PEPTIDE-DERIVED THERAPEUTICS TARGETING KDM5C FOR THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates to treatment of cancer. In particular, the present invention relates to peptide-derived therapeutics for the treatment of cancer.

BACKGROUND

Two of every five Canadians are diagnosed with cancer at some point in their lives (Canadian Cancer Society, Cancer Statistics 2016). For the majority of cancers, targeted therapies are not yet available. For example, systemic chemotherapy is the only treatment option for triple negative breast cancer after surgery. However, chemotherapy is highly toxic and cancer cells can eventually become resistant to the treatment.

It is known that one gene mutation or one protein dysfunction does not initiate the development of cancer, but rather it is the dysregulation of a system of proteins that initiate the process and drives progression. As a result, there is an urgent need to understand the mechanism of cancer progression and chemoresistance in order to develop strategies to overcome resistance. Lysine methylation is essential in regulating many biological processes that range from growth and proliferation to pathological conditions, such as neurodegenerative disease, intellectual disability, and cancer. Given the extensive regulatory importance realized for lysine methylation, any mutations or dysfunction in methyltransferase (KMT) or demethylase (KDM) enzymes (i.e. , the enzymes that catalyze the addition/removal of lysine methylation) can lead to deregulated cell function, tumourigenesis and chemotherapy resistance (Arrowsmith et al., 2012; Hanamoto et al., 2015; Rao and Dou, 2015).

The realization that lysine methylation plays a critical role in the development of many human diseases is perhaps not a surprising one. It is well established that dynamic post-translational modifications (PTMs) made to protein, such as phosphorylation and methylation, play a crucial role in the transmission of biological signals (Seo and Lee, 2004; Beck-Sickinger and Mori, 2006; Zhang et al., 2012). These small chemical protein modifications allow for cells to exert greater control over specific cellular processes, while dysfunction within this PTM network are common drivers of cancer development and progression (Jin and Zangar, 2009). Dysfunction in the dynamic lysine methylation network (currently consisting of >5000 different lysine methylation modifications) has been identified as a prominent contributor to the development of many different types of cancer. Given the involvement of lysine methylation in a growing number of different biological processes (Biggar and Li, 2015), methyl-modifying enzymes are emerging as a promising drug target.

To date, only a handful of KMT and KDM inhibitors have been discovered or developed, with almost all inhibitors currently within the preclinical stages of development (Hanamoto et al. , 2015). Indeed, given the similarity between catalytic domains among families of these enzymes, it has been difficult to develop a small molecule inhibitor that is specific for a dysfunctional enzyme without significant off-target effects. Given the potential for substantial off-target toxicity, there is a critical need for more refined, enzyme-specific, inhibitors to be developed. Peptide- based therapeutics may be designed with exquisite specificity for their targets. This results in fewer side-effects from treatment. Peptide-based drugs also offer good efficacy, tolerability, predicted metabolism, lower attrition rates, and the advantage of a standard synthesis protocol.

SUMMARY OF THE INVENTION

An object of the present invention is to provide peptide-derived therapeutics targeting KDM5C for the treatment of cancer. In one aspect of the present invention, there is provided a peptide that binds to KDM5C.

In another aspect of the present invention, there is provided a peptide that binds to KDM5C, wherein said peptide comprises the sequence:

X1X2X3X4X5X6X7X8 X ₉ ; where

X,=T or S

X ₂ =D, E or I

Xa=T, D or Q

X ₄ —Q, S, N or T

Xs=K or Nle

Xe=T

X ₇ =H

Xs=H

Xg=H; or a binding fragment thereof. In accordance with another aspect of the invention, there is provided a peptide that binds to

KDM5C and comprises the sequence selected from the group consisting of TDTTKTHHH;

TDTQKTHHH;TDTNKTHHH;TEDSKTHHH;TEDQKTHHH;TTQSKT

HHH;TDTSKTHHH;TEDTKTHHH;TEEQKTHHH;SDQQKTHHH;TT

QQKTHHH;SDQTKTHHH;TDDQKTHHH;TEENKTHHH;TEESKTH

HH;TTQTKTHHH;TDSTKTHHH;SETQKTHHH;SETSKTHHH;TDD

NKTHHH;TDTTnTHHH;TDTQnTHHH;TDTNnTHH H;TEDSnTHHH;

TEDQnTHHH;TTQSnTHHH;TDTSnTHHH;TEDTnTHHH;TEEQnTH

HH;SDQQnTHHH;TTQQnTHHH;SDQTnTHHH;TDDQnTHHH;TEE

NnTHHH;TEESnTHHH;TTQTnTHHH;TDSTnTHHH;SETQnTHHH;

SETSnTHHH;TDDNnTHHH;RTKQTARKSTGG;RTnQTARKSTGG;

GAKRHRKVLRDNI and GAKRHRnVLRDNI; wherein n= norLeucine (Nle) or a binding fragment thereof.

In accordance with another aspect of the invention, there is provided a peptide that binds to KDM5C, wherein said peptide comprises the sequence:

X1X2X3X4X5X6X7X8 X ₉ ; where

Xi=T

X ₂ =K, G, L, Q, V, E, H or I

X3— I , L, VorD

X4— L, M, V, F orS

X ₅ =V or K

X _e =v, G, R, L, K, F, H, T, A, P or N

X ₇ =H

Xs=H

Xg=H; or a binding fragment thereof

In accordance with another aspect of the invention, there is provided a peptide comprising the sequence selected from the group consisting of:

TDTNnTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TDTQKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TDTN KTH H H{6-am inohexanoic acid}GRKKRRQRRRPPQ;

TDTSnTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TEDSKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ; TDTSKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TDTT nTH H H{6-ami nohexanoic acid}GRKKRRQRRRPPQ;

TEDQnTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TTQSKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TEDQKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

GAKRHRnVLRDNI{6-aminohexanoic acid}GRKKRRQRRRPPQ; SETQnTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

SDQQKTHHH{6-aminohexanoic acid}GRKKRRQRRRPPQ;

TDTNnTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTQKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTNKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTSnTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TEDSKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTSKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTTnTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TEDQnTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TTQSKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TEDQKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; GAKRHRnVLRDNI{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; SETQnTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; SDQQKTHHH{6-aminohexanoic acid}FFLIPKGRRRRRRRRR; TDTNnTHHH{6-aminohexanoic aci d} R R WR R WR R WR R ;

TDTQKTHHH{6-aminohexanoic aci d} R R WR R WR R WR R ;

TDTNKTHHH{6-aminohexanoic acid}RRWRRWRRWRR;

TDTSnTHHH{6-aminohexanoic aci d} R R WR R WR R WR R ;

TEDSKTHHH{6-aminohexanoic acid}RRWRRWRRWRR;

TDTSKTHHH{6-aminohexanoic aci d} R R WR R WR R WR R ;

TDTT nTH H H{6-ami nohexanoic aci d} R R WR R WR R WR R ;

TEDQnTHHH{6-aminohexanoic acid}RRWRRWRRWRR;

TTQSKTHHH{6-aminohexanoic acid}RRWRRWRRWRR;

TEDQKTHHH{6-aminohexanoic acid}RRWRRWRRWRR;

GAKRHRnVLRDNI{6-aminohexanoic acid}RRWRRWRRWRR; SETQnTHHH{6-aminohexanoic aci d} R R WR R WR R WR R ; and SDQQKTHHH{6-aminohexanoic aci d} R R WR R WR R WR R . In other aspects of the present invention, there is provided methods of inhibiting the activity of KDM5C in a subject in need thereof or methods of treating a disease associate with increased KDM5C, including but not limited to cancer, in a subject in need thereof, comprising administering one or more of the peptides of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Peptide array screening for the systematic identification of peptide sequences that display interaction with KDM5C. Relative KDM5C interaction with an oriented peptide array library (OPAL). The relative intensity suggests a positive selection for the substituted residue at a given position of the peptide. The OPAL is derived from a library with equimolar mix of amino acids within a degenerate sequence with position-specific fixed amino acids. Degeneracy is then reduced in subsequent peptide arrays. Sequence motifs indicate amino acid preference at positions relative to the central lysine.

