Biomarker for familial platelet disorder and uses thereof

Abstract

Familial Platelet Disorder (FPD) is associated with germline RUNX1 mutations, establishing a preleukemic state and increasing the risk of developing acute leukemia. Conventional approaches lack intervention strategies that may delay or prevent leukemia progression in patients. The present disclosure integrates single-cell transcriptomics, flow cytometry, and spatial proteomics data analyses for a large cohort of FPD patient samples to show that RUNX1 mutations unleash inflammatory signaling in familial platelet disorder. Specifically, the receptor CD74 has been identified as a biomarker and a master regulator of downstream signaling pathways. Embodiments of the present disclosure provide diagnostic and treatment methods for FPD in patients.

Description

BIOMARKER FOR FAMILIAL PLATELET DISORDER AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 595,313, filed November 1 , 2023, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number R01 HL155426 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to biomarkers for familial platelet disorder (FPD) and their use. Particularly, the disclosure relates to useful methods of treating FPD in a subject.BACKGROUND OF THE DISCLOSURE

[0004] Familial Platelet Disorder (FPD) is an inherited autosomal dominant disorder caused by germline mutations in the RUNX1 gene.1 2FPD is characterized by life-long mild to moderate thrombocytopenia, platelet dysfunction, and an increased risk of developing hematological malignancies, primarily myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).364The median age of leukemia diagnosis in FPD is 29 years (ranging 6-77 years).45To this date, ~210 FPD families are known within the United States, with an expectation of over 5,500 families worldwide.3

[0005] The RUNX1 transcription factor plays a crucial role in normal and malignant hematopoiesis.6 7Monoallelic germline mutations of RUNX1 in FPD primarily result in haploinsufficiency, although some mutations exhibit dominant-negative function.8'10Overall, a reduction in RUNX1 activity leads to altered expression of target genes11that are involved in hematopoiesis, ribosome biogenesis,12cell cycle regulation,13and DNA damage response.14RUNX1 mutations establish a preleukemic state, leading to clonal hematopoiesis,15and subsequent development to leukemia upon acquisition of secondary mutations.16’17’18

[0006] Recent studies have also suggested that inflammation may contribute to the FPD phenotype19with individuals often exhibiting allergic and other inflammatory conditions such as eczema and reactive airways disease.20’15’21However, how RUNX1 mutations contribute toinflammation or impact the function of hematopoietic stem and progenitor cells (HSPCs) remains incompletely understood. This knowledge is crucial to successfully developing leukemic intervention strategies for FPD patients. Currently, there remains an unmet need for developing treatment methods that may delay or prevent leukemia progression in familial platelet disorder.SUMMARY OF THE DISCLOSURE

[0007] The current disclosure provides biomarkers for familial platelet disorder (FPD). The biomarkers can be used to develop diagnostic and treatment methods for FPD.

[0008] Embodiments provide for a method of diagnosing familial platelet disorder in a subject, the method comprising the steps of: (a) isolating CD34+ mononuclear cells from a biological sample obtained from the subject; (b) measuring in the isolated CD34+ mononuclear cells an expression level of CD74 receptor; (c) diagnosing the subject based on the measured expression level of CD74 receptor in the CD34+ mononuclear cells; and (d) managing familial platelet disorder associated with the subject. In some embodiments, the CD34+ mononuclear cells comprise CD34+ progenitor cells. In some embodiments, the biological sample comprises peripheral blood or bone marrow aspirate. In some embodiments, diagnosing the subject having familial platelet disorder includes detecting an upregulation in the expression level of CD74 receptor in the CD34+ progenitor cells. In some embodiments, managing familial platelet disorder in the subject comprises administering to the subject in need thereof a therapeutically effective amount of one or more therapeutic agents selected from the group of a CD74 expression inhibitor and an antagonist of CD74 function.

[0009] Embodiments also provide for a method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ mononuclear cells, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits or reduces expression of CD74 receptor or comprises an antagonist of CD74 function. In some embodiments, the agent comprises (S,R)-3-(4 hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1). In some embodiments, the agent comprises a monoclonal antibody, or binding fragment thereof that binds to CD74 receptor.

[0010] Embodiments also provide for a method of treating familial platelet disorder in a subject in need thereof, the method comprising: (a) isolating CD34+ mononuclear cells from a biological sample obtained from the subject, wherein the biological sample comprises peripheral blood or bone marrow aspirate; (b) detecting an upregulation of CD74 signaling in the isolated CD34+ mononuclear cells; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits CD74 expression or comprises an antagonist of CD74 signaling. In someembodiments, the agent comprises ISO-1 or an anti-CD74 monoclonal antibody. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an agent that inhibits PI3K / mTOR pathway downstream of CD74 signaling. In some aspects, the agent is selected from the group of Rapamycin, Vistusertib (AZD2014), and Idelalisib. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an agent that inhibits JAK / STAT pathway downstream of CD74 signaling. In some aspects, the agent comprises ruxolitinib.

[0011] The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A shows in vitro culture using CD34+cells for 14 days. The percentage of CD34+and CD33+ / 13+cells for FPD and HD (n=10-11) were assessed using flow cytometry and represented as a percentage of live cells. Statistical significance is calculated using students’ t- test and levels indicating differences between FPD and healthy control samples are represented as * p<0.05, ** p<0.01 , *** p<0.001.

