WO2020257587A1 - Methods of promoting erythropoiesis and overcoming erythropoitein resistance in patients - Google Patents

Abstract

Erythropoietin (Epo) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. The present disclosure is directed to methods for promoting erythropoiesis in a subject. The disclosure also provides methods for overcoming erythropoietin (Epo) resistance in a subject.

Description

METHODS OF PROMOTING ERYTHROPOIESIS AND OVERCOMING ERYTHROPOITEIN RESISTANCE IN PATIENTS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62/863,465, filed June 19, 2019, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Contract No. HL116436 awarded by National Institutes of Health. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The Sequence Listing in an ASCII text file, named as

37463_8815_02_PC_SequenceListing.txt of 1 KB, created on June 15, 2020, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

BACKGROUND

[0004] Erythropoiesis is a multiweek program coupling massive proliferation with progressive cellular differentiation ultimately enabling a tiny number of hematopoietic stem cells (HSCs) to yield millions of erythrocytes per second (Higgins, Clin Lab Med 35, 43-57, (2015)). Erythropoietin (Epo) is essential for red blood cell (RBC) production but this cytokine acts well after irreversible commitment of hematopoietic progenitor cells (HPCs) to an erythroid fate. It is not known if terminal erythropoiesis is tethered to the pool of available immature hematopoietic stem and progenitor cells (HSPCs).

[0005] It is estimated that a third of the world population suffers from anemia (Kassebaum et a , Blood 123, 615-624, (2014); Le, PLoS One 11, e0166635,

doi:10.1371/journal.pone.0166635 (2016)). Daily production of >200 billion erythrocytes is required to keep up with routine losses so even minor disequilibrium between blood loss and erythrocyte production can lead to anemia. Although many transient anemias are easily treated, therapy for chronic anemias is limited. RBC transfusions and Erythropoiesis Stimulating Agents (ESA) are not always effective, yet are linked to significant expense, inconvenience, potential toxicity, and generally transient utility (Sankaran and Weiss, Nat Med 21, 221-230, (2015); Huang and Tefferi, Eur J Haematol 83, 154-155, (2009)). Despite these limitations, development of new approaches to treat chronic anemia have been limited by the incomplete understanding of erythropoeisis.

[0006] A great deal of what is known about erythropoiesis relates to the terminal maturation of erythroid precursors leading to hemogobinization, enucleation and release of immature RBCs into the circulation (Wu et ah, Cell 83, 59-67, (1995); Hattangadi et ah Blood 118, 6258-6268, (2011)). In the setting of anemia or otherwise reduced oxygen delivery, juxtaglomerular renal Epo producing (REP) cells can increase Epo secretion many thousands-fold thereby serving as a feedback signal reporting erythrocyte need to the marrow. Nonetheless, Epo acts during a very narrow window of erythropoiesis, well after progenitor commitment to an exclusively erythoid fate. It is not known if these final steps of RBC maturation are coupled to the earlier stages of HSPC differentiation; a process that begins almost three weeks earlier when a HSC begins its march towards committed RBC precursors via a series of branching cell fate decisions.

SUMMARY OF THE DISCLOSURE

[0007] An aspect of this disclosure is directed at a method of promoting erythropoiesis in a subject in need thereof comprising administering a TGF signaling inhibitor to the subject.

[0008] In some embodiments, the subject suffers from anemia. In some embodiments, the anemia results from myelodysplastic syndrome, beta-thalassemia, myelofibrosis, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

[0009] In some embodiments, the subject is resistant to erythropoietin prior to

administration of the TGF signaling inhibitor.

[0010] In some embodiments, the subject has an elevated erythropoietin level associated with poor response to exogenous erythropoietin. [0011] In some embodiments, the method further comprises administering erythropoietin (EPO) to the subject before, during, or after the administration of the TGF signaling inhibitor.

[0012] In some embodiments, the TGF signaling inhibitor inhibits TGF signaling through a TGF receptor that is TGF R2 or AFK5 (TGF Rl). In some embodiments, the TGF signaling inhibitor is an antibody which antagonizes the interaction and binding between TGF and TGF receptor. In some embodiments, the TGF signaling inhibitor is a soluble polypeptide composed of the extracellular domain of a TGF receptor. In some embodiments, the TGF signaling inhibitor is an oligonucleotide selected from the group consisting of an antisense, RNAi, dsRNA, siRNA and ribozyme molecule. In some embodiments, the TGF signaling inhibitor is a small molecule organic compound. In some embodiments, the TGF signaling inhibitor antagonizes the activation of latent TGF to its active form capable of activating signaling via a TGF receptor.

[0013] Another aspect of the disclosure is directed at a method of improving

erythropoietin (EPO) sensitivity in a subject comprising administering a TGF signaling inhibitor to the subject.

[0014] In some embodiments, the subject suffers from anemia. In some embodiments, the anemia results from myelodysplastic syndrome, myelofibrosis, beta- thalassemia, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

[0015] In some embodiments, the subject is resistant to erythropoietin prior to

administration of the TGF signaling inhibitor.

[0016] In some embodiments, the subject has an elevated erythropoietic level associated with poor response to exogenous erythropoietin.

[0017] In some embodiments, the method further comprises administering erythropoietin (EPO) to the subject before, during, or after the administration of the TGF signaling inhibitor.

[0018] In some embodiments, the subject is not administered erythropoietin (EPO). [0019] In some embodiments, the TGF signaling inhibitor inhibits TGF signaling through a TGF receptor that is TGF R2 or ALK5 (TGF Rl). In some embodiments, the TGF signaling inhibitor is an antibody which antagonizes the interaction and binding between TGF and TGF receptor. In some embodiments, the TGF signaling inhibitor is a soluble polypeptide composed of the extracellular domain of a TGF receptor. In some embodiments, the TGF signaling inhibitor is an oligonucleotide selected from the group consisting of an antisense, RNAi, dsRNA, siRNA and ribozyme molecule. In some embodiments, the TGF signaling inhibitor is a small molecule organic compound. In some embodiments, the TGF signaling inhibitor antagonizes the activation of latent TGF to its active form capable of activating signaling via a TGF receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] FIGS. 1A - lO. Conditional deletion of TGFpi in megakaryocytes (MKs) increases apoptosis. (A) White blood cell (WBC), RBC, and platelet counts are shown for TGFpi^FUFL and TGFP 1 ^{Wk/ Wk} mice (n=16 per group). (B) Whole BM (Marrow)

(n=12 per group) and spleen (n=5 per group) cellularity are shown for TGFP 1 ^FL/FI (grey)

(black). (C) The number of Lin- (those not expressing mature lineage markers CD3, B220, CDl lb, Grl or Terl l9) marrow cells per leg (femur & tibia) is shown (n=13 per group). (D) Schematic of model for how excess immature HSPCs may yield normal marrow cellularity and blood cell counts due to apoptosis of surplus hematopoietic precursors. (E) Confocal immunofluorescence imaging of cleaved

Caspase3 (green) and Cdh5 (magenta) in bone marrow sections of TGFP 1 ^FL/FI and TGFpi^AMkMMk mice (n=3 per group). (F) Representative flow cytometry data of intracellular staining for cleaved Caspase3 in mice and for the fluorescence minus one (FMO) negative control. (G) Quantification of apoptotic cells as assessed by flow cytometry with intracellular staining for cleaved Caspase3 (n=4 & 3 for TGFP 1 ^FL/FI and TGF i^AMkMMk mice, respectively). (H) Representative flow cytometry data showing AnnexinV staining in marrow from TGFP 1 ^FL/FI (top row) and TGFP l ^AMk/AMk (bottom row) mice. (I) Quantification of the number of AnnexinV+ apoptotic cells in the marrow of TGFP 1 ^FL/FI and TGFP l ^AMk/AMk mice (n=9 per group). (J) Representative data for spleen as shown for marrow in H. (K) Quantification of the number of AnnexinV+ apoptotic cells in the spleen of TGFP 1 ^FL/FI and TGFP 1 ^AMk/AMk _mice (n=6 per group). (L) Representative flow cytometry data showing lineage markers (Lin=CD3, B220, CDl lb, Grl or Terl l9) plotted against AnnexinV in marrow from TGFP 1 ^FL/FI (top row) and TGFP 1 ^AMk/AMk (bottom row) mice. (M) Quantification of the number of AnnexinV+ apoptotic Lin+ and Lin- cells in the marrow of TGFpi^FUFL and TGFP 1 ^{Wk/ Wk} mice (n=8). (N) Gating and representative flow cytometry data showing AnnexinV staining in LKS- and LKS+ marrow populations for TGFpi^FL/FL (top row) and TGFP l ^AMk/AMk (bottom row) mice. (O) Quantification of the number of AnnexinV+ apoptotic LKS- and LKS+ cells in the marrow of TGFpi^FL/FL and TGFP 1 ^AMk/AMk mice (n=3 per group). All the quantified data are shown as mean ± SEM (* P<0.05, ** P<0.01, *** P<0.001, or if not shown, the comparison was not significant).

[0022] FIGS. 2A - 21. Increased hematopoietic stem and progenitor cells (HSPCs) in TGFpi^AMk/AMkmice have normal function. (A) Schematic for the competitive repopulation assay is shown. Marrow CD45.2 donor cells from either from TGFpi^FL/FL or TGFpi^AMk/AMk mice were mixed in a 1: 1 ratio with marrow donor cells from congenic CD45.1 mice, then transplanted into lethally irradiated mice (n=8 per group). CD45.2 chimerism in peripheral blood is shown at the indicated times after transplant (left).

