Methods and compositions for inhibiting clonal hematopoiesis

Abstract

Clonal hematopoiesis is an age-related condition caused by somatic mutations that confer a clonal selection advantage to hematopoietic stem cells. While clonal hematopoiesis is a benign condition, affected individuals have an increased risk of developing hematologic cancers (e.g., acute myeloid leukemia (AML)). In some embodiments, the present disclosure provides methods for inhibiting clonal hematopoiesis using a senolytic agent to target bone marrow stromal cell senescence. Some embodiments provide methods for inhibiting clonal hematopoiesis in a subject in need thereof, comprising administering a senolytic agent to the subject in an amount effective to inhibit bone marrow stromal cell senescence in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control.

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 402,828, filed August 31, 2022, which is incorporated by reference herein in its entirety.

[0002] (Reference to the Electronic Sequence Listing) The contents of the electronic sequence listing (J022770120WO00-SEQ-VLJ.xml; size: 4,597 bytes; and creation date: August 10, 2023) are incorporated herein by reference in their entirety.

[0003] (Government License Rights) This invention was made with government support under Grants DK118072, AG069010, and AG077925 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0004] (background) Clonal hematopoiesis is an age-related condition caused by somatic mutations that confer a clonal selective advantage to hematopoietic stem cells. Although clonal hematopoiesis is a benign condition, affected individuals have an increased risk of developing blood cancers (e.g., acute myeloid leukemia (AML)). The gene most frequently mutated in human clonal hematopoiesis and AML is DNA methyltransferase 3A (DNMT3A). The exact mechanism by which mutant DNMT3A genes confer clonal advantage and lead to the development of AML is unknown. Summary of the Invention [Means for solving the problem]

[0005] (overview) In some embodiments, the present disclosure provides a method for inhibiting clonal hematopoiesis using senolytic agents to target senescence in bone marrow stromal cells. The studies described herein demonstrate that a subset of bone marrow stromal cells acquires an enriched gene signature of cellular senescence and exhibits increased expression of senescence biomarkers in the context of Dnmt3a mutant hematopoiesis. Without being bound by theory, the data provided herein suggest that Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs) induce senescence in the bone marrow microenvironment, favoring clonal expansion and transformation to AML. Ex vivo coculture showed that bone marrow stromal cells cultured with Dnmt3a mutant HSPCs had increased expression of senescence markers and a senescence-associated secretory phenotype compared to bone marrow stromal cells cultured with control wild-type HSPCs. Functional studies conducted to assess the importance of bone marrow stromal cell senescence in Dnmt3a mutant transformation to AML showed that treatment with senolytic agents reduced peripheral blood myeloid cell overproduction concomitantly with Dnmt3a mutant cell transformation, suggesting that treatment with senolytic agents delays AML onset. Thus, the data provided herein demonstrate that Dnmt3a mutant hematopoietic cells can induce bone marrow stromal cell senescence and that targeting the senescent bone marrow microenvironment using senolytic agents is a viable strategy for, for example, inhibiting clonal hematopoiesis and preventing the onset of AML (e.g., transformation to AML).

[0006] Some aspects provide methods of inhibiting clonal hematopoiesis in a subject in need thereof, the method comprising administering a senolytic agent to the subject in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control.

[0007] In some embodiments, the subject exhibits one or more symptoms of acute myeloid leukemia.

[0008] In some embodiments, the subject has one or more known risk factors associated with acute myeloid leukemia.

[0009] In some embodiments, the subject has acute myeloid leukemia.

[0010] In some embodiments, the subject is 50 years of age or older.

[0011] In some embodiments, an effective amount of the senolytic agent inhibits myeloproliferation of the bone marrow stromal cells.

[0012] In some embodiments, the effective amount of the senolytic agent inhibits the progression of clonal hematopoiesis to a myeloid malignancy relative to a control.

[0013] In some embodiments, the subject's hematopoietic stem and progenitor cells (HSPCs) comprise a somatic mutation that confers a clonal selection advantage to the HSPCs.

[0014] In some embodiments, the mutation is a DNA methyltransferase 3A (DNMT3A) mutation.

[0015] In some embodiments, the method further comprises assaying a sample from the subject for the presence of the somatic mutation.

[0016] In some embodiments, the method further comprises assaying a sample from the subject for increased expression of a senescence biomarker compared to a control.

[0017] In some embodiments, the senescence marker is selected from senescence-associated β-galactosidase (SA-β-Gal), Cdkn2a (P16), Cdkn1a (P21), Cdkn1b (P27), IL-6 and IL-1α.

[0018] In some embodiments, the bone marrow stromal cells comprise adipo-Cxcl12-enriched reticular (CAR) cells and osteo-CAR cells.

[0019] In some embodiments, the senolytic agent is selected from dasatinib, quercetin, fisetin, 17-DMAG, navitoclax, and catechin.

[0020] In some embodiments, the senolytic agent is administered via an intravenous or intraosseous route.

[0021] Some embodiments relate to a method of inhibiting clonal hematopoiesis in a subject in need thereof, the method comprising: assaying a biological sample obtained from the subject for DNA methyltransferase 3A (DNMT3A) mutations; and administering to the subject a senolytic agent (optionally selected from dasatinib, quercetin, fisetin, 17-DMAG, navitoclax, and catechin) in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control.

[0022] Some embodiments relate to a method of inhibiting clonal hematopoiesis in a subject in need thereof, the method comprising: assaying a biological sample obtained from the subject for a biomarker of cellular senescence; and administering to the subject a senolytic agent (optionally selected from dasatinib, quercetin, fisetin, 17-DMAG, navitoclax, and catechin) in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control.

[0023] In some embodiments, the subject is at risk of developing a myeloid malignancy. A subject may be at risk of developing a myeloid malignancy, for example, if the subject has a family history of a myeloid malignancy.

[0024] In some embodiments, the biological sample comprises hematopoietic cells (optionally bone marrow stromal cells).

[0025] In some embodiments, the assaying step comprises performing single-cell RNA sequencing (RNA-seq) on the biological sample. [Brief explanation of the drawings]

[0026] [Figure 1A] Figures 1A-1B: Cellular senescence gene signatures are enriched in bone marrow stromal cells (BMSCs) from Dnmt3a mutant (Dnmt3aR878H / +) mice. (Figure 1A) Fold change in p21 gene expression in Adipo-CAR and Osteo-CAR BMSC populations at 12 weeks after transplantation of control wild-type or Dnmt3a mutant hematopoietic cells into wild-type recipient animals. (Figure 1B) Enrichment of cellular senescence pathways in Adipo-CAR and Osteo-CAR BMSC populations at 12 weeks after transplantation of control (WT) or Dnmt3a mutant hematopoietic cells into wild-type recipient animals. [Figure 1B] Same as above.