[0013] Figure 1 B shows colony formation ability of FPD (n=11) and HD (n=14) samples. CD34+cells were seeded at 1 ,000 cells / well density in duplicates using methocult H4434 (StemCell) and colonies were counted at day 14 (1°, left). Cells from the primary colony formation assays were collected and replated at the density of 100k cells / well and colonies were counted at day 14 (2°). Bar graph on the right shows CFU-GM and -M colony numbers of FPD and HD in primary plating. Statistical significance is calculated using students’ t-test and levels indicating differences between FPD and healthy control samples are represented as * p<0.05, ** p<0.01 , *** p<0.001.

[0014] Figures 2A-2B show results of colony formation ability of FPD and HD CD34+in the presence of inflammatory cytokines. Primary CD34+cells were seeded at the density of 1 ,000 cells / well and treated with LPS, CXCL8, CCL-2, CCL24, or IL-1 p at 10ng / mL. Colonies were counted at day 14 and harvested for secondary plating at 100k cells / well density. Statistical significance levels indicating differences between untreated and treated for FPD or healthy control samples were calculated via t-test and indicated by * p<0.05, ** p<0.01.

[0015] Figure 2C shows the number of CFU-GM colonies in FPD and HD samples with and without cytokine treatments. Statistical significance levels indicating differences between untreated and treated for FPD or healthy control samples were calculated via t-test and indicated by * p<0.05, ** p<0.01.

[0016] Figure 2D shows the results of secondary plating for FPD cells treated with indicated cytokines. Statistical significance levels indicating differences between untreated and treated for FPD or healthy control samples were calculated via t-test and indicated by * p<0.05, ** p<0.01.

[0017] Figure 3A shows surface expression levels of CD74 in CD34+cells of FPD (n=10) and HD (n=3) samples. Fresh bone marrow cells were analyzed for surface expression of CD34 and CD74 by flow cytometry. The populations are derived from live cells and the bar graph shows the percentage of CD74+in CD34+cells.

[0018] Figure 3B shows CD74 levels as analyzed using immunohistochemistry (IHC) staining. Transformed (MDS / CMML) and non-transformed FPD bone marrow slides were stained for CD74, and the levels were compared to healthy bone marrow. For each slide, 5 different regions of interest were analyzed, and data is shown as mean with standard error.

[0019] Figure 3C shows the results of CD74 knockout via CRISPR in colony formation assay. FPD (n=1) and HD (n=1) CD34+cells were subjected to CRISPR knockdown of CD74 and cultured in vitro. The colony formation ability of CD34+ cells upon CD74 knockdown was assessed by plating 1000 cells / well in H4434 methocult (StemCell) and counting on day 14 post culture.

[0020] Figures 3D-3E show results of colony formation ability of HD (n=8) and FPD (n=12) CD34+ cells upon treatment with MIF (10ng / ml_) and ISO-1 (25nM, HD n=5, FPD n=7). Bone marrow CD34+ cells were seeded at 1,000 cells / well density for primary plating and 100k cells / well for secondary plating. Statistical significance via student t-test indicates differences between FPD and healthy control samples.

[0021] Figure 3F shows an increase in megakaryocytes upon ISO-1 treatment in vitro. FPD MNCs (n=16) were treated with ISO-1 in three different concentrations for 7 days and megakaryocyte population percentage was measured using flow cytometry. Representative flow charts are shown.

[0022] Figure 3G shows phosphorylation levels of proteins in HD and FPD upon ISO-1 treatment. CD34+ cells were cultured in the presence of ISO-1 (100nM) for 48 hrs. Phosphorylation of indicated proteins was measured through intracellular flow cytometry. Data is shown as fold change differences in mean fluorescent intensity (MFI) of treated cells over untreated. Statistical significance via student t-test indicates differences between treated and untreated samples.

[0023] Figure 3H shows phosphorylation levels of indicated proteins upon CD74 CRISPR knockout in FPD CD34+ cells. Phosphorylation levels were measured using flow cytometry and data is shown as fold change values in sgCD74 over non-targeting control (sgNT). Statistical significance is calculated using students’ t-test and levels indicating differences between FPD andhealthy control samples, or vehicle treatment and treated groups as * p<0.05, ** p<0.01 , *** p<0.001.

[0024] Figure 4A presents dataset related to restoring phenotypic defects in FPD via targeting MIF or downstream PI3K / mTOR pathway. The representative violin plots show MFI values normalized to GAPDH for indicated proteins across FPD and HD samples.

[0025] Figure 4B shows the result of PI3K / mTOR pathway inhibition in FPD. MNCs from FPD (n=11) bone marrow samples (20-30,000 cells / well) were cultured in the presence of inhibitors at 3 different concentrations selected based on colorimetric cell viability assay. In vitro cell differentiation was assessed after 7 days of culture using flow cytometry analysis. Bar graph shows mean % of live cells for indicated populations.

[0026] Figure 4C shows the results of colony formation ability of FPD CD34+cells upon treatment with inhibitors. Colony formation was assessed by seeding CD34+cells at 1000 cells / well density and counting colonies at day 14 post-culture. Statistical significance is calculated using t-test and levels indicating differences between treatment versus baseline are shown as * p<0.05, ** p<0.01, *** p<0.001.

