Methods of regulating inflammation and autophagy using actriib ligand traps

Abstract

Methods for treating diseases or disorders associated with an increase in mTORC1 activity or inflammation, or elevated NT-proBNP, as well as methods of inducing autophagy in the treatment of diseases and disorders, with an ActRII ligand trap.

Description

Attorney Ref. No. 14247-847-228METHODS OF REGULATING INFLAMMATION AND AUTOPHAGY USING ACTRII LIGAND TRAPS1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of United States Provisional Patent Application No. 63 / 715,137, filed November 1, 2024, the entire contents of which is incorporated herein by reference and for all purposes.2. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14247-847-228_SEQLISTING.xml,” was created on October 16, 2025, and is 16,636 bytes in size.3. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to methods of treating diseases associated with increased mammalian target of rapamycin complex 1 (mTORCl) activity, or with certain mutations associated with Clonal Hematopoiesis of Indeterminate Potential (CHIP), with ActRII ligand traps.4. BACKGROUND

[0004] Certain disorders and diseases are associated with an increase in mTORCl activity, such as certain cancers, metabolic disorders, neurodegenerative diseases, fibrotic diseases, inflammatory diseases, autoimmune disorders, and certain genetic disorders. Such disorders and diseases are often also associated with increased inflammation. Targeting mTORCl and the symptoms of its upregulation has become an area of interest in healthcare, with ongoing research into how to effectively modulate its activity for therapeutic benefit.

[0005] MDS is one disease that is associated with increased inflammation, including increased activity of interferon signaling pathways, often leading to cardiovascular complications, which decrease survival outcomes in such patients. Methods for identifying patients at a high risk for developing complications before the patient presents with the complications would be beneficial, to allow early and / or aggressive treatment and to avoid or delay development of life-threatening complications.NAI-5004198159vl 1Attorney Ref. No. 14247-847-2285. SUMMARY

[0006] In one aspect, the present disclosure provides a method of treating a disease, disorder, or condition associated with an increase in mTORCl activity in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject, wherein the disease, disorder, or condition associated with an increase in mTORCl activity is non-hematological cancer, a hamartoma syndrome, a metabolic disorder, a neurodegenerative disease, a fibrotic disease, or an inflammatory or autoimmune disorder.

[0007] In another aspect, the present disclosure provides a method of treating cardiac hypertrophy, heart failure, atherosclerosis, ischemic heart disease, breast cancer, prostate cancer, cervical cancer, esophageal cancer, renal cell carcinoma, lung cancer, hepatocellular cancer, glioblastoma, Still’s Disease, psoriasis, lupus, rheumatoid arthritis, PTEN-related hamartoma syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Huntington’s Disease, Alzheimer’s Disease, Parkinson’s Disease, amyotrophic lateral sclerosis, fibrosis (e.g., of heart, liver, kidney, or lung), inflammatory bowel disease, scleroderma, adenomatous polyposis, lymphagioleiomyomatosis, multiple sclerosis, inflammatory bowel disease, multiple sclerosis, metabolic syndrome, cachexia, non-alcoholic fatty liver disease (NAFLD), obesity, or Type 2 diabetes in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject.

[0008] In another aspect, the present disclosure provides a method of treating a disease, condition, or disorder associated with an increase in inflammation in a tissue of a subject in need thereof, the method comprising administering an effective amount of an ActRII ligand trap to the subject to reduce inflammation in the tissue, wherein the tissue is a tissue other than bone tissue. In one embodiment, the tissue is adipose tissue, brain tissue, cardiac tissue, connective tissue, epithelial tissue, gastrointestinal tissue, tissue of the joint, kidney (renal) tissue, liver tissue, lung tissue, muscle tissue, nervous system tissue, skin tissue (epidermal and dermal tissue), or spleen tissue.

[0009] In certain embodiments of any of the aforementioned methods, the disease, condition, or disorder is Crohn's Disease, colitis, steatohepatitis, systemic lupus erythematosus, Sjogren’s syndrome, systemic sclerosis, rheumatoid arthritis, vasculitis, VEXAS syndrome, inflammatory lung disease (ILD), chronic obstructive pulmonary disease (COPD), chronic immune-mediated diseases, chronic inflammatory conditions due to mitochondrial dysfunction, or sepsis.

[0010] In another aspect, the present disclosure provides a method of inducing autophagy in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject.NAI-5004198159vl 2Attorney Ref. No. 14247-847-228In one embodiment, the subject has a disease, condition, or disorder selected from the group consisting of heart failure, NAFLD, Type 2 diabetes, obesity, chronic kidney disease (CKD), acute Kidney disease (AKD), inflammatory bowel disease (IBD), Alzheimer’s disease, Parkinson’s Disease, Huntington’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, lysosomal storage disorders (LSDs), muscular disorders and fatigue, lung cancer, breast cancer, prostate cancer, colorectal cancer, cervical cancer, esophageal cancer, renal cell carcinoma, and hepatocellular cancer.

[0011] In another aspect, the present disclosure provides a method of treating anemia, myelodysplastic syndrome (MDS), non-proliferative chronic myelomonocytic leukemia (CMML), or beta-thalassemia in a subject in need thereof, the method comprising administering an effective amount of an ActRII ligand trap to the subject, wherein the subject has one or more mutations associated with CHIP. In one embodiment, the method is for treating MDS.

[0012] In one embodiment, the subject further has a cardiac inflammation, cardiac hypertrophy, or a cardiac disorder.

[0013] In any of the aforementioned aspects and embodiments, the subject may be at least 60 years old, at least 65 years old, or at least 75 years old. In some embodiments, the one or more gene mutations associated with CHIP are selected from the group consisting of DNMT3A, TET2, ASXL1, JAK2, and SF3B1. In some embodiments, the subject has two or more mutations associated with CHIP. In some embodiments, the subject has elevated mTORCl levels. In some embodiments, the subject has elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels as compared to normal NT-proBNP blood levels.

[0014] In certain embodiments, the one or more gene mutations associated with CHIP are selected from the group consisting of DNMT3A, TET2, ASXL1, and SF3B1. In some embodiment, the one or more gene mutations associated with CHIP comprise ASXL1. In certain embodiments, the subject has a TET2 gene mutation.

[0015] In another aspect, the present disclosure provides a method of treating MDS in a subject with elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels as compared to normal NT-proBNP blood levels, the method comprising administering an effective amount of an ActRII ligand trap to the subject. In certain embodiments, the subject further has elevated expression levels of STING gene clusters (e.g., a z-score expression level of STING and / or one or more STING-related genes of at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1, or in terms of percentage, may be increased by at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or by more than 300%) or increased activation of STING NAI-5004198159vl oAttorney Ref. No. 14247-847-228 signaling. In certain embodiments, the subject has one or more TET2 and / or DNMT3A gene mutations. In certain embodiments, the subject has a TET2 gene mutation.

[0016] In another aspect, the present disclosure provides a method of reducing N-terminal pro-B- type natriuretic peptide (NT-proBNP) blood levels in a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject. In certain embodiments, the subject has one or more TET2 and / or DNMT3A gene mutations. In certain embodiments, the subject has a TET2 gene mutation.

[0017] In another aspect, the present disclosure provides a method of reducing inflammatory pathway activity in a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject, wherein the inflammatory pathway is the I16 / JAK / Stat3 pathway, a STING-dependent inflammatory pathway, or an interferon alpha-related pathway. In certain embodiments, the inflammatory pathway is a STING-dependent inflammatory pathway. In certain embodiments, the subject has one or more TET2 and / or DNMT3A gene mutations. In certain embodiments, the subject has a TET2 gene mutation.

[0018] In another aspect, the present disclosure provides a method of protecting the heart of a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject. In certain embodiments, the damage is caused by or related to MDS. In certain embodiments, the administering prevents inflammation and / or hypertrophy in cardiac tissue of the subject.

[0019] In certain embodiments, the dose of ActRII ligand trap that is administered is adjusted based on measured expression levels of certain biomarkers in the subject, for example, expression levels of NT-proBNP in a sample, such as a blood sample, collected from the subject.

[0020] In any of the aforementioned embodiments, the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is: i) 90% identical to SEQ ID NO:4; ii) 95% identical to SEQ ID NO:4; iii) 98% identical to SEQ ID NO:4; or iv) identical to SEQ ID NO:4. In some embodiments, the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is: i) 90% identical to SEQ ID NO:6; ii) 95% identical to SEQ ID NO:6; iii) 98% identical to SEQ ID NO:6; or iv) identical to SEQ ID NO:6. In some embodiments, the fusion protein comprises an L79D amino acid mutation. In some embodiments, ActRII ligand trap is a fusion protein comprising: i) a fragment of the extracellular domain of ActRIIB, wherein the fragment consists of the amino acid sequence of SEQ ID NO: 10; ii) a linker; and iii) an Fc of an IgG. In some embodiments, the ActRII ligand trap is a fusion protein of SEQ ID NO:4. In some embodiments, the ActRII ligand trap is a fusion protein of SEQ ID NO:6.NAI-5004198159vl 4Attorney Ref. No. 14247-847-228

[0021] In some embodiments, the subject is administered a pharmaceutical composition comprising a mixture of two or more of the ActRII ligand trap of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments, the subject is administered a pharmaceutical composition comprising a mixture comprising each of the ActRII ligand traps of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments, the ActRIIB ligand trap is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In some embodiments, the ActRII ligand trap is the only therapeutic agent administered to the subject.

[0022] In some embodiments, the cardiac hypertrophy or heart failure is not due to vascular calcification.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 depicts data demonstrating an observed reduction of serum B-type natriuretic peptide (BNP) levels in murine model of pressure overload (Transverse Aortic Constriction (TAC)- induced heart failure) after administration of the murine analogue of luspatercept (RAP-536).

[0024] FIG. 2 depicts a two-color heatmap indicating reduced mTORCl pathway activation and inflammatory signaling as evidenced by transcriptomics levels after administration of luspatercept to subjects with at least one of a DNMT3A, TET2, ASXL1 and SF3B1 mutation.

[0025] FIGs. 3A depicts a two-color heatmap indicating reduced mTORCl pathway activation as evidenced by proteomics analysis after administration of luspatercept to subjects with at least one of a DNMT3A, TET2, ASXL1 and SF3B1 mutation.

[0026] FIG. 3B depicts a two-color heatmap indicating reduced mTORCl pathway activation as evidenced by phosphoproteomics analysis after administration of luspatercept to subjects with at least one of a DNMT3A, TET2, ASXL1 and SF3B1 mutation.

[0027] FIG. 4 depicts downregulation of (a) GDF11 (one of the ActRII ligands inhibited by luspatercept) mediated mTORCl activity and (b) increase in autophagy markers following luspatercept treatment.

[0028] FIG. 5A depicts survival outcome as a function of NT -proBNP levels.

[0029] FIG. 5B depicts data that reveals a significant decrease in NT -proBNP levels after treatment with luspatercept as compared to epoetin alfa.

[0030] FIG. 5C depicts data that reveals the downregulation of inflammatory pathways in subjects treated with luspatercept and epoetin alfa.

[0031] FIG. 5D is a two-color heatmap showing results from a week 24 gene set variation analysis score for STING-dependent interferon stimulated genes (N=31).NAI-5004198159vl 5Attorney Ref. No. 14247-847-228

[0032] FIG. 5E depicts data correlating survival outcome according to STING gene clusters and NT-proBNP levels.

[0033] FIG. 5F depicts serum BNP levels (t-test, ****p < 0.0001) as an indicator for cardiac dysfunction. Data demonstrates that RAP-536 (murine analogue of luspatercept) significantly reduces cardiac dysfunction in transverse aortic constriction (TAC)-induced experimental heart failure model.

[0034] FIG. 5G is a two-color heatmap showing transcriptional downregulation of STING- dependent interferon-stimulated genes in cardiac tissue of mice.7. DETAILED DESCRIPTION

[0035] Provided herein are methods of treating diseases associated with mTORCl activity using ActRII ligand traps. Specific indications that can be treated using the ActRII ligand traps and methods disclosed herein are described in Section 7.2. Specific ActRII ligand traps and pharmaceutical compositions of ActRII ligand traps that can be used to treat the methods disclosed herein are described in Sections 7.3 and 7.4, respectively.7.1 General Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of the present disclosure. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0037] In some embodiments, chemical structures are disclosed with a corresponding chemical name. In case of conflict, the chemical structure controls the meaning, rather than the name.