[0027] [Figure 2A] Figure 2A-2B: BMSCs cultured with Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs) induce increased expression of senescence markers (P16, P21) (Figure 2A) and gene expression of β-galactosidase (β-gal) and SASP (IL-6, IL-1α) (Figure 2B) in BMSCs cultured for 7 days with control or Dnmt3a mutant HSPCs. [Figure 2B] Same as above.

[0028] [Figure 3A]Figures 3A-3B: Dnmt3a mutant HSPCs induce senescence in BMSCs. Senescence-associated β-galactosidase (β-gal) expression in BMSCs (Figure 3A) and endothelial cells (Figure 3B) from MxCre control or Dnmt3a mutant hematopoietic cells transplanted into wild-type recipient animals 12 weeks after transplantation. [Figure 3B] Same as above.

[0029] [Figure 4A] Figures 4A-4B: Dnmt3a mutant HSPCs induce increased expression of the anti-apoptotic proteins BCL2 and BCLxL in BMSCs. Expression of BCL2 and BCLxL proteins in BMSCs from MxCre control or Dnmt3a mutant hematopoietic cells transplanted into wild-type recipient animals 12 weeks after transplantation (Figure 4A). Number of BMSCs 12 weeks after transplantation of MxCre control (WT) or Dnmt3a mutant hematopoietic cells into wild-type recipient animals (Figure 4B). [Figure 4B] Same as above.

[0030] [Figure 5] Figure 5: Dnmt3a mutants expand the hematopoietic stem cell (HSC) pool. Frequency of donor-derived HSCs 12 weeks after transplantation of MxCre control (WT) or Dnmt3a mutant hematopoietic cells into wild-type recipient animals.

[0031] [Figure 6A]Figures 6A-6E: Navitoclax treatment attenuates Dnmt3a mutant peripheral blood myeloid cell overproduction. (Figure 6A) Dnmt3a mutant cells were transplanted into wild-type recipient mice. 12 weeks after transplantation, navitoclax or vehicle control was administered. Two weeks later, Npm1 mutation was induced in Dnmt3a mutant cells. (Figure 6B) Frequency of donor-derived mature Dnmt3a;Npm1 mutant myeloid cells in the peripheral blood of navitoclax-treated or vehicle control-treated transplanted mice. (Figure 6C) Engraftment of donor-derived mature Dnmt3a;Npm1 mutant myeloid cells in BM. (Figure 6D) β-gal expression in BMSCs, and (Figure 6E) monocyte counts in navitoclax-treated mice compared with vehicle-treated mice. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0032] (Detailed explanation) (clonal hematopoiesis) In some embodiments, provided herein are methods and compositions for inhibiting (e.g., preventing or suppressing) clonal hematopoiesis in a subject in need thereof by administering a senolytic agent(s) in an amount effective to inhibit senescence of bone marrow stromal cells (BMSCs) in the subject, thereby inhibiting clonal hematopoiesis in the subject. BMSCs reside in the bone marrow microenvironment, which is a collection of cells and structures that together support blood cell production in the bone marrow. Bone marrow has two cell fractions: a hematopoietic cell fraction and a non-hematopoietic cell fraction. The hematopoietic cell fraction expresses CD45 (CD45+). The non-hematopoietic cell fraction lacks expression of the hematopoietic and erythroid markers CD45 and Ter119 (CD45-Ter119-). This microenvironment also contains hematopoietic stem and progenitor cells (HSPCs), including hematopoietic stem cells (HSCs).

[0033] Bone marrow stromal cells (or stroma) comprise a heterogeneous population of cells that provide structural and physiological support to HSPCs. BMSCs also contain cells with stem cell-like characteristics, allowing them to differentiate into bone, cartilage, adipocytes, and hematopoietic supporting tissues. Additional cell types present in the bone marrow stroma include, but are not limited to, fibroblasts, macrophages, adipocytes, osteoblasts, osteoclasts, and endothelial cells. Bone marrow stromal cells also include adipo-Cxcl12-rich reticular (CAR) cells and osteo-CAR cells, endothelial cells (EC), arteriolar endothelial cells (EC-arteriolar), and sinusoidal endothelial cells (EC-sinusoidal). In some embodiments, the bone marrow stromal cells are adipo-Cxcl12-rich reticular (CAR) cells and / or osteo-CAR cells.

[0034] Hematopoietic stem cells are capable of self-renewal and have the ability to reconstitute all types of blood cells (including white blood cells, red blood cells, and platelets) to meet the changing blood cell demand in peripheral tissues. Hematopoietic stem and progenitor cells reside primarily in the bone marrow but can migrate to other hematopoietic and non-hematopoietic organs. When somatic mutations accumulate in HSCs and undergo positive selection (e.g., clonal selection advantage), this leads to clonal HSC expansion, called clonal hematopoiesis. As a result, all blood cells produced by clonal hematopoiesis have the same genetic mutations and a different genetic pattern from existing blood cells. Most people with clonal hematopoiesis do not show symptoms of the disease. However, people with clonal hematopoiesis have an increased risk of developing cardiovascular disease and blood cancers (e.g., myelodysplastic syndrome and acute myeloid leukemia). Although no single cause has been identified for clonal hematopoiesis, characteristics that may increase the risk of developing clonal hematopoiesis include age, smoking, male gender, and Caucasian race. Furthermore, radiation therapy and some chemotherapy regimens may be associated with clonal hematopoiesis. Aging is a major driver of clonal hematopoiesis, and while only 1% of people under 50 years of age have clonal hematopoietic somatic mutations, individuals over 65 years of age have a 10% risk, which jumps to approximately 20% in people over 90 years of age. The most frequently mutated genes include DNA methyltransferase 3A ("DNMT3A"), Tet methylcytosine dioxygenase 2 ("TET2"), Janus kinase 2 ("JAK2"), and ASXL transcription factor 1 ("ASXL1").

[0035] Therefore, "inhibiting clonal hematopoiesis" refers to the process of preventing or delaying clonal HSC proliferation. As discussed herein, certain mutations in HSPC induce senescence, favoring clonal proliferation and subsequent transformation into acute myeloid leukemia. Then, using senolytic agents to prevent this senescence prevents clonal proliferation.