[0027] Figure 5A shows in vivo effect of MIF / CD74 and mTOR pathway inhibition using Runx1R188Q / +-mutated mice. The representative bar graphs show the colony formation ability of WT and Het mice. LSK (lineage’Sca1+cKit+) cells were isolated from mice and seeded at 600 cells / well density in the presence of MIF (10ng / mL) and ISO-1 (50nM). Colonies counted at day 7 post culture and harvested for secondary plating at 100k cells / well density. Methocult M3434 (StemCell) was used.

[0028] Figure 5B shows increased platelet activation in ISO-1 and rapamycin / sirolimus treated Het mice. WT and Het mice (age and sex matched) were treated with ISO-1 and ruxolitinib daily, and rapamycin / sirolimus every other day for a duration of 8 weeks. Percentage of CD41+ / CD61+cells were measured in the peripheral blood of WT and Het mice upon thrombin (0.1 U / mL) treatment for 15 minutes. The bar graph shows percentage of all cells. Platelet activation marker (CD62P+) positivity was measured in CD41+ / CD61+cells upon thrombin (0.1 U / ml_) treatment. Statistical significance is calculated using t-test and are shown as * p<0.05, ** p<0.01 , *** p<0.001.

[0029] Figure 5C shows results of immunophenotyping in inhibitor-treated mice (WT and Het) at the end of experiments. BM cells were harvested and flow cytometry was used to determine percentages of monocyte (CD11 b+), neutrophils (Ly6-C7-G+), and macrophages (CD11 b7F4-80+) from live cells. Statistical significance is calculated using t-test and are shown as * p<0.05, ** p<0.01 , *** p<0.001.

[0030] Figure 6 shows colony formation ability of HD (n=2, duplicates) and FPD (n=2-4, duplicates) CD34+cells examined upon treatment with LPS (10ng / mL) with or without reparixin (CXCL8 inhibitor, 1uM), INCB3347 (CCL2 inhibitor, 1uM), SB332236 (CCL24 inhibitor, 1uM), and IL1 Ra (I L-1 p antagonist, 1uM). Colonies were counted at day 14 of culture. * p<0.05, ** p<0.01 , *** p<0.001.

[0031] Figures 7A-7D show the effect of inhibitors (AZD2014, Sirolimus, Ruxolitinib, and Idelalisib) against mTOR and PI3K / AKT pathways on the viability of FPD cells compared to healthy donor (HD) bone marrow cells as well as F?L / A / X1-mutated AML5 or ?L / A / X1-Wild-type AML2 cell lines when assessed using MTS cell viability assay at day 6 post-culture. The experiments were performed in triplicate, representing data from three independent experiments. The representative graphs plot % viability over concentration of inhibitor. The inhibitor doses that are the most effective in / L / / VX7-mutated AML5 cells but less toxic in healthy and FPD cells are selected and represented as grey box.

[0032] Figure 8A shows the area under curve (AUC) values of idelalisib, sirolimus, and ruxolitinib which are compared between RUNX1-wild type and -mutated AML samples using the BeatAML database.

[0033] Figure 8B shows the AML samples being separated into CD74-high and -low expressing categories, and the AUC values are compared for indicated inhibitors between these two groups.

[0034] Figures 8C-8F show levels of cytokines from FPD MNCs (n=4) upon treatment with AZD2014, sirolimus, idelalisib, and ruxolitinib for 48 hrs as measured using 65-plex Luminex assay. The bar graphs show a log of fold change in cytokine expression over vehicle. * p<0.05, ** p<0.01 , *** p<0.001.

[0035] Figure 9A shows flow analysis of bone marrow cells from WT (n=2) and Het (n=2) mice indicating upregulation of CD74 expression on lineage-negative cells in Het mice.

[0036] Figure 9B shows results from blood count data of the inhibitor-treated mice (WT and Het) at the end of the experiment.

[0037] Figure 9C shows results from blood count data of the inhibitor-treated mice (WT and Het) over the course of the experiment.DETAILED DESCRIPTION

[0038] Approximately, 4-19% of patients diagnosed with a myeloid malignancy at the age of 40 years or younger have a pre-disposing germline mutation.25'28FPD leads to one of the most common inherited forms of myeloid malignancies18'29and yet there are no available treatments. The identification of early cancer interception and prevention strategies has the potential tosignificantly improve the long-term quality of life and survival of FPD patients. In the present disclosure, CD74-mediated inflammatory and prosurvival pathways are identified as rationale druggable targets to rescue the hematopoietic dysfunction in FPD and potentially reduce leukemic risk.