[0038] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context otherwise, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0039] As used herein, the term "about" when used in conjunction with a number refers to any number within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the referenced number. In certain embodiments, the term "about" encompasses the exact number recited. In certain embodiments, the term "about" encompasses within 5% of the number recited.

[0040] As used herein, “ActRII” refers to activin receptor type II. As used herein, “ ActRIIB” refers to activin receptor type IIB. See, e.g., Attisano et al., 1992, Cell, 68:97-108, the contents of which are incorporated herein by reference in their entirety. GenBank™ accession number NM 001106.3 provides an exemplary human ActRIIB nucleic acid sequence. GenBank™ accession number NP 001097.2 provides an exemplary human ActRIIB amino acid sequence. The ActRIIB NAI-5004198159vlAttorney Ref. No. 14247-847-228 receptor may be from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification. Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms of the receptor. Members of the ActRIIB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0041] In certain embodiments, “treat”, “treating”, treatment” and the like refer to an action (such as administering an ActRII ligand trap) to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, or condition afflicting a subject.

[0042] The disclosure can be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments.7.2 Methods of Treatment7.2.1 mTORCl-associated Diseases, Disorders, and Conditions

[0043] Provided herein is a method of treating a disease, disorder, or condition associated with an increase in mTORCl activity in a subject in need thereof. In certain embodiments, the disease, disorder, or condition is a cancer, a hamartoma syndrome, a metabolic disorder, a neurodegenerative disease, a fibrotic disease, or an inflammatory or autoimmune disorder in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., an ActRII ligand trap disclosed in Section 7.3, or a pharmaceutical composition thereof, e.g., according to any embodiment disclosed in Section 7.4, to the subject. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a non-hematological cancer. In certain embodiments, the subject has elevated mTORCl levels. In certain embodiments, the mTORCl reduced signaling is of one or more of the pathways shown in FIG. 1, which will be described in further detail in the EXAMPLES.

[0044] The ActRII ligand trap may be administered to the subject to treat the disease, disorder, or condition associated with an increase in mTORCl activity according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0045] In certain embodiments, the ActRII ligand trap can be used in a method of treating cardiac hypertrophy, heart failure, atherosclerosis, ischemic heart disease, breast cancer, prostateNAI-5004198159vl 7Attorney Ref. No. 14247-847-228 cancer, cervical cancer, esophageal cancer, renal cell carcinoma, lung cancer, hepatocellular cancer, glioblastoma, Still’s Disease, psoriasis, lupus, rheumatoid arthritis, PTEN-related hamartoma syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Huntington’s Disease, Alzheimer’s Disease, Parkinson’s Disease, amyotrophic lateral sclerosis, fibrosis (e.g., of heart, liver, kidney, or lung), inflammatory bowel disease, scleroderma, adenomatous polyposis, lymphagioleiomyomatosis, multiple sclerosis, inflammatory bowel disease, multiple sclerosis, metabolic syndrome, cachexia, NAFLD, obesity, or Type 2 diabetes in a subject in need thereof. In certain embodiments, the cardiac hypertrophy or heart failure is not due to vascular calcification.

[0046] In certain embodiments, the subject is at least 60 years old, at least 65 years old, or at least 75 years old. In certain embodiments, the subject has elevated mTORCl levels. In certain embodiments, the subject has one or more gene mutations associated with CHIP.

[0047] In certain embodiments, the gene mutations associated with CHIP are somatic gene mutations. In certain embodiments, the gene mutation is a mutation of DNA (cytosine-5)- methyltransferase 3A (DNMT3 A), Tet methylcytosine dioxygenase 2 (TET2), Additional sex comb-like 1 (ASXL1), Janus kinase 2 (JAK2), Splicing factor 3B subunit 1 (SF3B1), tumor protein 53 gene (TP54), or Protein phosphatase ID (PPMID).

[0048] In certain embodiments, CHIP refers to the formation of a genetically distinct subpopulation of blood cells derived from hematopoietic stem cells (HSCs) or other early blood cell progenitors. The distinct subpopulation in the blood is characterized by a shared unique mutation in the cells’ DNA, and it is believed that this subpopulation is “clonally” derived from a single founding cell and is therefore made of genetic “clones” of the founder. CHIP mutations include mutations falling into the categories of epigenetic regulators (DNMT3a, TET2, and ASXL1), signaling proteins (JAK2), spliceosome components (SF3B1 and SRSF2), or members of the DNA damage response (TP53 and PPMID). Subjects with clonal hematopoiesis may have a mutation in a single gene or mutations in two or more genes. Subjects with one or more CHIP mutations may have an increased risk for blood cancer and cardiovascular complications, such as increased risk of heart attack or stroke.

[0049] In one embodiment, the subject has a mutation in one or more of DNMT3A, TET2, ASXL1, JAK2, and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A and / or TET2. In certain embodiments, the subject has a mutation in TET2. In certain embodiments, the subject has two or more mutations associated with CHIP. In certain embodiments, the subject has elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels as compared to normal NT-proBNP blood levels, e.g., less than 125 pg / mL for a person under 75 years of age or less than 450 pg / mL for a person 75 years of age or older. In certain embodiments, the subject hasNAI-5004198159vl oAttorney Ref. No. 14247-847-228 an NT-proBNP blood level of at least about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL. In certain embodiments, the subject also has one or more co-morbid conditions selected from anemia, a thalassemia, CMML, and MDS. In certain embodiments, the administering of the ActRII ligand trap is also effective to treat the co-morbid condition or conditions.

[0050] In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in two or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in three or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in each of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has elevated NT-proBNP blood levels as compared to normal NT-proBNP blood levels for an individual of the same gender, age, ethnicity, and body mass index. In certain embodiments, the subject has an NT-proBNP blood level that is about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, or more than 100% higher, such as 150% higher, 200% higher, or more than 250% higher than normal NT-proBNP blood levels. In certain embodiments, the subject has an NT-proBNP blood level of at least about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL, at least about 1000 pg / mL, at least about 1250 pg / mL, at least about 1500 pg / mL, at least about 1750 pg / mL, or at least about 2000 pg / mL. In certain embodiments, the subject also has one or more co-morbid conditions selected from anemia, a thalassemia, non-proliferative CMML, and MDS. In certain embodiments, the MDS is lower-risk MDS, e.g., according to the IPSS-R system having a score of less than about 3.7.2.2 NT-proBNP

[0051] In another aspect, the present disclosure provides a method of reducing NT-proBNP levels in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g, according to any embodiment disclosed in Section 7.4), according to any embodiment described in Section 7.2.7 (e.g, subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg). In certain embodiments, the subject has elevated NT-proBNP blood levels as compared to normal NT-proBNP blood levels for an individual of the same gender, age, ethnicity, and body mass index. In certain embodiments, the subject has an NT-proBNP blood level that is about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, or more than 100% higher, such as 150% higher, 200% higher, or more than 250% higher than normal NT-proBNP blood levels. In NAI-5004198159vl QAttorney Ref. No. 14247-847-228 certain embodiments, the subject has an NT-proBNP blood level that is about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL, at least about 1000 pg / mL, at least about 1250 pg / mL, at least about 1500 pg / mL, at least about 1750 pg / mL, or at least about 2000 pg / mL. In certain embodiments, administration of the ActRII ligand trap is effective to reduce NT-proBNP blood levels by at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 75% or at least about 85%. In certain embodiments, administration of the ActRII ligand trap is effective to reduce NT-proBNP blood levels by at least about 100 pg / mL, at least about 200 pg / mL, at least about 300 pg / mL, at least about 400 pg / mL, at least about 500 pg / mL, at least about 600 pg / mL, or at least about 700 pg / mL.

[0052] In certain embodiments, the subject has one or more of anemia, a thalassemia, nonproliferative CMML, and MDS. In certain embodiments, the present disclosure provides a method of reducing NT-proBNP levels in a subject with MDS, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., according to any embodiment disclosed in Section 7.4) according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0053] In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or more of DNMT3A, TET2, ASXL1, and SF3B1.7.2.3 Inflammation

[0054] In another aspect, the present disclosure provides a method of treating a disease, condition, or disorder associated with an increase in inflammation in a tissue of a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3, or a pharmaceutical composition thereof, e.g., according to any embodiment disclosed in Section 7.4, to the subject to reduce inflammation in the tissue, wherein the tissue is a tissue other than bone tissue.

[0055] The ActRII ligand trap may be administered to the subject to treat the disease, disorder, or condition associated with an increase in inflammation in a tissue according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0056] In certain embodiments, “inflammation,” may refer to a process undertaken by the body that releases chemicals that trigger an immune response to fight off infection or heal damaged tissue. Inflammation can damage healthy cells, tissues, and organs. This can lead to diseases such as cancer, heart disease, diabetes, asthma, Alzheimer’s disease, and autoimmune conditions.NAI-5004198159vl 10Attorney Ref. No. 14247-847-228Symptoms of inflammation include, but are not limited to, pain, fatigue, fever, sores, rashes, mood disorders (e.g., depression / anxiety), gastrointestinal issues, weight fluctuation, and frequent infections. Inflammation is involved in many diseases, such as autoimmune diseases (e.g., lupus, rheumatoid arthritis, psoriasis, ankylosing spondylitis), cardiovascular diseases (e.g., heart disease, high blood pressure), certain cancers, gastrointestinal diseases (e.g., Crohn’s disease, inflammatory bowel disease), lung diseases (e.g., asthma, chronic obstructive pulmonary disease), mental health conditions (depression, anxiety), metabolic diseases (e.g., Type 2 diabetes), and neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease). Modulation of inflammation can be detected via certain biomarkers, including C-Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR), Interleukins (e.g., IL-6, IL-1J3), Tumor Necrosis Factoralpha (TNF-a), fibrinogen, procalcitonin, white blood cell (WBC) count, MCP-1 (Monocyte Chemoattractant Protein-1), SAA (Serum Amyloid A), and Pentraxin-3 (PTX3). These markers may be quantified / measured through various known techniques, such as Western blotting, immunofluorescence, and microscopy.

[0057] In one embodiment, the tissue is adipose tissue, brain tissue, cardiac tissue, connective tissue, epithelial tissue, gastrointestinal tissue, tissue of the joint, kidney (renal) tissue, liver tissue, lung tissue, muscle tissue, nervous system tissue, skin tissue (epidermal and dermal tissue), or spleen tissue. In one embodiment, the tissue is cardiac tissue. In certain embodiments, the disease, condition, or disorder is Crohn's Disease, colitis, steatohepatitis, systemic lupus erythematosus, Sjogren’s syndrome, systemic sclerosis, rheumatoid arthritis, vasculitis, VEXAS syndrome, ILD, COPD, chronic immune-mediated diseases, chronic inflammatory conditions due to mitochondrial dysfunction, or sepsis.

[0058] In certain embodiments, the subject is at least 60 years old, at least 65 years old, or at least 75 years old. In certain embodiments, the subject has elevated mTORCl levels. In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or more of DNMT3A, TET2, ASXL1, JAK2, and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A and / or TET2. In certain embodiments, the subject has a mutation in TET2. In certain embodiments, the subject has two or more mutations associated with CHIP. In certain embodiments, the subject has elevated NT -proBNP blood levels as compared to normal NT -proBNP blood levels. In certain embodiments, the subject has an NT-proBNP blood level of at least about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL. In certain embodiments, the subject also has one or more co-morbid conditions selected from anemia, a thalassemia, non-proliferative CMML, and MDS. In certain embodiments, the administering of the ActRII ligand trap is also effective to treat the co-morbid condition or conditions.NAI-5004198159vl 1 IAttorney Ref. No. 14247-847-228

[0059] In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in two or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in three or more of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has a mutation in each of DNMT3A, TET2, ASXL1, and SF3B1. In certain embodiments, the subject has elevated NT-proBNP blood levels as compared to normal NT-proBNP blood levels for an individual of the same gender, age, ethnicity, and body mass index. In certain embodiments, the subject has an NT-proBNP blood level that is about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, or more than 100% higher, such as 150% higher, 200% higher, or more than 250% higher than normal NT-proBNP blood levels. In certain embodiments, the subject has an NT-proBNP blood level that is about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL, at least about 1000 pg / mL, at least about 1250 pg / mL, at least about 1500 pg / mL, at least about 1750 pg / mL, or at least about 2000 pg / mL. In certain embodiments, the subject also has one or more co-morbid conditions selected from anemia, a thalassemia, non-proliferative CMML, and MDS.