Figure 2. Inhibition of in vitro KDM5C H3K4Me3 demethylase activity. Average ± SEM are shown (n=3, biological).

Figure 3. Peptide inhibitor, EP4, dissociation constant with KDM5C. Interaction between EP4 and KDM5C binding analyzed by fluorescent polarization. The equilibrium dissociation constant (K _D ) was obtained for the complex.

Figure 4. Characterization of critical residues of the KDM5 inhibitor, EP4 by in vitro binding assay (left) Progressive C-terminal and N-terminal tandem truncation of EP4. Spot intensity (dark) indicates relative interaction with KDM5C as detected by chemiluminescence (right) Systematic mutation of the EP4 alters KDM5C binding activity. Relative binding preference of KDM5C was systematically determined in order to assess the possible amino acid mutations of the EP4 peptide that alter in vitro binding activity, either resulting in maintaining or strengthening (green), tolerable (yellow) or intolerable (red) KDM5C interaction. WT EP4 sequences are bolded.

Figure 5. In vitro target specificity of EP4 peptide within the KDM5 family. Relative recombinant KDM5 demethylation activity (KDM5A/B/C) towards H3K4me3 peptide in the presence of EP4 peptide. Data are averages +/- SEM (n=6 independent replicates). IC50 are reported as the [EP4] required to decrease KDM activity to 50% of control (i.e., no EP4) values.

Figure 6. Interaction with EP4 peptide with target KDM5C protein. The EP4 peptide was found to interact with the region of KDM5C that is responsible for cellular demethylation activity. These findings support biochemical data demonstrating that the EP4 can inhibit the demethylation activity of KDM5C and demonstrates that this inhibition occurs as a result of interaction with the KDM5C JmjC domain.

Figure 7. Cellular EP4 inhibitor delivery and viability. (A) Diagrammatic representation of EP4 inhibitor peptide showing N-terminally tagged 6-carboxyfluorescein and C-terminally tagged TAT cell delivery peptide. (B) Immunofluorescence microscopy showing internalization of EP4-TAT peptide (shown in green) to HCT 116 cells imaged 24 hr post-treatment with inhibitor. (C) Cell viability following 10mM treatment of EP4 peptide conjugated to cell penetrating peptides, TAT, PR9 and CPP.

Figure 8. Global cell cycle distribution in EP4-TAT treated HCT 116 cells. EP4-TAT increases global H3K4me3 levels in HCT 116 cells. (A) Two-parameter flow cytometric analysis of BrdU incorporation and DNA content was performed following a 24 hr exposure of 2mM and 5mM EP4-TAT to HCT 116 cells. (B) The proportion of propidium iodine stained cells were represented in each phase of the cell cycle cell cycle and (C) dose-response correlation of BrdU positive cells were represented. These values are expressed relative to untreated controls. Each value in B, C represents the mean (+/- SEM) determined from 3 independent experiments.

Figure 9. Comparison of KDM5 inhibitors, EP4-TAT and CPI-455. (A) KDM5 inhibitors (EP4- TAT and CPI-455) increase global H3K4me3 levels in HCT 116 cells. (B) Relative H3K4me3 levels were monitored in histone extracts from HCT 116 cells.

Figure 10. NCI60 cancer panel screen of EP4-TAT. EP4-TAT and TAT-alone peptides were sent to the National Cancer Institute - Developmental Therapeutics program for screening effect on cell growth on the NCI60 cancer panel. Cell growth was monitored post-treatment with peptide in a dose-responsive manner (n=4). EP4-TAT effects were normalized to TAT-alone effects to control for delivery peptide. Following drug addition, the plates were incubated for an additional 48 hours at 37°C, 5 % C02, 95 % air, and 100 % relative humidity. Cell growth was monitored by Sulforhodamine B (SRB) staining. Gl ₅₀ values are defined at the concentration of EP4-TAT that decreased cell growth by 50%.

Figure 11. EP4-TAT pre-treatment sensitizes non-small cell lung cancer cells to cisplatin treatment. (A) Non-small cell lung cancer cells (HOP-92) were exposed to 0.2mM EP4-TAT peptide for 24 hr prior to a 72 hr dose-response treatment with cisplatin. Cell viability is determined by resazurin assay and is relative to a 0.2mM TAT-alone control treatment. (B) Kaplan-Meier survival plot of KDM5C expression in NSCLC patients.

DETAILED DESCRIPTION

The present invention relates to peptide-derived therapeutics targeting enzymes in the lysine methylation pathway and the use of such therapeutics to treat diseases or disorders associated with dysfunction in lysine methylation. In particular, the present invention relates to peptide- derived therapeutics which target KDM5C and the uses thereof.

Peptides:

The present invention provides peptides that bind, optionally specifically bind, to KDM5C. In specific embodiments, the peptides of the present invention bind KDM5C with high affinity. In specific embodiments, the peptides bind to KDM5C and inhibit activity thereof. In certain embodiments, the peptides bind the catalytic core of KDM5C.

Exemplary peptides are set forth in the table below:

n= norLeucine (Nle)

In certain embodiments of the present invention, the peptides comprise the consensus sequence set forth below:

X1X2X ₃ X4X5THHH; where

Xi=T or S

X ₂ =D, E or I

X ₃ =T, D or Q

X4=Q, S, N or T

X ₅ =K or Nle

. (SEQ ID NO:65)

In certain embodiments of the present invention, there is provided a peptide comprising the sequence as set forth in any one of SEQ ID NOs: EP1-40.

In certain embodiments, the peptide inhibitors are modified from the natural substrate peptides of KDM5C. The natural substrate peptides of KDM5C include histones H3-K4 and H4-K20. In specific embodiments, the peptide inhibitors are histones H3-K4 and H4-K20 with Lys to nor- Leu mutations. In specific embodiments, the peptides comprise the sequence as set forth in any one of SEQ ID NO: EP41-44.