[0039] Inflammation drives tumor progression in multiple cancer types, including myeloid malignancies.30Increased inflammatory cytokines in the bone marrow niche can create genotoxic stress resulting in the dampening of growth signals in healthy HSPCs while promoting leukemic cell growth.31RUNX1 mutations have been shown to cause inflammation or inflammatory diseases in murine models and humans.22'32'36In particular, loss of RUNX1 in murine neutrophils caused increased secretion of cytokines such as TNF-a following activation of TLR4 signaling.19RUNX1 deletion in GMPs also caused inflammatory dysregulation through non-canonical type I IFN signaling.23A germline RUNX1- mutated mouse model (RunxlR1S8* / +) targeting an FPD mutation hotspot, also shows upregulation of inflammatory cytokines such as CCL11 and TNF-a in the bone marrow niche and increased myeloid skewing.22The present disclosure demonstrates that increased inflammatory stress is an early event in FPD HSPCs as they show increased levels of many inflammatory cytokines such as CCL24, CXCL8, GM-CSF, CCL2, and I L-1 p. FPD HSPCs show resistance to inflammation-induced cell growth suppression and increased myeloid bias.

[0040] Mechanistically, the present disclosure shows that an increase in many cytokines in FPD is due to increased CD74 signaling. CD74 is shown to also activate PI3K / AKT / mTOR pathway, important for cell proliferation and survival, as well as the release of numerous pro-tumorigenic cytokines such as IL-1 p, CCL2, CXCL8, and TNF-a, creating a feed-forward signaling loop.37'40Overexpression of CD74 is shown in multiple cancer types including AML, and the expression of CD74 / MIF is being evaluated in a clinical trial (NCT03918655) as a prognostic marker for AML progression.41'43Aspects of the current disclosure show that increased expression of CD74 and activation of PI3K / mTOR and JAK1 / 2 signaling provided an increased fitness advantage to FPD HSPCs. The present disclosure demonstrates the upregulation of JAK / STAT and PI3K / mTOR pathway upon activation of CD74 signaling using MIF stimulation and its reversal upon ISO-1 inhibition in FPD HSPCs. While PI3K / mTOR is known to be regulated by MIF / CD74 in other cell types, the direct regulation of the JAK / STAT signaling pathway via MIF / CD74 activation has not been shown in HSPCs. However, there is a correlation between overexpression of CD74 and STAT proteins in more aggressive tumors.44 45Aspects of the present disclosure further shows that the direct inhibition of CD74 signaling reversed phenotypic defects of FPD in vitro and in vivo and reduced cell-intrinsic inflammatory signaling pathways. Since the current inhibitors for directtargeting of CD74 signaling are not clinically approved,4647the regulatory-approved drugs that act downstream of CD74 signaling including PI3K / AKT / mT0R and JAK1 / 2 inhibitors were also tested.

[0041] Relevant to the goal of identifying treatments that could intercept leukemic transformation in RUNX1-FPD, previous studies have shown that inhibition of the PI3K / AKT / mTOR signaling have therapeutic value in leukemia.48Additionally, rescuing platelet counts and / or function has been shown with rapamycin treatment for immune thrombocytopenia4950, and idelal isib treatment for CLL patients.51The present disclosure demonstrates that PI3K / AKT / mTOR inhibitors rescued differentiation defects in human FPD progenitors by increasing megakaryocytes and a decreasing monocytes. Similar effects were observed upon targeting JAK1 / 2 using ruxolitinib. Interestingly, in vivo mTOR inhibition rescued megakaryocytic defects with a less than significant effect on myeloid skewing while JAK1 / 2 inhibition resulted in a more pronounced effect on myeloid differentiation and a minimal effect on megakaryopoiesis. Direct inhibition of CD74 signaling rescued both myeloid and megakaryocytic differentiation defects, and could be more effective being upstream of PI3K / mTOR and JAK signaling. A humanized CD74 antibody, milatuzumab (h LL1 ) has been tested in B-cell lymphoma, and showed efficacy in inhibiting leukemia cell growth in combination with anti-CD20 antibody. In addition, a monoclonal antibody against MIF, imalumab, also showed antitumor activity against solid tumors. While our data shows the efficacy of ISO-1 in inhibiting the MIF / CD74 pathway in RLWX7-mutated cells, future pre-clinical studies are needed to test the impact of milatuzumab or imalumab.

[0042] Based on the findings of increased inflammatory cytokines in the present disclosure, it indicates that dysregulated inflammatory signaling is a key contributor to leukemic risk in RUNX1- FPD. Deficiencies in efficient and durable engraftment occur when germline-mutated HSPCs have been used inadvertently as donor cells during hematopoietic stem cell transplantation (HSCT), suggesting abnormal interactions between HSPCs and stromal cells when either cell type has the germline mutation.2’52 53The present disclosure suggests that targeting inflammatory signals using CD74 pathway inhibitors can suppress inflammation and can potentially rescue some of these defects. To date, there are no cancer interception or prevention strategies for FPD patients who are at high risk for hematologic malignancies. The present disclosure also suggests that CD74 signaling could serve as a biomarker for disease severity or progression in FPD patients.

[0043] Provided herein are methods for diagnosing and treating familial platelet disorder in human subjects. The method comprises obtaining a biological sample from the subject, wherein the biological sample is peripheral blood or bone marrow aspirate; isolating CD34+ mononuclear cells from the biological sample obtained from the subject; detecting an upregulation of CD74signaling in the isolated CD34+ mononuclear cells; and administering to the subject a therapeutically effective amount of an agent that inhibits CD74 expression or comprises an antagonist of CD74 signaling.