[0036] In some embodiments, the subject's HSCs contain a somatic mutation that confers a clonal selection advantage to the HSCs. A clonal selection advantage occurs when somatic variants have a better fitness and are positively selected for compared to non-variants. A somatic variant is a cell that has a somatic mutation. A somatic mutation is a DNA change that occurs in the somatic cells of a multicellular organism. A somatic cell is any cell that is not a germline cell (e.g., a gamete, germ cell, or gametocyte). Because somatic mutations do not occur in germline cells, they are not passed on to offspring. Somatic mutations can occur spontaneously in response to stress or accumulate due to errors in DNA repair throughout the life cycle of an organism. In some embodiments, the somatic mutation is present in the HSPCs and confers a clonal selection advantage to the HSPCs. In some embodiments, the somatic mutation is a mutation in the DNMT3A gene. In some embodiments, the somatic mutation is a mutation in the mouse DNMT3A gene. In some embodiments, the somatic mutation is a mutation in the human DNMT3A gene. In humans, most mutations in DNMT3A are heterozygous point mutations at DNMT3A residue R882 (corresponding to R878 in mouse) within its catalytic domain, which are proposed to induce a dominant-negative loss of de novo methylation activity. In some embodiments, the somatic mutation is homologous (e.g., orthologous) to the somatic mutation at amino acid position 878 relative to wild-type mouse DNMT3A (UniProt Accession No. O88508; SEQ ID NO: 1). In some embodiments, the mutation is homologous (e.g., orthologous) to the R878H mutation in the mouse DNMT3A protein. In some embodiments, the somatic mutation is at amino acid position 882 relative to wild-type human DNMT3A (UniProt Accession No. Q9Y6K1; SEQ ID NO: 2). In some embodiments, the somatic mutation is an R882H mutation in the human DNMT3A protein.

[0037] (Wild-type mouse DNMT3A amino acid sequence)

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[0038] (Wild-type human DNMT3A amino acid sequence)

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[0039] II. Myeloid Malignancies (e.g., Hematologic Cancers) Embodiments of the present disclosure provide methods for inhibiting the progression of clonal hematopoiesis to myeloid malignancies (e.g., blood cancers). Myeloid malignancies are a group of disorders characterized by abnormal proliferation and differentiation of myeloid progenitor cells in the bone marrow and blood. The myeloid lineage in the hematopoietic system gives rise to various types of blood cells, including red blood cells, several types of white blood cells (such as neutrophils, eosinophils, and monocytes), and platelets. Acute myeloid leukemia (AML) is a rapidly progressive disease in which the bone marrow produces abnormal myeloblasts (a type of immature white blood cell), red blood cells, or platelets. It is the most common type of acute leukemia in adults. Chronic myeloid leukemia (CML) is characterized by increased, uncontrolled proliferation of myeloid cells in the bone marrow and their accumulation in the blood. It has a specific genetic marker known as the Philadelphia chromosome, which is the result of a chromosomal translocation. Myelodysplastic syndromes (MDS) are a diverse collection of conditions in which immature blood cells in the bone marrow fail to mature and become healthy blood cells. This can result in low numbers of one or more types of blood cells. MDS can progress to AML in some cases. Myeloproliferative neoplasms (MPNs) are diseases in which the bone marrow produces too many red blood cells, white blood cells, or platelets. Examples include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Chronic myelomonocytic leukemia (CMML) is a type of leukemia that begins in blood-forming cells in the bone marrow and infiltrates the blood. It affects monocytes, which can spread to other parts of the body.

[0040] Blood cancers (also called blood system cancers) begin in the bone marrow, where blood cells are produced. Normally functioning blood cells fight off infection and produce new blood cells. Blood cancers occur when abnormal blood cells grow (e.g., proliferate) uncontrollably and interfere with the function of normal blood cells. There are three major types of blood cancer: leukemia, lymphoma, and myeloma. Leukemia develops in the blood and bone marrow and occurs when the body produces too many abnormal white blood cells. When leukemia occurs, the bone marrow's ability to produce red blood cells and platelets is reduced. Non-Hodgkin's lymphoma and Hodgkin's lymphoma are blood cancers that develop in the lymphatic system from white blood cells called lymphocytes. Hodgkin's lymphoma is characterized by the presence of abnormal lymphocytes called Reed-Sternberg cells. Myeloma is a blood cancer of the blood's plasma cells, a type of white blood cell produced in the bone marrow. Symptoms of these blood cancers include fever and frequent infections, fatigue, nausea, unexplained weight loss, bone / joint pain, headache, shortness of breath, and lymphadenopathy. Blood cancers account for approximately 10% of cancer diagnoses, with more than 900,000 people diagnosed with blood cancer worldwide each year. In the United States, 68,000 people die from blood cancer each year. People with clonal hematopoiesis have an increased risk of developing blood cancers, particularly myelodysplastic syndromes (also called preleukemias) and acute myeloid leukemia. Transformation from clonal hematopoiesis to AML occurs, for example, through a process called myeloproliferation. Myeloproliferation is the uncontrolled overproduction of one or more types of bone marrow cells. In some embodiments, the methods provided herein are used to inhibit (e.g., prevent or delay) myeloproliferation that occurs during the transformation from clonal hematopoiesis to AML. In some embodiments, a senolytic agent described elsewhere herein is used in an amount effective to inhibit myeloproliferation (e.g., myeloproliferation of bone marrow stromal cells).

[0041] In some embodiments, the subject described herein has acute myeloid leukemia (AML). AML is a form of blood cancer characterized by the infiltration of bone marrow, blood, and other tissues by proliferative, clonal, and abnormally or poorly differentiated cells of the hematopoietic system. Major types of AML include, but are not limited to, AML with recurrent genetic abnormalities, AML with myelodysplasia-associated changes, therapy-related AML, and AML not otherwise specified. AML accounts for approximately 1% of all cancers and is the most common form of acute leukemia in adults. An estimated 20,050 people are diagnosed with AML in the United States each year. Approximately 11,540 deaths due to AML occur in the United States each year. The median age of diagnosis is 67 years, and 54% of diagnosed patients are 65 years old or older. In some embodiments, the subject described herein is 50 years old or older (e.g., 55, 60, 65, 70, 75, or 80 years old). In some embodiments, the subjects herein are 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, 70 years old or older, or 75 years old or older. In some embodiments, the subjects herein are 50 years old or older. In some embodiments, the subjects herein are 55 years old or older. In some embodiments, the subjects herein are 60 years old or older. In some embodiments, the subjects herein are 65 years old or older. In some embodiments, the subjects herein are 70 years old or older. In some embodiments, the subjects herein are 75 years old or older. In some embodiments, the subjects herein are 80 years old or older. The general therapeutic strategy for treating patients with AML has not changed substantially for more than 30 years. Current therapies include intensive induction therapy, consolidation therapy with intensive chemotherapy, and allogeneic hematopoietic cell transplantation. For elderly patients who cannot undergo intensive chemotherapy, supportive care is the only viable option, with a median survival of 5 to 6 months.Therefore, additional treatments for AML are needed.

[0042] In some embodiments, the subject exhibits one or more symptoms of AML. The symptoms of AML progress over several weeks. Symptoms of AML include, but are not limited to, fatigue, shortness of breath, fever, pale skin, sweating, weight loss, frequent infections, abnormal and frequent bleeding, bruising, bone and joint pain, and enlarged glands. In some embodiments, the subject exhibits at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten symptoms of AML. In some embodiments, the subject exhibits at least one symptom of AML. In some embodiments, the subject exhibits at least two symptoms of AML. In some embodiments, the subject exhibits at least three symptoms of AML. In some embodiments, the subject exhibits at least four symptoms of AML. In some embodiments, the subject exhibits at least five symptoms of AML. In some embodiments, the subject exhibits at least six symptoms of AML. In some embodiments, the subject exhibits at least seven symptoms of AML. In some embodiments, the subject exhibits at least eight symptoms of AML. In some embodiments, the subject exhibits at least 9 symptoms of AML, hi some embodiments, the subject exhibits at least 10 symptoms of AML.

