology is to reduce the immune reaction in humans that may result from administration of monoclonal antibodies generated by the non-human (e.g., mouse) hybridoma cell line.

[0069] Methods have been developed to replace light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact. Alternatively, "fully human" monoclonal antibodies are produced in mice transgenic for human immunoglobulin genes. Methods have also been developed to convert variable domains of monoclonal antibodies to more human form by recombinantly constructing antibody variable domains having both rodent and human amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDR is derived from mouse monoclonal antibodies, and the framework regions are derived from human amino acid sequences. It is thought that replacing amino acid sequences in the antibody that are characteristic of rodents with amino acid sequences found in the corresponding position of human antibodies will reduce the likelihood of adverse immune reaction during therapeutic use. A hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.

[0070] It is possible to create engineered antibodies, using monoclonal and other antibodies and recombinant DNA technology to produce other antibodies or chimeric molecules which retain the antigen or epitope specificity of the original antibody, i.e., the molecule has a binding domain. Such techniques may involve introducing DNA encoding the immunoglobulin variable region or the CDRs of an antibody to the genetic material for the framework regions, constant regions, or constant regions plus framework regions, of a different antibody. See, for instance, U.S. Pat. Nos. 5,091,513, and 6,881,557, which are incorporated herein by this reference.

[0071] By known means as described herein, polyclonal or monoclonal antibodies, binding fragments and binding domains and CDRs (including engineered forms of any of the foregoing), may be created that are specific to PFKFB3 protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.

[0072] Antibodies may be produced from any animal source, including birds and mammals. Particularly, the antibodies may be ovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition, newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by this reference. These techniques are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

[0073] Methods for producing polyclonal antibodies in various animal species, as well as for producing monoclonal antibodies of various types, including humanized, chimeric, and fully human, are well known in the art. Methods for producing these antibodies are also well known. For example, the following U.S. patents and patent publications provide enabling descriptions of such methods and are herein incorporated by reference: U.S. Patent publication Nos. 2004/0126828 and 2002/0172677; and U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275, 149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742, 159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861, 155; 5,871,907; 5,969, 108; 6,054,297; 6,165,464; 6,365, 157; 6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024. All patents, patent publications, and other publications cited herein and therein are hereby incorporated by reference in the present application.

[0074] It is fully expected that antibodies to PFKFB3 will have the ability to neutralize or counteract the effects of the PFKFB3 regardless of the animal species, monoclonal cell line or other source of the antibody. Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the "Fc" portion of the antibody. However, whole antibodies may be enzymatically digested into "Fc" (complement binding) fragment, and into binding fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen binding fragment will elicit an undesirable immunological response and, thus, antibodies without Fc may be particularly useful for prophylactic or therapeutic treatments. As described above, antibodies may also be constructed so as to be chimeric, partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.

C. PFKFB3 inhibitory small molecules

[0075] As used herein, a "small molecule" refers to an organic compound that is either synthesized via conventional organic chemistry methods (e.g., in a laboratory) or found in nature. Typically, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than about 1500 grams/mole. In certain embodiments, small molecules are less than about 1000 grams/mole. In certain embodiments, small molecules are less than about 550 grams/mole. In certain embodiments, small molecules are between about 200 and about 550 grams/mole. In certain embodiments, small molecules exclude peptides (e.g., compounds comprising 2 or more amino acids joined by a peptidyl bond). In certain embodiments, small molecules exclude nucleic acids.

[0076] For example, a small molecule PFKFB3 inhibitory may be any small molecules that is determined to inhibit PFKFB3 function or activity. Such small molecules may be determined based on functional assays in vitro or in vivo. PFKFB3 inhibitory molecules are known in the art and described in, for example, U.S. Patent publications 20130059879, 20120177749, 20100267815, 20100267815, and 20090074884, which are herein incorporated by reference.

[0077] Exemplary inhibitory compounds include: (lH-Benzo[g]indol-2-yl)-phenyl- methanone; (3H-Benzo[e]indol-2-yl)-phenyl-methanone; (3H-Benzo[e]indol-2-yl)-(4- methoxy-phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone; HC1 salt of (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone; (3H-Benzo[e]indol-2-yl)-(3-methoxy- phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-pyridin-3-yl-methanone; (3H-Benzo[e]indol-2- yl)-(2-methoxy-phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-(2-hydroxy-phenyl)- methanone; (3H-Benzo[e]indol-2-yl)-(4-hydroxy-phenyl)-methanone; (5-Methyl-3H- benzo[e]indol-2-yl)-phenyl-methanone; Phenyl-(7H-pyrrolo[2,3-h]quinolin-8-yl)-methanone; (3H-Benzo[e]indol-2-yl)-(3-hydroxy-phenyl)-methanone; (3H-benzo[e]indol-2-yl)-(2-chloro- pyridin-4-yl)-methanone; (3H-benzo[e]indol-2-yl)-(l-oxy-pyridin-4-yl)-methanone; Phenyl- (6,7,8, 9-tetrahydro-3H-benzo[e]indol-2-yl)-methanone; (3H-Benzo[e]indol-2-yl)-(4-hydroxy- 3-methoxylthenyl)-methanone; (3H-Benzo[e]indol-2-yl)-(4-benzyloxy-3-methoxy-phenyl)- methanone; 4-(3H-Benzo[e]indole-2-carbonyl)-benzoic acid methyl ester; 4-(3H- Benzo[e]indole-2-carbonyl)-benzoic acid; (4-Amino-phenyl)-(3H-benzo[e]indol-2-yl)- methanone; 5-(3H-Benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methyl; 5-(3H- Benzo[e]indole-2-carbonyl)-2-benzyloxy -benzoic Acidmethanone; (3H-Benzo[e]indol-2-yl)- (2-methoxy-pyridin-4-yl)-methanone; (5-Fluoro-3H-benzo[e]indol-2-yl)-(3-methoxy- phenyl)-methanone; (5-Fluoro-3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone; (4- Benzyloxy-3-methoxy-phenyl)-(5-fluoro-3H-benzo[e]indol-2-yl) - methanone; (5-Fluoro-3H- benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy-phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-(3- hydroxymethyl-phenyl)-methanone; Cyclohexyl-(5-fluoro-3H-benzo[e]indol-2-yl)- methanone; (5-Fluoro-3H-benzo[e]indol-2-yl)-(3-fluoro-4-hydroxy-phenyl) -methanone; (3H- Benzo[e]indol-2-yl)-p-tolyl-methanone; (3H-Benzo[e]indol-2-yl)-(3-methoxy -phenyl- methanol; (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanol; 3H-Benzo[e]indole-2-carboxylic acid phenylamide; 3H-Benzo[e]indole-2-carboxylic acid (3-methoxy-phenyl)-amide; (3H- Benzo[e]indol-2-yl)-(4-dimethylamino-phenyl)-methanone; (4-Amino-3-methoxy-phenyl)- (3H-benzo[e]indol-2-yl)-methanone; (4-Amino-3-methoxy-phenyl)-(5-hydroxy-3H- benzo[e]indol-2-yl)-methanone; (4-Amino-3-methoxy-phenyl)-(5-methoxy-3H- benzo[e]indol-2-yl)-methanone; N-[4-(3H-Benzo[e]indole-2-carbonyl)-phenyl]- methanesulfonamide; 3H-Benzo[e]indole-2-carboxylic acid (4-amino-phenyl)-amide; (4- Amino-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone; (4-Amino-2-fluoro-phenyl)- (5-methoxy-3H-benzo[e]indol-2-yl)-methanone; (4-Amino-3-fluoro-phenyl)-(5-methoxy-3H- benzo[e]indol-2-yl)-methanone; (4-Amino-2-methoxy-phenyl)-(5-methoxy-3H- benzo[e]indol-2-yl)-methanone; (4-Amino-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)- methanone; (4-Amino-3-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl) -methanone; (4-Amino-2-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl) -methanone; (4-Amino-3- fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone; (4-Amino-2-fluoro-phenyl)- (9-methoxy-3H-benzo[e]indol-2-yl)-methanone; (4-Amino-3-fluoro-phenyl)-(3H- benzo[e]indol-2-yl)-methanone; (4-Amino-2-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)- methanone; (4-Amino-phenyl)-(7-methoxy-3H-benzo[e]indol-2-yl)-methanone ; (4-Amino- phenyl)-(5-hydroxy-3-methyl-3H-benzo[e]indol-2-yl)-methanone ; (7-Amino-5-fluoro-9- hydroxy-3H-benzo[e]indol-2-yl)-(3-methyl-pyridin-4-yl)-metha none; (5-Amino-3H- pyrrolo[3,2-f]isoquinolin-2-yl)-(3-methoxy-pyridin-4-yl)-met hanone; (4-Amino-2-methyl- phenyl)-(9-hydroxy-3H-pyrrolo[2,3-c]quinolin-2-yl)-methanone ; and (4-Amino-phenyl)-(7- methanesulfonyl-3H-benzo[e]indol-2-yl)-methanone. [0078] Further exemplary inhibitory compounds include: l-Pyridin-4-yl-3-quinolin-4-yl- propenone; l-Pyridin-4-yl-3-quinolin-3-yl-propenone; l-Pyridin-3-yl-3-quinolin-2-yl- propenone; l-Pyridin-3-yl-3-quinolin-4-yl-propenone; l-Pyridin-3-yl-3-quinolin-3-yl- propenone; l-Naphthalen-2-yl-3-quinolin-2-yl-propenone; l-Naphthalen-2-yl-3-quinolin-3- yl-propenone; l-Pyridin-4-yl-3-quinolin-3-yl-propenone; 3-(4-Hydroxy-quinolin-2-yl)-l- pyridin-4-yl-propenone; 3 -(8-Hydroxy-quinolin-2-yl)-l-pyridin-3 -yl-propenone; 3-Quinolin- 2-yl- 1 -p-tolyl-propenone; 3 -(8-Hydroxy-quinolin-2-yl)- 1 -pyridin-4-yl-propenone; 3 -(8- Hy droxy-quinolin-2-yl)- 1 -p-tolyl-propenone; 3 -(4-Hy droxy-quinolin-2-yl)- 1 -p-tolyl- propenone; 1 -Phenyl-3 -quinolin-2-yl-propenone; 1 -Pyridin-2-yl-3 -quinolin-2-yl-propenone; l-(2-Hydroxy-phenyl)-3-quinolin-2-yl-propenone; l-(4-Hydroxy-phenyl)-3-quinolin-2-yl- propenone; 1 -(2- Amino-phenyl)-3 -quinolin-2-yl-propenone; 1 -(4- Amino-phenyl)-3 - quinolin-2 -yl-propenone; 4-(3-Quinolin-2-yl-acryloyl)-benzamide; 4-(3-Quinolin-2-yl- acryloyl)-benzoic acid; 3-(8-Methyl-quinolin-2-yl)-l-pyridin-4-yl-propenone; l-(2-Fluoro- pyridin-4-yl)-3 -quinolin-2 -yl-propenone; 3-(8-Fluoro-quinolin-2-yl)-l-pyridin-4-yl- propenone; 3-(6-Hydroxy-quinolin-2-yl)-l-pyridin-4-yl-propenone; 3-(8-Methylamino- quinolin-2-yl)- 1 -pyridin-4-yl-propenone; 3 -(7-Methyl-quinolin-2-yl)- 1 -pyridin-4-yl- propenone; and l-Methyl-4-[3-(8-methyl-quinolin-2-yl)-acryloyl]-pyridinium.