[0044] Aspects of the current disclosure are now described with additional details and options as follows: (I) Definitions; (II) Drug / lnhibitor Families; (III) Methods of Use; (IV) Examples; and (V) References. These sections do not limit the interpretation of the disclosure and are provided for organizational purposes only.(I) Definitions

[0045] Familial Platelet Disorder (FPD): FPD refers to a genetic, constitutional thrombocytopenia disease characterized by mild to moderately low platelet count, abnormal platelet function, easy bruising and / or prolonged bleeding, and an increased risk of developing a hematologic malignancy or blood cancers such as myelodysplastic syndrome and acute myeloid leukemia. FPD is associated with germline RUNX1 mutations, establishing a preleukemic state and increasing the risk of developing acute leukemia.

[0046] Biomarker: A biomarker refers to molecular, biological or physical attributes that characterize a physiological, cellular, or disease state and that can be objectively measured to detect or define disease progression or predict or quantify therapeutic responses. A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. A biomarker may be any molecular structure produced by a cell or organism. A biomarker may be expressed inside any cell or tissue; accessible on the surface of a tissue or cell; structurally inherent to a cell or tissue such as a structural component, secreted by a cell or tissue, produced by the breakdown of a cell or tissue through processes such as necrosis, apoptosis or the like; or any combination of these. A biomarker may be any protein, carbohydrate, fat, nucleic acid, catalytic site, or any combination of these such as an enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle, or any uni- or multimolecular structure or any other such structure now known or yet to be disclosed whether alone or in combination. In embodiments, a biomarker can be a receptor expressed on a surface of a cell, such as CD74 receptor. In embodiments, biomarkers disclosed herein can be used to diagnose FPD in a subject. In embodiments, biomarkers disclosed herein can be used to develop treatment methods for treating FPD in a subject.

[0047] Assays known to one of skill in the art can be used to measure a level of a biomarker. For example, the quantity of one or more biomarkers can be indicated as a value. The value canbe expressed numerically and result from assaying a sample, and can be derived, e.g., by measuring level(s) of the biomarker(s) in the sample by an assay performed in a laboratory, by measuring the ratio or ratios of the levels of two or more biomarkers, or from a dataset obtained from a provider such as a laboratory, or from a dataset stored on a server. The value may be qualitative or quantitative. As such, where detection is qualitative, the systems and methods provide a reading or evaluation, e.g., assessment, of whether or not the biomarker is present in the sample being assayed. The systems and methods may provide a quantitative detection of whether the biomarker is present in the sample being assayed, i.e. , an evaluation or assessment of the actual amount or relative abundance of the biomarker in the sample being assayed. In such cases, the quantitative detection may be absolute or, if the method is a method of detecting two or more different biomarkers in a sample, relative. As such, the term “quantifying” when used in the context of quantifying a biomarker in a sample can refer to absolute or to relative quantification. Absolute quantification can be accomplished by inclusion of known concentration(s) of one or more control biomarkers and referencing, e.g., normalizing, the detected level of the biomarker with the known control biomarkers (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different biomarkers to provide a relative quantification of each of the two or more biomarkers, e.g., relative to each other. The actual measurement of values of the biomarkers can be determined using any method known in the art.

[0048] Biological sample: "Sample" or "biological sample" refers to a biological material isolated from or derived from a subject. Embodiments of “derived from” refer to a biological sample being obtained from a subject or other source and including any modification to the sample, addition to the sample, or removal from the sample, as long as biomarkers of the present disclosure can be measured from the sample using the systems and methods of the present disclosure. The biological sample can contain any biological material suitable for detecting a mRNA, polypeptide or other marker of a physiologic or pathologic process in a subject, and can include fluid, tissue, cellular and / or non-cellular material obtained from the individual. In embodiments, a biological sample can include blood, serum, cells, plasma, cerebral spinal fluid, and urine. In embodiments, a biological sample can include serum. Serum from blood is a light yellow, clear liquid that remains after blood has clotted. Serum can be obtained by centrifuging clotted blood. Serum does not include an anti-coagulant. In embodiments, a biological sample can include plasma. Plasma is a light yellow, clear liquid that remains when blood clotting is prevented and can be obtained by centrifuging whole blood containing an anti-coagulant.

[0049] In embodiments, a biological sample may include cells. In embodiments, samples used in the methods of the present disclosure include mononuclear cells isolated from peripheral blood (PBMCs) or from bone marrow aspirates. Mononuclear cells can be isolated by any technique known in the art, including density centrifugation (e.g., with Ficoll-Paque). Density gradient centrifugation separates cells by cell density. In embodiments, PBMC can be isolated by leukapheresis. A leukapheresis machine is an automated device that takes whole blood from a donor and separates out the target PBMC fraction using high-speed centrifugation while returning the remaining portion of the blood, including plasma, red blood cells, and granulocytes, back to the donor.

[0050] Hematological Malignancy: Hematological malignancy refers to a general term for cancers that affects the blood or bone marrow.

[0051] Therapeutic Agent: An “agent” or a “therapeutic agent” refers to any biologically active compound capable of treating or inhibiting at least one disease state or condition. The agent or therapeutic agent is sufficient to yield a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic response. A therapeutic agent may comprise a wide variety of compounds such as drugs, inhibitors, small-molecule compounds, antibodies, antibody fragments, nanobodies, and the like. In some embodiments of the present disclosure, a therapeutic agent may include clinically approved drugs targeting cell signaling pathways such as PI3K / mTOR and JAK signaling. In some embodiments of the present disclosure, a therapeutic agent may include inhibitors or small-molecule antagonists of MIF / CD74 function. In some embodiments of the present disclosure, a therapeutic agent may include monoclonal antibodies against MIF and its receptor, CD74.