[0060] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to downregulate IL-6 gene and protein expression levels in the subject as compared to the IL-6 gene and protein expression levels in the subject prior to the administering. In certain embodiments, the IL-6 gene and / or protein expression levels are downregulated about 50% to about 75%, such as by about 50%, by about 55%, by about 60%, by about 65%, by about 70% or by 75%.

[0061] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to downregulate inflammatory pathways such as interferon alpha response and IL6 / JAK / STAT3 signaling in the subject as compared to the activity of these pathways and signaling the subject prior to the administering. Accordingly, provided herein is a method of reducing the activity of one or more inflammatory pathways in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., according to any embodiment disclosed in Section 7.4) according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg), to the subject. In certain embodiments, the inflammatory pathway is the interferon alpha pathway. In certain embodiments, the inflammatory pathway is associated with IL6 / JAK / STAT3 signaling. In certain embodiments, the inflammatory pathway is the stimulator of interferon genes (STING)- dependent inflammatory pathway.NAI-5004198159vl 1 9Attorney Ref. No. 14247-847-228

[0062] Accordingly, provided herein is a method of reducing the activity of STING-dependent inflammatory pathways in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., according to any embodiment disclosed in Section 7.4) to the subject according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg). In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce cGAS / STING pathway activation. In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce expression of STING-dependent genes in cardiac tissue. In certain embodiments, administration of the ActRII ligand trap to the subject treatment reduces the risk of death in the subject. In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or both of DNMT3A and TET2.

[0063] In certain embodiments, the subject has cardiac inflammation and hypertrophy, and one or both of a co-morbid condition selected from anemia, a thalassemia, non-proliferative CMML, and MDS and a mutation in one or both of DNMT3A and TET2. In such a subject, administration of an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., according to any embodiment disclosed in Section 7.4) according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg) to the subject may be effective to reduce the cardiac inflammation and hypertrophy, e.g., by reducing activity of any of the inflammatory pathways disclosed herein, such as interferon alpha, IL6 / JAK / STAT3, and / or STING.7.2.4 Autophagy

[0064] In another aspect, the present disclosure provides a method of inducing autophagy in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3, or a pharmaceutical composition thereof, e.g., according to any embodiment disclosed in Section 7.4, to the subject.

[0065] In certain embodiments, “autophagy” refers to the body’s process of destroying old and damaged cell parts through a lysosome-dependent regulated mechanism. Autophagy is essential for a cell to survive and function. Certain diseases may be related to inefficient autophagy including, but not limited to, cancer, Crohn’s disease, diabetes, heart disease, Huntington’s disease, kidney disease, liver disease, and Parkinson’s disease. Modulation of autophagy can be detected via certain biomarkers, including LC3 (Microtubule-associated protein lA / lB-light chain 3), P62 / SQSTM1, NAI-5004198159vl 1 oAttorney Ref. No. 14247-847-228Atg proteins, ULK1 (Unc-51 Like Autophagy Activating Kinase 1), mitochondrial markers, lysosomal markers, such as LAMP1, and total autophagic flux. These markers may be quantified / measured through various known techniques, such as Western blotting, immunofluorescence, and microscopy.

[0066] In certain embodiments, the subject has a disease, condition, or disorder selected from the group consisting of heart failure, NAFLD, Type 2 diabetes, obesity, CKD, AKD, IBD, Alzheimer’s disease, Parkinson’s Disease, Huntington’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, LSDs, muscular disorders and fatigue, lung cancer, breast cancer, prostate cancer, colorectal cancer, cervical cancer, esophageal cancer, renal cell carcinoma, and hepatocellular cancer.

[0067] The ActRII ligand trap may be administered to the subject to inducing autophagy according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0068] In certain embodiments, the subject is at least 60 years old, at least 65 years old, or at least 75 years old. In certain embodiments, the subject has elevated mTORCl levels. In certain embodiments, the subject has one or more gene mutations associated with CHIP, such as a mutation in one or more of DNMT3A, TET2, ASXL1, JAK2, and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A and / or TET2. In certain embodiments, the subject has a mutation in TET2. In certain embodiments, the subject has two or more mutations associated with CHIP. In certain embodiments, the subject has elevated NT -proBNP blood levels as compared to normal NT -proBNP blood levels. In certain embodiments, the subject has an NT-proBNP blood level of at least about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL. In certain embodiments, the subject also has one or more co-morbid conditions selected from anemia, a thalassemia, non-proliferative CMML, and MDS. In certain embodiments, the administering is also effective to treat the co-morbid condition or conditions. In certain embodiments, the cardiac hypertrophy or heart failure is not due to vascular calcification.7.2.5 CHIP Mutations as Biomarker for Treatment with ActRII Ligand Traps

[0069] In another aspect, the present disclosure provides a method of treating anemia, a thalassemia, non-proliferative CMML, or MDS in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g, according to any embodiment disclosed in Section 7.3, or a pharmaceutical composition thereof, e.g, according to any embodiment disclosed in Section 7.4, to the subject, wherein the subject has one or more gene mutations associated with CHIP. In certain embodiments, the one or more gene mutations associated with CHIP are in a gene selected from the NAI-5004198159vl I / IAttorney Ref. No. 14247-847-228 group consisting of DNMT3A, TET2, ASXL1, JAK2, and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A and / or TET2. In certain embodiments, the subject has a mutation in TET2. In certain embodiments, the subject has two or more mutations associated with CHIP. In certain embodiments, the subject has MDS.

[0070] The ActRII ligand trap may be administered to the subject to treat the anemia, a thalassemia, non-proliferative CMML, or MDS according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0071] In certain embodiments, the subject is at least 60 years old, at least 65 years old, or at least 75 years old. In certain embodiments, the subject has elevated NT -proBNP blood levels as compared to normal NT -proBNP blood levels.

[0072] Non-limiting examples of symptoms of anemia that can be treated using the methods disclosed herein include fatigue, loss of energy, rapid heartbeat, shortness of breath, headaches, difficulty concentrating, dizziness, pale skin, leg cramps, and insomnia. In certain embodiments, the subject may require regular red blood cell (RBC) transfusions to treat the anemia prior to or as an adjunctive therapy to the methods of treating disclosed and described herein. In certain embodiments, treatment of a subject with anemia according to the methods disclosed herein may reduce the transfusion burden (z.e., reduce the frequency of RBC transfusions) of the treated subject, e.g., from greater than four RBC units every 8 weeks to less than four RBC units every 8 weeks.

[0073] Non-limiting examples of symptoms of beta-thalassemia that can be treated using the methods disclosed herein include defective red blood cell production in the marrow, ineffective erythropoiesis, deficient hemoglobin levels, multiple organ dysfunction, iron overload, paleness, fatiguejaundice, and splenomegaly. The beta-thalassemia may be minor, intermedia, or major. The subject may be of any genotype, e.g., P+ / p+, P+ / p°, or p° / p°. Non-limiting examples of symptoms of non-proliferative CMML include splenomegaly, hepatomegaly, anemia, fatigue, shortness of breath, leukopenia, frequent infections, thrombocytopenia, easy bruising or bleeding, fever, weight loss, pain, pale skin, sweating, and loss of appetite. Non-limiting examples of symptoms of MDS include anemia, shortness of breath, fatigue, pale skin, leukopenia, frequent infections, neutropenia, thrombocytopenia, easy bruising or bleeding, weight loss, fever, loss of appetite, weakness, and bone pain.

[0074] MDS may be diagnosed in a subject according to the International Prognostic Scoring System (IPSS), which is utilized in the evaluation of prognosis in MDS. See, e.g., Greenberg et al., 1997, Blood, 89(6):2079-2088, and Erratum in Blood, 1998, 91 : 1100. The IPSS by Greenberg utilizes a criteria point system to characterize MDS patient outcomes as low risk (0 points; medianNAI-5004198159vl 1 cAttorney Ref. No. 14247-847-228 survival of 5.7 years); intermediate- 1 (0.5-1 point; median survival of 3.5 years); intermediate-2 risk (1.5-2.0 points; median survival of 1.2 years); or high risk (2.5-3.5 points; median survival of 0.4 years). The points system evaluates (i) the percentage of bone marrow blasts in the subject; (ii) the karyotype of the subject; and (iii) and cytopenias in the subject (defined as hemoglobin concentration of less than 10 g / dL, absolute neutrophil count of less than 1,800 / pL, and platelet count of less than 100,000 / pL).

[0075] In certain embodiments, the MDS is IPSS-defined low risk MDS. In certain embodiments, the MDS is IPSS-defined intermediate- 1 risk MDS In certain embodiments, the MDS is IPSS-defined intermediate-2 risk MDS. In certain embodiments, the MDS is IPSS-defined high risk MDS.

[0076] The IPSS was revised in 2012 to create a new set of criteria (“IPSS-R”) to characterize MDS patient outcomes as very low-risk (< 1.5 points), low-risk (> 1.5 - 3 points), intermediate-risk (>3 - 4.5 points), high-risk (> 4.5 - 6 points), or very high-risk (> 6 points). See, e.g., Greenberg et al., 2012 Sep 20, Blood, 120(12):2454-65, the contents of which are incorporated herein by reference in their entirety.

[0077] In certain embodiments, the MDS is IPSS-R-defined very low risk MDS. In certain embodiments, the MDS is IPSS-R-defmed low risk MDS. In certain embodiments, the MDS is IPSS-R-defined intermediate risk MDS. In certain embodiments, the MDS is IPSS-R-defmed high risk MDS. In certain embodiments, the MDS is IPSS-R-defmed very high risk MDS.

[0078] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce NT-proBNP levels in the blood of the subject as compared to the NT-proBNP levels in the blood of the subject prior to the administering. In certain embodiments, the NT- proBNP levels are reduced by at least about 50%, about 60%, or about 70%.

[0079] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce BNP levels in the blood of the subject as compared to the BNP levels in the blood of the subject prior to the administering. In certain embodiments, the BNP levels are reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70%. In certain embodiments, the BNP levels are reduced by at least about 60% or by at least about 70%.

[0080] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce cardiac hypertrophy in the subject. In certain embodiments, the reduction is measured by echocardiography. In certain embodiments, the administering causes about a 5%, about 10%, about 15%, or about 20% mass reduction in one or more ventricles of the heart or one or more atria of the heart.NAI-5004198159vl 16Attorney Ref. No. 14247-847-228

[0081] In certain embodiments, administration of the ActRII ligand trap to the subject is effective to downregulate the IL-6 gene and protein expression levels in the subject as compared to the IL-6 gene and protein expression levels in the subject prior to the administering. In certain embodiments, the IL-6 gene and / or protein expression levels are downregulated about 50% to about 75%, such as by about 50%, by about 55%, by about 60%, by about 65%, by about 70% or by about 75%.

[0082] In certain embodiments, administration of the ActRII ligand trap, such as one as disclosed in Section 7.3 (e.g., luspatercept), to the subject is effective to downregulate inflammatory pathways such as interferon alpha response and IL6 / JAK / STAT3 signaling in the subject as compared to the activity of these pathways and signaling the subject prior to the administering. In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce activation of stimulator of interferon genes (STING)-dependent inflammatory pathways in the subject. In certain embodiments, administration of the ActRII ligand trap to the subject is effective to reduce cGAS / STING pathway activation, which may in turn, reduce cardiac inflammation and hypertrophy. In certain embodiments, the ActRII ligand trap is effective to reduce expression of STING-dependent genes in cardiac tissue.

[0083] In certain embodiments, administration of the ActRII ligand trap to the subject treatment reduces the risk of death in the subject. Risk of death can be measured, e.g., by a hazard ratio, such as is described in Sashegyi et al., 2017 Apr, Oncologist, 22(4):484-486, the contents of which is incorporated herein by reference in their entirety.