[0043] In some embodiments, the subject has one or more risk factors associated with acute myeloid leukemia. A risk factor is a factor that affects the likelihood of developing a disease. Having one or several risk factors may not mean that the subject will develop AML. Conversely, a subject may develop AML without having any risk factors. Known risk factors for AML include, but are not limited to, aging (older age is a risk factor), male gender, smoking, exposure to certain chemicals (e.g., benzene, formaldehyde, diesel, or gasoline), treatment with chemotherapy drugs, exposure to radiation, having a blood disorder (e.g., polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis, or myelodysplastic syndrome), a family history of the disease, exposure to electromagnetic fields, and exposure to herbicides or pesticides. Additionally, there are several genetic syndromes that are risk factors for AML, such as Fanconi anemia, Bloom's syndrome, ataxia-telangiectasia, Diamond-Blackfan anemia, Schwachman-Diamond syndrome, Li-Fraumeni syndrome, neurofibromatosis type 1, and severe congenital neutropenia. In some embodiments, the subject has one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more) risk factors associated with acute myeloid leukemia. In some embodiments, the subject has one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has one or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has two or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has three or more risk factors associated with acute myeloid leukemia.In some embodiments, the subject has four or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has five or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has six or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has seven or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has eight or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has nine or more risk factors associated with acute myeloid leukemia. In some embodiments, the subject has ten or more risk factors associated with acute myeloid leukemia.

[0044] In some embodiments, the present disclosure provides a method for inhibiting the progression of clonal hematopoiesis to myeloid malignancies. The progression of clonal hematopoiesis to myeloid malignancies occurs due to clonal proliferation, which can progress to hematological abnormalities or leukemic transformation. Myeloid malignancies are a heterogeneous group of clonal disorders characterized by excessive proliferation, abnormal self-renewal, and / or abnormal differentiation of hematopoietic cells and myeloid progenitor cells. They include myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), and AMLs. Briefly, sources of mutagenic stress (e.g., chemotherapy, aging, or exogenous stress) can cause somatic mutations in clonal hematopoiesis. Selective pressure can select these somatic variants if they have better fitness compared to non-variants. Selective pressure can lead to clonal proliferation and the development of clonal hematopoiesis. In some cases, somatic variants can also directly lead to de novo AML formation. Clonal hematopoiesis may be followed by the development of cytopenias. In some cases, the development of AML due to clonal hematopoiesis may arise from moderate myelodysplastic syndrome (MDS), but may also bypass this progression.

[0045] (III.Cell aging) Aspects of the present disclosure relate to inhibiting senescence of bone marrow stromal cells. As described herein, "senescence" or "cellular senescence" (used interchangeably herein) refer to a cell fate with characteristic traits (e.g., irreversible replication arrest, sustained viability with resistance to apoptosis, and often increased metabolic activity). Cell signaling pathways that contribute to cells entering a senescent state can include signals related to tissue or cellular damage. For example, these signaling pathways may be mediated by DNA damage, telomeric uncapping or dysfunction, exposure to extracellular DNA, oncogene activation, replication stress or proliferation inducers (e.g., growth hormone / IGF-1), protein aggregates, misfolded proteins, failure of protein removal through reduced autophagy, the presence of advanced glycation end products (AGEs) resulting from reactions that reduce sugars at amino groups in proteins (e.g., hemoglobin A1c is an AGE), saturated lipids and other bioactive lipids (e.g., bradykinins, certain prostaglandins, etc.), reactive metabolites (e.g., Senescence-inducing factors may include, but are not limited to, stresses (e.g., oxygen-dependent oxidation (ROS), hypoxia or hyperxia), mechanical stress (e.g., bone-on-bone stress in osteoarthritis, or shear stress, such as occurs on the venous side of an arteriovenous fistula for hemodialysis or around atherosclerotic plaques), inflammatory cytokines (e.g., TNFα), damage-associated molecular patterns (DAMPs, e.g., released cellular contents that signal the destruction of neighboring cells), and pathogen-associated molecular patterns (PAMPs, e.g., bacterial endotoxins). Thus, inhibiting (e.g., preventing or delaying) bone marrow stromal cell senescence can be achieved, for example, by using senolytic agents to prevent irreversible replication arrest, sustained viability with resistance to apoptosis, and / or increased metabolic activity.

[0046] In some embodiments, the present disclosure provides a method for assaying a sample for the presence of somatic mutations. The types of mutations that can be assayed include single-base substitutions, insertions and deletions, duplications, and translocations. A method for detecting mutations can include isolating DNA from a sample and subjecting the DNA to PCR. After PCR, many assays can be performed to detect the presence of mutations, including, but not limited to, denaturing gradient gel electrophoresis (DGGE), constant denaturing gel electrophoresis (CDGE), temporal temperature gradient gel electrophoresis (TTGE), single-strand conformation polymorphism analysis (SSCP), protein truncation test (PTT), and high-resolution melting (HRM) analysis. In some embodiments, DGGE can be performed to detect the presence of mutations. In some embodiments, CDGE can be performed to detect the presence of mutations. In some embodiments, TTGE can be performed to detect the presence of mutations. In some embodiments, SSCP can be performed to detect the presence of mutations. In some embodiments, PTT can be performed to detect the presence of mutations. In some embodiments, HRM may be performed to detect the presence of mutations.

[0047] In some aspects, the present disclosure provides a method for assaying for increased expression of senescence markers. In some embodiments, assaying comprises detecting the expression of at least one (e.g., one, two, three, four, or more) senescence marker in a sample and comparing it to a control. In some embodiments, assaying comprises detecting the expression of at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) senescence marker in a sample. In some embodiments, assaying comprises detecting the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten senescence markers in a sample. In some embodiments, assaying comprises detecting the expression of at least one senescence marker in a sample. In some embodiments, assaying comprises detecting the expression of at least two senescence markers in a sample. In some embodiments, assaying comprises detecting the expression of at least three senescence markers in a sample. In some embodiments, assaying comprises detecting the expression of at least four senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least five senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least six senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least seven senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least eight senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least nine senescence markers in the sample. In some embodiments, assaying comprises detecting the expression of at least ten senescence markers in the sample.