Chimerism in of blood and bone marrow LKS+SLAM (Lin-, Kit+, Scal+, CD150+, CD48-) cells is shown 16 weeks after transplant for mice having received TGFpi^FL/FL (grey) or TGFP 1 ^AMk/AMk (black) donor cell grafts, as assessed by flow cytometry. (B) Schematic for secondary transplantation of WBM harvested from primary recipients is shown. CD45.2 chimerism in peripheral blood is shown at the indicated times after secondary transplant (left). Chimerism in of blood and in peripheral blood and marrow LKS+SLAM is shown 16 weeks after secondary transplant as for panel a (n=8 per group). (C) CFC enumeration of functional myeloid HPCs is shown for TGFpi^EL/FL and

TGFpi^AMk/AMk marrow cells (n=5 per group). The total number of CFU-GEMM (GEMM), CFU-GM (GM) and BFU-E are shown per leg (femur & tibia). (D) The number of spleen colony forming units (CFU-S12) 12 days after transplantation is shown per 100,000 donor cells transplanted (n=4 per group). (E) Gating and representative flow cytometry data is shown for the indicated HSPC populations. (F) Quantification of the number of marrow cells with the immunophenotype of CMP, GMP, MEP, EP, Pre-CFU-GM, Pre-CFU-GMP, Pre-MEP is shown for TGF i^HJFL and TGFp i ^AMk/AMk mice (n=12 per group). (G) The number of LKS⁺ (Lin Kit⁺Scal⁺), MPP and LKS+SLAM cells per leg is shown for TGFP 1 ^FL/FI and TGFP l^AMk/AMk mice (n=12 per group). (H) Gating and representative flow cytometry data is shown for LKS+SLAM cell cycle as reported by Ki67 and Hoechst staining. (I) Cell cycle of BM LKS+SLAM HSCs is shown for TGFP 1 ^FL/FI and

TGFpi^AMk/AMk as assessed by flow cytometry staining for DNA (DAPI) and Ki67 (mice n=6 per group). All the quantified data are shown as mean + SEM (* P<0.05, ** P<0.01, *** P O.OOl, or if not shown, the comparison was not significant).

[0023] FIGS. 3A - 3M. Surplus Epo-dependent erythroid precursors undergo apoptosis in vivo. (A) Gating and representative flow cytometry data is shown for AnnexinV staining of Terl 19+ erythroid precursors in marrow from TGFP 1 ^FL/FI (top row) and TGFP l^AMk/AMk (bottom row) mice. Quantification of the number of marrow,

AnnexinV-staining, apoptotic Terl 19+ erythroid cells is shown for TGFpi^FL/FL and TGFpi^AMk/AMk mice (n=12 per group). (B) Gating and representative flow cytometry data is shown spleen cells as described for panel a. Splenic Quantification of splenic apoptotic Terl 19+ erythroid cells is shown for TGFpi^FL/FL and TGFP 1 ^AMk/AMk mice (n=4 to 6 per group). (C) Flow cytometry gating strategy using LinE- (CD3, B220, Grl, CDl lb),

CD44, Terl 19, Hoechst (DNA) and AnnexinV to characterize erythroid maturation. (D) Quantification of erythroid precurors is shown (n=3 per group). Viable cells and

AnnexinV+ apoptotic cells for TGFpi^FL/FL (grey/green) and TGFP l ^AMk/AMk (black/blue) mice, respectively. (E) Representative flow cytometry showing staining of Kit and Epor within the AnnexinV+/Terl l9+ population identifies excess Epor+/Kit- erythroblasts within the TGFP 1 ^AMk/AMk marrow. (F) Plasma EPO levels are shown as assessed by ELISA (n=8 per group). (G) TGF i^FL/FL and TGF 1 ^{Wk/ Wk} mice were treated with 300U/kg Epo daily for 5 days and sacrificed on day 6 for analysis. (H) Representative AnnexinV staining of marrow erythroid precursor cells is shown before (top) and after (bottom) Epo treatment for TGFP 1 ^FL/FI (left) and TGFP 1 ^AMk/AMk (right) mice. The number of AnnexinV+ apoptotic Terl 19+ erythroid precursor cells is shown in marrow (I) and spleen (J) before and after Epo treatment (n=4 to 12 per group). (1) Apoptosis within maturing erythroid precursors is shown for marrow cells after Epo treatment (n=4 per group), as shown in panel d. Reticulocyte counts (L) and RBCs (M) are shown for TGFpi^FL/FL (grey/pink) and TGFP l ^AMk/AMk (black/red) mice before and after Epo (n=6 per group). All the quantified data are shown as mean + SEM (* P<0.05, ** P<0.01, *** P<0.001, or if not shown, the comparison was not significant).

[0024] FIGS. 4A-4M. Megakaryocytes direct TGFpi signaling in HSPCs but erythroid precursor maturation is independently regulated. (A) Gating and representative flow cytometry data assessing Smad2/3 phosphorylation (pSmad2/3) within the LKS+SLAM population is shown for TGFP 1 ^FL/FI and TGFP 1 ^AMk/AMk mice and for the FMO negative control. Smad2/3 phosphorylation (pSmad2/3) reported as the mean fluorescence intensity (MFI) within the indicated hematopoietic populations is shown for TGFP 1 ^FL/FI and TGFpi^AMkMMk mice (n=7 per group). (B) Schematic showing TGFpi^FUFL and

TGFpi^AMkMMk mice treated with 5pg/kg TGFpi daily for 5 days and sacrificed on day 6 for analysis. (C) MFI of pSmad2/3 staining is shown for Lin- HSPC subpopulations as quantified by flow cytometry before and after TGFpi treatment of TGFP 1 ^FL/FI (grey/light blue) and TGFP 1 ^AMk/AMk (black/dark blue) mice (n=3 to 4 mice per group). (D) RBC counts before and after treatment with TGFpi are shown for TGFpi^FL/FL and

TGFpi^AMkMMk mice (n= 6 ot 7 per group). The numbers of AnnexinV+ apoptotic cells in marrow (E) and spleen (F) are shown for TGFpi^EL/FL or TGFP 1 ^AMk/AMk mice (n=4 to 9 per group). (G) Apoptosis within maturing erythroid precursors is shown for marrow cells after TGFpi treatment (n=3 to 4 per group), as shown in panel 3d. (H) C57BL/6J mice were treated with either a TGFP-neutralizing antibody (ID 11) or isotype control antibody (13C4) at a dose of 10 mg/kg on days 1, 5 and 10 and then treated daily for 5 days with either PBS or Epo (300 U/kg). All mice were sacrificed for analysis on day 16. (I) MFI of pSmad2/3 staining is shown for Lin^neg HSPC subpopulations as quantified by flow cytometry of mice treated with 13C4 control antibody (dark green/tan) or ID 11 (light green/red) and then PBS or TGFpi (n=4 to 6 mice per group). (J) Red blood counts are shown for mice treated with 13C4 or 1D11 followed by either PBS or Epo (n=8 per group). (L) The total number Terl 19⁺ and the number of AnnexinV¹ apoptotic Terl 19⁺ erythroid precursors is shown for mice treated with 13C4 or 1D11 and then either PBS or Epo (n=4 to 6 per group).

[0025] FIGS. 5A-5C. Modular model of erythropoiesis compartmentalized by megakaryocyte TGFpi. (A) Schematic of proposed compartmentalized model of erythropoiesis. Megakaryocytic TGF 1 serves as a gatekeeper regulating the feed of committed erythroid progenitors to a maturation module regulated by Epo. Epo- dependent erythroblast survival is controlled by the need for RBC production sensed by oxygen delivery to renal Epo-producing cells. (B) Genetic deletion of TGFP in megakaryocytes, or use of TGFP ligand trap (1D11), licenses production of unneeded erythroid-committed progenitors. The excess erythroid precursors are not supported by homeostatic Epo, undergo apoptosis and fail to contribute to RBC production. (C) Excess erythroid precursors can be rescued to exogenous Epo or increased physiologic demand.

[0026] FIGS. 6A-6D. (A) Schema showing treatment of C57BL6/J mice with the TGFP neutralizing antibody (1D11) or isotype control antibody (13C4). Mice received antibody on days 1, 5 and 10 and were killed on day 11. Femur and tibia (leg) marrow was harvested for analysis. (B-D) The number of marrow cells is shown per leg for mice treated with 13C4 or ID 11 (n=4 per group). Lineage negative, Lin^neg (B), cells with the immunophenotype of hematopoietic progenitors (CMP, GMP, MEP, EP, Pre-CFU-GM, Pre-CFU-GMP, Pre-MEP) (C) and immature HSPCs ( LKS+ , LinnegKit+Scal+, MPP and LKS+SLAM) are shown (D). All the quantified data are shown as mean ± SD (* = P<0.05, ** = PC0.01, *** = PC0.001, or if not shown, the comparison was not significant).

DETAILED DESCRIPTION

[0027] It has been found that inhibiting the TGFP signaling promotes erythropoiesis and sensitizes the erythropoiesis response to erythropoietin (Epo). It has been demonstrated that Epo-resistant mice treated with ID 11 (an antibody that blocks the interaction and binding between TGFP and TGFP receptor), but not an isotype control antibody, display a strong erythropoietic response to a level of Epo after inhibition of the TGFP signaling. Accordingly, disclosed herein are methods for promoting erythropoiesis and sensitizing the erythropoiesis response to erythropoietin (Epo) by administration of a TGFP signaling inhibitor.

[0028] This disclosure is based on the inventors' finding that megakaryocyte-derived TGFpi compartmentalizes hematopoiesis by coupling haemangiopericytoma

haematopoietic progenitor cell (HPC, aka. haematopoietic stem cell) numbers to production of mature erythrocytes. The inventors have found that genetic deletion of TGFpi specifically in megakaryocytes (TGFP 1 ^AMk/AMIq increased functional

Hematopoietic stem/progenitor cells (HSPCs) including committed erythroid progenitors, yet total bone marrow and spleen cellularity and peripheral blood cell counts were entirely normal. Instead, excess erythroid precursors underwent apoptosis, predominantly those erythroblasts expressing the Epo receptor (Epor) but not Kit. Despite there being no deficiency of plasma Epo in TGF iAMk/AMk mice, exogenous Epo rescued survival of excess Epor-expressing erythroid precursors and triggered exuberant erythropoiesis. In contrast, exogenous TGF i caused anemia and failed to rescue erythroid apoptosis despite its ability to restore downstream TGF -mediated Smad phosphorylation in HSPCs. Thus, the inventors found that megakaryocytic TGF i regulates the size of the pool of immature HSPCs and in so doing, improves the efficiency of erythropoiesis by governing the feed of lineage-committed erythroid progenitors whose fate is decided by extramedullary renal Epo-producing cells sensing the need for new RBCs. Independent manipulation of distinct hematopoietic compartments offers a new strategy to overcome chronic anemias or possibly other cytopenias.

Methods for Promoting Erythropoiesis

[0029] An aspect of this disclosure is directed to methods for promoting erythropoiesis in a subject in need thereof comprising administering a TGF signaling inhibitor to the subject.