[0084] In another aspect, the present disclosure provides a method of treating MDS in a subject in need thereof, the method comprising administering an ActRII ligand trap, e.g., according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., according to any embodiment disclosed in Section 7.4) according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg), to the subject, wherein the subject has one or more gene mutations associated with CHIP. In certain embodiments the subject additionally has one or more of very low, low, or intermediate risk disease MDS, endogenous serum erythropoietin (sEPO) levels of less than 500 U / L, an Eastern Cooperative Oncology Group (ECOG) score of 0, 1, or 2, and is transfusion dependent. In certain embodiments, the subject is considered transfusion dependent when requiring RBC transfusions of 2 to 6 units per 8 weeks of treatment. In certain embodiments, the subject exhibits 2, 3 or each of these characteristics. In certain embodiments, the one or more gene mutations associated with CHIP are in a gene selected from the group consisting of SF3B1, TET2, ASXL1, and DNMT3A. In certain embodiments, the subject has a mutation in TET2. In one NAI-5004198159vl 17Attorney Ref. No. 14247-847-228 embodiment, the subject has a mutation in ASXL1. In certain embodiments, the subject has two or more mutations associated with CHIP. In certain embodiments, the subject has a mutation in DNMT3A and TET2. In certain embodiments, the subject has a mutation in DNMT3A and ASXL1. In certain embodiments, the subject has a mutation in TET2 and ASXL1. In certain embodiments, the subject has a mutation in TET2 and SF3B1. In certain embodiments, the subject has a mutation in ASXL1 and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A and SF3B1. In certain embodiments, the subject has three or more mutations associated with CHIP. In certain embodiments, the subject has a mutation in DNMT3A, TET2, and one or both of ASXL1 and SF3B1. In certain embodiments, the subject has a mutation in DNMT3A, TET2, ASXL1. and SF3B1. In certain embodiments, the subject additionally has a cardiac disorder. Non-limiting examples of cardiac disorders that the subject may have include coronary artery disease, heart failure, heart arrythmia, valve disease, or a congenital heart defect. In certain embodiments, the subject has a cardiac disorder and a TET2 mutation. In certain embodiments, the subject is ring sideroblast-negative (e.g., less than 15% of erythroblasts are ring sideroblasts in the subject). In certain embodiments, the subject has an NT -proBNP blood level of at least about 700 pg / mL, at least about 800 pg / mL, or at least about 900 pg / mL, at least about 1000 pg / mL, at least about 1250 pg / mL, at least about 1500 pg / mL, at least about 1750 pg / mL, or at least about 2000 pg / mL.

[0085] In certain embodiments, the subject has one or both of elevated NT-proBNP levels (e.g., > 2000 pg / mL) and elevated expression levels of STING gene clusters (e.g., a coordinated, increased level expression of the STING gene and other genes associated with the STING signaling pathway). Non-limiting examples of other genes associated with STING signaling include USP18, RSAD2, PARP14, MX1, IFITM3, EIF2AK2, and BST2. The subject may additionally or alternatively have increased STING signaling activity.

[0086] Expression of genes (including STING and those related) can be quantified by methods well-known in the art, for example, using Reverse Transcription-Quantitative Polymerase Chain Reaction (rt-qPCR) / spectrophotometry, RNA sequencing, microarrays, and / or Northern Blotting. Elevated expression levels may be as compared to a baseline expression level, e.g., in a control sample or healthy subject. Elevated STING and STING-related gene expression levels, in any embodiment disclosed herein, may be at least about 25% higher, at least about 50% higher, at least about 75% higher, at least about 100% higher, at least about 150% higher, at least about 200% higher, at least about 250% higher, or more than 300% higher than expression levels in the control sample. Expression levels can be converted to a standardized score, such as a z-score. For example, a z-score may be calculated according to the formula z = xp / o, wherein x is the expression value, p NAI-5004198159vl 1 oAttorney Ref. No. 14247-847-228 is the mean of the population, and o is the standard deviation. In certain embodiments, the mean is the mean expression level of, e.g., samples from a population of healthy subjects and the standard deviation is the standard deviation from the same population. In other embodiments, the mean is the mean expression level of, e.g., samples from a population that includes both healthy and non- healthy subjects and the standard deviation is the standard deviation from the same population. The mean is set as the “0” point (i.e., no change in expression), such as in FIGs. 5D and 5F. Positive values represent elevated gene expression levels, whereas negative values represent reduced gene expression levels. In certain embodiments, elevated STING and STING-related gene expression levels may be at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1.0. Administration of luspatercept to a subject may decrease STING and STING-related gene expression levels, e.g., to at least about -0.1, at least about -0.25, at least about -0.5, at least about -0.75, or at least about -1.0.

[0087] The activity of STING signaling may be measured by assessing molecular events downstream of STING by methods well known in the art, for example, by measuring phosphorylation of STING and its binding partner, TBK1. Phosphorylation in clinical tissue samples (e.g., biopsies) can be detected by techniques, such as Western Blotting. Measuring phosphorylated IRF3 (pIRF3) levels is another way to confirm STING activity. The STING pathway potently induces the production of type I IFNs (e.g, IFN-a, IFN-P), and thus IFN protein levels in serum, plasma, or tissue culture supernatants using an enzyme-linked immunosorbent assay (ELISA) can be used as an indicator of STING activity. Messenger RNA (mRNA) expression of ISGs, which are upregulated in response to IFN signaling, can also be quantified using quantitative polymerase chain reaction (qPCR) or RNA-sequencing. STING activation also drives the production of other inflammatory cytokines, such as TNF-a and IL-ip, which can be measured in clinical samples by ELISA. Phosphorylation of molecular targets downstream of STING may also converted to a standardized score, such as a z-score. A z-score of at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1.0 for the phosphorylated form of one or more downstream targets of the STING pathway may indicate increased activation of the STING pathway. Activation of inflammatory pathways can be measured in a similar manner.

[0088] In certain embodiments, survival outcome can be predicted based on the expression of STING-dependent genes. For example, in certain embodiments, the subject has both elevated NT- proBNP levels and elevated expression levels of STING gene clusters, which may indicate a significantly lower chance of survival and / or indicate a more severe form of the disease. In certain embodiments, administration of the ActRII ligand trap, such as one as disclosed in Section 7.3 NAI-5004198159vl 1 QAttorney Ref. No. 14247-847-228(e.g., luspatercept), to the subject treatment reduces the risk of death in such a subject. In certain embodiments, the z-score expression level of STING and STING-related genes in the subject to be treated with luspatercept may be at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1, or in terms of percentage, may be increased by at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or by more than 300%.

[0089] Expression levels of STING and STING-related genes may be quantified by routine methods known in the art, for example, by using advanced microscopy and image analysis. STING can be labelled with a fluorescent protein, imaged, e.g., with Single-Molecule Localization Microscopy (SMLM) or Photo-activated Localization Microscopy (PALM) and then analyzed with specialized software. Single-cell RNA sequencing can also be used to quantify in larger tissue or cell populations.

[0090] In certain embodiments, the subject is administered the ActRII ligand trap as a first line of therapy. In other embodiments, the subject has been administered another therapy prior to the ActRII ligand trap, such as an erythropoiesis-stimulating agent (ESA). In certain embodiments, the ESA was epoetin alfa. In certain embodiments, the epoetin alfa was not effective or sufficiently effective to treat the subject.7.2.6 Prevention and Prophylaxis

[0091] In certain embodiments, the ActRII ligand trap, such as one as disclosed in Section 7.3 (e.g., luspatercept), is administered as a preventative, or prophylactic measure, to prevent a subject who is at risk of developing a more severe form of the disease or condition, e.g., anemia, MDS, CMML, or beta-thalassemia, or a complication associated therewith, based on one or more criteria disclosed according to any embodiment herein.

[0092] For example, an ActRII ligand trap can be administered according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg) as a preventative, or prophylactic measure, to prevent a subject who is at risk of developing cardiac disorder. For example, the subject may have or display one or more of the following traits: i. diagnosed with one or more of anemia, MDS, CMML, or beta-thalassemia; ii. elevated NT -proBNP levels; iii. a genetic profile enriched with one or more mutations in TET2, SF3B1, DNMT3A and ASXL1;NAI-5004198159vl 20Attorney Ref. No. 14247-847-228 iv. elevated expression levels of STING gene clusters and / or increased activation of STING signaling.

[0093] The ActRII ligand trap can be any ActRII ligand trap according to any embodiment disclosed in Section 7.3 (e.g., luspatercept), or a pharmaceutical composition thereof (e.g., administered according to any embodiment disclosed in Section 7.4) according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg).

[0094] In certain embodiments, the subject’s condition is not severe at the time of prophylactic treatment with the ActRII ligand trap but is predicted to develop into a more severe disease. For example, a subject may have MDS, CMML, or beta-thalassemia, but does not yet show signs of any cardiovascular complications or elevated NT-proBNP levels, but have one or more mutations in TET2, SF3B1, DNMT3A and ASXL1 and / or elevated expression levels of STING gene clusters (e.g., a z-score expression level of STING and / or one or more STING-related genes of at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1, or in terms of percentage, be increased by at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or by more than 300%) and / or increased activation of STING signaling. The ActRII ligand trap can be, in such embodiments, administered to the subject to be cardioprotective and prevent cardiovascular damage that may otherwise result from progression of the MDS, CMML, or beta-thalassemia.

[0095] In certain embodiments, the subject has MDS, CMML, or beta-thalassemia and elevated NT-proBNP levels. In certain embodiments, the subject has MDS and has a genotype enriched with somatic mutations in TET2, SF3B1, DNMT3A and / or ASXL1. In certain embodiments, the subject has MDS and carries somatic mutations in TET2. In certain embodiments, the subject has MDS and increased activation of inflammatory pathways (e.g., interferon alpha response, IL6 / JAK / STAT3, or cGAS / STING signaling). In certain embodiments, the subject has MDS, elevated NT-proBNP levels, and elevated expression levels of STING gene clusters (e.g., a z-score expression level of STING and / or one or more STING-related genes of at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, or at least 1, or in terms of percentage, be increased by at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or by more than 300%).

[0096] In certain embodiments, subjects having these characteristics are more difficult to treat and are likely to develop a more severe and / or complicated form of disease but can still be treated effectively with the ActRII ligand trap (e.g., luspatercept). In certain embodiments, subjects havingNAI-5004198159vl 21Attorney Ref. No. 14247-847-228 these characteristics may be more effectively treated with the ActRII ligand trap (e.g., luspatercept), than subjects not having these characteristics.

[0097] In another aspect, the subject may have another disease or condition that is associated with cardiovascular complications, such as diabetes, kidney disease, sleep apnea, and certain autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus (lupus), psoriasis, scleroderma, and sarcoidosis), and a ActRII ligand trap such as one as disclosed in Section 7.3 (e.g., luspatercept) can be, in such embodiments, administered to the subject according to any embodiment described in Section 7.2.7 (e.g., subcutaneously every 21 days at a dose of about 1 mg / kg to about 1.75 mg / kg) to be cardioprotective and prevent cardiovascular damage that may otherwise accompany the disease or condition.7.2.7 Dosing and Administration

[0098] The ActRII ligand trap administered in accordance with any of the aforementioned methods may be according to any embodiment disclosed in Section 7.3. For example, in certain embodiments, the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is 90% identical to SEQ ID NO:4, 95% identical to SEQ ID NO:4; 98% identical to SEQ ID NO:4 or identical to SEQ ID NO:4. In certain embodiments, the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is 90% identical to SEQ ID NO:6, 95% identical to SEQ ID NO:6; 98% identical to SEQ ID NO:6 or identical to SEQ ID NO:6. In certain embodiments, the fusion protein comprises an L79D amino acid mutation. In certain embodiments, the ActRII ligand trap is a fusion protein comprising: i) a fragment of the extracellular domain of ActRIIB, wherein the fragment consists of the amino acid sequence of SEQ ID NO: 10; ii) a linker; and iii) an Fc of an IgG (e.g., an “ActRIIB ligand trap”). In certain embodiments, the ActRII ligand trap comprises a linker, such as a linker of SEQ ID NO: 11 or SEQ ID NO: 12. In certain embodiments, the ActRII ligand trap comprises a linker of SEQ ID NO: 11. In certain embodiments, the ActRII ligand trap is or comprises SEQ ID NO:4. In certain embodiments, the ActRII ligand trap is or comprises SEQ ID NO:6. In certain embodiments, the ActRII ligand trap is or comprises SEQ ID NO:8. In certain embodiments, an ActRIIB ligand trap that can be used with the methods disclosed herein is luspatercept.

