RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/120,596 filed on February 25, 2015, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to treating, preventing, or delaying the onset of bone marrow failure associated Fanconi Anemia.

GOVERNMENT INTEREST

[0003] This invention was made with government support under [ ] awarded by the []. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0004] Fanconi Anemia (FA) is an autosomal recessive DNA repair disorder characterized by congenital abnormalities, cancer predisposition, and progressive bone marrow failure (BMF). FA is caused by biallelic mutations in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway. Although the precise biochemical functions of the FA/BRCA pathway remain unclear, the pathway promotes homologous recombination (HR) repair. The FA/BRCA pathway also regulates cytokinesis, and pathway disruption results in increased binucleate bone marrow cells and apoptosis. FA cells are also uniquely hypersensitive to oxidative stress and apoptotic cytokines, such as IFNy and TNFa.

[0005] BMF of FA patients is attributable to impaired stem cell pool. FA patients develop progressive bone marrow failure during childhood, and frequently require an allogeneic or unrelated donor bone marrow transplant. All blood lineages are deficient in

FA patients suggests that the FA pathway regulates the function of hematopoietic stem and progenitor cells (HSPCs). CD34 ⁺ cells of FA patients, which are a human stem

cell/progenitor cell enriched population, were not only lower in the number, but also exhibited compromised clonogenicity. Similarly, mice with Fane mutations also displayed reduced numbers of hematopoietic stem cells (HSCs) with impaired reconstitution ability.

In addition, the FA pathway also controls hematopoietic development. Knockdown of FANCA and FANCD2 in human embryonic stem cells impaired embryonic hematopoiesis which could be rescued by FA gene complementation. Therefore, these studies link FA pathway with stem cell function.

[0006] The mechanism of bone marrow failure in FA remains elusive. A need exist for a better understanding of the mechanism of BMF in FA as well as therapies to treat BMF other than bone marrow transplant.

SUMMARY OF THE INVENTION

[0007] The invention provides methods of treating, preventing or delaying the onset of bone marrow failure in a patient having Fanconi Anemia (FA)by administering to the patient a composition comprising fresohmumab, ID 11 or an antigen binding fragment thereof.

[0008] The composition is administered before the patient is prepared for a bone marrow transplant, after the patient receives a bone marrow transplant or after the patient is prepared for a bone marrow transplant but prior to the bone marrow transplant.

[0009] Alternatively the composition is administered when the patient is having medical crisis, such as an infection. The infection is viral or bacterial. Optionally the method further includes administering androgen therapy or erythropoietin.

[00010] The composition is administered at a dose of 0.1 mg/kg to 150 mg/kg.

Preferably the composition is administers at a dose of 0.5 mg/kg to 10 mg/kg. More preferably, the composition is administered at a dose of 1 mg/kg to 5 mg/kg.

[00011] The composition is administered one to seven times per week. Preferably, the composition is administered three times per week. More preferably the composition is administered two times per week.

[00012] The composition is administered for a duration of 1 week to 1 year.

Alternatively, the composition is administered for a duration of one to two months. In other embodiments the composition is administered chronically. Prefereably, composition is administered intravenously.

[00013] Also include in the invention is a method of administering to a patient that has been prepared to receive a bone marrow transplant a composition comprising fresohmumab, 1D11 or an antigen binding fragment thereof. [00014] In another aspect the invention provides a method of expanding hematopoietic stem/progenitor cells comprising contacting a population of hematopoietic stem/progenitor cells with fresolimumab, 1D11 or an antigen binding fragment thereof.

[00015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[00016] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[00017] Figure 1. TGFp pathway inhibition corrects impaired stem cell function of FA mice.

A) TGF pathway inhibition by 1D11 and SD208 restores MMC resistance of Fancd2-/- stromal cells. Top panel: survival of WT or Fancd2-/- stromal cells in the presence of MMC. Error bars represent s.e.m. Bottom panel: Immunoblot of p-Smad2, showing the inhibition of TGF signaling in stromal cells treated by 1D11 and SD208. B) Acetaldehyde sensitivity of Lin- cells, from WT or Fancd2-/- mice, incubated with or without 1D11. C) Colony forming assay, showing acetaldehyde resistance of Fancd2-/- HSCs

(CD150+LSK cells) with 1D11 treatment. D) Schematic experimental design of the in vivo engraftment assay, using lentivirus shRNA transduction of Lin- BM cells from WT and Fancd2-/- mice. E) Deletion of Smad3 promotes the engraftment of Fancd2-/- cells (n=5 mice per group).

Figure 2. Fancd2-/- HSPCs are more sensitive to the suppressive function of TGFp.

A) Fancd2-/- HSPCs are more sensitive to TGF than WT HSPCs. Sorted Lin- cells from

WT and Fancd2-/- mice were treated with mTGFP (0.1, 1, 10 ng/mL). After 5 days culture,

LSK cells were counted and analyzed by flow cytometry. Data were shown after normalizing to untreated WT and Fancd2-/- group respectively. B) 1D11 and SD208 rescued the inhibitory effect of mTGF on Lin- cell growth. Sorted Lin- cells from WT and Fancd2-/- mice were treated with mTGF (Ing/mL) with or without 1D11 or SD208. Data are shown, after normalizing to untreated WT and Fancd2-/- group respectively. C) Clonogenic assay of WT and Fancd2-/- HSPCs treated with mTGF , 1D11 or SD208. D) Hemoglobin levels in MMC treated WT and Fancd2-/- mice, treated with or without 1D11 treatment. E) Red blood cells counts in MMC treated WT and Fancd2-/- mice, treated with or without 1D11 treatment.