[0052] Effective Amount: An “effective amount” or a “therapeutically effective amount” of an agent is an amount that upon administration to a subject in need thereof is sufficient to generate a desired response such as reducing or eliminating a sign or symptom of a condition or a disease. An effective amount also encompasses an effective amount of a first agent and an effective amount of a second agent administered in combination with the first agent. In some examples, the effective amount of the two combined agents is less than that of either agent when administered alone. In some examples, the effective amount of the two combined agents is more than that of either agent when administered alone.

[0053] Subject or patient: A “subject” or a “patient” refers to a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A "mammal" includes both human and non-human mammals, such as mice. In some embodiments of the present disclosure, a subject is a human patient, such as a patient diagnosed with familial platelet disorder (FPD). Insome embodiments of the present disclosure, a subject is a human patient yet to be diagnosed with FPD.

[0054] Treatment: Treatment refers to any therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, an improvement in the overall health or well-being of the subject, or by other clinical or physiological parameters associated with a particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. A “therapeutic” treatment is a treatment administered after the development of significant signs or symptoms of the disease. The terms “for treatment of”, “for use in the treatment of”, “for treating”, “for use in treating” and the like are understood to be synonymous and may be interchanged in describing and claiming the methods herein.(II) Inhibitor / Drug Families

[0055] The following table provides a summary of some inhibitor / drug families described in the present disclosure and exemplary drugs in each of these families.Table 1 : Families of therapeutic agents / inhibitors / drugs

[0056] In embodiments, MIF / CD74 inhibitors or small-molecule antagonists can be administered to treat a subject with familial platelet disorder. MIF / CD74 inhibitors include (S,R)-3-(4 hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), AV411 (Ibudilast), N- acetyl-p-benzoquinone imine (NAPQI), 4-iodo-6-phenylpyrimidine (4-IPP), P425, and CPSI- 1306.

[0057] In embodiments, PI3K / mTOR inhibitors in a combination therapy with MIF / CD74 antagonists can be administered to treat a subject with FPD. PI3K / mTOR inhibitors include Idelalisib, Rapamycin, Vistusertib (AZD2014), Temsirolimus, Everolimus, and Ridaforolimus.

[0058] In embodiments, JAK inhibitors in a combination therapy with MIF / CD74 antagonists can be administered to treat a subject with FPD. JAK inhibitors include Ruxolitinib (INCB018424), JAK Inhibitor I, Tofacitinib (CP-690550), Midostaurin, and TG101348.(Ill) Methods of Use / Treatment

[0059] Disclosed are methods of treating a subject with familial platelet disorder using therapeutic agents described herein. The agents can be administered by any appropriate route including orally or parenterally including buccally, sublingually, sublabially, by inhalation, intraarterially, intravenously, intraventricularly, intramuscularly, subcutaneously, intraspinally, intraorbitally, intracranially or intrathecal ly.

[0060] The administration of a therapeutic agent can be for prophylactic or therapeutic purposes. For prophylactic and therapeutic purposes, the treatments can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the treatment for a disease can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a disease or condition.

[0061] Therapeutic treatments can be distinguished from effective amounts based on the presence or absence of a research component to the administration. As will be understood by one of ordinary skill in the art, however, in human clinical trials effective amounts, prophylactic treatments and therapeutic treatments can overlap.

[0062] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by the subject, a physician, veterinarian, or researcher taking into account parameters such as physical, physiological and psychological factors including target, body weight, condition, previous or concurrent therapeutic interventions, and / or idiopathy of the subject.

[0063] An effective amount or concentration of a therapeutic agent can be any amount administered alone or in combination with additional therapeutic agents, is sufficient to achieve a desired effect in a subject. The effective amount of the agent will be dependent on several factors, including, but not limited to, the subject being treated and the manner of administration of the agent. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease or condition, or which is capable of reducing symptoms caused by any disease or condition.

[0064] In one example, a desired effect is to reduce or inhibit one or more symptoms associated with a disease or condition characterized by familial platelet disorder. The one or more symptoms do not have to be completely eliminated for the therapeutic agent to be effective. For example, an agent can decrease the sign or symptom by a desired amount, for example by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to how the sign or symptom would have progressed in the absence of the therapeutic agent or in comparison to currently available treatments.

[0065] The actual effective amount will vary according to factors such as the type of disease to be protected against / therapeutically treated and the particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like) time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of treatments for familial platelet disorder for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.

[0066] The present disclosure provides a method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ mononuclear cells, themethod comprising administering to the subject a therapeutically effective amount of an agent that inhibits or reduces expression of CD74 receptor or comprises an antagonist of CD74 function. In embodiments, the CD34+ mononuclear cells comprise CD34+ progenitor cells. In embodiments, the upregulation of CD74 signaling is determined by comparing percentage positivity of the CD34+ progenitor cells from the biological sample of the FPD subject with a biological sample from a healthy subject In embodiments, the biological sample is determined to have CD74 signaling upregulated if the percentage positivity of the CD34+ progenitor cells exceeds a threshold of 1.5 fold compared to a threshold obtained from the healthy subject.