[0099] In any of the methods disclosed herein, such as in Section 7.2, the ActRII ligand trap (such as disclosed in Section 7.3) may be administered to the subject, e.g., in a pharmaceutical composition (such as disclosed in section 7.4) parenterally. In certain embodiments, the ActRII ligand trap is administered to the subject subcutaneously. In one embodiment, the ActRII ligandNAI-5004198159vl 22Attorney Ref. No. 14247-847-228 trap is administered to the subject subcutaneously in the upper arm, abdomen, or thigh of the subject.

[0100] In certain embodiments, the ActRII ligand trap (e.g., an ActRIIB ligand trap according to any embodiment disclosed in Section 7.3 herein) is administered to the subject every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In certain embodiments, the ActRII ligand trap is administered to the subject once every 21 days.

[0101] In certain embodiments, the ActRII ligand trap (e.g., an ActRIIB ligand trap according to any embodiment disclosed in Section 7.3 herein) is administered to the subject at a dose of about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.25 mg / kg, about 1.33 mg / kg, about 1.50 mg / kg, about 1.75 mg / kg, or about 2.00 mg / kg. In certain embodiments, the dose of the ActRII ligand trap is about 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, or 1.75 mg / kg. In certain embodiments, the dose of the ActRII ligand trap is about 1 mg / kg to about 1.75 mg / kg. In certain embodiments, the dose of the ActRII ligand trap is about 1 mg / kg. In certain embodiments, the dose of the ActRII ligand trap is about 1.75 mg / kg.

[0102] In certain embodiments, the ActRII ligand trap (e.g., an ActRIIB ligand trap according to any embodiment disclosed in Section 7.3 herein) is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRIIB ligand trap is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that is at least 90% identical to SEQ ID NO:4 is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that comprises i) a fragment of the extracellular domain of ActRIIB, wherein the fragment consists of the amino acid sequence of SEQ ID NO: 10; ii) a linker; and iii) an Fc of an IgG is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that is at least 90% identical to SEQ ID NO:4 is administered to the subject subcutaneously once every 21 days (3 weeks) at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that is identical to SEQ ID NO:4 is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that is identical to SEQ ID NO:6 is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, an ActRII ligand trap that is identical to SEQ ID NO:4 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 NAI-5004198159vl nAttorney Ref. No. 14247-847-228 weeks) at a dose of about 1 mg / kg. In certain embodiments, an ActRII ligand trap that is identical to SEQ ID NO:6 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 1 mg / kg.

[0103] In certain embodiments, a mixture of ActRII ligand traps comprising two or more of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, a mixture of ActRII ligand traps comprising two or more of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 1 mg / kg. In certain embodiments, a mixture of ActRII ligand traps comprising two or more of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 1 mg / kg. In certain embodiments, a mixture of ActRII ligand traps comprising two or more of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg. In certain embodiments, a mixture of ActRII ligand traps comprising each of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 1 mg / kg. In certain embodiments, a mixture of ActRII ligand traps comprising each of the fusion proteins of SEQ ID NO:4, 6, and 8 is administered to the subject subcutaneously once every 21 days ( / .< ., 3 weeks) at a dose of about 1 mg / kg.

[0104] In certain embodiments, the ActRII ligand trap is not administered as part of a combination therapy with an mTOR or mTORCl inhibitor, such as rapamycin. In certain embodiments, the ActRII ligand trap is not administered as part of a combination therapy with a JAK2 inhibitor, such as momelotinib, ruxolitinb, pacritinib, or fedratinib. In certain embodiments, the ActRII ligand trap is not administered as part of a combination therapy with another ESA, such as recombinant erythropoietin. In certain embodiments, the ActRII ligand trap is not administered as part of a combination therapy with lenalidomide. In certain embodiments, the ActRII ligand trap is the only therapeutic agent administered to the subject. In certain embodiments, the subject may continue to receive other adjuvant non-pharmaceutical therapies, such as, but not limited to, RBC transfusion.

[0105] In certain embodiments, the dosing regimen for treating, e.g., according to any embodiment disclosed in any one of Sections 7.2.1 through 7.2.5, can be used prophylactically, e.g., such as disclosed in Section 7.2.6.

[0106] In certain embodiments, the dose of ActRII ligand trap that is administered is adjusted based on measured expression levels of certain biomarkers in the subject, for example, expression levels of NT-proBNP in a sample, such as a blood sample, collected from the subject. For example, NAI-5004198159vl ,1Attorney Ref. No. 14247-847-228 in certain embodiments, an initial dose of ActRII ligand trap that is administered to a subject according to any embodiment disclosed in any one of Sections 7.2.1 through 7.2.5 can be used prophylactically, e.g., such as disclosed in Section 7.2.6. After a first period of time or certain number of administrations of ActRII ligand trap, expression levels of NT -proBNP can be measured in the subject. If expression levels of NT-proBNP are decreased too much, e.g., below normal levels, an adjusted dose that is lower than the initial dose may be administered to the subject. For example, the subject may be initially administered about 1.0 mg / kg of the ActRII ligand trap, which could be reduced to about 0.75 mg / kg, about 0.60 mg / kg, or about 0.50 mg / kg if expression levels of NT-proBNP are decreased too much in the subject after, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations of ActRII ligand trap.

[0107] In another embodiment, an initial dose of ActRII ligand trap is administered to a subject according to any embodiment disclosed in any one of Sections 7.2.1 through 7.2.6, e.g., diagnosed with one or more of anemia, MDS, CMML, or beta-thalassemia, and, after a first period of time or certain number of administrations of ActRII ligand trap, expression levels of NT-proBNP can be measured in the subject. If expression levels of NT-proBNP are not sufficiently decreased e.g., are reduced by less than 30%, less than 20%, or less than 10%, an adjusted dose that is greater than the initial dose may be administered to the subject. For example, the subject may be initially administered about 0.50 mg / kg of the ActRII ligand trap, which could be increased to about 0.75 mg / kg, 1.0 mg / kg, 1.25 mg / kg, about 1.5 mg / kg, about 1.75 mg / kg, or about 2.0 mg / kg if expression levels of NT-proBNP are not sufficiently decreased after, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations of ActRII ligand trap.

[0108] Accordingly, provided herein is a method of treating a subject in need thereof, the method comprising taking an initial baseline measurement of NT-proBNP in a sample of the subject, administering an ActRII ligand trap at an initial dose for a certain period of time or number of administrations, then measuring an expression level of NT-proBNP in a sample of the subject. The method may further comprise adjusting the dose to an “adjusted” dose based on the difference between the initial baseline NT-proBNP measurement (and / or a control sample) and the measurement after the certain period of time / number of administrations, e.g., as described above.7.3 ActRII Ligand Traps

[0109] In certain embodiments, the ActRII ligand traps described in this section can be used in the methods provided herein (e.g., in Section 7.2). In certain embodiments, the ActRII ligand trap is an ActRIIB ligand trap. In certain embodiments, an ActRIIB ligand trap that can be used with the methods disclosed herein is luspatercept.NAI-5004198159vl 25Attorney Ref. No. 14247-847-228

[0110] In certain embodiments, the ActRII ligand trap comprises a polypeptide derived from (e.g., comprises one or more mutations) or comprising all or a fragment of the ActRIIB receptor. In certain embodiments, the ActRII ligand trap is a soluble peptide comprising all or a fragment of the extracellular domain of ActRIIB or a variant polypeptide thereof that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the extracellular domain of ActRIIB. In certain embodiments, the ActRII ligand trap is a soluble peptide comprising all or a portion of the ligand-binding portion of ActRIIB. In certain embodiments, the ligand binding portion comprises amino acids 19-134 of SEQ ID NOs: l or 2. In certain embodiments, the ligand binding portion comprises amino acids 25 to 131 of SEQ ID NOs:l or 2. In certain embodiments, an ActRII ligand trap comprises amino acids 25 to 131 of SEQ ID NO: 1 or SEQ ID NO:2 or a variant polypeptide thereof that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0111] The numbering of amino acids for all of the ActRIIB -related sequences described herein is based on the amino acid numbering for SEQ ID NO: 1 and SEQ ID NO:2 (which only differ in the amino acid expressed at position 79), unless specifically stated otherwise. For example, if an ActRII amino acid sequence is described as having a substitution / mutation at amino acid position 79, then it is to be understood that position 79 refers to the 79thamino acid in SEQ ID NO: 1 or SEQ ID NO:2, from which the ActRII ligand trap is derived.

[0112] In certain embodiments, an ActRII ligand trap comprises all or a fragment of the extracellular domain of ActRIIB or a variant polypeptide thereof comprising a leucine at the position corresponding to amino acid 79 of the ActRIIB precursor amino acid sequence, e.g., SEQ ID NO: 1 or 2. In certain embodiments, an ActRII ligand trap comprises all or a fragment of the amino acid sequence of SEQ ID NO:2 or a variant polypeptide thereof that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0113] In certain embodiments, an ActRII ligand trap comprises a polypeptide of SEQ ID NO: 10.

[0114] In certain embodiments, the ActRII ligand trap that may be used according to the methods described herein is a fusion protein comprising the extracellular domain of ActRIIB, or a variant polypeptide thereof that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the extracellular domain of ActRIIB, and a heterologous portion comprising a constant region from an IgG heavy chain (or a fragment thereof), such as an Fc domain. The extracellular domain of ActRIIB or variant thereof and heterologous portion may be bound directly together or be joined by a linker. Exemplary linkers include short polypeptide sequences comprising 2 to 10, 2 to 5, 2 to 4, or 2 to 3 amino acid residues (e.g., glycine residues),NAI-5004198159vlAttorney Ref. No. 14247-847-228 such as a Gly-Gly-Gly (GGG, SEQ ID NO: 11) linker. In another specific embodiment, the linker comprises the amino acid sequence Thr-Gly-Gly-Gly (TGGG, SEQ ID NO: 12). In certain embodiments, the linker is a bond.

[0115] In certain embodiments, the ActRII ligand trap that may be used according to any method disclosed herein, e.g., in Section 7.2, comprises all or a fragment of the extracellular domain of ActRIIB (e.g., SEQ ID NO: 10) fused directly or indirectly to an Fc portion of an antibody. In certain embodiments, the ActRII ligand trap is a fusion protein comprising a polypeptide of SEQ ID NO: 10 and the Fc portion of an IgG (or a fragment thereof). In certain embodiments, the ActRII ligand trap is a fusion protein comprising a polypeptide of SEQ ID NO: 10 and the Fc portion of human IgG (or a fragment thereof). In certain embodiments, the ActRII ligand trap is a fusion protein comprising a polypeptide of SEQ ID NO: 10 and the Fc portion of IgGl. In another embodiment, the ActRII ligand trap is a fusion protein comprising: (i) a fragment of the extracellular domain of ActRIIB, wherein the fragment consists of the sequence of amino acid 25-131 of SEQ ID NO:2 (e.g., SEQ ID NO: 10); (ii) a linker; and (iii) an Fc of an IgG (or a fragment thereof). In certain embodiments, the fusion protein comprises a linker. In certain embodiments, the linker is GGG (SEQ ID NO: 11).

[0116] In one embodiment, the ActRII ligand trap is, comprises, or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4. Said differently, in certain embodiments, the ActRII ligand trap that may be used according to any method disclosed herein, e.g., in Section 7.2, comprises amino acids 25-131 of SEQ ID NO:2 or exhibits at least an 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or sequence homology to amino acids 25-131 of SEQ ID NO:2. In certain embodiments the ActRII ligand trap is identical to the amino acid sequence of SEQ ID NO:4.