[00018] Figure 3. Inhibition of TGF-β Pathway Rescues Physiological Stress- induced Bone Marrow Failure in FA Mice

(A) TGF-β pathway inhibition by 1D11 prevents pLpC induced DNA damage in HSCs in vivo. WT and Fancd2-/- mice were injected intraperitoneally with pLpC (5mg/kg) and 1D11 or isotype control antibody (lOmg/kg). Forty-eight hours after the treatments, HSCs were sorted for DNA damage analysis by immunofluorescence staining or by the single cell comet tail assay. (B) Percentages of HSCs with γΗ2ΑΧ foci) and (C) 53BP1 foci are shown. Hundred to 150 cells were counted for each sample. (D) Olive tail moment in a comet assay demonstrating that 1D11 significantly reduces LpC induced DNA damage in Fancd2-/- HSCs. Ninety-two to 196 HSCs from each group were scored. (E) Schematic of pLpC-induced bone marrow failure mouse model. (F) Peripheral blood analysis of pLpC plus isotype or 1D11-treated WT and Fancd2-/- mice as shown in (E). Red blood cell (RBC) counts, white blood cell counts (WBCs) and hemoglobin levels are shown (n=4-5 mice per group). (G) CFU-S content in the bone marrow of WT or Fancd2-/- mice after four weeks of pLpC plus isotype or 1D11 treatment as shown in (E). (H) Inhibition of TGF- β pathway rescues pI;pC-mediated functional defects of HSPCs. WT and Fancd2-/- mice were exposed to pLpC plus isotype or ID 11 for four weeks as shown in (E) and bone marrow cells were transplanted into lethally irradiated recipients (CD45.1) along with 1x105 helper cells (CD45.1). Donor-derived cells (CD45.2) in peripheral blood were analyzed at 4 weeks post transplantation (n=4~5 recipient mice per group). (I, J) 1D11 rescues pLpC-induced DNA damage in HSPCs. (I) Representative images and (J)

quantification of γΗ2ΑΧ foci in HSPCs from WT and Fancd2-/- mice after four weeks treatment with pLpC plus isotype or 1D11 as shown in (E). Error bars represent s.e.m. See also Figure 7. [00019] Figure 4. TGF-β pathway Inhibition Rescues Acetaldehyde-induced Genotoxicity in HSPCs from FA Mice.

(A) Colony forming assay showing acetaldehyde resistance of Fancd2-/- HSCs (CD48- CD150+LSK cells) with 1D11 treatment. HSCs from WT and Fancd2-/- mice were exposed 2mM acetaldehyde for 4h and then cultured in methy cellulose medium containing 10 μg/mL 1D11 or its isotype control antibodies for 10 days. Survival of the hematopoietic progenitors was determined by colony quantification. (B) Inhibition of TGF-β pathway enhances acetaldehyde resistance of HSCs. Lin- cells from bone marrow (BM) of WT and Fancd2-/- mice were pretreated with 1D11 or isotype control antibody for 30min, followed by exposure to 2mM acetaldehyde for 4h. After 24h in culture with 1D11 or isotype control antibody, equal numbers of cells were transplanted into lethally irradiated recipients (CD45.1) along with 1x105 helper cells (CD45.1). Donor-derived cells (CD45.2) in peripheral blood were analyzed by flow cytometric analysis at 4 and 16 weeks post transplantation (n=4~5 recipient mice per group). (C, D, E, F) ID 11 rescues acetaldehyde- induced DNA damage in HSCs and Lin- cells. Representative images (C) and quantification (D) of γΗ2ΑΧ foci in HSCs from WT and Fancd2- /- mice are shown. HSCs were pretreated with 1D11 or isotype control antibody followed by exposure to acetaldehyde for 4h, and harvested for immunofluorescence at the indicated time points. Hundred to 200 cells with more than 5 foci were counted for each sample. (E) Representative images of alkaline comets of bone marrow Lineage-negative cells from WT and Fancd2-/- mice. Comet tails of Fancd2-/- cells in the isotype plus acetaldehyde treatment group are highlighted by a star. (F) Olive tail moment demonstrating that 1D11 significantly prevents acetaldehyde-induced DNA damage in Fancd2-/- bone marrow in vitro. Ninety-eight to 275 cells from each group were scored. Error bars represent s.e.m. See also Figure6.

[00020] Figure 5. TGF-β Pathway Inhibition Upregulates HR and Downregulates NHEJ in HSCs from Fancd2-/- mice. FA

(A) TGF-β pathway inhibition induces expression of the majority genes involved in DNA damage repair in HSCs from WT mice. HSCs were sorted from WT mice after 48h treatment with 1D11 or isotype control antibody, and used for qRT-PCR analysis. (B) The expression levels of DNA damage repair genes in Fancd2-/- and WT HSCs. Some genes involved in HR and NHEJ pathways were pointed out by arrow. (C) Blockade of TGF-β pathway induces HR gene expression and downregulates NHEJ gene expression in HSCs from Fancd2-/- mice. HSCs were sorted from Fancd2-/- mice after 48h treatment with 1D11 or isotype control antibody, and used for qRT-PCR analysis. Some genes involved in HR and NHEJ pathways were pointed out by arrow. (D, E) Gene expression of

representative NHEJ (D) and HR (E) genes Lig4, Prkdc, Brca2, mdXrccl in HSCs. (F) The frequency of HSCs in Fancd2-/- mice after 48h treatment with 1D11 or isotype control antibody (n=4 mice per group). (G) Schematic of acetaldehyde sensitivity assay in bone marrow HSPCs from WT or Fancd2-/- mice. (H) 1D11 does not protect the Fancd2-/- HSPCs from genotoxic stress when HR is inhibited. HSPCs from WT or Fancd2-/- mice were exposed to 1D11 and RAD51 inhibitors (10 μΜ) for 30 min followed by exposure to acetaldehyde for 4 hrs. The cells were then washed and cultured in presence of 1D11 and RAD51 inhibitors for five days and survival was determined. Error bars represent mean ± s.e.m. *p < 0.05; **p < 0.01. See also Figure 8.

[00021] Figure 6. Inhibition of TGF-β Pathway Rescues Acetaldehyde-induced Genotoxicity in HSPCs from FA Mice.