[0079] Further exemplary inhibitory compounds include: PFK15 (l-(4-pyridinyl)-3-(2- quinolinyl)-2-propen-l-one); (2S)-N-[4-[[3-Cyano-l-(2-methylpropyl)-lH-indol-5- yl]oxy]phenyl]-2-pyrrolidinecarboxamide 3PO (3-(3-Pyridinyl)-l-(4-pyridinyl)-2-propen-l- one); (2S)-N-[4-[[3-Cyano-l-[(3,5-dimethyl-4-isoxazolyl)methyl]-lH -indol-5- yl]oxy]phenyl]-2-pyrrolidinecarboxamide; and Ethyl 7-hydroxy-2-oxo-2H-l-benzopyran-3- carboxylate.

III. THERAPEUTIC METHODS [0080] The methods described herein may be used to treat or prevent protein misfolding disorders by inhibition of PFKFB3.

[0081] The diseases amenable for treatment include, but are not limited to those tabulated below with their major aggregating protein.

IV. PHARMACEUTICAL COMPOSITIONS

[0082] Embodiments include methods for treating cancer with compositions comprising a PFKFB3 inhibitor. Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous injection. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, or about 25% to about 70%. In some embodiments, the compositions are administered orally. [0083] Typically, compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0084] The manner of application may be varied widely. Any of the conventional methods for administration of a pharmaceutical composition are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject. [0085] In many instances, it will be desirable to have multiple administrations of at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations may range from 2 day to twelve week intervals, more usually from one to two week intervals. The course of the administrations may be followed by assays for PFKFB3 activity.

[0086] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.

[0087] The PFKFB3 inhibitors can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intradermal, intramuscular, sub-cutaneous, or even intraperitoneal routes. In some embodiments, the composition is administered by intravenous injection. The preparation of an aqueous composition that contains an active ingredient will be known to those of skill in the art in light of the current disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0088] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0089] The compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0090] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0091] Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0092] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0093] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0094] Typically, for a human adult (weighing approximately 70 kilograms), from about 0.1 mg to about 3000 mg (including all values and ranges there between), or from about 5 mg to about 1000 mg (including all values and ranges there between), or from about 10 mg to about 100 mg (including all values and ranges there between), of a compound are administered. It is understood that these dosage ranges are by way of example only, and that administration can be adjusted depending on the factors known to the skilled artisan.

[0095] In certain embodiments, a subject is administered about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,

9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (meg) or μg/kg or micrograms/kg/minute or mg/kg/min or micrograms/kg/hour or mg/kg/hour, or any range derivable therein. [0096] A dose may be administered on an as needed basis or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivable therein). A dose may be first administered before or after signs of a condition. In some embodiments, the patient is administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of the condition (or any range derivable therein). The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of an the condition have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days after symptoms of an infection have disappeared or been reduced. V. COMBINATION THERAPY

[0097] The compositions and related methods, particularly administration of a PFKFB3 inhibitor may also be used in combination with the administration of conventional therapies, such as those known in the art and/or described below. For example, the current methods and compositions may be used in combination with tradition therapies for treating a protein misfolding disease such as type 2 diabetes. Traditional therapies for type 2 diabetes include metformin, sulfonylureas, such as glyburide, glipizide, and glimepiride (Amaryl), meglitinides such as repaglinide and nateglinide, thiazolidinediones such as rosiglitazone and pioglitazone, DPP-4 inhibitors such as sitagliptin, saxagliptin, and linagliptin, GLP-1 receptor agonists such as exenatide and liraglutide, SGLT2 inhibitors such as canagliflozin and dapagliflozin, insulin therapy such insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, and insulin isophane, and aspirin therapy.

[0098] Actual dosage levels of the active ingredients in the methods of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors, including the activity of the chemotherapeutic agent selected, the route of administration, the time of administration, the rate of excretion of the therapeutic agent, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0099] Administration of pharmaceutical compositions to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary.

[0100] Various combinations with the PFKFB3 inhibitor and a traditional therapy may be employed, for example, a PFKFB3 inhibitor is "A" and the traditional therapy (or a combination of such therapies) given as part of a treatment for a protein misfolding disorder, is "B": A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0101] Administration of pharmaceutical compositions to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. VI. KITS

[0102] Certain aspects concern kits containing compositions described herein or compositions to implement methods described herein.

[0103] In various aspects, a kit is envisioned containing therapeutic agents and/or other therapeutic and delivery agents. In some embodiments, a kit for preparing and/or administering a therapy described herein may be provided. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions, therapeutic agents and/or other therapeutic and delivery agents. In some embodiments, the kits comprise lipid delivery systems. In some embodiments, the lipid is in one vial, and the therapeutic agent is in a separate vial. The kit may include, for example, at least one inhibitor of PFKFB3 expression/activity, one or more lipid component, as well as reagents to prepare, formulate, and/or administer the components described herein or perform one or more steps of the methods. In some embodiments, the kit may also comprise a suitable container means, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made from sterilizable materials such as plastic or glass.