[0067] In embodiments, the inhibitor or small-molecule antagonist of MIF / CD74 signaling may be selected from the group of (S,R)-3-(4 hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), AV411 (Ibudilast), N-acetyl-p-benzoquinone imine (NAPQI), 4-iodo-6- phenylpyrimidine (4-IPP), P425, and CPSI- 1306. In some cases, the agent inhibiting MIF / CD74 signaling may comprise ISO-1 , and wherein ISO-1 may be administered to a subject in need thereof at a dose of from about 1 mg / kg to about 10 mg / kg. Alternatively, monoclonal antibodies or antibody fragments against CD74 receptor (e.g., Milatuzumab) or against MIF (e.g., Imalumab, BaxG03, BaxB01 , and BaxM159) may also be used as inhibitors of MIF / CD74 signaling. Bioengineered anti-MIF nanobodies are also developed and demonstrated the effective inhibitory effect on MIF-mediated inflammatory responses during multiple pathological states. Several preclinical studies and clinical trials have been conducted to evaluate the safety and biological activity of anti-MIF antibodies and MIF antagonists65. MIF antagonists have been reported to be efficient inhibitors of MIF’s tautomerase activity. The major classes of MIF inhibitory chemical compounds are the derivatives of dopachrome, acetaminophen, isoxazolines, acetylenic compounds, phenyl pyrimidine, isothiocyanate, benzoxazinone, benzoxazol-2-one, chromene, isoxazoline, pyrimidazole, isocoumarin, phenyl pyruvic acid, Schiff bases, cinnamates, plant- derived compounds, oxygen heterocycles, and curcumin65.

[0068] The present disclosure also provides a method of treating familial platelet disorder (FPD) in a subject in need thereof, the method comprising: obtaining a biological sample from the subject, wherein the biological sample comprises peripheral blood or bone marrow aspirate; isolating CD34+ mononuclear cells from the biological sample obtained from the subject; detecting an upregulation of CD74 signaling in the isolated CD34+ mononuclear cells; and administering to the subject a therapeutically effective amount of an agent that inhibits CD74 expression or comprises an antagonist of CD74 signaling. In embodiments, the CD34+ mononuclear cells comprise CD34+ progenitor cells.

[0069] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an agent that inhibits PI3K / mTOR pathway downstream of CD74 signaling in combination with MIF / CD74 antagonist. In some cases, the agent inhibiting PI3K / mTOR pathway is selected from the group of Rapamycin / Sirolimus, Vistusertib (AZD2014), and Idelalisib. It is to be noted that several embodiments of the present disclosure utilize rapamycin inhibitor. Rapamycin, which is also known as Sirolimus, is an inhibitor of PI3K / mTOR pathway. In the present disclosure, the terms “rapamycin” and “sirolimus” have been used interchangeably throughout the disclosure.

[0070] In some embodiments, rapamycin or sirolimus may be administered to a subject in need thereof at a dose of from about 0.1 mg to about 10 mg per day. In other embodiments, rapamycin may be administered at a dose of from about 1 mg to about 7.5 mg per day. In further embodiments, rapamycin may be administered at a dose of from about 1 mg to about 5 mg per day. In some embodiments, these daily doses may be administered on days following an initial dose of from about 2.5 mg to about 15 mg. In other embodiments, the initial dose may be administered at from about 3 mg to about 10 mg.

[0071] In some embodiments, particularly including child subjects, rapamycin or sirolimus may be administered at a daily dose of from about 1 mg / m2 / day to about 5 mg / m2 / day. In other separate embodiments, the dosing is, respectively, from about 1 mg / m2 / day to about 4 mg / m2 / day; from about 1 mg / m2 / day to about 3 mg / m2 / day; and from about 1 mg / m2 / day to about 2 mg / m2 / day.

[0072] In some embodiments, idelalisib may be administered to a subject in need thereof at a dose of from about 1 mg BID (twice daily) to about 200 mg BID. In other embodiments, idelalisib may be administered at a dose of from about 10 mg BID to about 200 mg BID. In other embodiments, idelalisib may be administered at a dose of from about 25 mg BID to about 150 mg BID. In further embodiments, idelalisib may be administered at a dose of from about 50 mg BID to about 150 mg BID. In additional embodiments, idelalisib may be administered at a dose of from about 100 mg BID (twice daily) to about 150 mg BID.

[0073] In some embodiments, vistusertib may be administered to a subject in need thereof at a dose of from about 1 mg BID to about 150 mg BID. In other embodiments, vistusertib may be administered at a dose of from about 10 mg BID to about 120 mg BID. In other embodiments, vistusertib may be administered at a dose of from about 20 mg BID to about 100 mg BID. In other embodiments, vistusertib may be administered at a dose of from about 25 mg BID to about 75 mg BID. In other embodiments, vistusertib may be administered at a dose of from about 40 mg BID to about 60 mg BID. In other embodiments, vistusertib may be administered at a dose of about 50 mg BID.