In certain embodiments of the present invention, there is provided peptides comprising variant sequences other than those specifically disclosed herein, which comprise significant sequence identity (e.g. 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity) to the amino acid sequence provided that such peptides retain the ability to inhibit KDM5C activity. Such peptides can comprise one or more amino acid substitutions, additions, deletions, or insertions as compared to the parent amino acid sequence. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g. Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g. Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc. In certain embodiments, naturally occurring amino acids in the peptides are replaced with amino acid analogs and derivatives thereof.

A worker skilled in the art could readily determine amino acid substitutions or truncations which impact binding activity of the peptides of the present invention. In certain embodiments, there is provided the EP4 peptide (i.e. the peptide comprising T E D S K T H H H) comprising one or more substitutions.

Figure 4 provides details with respect to the impact of substitutions on the binding activity of EP4. Following this systematic approach, position-specific tolerable mutations were identified. Accordingly, in certain embodiments, there is provided a peptide that binds to KDM5C, wherein said peptide comprises the sequence (SEQ ID NO:66):

X1X2X3X4X5X6X7X8 X ₉ ; where

Xi=T

X ₂ =K, G, L, Q, V, E, H or I

X3— I , L, V or D

X4— L, M, V, F or S

Xs=V or K

Xe=V, G, R, L, K, F, H, T, A, P, I or N

X ₇ =H

Xs=H

X ₉ =H.

In certain embodiments of the present invention, there is provided peptides comprising a fragment of the sequences specifically disclosed herein comprising at least 5 contiguous amino acids, provided that such peptides retain the ability to inhibit KDM5C activity. In certain embodiments of the present invention, the peptides or fragments thereof comprise additional amino acids at the N and/or C terminus. In certain embodiments, the peptides of the present invention comprise A or AA at the N terminus. In certain embodiments, the peptides of the present invention comprise A or AA at the C terminus. In certain embodiments, the peptides of the present invention comprise A or AA at the N and C terminals. In certain embodiments of the present invention, there is provided a conjugate or fusion protein comprising the peptide of the present invention and heterologous amino acid sequence.

In certain embodiments, the peptides of the present invention includes a linker sequence. Linkers are known in the art and are generally classified into 3 categories according to their structures: (1) flexible linkers, (2) rigid linkers, and (3) in vivo cleavable linkers. Besides the basic role in linking the functional peptides together (as in flexible and rigid linkers) or releasing free functional peptide inhibitor /n vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of inhibitor peptides, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.

Linker Model _ Advantages _ Example(s) _

Flexible Allows for interaction between (GGGGS)n, (G)n, 6- functional peptide and delivery aminohexanoic acid (i.e. , mechanism (i.e., functional units) ahx)

Increases separation between

functional units

Rigid Maintain distance between functional (EAAAK)n, (XP)n

units

Cleavable Allows for in vivo separation of Disulphide, protease

functional units sensitive peptide

sequences

In certain embodiments, the peptides of the present invention further comprise a 6- aminohexanoic acid linker. The chemical structure of the linker is set forth below:

In specific embodiments, a cell penetrating peptide is conjugated to the peptide of the invention via a linker sequence. In certain embodiments of the present invention, the peptides comprise other modifications including, without limitation, glycosylations, acetylations, phosphorylations, PEG, D-amino acids, nanoparticles, solid lipid nanoparticles, esterification, N-acetylation or may be formulated with liposomes, nano-emulsions, mucoadhesive polymers, nanoparticles, solid lipid nanoparticles.

It is known in the art that peptide modifications may improve therapeutic peptide delivery by increasing stability, inhibiting enzyme activity, enhancing absorption and/or cell targeting.

Mechanisms of therapeutic peptide delivery

_ Goal _ Peptide modification/formulations

Stomach

Increased stability PEG, D-amino acids, nanoparticles, solid lipid

nanoparticles

Small intestine

Increased stability cyclization, PEG, lipidation, D-amino acids, polymer matrices, nanoparticles, esterification, /V-acetylation

Enzyme inhibitors soybean trypsin inhibitor, aprotinin, puromycin,

bacitracin

Absorption enhancers chitosans, fatty acids, lectins, Zonula occludens

toxin, cell penetrating peptides, liposomes, nanoemulsions, mucoadhesive polymers, nanoparticles, solid lipid nanoparticles

Circulation

Increased stability PEG, hyper-glycosylation, liposomes, nanoparticles Cell targeting Antibody, cell penetrating peptides

The peptides of the present invention may be coupled, either directly or via a linker, to a cell penetrating motif or other moiety so as to more efficiently facilitate the delivery of the peptide to the interior of a cell. Thus, the peptide can be provided as part of a composition or conjugate comprising the peptide and cell penetrating motif or other moiety. Any of various cell penetrating motifs and or other moieties useful for these purposes can be used. By way of illustration, suitable cell penetrating motifs and other relevant moieties (e.g. cell-membrane anchoring moieties) include lipids and fatty acids, cell penetrating peptides, and other types of carrier molecules (e.g. Pep-1).

In certain embodiments, the peptides of the present invention are coupled either directly or via a linker to a cell penetrating peptide. A repository of cell penetrating peptide can be found at crdd.osdd.net/Raghava/cppsite/index.html. Exemplary cell penetrating peptide are set forth in the table below:

CPP name Sequence Origin Class

TAT48-60 GRKKRRQRRRPPQ (SEQ ID NO:45) HIV-1 TAT protein Cationic

TAT49-57 RKKRRQRRR (SEQ ID NO:46) HIV-1 TAT protein Cationic

Penetratin, Antennapedia Drosophila

RQIKIWFQNRRMKWKK (SEQ ID NO:47) Cationic pAntp(43-58) melanogaster

Polyarginines Rn Chemically synthesized Cationic

VKRGLKLRHVRPRVTRMDV (SEQ ID

DPV1047 Chemically synthesized Cationic

NO:48)

PR9 FFLIPKGRRRRRRRRR (SEQ ID NO:49) Chemically synthesized Cationic

Mut6DPT (CPP) RRWRRWRRWRR (SEQ ID NQ:50) Chemically synthesized Cationic

GALFLGFLGAAGSTMGAWSQPKKKRKV HIV glycoprotein 41/SV40 T

MPG Amphipathic

(SEQ ID NO:51 ) antigen NLS

KETWWETWWTEWSQPKKKRKV (SEQ ID Tryptophan-rich

Pep-1 Amphipathic

NO:52) cluster/SV40 T antigen NLS

Vascular endothelial

pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO:53) Amphipathic cadherin

MVRRFLVTLRIRRACGPPRVRV (SEQ ID

ARF(1-22) p14ARF protein Amphipathic

NO:54)

MVKSKIGSWILVLFVAMWSDVGLCKKRP N terminus of unprocessed

BPrPr(1-28) Amphipathic

(SEQ ID NO:55) bovine prion protein

MAP KLALKLALKALKAALKLA (SEQ ID NO: 56) Chemically synthesized Amphipathic

GWTLNSAGYLLGKI NLKALAALAKKI L Chimeric galanin-

Transportan Amphipathic

(SEQ ID NO:57) mastoparan

LSTAADMQGVVTDGMASGLDKDYLKPDD

p28 Azurin Amphipathic

(SEQ ID NO:58)