[0048] In some embodiments, the detection may involve staining the senescence marker with a fluorescent marker and visualizing the results measured using a plate reader or by flow cytometry or epifluorescence microscopy. The senescence marker may be any senescence-associated secretory phenotype (SASP) marker. Senescence markers include senescence-associated β-galactosidase (SA-β-Gal), cyclin-dependent kinase inhibitor 2a (Cdkn2a) (P16), Cdkn1a (P21), Cdkn1b (P27), interleukin-6 (IL-6), IL-1α, IL-7, IL-1β, IL-13, IL-15, IL-8, growth-regulated oncogene (GRP), and IFN-γ. Oncogene) α (GRO-α), GRO-β, GRO-g, monocyte chemoattractant protein-2 (MCP-2), MCP-4, MIP-1α, MIP-3α, HCC-4, eotaxin, eotaxin-3, TECK, ENA-78, I-309, I-TAC, GM-CSE, G-CSE, IFN-γ, BLC, MIF, amphiregulin, epiregulin, heregulin, EGF, bFGF, HGF, KGF (FGF7), VEGF, angiogenin, SCF, SDF-1, PIGF, NGF, insulin-like growth factor binding protein 2 (IGFBP-2), IGFBP-3, IGF The expression of a protein may be selected from, but is not limited to, BP-4, IGFBP-6, IGFBP-7, matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-10, MMP-12, MMP-13, MMP-14, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2, PAI-1, PAI-2; tPA; uPA, cathepsin B, ICAM-1, ICAM-3, OPG, sTNFRI, TRAIL-R3, Fas, sTNFRII, Fas, uPAR, SGP130, EGF-R, PGE2, nitric oxide, reactive oxygen species, fibronectin, collagen, and laminin (see, e.g., Xu et al., Front Pharmacol., 2020;11:601325). In some embodiments, the method includes assaying a sample from the subject for increased expression of SA-β-Gal compared to a control.In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a P16 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a P21 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a P27 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-6 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-1α control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-7 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-1β control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-13 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an IL-15 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for IL-8. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for GRO-α. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for GRO-β. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for GRO-g. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for MCP-2. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for MCP-4.In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a MIP-1α control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a MIP-3α control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a HCC-4 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an eotaxin control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an eotaxin-3 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a TECK control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an ENA-78 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an I-309 control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to an I-TAC control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a GM-CSE control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a G-CSE control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an IFN-γ control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a BLC control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a MIF control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an amphiregulin control.In some embodiments, the method includes assaying a sample from the subject for increased expression of epiregulin compared to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of heregulin compared to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an EGF control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a bFGF control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an HGF control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a KGF (FGF7) control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a VEGF control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an angiogenin control. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to an SCF control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for SDF-1. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for PIGF. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for NGF. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for IGFBP-2. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for IGFBP-3. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for IGFBP-4.In some embodiments, the method includes assaying a sample from the subject for increased expression of IGFBP-6 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of IGFBP-7 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-1 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-3 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-10 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-12 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-13 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of MMP-14 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of TIMP-1 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for TIMP-2. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for PAI-1. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for PAI-2. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for tPA. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for uPA. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for cathepsin B.In some embodiments, the method includes assaying a sample from the subject for increased expression of ICAM-1 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of ICAM-3 relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of OPG relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression of sTNFRI relative to a control. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for TRAIL-R3. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for Fas. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for sTNFRII. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for Fas. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for uPAR. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for SGP130. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for EGF-R. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for PGE2. In some embodiments, the method includes assaying a sample from the subject for increased expression relative to a control for nitric oxide. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a control for reactive oxygen species. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a control for fibronectin. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a control for collagen. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to a control for laminin. In some embodiments, the method includes assaying a sample from the subject for increased expression compared to controls for senescence-associated β-galactosidase (SA-β-Gal), Cdkn2a (P16), Cdkn1a (P21), Cdkn1b (P27), IL-6, and IL-1α.

[0049] In some aspects, the present disclosure provides methods related to the use of senolytic agents to inhibit senescence in bone marrow stromal cells. A senolytic agent is an agent that selectively kills senescent cells. In some embodiments, the senolytic agent is a class of drugs, small molecules, or compounds that selectively kill senescent cells. The senolytic agent can also be in the form of a vaccine or a gene editing method (e.g., using CRISPR / Cas to modify specific genes to induce apoptosis). In some embodiments, the senolytic agent can delay, prevent, alleviate, or reverse age-related diseases. In some embodiments, the senolytic agent can delay, prevent, alleviate, or reverse age-related diseases. In some embodiments, the senolytic agent can delay, prevent, alleviate, and / or reverse age-related diseases.

[0050] In some embodiments, the senolytic agent is a drug that selectively kills senescent cells. In some embodiments, the senolytic agent is a small molecule that selectively kills senescent cells. In some embodiments, the senolytic agent is a compound that selectively kills senescent cells. In some embodiments, the senolytic agent is a drug, small molecule, or compound that selectively kills senescent cells. In some embodiments, the senolytic agent is a drug, small molecule, and compound that selectively kills senescent cells. In some embodiments, the senolytic agent is a drug, small molecule, and / or compound that selectively kills senescent cells. In some embodiments, the senolytic agent is a vaccine or a method of gene editing (e.g., CRISPR / Cas). In some embodiments, the senolytic agent is a vaccine and a method of gene editing (e.g., CRISPR / Cas). In some embodiments, the senolytic agent is a vaccine and / or a method of gene editing (e.g., CRISPR / Cas). In some embodiments, the senolytic agent is a vaccine. In some embodiments, the senolytic agent is a method of gene editing (e.g., CRISPR / Cas).

[0051] The senolytic agent may be selected from, but is not limited to, dasatinib, quercetin, fisetin, piperlongumine, azithromycin, roxithromycin, navitoclax (ABT-263), luteolin, curcumin, curcumin analog EF24, A1331852, A1155463, geldanamycin, tanespimycin, nutlin-3a, FOXO4-associated peptide, BCL-2 inhibitor, Src inhibitor, USP7 inhibitor, SSK1, BIRC5 knockout, GLS1 inhibitor, anti-GPNMB vaccine, cardiac glycoside (e.g., ouabain, proscillaridin a, digoxin), 25-hydroxycholesterol (25HC), procyanidin C1, 17-DMAG (alvespimycin), catechin (e.g., SUNPHENON®), and EF-24.

[0052] In some embodiments, the senolytic agent has Formula I: [ka] The compound is dasatinib (SPRYCEL®) (CAS number 302962-49-8)) as shown in

[0053] In some embodiments, dasatinib is administered to a subject at a dose of about 100 mg to about 150 mg daily, weekly, or monthly.

[0054] In some embodiments, the senolytic agent has Formula II: [ka] The compound is quercetin (CAS number 117-39-5) as shown in

[0055] In some embodiments, quercetin is administered to a subject at a dose of about 100 mg to about 500 mg daily, weekly, or monthly.

[0056] In some embodiments, the senolytic agent has Formula III: [ka] The compound is 17-DMAG (alvespimycin) (CAS number 467214-21-7) as shown in

[0057] In some embodiments, alvespimycin is administered to a subject at a dose of about 50 mg to about 1000 mg (eg, about 500 mg to about 100-mg) daily, weekly, or monthly.