[0030] As used herein, the phrase "promoting erythropoiesis" refers to causing an increase in red blood cell (erythrocyte) count in a subject to whom the subject method is applied to as compared to a control red blood cell count, e.g.,the red blood cell count of the subject before the method was applied to the subject. In some embodiments, the methods of the disclosure result in an increase of at least 50%, 100%, 200%, 300%, 400%, 500%, or more in red blood cell counts. In some embodiments, the methods of the disclosure result in an increase of at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold or more in red blood cell counts.

[0031] As disclosed herein, a subject in need (of promoting erythropoiesis) is a subject that has a defect in erythropoiesis, which may result from, e.g., being unresponsive to Epo). Such subject may manifest a low red blood cell count or a low number of functional red blood cells.

[0032] In some embodiments, a subject in need (of promoting erythropoiesis) has a low red blood cell count. In some embodiments, the subject is an adult male and the subject's red blood cell count is less than 4.7 million red blood cells per milliliter of blood. In some embodiments, the subject is an adult female and the subject's red blood cell count is less than 4.2 million red blood cells per milliliter of blood. In some embodiments, the subject is a child between ages 5 and 18 and the subject's red blood cell count is less than 4 million red blood cells per milliliter of blood. [0033] In some embodiments, a subject in need (of promoting erythropoiesis) has a low number of functional red blood cells. Functional red blood cells are red blood cells capable of transporting oxygen to tissues and cells. In some embodiments, the subject is an adult male and the subject's functional red blood cell count is less than 4.7 million red blood cells per milliliter of blood. In some embodiments, the subject is an adult female and the subject's functional red blood cell count is less than 4.2 million red blood cells per milliliter of blood. In some embodiments, the subject is a child between ages 5 and 18 and the subject's functional red blood cell count is less than 4 million red blood cells per milliliter of blood.

[0034] In some embodiments, the subject suffers from anemia. In some embodiments, the anemia is chronic. In some embodiments, the anemia results from myelodysplastic syndrome, beta-thalassemia, myelofibrosis, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

[0035] In some embodiments, the subject who suffers from chronic anemia and has high plasma Epo levels. As used herein, the phrase "high plasma Epo levels" refers to endogenous plasma Epo levels that are higher than normal levels of plasma Epo levels. Normal levels (a.k.a., "normal range") of plasma Epo depends on the age and sex of the subject, as shown below.

[0036] Table 1: Normal endogenous Epo level ranges found in human plasma (minimum- maximum).

Age Male (mlU/mL) Female (mlU/mL)

< 1 Year Not established Not established

1-3 Years 1.7-17.9 2.1-15.9

4-6 Years 3.5-21.9 2.9-8.5

7-9 Years 1.0-13.5 2 1 8.2

10-12 Years 1.0-14.0 1.1-9.1

13-15 Years 2.2-14.4 3.8-20.5

16-18 Years 1.5-15.2 2.0-14.2

> 18 Years 2.6-18.5 2.6-18.5

[0037] In some embodiments, the endogenous Epo levels of a subject is at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the maximum normal plasma Epo levels based on the sex and age of the subject, as shown above. In some embodiments, the endogenous Epo levels of a subject is at least 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% higher than the maximum normal plasma Epo levels based on the sex and age of the subject, as shown above. In some embodiments, the endogenous Epo levels of a subject is at least 2 folds, 3 folds, 4 folds, 5 folds, 8 folds, 10 folds, 15 folds, 20 folds, 25 folds or 30 folds higher than the maximum normal plasma Epo levels as shown above.

[0038] In some embodiments, "high plasma Epo levels" refers to endogenous Epo in the plasma levels of higher than 18.5 milliunits per milliliter (mU/mL). In some

embodiments, "high plasma Epo levels" refers to endogenous Epo in the plasma levels of higher than 40 mU/mL, 50 mU/mL, 60 mU/mL, 70 mU/mL, 80 mU/mL, 90 mU/mL, 100 mU/mL, 125 mU/mL, 150 mU/mL, 180 mU/mL, 200 mU/mL, 250 mU/mL , 300 mU/mL, 350 mU/mL, 400 mU/mL, 450 mU/mL, 450 mU/mL, 500 mU/mL, 550 mU/mL, or 600 mU/mL.

[0039] In chronic anemia patients with high plasma Epo levels, Epo does not promote erythropoiesis efficiently. These patients do not respond to administration of exogenous Epo either. In some embodiments, the method comprises administering to the subject a TGF signaling inhibitor without administering additional (exogenous) Epo to the patient.

[0040] In some embodiments, the method comprises administering erythropoietin to the subject before, during, or after the administration of the TGF signaling inhibitor. In some embodiments, erythropoietin is administered to the subject less than an hour before, at least 1 hour before, at least 2 hours before, at least 3 hours before, at least 6 hours before, at least 9 hours before, at least 12 hours before, at least 15 hours before, at least 18 hours before, at least 24 hours before, or at least 36 hours before the administration of the TGF signaling inhibitor. In some embodiments, erythropoietin is administered to the subject less than an hour after, at least 1 hour after, at least 2 hours after, at least 3 hours after, at least 6 hours after, at least 9 hours after, at least 12 hours after, at least 15 hours after, at least 18 hours after, at least 24 hours after, or at least 36 hours after the administration of the TGF signaling inhibitor.

[0041] Another aspect of this disclosure is directed to methods for treating subject who has low endogenous Epo levels, and the method comprises administering to the subject a TGF signaling inhibitor followed by or concurrently with administering Epo. As used herein, the phrase "low endogenous Epo levels" refers to an endogenous Epo level that is less than the minimum Epo level in the normal range, as described above. In some embodiments, the endogenous Epo levels of a subject is at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 95% lower than the maximum normal plasma Epo level as shown above. In some embodiments, the endogenous Epo levels of a subject is at least 2 folds, 3 folds, 4 folds, 5 folds, 8 folds or 10 folds lower than the maximum normal plasma Epo level as shown above.

[0042] In some embodiments, the phrase "low endogenous Epo levels" refers to endogenous Epo levels less than 4 mU/mL. In some embodiments, the phrase "low endogenous Epo levels" refers to endogenous Epo levels less than 2.6 mU/mL. In some embodiments, "low endogenous Epo levels" refers to 2 mU/mL, 1.5 mU/mL, 1 mU/mL, 0.5 mU/mL, 0.2 mU/mL or lower endogegous plasma Epo levels.

Methods for Improving Eythropoietin (Epo) Sensitivity

[0043] Another aspect of this disclosure is directed to methods for improving

erythropoietin sensitivity in a subject comprising administering a TGF signaling inhibitor to the subject. A method that improves erythropoietin sensitivity in a subject improves the responsiveness of the subject to erythropoietin (Epo). When the responsiveness to Epo is improved/increased, less exogenous Epo is needed to achieve the same effect in promoting erythropoiesis. In some embodiments, the subject is resistant to Epo, and the method of the disclosure overcomes Epo resistance. In some embodiments, the phrase "resistant to Epo" refers to a subject who does not respond to exogenous Epo administration by increasing red blood cell production, i.e., in this subject exogenous Epo administration does not result in increased rate of red blood cell production. In some embodiments, a subject who is resistant to Epo partially responds to Epo administration· In some embodiments, the partial response to Epo is at most 75%, 50%, 25%, 10% or 5% of the response of a healthy control, wherein the same dose of exogenous Epo is administered to the subject and the healthy control. In some embodiments, response to exogenous Epo is determined by monitoring red blood cell counts before and after an exogenous Epo administration. In some embodiments, if a subject is responsive to Epo, the subject’s red blood cell counts increase following an exogenous Epo administration. In some embodiments, the increase in red blood cell counts is at least 30%, 40%, 50%, 80%, 100%, 150%, 200%, 250%, or 300% higher than the blood cell counts before the administration of exogenous Epo. In some embodiments, a subject that is responsive to Epo produces more red blood cells when an increasing amount of Epo is administered. In some embodiments, if a subject is resistant to Epo, the subject’s red blood cell counts do not increase following an exogenous Epo administration. In some embodiments, if a subject is resistant to Epo, the subject’s red blood cell counts do not increase even an increasing amount of Epo is administered.

[0044] In some embodiments, the subject suffers from anemia. In some embodiments, the anemia is chronic. In some embodiments, the anemia results from myelodysplastic syndrome, beta-thalassemia, myelofibrosis, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

[0045] In some embodiments, the subject is unresponsive to erythropoietin prior to administration of the TGF signaling inhibitor. In some embodiments, the subject has a high erythropoietin level associated with poor response to exogenous erythropoietin.

[0046] In some embodiments, erythropoietin is administered to the subject before, during, or after the administration of the TGF signaling inhibitor. In some embodiments, erythropoietin is administered to the subject less than an hour before, at least 1 hour before, at least 2 hours before, at least 3 hours before, at least 6 hours before, at least 9 hours before, at least 12 hours before, at least 15 hours before, at least 18 hours before, at least 24 hours before, or at least 36 hours before the administration of the TGFP signaling inhibitor. In some embodiments, erythropoietin is administered to the subject less than an hour after, at least 1 hour after, at least 2 hours after, at least 3 hours after, at least 6 hours after, at least 9 hours after, at least 12 hours after, at least 15 hours after, at least 18 hours after, at least 24 hours after, or at least 36 hours after the administration of the TGFP signaling inhibitor.

[0047] In some embodiments, the subject is not administered erythropoietin.

"TGFp pathway inhibitors" or "TGFp signaling inhibitors"

[0048] "TGFP pathway inhibitors" or "TGFP signaling inhibitors" as used herein, refer to molecules which inhibit the signal transduction mediated by TGFp. TGFP signaling inhibitors include molecules which inhibit the level and/or activity of TGFP such as agents that block the upstream synthesis and activation of latent TGFP to form active TGFP; agents that prevent the release/secretion of latent or active TGFP from megakaryocytes; agents that block the interaction between TGFP with its receptors, and agents that inhibit the downstream signaling cascade, such as molecules which inhibit the level and/or activity of downstream targets of TGFP signaling, for example, p57, among others (including those in the Smad-dependent pathway of TGFP signaling). TGFP-pathway inhibitors also include molecules that inhibit the function or activity of TGFP receptors. See, e.g., Nagaraj and Datta, Exp. Opin. lnvestig. Drugs 19(1): 77-91 (2010); Korpal and Yang, Eur J Cancer 46: 1232-1240 (2010); and Akhurst et ak, Nature Reviews 11:791 (2012); all of which are incorporated herein by reference in entirety.