DPKGDPKGVTVTVTVTVTGKGDPKPD

VT5 Chemically synthesized Amphipathic

(SEQ ID NO:59)

RRIRPRPPRLPRPRPRPLPFPRPG (SEQ Bactenecin family of

Bac 7 (Bac 1-24) Amphipathic

ID NO:60) antimicrobial peptides

C105Y CSIPPEVKFNKPFVYLI (SEQ ID NO:61 ) a1 -Antitrypsin Hydrophobic

Derived from synthetic

PFVYLI PFVYLI (SEQ ID NO:62) Hydrophobic

C105Y

Pep-7 SDLWEMMMVSLACQY (SEQ ID NO:63) CHL8 peptide phage clone Hydrophobic

Repository can be found at crdd.osdd.net/Raghava/cppsite/index.html

In certain embodiments of the present invention, a TAT cell penetrating peptide is linked either directly or via a linker to the peptides of the present invention. In certain embodiments of the present invention, a TAT cell penetrating peptide comprising the sequence GRKKRRQRRRPPQ is linked to the peptides of the present invention directly or via a linker. In certain embodiments of the present invention, a TAT cell penetrating peptide comprising the sequence GRKKRRQRRRPPQ is linked to the peptides of the present invention via a 6-aminohexanoic acid linker. In certain embodiments, the cell penetrating peptide is linked to the N-terminus of the peptide either directly or indirectly via a linker. In certain embodiments, the cell penetrating peptide is linked to the C-terminus of the peptide either directly or indirectly via a linker.

In specific embodiments of the present invention, there is provided a peptide-derived inhibitor comprising the sequence set forth in the table below:

KDM5C peptide inhibitor sequence complete with delivery peptide.

The peptides of the present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art.

Recombinant techniques may also be used to generate the peptides of the present invention. Such recombinant techniques are known in the art. Accordingly, the present invention also provides a nucleic acid encoding the amino acid sequence of the peptide, and conjugates comprising the peptide. The nucleic acid can comprise DNA or RNA, and can be single or double stranded. Furthermore, the nucleic acid can comprise nucleotide analogues or derivatives (e.g. inosine or phophorothioate nucleotides and the like). The nucleic acid can encode the amino acid sequence of the peptide as part of a fusion protein comprising such sequence and a cell penetrating motif. The nucleic acid encoding the amino acid sequence of the peptide can be provided as part of a construct comprising the nucleic acid and elements that enable delivery of the nucleic acid to a cell, and/or expression of the nucleic acid in a cell. Such elements include, for example, expression vectors and transcription and/or translation sequences. Suitable vectors, transcription/translation sequences, and other elements, as well as methods of preparing such nucleic acids and constructs, are known in the art.

Accordingly, in certain embodiments polynucleotide encoding and expressing one or more peptide(s) of the invention. In another preferred embodiment, the polynucleotide is inserted in a vector. Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a prokaryotic or eukaryotic cell. The polynucleotide is inserted into an expression vector in proper orientation and correct reading frame for expression. In certain embodiments, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Recombinant vectors are known in the art and include but are not limited to plasmids and viral vectors. Viral vectors include but are not limited to oncolytic viral vectors, lentivirus and adenovirus vectors.

Pharmaceutical Compositions:

The peptides and peptide derived inhibitors of the present invention be formulated as a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises one or more peptides and peptide derived inhibitors of the invention alone or in combination with one or more other active agents and a pharmaceutically acceptable carrier.

Polynucleotides and vectors encoding the peptides of the invention may also be formulated as pharmaceutical compositions. In certain embodiments, the pharmaceutical composition comprises one or more polynucleotides or one or more vectors of the present invention alone or in combination with one or more other active agents and a pharmaceutically acceptable carrier.

The pharmaceutical composition may comprise one or more other pharmaceutically active agents or drugs. Examples of such other pharmaceutically active agents or drugs that may be suitable for use in the pharmaceutical composition include anticancer agents. Suitable anticancer agents include, without limitation, alkylating agents; nitrogen mustards; folate antagonists; purine antagonists; pyrimidine antagoinists; spindle poisons; topoisomerase inhibitors; apoptosis inducing agents; angiogenesis inhibitors; podophyllotoxins; nitrosoureas; cisplatin; carboplatin; interferon; asparginase; tamoxifen; leuprolide; flutamide; megestrol; mitomycin; bleomycin; doxorubicin; irinotecan; and taxol, geldanamycin and various anti-cancer peptides and antibodies.

The carrier may be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier may be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the active agents, as well as the method of administration. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods. The following formulations for oral, aerosol, topical, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the compound of the invention are known, and, formulations appropriate for each of these routes of administration are known in the art.

In certain embodiments, one or more peptides of the present invention are conjugated, directly or indirectly, to a carrier. Appropriate carriers are known in the art and include but are not limited to proteins including but not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA); virus-like particles and viruses.

Methods of Treatment

The present invention also provides methods of inhibiting KDM5C activity. This method comprises bringing KDM5C into contact with a peptide, peptide derived inhibitor or a pharmaceutical composition of the present invention. This contact may occur in vivo or in vitro. Accordingly, in certain embodiments, the present invention provides methods of inhibiting the activity of KDM5C in a subject in need thereof, by administering one or more peptide(s), one or more peptide(s) derived inhibitor(s), one or more polynucleotide(s) or vector(s) encoding one or more peptide(s) or one or more pharmaceutical composition(s) of the present invention alone or in combination with one or more other active agents. The subject may be a mammal. In certain embodiments, the subject is a human.

The present invention also provides methods of treatment of disease associated with increased KDM5C activity. Accordingly, in certain embodiments, the present invention provides methods of treatment of disease associated with increased KDM5C activity in a subject in need thereof, by administering to the with one or more peptide(s), one or more peptide(s) derived inhibitor(s), one or more polynucleotide(s) or vector(s) encoding one or more peptide(s) or one or more pharmaceutical co position(s) of the present invention alone or in combination with one or more other active agents.

In certain embodiments, the disease associated with increased KDM5C activity is a proliferative disease. In certain embodiments, the proliferative disease is cancer. Accordingly, in certain embodiments, the present invention provides methods of treatment of a cancer associated with increased KDM5C activity in a subject in need thereof, by administering one or more peptide(s), one or more peptide(s) derived inhibitor(s), one or more polynucleotide(s) or vector(s) encoding one or more peptide(s) or one or more pharmaceutical composition(s) of the present invention alone or in combination with one or more other active agents.