[0058] In some embodiments, the senolytic agent has Formula IV: [ka] The compound is fisetin (CAS number 528-48-3) as shown in

[0059] In some embodiments, fisetin is administered to a subject at a dose of about 100 mg to about 1000 mg (eg, about 500 mg to about 100-mg) daily, weekly, or monthly.

[0060] In some embodiments, the senolytic agent has Formula V: [ka] The compound is catechin (SUNPHENON®) (CAS number 7295-85-4) as shown in

[0061] In some embodiments, catechin (SUNPHENON®) is administered to a subject at a dose of about 50 mg to about 500 mg (eg, about 500 mg to about 100-mg) daily, weekly, or monthly.

[0062] In some embodiments, the senolytic agent has Formula VI: [ka] The compound is navitoclax (CAS number 923564-51-6) as shown in

[0063] In some embodiments, navitoclax is administered to a subject at a dose of about 50 mg to about 1000 mg (eg, about 500 mg to about 100-mg) daily, weekly, or monthly.

[0064] III. Treatment Methods A "subject in need thereof" refers to a subject in need of treatment for clonal hematopoiesis (e.g., AML, bone marrow stromal cell senescence). In some embodiments, a subject in need thereof may be a subject who is "non-responsive" or "resistant" to standard treatment for clonal hematopoiesis (e.g., AML, bone marrow stromal cell senescence). In some embodiments, the terms "non-responsive" and "resistant" refer to a subject's response to treatment that is not clinically sufficient to alleviate one or more symptoms associated with the neurological disease or disorder.

[0065] A subject generally refers to a mammal. The mammal may be, for example, a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. In some embodiments, the subject is a human. In some embodiments, the subject is a primate. In some embodiments, the subject is a primate. In some embodiments, the subject is a mouse. In some embodiments, the subject is a rat. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat. In some embodiments, the subject is a cow. In some embodiments, the subject is a horse. In some embodiments, the subject is a goat. In some embodiments, the subject is a camel. In some embodiments, the subject is a sheep. In some embodiments, the subject is a pig.

[0066] In some embodiments, the subject has AML. In some embodiments, the subject is at risk of blood cancer (e.g., AML). In some embodiments, the subject exhibits one or more symptoms of AML. In some embodiments, the subject exhibits one or more risk factors for AML.

[0067] In some embodiments, the subject is about 50 (e.g., about 48, about 49, about 50, about 51, about 52, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95) years old or older. In some embodiments, the subject is about 48, about 49, about 50, about 51, about 52, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 years old or older. In some embodiments, the subject is about 48 years old. In some embodiments, the subject is about 49 years old. In some embodiments, the subject is about 50 years old. In some embodiments, the subject is about 51 years old. In some embodiments, the subject is about 52 years old. In some embodiments, the subject is about 55 years old. In some embodiments, the subject is about 60 years old. In some embodiments, the subject is about 65 years old. In some embodiments, the subject is about 70 years old. In some embodiments, the subject is about 75 years old. In some embodiments, the subject is about 80 years old. In some embodiments, the subject is about 85 years old. In some embodiments, the subject is about 90 years old. In some embodiments, the subject is about 70 years old. In some embodiments, the subject is about 95 years old.

[0068] In some embodiments, subjects at risk for hematological cancer (e.g., AML) exhibit signs of clonal hematopoiesis. This includes subjects with age-related mutations (mainly DNMT3A, TET2, and ASXL1) associated with clonal hematopoietic proliferation and malignant disease. In some embodiments, the subject has a mutation in DNMT3A. In some embodiments, the subject has a mutation in TET2. In some embodiments, the subject has a mutation in ASXL1.

[0069] In some embodiments, the inhibition of clonal hematopoiesis can be measured relative to a control. A "control" can be a subject that does not have a somatic mutation (e.g., DNMT3A) that results in aberrant clonal hematopoiesis. A control can be a subject that exhibits low levels of clonal hematopoiesis. A control can be a subject that does not exhibit symptoms of a myeloid malignancy. A control can be a subject that exhibits little to no expression of senescence markers, or expression above or below a threshold.

[0070] An "effective amount" is an amount that alleviates one or more symptoms associated with a particular condition (e.g., clonal hematopoiesis and / or AML (or other hematologic cancer)). In some embodiments, an effective amount inhibits myeloproliferation of bone marrow stromal cells. In some embodiments, an effective amount inhibits the progression of clonal hematopoiesis to a myeloid malignancy. "Inhibition" refers to at least 20% inhibition compared to a control (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In some embodiments, inhibition is at least 20% inhibition compared to a control (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In some embodiments, inhibition is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to a control. In some embodiments, the inhibition is at least 20% inhibition compared to a control. In some embodiments, the inhibition is at least 30% inhibition compared to a control. In some embodiments, the inhibition is at least 40% inhibition compared to a control. In some embodiments, the inhibition is at least 50% inhibition compared to a control. In some embodiments, the inhibition is at least 60% inhibition compared to a control. In some embodiments, the inhibition is at least 70% inhibition compared to a control. In some embodiments, the inhibition is at least 80% inhibition compared to a control. In some embodiments, the inhibition is at least 90% inhibition compared to a control.

[0071] In some embodiments, the senolytic agent is formulated in one or more compositions (e.g., separate compositions or the same composition) for administration to a subject. The compositions may take any form (e.g., liquid, aerosol, solution, inhalant, mist, spray; or solid, powder, ointment, paste, cream, lotion, gel, patch, etc.) suitable for administration by any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intraosseous, intrapleural, intrathecal, transdermal, transmucosal, rectal, etc.). In some embodiments, the composition is in the form of a liquid, aerosol, solution, inhalant, mist, spray, or solid, powder, ointment, paste, cream, lotion, gel, or patch. In some embodiments, the composition is in the form of a liquid, aerosol, solution, inhalant, mist, spray, or solid, powder, ointment, paste, cream, lotion, gel, and / or patch. In some embodiments, the composition is in the form of a liquid. In some embodiments, the composition is in the form of an aerosol. In some embodiments, the composition is in the form of a solution. In some embodiments, the composition is in the form of an inhalant. In some embodiments, the composition is in the form of a mist. In some embodiments, the composition is in the form of a spray. In some embodiments, the composition is in the form of a solid. In some embodiments, the composition is in the form of a powder. In some embodiments, the composition is in the form of an ointment. In some embodiments, the composition is in the form of a paste. In some embodiments, the composition is in the form of a cream. In some embodiments, the composition is in the form of a lotion. In some embodiments, the composition is in the form of a gel. In some embodiments, the composition is in the form of a patch.