[0049] TGFP signaling inhibitors suitable for use in the present methods include large molecule inhibitors (such as monoclonal antibodies, and soluble TGFP antagonists such as polypeptides composed of the extracellular domain of a TGFP receptor), antisense oligonucleotides, and small molecule organic compounds.

[0050] In some embodiments, a large molecule TGF P-pathway inhibitor is used in the present methods, which includes antibodies, antibody derivatives and antigen-binding antibody fragments that antagonize TGF-P ligand binding to TGF-P receptors. Examples of such antibodies are disclosed, for example, in U.S. Patent No. 7,723,486, and EP 0945464, the entire contents of which are incorporated by reference. Such antibodies, derivatives and fragments thereof can be generated against one, two, or all TGF-b isoforms (i.e., TGF-bI, TGF^2, and/or TGF^3) or against one or more TGF-b receptors (e.g., TGF^RI, TGF^RII, and/or TGF^RIII). Antibodies are preferentially generated against the regions of TGF-b or TGF-b receptors that are involved in ligand binding and/or signal transduction. Preferred TGF-b epitopes for binding include amino acids 56 to 69 of TQR-b2 (TQHSRVLSLYNTIN ; SEQ ID NO: 1) with a three amino acid (CGG) extension at the N-terminus, even more preferably amino acids 60 to 64 of TORb2 (RVLSL, SEQ ID NO: 2). Other useful epitopes include amino acids 56 to 69 of TORb 1 (CGG- TQYSKVLSLYNQHN; SEQ ID NO: 3). Antibodies that interfere with activation of latent TOEb either by directly binding to latent TOEb so as to prevent its activation (Scholar Rock has such an agent https://scholarrock.com/pipeline/scholar-rock-pipeline/) or blocking cellular proteins that bind to or are otherwise involved in activation of latent TOEb (e.g. antibodies to integrins ITGAV/ITGB6).

[0051] Specific examples of suitable antibodies include Lerdelimumab (CAT- 152) (Cambridge Antibody Technology) (Mead et ak, Invest Ophthalmol Vis Sci 44(8):3394- 3401 (2003)), Metelimumab (CAT-192) (Cambridge Antibody Technology) (Benigni et ak, J Am Soc Nephrol 14(7): 1816-1824 (2003)), GC-1008 (Genzyme Corp. and

Cambridge Antibody Technology) (Lahn et ak, Expert Opin Investig Drugs 14(6):629-643 (2005)), ID11 (Genzyme Corp.) (Nam et ak, Cancer Res 66(12):6327-35 (2006)), SR-2F (National Cancer Institute) (Lahn et ak (2005), supra), and 2G7 (Genentech Inc.)

(Muraoka-Cook et ak, Clin Cancer Res 1 l(2):937s-943s (2005)), all of which reviewed by Nagaraj et ak, Expert Opinion Investig Drugs 19(1): 77-91 (2010); LY2382770 (Eli Lilly); IMC-TR1, an anti-TC^RII blocking antibody (ImClone LLC) (ClinicalTrials.gov, NCT01646203); and STX-100 (Stromedix), an antibody that blocks a\^6 integrins that are believed to be involved in the activation of latent TϋRb. See, also, review by Akhurst et ak, Nature Reviews 11:791 (2012).

[0052] Various methods for the preparation of antibodies are known in the art (see, Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). For example, a TGF-b or a TGF-b receptor or a fragment thereof can be administered to a recipient mammal to generate an immune response. Polyclonal as well as monocolonal antibodies that are specific for the TGF-b or TGF-b receptor can be generated from such an immunized mammal. For example, monoclonal antibodies specific for TGF-b or TGF-b receptor protein may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.

[0053] Antibodies that bind specifically to TGF-b or TGF-b receptor protein can also be produced by recombinant means, including recombinantly produced chimeric and humanized antibodies. Humanized or human antibodies are preferred for use in therapeutic contexts to avoid unwanted immunogenicity caused by antibody molecules. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody sequences for corresponding human antibody sequences, are well documented in the art (see for example, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988), Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285(1993); and Sims et al., J.

Immunol. 151: 2296 (1993)). Fully human monoclonal antibodies of the invention can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human monoclonal antibodies of the invention can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci (see also, Jakobovits, Exp. Opin. Invest. Drugs 7(4): 607-614 (1998); U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and 6,114,598 issued 5 Sep. 2000).

[0054] Specificity and affinity of an antibody for TGF-b or TGF-b receptor can be assessed by many techniques known in the art. For example, the specificity may be determined by ELISA. Wells of a multi-well plate are coated with TGF-b or TGF-b receptor protein, using methods known in the art. Anti- TGF-b or TGF-b receptor protein antibodies are added, and reactivity with TGF-b or TGF-b receptor protein is determined by antibody binding affinity. Other means of determining specificity, well known to those of skill in the art, include FACS analysis and immunochemistry.

[0055] Antibody formulations of the invention are administered via any route capable of delivering the antibodies to a patient. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Typically, an antibody is given at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg antibodies per week are effective and well tolerated.

[0056] In other embodiments, the TGF-b signaling inhibitor is a soluble TGF antagonist such as a polypeptide composed of the extracellular domain of a TGF receptor (referred to by some as "ligand trap"). Examples of soluble TGF antagonists include TGF^RIFFc (a stabilized soluble protein from Biogen Idee; see Muraoka et al., J Clin Invest

109(12): 1551-1559 (2002)), and betag 1 y c a n/TG F- b R 111 (a recombinant soluble protein comprising the extracellular domain of the TGF^3 receptor; see Bandyopadhyay et ah, Cancer Res 59(19):5041-5046 (1999)), both reviewed by Nagaraj et al. (2010, supra).

[0057] In other embodiments, the TGF-b signaling inhibitor is an oligonucleotide inhibitor, such as but not limited to antisense oligonucleotides, RNAi, dsRNA, siRNA and ribozymes. Such antisense oligonucleotides antagonize TGF-b signaling.

[0058] As used in the specification, "antisense oligonucleotide" refers to a stretch of single- stranded DNA or RNA, usually chemically modified, whose sequence (3'-5') is complementary to the sense sequence of a molecule of mRNA. Antisense molecules thereby effectively inhibit gene expression by forming RNA/DNA duplexes, and offer a more targeted option for cancer therapy than chemotherapy or radiation. Antisense is believed to work by a variety of mechanisms, including physically blocking the ability of ribosomes to move along the messenger RNA, and hastening the rate at which the mRNA is degraded within the cytosol.

[0059] The antisense oligonucleotide may be a 5-10-5 gap-mer methoxyl ethyl modified (MOE) oligonucleotide corresponding to the sequence of a TGF-b isoform. The antisense oligonucleotides according to the present invention are typically between 7 and 100 nucleotides in length. In one embodiment, the antisense oligonucleotides comprise from about 7 to about 50 nucleotides, or nucleotide analogs. In another embodiment, the antisense oligonucleotides comprise from about 7 to about 35 nucleotides, or nucleotide analogs. In other embodiments, the antisense oligonucleotides comprise from about 12 to about 35 nucleotides, or nucleotide analogs, and from about 15 to about 25 nucleotides, or nucleotide analogs. In one embodiment, this oligonucleotide has a phosphorothioate backbone throughout.

[0060] It is understood in the art that an antisense oligonucleotide need not have 100% identity with the complement of its target sequence in order to be effective. The antisense oligonucleotides in accordance with the present invention, therefore, have a sequence that is at least about 70% identical to the complement of the target sequence. In one embodiment of the present invention, the antisense oligonucleotides have a sequence that is at least about 80% identical to the complement of the target sequence. In other embodiments, they have a sequence that is at least about 90% identical or at least about 95% identical to the complement of the target sequence, allowing for gaps or mismatches of several bases. Identity can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software.

[0061] In order for the antisense oligonucleotides of the present invention to function in inhibiting TGF-b, it is desirable that they demonstrate adequate specificity for the target sequence and do not bind to other nucleic acid sequences in the cell. Therefore, in addition to possessing an appropriate level of sequence identity to the complement of the target sequence, the antisense oligonucleotides for use in the present invention should not closely resemble other known sequences. The antisense oligonucleotides of the present invention, therefore, should be less than 50% identical to any other mammalian nucleic acid sequence.

[0062] Specific examples of antisense oligonucleotides useful as TGF signaling inhibitors include AP- 12009 (Antisense Pharma) (Schlingensiepen et al., Recent Results Cancer Res 177:137-150 (2008)), AP-11014 (Antisense Pharma) (Saunier et al., Curr Cancer Drug Targets 6(7):565-578 (2006)), and NovaRx (NovaRx) (Lahn et al., Expert Opin Investig Drugs 14(6):629-643 (2005)), as reviewed by Nagaraj et al., Expert Opinion Investig Drugs 19(1): 77-91 (2010), all the above publications incorporated herein by reference.

[0063] Inhibition of TGF-b may also be achieved using RNA interference or "RNAi". RNAi or double- stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates. RNA interference mediated by siRNAs is known in the art to play an important role in post-transcriptional gene silencing (Zamore, Nature Struc. Biol., 8:746-750, 2001). In nature, siRNA molecules are typically 21-22 base pairs in length and are generated when long double-stranded RNA molecules are cleaved by the action of an endogenous ribonuclease. RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell a suitable precursor (e.g. vector, etc.) of such an siRNA or siRNA-like molecule. An siRNA may then associate with other intracellular components to form an RNA-induced silencing complex (RISC). Transfection of mammalian cells with synthetic siRNA molecules having a sequence identical to a portion of a target gene leads to a reduction in the mRNA levels of the target gene (Elbashir et al., Nature, 411:4914498, 2001).

[0064] Oligonucleotide TGF-b inhibitors can be siRNA molecules that are targeted to a TGF-b ligand or receptor gene such that the sequence of the siRNA corresponds to a portion of said gene. RNA molecules used in the present invention generally comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion.

The total number of nucleotides in the RNA molecule is typically less than 49 in order to be effective mediators of RNAi. In some embodiments, the number of nucleotides is 16 to 29, and in some specific embodiments, 18 to 23, and in other specific embodiments 21-23. In certain embodiments of the invention, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecules are about 21-23 nucleotides in length. In an embodiment, siRNA or siRNA-like molecules comprise and 19-21 bp duplex portion, each strand having a 2 nucleotide 3' overhang. In certain embodiments of the invention, the siRNA or siRNA-like molecule is substantially identical to a TGF-b -encoding nucleic acid or a fragment or variant thereof. The double- stranded siRNA molecules can further comprise poly-T or poly-U overhangs at the 3' and 5' ends to minimize RNase-mediated degradation of the molecules. Design and construction of siRNA molecules is known in the art (see, for example, Elbashir, et al, Nature 411:494498, 2001; Bitko and Barik, BMC Microbiol., 1:34, 2001). In addition, kits that provide a rapid and efficient means of constructing siRNA molecules by in vitro transcription are also commercially available (Ambion, Austin, Tex.; New England Biolabs, Beverly, Mass.).