[00022] (A) Acetaldehyde sensitivity of Lin- cells from bone marrow of WT or Fancd2- /- mice incubated with isotype control or 1D11 antibody (10μg/mL). Cells were exposed to 2-8 mM acetaldehyde for 4 hours and survival was determined by counting live cell numbers after 4 days in culture. Error bars represent mean ± s.e.m. (B) Representative FACS plots of the peripheral blood samples after staining with antibodies against CD45.1 or CD45.2 are shown. Lin- cells from WT or Fancd2-/- murine bone marrow were exposed to acetaldehyde for 3 hrs and then cultured with isotype or 1D11 antibody before

transplantation into lethally irradiated recipients. The peripheral blood from recipients was analyzed for donor cell engraftment at 4 weeks after transplantation by staining with CD45.1 and CD45.2 antibodies. The percentage of donor-derived CD45.2+ cells or recipient-derived CD45.1+ cells in peripheral blood of recipients are shown in FACS plots. The average percentage of CD45.2+ cells are shown in the plot. (C) Lineage distribution of donor-derived cells (CD45.2) in peripheral blood of recipients as detected after staining the blood samples with antibodies against lineage markers in the transplantation experiments described in (B). (n=5 mice per group). Error bars represent mean ± s.e.m. (D) DNA repair kinetics in WT and Fancd2-/- Lin- immature cells as shown by the percentage of cells with γΗ2ΑΧ foci. Lin- cells were exposed to 4mM acetaldehyde for 4 hrs and allowed to recover in presence of isotype or 1D11 antibody. The cells were analyzed for γΗ2ΑΧ foci and 30- 100 cells were counted in each time point. (E) Schematic of acetaldehyde and ID11/isotype antibody treatments in vitro. Lineage-negative cells from bone marrow of WT and Fancd2- /- mice were pretreated with 1D11 or isotype for 30 min followed by 4h treatment with 4mM acetaldehyde. After washing out, the cells were allowed to recover for 4h in presence of 1D11 or isotype antibody. DNA damage was assessed by a single cell comet tail assay.

[00023] Figure 7. Inhibition of TGF-β Pathway Promotes DNA Repair in HSPCs from pI:pC-treated FA Mice.

(A) Physiological stress induced by pI:pC activates TGF-β pathway in HSPCs. Phospho- Smad2/3 levels in HSPCs of pI:pC- or TGF i-treated mice were analyzed by flow cytometry. (B) Representative immunofluorescence staining showing γΗ2ΑΧ and 53BP1 foci in HSCs from WT and Fancd2-/- mice treated with pI:pC (5mg/kg) plus 1D11 or isotype antibody (lOmg/kg) for 48h. (C) Representative images of alkaline comets of HSCs from WT and Fancd2-/- mice are shown. (D) Total BM cell numbers in WT and Fancd2-/- mice treated with pI:pC plus 1D11 or isotype antibody. (E) DCF-DA staining showing that inhibition of TGF-β pathway does not reduce pI:pC-induced reactive oxygen species (ROS) levels in Fancd2-/- HSPCs. Mice were treated with pI:pC plus 1D11 or isotype antibody and after 48 hrs, HSPCs were analyzed. (F) Inhibition of TGF-β pathway partially prevents pI:pC-induced exit from quiescence of HSCs. (n=3 mice per group). (G) Representative immunofluorescence staining showing γΗ2ΑΧ foci in HSPCs from WT and Fancd2-/- mice treated with pI:pC plus 1D11 for four weeks.

[00024] Figure 8. Inhibition of TGF-β Pathway in murine HSPCs Promotes DNA Repair Activity.

(A) Experimental scheme for inhibition of TGF-β Pathway in mice. WT or Fancd2-/- mice were treated with isotype or 1D11 antibody (lOmg/kg) for 48h, and bone marrow HSCs were sorted for gene expression profile and cell cycle analysis. (B) Increased number of Fancd2-/- HSCs in S-G2M phase of cell cycle after 1D11 treatment. Cell cycle analysis of HSCs from WT or Fancd2-/- mice treated with isotype or 1D11 antibody for 48h is shown. Error bars represent mean ± s.e.m. (n=4 mice per group). (C) Representative immunoblots showing 1D11 efficiently inhibits the level of p-Smad2 in hematopoietic progenitors from Fancd2-/- mice. (D) RAD51 inhibitors, RI-1 and B02, significantly decrease HR efficiency. Homologous recombination assay was measured in U20S cells with DR-GFP reporter after treatment with 10μΜ RI-1 and 10μΜ B02. The representative of two independent experiments is presented; error bars represent mean ± s.e.m. (E, F) RAD51 inhibitors, RI-1 and B02, efficiently block RAD51 foci formation. GM6914 (FA- A) cells or corrected GM6914+FANCA cells were treated with ΙμΜ MMC and ΙΟμΜ inhibitors for 6h before immunofluorescence analysis. Representative images (E) and quantification (F) of RAD51 foci are shown. (G) RAD51 inhibitors do not show cytotoxicity in HSPCs. HSPCs from WT or Fancd2-/- mice were treated with B02 (ΙΟμΜ) or RI-1 (ΙΟμΜ) in vitro for five days and survival was determined. (H) Colony forming assay showed that RAD51 inhibitors block the protective function of 1D11 after genotoxic stress in Fancd2-/- HSPCs. HSPCs from WT or Fancd2-/- mice were exposed to 1D11 (lC^g/mL) and RAD51 inhibitors (ΙΟμΜ) for 30 min followed by exposure to acetaldehyde for 4 hrs. The cells were then washed and cultured in presence of 1D11 and RAD51 inhibitors for 7-9 days, and hematopoietic colonies were counted.

DETAILED DESCRIPTION OF THE INVENTION

[00025] The invention is based in part upon the surprising discovery that inhibition of the TGF β signaling pathway will rescue the growth of bone marrow cells in patients with Fanconi Anemia (FA).

[00026] More specifically, it was discovered that blocking the transforming growth factor β (TGF ) pathway, by either genetic deletion or pharmacologic inhibition, significantly enhanced FA cellular growth and improved cellular survival in the presence of DNA interstrand cros slinking agents.

[00027] Blocking the TGF-β pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-β signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth.

[00028] In humans, three isoforms of TGF , TGF i , TGF 2 and TGF 3, are known to exist. (Swiss Prot accession numbers P001137, P08112 and P10600 (respectively)). In their biologically active state, these three isoforms are 25 kDa homodimers comprising two 112 amino acid monomers joined by an inter-chain disulfide bridge. TGF i differs from TGF 2 by 27 amino acids, and from TGF 3 by 22 amino acids. The differences are mainly conservative amino acid changes. The three-dimensional structure of TGFfi has been determined by X-ray crystallography and the receptor binding regions have been defined. Both human TGF s and mouse TGF s are similar. The human TGF i has one amino acid difference from a mouse TGF i. Human TGF 2 has only a three amino acid difference from mouse TGF ¾ and human and mice TGF 3 are identical. One neutralizing mouse monoclonal antibody that binds TGF i, TGF 2 and TGF 3 isoforms is known as 1D11 and is available from R&D Systems- (Catalog No. MAB-1835) through the ATCC (Accession No. HB 9849). Fresolimumab, also known as GC1008, (CAS Registry Number: 948564-73- 6) is a humanized monoclonal IgG4 antibody that neutralizes all TGF isoforms and is suitable for therapeutic use in humans.