[0072] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of an aqueous solution or powder for aerosol administration by inhalation (either through the mouth or nose) or insufflation; in the form of a tablet or capsule for oral administration; in the form of a sterile aqueous solution or dispersion suitable for administration either by direct injection or by addition to sterile infusion fluid for intravenous infusion; or in the form of a lotion, cream, foam, patch, suspension, solution, or suppository for transdermal or transmucosal administration. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of an aqueous solution and / or powder for aerosol administration by inhalation and / or insufflation. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of an aqueous solution for aerosol administration by inhalation. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of an aqueous solution for aerosol administration by insufflation. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a powder solution for aerosol administration by inhalation. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a powder solution for aerosol administration by insufflation. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a tablet and / or capsule for oral administration. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of tablets for oral administration. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of capsules for oral administration. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a sterile aqueous solution and / or dispersion suitable for administration either by direct injection or by addition to sterile infusion fluids for intravenous infusion. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a sterile aqueous solution for administration by direct injection. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a sterile aqueous solution for administration by addition to sterile infusion fluids for intravenous infusion. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a dispersion suitable for administration by direct injection. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a dispersion suitable for administration by addition to sterile infusion fluids for intravenous infusion.In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a lotion for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a cream for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a foam for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a patch for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a suspension for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a solution for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a suppository for transdermal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a lotion for transmucosal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a cream for transmucosal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a foam for transmucosal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a patch for transmucosal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a suspension for transmucosal administration. In some embodiments, the pharmaceutical composition of the present disclosure may be in the form of a solution for transmucosal administration. In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of suppositories for transmucosal administration.

[0073] In some embodiments, the route of administration of the senolytic agent may be intravenous. In some embodiments, the route of administration of the senolytic agent may be intraosseous. In some embodiments, the senolytic agent may be administered either continuously or intermittently. In some embodiments, the senolytic agent may be administered continuously. In some embodiments, the senolytic agent may be administered intermittently. [Example]

[0074] Example 1. Bone marrow stromal cells (BMSCs) acquire an enriched gene signature of cellular senescence in the context of Dnmt3a mutant hematopoiesis. Clonal hematopoiesis (clonal hematopoiesis) is an age-related condition caused by somatic mutations that confer a clonal selective advantage to hematopoietic stem cells (HSCs). The gene most frequently mutated in human clonal hematopoiesis and acute myeloid leukemia (AML) is DNA methyltransferase 3A (DNMT3A). The precise mechanism by which DNMT3A mutations confer clonal advantage and lead to the development of AML is unknown. We performed single-cell RNA sequencing (RNA-seq) of hematopoietic bone marrow (BM) and non-bone marrow (BM) fractions from a mouse model of DNMT3A hotspot mutations. Surprisingly, we found that a subset of bone marrow stromal cells (BMSCs) acquired an enriched gene signature of cellular senescence and had increased P21 expression in the context of Dnmt3a mutant hematopoiesis (Figure 1A-B).

[0075] First, we investigated the effects of Dnmt3a mutant hematopoietic cells on wild-type non-hematopoietic cells in the BM microenvironment. We transplanted Dnmt3a mutant (R878H / +) or control (wild-type Dnmt3a) BM cells into middle-aged wild-type recipient mice and performed single-cell RNA-seq on enriched hematopoietic (CD45+) and non-hematopoietic (CD45- Ter119-) BM cell fractions. By examining markers of senescence in the profiled BMSC subsets, we observed a strong enrichment of cellular senescence gene signatures in Osteo-CAR ("Cxcl12-enriched reticular") and Adipo-CAR cells after exposure to Dnmt3a mutant hematopoietic cells (Figure 1A). This gene expression program included increased expression of the canonical senescence-associated gene Cdkn1a (p21) in mutant Osteo-CAR and Adipo-CAR cells compared to controls (Figure 1B). Furthermore, endothelial cells (ECs), arteriolar endothelial cells (ECs), and sinusoidal endothelial cells (ECs) did not show increased Cdkn1a (p21) expression. This led us to hypothesize that Dnmt3a mutant HSCs remodel the BM microenvironment through the induction of senescence.

[0076] Example 2. Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs) induce senescence and favor clonal expansion and transformation to acute myeloid leukemia. We tested whether Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs) induce senescence in the BM microenvironment, favoring clonal expansion and transformation to AML. Ex vivo coculture showed that BMSCs cultured with Dnmt3a mutant HSPCs have increased expression of senescence markers (P16, P21, β-gal) and a senescence-associated secretory phenotype (IL-6, IL-1a) compared with BMSCs cultured with control wild-type (WT) HSPCs (Figures 2A-B). A similar phenotype was observed with Dnmt3a mutant cells transplanted into WT recipient mice; this resulted in increased β-gal staining in BMSCs, which was not observed in endothelial cells (Figures 3A-B).

[0077] Using ex vivo coculture of BMSCs with either Dnmt3a mutant (R878H / +) HSPCs or control HSPCs, we found that after 7 days ("day 7"), Dnmt3a mutant HSPCs caused increased expression of the senescence markers Cdkn2a (p16), Cdkn1a (p21), and β-galactosidase (β-gal) in primary wild-type BMSCs compared with BMSCs cultured with control HSPCs (Figure 2A). Increased expression of Il6 and Il1α, components of the senescence-associated secretory phenotype (SASP), was also observed in BMSCs cultured with R878H / + HSPCs compared with BMSCs cultured with control HSPCs (Figure 2B).

[0078] In vivo transplantation experiments were performed using either control (wild-type Dnmt3a) or Dnmt3a mutant (R878H / +) BM hematopoietic cells. The data showed that 12 weeks after transplantation of Dnmt3a mutant or control BM cells, BMSCs transplanted with Dnmt3a mutant hematopoietic cells showed increased intracellular β-gal compared with BMSCs transplanted with control BM hematopoietic cells (Fig. 3A), but not in endothelial cells transplanted with either control or Dnmt3a mutant hematopoietic cells (Fig. 3B).

[0079] Furthermore, we found that BCL2 and BCLxL expression was increased in BMSCs without changing the total number of BMSCs (Figures 4A-B). In these mice, Dnmt3a mutant HSCs proliferated compared with control HSCs (Figure 5).

[0080] In vivo transplantation experiments also demonstrated increased intracellular expression of the senescence-associated anti-apoptotic proteins Bcl-2 and Bcl-xL in BMSCs 12 weeks after transplantation of Dnmt3a mutant cells compared with transplantation of control BM cells (Figure 4A), without altering the total number of BMSCs (Figure 4B). Furthermore, in these animals, Dnmt3a mutant HSCs proliferated compared with control HSCs (Figure 5). This supports a positive correlation between BMSC senescence and the proliferation of Dnmt3a mutant HSCs in vivo.

[0081] Example 3. Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs) induce senescence and favor clonal expansion and transformation to acute myeloid leukemia. Next, we assessed the functional importance of BMSC senescence in the transformation of Dnmt3a mutants into AML. Dnmt3a mutant cells were transplanted into wild-type recipient mice and treated with the senolytic navitoclax or vehicle control. Subsequently, we investigated the functional significance of BMSC senescence in the transformation of Dnmt3a mutants into AML. c We observed that navitoclax treatment reduced peripheral blood myeloid cell overproduction concomitantly with the transformation of Dnmt3a;Npm1 mutant cells, suggesting that senolytic agents may delay AML development (Figures 6A-6E).