[0104] The kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill. The instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.

[0105] In some embodiments, kits may be provided to evaluate the expression of PFKFB3 or related molecules. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers and probes, nucleic acid amplification, and/or hybridization agents. In a particular embodiment, these kits allow a practitioner to obtain samples in blood, tears, semen, saliva, urine, tissue, serum, stool, colon, rectum, sputum, cerebrospinal fluid and supernatant from cell lysate. In another embodiment, these kits include the needed apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assays can also be included in the kits. [0106] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. The components may include probes, primers, antibodies, arrays, negative and/or positive controls. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, 10x, or 20x or more.

[0107] The kit can further comprise reagents for labeling PFKFB3 in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine-reactive dye or any dye known in the art.

[0108] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquotted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits may also include a means for containing the nucleic acids, antibodies or any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0109] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. [0110] Alternatively, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg or at least or at most those amounts of dried dye are provided in kits in certain aspects. The dye may then be resuspended in any suitable solvent, such as DMSO. [0111] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. [0112] The kits may include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.

[0113] A kit may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

VII. EXAMPLES

[0114] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. EXAMPLE 1: Targeting PFKFB3 rescues β-cells from islet amyloid pancreatic polypeptide (LAPP) toxicity

[0115] Type 2 diabetes (T2D) is characterized by a progressive defect in insulin secretion in the setting of relative insulin resistance (Cantley and Ashcroft, 2015). The mechanisms that initiate β-cell dysfunction in T2D remain unclear, partly because there is no means to access the human pancreas preceding diabetes. Pathological studies in individuals that have T2D reveal a partial β-cell deficit with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed and secreted with insulin by β-cells (Butler et al., 2003; Clark et al., 1987; Clark et al., 1988; Junker et al., 1977). In common with other protein misfolding diseases, the most toxic forms of IAPP aggregates are small membrane permeant oligomers (Gurlo et al., 2010; Janson et al., 1999; Kegulian et al., 2015). β-cell dysfunction in T2D shares characteristics of affected cells in other protein misfolding diseases, for example Alzheimer's disease and Parkinson's disease.

[0116] These characteristics include disruption of the mitochondrial network leading to reactive oxygen species (ROS)-mediated DNA damage, endoplasmic reticulum stress and a chronic inflammatory response, including activation of HIFla (Byun et al., 2015; Cook et al., 2012; Gurlo et al., 2010; Zhang et al., 2007). Both glucose and lipid metabolism are abnormal, along with aberrant Ca2+ signaling leading to calpain hyperactivation (Atherton et al., 2014; Huang, 2010). The autophagy-lysosome pathway and ubiquitin-proteasome pathway are altered in both β-cells in T2D and affected cells in neurodegenerative diseases, consistent with the accumulation of misfolded proteins (Cook et al., 2012; Costes et al., 2014; Costes et al., 2011; Rivera et al., 2011).

[0117] In the current example, the inventors employed a variety of tools to investigate the mechanisms subserving the toxic effects of human IAPP in β-cells of HIP rats, a transgenic model of type 2 diabetes. It is established that hIAPP induces major metabolic and mitochondrial network changes through the activation of the HIFla/PFKFB3 stress pathway that, whereas in the short term are aimed to preserve β-cell survival, longer term result in cell death mediated by cytosolic Ca2+ accumulation. The inventors further identify novel strategies to protect β-cells from IAPP toxicity by either constraining the HIFla/PFKFB3 induced metabolic changes or by inhibiting the cytosolic accumulation of Ca2+ consequent to the metabolic changes.

B. RESULTS

[0118] hIAPP toxic oligomers induce mitochondrial network fragmentation with reduced mitochondrial function, β-cells in T2D are characterized by a fragmented mitochondrial network, mitochondrial dysfunction and disrupted Ca2+ dynamics (Anello et al., 2005; Gurlo et al., 2016a; Gurlo et al., 2010; Lu et al., 2010). The inventors visualized β-cell mitochondria with Tom20 immunostaining in pancreatic sections from human T2D donors (FIG. 6A and B) and found that they were more fragmented and less dense (p<0.005) compared to β-cell mitochondria from non-diabetic (ND) donors (FIG. 6C), consistent with previous work (Anello et al., 2005). To investigate the potential role of toxic hIAPP oligomers in inducing these abnormalities, the inventors used INS 832/13 cells overexpressing hIAPP, a previously validated in vitro model (Gurlo et al., 2010). Furthermore, since the mitochondrial network and the dynamics of Ca2+ changes vary through the cell cycle, the inventors synchronized INS 832/13 cells at the Gl/S stage of the cell cycle (Oh post-aphidicolin release), as confirmed by FACS analysis (FIG. 13A and B) (Mitra et al., 2009). It was found that hIAPP induced fragmentation of the mitochondrial network (FIG. 6D and E). While mitochondrial network of control β-cells (CTRL) was extensively reticular and tubular throughout the entire cytoplasm (-70% of the cells), it became fragmented in cells overexpressing hIAPP (-50% of cells), with perinuclear clustering and a bipolar distribution being apparent (FIG. 6D and E). hIAPP toxicity was confirmed by cell death, as measured with flow cytometry at 12h postrelease from aphidicolin block by increase in subGl peak from 1% to 50% (FIG. 13B). Therefore, it was concluded that, in common with other amyloidogenic proteins, hIAPP induces significant mitochondrial network fragmentation (Gurlo et al., 2010; Jung and Lee, 2010; Lu et al., 2010; Ma et al., 2012). The inventors next investigated the impact of hIAPP overexpression on regulators of mitochondrial fission (dynamin related protein 1 ((Drpl)) and fusion (mitofusins 1/2 (MFN1/2) and optical atrophy related 1 (Opal)).

[0119] Immunoblotting of cell extracts from hIAPP overexpressing INS 832/13 cells revealed reduced protein levels of MFN2 (0.4 and 0.3-fold from 2 independent experiments FIG. 14 A) but no change in Drpl, implying reduced fusion was at least partly responsible for hIAPP induced mitochondrial fragmentation (FIG. 14A). This was further supported by the finding that overexpression of the dominant negative Drpl mutant K48A failed to protect against hIAPP-induced mitochondrial fragmentation (FIG. 14B). [0120] Given that hIAPP induced mitochondrial fragmentation, the inventors set out to determine whether changes in mitochondrial shape due to hIAPP were associated with changes in mitochondrial function by measuring cellular oxygen consumption and mitochondrial membrane potential.

[0121] The inventors measured the oxygen consumption rate (OCR) of islets isolated from 5-6 month old prediabetic HIP rats versus those from WT. The inventors observed a 30% decrease in OCR in response to 20 mM glucose in HIP rat islets (p<0.01) (FIG. 7A and B). To investigate if the hIAPP-induced decrease in mitochondrial function was mediated by a loss of mitochondrial membrane potential, the inventors treated INS 832/13 cells synchronized at Gl/S and S (Oh and 4h post-release from aphidicolin block) with tetramethylrhodamine, ethyl ester (TMRE) and performed FACS analysis. Visualizing the overlay of TMRE fluorescence from CTRL and hIAPP -transduced INS 832/13 cells indicated that there was no difference in the mitochondrial membrane potential of the two (not shown). However, the inhibition of mitochondrial ATPase activity by incubation with oligomycin (5 mM) or by restricting the availability of glucose due to exposure to 2-deoxyglucose (DOG, 2 mM) decreased the number of viable hIAPP overexpressing β-cells (FIG. 7C and D) while not affecting the number of viable control cells. This indicates that mitochondrial membrane potential is more vulnerable to additional toxic stimuli due to presence of hIAPP. [0122] hIAPP toxicity increases aerobic glycolysis disengaged from mitochondrial oxidative phosphorylation. Next, the inventors investigated the metabolic changes induced by hIAPP toxicity. Analysis of the microarray data (referred to also in Schludi et al., JCI Insight in press, GEO Accession number GSE90779) from WT and HIP transgenic rats showed upregulation of a subset of genes involved in aerobic glycolysis (lactate dehydrogenase A and C, LDHA and LDHC; phosphofructokinase L, PFKL; pyruvate kinase M2, PKM2 and 6- phosphofructo-2-kinase fructose 2,6 biphosphatase, PFKFB3) concomitant with the downregulation of genes involved in the TCA cycle (pyruvate carboxylase, PC; malate dehydrogenase, MDH; fumarate hydratase, FH; succinate dehydrogenase, SDH (FIG. 8 A). Moreover, while PFKFB l and PFKFB4 were both downregulated in the HIP islets (not shown), PFKFB3 was strongly upregulated (20- and 9.5-fold in two independent experiments, respectively), indicating a selective metabolic response upon hIAPP stress. Interestingly, HIFla was upregulated while its negative regulator Von-Hippel Lindau tumor suppressor was downregulated (FIG. 8 A). Consistent with this, a subset of HIFla targets were found to be differentially expressed, corroborating the engagement of the HIFla/PFKFB3 stress pathway in the response (FIG. 8 A).