The types of cancer include but are not limited to a cancer selected from the group consisting of acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g. lymphangiosarcoma, lymphangioendothelio sarcoma, hemangio sarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g. cholangiocarcinoma); bladder cancer; breast cancer (e.g. adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast, triple negative breast cancer (TNBC), ER positive breast cancer, ER negative breast cancer, PR positive breast cancer, PR negative breast cancer, ER/PR positive breast cancer, ER/PR negative breast cancer, HER2 positive breast cancer, HER2 negative breast cancer); brain cancer (e.g. meningioma, glioblastomas, glioma (e.g. astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g. cervical adenocarcinoma, squamous cell carcinoma of the cervix); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g. colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g. Kaposi' s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g. uterine cancer, uterine sarcoma); esophageal cancer (e.g. adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g. intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g. stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g. head and neck squamous cell carcinoma, oral cancer (e.g. oral squamous cell carcinoma), throat cancer (e.g. laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain disease (e.g. alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g. nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g. hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g. bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g. systemic mastocytosis); muscle cancer; myelodysplasia syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g. polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g. neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g. gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g. bone cancer); ovarian cancer (e.g. cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g. pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g. Paget' s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g. prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g. squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g. appendix cancer); soft tissue sarcoma (e.g. malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g. seminoma, testicular embryonal carcinoma); thyroid cancer (e.g. papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g. Paget' s disease of the vulva).

In specific embodiments of the present invention, there is provided a method of treatment of a cancer in a subject in need thereof, by administering to the with a peptide, peptide derived inhibitor or a pharmaceutical composition of the present invention, wherein the cancer is selected from the group consisting of bladder, non-small lung carcinoma, small cell lung carcinoma, leukemia, liver, breast, colon, and pancreatic cancer.

In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, one or more peptide(s), one or more peptide(s) derived inhibitor(s), one or more polynucleotide(s) or vector(s) encoding one or more peptide(s) or one or more pharmaceutical composition(s)of the present invention are used in combination with additional pharmaceutical agents in the methods of the present invention.

The additional pharmaceutical agents may include but are not limited to anti-cancer agents. Anti-cancer agents encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g. tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immuno stimulants and/or immunodulatory agents (e.g. IL- 1 , 2, 4, 6, or 12), immune cell growth factors (e.g. GM- CSF) and antibodies (e.g. HERCEPTIN (trastuzumab), T-DM1 , AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), anti androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD- MA), phthalocyanine, photo sensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (Abraxane), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor- activated pro-drug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g. 2'-paclitaxel methyl 2- glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), antimetabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. I- methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca<2+> ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g. axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU 11248), toceranib (PALLADIA®), vandetanib (ZACTEVIA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD- 2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (V ARGATEF® ) , AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981 , tivozanib (AV-951), OSI-930, MM-121 , XL- 184, XL-647, and/or XL228), proteasome inhibitors (e.g. bortezomib (VELCADE)), mTOR inhibitors (e.g. rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, caraiinomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,, aminopterin, and hexamethyl melamine.

EXAMPLE The example below details the development of peptide inhibitors that target KDM5C. The focus on the KDM5 family (histone H3K4me2/3 demethylases) results from growing evidence for a causal role of these KDMs in a number of different human cancers, contributing to cancer cell proliferation and drug resistance. KDM5C in ovarian, breast, prostate, and colon cancer, in addition to other tumors, is highly expressed and involved in the regulation of tumor-related gene expression through the abnormal demethylation of histone H3K4me2/3. Colon cancer is the second most common among malignant solid tumors. Chemotherapy is a standard treatment for this disease; however, the number of effective chemotherapy drugs available to treat colon cancer is limited. Recent studies have shown that KDM5C may have a specific role in drug resistance in colon cancer, and that KDM5C is overexpressed in some lung, gastric and cervical cancers (Lin et al., 2018). The importance of KDM5 H3K4me2/3-specific demethylase activity towards its role in cell proliferation and drug resistance in cancer has not been resolved so far, and it’s unclear how much of KDM5’s role in cancer is attributable to histone-specific demethylation. KDM5C regulation is extensive and has been reported to downregulate the genes that regulate cell proliferation, including the tumor protein p53, PCNA, MKI67, and the cyclin-dependent inhibitor, p21 , thereby promoting cell proliferation (Xu et al., 2017; Stein et al.,

2014). In agreement with these findings, unpublished preliminary research from the Biggar lab also suggests that KDM5C may also function through the demethylation of the tumor protein, p53 at trimethylated lysine K370me3, effectively decreasing p53 activity and decreasing the expression of p21 and PCNA. Furthermore, KDM5C downregulates BMP7 in liver cancer and BRMS1 in breast cancer to promote invasion, inhibits the von-Hippel Lindau (VHL) tumor suppressor gene, and has been shown to downregulate ABCC1 expression thereby promoting drug resistance in colon cancer (Lin et al., 2018; Stein et al, 2014; Ji et al., 2015; Wang et al.,

2015). Interestingly, this presents a mechanism whereby KDM5C inhibition may decrease drug cancer cell resistance in colon cancer, but may also be a conserved mechanism for the treatment of other KDM5C-dependent cancers. As a result of its established role in cancer development and progression, there has been significant interest in the development of KDM5- specific inhibitors. To date, one highly regarded inhibitor of the KDM5 family of demethylases is available under the name CPI-455 and has been shown to increase global H3K4me3 methylation levels, however, this inhibitor unfortunately has weak activity towards KDM5 inhibition in vivo with an IC50 of ~25 uM (Vinogradova et al., 2016).

Materials and Methods KDM5C Construct Information

The plasmid used to product recombinant KDM5C was:

Vector Name: pFB-CT10HF-LIC

Construct ID: JARID1CA-c022

The sequence of recombinant KDM5C is set forth below (SEQ ID NO:64):