[0065] The present invention further contemplates use of ribozyme oligonucleotide inhibitors of TORb signaling. Ribozymes are RNA molecules having an enzymatic activity that enables the ribozyme to repeatedly cleave other separate RNA molecules in a nucleotide- sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any mRNA transcript, and efficient cleavage can be achieved in vitro (Kim et al., Proc. Natl. Acad. Sci. USA, 84:8788, 1987; Haseloff and Gerlach, Nature, 334:585, 1988; Cech, JAMA, 260:3030, 1988; Jefferies et al., Nucleic Acids Res., 17:1371, 1989).

Typically, a ribozyme comprises two portions held in close proximity: an mRNA binding portion having a sequence complementary to the target mRNA sequence, and a catalytic portion which acts to cleave the target mRNA. A ribozyme acts by first recognizing and binding a target mRNA by complementary base-pairing through the target mRNA binding portion of the ribozyme. Once it is specifically bound to its target, the ribozyme catalyzes cleavage of the target mRNA. Such strategic cleavage destroys the ability of a target mRNA to direct synthesis of an encoded protein. Having bound and cleaved its mRNA target, the ribozyme is released and can repeatedly bind and cleave new target mRNA molecules.

[0066] In further embodiments, the TGF signaling inhibitor utilized in the methods of this invention is a small molecule, synthetic or naturally occurring organic compound. As used herein, a small molecule compound is defined as a molecule of less than 1200 Daltons, preferably less than 1000 Daltons, or preferably less than 800 Daltons.

[0067] Examples of suitable small molecule TGF -pathway inhibitors include LY-550410 (Eli Lilly) (Yingling et al., Nat Rev Drug Discov 3(12) : 1011-1022 (2004)), LY-580276 (Eli Lilly) (Sawyer, Curr Med Chem Anticancer Agents 4(5):449-455 (2004)), LY-364947 (Eli Lilly) (Sawyer et al., J Med Chem 46(19):3953-3956 (2003)), LY-2109761 ((Eli Lilly) (Sawyer, Curr Med Chem Anticancer Agents 4(5):449-455 (2004)), LY-2157299 (Eli Lilly) (Yingling et al., Nat Rev Drug Discov 3(12):1011-1022 (2004)), LY-573636 (Eli Lilly), SB-505124 (GlaxoSmithKline) (Saunier et al., Curr Cancer Drug Targets

6(7):565-578 (2006)), SB-431542 (GlaxoSmithKline) (Hjelmeland et al., Mol Cancer Ther 3(6):737-745 (2004)), GW788388 (GlaxoSmithKline), SD-208 (Scios Inc.) (Uhl et al., Cancer Res 2004;64(21):7954-7961 (2004)), SD-093 (Scios Inc.) (Subramanian et al., Cancer Res 64(15):5200-5211 (2004)), Ki-26894 (Kirin Brewery Co.) (Ehata et al.,

Cancer Sci 98(1): 127-133 (2007)), Sml6 (Biogen Idee; see also Suzuki et al., Cancer Res 67(5):2351-2359 (2007)), NPC-30345 (Scios Inc.) (Dumont et al., Cancer Cell

2003;3(6):531-6 (2003)), A-83-01 (Kyoto Pharma) (Tojo et al., Cancer Sci 96(11):791- 800 (2005)), SX-007 (Scios Inc.) (Tran et al., Neuro Oncol 9(3):259-70 (2007)), IN- 1130 (In2Gen Co.) (Moon et al., Kidney Int 70(7): 1234-1243 (2006)), and pyrrole-imidazole polyamide. See, also, Akhurst et al., Nature Reviews 11:791 (2012). Vactosertib

(MedPacto) is another TGF Rl (ALK5) inhibitor in clinical development.

[0068] Additional exemplary small molecule TGF -pathway inhibitors include compounds having the following generic formula, as disclosed in U.S. Patent No.

7,087,626 (to Eli Lilly, incorporated herein by reference):

or the formula as U.S. Patent No. 7,087,626 (to Eli Lilly, incorporated herein by reference):

or another formula disclosed in U.S. Patent No. 7,087,626 (to Eli Lilly, incorporated herein by reference):

or yet another formula disclosed in U.S. Patent No. 7,087,626 (to Eli Lilly, incorporated herein by reference):

[0069] A particular example of a TGF -pathway inhibitor compound LY2157299, is disclosed in U.S. Patent No. 7,265,225 (to Eli Lilly, incorporated herein by reference):

[0070] Other exemplary small molecule TGF -pathway inhibitors include compounds with the following generic formula as disclosed in U.S. Patent No. 6,476,031 (to Scios Inc., incorporated herein by reference):

or the pharmaceutically acceptable salts thereof.

[0071] A particular example of a compound of this formula, SD-093, is disclosed in U.S. Patent No. 6,476,031 (to Scios Inc.):

[0072] Another example is SD-208 (Scios) having the formula:

Cl

SD2

[0073] Other exemplary small molecule TGF -pathway inhibitors include compounds as disclosed in U.S. Patent No. 7,407,958, in particular the compound IN-1130:

[0074] Other exemplary small molecule TGF -pathway inhibitors include compounds as disclosed in U.S. Patent No. 7,053,095 (Pfizer Inc.), in particular the compound:

[0075] Other exemplary small molecule TGF -pathway inhibitors include, but are not limited to, compounds with the following formulas:

SB-431542 (GlaxoSmithKline)

[0076] Additional inhibitors described by Nagaraj (2010, supra ) and Akhurst (2012, supra ) also suitable for use herein include Trx-xFoxHlb and Trx-Lefl (Smad-interacting peptide aptamers) (Cui et al., Oncogene 24(24):3864-3874 (2005)), Distertide (pi 44) ( a peptide based on TbMII that blocks ligand binding to receptors, from Digna Biotech), pl7 (peptide derived from phage display that targets TGF i binding to receptor, from Digna Biotech), LSKL (a peptide based on thrombospondin and specifically blocks TGF activation).

Additional TGF-b receptor inhibitors

[0077] Galunisertib (LY2157299) and others disclosed in Herbertz S et al. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther. 2015 Aug 10;9:4479-99. doi: 10.2147/DDDT.S86621. eCollection 2015.

[0078] Those disclosed in Bonafoux D, Lee WC. Strategies for TGF-beta modulation: a review of recent patents. Expert Opin Ther Pat. 2009 Dec;19(12):1759-69. doi:

10.1517/13543770903397400.

[0079] Repsox, LY2109761, LY364947 and K02288 disclosed for example in Ide M, et al. Transforming growth factor b-inhibitor Repsox downregulates collagen expression of scleroderma dermal fibroblasts and prevents bleomycin-induced mice skin fibrosis. Exp Dermatol. 2017 Nov;26(l l):1139-1143. doi: 10.1111/exd.l3366. Epub 2017 Aug 25.

TGF-b pathway inhibitors with target unknown

[0080] Compounds disclosed in Jeong JH, et al. Novel TGF-bI inhibitor antagonizes TGF^l-induced epithelial-mesenchymal transition in human A549 lung cancer cells. J Cell Biochem. 2019 Jan;120(l):977-987. doi: 10.1002/jcb.27460. Epub 2018 Sep 14.

Direct TGF-bI inhibitors

[0081] TOEb-heuίGhϋzί^ antibody ID 11 - This antibody neutralizes all three isoforms (used in the Example below, available from Thermo-Fisher (Catalog MA5-23795) (Isoform information from Liang X et al. Anti-TGF-b Antibody, 1D11, Ameliorates Glomerular Fibrosis in Mouse Models after the Onset of Proteinuria. PLoS One. 2016 May 17;l l(5):e0155534. doi: 10.1371/joumal.pone.0155534. eCollection 2016.) [0082] Fresolimumab (GC1008) mAh inhibits all three isoforms— Griitter C et al. A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions. Proc Natl Acad Sci U SA. 2008 Dec 23;105(51):20251-6. doi: 10.1073/pnas.0807200106. Epub 2008 Dec 10.

[0083] Metelimumab (CAT- 192) mAh specific for isoform 1— Sorbera, L.A.

Metelimumab. Drugs Fut 2004, 29(11): 1081 DOI: 10.1358/dof.2004.029.11.860002

[0084] TGF-b 1-specific, humanized, neutralizing mAh (TGF-bI mAb) developed by Eli Lilly— Voelker J et al. Anti-TGF-bI Antibody Therapy in Patients with Diabetic Nephropathy. J Am Soc Nephrol. 2017 Mar;28(3):953-962. doi:

10.1681/ASN.2015111230. Epub 2016 Sep 19.

[0085] Those disclosed in Bonafoux D, Lee WC. Strategies for TGF-beta modulation: a review of patents. Expert Opin Ther Pat. 2009 Dec;19(12):1759-69. doi:

10.1517/13543770903397400.

Administration and Dose

[0086] A suitable TGFb-pathway inhibitor can be combined with one or more

pharmaceutically acceptable carriers for administration. As used herein, a

pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents and the like. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the TOEb- pathway inhibitor or the chemotherapeutic drug, its use in practicing the methods of the present invention is appropriate. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include water, saline solutions, alcohol, sugar, gel, oils, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, preservatives and the like, or combinations thereof. In accordance with the present invention, the active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder, syrup, suspensions that are suitable for injections, implantations, inhalations, ingestions or the like.

[0087] According to the present disclosure, a suitable TGFb-pathway inhibitor can be administered to a patient via various routes, including the sublingual, oral, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular) route.

[0088] Generally speaking, the daily dose of a suitable TGF -pathway inhibitor will be dictated by its mode of action, pharmacokinetics and pharmacodynamics. The precise dose can be determined by the treating physician, taking into consideration of the patient's blood cell levels, the route of administration and other physical parameters such as age, weight and overall well being.