[00029] Bone marrow stromal cells derived from Fancd2-deficient mouse exhibited hyperactive noncanonical TGF -Erk pathway, and inhibition of this pathway also restored resistance to genotoxic agents. Moreover, inhibition of the canonical TGF -Smad pathway rescued the proliferation defect of hematopoietic stem/progenitor cells (HSPCs) from Fancd2-deficient mice and human FA patients. Mechanistically, hyperactive TGFfi signaling in FA cells resulted in elevated non-homologous end joining (NHEJ) activity and reduced homologous recombination (HR) repair. The activation of HR repair by TGFfi inhibition accounts, at least in part, for the improvement in cellular growth. Taken together, inhibition of the TGFfi signaling pathway will provide a therapeutic strategy in the clinical treatment of FA patients with bone marrow failure.

[00030] Despite the elucidation of the FA/BRCA pathway ² ' ³ , the pathophysiological mechanism of BMF in FA has remained elusive. Research has been hampered by the fact that FA pathway-deficient mice do not spontaneously develop bone marrow failure ¹² . Recent studies have demonstrated that HSPCs from FA patients and FA mice have a hyperactive p53/p21 axis, resulting, at least in part, in the increased BMF ⁹ . The

hyperactivation of p53/p21 appears to result from unresolved DNA replication stress, endogenous DNA damage, and other cellular stresses. The progressive impairment of HSPC by p53-mediated cell cycle arrest and apoptosis also accounts for the observed delay in onset of BMF in FA patients.

[00031] Here, we describe a novel mechanism for BMF in FA patients and mouse models - namely, the hyperactivation of the TGFfi pathway in FA HSPCs and stromal bone marrow fibroblasts. Using an unbiased shRNA screen, we initially identified hyperactive components of the TGFfi signaling pathway, which suppress the growth of FA patient- derived cell lines. Inhibition of TGF pathway, in HSPCs and in primary bone marrow stromal cells, partially rescued the growth and crosslinker hypersensitivity of these cells. Bone marrow failure is treated, prevented or delayed, by administering to a subject having Fanconi Anemia a composition comprising fresolimumab, 1D11 or an antigen binding fragment thereof. . In other aspects, fresolimumab, 1D11 or an antigen binding fragment threod is administered to a subject that is to receive a bone marrow transplant.

[00032] Treatment is efficacious if the treatment leads to clinical benefit such as, an increase in bone marrow stems cells and/or bone marrow stromal fibroblast cells in the patient. When treatment is applied prophylactically, "efficacious" means that the treatment retards or prevents bone marrow failure or alleviates a clinical symptom of bone marrow failure such as decreasing blood count. Efficaciousness is determined in association with any known method for diagnosing or treating bone marrow failure.

[00033] Fresolimumab, 1D11 or an antigen binding fragment thereof is administered before the patient is prepared for a bone marrow transplant, after a bone marrow transplant or both. Alternatively, fresolimumab, 1D11 or an antigen binding fragment thereof is administered after the patient is prepared for a bone marrow transplant but before the bone marrow transplant. By prepared for a bone marrow transplant is meant that the paint has had undergone a conditioning regimen such a chemotherapy, total body irradiation, or both to weaken or destroy the unhealthy bone marrow.

[00034] In other embodiments, fresolimumab, 1D11 or an antigen binding fragment thereof is administered to subject during a medical crisis such as a bacterial or viral infection.

[00035] Therapeutic Administration

[00036] The invention includes administering to a subject having Fanconi Anemia a composition comprising a fresolimumab (GC1008), 1D11 or an antigen binding fragment thereof.

[00037] An effective amount of a therapeutic compound is preferably from about 0.1 mg/kg to about 150 mg/kg. More preferably, 0.5 mg/kg to 10 mg/kg, even more preferably 1 mg/kg to 5 mg/kg. Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other therapeutic agents for treating, preventing or alleviating bone marrow failure such as androgen therapy or erythropoietin. [00038] A therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from Fanconi Anemia by standard methods.

[00039] Doses may be administered once, or more than once. In some embodiments, it is preferred that the therapeutic compound is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week for a predetermined duration of time, most preferably 3 times per week although less frequent dosing may be preferred if targeting the blood compartment as in Fanconia Anemia. The predetermined duration of time may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to 1 year, preferably for 1 to two months. In some cases, chronic administration may be desired, especially in the treatment of a condition lasting more than three months like Fanconia Anemia. The terms "chronic administration" or "administered chronically" mean prolonged drug administration for a duration of greater than three months.

[00040] The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, the compound is administered orally, rectally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intramuscularly, and intravenously, preferably intravenously. The inhibitors are optionally formulated as a component of a cocktail of therapeutic drugs. Examples of formulations suitable for parenteral administration include aqueous solutions of the active agent in an isotonic saline solution, a 5% glucose solution, or another standard pharmaceutically acceptable excipient. Standard solubilizing agents such as PVP or cyclodextrins are also utilized as

pharmaceutical excipients for delivery of the therapeutic compounds.

[00041] The therapeutic compounds described herein are formulated into compositions for other routes of administration utilizing conventional methods. For example, the therapeutic compounds are formulated in a capsule or a tablet for oral administration.

Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a therapeutic compound with a solid carrier and a lubricant.

Examples of solid carriers include starch and sugar bentonite. The compound is administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, conventional filler, and a tableting agent. Other formulations include an ointment, suppository, paste, spray, patch, cream, gel, resorbable sponge, or foam. Such formulations are produced using methods well known in the art.