MEPGSDDFLPPPECPVFEPSWAEFRDPLGYIAKIRPIAEKSGICKIRPPADWQPPFAVEV DNFR

FTPRIQRLNELEAQTRVKLNYLDQIAKFWEIQGSSLKIPNVERRILDLYSLSKIVVE EGGYEAICK

DRRWARVAQRLNYPPGKNIGSLLRSHYERIVYPYEMYQSGANLVQCNTRPFDNEEKD KEYKP

HSIPLRQSVQPSKFNSYGRRAKRLQPDPEPTEEDIEKNPELKKLQIYGAGPKMMGLG LMAKDK

TLRKKDKEGPECPPTVVVKEELGGDVKVESTSPKTFLESKEELSHSPEPCTKMTMRL RRNHSN

AQFIESYVCRMCSRGDEDDKLLLCDGCDDNYHIFCLLPPLPEIPKGVWRCPKCVMAE CKRPPE

AFGFEQATREYTLQSFGEMADSFKADYFNMPVHMVPTELVEKEFWRLVNSIEEDVTV EYGADI

HSKEFGSGFPVSDSKRHLTPEEEEYATSGWNLNVMPVLEQSVLCHINADISGMKVPW LYVGM

VFSAFCWHIEDHWSYSINYLHWGEPKTWYGVPSLAAEHLEEVMKKLTPELFDSQPDL LHQLVT

LMNPNTLMSHGVPVVRTNQCAGEFVITFPRAYHSGFNQGYNFAEAVNFCTADWLPAG RQCIE

HYRRLRRYCVFSHEELICKMAACPEKLDLNLAAAVHKEMFIMVQEERRLRKALLEKG ITEAERE

AFELLPDDERQCIKCKTTCFLSALACYDCPDGLVCLSHINDLCKCSSSRQYLRYRYT LDELPAM

LHKLKV

Purification of recombinant target proteins

Expression and purification of KDM5C

SF9 cells (500 ml_ at 10 ⁶ cells/mL) were infected with KDM5C-His ₆ baculovirus at a 1 :100 ratio. After 60 hr post-infection, cells were harvested by centrifugation and the pellet was frozen on liquid nitrogen. Cells were lysed in P5 buffer (50mM NaHP04 pH 7, 500 mM NaCI, 10% glycerol, 0.05% TritonX-100, 0.5 M DTT, 5 mM Imidazole and protease inhibitors) and homogenized by 20 passes through a dounce homogenizer (pestle A) followed by sonication (three times each for 30 sec at 40% intensity). The dounce homogenized cell lysate was incubated with 1 mM MgCI ₂ and 2.5 U/ml benzonase nuclease at 4°C for 1 hr followed by centrifugation at 18,000 g for 45 min. The soluble cell lysate was incubated with prewashed 200 mI_ HisPur™ Ni-NTA Resin with P5 buffer for 1 hr at 4°C. The beads were pelleted by centrifugation at 800 xg for 2 min, the supernatant was removed and the beads were washed 4 times each of 5 min with P40 buffer under rotation at 4°C. Finally, the protein was eluted with P500 buffer. The purified protein was dialyzed in storage buffer (20 mM HEPES pH 7.5, 150mM NaCI, 10% glycerol, 1 mM DTT), snap frozen on liquid nitrogen and stored in small aliquots at -80°C.

Synthesis of Oriented Peptide Array Library (OPAL) The peptide libraries were synthesized on cellulose membrane using the ResPep SL automatic peptide and SPOT array synthesizer (Intavis). An extra fine needle tip was used to achieve a density of 600 peptides per SPOT membrane (8 *12 cm). The following oriented peptide library arrays were synthesized for binding dependent interactions: AXXXX[Lys]XXXXA and AXXXX|nor-Leu]XXXXA; where is a mixture of 19 amino acids (except Cys), and the brackets ([/]) encase the amino acids that were preferred by the protein of interest. To generate oriented peptide library pools, each degenerated position was scanned with any of the 19 amino acids (excluding Cys).

Target protein binding assays

The OPAL was designed sequentially starting from the most degenerate to highly specific peptide against our target protein as described above. The potential inhibitor peptides were initially screened based on the binding affinity between the peptides and target proteins. All the steps were carried out at room temperature unless otherwise stated. The OPAL cellulose macro arrays are presoaked in 100% ethanol followed by 50% ethanol for 15 min with constant rocking. The membrane is then washed with distilled water three times each of 15 min. The processed membrane is first blocked with 5% nonfat dry milk in Tris buffered saline containing 0.05 % Tween 20 (TBST) for 1 hr at room temperature. Finally, the array was equilibrated with peptide binding buffer (50 mM Tris-CI, 350 mM NaCI, 10% glycerol, 0.5 mM DTT and 0.05% Tween20). The array was then incubated with 1 mM of target protein overnight at 4 °C under rotation. The excess protein was washed away by three consecutive 10 min washes with TBST. Each array was then incubated with HRP conjugated anti-His antibody (1 :5000) in TBST for 1 hr. The array was then washed thrice each of 10 min. The signals were detected using chemiluminescence. The signal intensities observed were subjected to densitometry analysis using ImageJ software protein array analyzer.

In vitro lysine demethylase activity inhibition assay

Inhibition of in vitro demethylase activity by the peptides was analysed using Succinate-Glo™ JmjC demethylase assay kit (Promega). The experiment was performed in low-volume 384-well plates at room temperature as per the manufacturer’s instruction.

Fluorescent polarization

Recombinant KDM5C protein was serially diluted in a 384-well plate, followed by the addition of fluorescein-labeled inhibitor peptide in PBS buffer. The mixtures were incubated in the dark for 30 min prior to fluorescent polarization measurements at room temperature on an EnVision Multilabel Plate Reader (PerkinElmer) with the excitation set at 480 nm and emission at 535 nm. Binding curves were generated by fitting the binding data to a hyperbolic nonlinear regression model using Prism 3.0 (GraphPad software, Inc., San Diego, CA), which also produced the corresponding dissociation constants (K _d ).

Delivery of inhibitor peptide to the cell line

Synthesis of three different cell penetrating peptide peptide with sequence Gly-Arg-Lys-Lys-Arg- Arg-GIn-Arg-Arg-Arg-Pro-Pro-GIn (i.e. , TAT), Phe-Phe-Leu-lle-Pro-Lys-Gly-(Arg) ₉ (i.e., PR9) and (Arg-Arg-Trp) ₃ -Arg-Arg (i.e., MutD6 or CPP) were carried out by solid phase synthesis on a ResPep SL peptide synthesizer (INTAVIS)) following the Fmoc chemistry protocol. A 6- carboxyfluorescein (FITC derivative, referred to as only FITC in this document) was added to the C-terminal end of the peptides for ligation to a fluorochrome FITC and was separated by the addition of a 6-aminohexanoic acid group to provide both (1) fluor flexibility and (2) reduce steric constraints of the molecule.

To evaluate the internalization of FITC-labelled peptides, exponentially growing HCT 116 cells were seeded on 6 well plate at a density of 2x10 ⁵ cells per well and incubated overnight. After overnight incubation, the media (DMEM with penstrep and 10% FBS) was replaced with fresh media supplemented with 10 mM FITC-labelled inhibitor peptides. Following the incubation (24 hr), cells were washed three times with ice cold PBS to remove the excess extracellular complexes. Cells were then stained with Hoechst dye (1 :2000 dilution from 10 mg/ml_ stock in PBS) directly adding sufficient staining solution to the well. Cells were incubated for 10 min with the dye, protected from light. The staining solution is discarded and the cells were washed 3 times with PBS and imaged directly under fluorescent microscope.

Cell Viability assay and IC ₅₀ determination

Cell viability was measured using the Resazurin reduction assay which indirectly quantifies living cells through the metabolically active reduction of resazurin to fluorescent resorufin. This assay allows to maintain cells viability and, therefore, to monitor cell growth with time. Exponentially growing cells were seeded into 96-well plates at the density of 2.0 x 10 ⁴ cells/mL and incubated overnight. The media was replaced with fresh media prior to inhibitor treatment. Cells were treated with 0.2 mM of inhibitor peptide for 24 hr. The inhibitor peptide was diluted in the cell culture media (DM EM -/-) in the absence of serum from a 5 mM stock. All the treated cells were compared to the control (TAT alone peptide with equivalent quantity of DMSO) which were considered as 100% viable. One set of wells also prepared with medium only for background subtracting and instrument gain adjustment. The experiments were carried out in triplicate and expressed as mean ±SD. A 10% resazurin solution (0.15mg/ml stock dissolved in PBS, filter sterilized and stored protected from light at 4 °C) was then added to each well and incubated for 2 hr. The fluorescence was recorded using a multiwell plate reader (Perkin Elmer) at Ex. 560 nm and Em. 610 nm.