[0117] In one embodiment, the ActRII ligand trap is, comprises, or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6. In certain embodiments, the ActRII ligand trap is identical to the amino acid sequence of SEQ ID NO:6.

[0118] In one embodiment, the ActRII ligand trap is, comprises, or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the ActRII ligand trap is identical to the amino acid sequence of SEQ ID NO:8.

[0119] In certain embodiments, the methods described herein use isolated or purified polypeptides derived from ActRIIB, / .< ., ActRIIB polypeptides that are isolated from, or otherwise substantially free of, other proteins can be used with the methods and compositions described herein. ActRIIB polypeptides will generally be produced by expression from recombinant nucleic acids. In certain aspects, the ActRIIB polypeptides used in the methods described herein are NAI-5004198159vl 07Attorney Ref. No. 14247-847-228 encoded by isolated and / or recombinant nucleic acids, including fragments, functional variants and fusion proteins disclosed herein. The subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, e.g., in methods for making ActRII ligand traps or as direct therapeutic agents (e.g., in a gene therapy approach). In certain embodiments, ActRIIB polypeptides may further be “processed” and comprise post-translational modifications in addition to any that are naturally present in the ActRIIB polypeptides. Such modifications may include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.

[0120] In one embodiment, ActRII ligand traps that may be used according to any method disclosed herein, e.g., in Section 7.2, may be produced according to methods known in the art, such as those disclosed, e.g., in WO 2016 / 182280 Al or WO 2016 / 09077 Al. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, such as allelic variants. In certain embodiments, the ActRII ligand traps that may be used according to any method disclosed herein, e.g., in Section 7.2, may be produced from isolated or recombinant nucleic acid sequences in the art.

[0121] In further embodiments, the nucleic acid sequences can be isolated, recombinant, and / or fused with a heterologous nucleotide sequence, or in a DNA library.7.4 Pharmaceutical Compositions

[0122] In certain embodiments, an ActRII ligand trap, as disclosed herein is administered to a subject as part of a pharmaceutical composition comprising the ActRII ligand trap, such as an ActRIIB ligand trap, and one or more pharmaceutically acceptable excipients. In certain embodiments, the ActRII ligand trap is present in the pharmaceutical composition in a therapeutically effective amount. A pharmaceutical composition comprising the ActRII ligand trap, such as an ActRII ligand trap, and one or more pharmaceutically acceptable excipients can be formulated to be compatible with the intended route of administration as described herein. In one embodiment, the pharmaceutical composition comprises an ActRII ligand trap of SEQ ID NO:4, 6, or 8 and one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition comprises a mixture of two or more ActRII ligand trap of SEQ ID NO:4, 6, or 8 and one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition comprises a mixture comprising each of ActRII ligand traps of SEQ ID NO:4, 6, or 8 and one or more pharmaceutically acceptable excipients.

[0123] Examples of suitable pharmaceutically acceptable excipients include, but are not limited to, antioxidants (e.g., ascorbic acid), preservatives (e.g., benzyl alcohol, methyl parabens, p-NAI-5004198159vl 28Attorney Ref. No. 14247-847-228 hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, buffers, lubricants, fillers, and / or diluents. Typical buffers that can be used include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. Buffer components can also include water-soluble reagents, such as phosphoric acid, tartaric acids, succinic acid, citric acid, acetic acid, and salts thereof.

[0124] A pharmaceutical composition may contain other pharmaceutically acceptable excipients, e.g., for modifying or maintaining the pH, osmolarity, viscosity, or stability of the pharmaceutical composition. In a specific embodiment, the pharmaceutical composition comprises an aqueous buffer. In certain embodiments, the pharmaceutical composition may comprise a physiological saline solution. In a specific embodiment, the saline solution is, e.g., a sodium chloride solution.

[0125] Pharmaceutical compositions provided herein may contain still other pharmaceutically acceptable formulation agents, such as those described, e.g., in Remington’s Pharmaceutical Sciences, 23rdEd. (2020, Elsevier, Amsterdam, The Netherlands).

[0126] In one embodiment, the ActRII ligand trap is formulated for subcutaneous administration. In another embodiment, the ActRII ligand trap is packaged in a container as a sterile, preservative-free lyophilized powder or cake. In certain embodiments, the container comprises about 25 mg of the ActRII ligand trap. In certain embodiments, the container comprising about 25 mg of the ActRII ligand trap comprises a total of about 37.5 mg of protein. In certain embodiments, the ActRII ligand trap in the container is reconstituted with a volume of water for injection, such that the final concentration of the reconstituted ActRII ligand trap in the water for injection is about 50 mg / mL with a pH of approximately 6.5. In certain embodiments, the ActRII ligand trap in the container comprising about 25 mg of the ActRII ligand trap is or is intended to be reconstituted with about 0.68 mL of water for injection prior to administration to a subject. In certain embodiments, the reconstituted ActRII ligand trap is intended to be administered to a subject within about 10 hours of reconstitution.

[0127] In certain embodiments, the container comprises about 75 mg of the ActRII ligand trap. In certain embodiments, the container comprising about 75 mg of the ActRII ligand trap comprises a total of about 87.5 mg of protein. In certain embodiments, the ActRII ligand trap in the container is reconstituted with a volume of water for injection, such that the final concentration of the reconstituted ActRII ligand trap in the water for injection is about 50 mg / mL with a pH of approximately 6.5. In certain embodiments, the ActRII ligand trap in the container comprising about 75 mg of the ActRII ligand trap is or is intended to be reconstituted with about 1.6 mL ofNAI-5004198159vl 29Attorney Ref. No. 14247-847-228 water for injection prior to administration to a subject. In certain embodiments, the reconstituted ActRII ligand trap is intended to be administered to a subject within 10 hours of reconstitution.

[0128] In certain embodiments, the container comprises the ActRII ligand trap at a concentration of about 50 mg / mL in a 10 mM citrate buffer-based solution, wherein the 10 mM citrate-buffer based solution comprises 10 mM citrate, pH 6.5, 9% sucrose, and 0.02% polysorbate 80. In certain embodiments, the container is intended for storage at between 2 °C and 8 °C, e.g., for at least about 18 months. In certain embodiments, the container is a glass vial (e.g., having a volume of 3 mL) with a butyl-coated stopper. In certain embodiments, the stopper is a rubber stopper. In certain embodiments, the rubber stopper is secured in place by a crimped flip cap (e.g., a crimpled aluminum flip cap). In certain embodiments, the glass vial comprises about 25 mg of the ActRII ligand trap, and the plastic button is red. In certain embodiments, the glass vial comprises about 75 mg of the ActRII ligand trap, and the plastic button is white. In specific embodiments, the ActRII ligand trap is a fusion protein comprising the amino acid sequence of SEQ ID NO:4. In specific embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence of SEQ ID NO:4. In specific embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the glass vial comprises about 75 mg of the ActRII ligand trap, and the plastic button is white. In specific embodiments, the ActRII ligand trap is a fusion protein comprising the amino acid sequence of SEQ ID NO:6. In specific embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence of SEQ ID NO:6. In specific embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:6.

[0129] In a specific embodiment, the ActRII ligand trap is packaged in a container as a sterile, preservative-free lyophilized powder or cake. In a specific embodiment, the container comprises about 56 mg of ActRII ligand trap, about 0.19 mg of citric acid monohydrate, about 3.03 mg of trisodium citrate dehydrate, about 0.24 mg of polysorbate 80, and about 100.80 mg of sucrose. In specific embodiments, the ActRII ligand trap is a fusion protein comprising the amino acid sequence of SEQ ID NO:4. In specific embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:4. In specific embodiments, the ActRII ligand trap is a fusion protein comprising the amino acid sequence of SEQ ID NO:6. In certain embodiments, the ActRII ligand trap is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:6. In certain embodiments, the ActRII ligand trap comprises a mixture of fusion proteins, each independently comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:4, 6, or 8. In specific embodiments, the ActRII ligand trap comprises aNAI-5004198159vl 30Attorney Ref. No. 14247-847-228 mixture of fusion proteins of SEQ ID NO:4, 6, and 8. In specific embodiments, the ActRII ligand trap comprises luspatercept.

[0130] In certain embodiments, the ActRII ligand trap is substantially pure in a pharmaceutical composition. Specifically, at most 20%, 10%, 5%, 2.5%, 1%, 0.1%, or at most 0.05% of the compounds in the pharmaceutical composition are compounds other than the ActRII ligand trap and the pharmaceutically acceptable excipient.8. EXAMPLESExample 1: Cell and Cardiac Tissue Lysates

[0131] AC 16 human cardiomyocytes (SCC109, Millipore Sigma Aldrich) were cultured inDMEM / F-12 media per manufacturer’s recommendations. Recombinant human GDF11 protein (Catalog # 1958-GD-010 / CF; R&D Systems) and luspatercept / ACE-536 (Catalog # HY-P99720; MedChemExpress) at concentrations of 100 ng / mL and 10 pg / mL, respectively, were used for both autophagy detection and Simple Western analysis. For Simple Western analysis, AC16 cells were plated in 10 cm polystyrene tissue culture dishes and treated with GDF11 alone or with a premixed cocktail of GDF11 and luspatercept. After 48 and 72 hours of treatment, cells were harvested by scraping and were washed with Dulbecco's phosphate-buffered saline (DPBS, Catalog # 14190235; Thermo Fisher Scientific) by centrifuging at 1500 rpm for 10 minutes at 40°C. Cell pellets were resuspended in Radio-Immunoprecipitation Assay (RIP A) lysis and extraction buffer (Catalog # PI89900; Themo Fisher Scientific) with a protease and phosphatase inhibitor cocktail (Catalog # 78441; Thermo Fisher Scientific), and kept on ice for 40 minutes with intermittent vortexing to completely lyse the cells, followed by centrifugation at 12000 rpm for 15 minutes at 40°C. Supernatants were collected and used for Simple Western analysis.

[0132] For tissue lysate preparation, snap-frozen cardiac tissues from the TAC study were manually crushed in RIPA lysis and extraction buffer with the protease and phosphatase inhibitor cocktail using disposable pellet pestles (Catalog # 12-141-368; Themo Fisher Scientific). Tissue lysates were kept on a shaker at 40°C for 2 hours followed by centrifugation at 12000 rpm for 15 minutes at 40°C. Supernatants were collected and subjected to Simple Western analysis.

[0133] Autophagy Detection: CYTO-ID® autophagy detection kit (Catalog # ENZ-51031- K200; Enzo Life Sciences) was used to determine cellular autophagy. The dye in the kit fluoresces green upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). Briefly, 5000 AC 16 cells were seeded in flat-bottom 96 well plates. After 18 hours, cells were treated with either GDF11 alone or a premixed cocktail of GDF11 and luspatercept. After 48 hours (about 2 days) of treatment, cells were treated with a IX cocktail ofNAI-5004198159vl 31Attorney Ref. No. 14247-847-228CYTO-ID® Green Detection Reagent for 30 minutes in a CO2 incubator per manufacturer’s protocol. Cells were washed with complete DMEMZF-12 media prior to imaging in INCUCYTE® 52089. Data was analyzed using INCUCYTE® software to calculate mean and total green fluorescence intensity and area.