[00042] Therapeutic compounds are effective upon direct contact of the compound with the affected tissue. Accordingly, the compound is administered topically. Alternatively, the therapeutic compounds are administered systemically. For example, the compounds are administered by inhalation. The compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00043] Additionally, compounds are administered by implanting (either directly into an organ or subcutaneously) a solid or resorbable matrix which slowly releases the compound into adjacent and surrounding tissues of the subject.

[00044] Definitions

[00045] The term "TGF " or "transforming growth factor-beta" refers to the family of molecules described that have either the full-length, native amino acid sequence of any of the humans TGF isoforms.

[00046] A 'TGF antibody'" or antigen binding fragment thereof refers to an antibody that binds to any of the isoforms of TGF , preferably binding to either TGF i, TGF 2, or TGF 3, or to any combination thereof

[00047] The term "polypeptide" refers, in one embodiment, to a protein or, in another embodiment, to protein fragment or fragments or, in another embodiment, a string of amino acids. In one embodiment, reference to "peptide" or "polypeptide" when in reference to any polypeptide of this invention, is meant to include native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992). [00048] As used interchangeably herein, the terms "oligonucleotides", "polynucleotides", and "nucleic acids" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, all as described herein.

[00049] The term "homology", when in reference to any nucleic acid sequence indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence. Homology may be determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid or amino acid sequence homology may include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.

[00050] As used herein, the term "substantial sequence identity" or "substantial homology" is used to indicate that a sequence exhibits substantial structural or functional equivalence with another sequence. Any structural or functional differences between sequences having substantial sequence identity or substantial homology will be de minimus; that is, they will not affect the ability of the sequence to function as indicated in the desired application. Differences may be due to inherent variations in codon usage among different species, for example. Structural differences are considered de minimus if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure. Such characteristics include, for example, the ability to hybridize under defined conditions, or in the case of proteins, immunological crossreactivity, similar enzymatic activity, etc. The skilled practitioner can readily determine each of these characteristics by art known methods.

[00051] Additionally, two nucleotide sequences are "substantially complementary" if the sequences have at least about 70 percent or greater, more preferably 80 percent or greater, even more preferably about 90 percent or greater, and most preferably about 95 percent or greater sequence similarity between them. Two amino acid sequences are substantially homologous if they have at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% similarity between the active, or functionally relevant, portions of the polypeptides.

[00052] To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[00053] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G, Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). [00054] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, "ameliorated" or "treatment" refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.

[00055] Thus, treating may include suppressing, inhibiting, preventing, treating, delaying the onset of or a combination thereof. Treating refers inter alia to increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative

therapeutics, or a combination thereof. "Suppressing" or "inhibiting", refers inter alia to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof. The symptoms are primary, while in another embodiment, symptoms are secondary. "Primary" refers to a symptom that is a direct result of the proliferative disorder, while, secondary refers to a symptom that is derived from or consequent to a primary cause. Symptoms may be any manifestation of a disease or pathological condition.

[00056] As used herein, "an ameliorated symptom" or "treated symptom" refers to a symptom which approaches a normalized value, e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests. [00057] As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

[00058] As used herein, the term "safe and effective amount" or "therapeutic amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

[00059] The terms "patient" "subject" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

[00060] By the term "modulate," it is meant that any of the mentioned activities, are, e.g., increased, enhanced, increased, augmented, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an antagonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values.

[00061] As used herein, "molecule" is used generically to encompass any vector, antibody, protein, drug and the like which are used in therapy and can be detected in a patient by the methods of the invention. For example, multiple different types of nucleic acid delivery vectors encoding different types of genes which may act together to promote a therapeutic effect, or to increase the efficacy or selectivity of gene transfer and/or gene expression in a cell. The nucleic acid delivery vector may be provided as naked nucleic acids or in a delivery vehicle associated with one or more molecules for facilitating entry of a nucleic acid into a cell. Suitable delivery vehicles include, but are not limited to: liposomal formulations, polypeptides; polysaccharides; lipopolysaccharides, viral formulations (e.g., including viruses, viral particles, artificial viral envelopes and the like), cell delivery vehicles, and the like.

EXAMPLES

[00062] EXAMPLE 1: GENERAL METHODS

[00063] Animals

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[00064] We generated Fancd2 ^{" "} mice as previously described . C57BL/6J-CD45.1, and C57BL/6J-CD45.2 mice were obtained from mice were purchased from The Jackson Laboratory. All mice were in C57BL/6J background, and were bred and maintained in a temperature- and humidity-controlled environment and given unrestricted access to 6% chow diet and acidified water. We treated WT and Fancd2 ^_/" mice with 0.3mg/kg MMC to induce bone marrow failure. The neutralizing anti-mouse TGFfi monoclonal antibody 1D11 was provided by Genzyme, and administrated mice at 10 mg/kg by intraperitoneal injections three time per week. Animal experiments were performed following the approved protocol of the Animal Care and Use Committee at the Dana Farber Cancer Institute.

[00065] Hematopoietic stem/progenitor cell culture and flow cytometry analysis.

[00066] For mouse hematopoietic stem/progenitor cell culture, Lin ^" cells were cultured in vitro in StemSpan SFEM media with lOng/mL SCF, 20ng/mL IGF-2, 20ng/mL TPO, lOng/mL heparin, and lOng/mL a-FGF. Half media was changed every three days. For human cord blood cells, CD34 ⁺ cells were isolated using CD34 microbead kit (Miltenyi Biotec). Cells were cultured in StemSpan SFEM media with lOOng/mL hSCF, lOOng/mL FLT3 ligand, lOng/mL TPO, lOng/mL IL-6. Cells were treated with lOug/mL 1D11 or 10 μΜ SD208.

[00067] To perform flow cytometry analysis, Lin ^" cells were collected at day 2 and 5 post transduction and suspended in staining medium (PBS with 2% heat-inactivated calf serum), and incubated with PE-conjugated c-Kit and APC-conjugated Sca-1 antibodies were added to the cells for 30 min at 4°C in the dark. Stem cell population (GFP ⁺ Lin ^" c-Kit ⁺ Sca-1 ⁺ ) was analyzed by FACS. All these antibodies were purchased from eBioscience. [00068] Functional cell-based assays

[00069] For survival assays, cells were seeded at a density of lxl 0 ³ cells per well in 96- well plates. After 72 hours of culture in indicated concentrations of MMC or post exposure of acetaldehyde, viability was assessed using CellTiterGlo reagent (Promega). In order to assess clonogenicity, cells were seeded at a low density (500-1000 cells per well) in 6-well plates and allowed to form colonies. The cells were then fixed in methanol/20% acetic acid and stained with 1% crystal violet. Colony formation was assessed by solubilizing crystal violet stain with methanol and quantifying UV absorbance for each condition.