[0082] Using navitoclax, a senolytic agent that targets Bcl-2 / Bcl-xL, we assessed the extent to which elimination of senescent cells triggered by Dnmt3a mutant hematopoiesis in vivo delays the progression from clonal hematopoiesis to myeloid malignancies. Briefly, Dnmt3a mutant cells (CD45.2 + ;Dnmt3aR787H / + MxCre;Npm1 frt-cA / + Flpo ERT BM-derived cells from mice) were transfected into recipient mice (CD45.1 + The senolytic agent was transplanted into recipient mice (Fig. 6A). Twelve weeks later ("pre-senolytic agent"), recipient mice were administered either a vehicle control or the senolytic agent navitoclax by oral gavage to selectively eliminate senescent cells. Navitoclax was administered at a dose of 50 mg / kg / day for 1 week, followed by 2 weeks of no treatment, followed by 1 week of treatment. Blood samples were collected from pre-senolytic agent mice before administration of the vehicle control or navitoclax. After navitoclax treatment ("post-senolytic agent"), tamoxifen was used to detect the AML driver mutation Npm1 cA / + ("TAM") to initiate progression to myeloid malignancies. Blood samples were collected from post-senolytic agent and TAM mice. Tamoxifen was administered for one week. Blood samples were taken from tamoxifen-treated mice ("post-Npm1"). The data showed that in the peripheral blood ("PB"), tamoxifen administration ("TAM") significantly increased myeloid cell (CD45.2) expression in both vehicle- and navitoclax-treated mice compared to post-senolytic agent samples. + However, at subsequent sampling times ("post-Npm1"), navitoclax-treated mice showed an increased frequency of myeloid cells (CD45.2) in their peripheral blood (PB). + At 5 months after tamoxifen treatment ("harvest"), navitoclax-treated mice had a decreased frequency of donor (CD45.2) in the BM compared to vehicle-treated controls (Figure 6B). + ) cells (Figure 6C), maintained lower BMSC senescence (measured by β-gal) (Figure 6D), and had reduced monocyte ("white blood cell" or "WBC") numbers (Figure 6E). + ;Dnmt3a R787H / + MxCre;Npm1 frt-cA / + Flpo ERTAlthough cell-transplanted recipient mice did not yet develop full-blown myeloid malignancies, these results support that elimination of senescent cells induced by Dnmt3a mutant hematopoiesis using senolytic agents delays bone marrow proliferation during the transformation of clonal hematopoiesis to AML.

[0083] Taken together, we demonstrated that Dnmt3a mutant hematopoietic cells can induce BMSC senescence, and we recommend targeting the senescent BM microenvironment as a strategy to reduce clonal hematopoiesis and prevent transformation to AML.

[0084] All references, patents, and patent applications disclosed herein are each incorporated by reference with respect to the subject matter cited, which in some cases may include the entire document. The indefinite articles "a" and "an," as used in this application in the specification and claims, unless expressly stated otherwise, should be understood to mean "at least one."

[0085] Unless expressly stated otherwise, it should also be understood that in any method claimed in this application that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0086] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," etc., are to be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

[0087] The terms "about" and "substantially" before a numerical value mean ±10% of the stated numerical value.

[0088] Where a range of values ​​is provided, each value therebetween, inclusive of the upper and lower limits of the range, is specifically contemplated and described herein.

Claims

1. 1. A method of inhibiting clonal hematopoiesis in a subject in need thereof, comprising: administering a senolytic agent to a subject in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control. A method comprising:

2. 10. The method of claim 1, wherein the subject exhibits one or more symptoms of acute myeloid leukemia.

3. 3. The method of claim 1 or 2, wherein the subject has one or more known risk factors associated with acute myeloid leukemia.

4. 10. The method of any one of the preceding claims, wherein the subject has acute myeloid leukemia.

5. 10. The method of any one of the preceding claims, wherein the subject is 50 years of age or older.

6. 10. The method of any one of the preceding claims, wherein an effective amount of the senolytic agent inhibits myeloproliferation of the bone marrow stromal cells.

7. 10. The method of any one of the preceding claims, wherein an effective amount of the senolytic agent inhibits the progression of clonal hematopoiesis to a myeloid malignancy compared to a control.

8. 10. The method of any one of the preceding claims, wherein the subject's hematopoietic stem and progenitor cells (HSPCs) comprise a somatic mutation that confers a clonal selection advantage to the HSPCs.

9. 9. The method of claim 8, wherein the mutation is a DNA methyltransferase 3A (DNMT3A) mutation.

10. assaying a sample from said subject for the presence of said somatic mutation.

10. The method of claim 8 or 9, further comprising:

11. Assaying a sample from said subject for increased expression of aging biomarkers compared to a control.

10. The method of any one of the preceding claims, further comprising:

12. 12. The method of claim 11, wherein the senescence marker is selected from senescence-associated β-galactosidase (SA-β-Gal), Cdkn2a (P16), Cdkn1a (P21), Cdkn1b (P27), IL-6, and IL-1α.

13. 2. The method of any one of the preceding claims, wherein the bone marrow stromal cells comprise adipo-Cxcl12-enriched reticular (CAR) cells and osteo-CAR cells.

14. 10. The method of any one of the preceding claims, wherein the senolytic agent is selected from dasatinib, quercetin, fisetin, 17-DMAG, navitoclax, and catechin.

15. 10. The method of any one of the preceding claims, wherein the senolytic agent is administered via an intravenous or intraosseous route.

16. 1. A method of inhibiting clonal hematopoiesis in a subject in need thereof, comprising: Assaying a biological sample obtained from the subject for DNA methyltransferase 3A (DNMT3A) mutations; and administering a senolytic agent to a subject in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control. A method comprising:

17. 1. A method of inhibiting clonal hematopoiesis in a subject in need thereof, comprising: assaying a biological sample obtained from the subject for a biomarker of cellular senescence; and administering a senolytic agent to a subject in an amount effective to inhibit senescence of bone marrow stromal cells in the subject, thereby inhibiting clonal hematopoiesis in the subject relative to a control. A method comprising:

18. 18. The method of claim 16 or 17, wherein the subject is at risk of developing a myeloid malignancy.

19. The method of any one of claims 16 to 18, wherein the biological sample comprises hematopoietic cells.

20. 20. The method of any one of claims 16 to 19, wherein the assaying step comprises performing single-cell RNA sequencing (RNA-seq) on the biological sample.

21. 21. The method of any one of claims 16 to 20, wherein the senolytic agent is selected from dasatinib, quercetin, fisetin, 17-DMAG, navitoclax, and catechin.

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