[0123] To evaluate if hIAPP alters cellular metabolism, the inventors performed an unbiased metabolomics analysis in INS 832/13 cells overexpressing hIAPP versus rIAPP (used as a negative control). The changes in the metabolite composition induced by hIAPP included decreased levels of fructose 1,6-biphosphate (F16BP), glycerol-3 -phosphate (G3P), phosphoenolpyruvate (PEP), and increased levels of the glycolytic end products lactate and alanine, consistent with there being enhanced flux through glycolysis (FIG. 15 A). Interestingly, while a-ketoglutarate (a-KG) was reduced in cells overexpresing hIAPP, its intermediate 2-hydroxyglutarate (2-HG), which may divert glucose from entry into the TCA cycle (Harris, 2015; Oldham et al., 2015), was increased (FIG. 15A). Furthermore, the GSH/GSSG ratio was decreased, indicating redox stress in cells overexpressing hIAPP (FIG. 15 A). Together, the impairment of mitochondrial function shown by the Seahorse analysis, the reduced expression of the key TCA enzymes the inventors found in the microarray analysis, and the accumulation of succinate and 2-hydroxyglutarate were all in line with the molecular pathways of HIFla stabilization (Koivunen et al., 2007; Oldham et al., 2015; Xu et al., 2011).

[0124] To investigate the impact of hIAPP on the utilization of glucose, the inventors compared the mass isotopologue distribution (MID) of TCA intermediates using [U-13C6]- labeled glucose in INS 832/13 cells transduced with hIAPP vs. rIAPP adenoviruses. The intracellular M6 fraction of glucose was near 100%, demonstrating a high efficiency of labeling of glucose and its downstream metabolites (FIG. 15B). hIAPP did not alter the glucose labeling pattern as there was no difference in the M6 fraction compared to control rIAPP overexpressing cells (FIG. 15B). Linkage between glycolysis and the TCA cycle was disrupted by hIAPP as demonstrated by a relative decrease in the flux of pyruvate to different glucose-derived metabolites (with M0 - indicating no labeled carbons, and Ml to M5 - one to more labeled carbons) (FIG. 15B). The M2 fractions of fumarate (Fum) and a-ketoglutarate (a-KG) derived from the conversion of Pyr to acetyl-CoA after the first round of the TCA cycle, were decreased in hIAPP compared to rIAPP overexpressing cells. The M3 fraction of aspartate (Asp) (surrogate marker for oxaloacetate (OAA)), fumarate (Fum) and malate (Mai) were also reduced, suggesting decreased pyruvate anaplerosis in hIAPP compared to rIAPP overexpressing cells (FIG. 15B). In addition, M5 citrate was lower, suggesting a significant decrement in the use of OAA in consecutive rounds of the TCA cycle in the hIAPP cells (FIG. 15B). Interestingly, the M0 (no labelled carbons) fraction of most TCA metabolites was increased in hIAPP cells, suggesting that another source of energy besides glucose is metabolized through the TCA cycle. Whereas the key pentose phosphate pathway (PPP) metabolite, glucose-6-phosphate/fructose-6-phosphate (G6P-F6P), was reduced (FIG. 15 A), inositol monophosphate (FMP) and the M5 isotopologues of ADP and ATP nucleotides were higher in hIAPP overexpressing cells, indicating increased contribution of de novo purine synthesis via the pentose phosphate pathway (FIG. 15C).

[0125] To corroborate the disengagement of glycolysis from the TCA cycle in β-cells overexpressing hIAPP, the inventors measured lactate production rate of isolated islets from WT and HIP rats. LDHA and MCT1 transcriptional expression were increased (p<0.05, FIG. 8B) in HIP versus WT islets as well as the lactate production rate; the latter was 20-fold higher in HIP islets (p<0.05 vs WT), revealing a metabolic switch to aerobic glycolysis (FIG. 8C).

[0126] Taken together, these results indicate that the linkage of glycolysis to mitochondrial oxidative phosphorylation and pyruvate anaplerosis in β-cells is disrupted by hIAPP toxicity (FIG. 15E). Of interest, the inventors' findings in β-cells expressing hIAPP mirror those previously reported in neurons exposed to toxic oligomers of amyloidogenic proteins, with increased aerobic glycolysis and lactate production (Newington et al., 2011), implying disengagement of glycolysis from mitochondrial respiration by protein misfolding. The inventors next sought to reveal the possible mechanism linking the presence of hIAPP toxic oligomers in the cell with the observed metabolic changes.

[0127] Increased expression of PFKFB3 in β-cells due to hIAPP-induced stress. The inventors reasoned that activation of aerobic glycolysis required enhanced glucose flux through glucokinase (a type IV hexokinase with high Km and low affinity for glucose). These features are promoted by 6-phosphofructo-2-kinase 2,6- fructose biphosphatase (PFKFB3) (Arden et al., 2008), an allosteric activator of the rate-limiting enzyme of glycolysis - phosphofructokinase 1 (PFKl) (Hue and Rider, 1987; Rider et al., 2004) and activator of β- cell glucokinase (Arden et al., 2008).

[0128] For detection of PFKFB3 the inventors utilized previously reported antibody (Lu et al., 2015), specificity of which the inventors confirmed by silencing PFKFB3 and measuring transcript and protein levels by western blot, qRT-PCR, and immunofluorescence (FIG. 16A- C). The inventors found that PFKFB3 protein levels were increased in isolated islets from HIP versus WT rats especially prior to diabetes onset at 6 months of age (FIG. 8D) consistent with a role of hIAPP in inducing glycolysis through enhanced PFKFB3 expression. The protein levels of PFKFB3 and PFKl were also increased in islets from hIAPP transgenic mice before the onset of hyperglycemia (2.6- and 2-fold vs WT, respectively) (not shown). PFKFB3 was also upregulated in hIAPP overexpressing INS 832/13 cells as shown by western blot (1.7-fold) and confirmed by immunocytochemistry (FIG. 16A-C). Most importantly, the inventors observed increase in PFKFB3 immunoreactivity in the nuclei of β- cells of humans with T2D compared to non-diabetic subjects, similar to what was observed in HIP rats (FIG. 9A-C) (p<0.05 vs. WT and p<0.005 vs ND). The finding of increased PFKFB3 expression was supported by finding higher levels of its upstream regulator, hypoxia inducible factor-la (HIFla) in the nuclear fraction of isolated islets from T2D humans (FIG. 9D). [0129] Recently, γΗ2Α.Χ was found to be a key for the activation of HIFla and its downstream targets, including PFKFB3 (Rezaeian et al., 2017). Interestingly, β-cells overexpressing hIAPP, coincident with increased aerobic glycolysis and fragmented mitochondria, also showed increased nuclear γΗ2Α.Χ (FIG. 8E and 16E). Increased frequency of γΗ2Α.Χ positive β-cells was also found in islets of pre-diabetic HIP rats (p<0.05 vs WT) (FIG. 8E) and humans with T2D (Tornovsky-Babeay et al., 2014).