Inhibition of target enzyme activity in HCT 116 cell line

The EP4-TAT inhibitor from all the above experiments were tested for histone methylation status in HCT 116 cells. 3x10 ⁶ cells were plated in 10cm dish and incubated overnight. Cells were then treated with the inhibitors at various concentrations (0.001 to 15 mM). Cells (5x10 ⁶ cells/mL), 24 hr of post dosing, were collected in 15 mL falcon tube and centrifuged at 300 xg for 10 min. The supernatant was discarded and the cells are washed with iced cold PBS. The cell pellet is flash-frozen in liquid nitrogen and stored at -80°C. Histone isolation is done using standard protocol. To summarize, cells were re-suspended in 1 mL hypotonic lysis buffer (10 mM Tris-HCI pH 8, 1 mM KCI, 1.5 mM MgCI ₂ and 1 mM DTT) containing protease inhibitor. The cells were transferred to 1.5 mL tube and incubated for 30 min on rotor at 4°C to promote hypotonic swelling and lysis. The intact nuclei are collected by centrifugation at 10,000 xg for 10 min in a cooled tabletop centrifuge. The supernatant is entirely discarded and pellets were re suspended completely in 600 pL 0.4N H ₂ S0 ₄ and incubated overnight on rotor at 4°C. The nuclear debris were removed by centrifugation at 16,000 xg for 10 min. The supernatant containing the histones were transferred to a fresh 1.5 mL tube and precipitated by adding 195 pL TCA (33%) drop by drop. The reaction is incubated at 4°C overnight under rotation. The histones were pelleted by centrifugation at 16,000 xg for 10 min. After complete removal of the supernatant carefully, the histone pellets were washed with ice-cold acetone to remove the left over acids without disturbing the pellet. Finally, the pellets were air dried for 30 min at room temperature. The histone pellets were dissolved in 100 pL milliQ water and stored frozen at - 20°C. Samples of 1 , 3 and 5 pL of histones were separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue and characterized on the quality and concentration of the histone. The locations of the linker histone H1 and the core histones H3, H2B, H2A and H4 were noted. For western blot, of total of 1 mI_ of histones were separated on 15% SDS-PAGE and transferred overnight at 15V on PVDF membrane. Following blocking with 5% nonfat dry milk in 1X TBST for 1 hr, the membrane containing histones lanes treated with inhibitor, were probed with H3K4Me3 (Abeam) primary antibody (1 :2500) in 1X PBST. Both the membranes were incubated overnight at 4°C under rotation. Following this incubation, membranes were washed in 1X TBST and 1X PBST respectively for 30 min, followed by incubation with secondary antibody for an additional 1 hr. The membranes were further washed for 30 min as before. Histone proteins were detected by Supersignal™ West Pico PLUS Chemiluminescent substrate (ThermoFisher Scientific) using the Chemidoc XRS+ imaging system (BioRad).

Flow Cytometry

A total of 0.3x10 ⁶ HCT 116 cells were plated in 6 well-plate and incubated overnight. Cells were treated with the inhibitor, DMSO and TAT alone controls. For each condition, approximately 1X10 ⁶ HCT 116 cells were collected along with the floating cells in the media by centrifugation at 300 xg for 10 min. The cells were then washed with 5 mL of ice-cold PBS and re-suspended in 0.5 mL of ice-cold PBS. The cells were slowly dropped into 4.5 mL of vortexing ice-cold 70% ethanol for rapid dispersion. The sample was incubated on ice for 45 min and then fixed at -20 °C overnight. The fixed cells were centrifuged at 4°C at 300 xg for 10 min. The resultant cell pellet was re-suspended to 200 pL of the stain master mix (133.7 pL of 1 mg/mL propidium iodide (PI), 1 pL of 10 mg/mL RNase A and PBS 865.3 pL). The Pl-treated cells were incubated at 37°C for 30 min and then analyzed by a flow cytometry (BD AccuriTM C6 Plus). The BD Accuri C6 Plus software version FCS 3.1 was used for apoptosis and cell cycle analysis.

NCI60 Cancer Cell Screen

Following the receipt at the NCI, cell-active EP4-TAT was be tested for its effects on cell viability in a dose-responsive manner in the complete panel. The effect of EP4-TAT on cell viability was carried out using Sulforhodamine B (SRB) staining in all 60 cell lines (n=4). Using several measurements [time zero (T _z ), control growth (C), and growth at the five inhibitor concentrations (T _j )], the percentage growth will be calculated at each inhibition concentration. Three dose- response parameters are calculated: (1) Gl ₅₀ (drug concentration resulting in a 50% reduction in the net protein increase, [(T _r T _z )/(C-T _z )] x 100 = 50), (2) TGI (drug concentration resulting in total growth inhibition, T, = T _z ), and (3) LC ₅₀ ([(T -T _z )/T _z ] x 100 = -50). Experimental Results

Identification of potent high affinity target binding peptides

A high affinity peptide screen was carried out against target protein KDM5C. The method involves the sequential synthesis and printing of OPALs. Figure 1 shows the binding of KDM5C to the unselective degenerate peptide arrays. The intensity of dark spots represents the binding affinity which is quantified by ImageJ protein array analyzer.

Further the best hits from the arrays were then used to design sequence-selective peptides followed next by the sequence-specific high affinity peptides. At the end of the experiment, 44 KDM5C specific potential high affinity peptides were selected.

In vitro validation: KDMase inhibition

All the potential KDM5C inhibitors selected from the OPAL screen were tested for inhibition of KDM5C demethylation activity in vitro. KDM5C demethylation activity was carried out using a H3K4me3 substrate peptide. A total of 20 (EP1-EP20) inhibitor peptides, representing all lysine- derived versions of top inhibitor candidates from the OPAL screen, were tested for inhibitory activity in a dose-responsive manner (Figure 2). EP4 was selected from this screen as it displayed the top-most inhibitory activity in vitro.

In order to quantify the dissociation kinetics of EP4 with KDM5C, fluorescent polarization was performed, and it was determined that EP4 bound to KDM5C with an experimental Kd of 0.11 +/- 0.03 nM (Figure 3). EP4 was further shown to display in vitro specificity towards the inhibition of KDM5C activity in a panel of KDM5 paralogs (Figure 4).

Characterization of critical binding residues of EP4

The position and contribution of critical residues within the EP4 inhibitor were assessed by peptide array and in vitro recombinant KDM5C binding assay. Progressive C-terminal and N- terminal tandem truncations of EP4 sequence were used to assess the individual residue contribution of EP4 to KDM5C binding. Relative binding was qualitatively determined by chemiluminescence (Figure 5; left). A systematic mutation of the EP4 was also carried out to in order to assess possible amino acid mutations that alter in vitro KDM5C binding activity, either resulting in a maintenance or strengthening (green; greater than 100%(relative EP4 binding)), tolerable (yellow; 80% percentile) or intolerable interaction (red; 50% percentile) (Figure 5; right). The position-specific tolerable mutations that can be made to EP4 were determined by those amino acid substitutions that retained at least 100% of WT EP4 relative binding activity.