[00070] ELISA

[00071] Cell culture supernatant were harvested, and the TGF i level was assessed using TGF-βΙ Multispecies ELISA Kit (Invitrogen) following the manual procedure.

[00072] Immunofluorescence

[00073] Cells were grown on coverslip for 24 hours before treated with MMC. Cells were fixed with 4% (w/v) paraformaldehyde for lOmin at room temperature, washed three times with PBS, followed by extraction with 0.3% Triton X-10 for lOmin on ice. The incubation with the primary antibody (anti-RAD51, Santa Cruz) was done at 37°C.

[00074] Comet Assay

[00075] To evaluate MMC induced DNA cross-link damage and repair, a modified alkaline Comet assay was performed ³⁷ ' ³⁸ . Briefly, cells were seeded into 6-well plates at 20% confluence and treated with MMC for 6 hours, and washed and release for 24 and 48 hours. Cells were collected, placed on slide coated with agarose, and lysed according to manufacturer's protocol of Trevigen's Comet Assay Kit (Trevigen). After lysis, the slides were irradiated to induce strand breaks with 5Gy γ-radiation. Electrophoresis was conducted, and comets were visualized using an Axio Imager Zl fluorescence microscope with an AxioCam MRm CCD camera (Zeiss, Thornwood, NY).

[00076] Real time RT-PCR

Total RNA was isolated using the RNeasy Mini kit (Qiagen, CA). cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Life Technologies). All real time PCR reactions were done using Vii A 7 PCR machine. 20μ1. reaction system was composed of ΙΟμΙ. SYBR Green, 2.5μ1. 20uM primer mixture, lOng cDNA and nuclease-free water. All experiments were performed in triplicate. Gapdh was the internal control. The primer sequences were shown as follows: Tgfbl sense: CAGCTCCTCATCGTGTTGGTG (SEQ ID NO: 11); Tgfbl antisense: GCACATACAAATGGCCTGTCTC (SEQ ID NO: 12); Smad3 sense: CACGCAGAACGTGAACACC (SEQ ID NO: 13); Smad3 antisense:

GGCAGTAGATAACGTGAGGGA (SEQ ID NO: 14); Gapdh sense:

TGGATTTGGACGCATTGGTC (SEQ ID NO: 15); Gapdh antisense:

TTTGCACTGGTACGTGTTGAT (SEQ ID NO: 16).

[00077] Murine Bone Marrow Transplantation

[00078] Donor cells (CD45.2 ⁺ ) were transplanted into lethally irradiated (10 Gy dose) recipient (congenic B6-CD45.1 ⁺ mice) along with lxl0 ⁵ cells competitive bone marrow cells from congenic B6-CD45.1 ⁺ mice. Peripheral blood from recipient mice was analyzed for donor cell engraftment as described (Parmar et al, 2010).

[00079] Colony-forming Unit-spleen (CFU-S) Assay

[00080] Recipient mice (wild-type, 8-12 weeks old) were irradiated with a split dose of 1100 rad (550 rad each, 4 hours apart) before transplantation. Forty thousand bone marrow cells from donor mice were transplanted into each recipient mouse. Ten to 12 days posttransplantation, spleens were harvested and fixed with Bouin fixative solution.

[00081] In Vivo Xenograft Assay

[00082] Human cord blood derived CD34 ⁺ cells were transduced with lentivirus encoding shFANCD2 or shControl as described (Ceccaldi et al, 2012). While cells were selected with 2μg/mL puromycin, cells were also treated with 10μg/mL GC1008. After 48h treatment, 2xl0 ⁵ cells were transplanted into sub-lethally irradiated (2.5Gy) NSG mice. The recipient mice were treated with lOmg/kg GC1008 at 3 doses per week for two weeks. Two and eight weeks after transplantation, human cells in peripheral blood (PB) were analyzed using anti-human CD45 antibody (eBioscience, 17-0459-42) by flow cytometry.

[00083] TGF-β Pathway Activity Using Luciferase Reporter Assay

[00084] 293T cells were transiently transfected with a TGF-β responsive luciferase promoter (CAGA-luc) plasmid (kindlyprovided by H.Y. Lin, Massachusetts General Hospital) along with FANCD2 or control vector. Cells were harvested at 48h after transfection and luciferase activity was determined using the Dual-Lucifease Reporter Assay system (Promega).

[00085] Traffic Light Reporter (TLR) Assay

[00086] Genome engineering experiments were performed as previously described

(Certo et al, 2011). Briefly, single copy of TLR cell lines including FANCA-/- fibroblast cells (GM6914) and FANCA corrected GM6914 with or without shSMAD3 were generated by transducing cells with TLR-BFP reporter lentivirus, typically yielding -5% transduction based on fluorescence. Two days after transduction, transduced cells (BFP+) were sorted by FACS. To generate double strand break, cells were seeded at 2x105 cells per well in 6-well plate 24h before transduction, and cells were transduced with lentivirus containing I-Scel alone or I-Scel plus GFP donor template. For SD208 treatment, cells were treated with ΙΟμΜ SD208 after 3-4h post-transduction. All transductions were carried out in

[00087] the presence of 8 μg/mL polybrene. Twenty-four hours after transduction, medium was changed. Genome engineering events were analyzed by flow cytometry at 72 hours after transduction. NHEJ is represented bymCherry fluorescence, and HR by GFP fluorescence.

[00088] Drug Sensitivity Assays

[00089] For survival assays, cells were seeded at a density of 1x103 cells per well in 96- well plates. After 3-6 days of culture in indicated concentrations of MMC or post exposure of acetaldehyde, viability was assessed using CellTiterGlo reagent (Promega). In order to assess clonogenicity, cells were seeded at a low density (500-1000 cells per well) in 6-well plates and allowed to form colonies. The cells were then fixed in methanol/20% acetic acid and stained with 1% crystal violet. Colonies were counted after crystal violet staining.