[0130] Taken together, these findings indicate that hIAPP decouples glycolysis from oxidative respiration due to yH2A.X-associated accumulation of nuclear HIFla in INS 832/13 cells and β-cells from hIAPP transgenic rodents. To establish if the induction of PFKFB3 plays a role in hIAPP-induced β-cell toxicity, the inventors next set out to suppress PFKFB3 expression in β-cells overexpressing hIAPP.

[0131] Downregulation of PFKFB3 restores control metabolite composition and TCA flux via acetyl-CoA and stimulates PPP from lower isotopologue fractions. [0132] PFKFB3 silencing restored most metabolites to their control levels (FIG. 10A). ATP levels and ADP/ATP and AMP/ ATP ratios were also normalized, as well as lactate and palmitate levels, and precursors of nucleotide synthesis and metabolites of the thiopyruvate (homocysteine) pathway (FIG. 10A).

[0133] To investigate the impact of inhibition of PFKFB3 on the metabolic fate of glucose, the inventors analyzed the isotopologue distribution of [U-13C6]-labeled glucose in INS 832/13 cells overexpressing rIAPP or hIAPP, in the presence or absence of PFKFB3.

[0134] PFKFB3 silencing led to a decrease of pyruvate anaplerosis via OAA as demonstrated by a relative decrease in the M3 fractions of Mai, Asp, and Fum (FIG. 10B and FIG. 17A). However, the conversion of acetyl-CoA to Cit, a-KG and Fum via the generation of M2 isotopologues was increased, implying re-engagement of glycolysis with the TCA cycle (FIG. 10B). The M6 isotopologues of F16BP and G6P-F6P were suppressed by PFKFB3 silencing in cells overexpressing hIAPP (not shown). M3 fractions of G3P, PEP, Pyr and alanine were reduced (not shown), as well as the lactate levels (FIG. 10A) confirming that the PFKFB3 silencing reduced flux through the main glycolytic intermediates, as expected from the inventors' hypothesis.

[0135] M2 to M5- of G6P-F6P, M3 of ribulose-5-phosphate (R5P) and Ml to M6 isotopologues of sedoheptulose-7-phosphate (S7P) were increased in hIAPP overexpressing cells when PFKFB3 was silenced, indicating that the PPP was favored during β-cell rescue (FIG. 10B). [0136] The inventors concluded that inhibition of PFKFB3 promotes a partial re- engagement of glycolysis with the mitochondrial TCA cycle in β-cells overexpressing hIAPP by reinforcing metabolic flux through the acetyl-CoA while permitting further PPP. [0137] Since hIAPP remodels β-cell metabolism and ultimately leads to cell death (Gurlo et al., 2016a; Gurlo et al., 2016b; Huang et al., 2007; Saisho et al., 2011), the inventors next investigated the impact of PFKFB3 inhibition on the mitochondrial network integrity, DNA damage and cell death in cells overexpressing hIAPP. [0138] Inhibition of PFKFB3 restores mitochondrial morphology and reduces genotoxic stress and β-cell death. The re-engagement of glycolysis to the TCA cycle due to PFKFB3 silencing in INS 832/13 cells overexpressing hIAPP was associated with an increased number of cells exhibiting a fused-to-intermediate mitochondrial morphology (FIG. 11 A), a form known to maximize ATP production (Gomes et al., 2011; Tondera et al., 2009). [0139] Restoration of the mitochondrial network was accompanied by the suppression of hIAPP induced genotoxic stress as evaluated by propidium iodide uptake and γΗ2Α.Χ staining (FIG. 16E and F).

[0140] PFKFB3 suppression reduced cell death in INS 832/13 cells overexpressing hIAPP, as demonstrated by the reduction of the subGl peak in the flow cytometry histogram (46.3 ± 2.5% vs 20.7 ± 0.1%, p<0.005) (FIG. 1 IB). Similarly, the levels of cleaved caspase 3 and the caspase 3 derived PARP-1 fragment (89 KDa) were reduced upon PFKFB3 silencing (FIG. 11C).

[0141] Altogether, these results underlie the important role of PFKFB3 in mediating the toxic actions of hIAPP in β-cells. [0142] Since the hyperactivation of Ca2+-dependent protease calpain plays a proximal role in β-cell dysfunction and loss under conditions of hIAPP toxicity (Gurlo et al., 2016a; Huang et al., 2010), the inventors next investigated whether hIAPP affects Ca2+ levels in different subcellular compartments and whether these changes were abrogated by PFKFB3 inhibition (Gurlo et al., 2016a; Huang et al., 2010). [0143]

[0144] PFKFB3 silencing restores normal cytosolic levels of calcium. While it is known from previous work in the inventors' laboratory that hIAPP induces calpain hyperactivation, which is also a characteristic of β-cells in humans with T2D, the subcellular compartment(s) that exhibit aberrant Ca2+-induced calpain hyperactivation are unknown. One possibility is that enhanced glycolysis due to hIAPP leads to unregulated (e.g. independent of extracellular glucose) K(ATP) channel closure and therefore unregulated, sustained Ca2+ entry in the cell through voltage-gated Ca2+ channels. In INS 832/13 cells at GO, the presence of hIAPP led to an increase in cytosolic Ca2+ transients (p<0.001 vs CTRL), while not affecting ER or mitochondrial Ca2+ (FIG. 1 ID). Silencing PFKFB3 using a specific siRNA restored Ca2+ to control levels in the cytosol despite hIAPP overexpression (FIG. 11D). Interestingly, PFKFB3 silencing in non-stressed β-cells also reduced Ca2+ levels in the ER (p<0.005 vs CTRL) (FIG. 1 ID), supporting the proposed link between ER Ca2+ levels and flux through aerobic glycolysis (James et al., 2013).

[0145] The inventors reasoned that if hIAPP stimulates Ca2+ entry through membrane depolarization, which partly involves the PFKFB3 metabolic pathway, then using a K(ATP) small molecule opener, tifenazoxide NN-414, should mimic PFKFB3 inhibition. Interestingly, the pharmacologically-induced opening of K(ATP) channels resulted not only in the same FACS profile of β-cells as with PFKFB3 silencing (FIG. 18A and B), but it also appeared to restore the fused mitochondrial networks (FIG. 18C and D). These results indicate that aerobic glycolysis-dependent pathways of epistatic control of K(ATP) channel closure and membrane depolarization are linked to enhanced Ca2+ transients in β-cells overexpressing hIAPP (working model in FIG. 12B).

[0146] Inversely, γΗ2Α.Χ foci increased and also coincided with mitochondrial fragmentation upon closure of K(ATP) channels with glybenclamide (Gly) as applied alone or in combination with hIAPP (FIG. 18C and D). In line with these findings, glybenclamide maintained, while NN-414 reduced, the proportion of apoptotic cells observed in hIAPP background as measured by FACS analysis (FIG. 18A and B).

[0147] These results indicate that an important mechanism by which PFKFB3 silencing improves the survival of β-cells overexpressing hIAPP is through the suppression of abnormal cytosolic Ca2+ transients. This implies that hIAPP toxicity is mediated by an adverse cycle of sustained diversion of pyruvate to lactate and its deleterious impact on cellular Ca2+ dynamics. The impact of restoring the latter to resolve the actions of hIAPP toxicity on mitochondrial network and function, reduced flux through aerobic glycolysis and, with this, re-coupling of insulin secretion to glucose sensing via mitochondrial respiration implies a proximal role of the aberrant hIAPP induced Ca2+ signaling (FIG. 12A and B, scheme and working model). C. DISCUSSION

[0148] In this study the inventors uncovered that stress induced by hIAPP toxic oligomers recapitulates the metabolic phenotype reported in β-cells in T2D (Anello et al., 2005; Puri et al., 2009; Sivitz and Yorek, 2010) as well as in neurons in neurodegenerative diseases (Cook et al., 2012; Dunn et al., 2014; Newington et al., 2011; Zhang et al., 2007).