[0089] In some embodiments, an effective amount of Epo is about 0.2 mg/kg to 100 mg/kg. In other embodiments, the effective amount of Epo is about 0.2mg/kg, 0.5mg/kg, lmg/kg, 8mg/kg, lOmg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, lOOmg/kg, 150mg/kg, 175mg/kg or 200mg/kg of TGF -pathway inhibitor. As used in this disclosure, the term "about" refers to a variation within approximately ±10% from a given value.

[0090] In some embodiments, an effective amount of a TGF -pathway inhibitor is about 0.2 mg/kg to 100 mg/kg. In other embodiments, the effective amount of a TGF -pathway inhibitor is about 0.2mg/kg, 0.5mg/kg, lmg/kg, 8mg/kg, lOmg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, lOOmg/kg, 150mg/kg, 175mg/kg or 200mg/kg of TGF -pathway inhibitor.

[0091] .In some embodiments, the TGF -pathway inhibitor and Epo are administered consecutively. In some embodiments, the TGF -pathway inhibitor and Epo are administered separately. In some embodiments, the TGF -pathway inhibitor and Epo are administered simultaneously.

[0092] In some embodiments, the TGF -pathway inhibitor and Epo are administered in one composition. In one embodiment, a composition comprising a TGF -pathway inhibitor and Epo is not administered to the subject continuously; rather it is administered intermittently. In a specific embodiment, intermittent administration is performed once every other day, every three days, every four days, every five days, or once a week. In another specific embodiment, intermittent administration is performed once every hour, every two hours, every three hours, every six hours, every ten hours, or every twelve hours. [0093] A TGF -pathway inhibitor or Epo can be admixed with a pharmaceutically acceptable carrier to make a pharmaceutical preparation in any conventional form including, inter alia, a solid form such as tablets, capsules (e.g. hard or soft gelatin capsules), pills, cachets, powders, granules, and the like; a liquid form such as solutions, suspensions; or in micronized powders, sprays, aerosols and the like.

[0094] The pharmaceutical compositions of the present disclosure can be used in liquid, solid, tablet, capsule, pill, ointment, cream, nebulized or other forms as explained below. In some embodiments, the composition of the present disclosure may be administered by different routes of administration such as oral, oronasal, parenteral or topical.

[0095] "Oral" or "peroral" administration refers to the introduction of a substance into a subject's body through or by way of the mouth and involves swallowing or transport through the oral mucosa (e.g., sublingual or buccal absorption) or both.

[0096] "Oronasal" administration refers to the introduction of a substance into a subject's body through or by way of the nose and the mouth, as would occur, for example, by placing one or more droplets in the nose. Oronasal administration involves transport processes associated with oral and intranasal administration.

[0097] "Parenteral administration" refers to the introduction of a substance into a subject's body through or by way of a route that does not include the digestive tract. Parenteral administration includes subcutaneous administration, intramuscular administration, transcutaneous administration, intradermal administration, intraperitoneal administration, intraocular administration, and intravenous administration.

[0098] "Topical administration" means the direct contact of a substance with tissue, such as skin or membrane, particularly the oral or buccal mucosa.

[0099] The pharmaceutical preparations of the present disclosure can be made up in any conventional form including, inter alia ,: (a) a solid form for oral administration such as tablets, capsules (e.g. hard or soft gelatin capsules), pills, sachets, powders, granules, and the like; (b) preparations for topical administrations such as solutions, suspensions, ointments, creams, gels, micronized powders, sprays, aerosols and the like. The pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.

[00100] For topical administration to the skin or mucous membrane the

aforementioned composition is preferably prepared as ointments, tinctures, creams, gels, solution, lotions, sprays; aerosols and dry powder for inhalation, suspensions, shampoos, hair soaps, perfumes and the like. In fact, any conventional composition can be utilized in this invention. Among the preferred methods of applying the composition containing the agents of this invention is in the form of an ointment, gel, cream, lotion, spray; aerosol or dry powder for inhalation. The pharmaceutical preparation for topical administration to the skin can be prepared by mixing the aforementioned active ingredient with non-toxic, therapeutically inert, solid or liquid carriers customarily used in such preparation. These preparations generally contain 0.01 to 5.0 percent by weight, or 0.1 to 1.0 percent by weight, of the active ingredient, based on the total weight of the composition.

[00101] In preparing the topical preparations described above, additives such as preservatives, thickeners, perfumes and the like conventional in the art of pharmaceutical compounding of topical preparation can be used. In addition, conventional antioxidants or mixtures of conventional antioxidants can be incorporated into the topical preparations containing the aforementioned active agent. Among the conventional antioxidants which can be utilized in these preparations are included N-methyl-a-tocopherolamine, tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin and the like.

[00102] Cream-based pharmaceutical formulations containing the active agent, used in accordance with this invention, are composed of aqueous emulsions containing a fatty acid alcohol, semi-solid petroleum hydrocarbon, ethylene glycol and an emulsifying agent.

[00103] Ointment formulations containing the active agent in accordance with this invention comprise admixtures of a semi-solid petroleum hydrocarbon with a solvent dispersion of the active material. Cream compositions containing the active ingredient for use in this invention preferably comprise emulsions formed from a water phase of a humectant, a viscosity stabilizer and water, an oil phase of a fatty acid alcohol, a semi solid petroleum hydrocarbon and an emulsifying agent and a phase containing the active agent dispersed in an aqueous stabilizer-buffer solution. Stabilizers may be added to the topical preparation. Any conventional stabilizer can be utilized in accordance with this invention. In the oil phase, fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components are derived from the reduction of a long-chain saturated fatty acid containing at least- 14 carbon atoms.

[00104] Also, conventional perfumes and lotions generally utilized in topical preparation for the hair can be utilized in accordance with this invention. Furthermore, if desired, conventional emulsifying agents can be utilized in the topical preparations of this invention.

[00105] The present methods can be used to treat any mammalian subject, including humans, nonhuman primates, companion animals (such as dogs, cats), horses, cows, pigs, sheep, among others.

[00106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[00107] The specific examples listed below are only illustrative and by no means limiting.

EXAMPLES

Example 1: Materials and Methods

Animals

[00108] Transgenic C57BL/6 mice with Cre recombinase driven from the regulatory elements of Cxcl4 (Platelet factor 4, Pf4), C57BL/6-Tg(Pf4-cre)Q3Rsko/J (Pf4-cre)(Tiedt et al., Blood 109, 1503-1506, (2007)), were crossed with mice harboring a Boxed TGFpi locus (Azhar et al. 2009) mice (Tgfbl^{tm2 1Doe}, TGF i^FUFL mice) to generate Pf4- Cre/TGFP 1 loxP/loxP mice with selective deletion of TGFpi in megakaryocytes and platelets (TGFP 1 ^AMk/AMk mice). Animals were maintained in the Weill Cornell Medicine Animal Facility. All protocols were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Immunophenotypic analysis of BM HSPCs and HSCs by flow cytometry

[00109] Mice were killed by CO2 asphyxiation and bones (femur, tibia, +/- humeri) were dissected free of muscle and tendons and crushed in DMEM using a mortar and pestle.

[00110] The resulting cell suspension was filtered through a 40-pm mesh and washed in PEB (2 mM EDTA, 0.2% BSA in PBS, pH 7.4). Spleens were isolated, and then minced before grinding through a 40-pm mesh to generate single-cell suspensions. Lin cells were purified using a biotinylated lineage cell depletion cocktail (Miltenyi Biotec).

Hematopoietic populations were identified as following: LT-HSCs, LKS+; short-term HSCs, LKS⁺; MPPs, LKS⁺; common myeloid progenitors (CMPs), LKS- FcRlow;

granulocyte macrophage progenitors (GMPs), LKS-’ FcR⁺; and megakaryocyte erythroid progenitors (MEPs), LKS- FcR- MEPs. To identify LKS + SLAM cells, lineage-depleted cells were stained with APC-conjugated CD117 (BD), PECy7-conjugated Sca-1

(BioLegend), Alexa Fluor 700-conjugated CD48 (BioLegend) and PE-conjugated CD150 (BioLegend).

[00111] To analyze cell cycle, lineage-depleted cells were stained with streptavidin (BioLegend), APC-conjugated CD117 (BD), and PECy7-conjugated Sca-1 (BioLegend), and then fixed and permeabilized using CytoFix/Perm (BD) before staining with

PerCP/C5.5 -conjugated Ki67 (BD) and Hoechst 33342 (Invitrogen). To analyze pSmad2/3 signal, cells were stained with streptavidin (BioLegend), APC-conjugated CD117 (BD), and PECy7-conjugated Sca-1 (BioLegend), and then fixed and

permeabilized using CytoFix/Perm (BD) before staining with first with pSmad2/3 primary antibody (Cell Signaling Technology) and then with secondary antibody CY3 (Jackson Labs).

[00112] Analysis gates were set based upon the fluorescence minus one (FMO) fluorophore. Multidimensional FACS analysis was performed using a BD LSRII equipped with five lasers (BD). FlowJo and FCS express were used to analyze flow cytometry data (Tree Star).

[00113] To confirm specificity of pSmad2/3 staining, C57BL/6J mice were treated with either the TGF -neutralizing antibody 1D11 (I; 10 mg/kg) or a non targeted control antibody 13C4 (C; 10 mg/kg) on day 5, 10 and 15. Mice were sacrificed on day 16 for analysis. Smad2/3 phosphorylation was assessed by intracellular flow cytometry as described previously. Stem and progenitors cell assays

[00114] Primitive myeloid progenitors were enumerated using the day 12 CFU-S (CFU- S12) assay. Recipient mice were sublethally irradiated using a Cs-y-ray source and injected with 10⁵ BM cells. Spleens were isolated 12 days after transplantation and fixed in Bouin’s solution. The number of macroscopic spleen colonies were counted and expressed as the number of CFU-S colonies per 10⁵ donor cells. Clonogenic myeloid progenitors were assessed by standard methylcellulose CFC assays (MethoCult GF M3434; Stem Cell Technologies) using 1.5 x 10⁴ BM mononuclear cells (BMMCs) per well (6-well plate). Colonies were scored after 7 days of incubation and expressed as the number of CFUs per 1.5 x 10⁴ BMMCs before normalizing to total leg (femur/tibia) cell counts.