[00090] Chromatin Immunoprecipitation (ChIP) Assay

[00091] The ChIP assay was performed as described previously (Park et al., 2013). Briefly, 10x106 cells were treated with MMC (ΙμΜ) for 8 hours. Cells were chemically crosslinked with 1% formaldehyde for 15 min at room temperature. Cells were rinsed twice with lx PBS and harvested in Famham lysis buffer (5mM PIPES pH8.0, 85mM KC1, 0.5% NP-40, and protease inhibitor cocktail). After washing, cells were resuspended in sonication buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail), and sonicated withl0x30 seconds pulses, 5 min in total, 18-21 Watts of power. After sonication, 5% samples were used as input. Sonicated samples were further divided in half, and incubated overnight with 100 μΐ. of Dynal Protein G magnetic beads that had been preincubated with anti-FANCD2 (Novus Biologicals, NB 100-316) or IgG control antibody. Beads were washed 5x with LiCl wash buffer (lOOmM Tris pH 7.5, 500mM LiCl, 1% NP- 40, 1% sodium deoxycholate), and lx TE buffer containing 50mM NaCl. Bound complexes were eluted from the beads by heating at 65°C for 1 hour (vortexing every 5min). Crosslinking was reversed by incubating samples at 65°C for overnight. DNA was purified and then analyzed by real-time PCR using SMAD1 promoter primers (primer #1 : 5' AAGGC AGGAGAATTGCTTGA-3 ' , 5'-CCTTCACCTTCTGCCATGAT-3'; primer#2: 5'- C AAGGGAGGGTTTC AAC AG-3 ' , 5'- TGAGCACTTACTGGTCAATTCG-3 ').

[00092] Example 2: TGFp pathway inhibition corrects compromised stem cell function of FA mice

[00093] Bone marrow failure in FA results from impaired stem cell function caused by

2 28 32

endogenous genotoxins ' ' . Additionally, compared to WT HSPCs, Fancd2-/- HSPCs are hypersensitive to TGFP (Figure 2A). Therefore, we hypothesized that inhibition of TGFP pathway may correct the defective function of stem cells collected from FA mice. To block the TGF pathway, we use the anti-murine TGFP monoclonal antibody 1D11, because it has been shown to promote the regeneration of bone marrow in murine models. Blockage of TGF using this neutralizing antibody 1D11 promotes multilineage hematopoietic regeneration in the 5-FU-treated myelosuppression murine model ²¹ . Treatment with 1D11 antibody significantly blocked the activation of the TGF pathway in murine stromal cells, and rescued MMC sensitivity of Fancd2 ^_/" stromal cells (Figure 1A). Importantly, 1D11 treatment also corrected the suppressive effects caused by either exogeneous TGFpi (Figure 2B) or endogenous TGFP signaling (Figure 2C). These data demonstrate that 1D11 can efficiently block the TGFP pathway.

[00094] Next, we investigated the potential effect of 1 D 11 on Fancd2 ^_/" HSPCs in the presence of acetaldehyde. As expected, both Lin ^" cells and HSCs from Fancd2 ^_/" mice were more sensitive to acetaldehyde than congenic controls; in contrast, 1D11 treatment counteracted the genotoxic effects of acetaldehyde, and significantly rescued the growth and clonogenic capacity of Fancd2 ^_/" HSPCs (Figure IB and C). We further examined whether TGFP pathway inhibition could rescue bone marrow failure in vivo using an MMC-induced bone marrow failure model in FA mice. 1D11 treatment significantly rescued hemoglobin levels and red blood cell counts in Fancd2-/- mice treated with MMC (Figure 2D and E). To further assess the effect of TGFP pathway inhibition on FA HSCs in vivo, we performed an engraftment assay, using control or shSmad3 lentivirus-transduced Lin ^" cells from WT and Fancd2 ^_/" mice (Figure ID). Smad3-deleted Fancd2 ^_/" cells engrafted more efficiently than control cells (Figure IE). Taken together, our data indicate that inhibition of the TGFP pathway by ID 11 restores resistance of HSCs from FA mice to endogenous genotoxins, and partially corrects impaired stem cell function.

[00095] EXAMPLE 3: TGF-B PATHWAY INHIBITION RESCUES ACETALDEHYDE- INDUCED GENOTOXICITY IN HSPCS FROM FA MICE

[00096] DNA damage induced by the endogenous genotoxic agents, such as

acetaldehyde, causes the attrition of HSCs, leading to spontaneous bone marrow failure in murine FA models (Garaycoechea et al., 2012; Langevin et al, 2011). We reasoned that blockade of the TGF-β pathway may contribute to the repair of acetaldehy de-induced DNA damage in FA HSCs. As expected, bone marrow cells irom Fancd2 ^{~ ~} mice were sensitive to acetaldehyde exposure. The neutralizing anti-TGF-β antibody ID 11 treatment counteracted the genotoxic effects of acetaldehyde, and rescued the growth and clonogenic capacity of Fancd2 ^~/~ HSCs or Lin ^" cells from bone marrow (Figure 4A and 6A). To directly measure the protective effect of 1D11 on the survival of HSPCs after acetaldehyde exposure, a stem cell transplant was performed. The percentage of donor-derived cells at 4 and 16 weeks post transplantation was higher in recipients of ID 11 treated Fancd2 ^~/~ group than those in the isotype control group (Figure 4B and 6B), and 1D11 did not significantly affect the lineage differentiation of HSPCs (Figure 6C). Furthermore, γΗ2ΑΧ foci, a marker for DNA damage, resolved more rapidly in 1D11 treated Fancd2 ^~/~ cells, suggesting an improvement of DNA repair (Figure 4C, 4D, and 6D). Repair of DNA damage was further confirmed by comet assay (Figure 4E, 4F, and 6E). 1D11 treated Fancd2 ^~/~ cells also showed improvement in DNA repair when DNA damage was directly assessed by alkaline comet assay and confirmed that acetaldehy de-induced γΗ2ΑΧ foci indeed correspond to unrepaired double strand breaks (Figure 4E, 4F, and 6E), Taken together, pharmacologic inhibition of the TGF-β pathway by 1D11 promotes resistance of FA cells to endogenous genotoxic agents, and partially corrects the impaired stem cell function by promoting DNA repair.