[0149] The main adaptive metabolic response is the disengagement of glycolysis from the mitochondrial TCA cycle along with fragmentation of the mitochondrial network. The inventors also establish that in common with some neurodegenerative diseases, this metabolic adaptation is mediated at least in part by the activation of the HIFla/PFKFB3 stress pathway. The HIFla stress response provides short term survival benefit in response to acute stress such as a hypoxic event. However, given the unique dependence of β-cell function on tight engagement of the glycolytic pathway with the TCA cycle, this adaptive change predictably causes impairment of β-cell function with relatively high insulin secretion at baseline glucose values (because of ATP generated by unrestrained glycolysis) but a deficient response to glucose stimulation, both characteristics of β-cells in T2D.

[0150] The increased flux through glycolysis under conditions of hIAPP toxicity is mediated by the actions of PFKFB3 to enhance flux through glucokinase (Agius, 1998; Ghesquiere et al., 2014; Massa et al., 2004) with the product pyruvate predominately redirected to lactate. The latter regenerates NAD for the maintenance of the redox state in mitochondria (Dawson et al., 1979). Also, under hIAPP toxicity the enhanced glucose flux through glycolysis was partitioned to a greater extent than in healthy β-cells through the pentose phosphate pathway (PPP), typically activated under conditions of stress or in the cell cycle to provide nucleotide precursors for DNA synthesis (and/or repair) and reducing equivalents. Therefore, β-cells exposed to hIAPP induced stress adopt a metabolic pattern that mimics the so called Warburg effect reported in cancer cells (Vander Heiden et al., 2009).

[0151] The inventors sought to test the postulate that suppression of accelerated glycolysis (by silencing PFKFB3) or by suppressing cytosolic Ca2+ uptake by inhibiting K(ATP) channel mediated membrane depolarization would protect β-cells against hIAPP toxicity. Silencing of PFKFB3 was indeed beneficial to both β-cell viability and function in the context of hIAPP toxicity. Silencing of PFKFB3 suppressed hIAPP-increased glycolytic flux, restored pyruvate incorporation into TCA to generate Ac-CoA, while still permitting the adaptive increase in the PPP pathway. The latter supports not only DNA repair but also increased formation of NAD and NADP for protection of β-cells against ROS to which they are particularly vulnerable (Lushchak, 2012). Moreover, higher availability of NAD+ by PFKFB3 downregulation may contribute to the observed restoration of the mitochondrial networks since Opal is activated via sirtuin 3 (Samant et al., 2014) in an NAD+ dependent manner. Increased NAD and NADP availability and the restored fused mitochondrial networks by PFKFB3 inhibition was predictably accompanied by reduced genotoxic stress as revealed by the γΗ2Α.Χ. Interestingly, reducing the levels of PFKFB3 and aerobic glycolysis restored cytosolic Ca2+ to levels observed in cells expressing nontoxic rIAPP. This implies that inhibition of aerobic glycolysis may reduce β-cell death by reinstating the Ca2+ homeostasis.

[0152] To address this, the inventors employed a highly specific K(ATP) channel inhibitor NN-414 in β-cells exposed to toxic hIAPP oligomers. Remarkably, this strategy was equally as effective as suppression of PFKFB3 in protecting β-cells against hIAPP toxicity, supporting the postulate that the unrestrained ATP production by accelerated glycolysis plays a proximal role in hIAPP induced toxicity. This conclusion is further supported by findings in β-cells in humans with congenital hyperinsulinism caused by glucokinase mutations that result in enhanced flux through glycolysis. In childhood such individuals are vulnerable to hypoglycemia due to the dysregulated insulin secretion but with time they are vulnerable to diabetes due to β-cell loss mediated through enhanced Ca2+ signaling assumed to be secondary to unrestrained glycolysis (Tornovsky-Babeay et al., 2014).

[0153] Altogether, these results indicate that β-cells exposed to the hIAPP protein misfolding are again seen to share much in common with neurons similarly afflicted by toxic aggregates of locally expressed amyloidogenic proteins. In both β-cells and neurons, a comparable adaptive stress response is engaged that is designated to achieve short term benefit of protecting cells from acute injury at the expense of function but given the sustained nature of the toxicity, with time the stress response contributes to cell loss. This data demonstrates that PFKFB3 inhibition is beneficial to β-cells and neurons exposed to toxic oligomer induced stress by restoring glucose metabolism and its engagement to an interconnected mitochondrial network.

D. Methods

[0154] Cell culture. The rat insulinoma cell line INS 832/13 was provided by Dr. Christopher Newgard (Duke University, Durham, NC). INS 832/13 cells were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 100 IU/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), 10% heat- -inactivated fetal calf serum (FCS) (Gemini Bio—Products, West Sacramento, CA, USA), and 50 μΜ β-mercaptoethanol (Sigma, St. Louis, MO, USA) at 37 °C in a humidified 5% C0 ₂ atmosphere.

[0155] Cell cycle synchronization. INS 832/13 cells were plated in culture medium with 10% FCS for 24h. Medium was then replaced with fresh medium containing 0.1% FCS for 56h to allow cells to reach the GO out-of-cycle state. Synchronization of cells in Gl/S, S and G2/M stages of cell cycle was carried out as follows: after 24h in medium containing 10% FCS, cells were maintained in culture medium + 0.1% FCS for 56h. Medium was replaced with fresh medium + 10% FCS and, 12h later, aphidicolin was added. After 12h treatment with aphidicolin (Sigma A0781, St. Louis, MO, USA), the medium was replaced with medium containing 10% FCS w/o aphidicolin and the cells were collected at Oh, 4h, 12h after aphidicolin release. Cell cycle distribution was determined based on flow cytometry profiling of DNA content.

[0156] Cell Cycle Distribution Analysis by Flow Cytometry. Cells were trypsinized, washed with ice-cold PBS, and fixed in 80% methanol at—20 °C for at least 2h. Cells were stained with propidium iodide (50 μg/ml) in presence of RNase A (50 μg/ml) in PBS for 30 min at 37 °C after methanol was removed by centrifugation at 2000 g for 2 min. DNA content analysis was performed using NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) equipped with the NovoExpress software.

[0157] Scheme of treatments. In experiments involving cells synchronized in GO, adenoviruses, siRNA, plasmids or drugs were applied 36 hr before the end of 56h culture in medium containing 0.1% FCS.

[0158] Adenoviruses. Cells were transduced with rodent IAPP (rIAPP) or human IAPP (hIAPP) adenoviruses (Huang et al 2010) (100 MOI [multiplicity of infection]) for 24h or 36h. Small interfering RNA. PFKFB3 small interfering RNAs (siRNAs) (L--095107-02- -0005) were purchased from Dharmacon, Lafayette, CO, USA.

[0159] Plasmids. Drpl K48A plasmid containing a dominant negative mutation in Drpl gene was kindly provided by Dr. Takehiro Yasukawa (University College London, London, UK).

[0160] Drugs. The potassium channel opener, NN— 414 (6— chloro— 3— (1- -methylcyclopropyl)amino-4H~thieno[3,2~e]~l,2,4~thiadiazine 1,1—dioxide) (Sigma SML0553, St. Louis, MO, USA), the sulfonylurea derivate, Glybenclamide (Sigma G0639, St. Louis, MO, USA) were dissolved in dimethylsulfoxide (DMSO) to prepare 3 mM and 25 mM stock solutions. The working concentrations were 3 μΜ for NN414 and 100 μΜ for Glybenclamide. Oligomycin (5 mM) (Sigma 04876, St. Louis, MO, USA) and 2-deoxy- -glucose (2—DOG, 1 mM) (Sigma D6134, St. Louis, MO, USA) were used in experiments evaluating the mitochondrial membrane potential. Final concentration of DMSO in medium was <0.04%.

[0161] Mitochondrial membrane potential. Cells synchronized in Gl/S or S phase of cell cycle were washed with PBS and trypsinized. One million cells from each sample were incubated for 15 min at 37 °C with tetramethylrhodamine ethyl ester (TMRE) (10 nM, Sigma 87917, St. Louis, MO, USA). Afterwards cells were centrifuged at 2000 g for 2 min, TMRE solution was removed and cells were resuspended in fresh culture medium. Mitochondrial membrane potential was measured using NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). Data were analyzed by NovoExpress software.