HSC transplantation

[00115] Recipient mice were irradiated with 9 Gy using a ¹³⁷Cs-y-ray source. 3 hours after irradiation, whole bone marrow cells (WBMCs) from donor animals were infused via the tail vein of recipient animals. Competitive repopulation was used to assess the relative number of HSCs in WBM from TGF i^AMk'^AMk and TGF i^FUFL mice. TGFp i' ^k/AMk or TGFP 1 ^FL/FI WBMCs were mixed with an equal number of WBMCs from congenic CD45.1^+/+ control mice. The 1:1 (test/competitor) mixture (10⁶ cells total) was injected into lethally irradiated mice via tail vein. Peripheral blood was analyzed for lymphoid (CD3/B220) and myeloid (Grl/CDl lb) engraftment by flow cytometry at various times after transplantation. Animals were sacrificed after 16 weeks and marrow harvested for immunophenotyping of HSPC populations and for serial transplantation. Serial transplantation of secondary recipients was performed as for primary recipients except the WBM donor cells were not mixed with additional congenic competitor cells.

Cell count analysis

[00116] To assess blood count recovery, peripheral blood (50 pi) was collected into EDTA-coated capillary tubes (Thermo Fisher Scientific). Differential blood counts were measured using an automated AD VIA 120 Multispecies Hematology Analyzer (Bayer Healthcare) calibrated for murine blood.

Apoptosis analysis

[00117] Apoptosis in mononuclear cell suspensions was assessed using the Annexin V- FITC Apoptosis Kit (BioLegend, 640914) according to manufacturer’s instructions and analyzed via flow cytometry. Cleaved Caspase3 was assessed in surface stained mononuclear cells suspensions permeabilized using CytoFix/Perm (BD). After permeabilization, cells were stained with cleaved-Caspase3 primary antibody (Cell Signaling Technology), washed and then stained with secondary antibody (anti-rabbit)

CY3 (Jackson Labs). The cells were then analyzed by flow cytometry.

Imm unoflourescence

[00118] Femurs were fixed in 4% PFA overnight, and then decalcified using 10% EDTA before freezing in OCT (Sakura Finetek). Immunofluorescence staining was performed on frozen sections. After blocking with blocking buffer (3% BSA, 3% Serum and 0.03% tween), sections were incubated in primary antibodies overnight at 4°C using anti-cleaved Caspase3 (Cell Signaling Technology) antibody. After washing sections were stained with secondary antibody CY3 (Jackson Labs). The specificity of staining was confirmed in sequential sections using the secondary antibody alone. Images were acquired using a Zeiss spinning disk confocal microscope or a Zeiss 710 laser scanning confocal microscope.

In vivo stimulation of erythropoiesis

[00119] Human recombinant EPO (300 U/kg body weight, ~6U per mouse) was injected subcutaneously for 5 consecutive days. On day 6, blood was sampled and mice were killed for analysis.

[00120] To induce hemolysis, TGF i^AMkMMkor TGP i^FUFL mice were injected intraperitoneally with phenylhydrazine hydrochloride (PHZ) at 60 mg/kg (Sigma- Aldrich) on day 1 and 2. Blood was sampled on days 3, 5, 8 and 14 from staggered cohorts of mice.

[00121] To assess the ability of exogenous TGF i to rescue megakaryocytic TGF i knockout, mice were treated with TGF i (5mg/kg/day) subcutaneously for 5 consecutive days as indicated. On day 6, blood was sampled and mice were killed for analysis.

Imm unoblotting

[00122] Mononuclear cell suspensions were washed in PEB, pelleted and then lysed in TBS containing 2 mM EDTA, IX Laemmli Sample Buffer, 1% NP-40, with phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium pyrophosphate) and protease inhibitor cocktail tablets (Roche). Samples were separated on 10% NuPAGE gels (Invitrogen), transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore) and blocked with 5% nonfat dried milk in PBS with 0.1% Tween-20. Primary and secondary antibodies were diluted in blocking solution. Primary antibodies against cleaved Caspase3 (Cell Signaling Technology), pSmad2 (Cell Signaling Technology) Smad2/3 (Cell Signaling Technology), and b-actin (Cell Signaling Technology) were used. Secondary peroxidase-conjugated anti-rabbit antibody (EMD Millipore) was used before

chemiluminescent visualization using the SuperSignal West Femto Substrate (Thermo Fisher Scientific). Signal intensities were quantified using ImageJ (NIH).

Statistical analysis

[00123] All data are expressed as mean ± SEM. Student’s t test (two-tailed) was used to analyze the statistical differences between groups, with the p-values indicated in the plots (* P < 0.05, ** P < 0.01, *** P < 0.001, or if not shown, the comparison was not significant).

Example 2: Conditional deletion of TGFpi in megakaryocytes (MKs) increases bone marrow apoptosis.

[00124] Many HSCs reside in close proximity to megakaryocytes where they can be maintained in a quiescent state by niche signals such as Tgl^'P 1 and Cxcl4(Zhao et al. 2014; Bruns et al. 2014). Although genetic ablation of Mks using an inducible diphtheria toxin receptor (iDTR) under control of Mk-specific Cxcl4-cre driver releases HSCs from quiescence and licenses expansion of the HSC pool, it is not known how this affects blood cell production or other aspects of hematopoiesis. TORbI was selectively deleted in megakaryocytes (TGFP 1 ^AMk/AML _ancj found that peripheral blood counts were entirely normal in TGFP 1 ^AMk/AMk _il3ice compared to TGFpi^FL/FL littermate controls despite the pool of primitive hematopoeitic cells being expanded (FIG. 1 A). Sequestration of maturing cells within hematopoietic tissues could not explain this discrepancy because total bone marrow and spleen cellularity was normal in TGFP 1 ^AMk/AMk _jee (FIG. IB). Excess HSCs in TGFP 1 ^MI'^{/ MI}' mice appeared capable of robust differentiation because the number of immature lineage-negative (Lin-) hematopoietic progenitor cells was increased in the marrows of TGFP 1 ^AMk/AMk _mice (FIG. 1C). Thus, it remained unexplained why the expanded number of HSCs and HPCs do not increase blood counts and marrow cellularity.

[00125] Hematopoietic cell population size is determined by the balance of cell gain (proliferation/self-renewal and differentiation) and cell loss (apoptosis and differentiation). Many late acting hematopoietic cytokines are not required for lineage commitment yet provide essential proliferation, differentiation and survival signals during maturation of hematopoeitie precursors(Wu et al. 1995; Lieschke et al. 1994). It was wondered if the excess progenitors observed in the TGFP 1 ^AMk/AMk mice failed to increase blood counts because their progeny were unneeded, and inadequately supported by homeostatic levels of late acting cytokines (FIG. ID). Indeed, bone marrow apoptosis was markedly increased in the TGFP 1 ^AMk/AMk mice compared to controls, as reported by cleaved caspase 3 (FIG. lE-l G) or AnnexinV binding (FIG. IH-IK). The excess apoptotic cells seemed largely restricted to hematopoietic cells expressing the lineage markers (Lin) Grl, GDI lb, CD3, B220, or Terl l9 (FIG. 1L-1M). Annexin V staining of lineage-marker negative (Lin-), Kit-f Seal- (LKS-) HPCs and LKS+ HSPCs was rare in both TGFfil ^lMI ' ^lMk mice and littermate controls (FIG. IN- 10). These results support an interpretation that excess, uneeded hematopoietic precursors are pruned by apoptosis during hematopoietic differentiation.

Example 3: Increased hematopoietic stem and progenitor cells in TGFpi^AMk/AMk mice have normal function.

[00126] To rule out the possibility that paogeny of HSPCs from r(}rb]^AM!z/AM!ί mice are intrinsically defective, competitive repopulation assays were performed by co

transplanting bone marrow from TGFP 1 ^AMk/AMk _il3ice or TGFpi^FL/FL littermates competitively with CD45.1 congenic donor cells. As expected, TGFpi^FL/FL donor cells engrafted and contributed to multi-lineage hematopoiesis indistinguishably from the congenic CD45.1 controls (FIG. 2A) In contrast, TGFfil ^{IMk/ iMk} donor cells outcompeted control cells confirming that 7^’6'/ /;/ ^{IM// IM/} donors are enriched for functional HSCs and indicating that these TGFfil ^{iM1J iMk} HSCs yield progeny fully capable of reconstituting hematopoiesis after transplant. Serial transplantation of recipients demonstrated HSC self renewal and contribution to hematopoiesis was intrinsically normal in the TGFpi^AMkMMk donor cells (FIG. 2B).

[00127] Ex vivo HPC function appeared normal in TGFfil ^{IM/ lMk} mice suggesting that these cells were not intrinsicly defective in TGFfil ^{IMk/ lMk} mice (FIG. 2C). The number of granulocytic-monocytic progenitors (GMP), burst forming unit erythroid progentitors (BFU-E), colony forming unit erythroid (CFU-E) and granulocyte, erythroid, monocyte, megakaryocyte progenitors (CFU-GEMM) were all increased in the marrow of

TGFbl^DMk/DMk mice. Similarly, the 12 day colony forming unit spleen (CFU-S12) assay showed a greater number of functionally normal MPPs in TGFfil ^{lMk/ lMk} donor marrow compared to TGFP 1 ^FL/FI littermate controls (FIG. 2D). Quantified HSPC

immunophenotypes correlated well with functional measures again demonstrating that the number of HSPCs is increased in 7^’G'/ /;/ ^{IM// IM/} mice (FIG. 2E-2G). The larger number of HSPCs and their more proliferative character in 7^’G'/ /; / ^{l ft/ IM/} mice (FIG. 2H-2I) indicate that megakaryocytic TGF i normally curbs the HSPC pool size. Yet progeny of these excess HSPCs appear to undergo apoptosis rather than contribute to bone marrow cellularity and mature blood cell counts.

Example 4: Excess erythroid precursors undergo apoptosis in vivo

[00128] The islands of apoptotic cells observed in bone marrow of TGF i^AMk/AMkmice (FIG. IE) are reminiscent of erythroid islands suggesting that excess erythroid precursors may be contributing to the formation of these clusters. Indeed, the proportion of Annexin V-staining apoptotic Terl l9+ erythroid precursor cells (EPCs) was ~6-fold higher in the marrow and spleen of TGFP l ^AMk/AMk mice compared to TGFP 1 ^FL/FI littermate controls (FIG. 3A & FIG. 3B, respectively). Apoptosis was quantified within well-defined populations of maturing erythroid precursors (Mori et al. 2015; Hu et al. 2013; Chen et al. 2009) (FIG. 3C) using using Annexin V. Although the number of immature erythroid precursors— such as pro/basophilic (El), polychromatophilic (Eli), and

orthochromatophilic erythroblasts (EIII)— were increased in TORb 1 ^{MI / MI}' mice, the excess seemed largely comprised of cells staining for the apoptotic marker Annexin V (FIG. 3D). In contrast, the number of mature reticulocytes and erythrocytes (EIV/EV) in TGF 1 ^{MI / MI}' mice was indistinguishable from TGFP 1 ^FL/FL. These results suggest that excess erythroid committed precursors are culled during early maturation thereby producing normal numbers of erythrocytes and normal red blood cell counts (RBCs).