[0134] Simple Western Analysis: Lysates were probed with Lampl (CST; 46843S; 1 :40), Beclin-1 (Novus Biologicals; NB500249; 1 :40), pS6 (CST; 4858S; 1 :40), Lampl (CST; 9091S; 1 :40), pAkt (CST; 4060S; 1 :40), Akt (CST; 9272S; 1 :40), LC3 (CST; 12741 S; 1 :40), p70 S6 kinase, (CST; 9202S; 1 :40), S6 (CST; 2317S; 1 :40), pmTOR (CST; 2971 S; 1 :40), mTOR (CST; 2972S; 1 :40), p62 / SQSTMl (Novus Biologicals; NBP1-49954; 1 :40), GAPDH (CST; 2118S; 1 :500) and IL6 (Novus Biologicals; NBP216957; 1 :40) antibody. For analysis, either anti-rabbit detection kit (ProteinSimple DM-001) or Anti -Mouse Detection Kit (ProteinSimple DM-002) was selected and run in a 12-230-kDa Separation Module (ProteinSimple SM-W004) using manufacturer’s protocol. In brief, lysates were diluted to 1 pg / pL in Wes™ sample buffer (ProteinSimple 042-195) and mixed with Fluorescent Master Mix (1 :4, ProteinSimple PS-FL01-8). Samples were denatured at 95°C for 5 minutes prior to loading into the WES™ capillary plate. Subsequently, the antibody diluent (ProteinSimple 042-203), the primary and secondary antibodies, and the chemiluminescent substrate were added into the WES™ capillary plate. The protocol used was:(l) separation at 395 V for 30 minutes, (2) blocking for 5 minutes, (3) incubation with primary antibodies for 30 minutes, (4) incubation with the appropriate secondary antibody for 30 minutes and (5) chemiluminescence detection (luminol / peroxide) for 15 minutes. The obtained results were analyzed with software from Compass (San Jose, CA, USA).Example 2: Mice Experiments

[0135] C57 / B16 mice (18 weeks old) with intact TET2 gene (WT), TET2+ / - (Het), and TET2- / -(Homo) were procured from Jackson Laboratories, Bar Harbor, ME, USA and were fed a healthy diet ad libitum. After trans-aortic constriction (TAC) surgery (described in further detail below), vehicle or RAP-536 (10 mg / kg) were subcutaneously administered to each mouse twice a week (biw) for 7 weeks. Mice weight and survival was monitored throughout the study. Endpoints included cardiac function and dimensions, pulmonary edema, pleural effusion, survival rate, and plasma BNP concentration, as well as organ weights and fibrosis. Echocardiography (described further below) was performed to assess cardiac hypertrophy and function. At the end of treatment (56 days post TAC surgery), mice were anesthetized with 2.5% isoflurane and bled via the carotid artery. Plasma was collected and serum was stored for pharmacodynamic biomarker analysis andNAI-5004198159vl 32Attorney Ref. No. 14247-847-228 stored for analyzed for brain natriuretic peptide (BNP) and cytokine levels, drug exposure, and Pro- C6 and ARGS levels.

[0136] The chest cavity was then exposed and was carefully examined to determine the presence of pleural effusion, which was defined by a volume of fluid accumulation greater than 30 pL. The hearts and lungs were harvested, grossly examined, and weighed immediately. The hearts were sectioned. A portion was set aside for APEX RNA analysis. A portion was fixed in 10% neutral buffered formalin for 24 hours, then transferred into 70% ethanol to be processed. Collagen content was determined by picrosirius red staining, and the remaining portion was stored at -80°C for Simple Western analysis. The right tibia was dissected, and its length (TL) was measured to normalize the heart weight (HW) and lung weight (LW). The LW was measured again after air drying for approximately 60 hours. Blood samples taken at the end of the study were analyzed via LUMINEX® panel (see, e.g., Chirinos et al., 2020 Mar 24, J Am Coll Cardiol., 75(11): 1281-1295, the contents of which are incorporated herein by reference in their entirety), which assessed forty- nine (49) biomarkers associated with fibrosis, myocardial injury, atrial stretch, myocardial hypertrophy, inflammation and oxidative stress, renal markers, and endothelial dysfunction. Serum BNP levels were also assessed, and western blot analyses of heart tissues were also performed.

[0137] Transverse Aortic Constriction (TAC) surgery: Mice were anesthetized with isoflurane mixed with 0.5% L / min O2, endotracheally intubated with a 20-gauge catheter, and ventilated at a 300-pL stroke volume and 140 strokes per minute using a dual mode veterinary ventilator (Microvent 1, Hallowell EMC, Pittsfield, MA). An approximately 1.0-1.5 cm incision was made from the suprasternal notch to the second rib, and the sternum retracted. The thymus was gently separated to expose the aortic arch. The aortic arch was then ligated by tying a 7-0 silk suture ligature against a 27-gauge steel banding adapter (0.40 mm outer diameter) between the right innominate and left common carotid arteries. The banding adapter was then quickly removed to yield a constriction around the estimated 70% of aortic flow reduction. After aortic constriction, the rib cage was closed with a 6-0 non-absorbable silk suture. The sham-operated animals underwent the same surgical procedures without TAC. The animals were allowed to recover in a warm box post-surgery and were returned to their home cages after they were fully awake. Their body temperature was maintained at 37 °C with a heating pad throughout the surgical procedure.

[0138] Echocardiography: A week before (baseline) and at 4- and 8-weeks after TAC surgery, mice were anesthetized with isoflurane (2-3% for induction and 1% for maintenance).Echocardiographic assessments of left ventricular function and dimensions were conducted using the VISUALSONICS® VEVO® 3100 echocardiographic system equipped with a 30-MHz transducer (FUJIFILM® VISUALSONICS® Inc., Toronto, Canada). The flow velocities of the left NAI-5004198159vl ,Attorney Ref. No. 14247-847-228 common carotid (LC) and right common carotid (RC) arteries were acquired with pulsed wave doppler at 4 weeks post-TAC, and the LC and RC peak flow velocity was measured to calculate the percentage constriction of TAC. Animals with an RC / LC ratio < 2.0 were considered to have failed TAC. A 2-dimensional (2-D) parasternal short-axis view of the left ventricle (LV) was obtained at the level of the papillary muscle. The cardiac function and dimensions, including ejection fraction (EF), LV anterior and posterior wall thickness (LVAW and LVPW, respectively), LV mass, and LV volume, were all derived from the M-mode image using the auto LV measurement tool of the VEVOLAB® software.

[0139] Pulsed Wave (PW) Doppler Mode waveform of mitral valve flow was obtained from the apical four chamber view to determine mitral isovolumic relaxation time (IVRT) and peak early and atrial filling (E and A) waves. A tissue doppler mode image to assess mitral valve annulus from the apical four chamber view was taken to measure E and A waves, and the ratio (E / A) was calculated as an indication of myocardium health.

[0140] Using the above-described murine model of pressure overload TAC-induced heart failure (which has been shown to have elevated mTORCl and inflammatory signaling), the murine analogue of luspatercept (RAP-536) was shown to significantly reduces cardiac hypertrophy both in WT and TET2-KO mice (21% and 16% left ventricular mass reduction by echocardiography, p = 0.001 and p = 0.01 respectively). Moreover, we also observed reduction of serum BNP levels (59% reduction in WT and 69% in TET2 -KO, p < 0.0001 and p = 0.0002 respectively), as shown in FIG.1Example 3: Luspatercept Modulates mTORCl and Inflammatory Signaling Pathways in Human Subjects with Certain Blood Cell Mutations

[0141] Subjects having a diagnosis of MDS requiring red blood cell transfusions (2-6 packed red blood cell units per 8 weeks for >8 weeks prior to treatment) were administered luspatercept subcutaneously once every 3 weeks starting at 1.0 mg / kg body weight with titration up to 1.75 mg / kg.

[0142] Transcriptomics (bulk RNA-SEQ), proteomics, and phospho-proteomics (bone marrow) were assessed at baseline and at week 24 from blood samples collected from subjects. Genomic DNA was isolated from bone marrow mononuclear cells (BMMCs), and myeloid specific 36 somatic genes mutations were identified by targeted next-generation sequencing (400X; sensitivity 3%) (MLL laboratory information system) at baseline. Hematologic parameters (e.g., hemoglobin (Elb), complete blood counts) and cytokine / chemokine levels (peripheral blood) were also assessed at baseline and at week 24.NAI-5004198159vl 34Attorney Ref. No. 14247-847-228

[0143] Results: Eighty-five percent (123 / 145) of luspatercept-treated subjects had at least one mutation in DNMT3A, TET2, ASXL1, and / or SF3B1, and a modest myeloid biased expansion at baseline (R = 0.3, p = <0.0002). Luspatercept treatment significantly reduced anemia from baseline in these patients with CHIP-related mutations. Additionally, significant downregulation of NT- proBNP levels (A median= 590 pg / mL in responders, p = 0.02) was observed in subjects who received luspatercept. In responding subjects, luspatercept treatment reduced mTORCl pathway activation and inflammatory signaling at transcriptomic level as shown in FIG. 2. Additionally, luspatercept treatment reduced mTORCl pathway activation in a proteomic analysis (FIG. 3A) and phosphoproteomic analysis (FIG. 3B). Consistent with this, inhibition of GDF11 -mediated mTORCl activity and activation of autophagy / lysosome markers was also observed following luspatercept treatment (indicating possible sequestration of GDF11 by luspatercept) as shown in FIG. 4. Furthermore, upregulation of an anti-inflammatory regulator (growth / differentiation factor 15) following treatment with luspatercept was observed.Example 4: NT-proBNP Levels and STING Signaling Predicts Survival in Lower-Risk Myelodysplastic Syndromes

[0144] Patients with MDS exhibit increased activation of inflammatory and interferon signaling pathways (see, e.g., Villaume et al. 2025 Feb 1, Haematologica, 110(2):283-299, the contents of which are incorporated herein by reference in their entirety), which confers significant risk for cardiovascular disease (CVD). MDS subjects also have been shown to have elevated NT-proBNP levels (a biomarker for cardiovascular morbidity and mortality). Patients who survived five years after their diagnosis had a cumulative risk of death due to CVD comparable to that from MDS or leukemia (see, e.g., Brunner et al., 2017 Oct 18, Blood Adv., l(23):2032-2040, the contents of which are incorporated herein by reference in their entirety). Despite this clinical burden, no therapies specifically target CVD complications in MDS patients. Clinical data is used herein to identify predictive biomarkers of survival in LR-MDS patients. Preclinical models reveal the effect of luspatercept on cardiovascular dysfunction following pressure overload induced heart failure.

[0145] Subjects included those with the following attributes:* a documented diagnosis of MDS according to WHO 2016 classification that meets revised international prognostic scoring system (IPSS-R) classification of very low, low, or intermediate risk disease, and had < 5% blasts in bone marrow;* an endogenous serum erythropoietin (sEPO) level of < 500 U / L;* required red blood cell (RBC) transfusions, as documented by an average transfusion requirement of 2 - 6 units / 8 weeks of packed red blood cells (pRBCs); andNAI-5004198159vl 35Attorney Ref. No. 14247-847-228• an Eastern Cooperative Oncology Group (ECOG) score of 0, 1, or 2.

[0146] Subjects were excluded if they had a known history of acute myeloid leukemia, clinically significant anemia due to iron, vitamin B 12, or folate deficiencies, or autoimmune or hereditary hemolytic anemia, or hypothyroidism, or any type of known clinically significant bleeding or sequestration or drug induced anemia, or uncontrolled hypertension.

[0147] A first arm of subjects was administered a starting dose of 1.0 mg / kg luspatercept by subcutaneous injection every 3 weeks (21 days; Q3W). Dose levels were increased in a stepwise manner beyond the starting dose to 1.33 mg / kg, and up to a maximum of 1.75 mg / kg.

[0148] A second arm of subjects was administered a starting dose of 450 lU / kg (maximum total starting dose is 40,000 IU) of epoetin alfa by subcutaneous injection once every week (7 days; QW). Dose levels were increased in a stepwise manner beyond the starting dose to 787.5 lU / kg, and up to a maximum of 1,050 lU / kg (with a maximum total dose of 80,000 IU).

[0149] NT -proBNP levels and survival probability was analyzed in all subjects, revealing that baseline elevated NT-proBNP levels were prognostic for survival (>2000 pg / mL, n=354), as shown in FIG. 5A. The incidence of cardiac disorders was also analyzed and stratified by the most frequent MDS-related somatic mutations (SF3B1, TET2, ASXL1, and DNMT3A), which together accounted for 87% of evaluable subjects (n=306 / 350). Surprisingly, adverse events (AEs) reported as cardiac disorders (-20%) were enriched in subjects with somatic mutations in TET2, SF3B1, DNMT3A, and ASXL1 (-89% of total cardiac AEs). Most importantly, out of all deaths reported, 18% were in subjects with cardiac disorders, and those subjects were particularly enriched with TET2 mutations (61%).