[0162] Mitochondrial network. INS 832/13 cells were grown on coverslips and incubated with the cell—permeant mitochondria—specific red fluorescent probe MitoTracker Red CMXRos (MTR) (Cell Signaling Technology 9082P, Danvers, MA, USA,) at a final concentration of 50 nM at 37 °C for the last 30 min in culture. Cells were then washed with PBS and fixed in 100% methanol at -20 °C for 20 min. Images were taken under a 63X objective with the AxioImager.M2a fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with the optical sectioning system ApoTome.2 and software ZEN2. At least 500 cells per group were analyzed to quantify the mitochondrial architecture. Mitochondrial morphology was classified as fused~to~intermediate if fused mitochondria occupied >50% of the mitochondrial area and fragmented if fragmented mitochondria were present in >50% of the mitochondrial area. Mitochondrial morphology was independently scored by two observers (CM. and K. V ). [0163] Calcium measurements. Cells were seeded on glass coverslips and synchronized in GO. For cytosolic free calcium measurements, cells were loaded with 2.5 μΜ fura 2- AM for 45 min in medium containing 11 mM glucose. For mitochondrial and ER free calcium measurements, cells were transduced with adenovirus expressing specific probes. For all measurements, cells on coverslips were placed into a 1 ml perifusion chamber containing saline with 11 mM glucose for 7 min, followed by 10 min of perifusion at 0.3 ml/min. Experiments were carried out at 32-34°C using in-line solution and chamber heaters (Warner Instruments, Hamden, CT, USA). Excitation was provided by a TILL Polychrome V monochromator . Excitation (x) or emission (m) filters (ETtype;; Chroma Technology, Bellows Falls, VT, USA) were maintained in combination with a FF444/521/608- -DiOldichroic (Semrock, Lake Forest, IL, USA) as follows: fura2, 340/10x and 380/10x, 535/30m (R340x/380x -535m);; D4ER, 430/24x, 470/24m and 535/30m (430x - R535m/470m);; Mito—Pericam, 480/410. Fluorescence emission was collected with a QuantEM:512SC camera (PhotoMetrics, Tucson, AZ, USA) or an ORCA-1 camera (Hamamatsu, Skokie, IL, USA) at 0.125-0.2 Hz. 80-130 cells were analyzed per/sample and data were analyzed using Metafluor software (Molecular Devices, Sunnyvale, CA, USA).

[0164] Immunocytochemistry and morphometrical analysis. 300,000 cells were seeded on coverslips in 6~well plate and synchronized as previously described. Cells were fixed with 4% PFA for 10 min at room temperature. After washing, cells were permeabilized with 0.4% Triton X-100/Tris~buffered saline for 15 min at room temperature, blocked with 3% bovine serum albumin, 0.2% Triton X~100/Tris~buffered saline for 1 hr at room temperature and incubated overnight at 4 °C with primary antibodies. Secondary antibodies were applied for lh at room temperature. For the PI staining, cells were incubated with 0.5 μΜ propidium iodide (PI, Molecular Probes, Eugene, OR, USA) for 20 min at 37 °C as previously described (Huang et al., 2010) and then fixed with 4% PFA. Coverslips or slides were mounted using Vectashield with DAPI (Vector Laboratories, H-1200, Burlingame, CA, USA). The frequency of cell death was evaluated after staining with PI or with MTR and antibody against γΗ2Α.Χ. 25 fields per section were imaged using a Leica DM6000 fluorescent microscope (Wetzlar, Germany) with a 20X objective equipped with a OpenLab 5.5 software (Improvision, Coventry, UK). Only cells that had two—thirds or more of the nuclear area covered by PI or γΗ2Α.Χ staining were considered positive. The frequency was expressed as percentage of cells expressing the marker of interest over the total cells counted. Image analysis was performed blindly by two independent investigators (K.V. and CM.). [0165] U- ¹³ C— glucose tracing and HPLC--MS analysis. For metabolomic analysis, cells were incubated in medium containing [U--13C6] glucose (Cambridge Isotope Laboratories CML1396, Tewksbury, MA, USA) for 24h. To extract intracellular metabolites, cells grown in 6— well plate were briefly rinsed with 2 ml of ice-cold 150 mM ammonium acetate (pH=7.3), before addition of 1 ml of ice— cold 80% methanol. Cells were scraped and transferred into Eppendorf tubes and then 5 nM D/L— norvaline was added. After vortexing at maximum velocity, samples were spun at 20,000g for 5 min at 4 °C. Supernatant was then moved into a glass vial, dried using speedvac centrifuge, and reconstituted in 50 μΐ 70% acetonitrile. 5 μΐ of each sample was injected onto a Luna H ₂ (150 mm x 2 mm, Phenomenex, Torrance, CA, USA) column. Samples were analyzed with an UltiMate 3000RSLC (Thermo Scientific, Waltham, MA, USA) coupled to a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA). The Q Exactive was run with polarity switching (+3.00kV /—2.25 kV) in full scan mode with an m/z range of 70—1050. Separation was achieved using 5 mM NH4AcO (pH 9.9) and ACN. The gradient started with 15% NH4AcO and reached 90% over 18 min, followed by an isocratic step for 9 min and reversal to the initial 15% NH4AcO for 7 min.

[0166] Animal models. Animal studies were performed in compliance with the guidelines of the UCLA Office of Animal Research Oversight. 9 months old WT, rIAPP (rTG) or hIAPP (hTG) transgenic male mice and WT, hIAPP (HIP) transgenic male rats between 2 to 6 months of age were generated in Dr. Peter Butler's laboratory (UCLA, Los Angeles, CA), as previously described (Butler et al., 2004;; Soeller et al., 1998). Littermates of the same sex were randomly assigned to experimental groups.

[0167] Islet isolation. After an overnight fast, animals were euthanized using isoflurane. The bile duct was cannulated, and a Hanks' balanced salt solution (HBSS) (Invitrogen,

Carlsbad, CA, USA) containing 0.23 mg/ml liberase (Roche 05401020001, Basel, SUI), and

0.1 mg/ml DNase (Roche 10104159001, Basel, Switzerland) was injected in the pancreas.

The pancreas was then removed and transferred into a glass vial containing ice-cold liberase solution, digested for 20 min at 37 °C, and dispersed by shaking for 30 s. Islets were manually picked and cultured in RPMI 1640 medium (11 mM glucose) supplemented with

100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% FCS. Islets were studied within 2 days of isolation.

[0168] Lactate measurements. Medium from cultured rodent islets was sampled every hour within 4 hours and lactate was analyzed using an enzymatic assay (Trinity Biotech 732- 10, Bray, Ireland) according to the manufacturer ^' s instructions. Islets were collected for protein extraction. Lactate production (per μg protein) was expressed as the hourly change in the accumulated amount of lactate.

[0169] Mitochondrial function. Oxygen consumption rate (OCR) was determined using the Seahorse XF Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). After an overnight recovery, isolated islets from WT and HIP rats were seeded (25—50 islets per well) into the V7 plate (Seahorse Bioscience, North Billerica, MA, USA). To assess mitochondrial function, OCR was measured at the basal state and after stimulation with 20 mM glucose and sequential injection of oligomycin (ATP synthase inhibitor), carbonyl cyanide-p trifluoromethoxyphenylhydrazone (FCCP; uncoupler), and rotenone (complex I inhibitor).

[0170] Human subjects. Pancreata were procured from brain dead organ donors by the JDRF Network for Pancreatic Organ Donors with Diabetes (nPOD), administered by the University of Florida, Gainesville, Florida. All procedures were in accordance with federal guidelines for organ donation and the University of Florida Institutional Review Board. Three pancreata from individuals with type 2 diabetes (T2D) (6186, 6275, 6255) and 3 from nondiabetic (ND) (6104, 6288, 6020) controls matched by age, sex and BMI were examined in this study.