[00097] EXAMPLE 4: INHIBITION OF TGF-B PATHWAY RESCUES PHYSIOLOGICAL STRESS-INDUCED BONE MARROW FAILURE IN FA MICE

[00098] Physiological stress-induced DNA damage is a source of DNA damage in HSCs in FA. The accumulation of physiological stress-induced DNA damage results in a collapse of the hematopoietic system in a murine FA model (Walter et al, 2015). Interestingly, polyinosinic:polycytidylic acid (pI:pC) exposure caused activation of TGF-β pathway in HSPCs (Figure 7A). To determine whether inhibition of TGF-β pathway also counteracts the genomic instability of HSCs caused by physiological stress, we simultaneously treated WT and Fancd2 ^~/~ mice for 48h with pI:pC (to induce stress) and IDl l (to block the TGF-β pathway). As previously described (Walter et al, 2015), administration of pI:pC induced DNA damage in HSCs and caused increased levels of γΗ2ΑΧ and 53BP1 foci (Figure 1A- 1D and 7B); inhibition of the TGF-β pathway by IDl l markedly reduced the pI:pC- induced DNA damage (Figure 1A-4D, 7B, and 7C). Importantly, inhibition of the TGF-β pathway rescued the viability oiFancd2 ^~ total bone marrow cells following pI:pC treatment (Figure 7D). Consistent with the previous study (Walter et al, 2015), pI:pC exposure induced oxidative stress in HSPCs from Fancd2 ^_/" mice, however, 1D11 did not affect the level of oxidative stress (Figure 7E). As expected, pI:pC exposure induced proliferation of HSPCs, resulting in a concomitant reduction in the frequencies of quiescent cells. Interestingly, 1D11 treatment reduced pI:pC-induced cell cycling and increased the percentage of quiescent HSPCs, perhaps accounting for the improved DNA repair (Figure 7F). Thus, TGF-β pathway inhibition counteracts physiological stress-induced DNA damage in HSCs.

[00099] To determine whether TGF-β pathway inhibition rescues pI:pC induced bone marrow failure, we treated WT and Fancd2 ^~/~ mice with pI:pC and 1D11 (Figure IE). Interestingly, four weeks of pI:pC treatment caused peripheral blood pancytopenia in isotype control antibody -treated Fancd2 ^~/~ mice with reduction in red blood cells (RBC), white blood cells (WBC) and hemoglobin levels (Figure IF). However, administration of IDl l significantly prevented these peripheral blood abnormalities (Figure IF). To examine the bone marrow HSC function, we next transplanted bone marrow cells from these pI:pC (plus or minus IDl l) treated donor mice into lethally irradiated recipients and evaluated spleen colony -forming units (CFU-S), thus allowing the quantification of the surviving multipotent short-term HSCs. As expected, pI:pC treatment caused a marked reduction in the CFU-S content oiFancd2 ^~ bone marrow, and IDl l improved this defect (Figure 1G). Consistent with these results, in a competitive repopulation experiment, recipient mice transplanted with bone marrow from IDl l treated Fancd2-/- mice showed improved short- term donor cell engraftment compared to the recipients transplanted with isotype control (Figure 1H). Bone marrow HSPCs from Fancd2 ^{" "} mice treated with IDl l also exhibited less DNA damage upon pI:pC exposure (Figure II, 1J, and 7G). Together, these data suggest that TGF-β pathway inhibition rescues pI:pC-induced bone marrow failure in FA mice and may be effective in preventing the bone marrow collapse of FA patients observed after viral infections.

[000100] EXAMPLE 5: TGF-B PATHWAY INHIBITION UPREGULATES HR AND

DOWNREGULATES NHEJ IN HSCS OF FA MICE

[000101] To determine the molecular mechanism by which TGF-β pathway regulates DNA repair and rescues genotoxicity in HSCs, we next analyzed the expression of DNA repair genes in HSCs. Genes associated with NHEJ, HR, nucleotide excision repair (NER), mismatch repair (MMR), and the DNA damage response (DDR) were evaluated.

Interestingly, over 70% of DNA repair genes examined were significantly upregulated in HSCs from WT mice exposed to 1D11, the neutralizing anti-murine TGF-β monoclonal antibody (Figure 6A and 8A). In contrast, many genes involved in NHEJ were upregulated and many genes involved in HR were downregulated in HSCs from Fancd2 ^_/" mice, when compared to WT HSCs (Figure 5B). This skewed expression pattem in Fancd2 ^~/~ HSCs was reversed by TGF-β pathway inhibition, as ID 11 antibody treatment induced expression of HR genes such as Brca2 and Xrccl in Fancd2 ^~/~ HSCs, and caused a concomitant reduction of NHEJ gene expression, such as Lig4 and Prkdc (Figure 5C-5E). Previous studies have shown that HR is upregulated when HSCs are driven into the cell cycle, thereby resulting in repair of double strand breaks (Beerman et al., 2014). Accordingly, the increase in HR activity in FA HSCs following TGF-β pathway inhibition may result, at least in part, from the release from cell cycle arrest (Figure 8B), subsequently leading to a higher frequency of HSCs in FA mice (Figure 5F). To confirm that inhibition of TGF-β pathway in HSCs creates an HR-competent state and thereby functionally protects them from genotoxicity, we inhibited both HR and TGF-β pathway and examined the survival of cells after

acetaldehyde exposure (Figure 5G). We used pharmacologic inhibitors which blocked HR, resulting in the reduction of RAD51 foci and the inhibition of DR-GFP plasmid

recombination in a reporter assay (Figure 8D-8F). As expected, 1D11 exposure inhibited TGF-β pathway signaling in bone marrow from FA mice (Figure 8C). Interestingly, 1D11 did not protect the HSPCs from acetaldehy de-induced genotoxicity when HR was inhibited by pharmacologic inhibitors of RAD51 (Budke et al, 2012; Huang et al, 2012) (Figure 5H, 8G and 8H). Collectively, these data indicate that TGF-β pathway inhibition upregulates HR and downregulates NHEJ in HSPCs of Fancdl ^" mice and functionally promotes their survival.

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