EP4 peptide interacts with the KDM5C catalytic domain

KDM5C quaternary structure, comprising the JmjN, JmjC and ZF domains, was modelled for predicting different binding sites using refined protein structure to identify spatial properties, backbone positioning, and protein-side-chain conformation that concurrently strengthens global topologies and protein modeling structural properties. Our structural model with the lowest score had a 0.110 RMSD(A). Each side-chain residue was created using the most plausible rotamer from the Dunbrack backbone-dependent rotamer library from UCSF Chimera. Auto-optimization for EP4 peptide is conducted with Avogadro to monitor poor contacts and improper bonds. For initial modelling of KDM5CA-EP4 complex, the inhibitor positing should substitute other co factors in the investigational framework. This was achieved by superimposing template structure on the KDM5C catalytic core structure to replace template cofactors with other enzymatic cofactors. Figure 6 shows the superimposed structure of the catalytic core KDM5C bound with inhibitor peptide. Different binding motifs were analyzed within KDM5C-A for predicting binding pocket for EP4 using Autodock 4 that employs Mozyme function of MOPAC2009 enabling fast, semi-empirical quantum mechanical calculation of the protein charge.

By this method, 200 clustered KDM5CA-EP4 protein peptide complex structures are

synthesized and the top structure with the lowest z-score of -1.6 is predicted to yield the best possible binding interaction for EP4 peptide with KDM5C (Figure 6). Further analyzation of the complex using Discovery studio visualizer detected the formation of salt bridges between the protein-peptide complex possessing heavy attractive charge and providing high stability within the complex. We also investigated 2D and 3D structural modelling of standard and mutated EP4 Inhibitory peptide sequences, EP4-2/8, EP4-T1A/H9A, EP4-T1A, EP4-H9A that revealed less significant interactions with KDM5C as compared to standard EP4 peptide structure, suggesting that any single deletion or additional modifications to EP4 sequence disrupts the integrity of the protein-peptide complex, thereby weakening the interactions.

Inhibitors were delivered and reduced colorectal carcinoma cell viability

Our EP4 KDM5C inhibitor peptide were tested for a decrease in cell viability. Individual peptide was tagged initially with 3 different cell penetrating peptide in order to decide the best cell delivery peptide (Figure 7A). All the peptides were tagged with fluorophore FITC on the C terminal end. 10 mM of the FITC tagged peptides after 24 hr post treatment when visualized under the fluorescent microscope shown to be successfully delivered into the nucleus which is seen as green foci (Figure 7B).

The cell penetrating peptide tagged EP4 inhibitors are then tested for loss of cell viability on colorectal carcinoma HCT 116 cell line at a 10 mM dose (Figure 1C). EP4 peptide with a TAT tag showed maximum and consistent loss of cell viability. Cell penetrating peptide CPP by itself showed 50% loss of cell viability, followed by PR9 (30% loss) whose overall performance was also low compared to the TAT-inhibitors and hence discontinued further.

Flow cytometry

To determine whether loss in cell viability was associated with increased apoptosis, HCT 116 cells were treated with 2 mM of EP4-TAT and analyzed by PI staining. Representative data from flow cytometry analysis are shown in Figure 8. Frequency distribution histogram shows an increasing percentage of subG1 peak in 2 mM EP4-TAT treated cells (52.8%) as compared to the untreated control (11.0%).

Histone lysine methylation status is inhibitor-responsive in colorectal carcinoma

HCT 116 colorectal carcinoma cell line was treated with the KDM5C inhibitor EP4-TAT to test dynamic changes in H3K4 tri-methylation levels in comparison to the commercial KDM5 inhibitor, CPI-455. The demethylation of histone H3K4 tri-methylation was found to be inhibited when treated with 25 nM of the KDM5C inhibitor (Figure 9A). EP4-TAT was deemed to be more effective for cellular KDM5C inhibition than that of the CPI-455 alternative (Figure 9B).

EP4-TAT performance on the NCI60 cancer panel screen

To determine the therapeutic breadth of our EP4-TAT peptide, we utilized the National Cancer Institute Developmental Therapeutics Program (NCI DTP) to screen EP4-TAT (vs. TAT alone) on a panel of 60 cancer cell lines (NCI60; representing leukemia, melanoma, and lung, colon, brain, ovary, breast, prostate, and kidney cancers). EP4-TAT was found to have a growth inhibitory (Gl ₅₀ ) effect in 7 lines, spanning CNS (SNB-75) and renal (A498, RXF393, UO-31), and non-small cell lung cancer (NSCLC; HOP-92, NCI-226, EKVX) (Figure 10).

EP4-TAT increases chemo-sensitivity in NSCLC cells As KDM5C has also been reported to facilitate drug resistance, we explored the potential of EP4-TAT to increase sensitivity to chemotherapies. We have focused this study towards cisplatin treatment as KDM5C inhibition is reported to reduce resistance to platinum-based drugs ()and EP4-TAT responsive NCI60 cancers (Figure 10) are generally cisplatin-responsive. Further, an EP4-TAT responsive NSCLC line (HOP-92) robustly displayed a decreased cisplatin IC ₅ o following pre-exposure to EP4-TAT (Figure 11A). In patients of NSCLC, a higher KDM5C expression in is prognostic of a lower median survival (Figure 11 B).

Top KDM5C inhibitors screened from OPAL array.

SEQ ID NO: Experimental Peptide Sequence

1 EP 1 TDTTKTH H H

2 EP 2 TDTQKTH H H

3 EP 3 TDTN KTH H H

4 EP 4 TE DS KTH H H

5 EP 5 TE DQKTH H H

6 EP 6 TTQSKTH H H

7 EP 7 TDTS KTH H H

8 EP 8 TE DTKTH H H

9 EP 9 TE EQKTH H H

10 EP 10 SDQQKTH H H

11 EP 11 TTQQKTH H H

12 EP 12 SDQTKTH H H

13 EP 13 TDDQKTH H H

14 EP 14 TE EN KTH H H

15 EP 15 TE ESKTH H H

16 EP 16 TTQTKTH H H

17 EP 17 TDSTKTH H H

18 EP 18 SETQKTH H H

19 EP 19 SETS KTH H H

20 EP 20 TD DN KTH H H

21 EP 21 TDTTnTH H H

22 EP 22 TDTQnTH H H

23 EP 23 TDTN nTH H H

24 EP 24 TE DS nTH H H

25 EP 25 TE DQnTH H H

26 EP 26 TTQS nTH H H

27 EP 27 TDTS nTH H H

28 EP 28 TE DTnTH H H

29 EP 29 TE EQnTH H H

30 EP 30 S DQQnTH H H

31 EP 31 TTQQnTH H H

32 EP 32 SDQTnTH H H

33 EP 33 TD DQnTH H H

34 EP 34 TE EN nTH H H

35 EP 35 TE ESnTH H H

36 EP 36 TTQTnTH H H

37 EP 37 TDSTnTH H H

38 EP 38 SETQnTH H H

39 EP 39 SETS nTH H H 40 EP 40 T D D N n T H H H

41 EP 41 R T K Q T A R K S T G G

42 EP 42 R T n Q T A R K S T G G

43 EP 43 G A K R H R K V L R D N I

44 EP 44 G A K R H R n V L R D N I

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