[0171] Human islets. Human pancreatic islets were from the Islet Cell Resource Consortium. They were derived from 1 T2D brain—dead organ donor from the University of Pennsylvania, 1 T2D donors from Southern California Islet Cell Resources Center (City of Hope) (see table— HI128), 1 T2D from the University of Wisconsin (see table— HI130), 1 non diabetic brain— dead organ donor from Southern California Islet Cell Resources Center (City of Hope) (see table— HI126) and 1 non diabetic brain— dead organ donor from the University of Pennsylvania (see table— HI131). Dithizone staining was performed to assess the islet purity that was 90-95%. The donors, aged 25-60 years, were heart— beating cadaver organ donors. Islets were cultured in RPMI 1640 medium (5.5 mM glucose) containing 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) for one day and then processed for western blotting analysis.

[0172] Methods details. Antibodies. The following antibodies were used: anti-PFKFB3 (Abeam 181861, Cambridge, UK, 1 :200 for IF, 1 : 1000 for WB) anti-yH2A.X (Cell Signaling Technology 2577S, Danvers, MA, USA, 1 :200 for IF, 1 : 1000 for WB in human tissue), anti-yH2A.X (Abeam 26350, Cambridge, UK, 1 :200 for IF, 1 : 1000 for WB in rodent tissue), anti-Tom20 (Santa Cruz Biotechnology sc-11415, Dallas, TX, USA, 1 :200 for IF), anti-HIFla (NOVUS Biologicals NB100-105, Littleton, CO, USA, 1 : 1000 for WB), anti-MFN2 (Cell Signaling Technology 9482S, Danvers, MA, USA, 1 : 1000 for WB), anti- -Opal (BD [0173] Transduction 612606, San Diego, CA, USA, 1 : 1000 for WB), anti-Drpl (Cell Signaling Technology 8570S, Danvers, MA, USA, 1 : 1000 for WB), anti-nucleolin (Santa Cruz Biotechnology sc— 13057, Dallas, TX, USA, 1 : 1000 for WB), anti— cleaved caspase 3 (Cell Signaling Technology 9661 S, Danvers, MA, USA, 1 : 1000 for WB), anti-PARPl (Cell Signaling Technology 9542S, Danvers, MA, USA, 1 : 1000 for WB), anti-insulin (DAKO A0564, Glostrup, Denmark, 1 :200 for IF) and anti-GAPDH (Cell Signaling Technology 2118S, Danvers, MA, USA, 1 : 1000 for WB). Secondary antibodies for immunofluorescence staining were F(ab')2 conjugates with Cy3 or FITC purchased from Jackson Laboratories and used at dilution of 1 :200. qRT-PCR. The levels of PFKFB3, LDHA and MCT1 mRNA were quantified by qRT— PCR. Total RNA was isolated using a RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 250 ng of total RNA from each sample was denatured at 65 °C and then reverse transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) at 50 °C for lh. Real-time quantitative polymerase chain reaction (qPCR) was performed using ABI7900HT (Applied Biosystems™, Foster City, CA, USA) with initial denaturation at 95 °C for 20 s, followed by 45 cycles of 94 °C for 1 s and 60 °C for 20 s, then continued with a dissociation stage. Each qPCR reaction contained 1 ^χ Fast SYBR® Green Master Mix (Applied Biosystems™, Foster City, CA, USA), 1 μΜ of each primer, and 400 ng cDNA. Relative mRNA expression of target gene was determined using the comparative cycle threshold (Ct) method, where the amount of target cDNA was normalized to the internal control, GAPDH cDNA. The primers used were : PFKFB3 (fwd: CACGGCGAGAATGAGTACAA (SEQ ID NO:3), rev: TTCAGCTGACTGGTCCACAC (SEQ ID NO:4)) (Arden et al., 2008); LDHA (fwd: TGC TGG AGC CAC TGT CG (SEQ ID NO:5), rev: CTG GGT TTG AGA CGA TGA GC (SEQ ID NO:6)) (Laybutt et al., 2003); MCT1 (fwd: ATG TAT GCC GGA GGT CCT ATC (SEQ ID NO:7), rev: CCA ATG GTC GCT TCT TGT AGA (SEQ ID NO:8)) (Smith and Drewes, 2006) and GAPDH (fwd: ATG ACT CTA CCC ACG GCA AG (SEQ ID NO:9), rev: CTG GAA GAT GGT GAT GGG TT (SEQ ID NO: 10)). [0174] Tissue immunostaining and morphometrical analysis. 4-μm paraffin tissue sections from human or rodent samples were exposed to toluene for 10 min and, then, to 100% ethanol for other 10 min, 95% ethanol and 70% ethanol for 5 min each, and water. Sections were transferred in heat— induced antigen retrieval solution in citrate buffer at pH 6.0, using microwave and then cooled to room temperature for 1 hour, then soaked in Soaking Buffer (TBS, 0.4% TX100) for 30 minutes on ice, and washed once with TBS. After blocking the unspecified binding sites with a blocking solution (TBS, 3% BSA, 0.2% TX100) for 1 hour, the slides were incubated with the primary antibodies diluted in Antibody Buffer (TBS, 3% BSA, 0.2% Tween-20) overnight at +4°C. After washing in TBST, slides were incubated with secondary antibodies diluted in Antibody Buffer for 1 hour at room temperature. Slides were then mounted using Vectashield with DAPI. The presence of PFKFB3 in the islets was evaluated in pancreatic sections immunostained for PFKFB3 and insulin. Images of 25 islets per sample were taken using a Leica DM6000 fluorescent microscope (Wetzlar, Germany) with a 20X objective equipped with a OpenLab 5.5 software (Improvision, Coventry, UK). The frequency of nuclear PFKFB3 staining was expressed as a percentage of β—cells expressing PFKFB3 only in the nuclei. Nuclei were considered positive for PFKFB3 staining only if two-thirds of their area was occupied by multiple bright puncta of the marker of interest. The frequency of both cytoplasmic and nuclear PFKFB3 staining was expressed as percentage of β-cells expressing the PFKFB3 in the cytoplasm and nuclei. Image analysis was performed blindly by two independent investigators (K.V. and CM.). To visualize mitochondria in human pancreatic tissue, sections were stained with Tom20. The mitochondrial area was quantified using the Image—Pro Premier 9.1 software (Rockville, MD, USA) and expressed as Tom20 positive area, inside the insulin positive area in the islet, divided by the number of β-cells.

[0175] Western blotting. To prepare whole cell extracts, cells or islets were incubated for 20 min on ice in NP40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM MgC12, 0.5% NP--40, 1 mM DTT, 5 mM NaF, 1 mM Na3V04, and protease inhibitor cocktail (Sigma P2714, St. Louis, MO, USA), sonicated, and spun at 10,000 g at 4 °C for 10 min. To separate cytoplasmic and nuclear protein fractions, after incubation in NP40 lysis buffer, samples were centrifuged at 3,500 g at 4 °C for 10 min. Then, supernatant representing the cytoplasmic part was transferred in another eppendorf whereas the pellet (nuclear part) was resuspended in RIPA lysis buffer. Protein concentration was determined using the DC protein assay kit (Bio- -Rad, Irvine, CA, USA). Proteins (30-35 μg/lane) were separated by SDS--PAGE (4-20%) and, then, transferred onto polyvinylidene fluoride membranes (Bio— Rad, Irvine, CA, USA) by semi-dry electroblotting. After blocking with 5% milk for lh, membranes were probed overnight at 4 °C with primary antibodies. Then, membranes with transferred protein were incubated with horseradish peroxidase-conjugated secondary antibodies for lh at room temperature (Invitrogen, Carlsbad, CA, USA). Proteins were visualized using ECL reagents from Bio— Rad and expression levels were quantified using the Labworks software (UVP).

[0176] Statistical analysis. Results are expressed as the means ± SEM. The statistical analysis was performed by two— tailed t test or one— way ANOVA using GraphPad Prism V software (La Jolla, California, USA). A value of P < 0.05 was taken as an evidence of statistical significance.

TABLE 1. Characteristics of pancreatic tissue donors

TABLE 2, Characteristics of human islets donors

[0177] Although certain embodiments have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention. Further, where appropriate, aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. Any reference to a patent publication or other publication is a herein a specific incorporation by reference of the disclosure of that publication. The claims are not to be interpreted as including means-plus- or step-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase(s) "means for" or "step for," respectively.

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