Example 5: Excess erythroid precursors are rescued by exogenous Epo

[00129] Apoptotic Terl 19+ erythroid precursors within TGFP 1 ^AMk/AMk marrow predominantly expressed the erythropoietin (Epo) receptor (Epor) but not Kit (FIG. 3E). Epo provides a survival signal to Epor+ erythroid precursors allowing them to escape apoptosis and continue differentiation (Ingley, Tilbrook, and Klinken 2004; Socolovsky et al. 2001 ; Malik et al. 2013). Excess apoptosis of erythroid precursors in TGF i^AMk/AMk mice was not due to subnormal plasma Epo levels (FIG. 3F). It was reasoned that surplus, unneeded Epor+ cells may not be supported by physiologic Epo levels. To test this, the inventors treated mice with exogenous Epo (300 U/kg) (FIG. 3G). Strikingly, exogenous Epo rescued the excess apoptosis of erythroid precursors in TGFpi^AMk/AMk marrow and spleen (FIGs. 3H-3K). TGF i^AMk/AMkmice responded to Epo much more robustly compared to TGFP 1 ^FL/FI littermate controls (FIGs. 3F-3M) suggesting that the rescued apoptosis resulted in increased erythropoietic output. Using phenylhydrazine (PHZ) induced hemolysis as an anemia model, it was found that the erythropoietic response of TGFpi^AMkMMk mice was much more brisk and robust than that of TGFP 1 ^FL/FI littermate controls. These results demonstrate that although genetic deletion of TGFpi in megakaryocytes expands the number of committed erythroid progenitors, the resultant glut of their erythroid precursor progeny requires non-homeostatic Epo levels to promote survival and maturation to erythrocytes.

Example 6: The dominant source of TGFpi signaling in HSPCs is produced by Megakaryocytes

[00130] Megakaryocytic TGFpi is necessary for robust phosphorylation of receptor activated Smad2 and Smad3 (Smad2/3) in HSPCs, as assessed by flow cytometry with intracellular staining (FIG. 4A) and western blot of Fin- marrow cells (FIG. 4B). In contrast, it was detected little phospho-Smad2/3 (pSmad2/3) in erythroid precursors in either the TGFpi^^mice or TGFpi^FUFL littermate controls (FIG. 4A). Similarly, western blot of Fin-i- (cells expressing mature hematopoietic lineage markers) marrow cells showed little difference in pSmad2/3 between the two genotypes (FIG. 4B).

Pretreating mice with a TGFpi neutralizing antibody (ID 11) reduced the mean fluorescence intensity (MFI) of pSmad2/3 in HSPCs compared to an isotype control (13C4) antibody confirming the specificity of the phospho-flow cytometry assay. HSPCs in TGFp 1 ^AMk/AMk mice were capable of normal TGFpi signaling because exogenous TGFpi induced strong phosphorylation of Smad2/3 restoring levels to those seen in TGFp 1 ^FL/FI controls (FIG. 4C-4D). Thus, megakaryocytes serves as the major source of TGFP triggering Smad2/3 phosphorylation in HSPCs.

Example 7: Exogenous TGFpi cannot rescue the erythroid phenotype of

TGFpi^AMk/AMk mice

[00131] It is possible that TGFpi sensitizes erythroid precursors to Epo, and if true, exogenous TGFpi should rescue late erythroid precursor dropout in TGFP 1 ^{Wk/ Wk} mice. To test this, the mice were treated with TGFpi for 5 days and assessed erythroid response (FIG. 4C). Although exogenous TGFpi reestablished pSmad2/3 in TGFp i ' ^{k/, k} HSPCs (FIG. 4D), it did not trigger an erythroid response. Rather, exogenous TGFpi induced mild anemia in the TGF i^AMk/AMk mice (FIG. 4E) coupled with worsened apoptosis in the marrow and spleen (FIGs. 4F-4G). In contrast, exogenous TGFpi did not induce detectable changes in RBCs or apoptosis in TGFP 1 ^FL/FI controls (FIGs. 4E-4G).

Apoptosis of erythroid precursors was unchanged by exogenous TGFpi in TGFP 1 ^AMk/AMk mice and littermate controls. These findings suggest that the activity of megakaryocytic TGFpi is compartmentalized within the marrow with predominant effects on immature HSPCs while excluding their progeny.

Example 8: TGFp blockade stimulates overproduction of apoptotic erythroid committed precursors that can be coupled to RBC production using low-dose Epo

[00132] Because the inventors found that the megakaryocytes are responsible for the majority of TGFP signaling in HSPCs, it is possible that blockade of TGFP signaling could phenocopy these effects by inducing overproduction of erythroid committed precursors. To test this, C57BL6/J (B6) mice were pretreated with a TGFpi neutralizing antibody (1D11) or non-targeting, isotype control antibody (13C4) and then either PBS or low-dose Epo (FIG. 4 H). TGFP neutralization by ID 11 reduced pSmad2/3 MFI in HSPCs in wild-type mice whereas the 13C4 control had no effect, demonstrating on-target activity (FIG. 41). As the inventors found in TGF/ii AMk/AMk mice, B6 mice treated with endogenous TGFP neutralized by 1D11 had an expanded pool of Lin- and HSPCs in marrow compared to those receiving the control 13C4 antibody (FIG. 6B). Low dose Epo triggered a brisk erythropoietic response in mice treated with 1D11 but not those treated with the 13C4 control (FIG. 4J). Consistent with this finding, exogenous Epo rescued the erythroid precursor dropout observed in B6 mice treated with ID 11 but did not affect the low apoptosis observed in mice treated with the 13C4 control (FIGS. 4K-4L). Therefore, the boundary of megakaryocytic TGFpi activity is compartmentalized within the marrow with predominant effects on immature HSPCs while excluding their progeny (FIGS. 5A- 5B).

Example 9

[00133] Erythropoiesis is subject to modular regulation. Epo acts during a very limited stage of differentiation— supporting early erythroid precursors (CFUe/pre-erythroblasts) as they gear up for iron accumulation, heme synthesis and globin gene transcription. Accordingly, many causes of anemia are unresponsive to exogenous Epo. TGF superfamily activin receptor (Acvr2a/b) ligand traps have shown activity treating chronic Epo-unresponsive anemia in myelodysplastic syndrome (MDS) and beta-thalassemia and are thought to promote erythroid maturation after Epo signaling. Here, the instant disclosure shows that the number of committed erythroid progenitors is controlled by the availability of megakaryocytic TGF i; adding a new level of erythroid regulation prior to the Epo restriction point. Thus, megakaryocytic TGFp i is a gate-keeper matching immature hematopoiesis to the production of mature effector cells. Mks and eythroids share a common progenitor, but Mks also have a direct ontologic link to a subset of Mk- biased HSCs It is important to understand the evolutionary reasoning for Mks being placed at the helm of hematopoiesis and to explore the potential of manipulating this pathway clinically.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting erythropoiesis in a subject in need thereof comprising administering a TGF signaling inhibitor to the subject.

2. The method of claim 1, wherein the subject suffers from anemia.

3. The method of claim 2, wherein the anemia results from myelodysplastic syndrome, beta-thalassemia, myelofibrosis, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

4. The method of claim 2 or 3, wherein the subject is resistant to erythropoietin prior to administration of the TGF signaling inhibitor.

5. The method of claim 2 or 3, wherein the subject has an elevated erythropoietin level associated with poor response to exogenous erythropoietin.

6. The method according to any one of claims 1-5, further comprising administering erythropoietin (EPO) to the subject before, during, or after the administration of the TGF signaling inhibitor.

7. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor inhibits TGF signaling through a TGF receptor that is TGF R2 or ALK5 (TGF RI).

8. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor is an antibody which antagonizes the interaction and binding between TGF and TGF receptor.

9. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor is a soluble polypeptide composed of the extracellular domain of a TGF receptor.

10. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor is an oligonucleotide selected from the group consisting of an antisense, RNAi, dsRNA, siRNA and ribozyme molecule.

11. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor is a small molecule organic compound.

12. The method according to any one of claims 1-6, wherein the TGF signaling inhibitor antagonizes the activation of latent TGF to its active form capable of activating signaling via a TGF receptor.

13. A method of improving erythropoietin (EPO) sensitivity in a subject comprising administering a TGF signaling inhibitor to the subject.

14. The method of claim 13, wherein the subject suffers from anemia.

15. The method of claim 14, wherein the anemia results from myelodysplastic syndrome, myelofibrosis, beta-thalassemia, chronic kidney disease, chemotherapy-induced anemia in subjects with cancer, inflammatory bowel disease including Crohn's disease and ulcerative colitis, or myelodysplasia from the treatment of cancer with chemotherapy or radiation.

16. The method of claim 14 or 15, wherein the subject is resistant to erythropoietin prior to administration of the TGF signaling inhibitor.

17. The method of claim 14 or 15, wherein the subject has an elevated erythropoietic level associated with poor response to exogenous erythropoietin.

18. The method according to any one of claims 13-17, further comprising

administering erythropoietin (EPO) to the subject before, during, or after the

administration of the TGF signaling inhibitor.

19. The method according to any one of claims 13-17, wherein the subject is not administered erythropoietin (EPO).

20. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor is an antibody which antagonizes the interaction and binding between TGF and TGF receptor.

21. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor is a soluble polypeptide composed of the extracellular domain of a TGF receptor.

22. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor is an oligonucleotide selected from the group consisting of an antisense, RNAi, dsRNA, siRNA and ribozyme molecule.

23. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor inhibits TGF signaling through a TGF receptor that is TGF R2 or ALK5 (TGF RI).

24. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor is a small molecule organic compound.

25. The method according to any one of claims 13-19, wherein the TGF signaling inhibitor antagonizes the activation of latent TGF to its active form capable of binding to a TGF receptor or activating signaling via a TGF receptor.