[0150] The effect of treatment (luspatercept or epoetin alfa) on NT-proBNP levels in the subjects was also analyzed. Compared to epoetin alfa, luspatercept significantly lowered NT- proBNP levels (A median = 619 pg / mL in responding subjects, P = 0.002), as shown in FIG. 5B, including a 61% median reduction from baseline in ring-sideroblasts (RS)-negative LR-MDS patients (n=9, p = 0.03), a subgroup previously shown to be associated with poor prognosis. In subjects with or without heart failure, chronic inflammation plays a crucial role in elevating NT- proBNP levels (see, e.g., Fish-Trotter et al., 2020 Jul, Circ Heart Fail., 13(7):e006570, the contents of which are incorporated herein by reference in their entirety). To identify potential patho- mechanistic pathways involved in LR-MDS, analysis of bone marrow mononuclear cells at baseline and week 24 were performed in samples collected from each subject. It was observed that luspatercept treatment was associated with downregulation of inflammatory pathways (e.g.,NAI-5004198159vl 36Attorney Ref. No. 14247-847-228 interferon alpha response, IL6 / JAK / STAT3 signaling). The same effect was not seen after epoetin alfa treatment, as shown in FIG. 5C.

[0151] The upstream inflammatory response pathways were systematically analyzed in those subjects treated with luspatercept, which revealed that luspatercept treatment reduced stimulator of interferon genes (STING) dependent inflammatory pathways activation in responding subjects, as shown in FIG. 5D. Innate immune cyclic GMP-AMP synthase (cGAS) and STING pathways that recognizes cytosolic and mitochondrial DNA, leading to interferon stimulated gene (ISG) transcription, have recently been shown to be activated in TET2 and DNMT3 A mutant / deficient human cells (see, e.g., Villaume et al.). The cGAS / STING pathway also plays an essential pathogenic role in cardiac inflammation and hypertrophy in mice models with pressure overload- induced heart failure (see, e.g., Hu et al., 2020 Jun 1, Am J Physiol Heart Circ Physiol., 318(6):H1525-H1537, the contents of which are incorporated herein by reference in their entirety). Using cox regression model, a STING-dependent gene signature comprising seven genes that predicted survival in lower-risk MDS patients irrespective of treatment arm was identified. Next, a multivariable analysis was performed to investigate whether STING gene clusters and NT -proBNP levels together with treatment (Tx) arm were associated with survival outcomes. Subjects with elevated expression of STING gene clusters and elevated NT-proBNP levels (<2000 pg / mL versus >2000 pg / mL) at baseline carried a significantly higher risk (HR 2.8, p = 0.004 and HR 2.36, p = 0.01 respectively). Luspatercept treatment significantly reduced the risk compared to epoetin alfa (p = 0.02, HR: 0.47), as shown in FIG. 5E. This may be due to reduction of NT- proBNP levels and inhibition of cGAS / STING pathway activation.

[0152] To evaluate these clinical findings, the effect of murine version of luspatercept (RAP- 536) both in wild type (WT) and TET2 knockout (TET2-KO) C57BL / 6 mice in a transverse aortic constriction (TAC)-induced pressure overload murine model that was previously reported to have elevated cGAS / STING signaling (by Hu et al.) was investigated. RAP-536 significantly reduced serum BNP levels (WT: 59%, p < 0.0001; TET2-KO: 69%, p = 0.0002) and downregulated STING-dependent interferon-stimulated genes in cardiac tissue, as shown in FIG. 5F and 5G.

[0153] In summary, this study links STING signaling to the survival LR-MDS patients and, together with elevated NT-proBNP levels, can predict survival in lower-risk MDS patients. Luspatercept shows superior effect in MDS patients with both elevated NT-proBNP levels and inflammatory signaling. Most importantly, luspatercept may exert mutation-agnostic, direct cardioprotective effects via modulation of STING dependent signaling.NAI-5004198159vl 37Attorney Ref. No. 14247-847-2289. DESCRIPTION OF THE SEQUENCESTable 1. Sequence Information.NAI-5004198159vl 38Attorney Ref. No. 14247-847-228

[0154] Throughout this application, various publications, patents, patent applications and other documents have been referenced. The disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference in this application for all purposes, including in order to more fully describe the state of the art to which this the subject matter disclosed herein pertains. Although the disclosed subject matter has been described with reference to the examples provided above, it should be understood that various modifications could be made without departing from the spirit of the disclosed subject matter. Many variations will become apparent to those skilled in the art upon review of this specification.NAI-5004198159vl O Q

Claims

Attorney Ref. No. 14247-847-228CLAIMSWhat is claimed is:

1. A method of treating a disease, disorder, or condition associated with an increase in mTORCl activity in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject, wherein the disease, disorder, or condition associated with an increase in mTORCl activity is non-hematological cancer, a hamartoma syndrome, a metabolic disorder, a neurodegenerative disease, a fibrotic disease, or an inflammatory or autoimmune disorder.

2. A method of treating cardiac hypertrophy, heart failure, atherosclerosis, ischemic heart disease, breast cancer, prostate cancer, cervical cancer, esophageal cancer, renal cell carcinoma, lung cancer, hepatocellular cancer, glioblastoma, Still’s Disease, psoriasis, lupus, rheumatoid arthritis, PTEN-related hamartoma syndrome, tuberous sclerosis complex, Peutz- Jeghers syndrome, Huntington’s Disease, Alzheimer’s Disease, Parkinson’s Disease, amyotrophic lateral sclerosis, fibrosis (e.g., of heart, liver, kidney, or lung), inflammatory bowel disease, scleroderma, adenomatous polyposis, lymphagioleiomyomatosis, multiple sclerosis, inflammatory bowel disease, multiple sclerosis, metabolic syndrome, cachexia, non-alcoholic fatty liver disease, obesity, or Type 2 diabetes in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject.

3. A method of treating a disease, condition, or disorder associated with an increase in inflammation in a tissue of a subject in need thereof, the method comprising administering an effective amount of an ActRII ligand trap to the subject to reduce inflammation in the tissue, wherein the tissue is a tissue other than bone tissue.

4. The method of claim 3, wherein the tissue is adipose tissue, brain tissue, cardiac tissue, connective tissue, epithelial tissue, gastrointestinal tissue, tissue of the joint, kidney (renal) tissue, liver tissue, lung tissue, muscle tissue, nervous system tissue, skin tissue (epidermal and dermal tissue), or spleen tissue.

5. The method of claim 3 or claim 4, wherein the disease, condition, or disorder is Crohn's Disease, colitis, steatohepatitis, systemic lupus erythematosus, Sjogren’s syndrome, systemic sclerosis, rheumatoid arthritis, vasculitis, VEXAS syndrome, inflammatory lung disease (ILD), chronic obstructive pulmonary disease (COPD), chronic immune-mediated diseases, chronic inflammatory conditions due to mitochondrial dysfunction, or sepsis.NAI-5004198159vl 40Attorney Ref. No. 14247-847-2286. A method of inducing autophagy in a subject in need thereof, the method comprising administering an ActRII ligand trap to the subject.

7. The method of claim 3, wherein subject has a disease, condition, or disorder selected from the group consisting of heart failure, non-alcoholic fatty liver disease, Type 2 diabetes, obesity, chronic kidney disease (CKD), acute Kidney disease (AKD), inflammatory bowel disease (IBD), Alzheimer’s disease, Parkinson’s Disease, Huntington’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, lysosomal storage disorders (LSDs), muscular disorders and fatigue, lung cancer, breast cancer, prostate cancer, colorectal cancer, cervical cancer, esophageal cancer, renal cell carcinoma, and hepatocellular cancer.

8. A method of treating anemia, myelodysplastic syndrome (MDS), non-proliferative chronic myelomonocytic leukemia (CMML), or beta-thalassemia in a subject in need thereof, the method comprising administering an effective amount of an ActRII ligand trap to the subject, wherein the subject has one or more mutations associated with Clonal Hematopoiesis of Indeterminate Potential (CHIP).

9. The method of claim 8, wherein the subject further has a cardiac inflammation, cardiac hypertrophy, or a cardiac disorder.

10. The method of claim 8 or claim 9, wherein method is for treating MDS.

11. The method of any one of the preceding claims, wherein the subject is at least 60 years old, at least 65 years old, or at least 75 years old.

12. The method of any one of the preceding claims, wherein the one or more gene mutations associated with CHIP are selected from the group consisting of DNMT3A, TET2, ASXL1, JAK2, and SF3B1.

13. The method of any one of the preceding claims, wherein the one or more gene mutations associated with CHIP are selected from the group consisting of DNMT3A, TET2, ASXL1, and SF3B1.

14. The method of any one of the preceding claims, wherein the one or more gene mutations associated with CHIP comprise ASXL1.NAI-5004198159vl 41Attorney Ref. No. 14247-847-22815. The method of any one of the preceding claims, wherein the subject has two or more mutations associated with Clonal Hematopoiesis of Indeterminate Potential (CHIP).

16. The method of any one of the preceding claims, wherein the subject has elevated mTORCl levels.

17. The method of any one of the preceding claims, wherein the subject has elevated N- terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels as compared to normal NT- proBNP blood levels.

18. A method of treating MDS in a subject with elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels as compared to normal NT-proBNP blood levels, the method comprising administering an effective amount of an ActRII ligand trap to the subject.

19. The method of claim 18, wherein the subject further has elevated expression levels of STING gene clusters.

20. A method of reducing N-terminal pro-B-type natriuretic peptide (NT-proBNP) blood levels in a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject.

21. A method of reducing inflammatory pathway activity in a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject, wherein the inflammatory pathway is the I16 / JAK / Stat3 pathway, a STING-dependent inflammatory pathway, or an interferon alpha-related pathway.

22. The method of claim 21, wherein the inflammatory pathway is a STING-dependent inflammatory pathway.

23. The method of any one of the preceding claims, wherein the subject has one or more TET2 and / or DNMT3 A gene mutations.

24. The method of claim 23, wherein the subject has a TET2 gene mutation.

25. A method of protecting the heart of a subject, the method comprising administering an effective amount of an ActRII ligand trap to the subject.

26. The method of claim 25, wherein the damage is caused by or related to MDS.NAI-5004198159vl 42Attorney Ref. No. 14247-847-22827. The method of claim 25, wherein the administering prevents inflammation and / or hypertrophy in cardiac tissue of the subject.

28. The method of any one of the preceding claims, wherein the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is: i) 90% identical to SEQ ID NO:4; ii) 95% identical to SEQ ID NO:4; iii) 98% identical to SEQ ID NO:4; or iv) identical to SEQ ID NO:4.

29. The method of any one of the preceding claims, wherein the ActRII ligand trap is a fusion protein comprising an amino acid sequence that is: i) 90% identical to SEQ ID NO: 6; ii) 95% identical to SEQ ID NO:6; iii) 98% identical to SEQ ID NO: 6; or iv) identical to SEQ ID NO: 6.

30. The method of claim 28 or 29, wherein the fusion protein comprises an L79D amino acid mutation.

31. The method of any one of the preceding claims, wherein the ActRII ligand trap is a fusion protein comprising: i) a fragment of the extracellular domain of ActRIIB, wherein the fragment consists of the amino acid sequence of SEQ ID NO: 10; ii) a linker; and iii) an Fc of an IgG.

32. The method of any one of the preceding claims, wherein the ActRII ligand trap is a fusion protein of SEQ ID NO:4.

33. The method of any one of the preceding claims, wherein the ActRII ligand trap is a fusion protein of SEQ ID NO:6.

34. The method of any one of the preceding claims, wherein the subject is administered a pharmaceutical composition comprising a mixture of two or more of the ActRII ligand traps of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.NAI-5004198159vl 43Attorney Ref. No. 14247-847-22835. The method of any one of the preceding claims, wherein the subject is administered a pharmaceutical composition comprising a mixture comprising each the ActRII ligand traps of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

36. The method of any one of the preceding claims, wherein the ActRIIB ligand trap is administered to the subject subcutaneously once every 21 days at a dose of about 0.5 mg / kg to about 1.75 mg / kg.

37. The method of any one of the preceding claims, wherein the ActRII ligand trap is the only therapeutic agent administered to the subject.

38. The method of any one of the preceding claims, wherein the cardiac hypertrophy or heart failure is not due to vascular calcification.NAI-5004198159vl 44

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