[0074] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an agent that inhibits JAK / STAT pathway downstream of CD74 signaling in combination with MIF / CD74 antagonist. In some cases, the agent inhibiting JAK / STAT pathway comprises ruxolitinib. In some cases, other inhibitors of Janus Kinase family may also be used such as JAK Inhibitor I or Tofacitinib.

[0075] In some embodiments, ruxolitinib may be administered to a subject in need thereof at a dose of from about 1 mg BID to about 150 mg BID. In other embodiments, ruxolitinib may be administered to a subject in need thereof at a dose of from about 1 mg BID to about 100 mg BID. In some embodiments, ruxolitinib may be administered to a subject in need thereof at a dose of from about 1 mg BID to about 75 mg BID. In separate embodiments, ruxolitinib may be administered in dose ranges, respectively, of from about 5 mg BID to about 25 mg BID; from about 25 mg BID to about 50 mg BID; from about 50 mg BID to about 75 mg BID; from about 20 mg BID to about 30 mg BID; and from about 40 mg BID to about 60 mg BID. In other embodiments, ruxolitinib is administered at doses, respectively, of 5 mg BID, 10 mg BID, 20 mg BID, 25 mg BID, 30 mg BID, 40 mg BID, 50 mg BID, 60 mg BID, 70 mg BID, and 75 mg BID.

[0076] In some aspects, the present disclosure provides a method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ progenitor cells, the method comprising administering to the subject a therapeutically effective amount of a first agent that reduces expression of CD74 receptor or comprises an antagonist of CD74 signaling, and a therapeutically effective amount of a second agent that inhibits PI3K / mTOR pathway downstream of CD74 signaling. In embodiments, the first agent and the second agent are administered to the subject simultaneously or separately. In some embodiments, the first agent comprises ISO-1 or an anti-CD74 monoclonal antibody, and the second agent comprises Rapamycin, Vistusertib (AZD2014), or Idelalisib.

[0077] In some aspects, the present disclosure provides a method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ progenitor cells, the method comprising administering to the subject a therapeutically effective amount of a first agent that reduces expression of CD74 receptor or comprises an antagonist of CD74 signaling, and a therapeutically effective amount of a second agent that inhibits JAK / STAT pathway downstream of CD74 signaling. In embodiments, the first agent and the second agent are administered to the subject simultaneously or separately. In some embodiments, the first agent comprises ISO-1 or an anti-CD74 monoclonal antibody, and the second agent comprises ruxolitinib. 1

[0078] As described above, an effective amount is one in which any toxic or detrimental side effects of the compound and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. Determination of effective amount is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease or condition symptoms in the subject. Suitable models in this regard include, for example, murine, rat, rabbit, porcine, feline, non-human primate, and other accepted animal model subjects known in the arts. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the treatments for FPD patients.

[0079] The examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the scope of the disclosure.(IV) Examples

[0080] Example 1 : Materials and Methods

[0081] Statistical analysis: For in vitro experiments using patient samples, statistical analyses and graphing of the data was performed in Microsoft Excel and GraphPad Prism, version 8 (GraphPad Software) with the two-tailed, unpaired Student’s t-test, or two-way ANOVA test; adjusted p-values were calculated using t-test and Bonferroni correction. Flow-cytometry data were analyzed with FlowJo software. Data are expressed as means and standard errors. No samples were excluded from the analyses, and no randomization of samples was performed. A p-value and adjusted p-value of less than 0.05 was considered to indicate statistical significance.

[0082] Patient sample processing: All samples were obtained from patients evaluated at National Institute of Health (NIH) and Oregon Health & Science University (OHSU). Patients were consented using guidelines approved by the Institutional Review Boards at each location. Fresh bone marrow (BM) and peripheral blood (PB) samples from FPD patients and healthy donors were obtained from a natural history study under an NIH IRB-approved protocol (NCT03854318) and OHSU IRB.21Bone marrow mononuclear cells (MNCs) and purified CD34+ cells from normal donors were purchased commercially (Lonza, Walkersville, MD). MNCs from patient samples were isolated by centrifugation through a Ficoll-Paque density gradient54, and red blood cells were lysed using an aqueous solution of 0.15 M NH4CI, 10 mM KHCO3 and 0.1 mM EDTA. Plasma samples were prepared by centrifuging bone marrow and peripheral blood samples at 2500 rpmfor 10 min. CD34+ or CD14+ cells were enriched using an immunomagnetic column (Miltenyi Biotech, San Diego, CA) and purity was evaluated using a FACSAria I Hu flow cytometer (BD Biosciences, San Jose, CA). Primary MNCs and CD34+ cells were either freshly analyzed without prior treatment or cultured overnight in cytokine-free media with 20% BIT (bovine insulin transferrin, Stem Cells Technologies, Vancouver, BC, Canada). The isolated cells were used in subsequent experiments accordingly.

[0083] In vitro patient sample assays: Isolated mononuclear cells and HSPCs from bone marrow samples were cultured in IMDM medium supplemented with 20% FBS, 2mM L-glutamine, and 100U / mL penicillin-streptomycin. IL-3 (25 ng / mL), IL-6 (20 ng / mL), SCF (100 ng / mL), FLT3- ligand (50 ng / mL), TPO (25 ng / mL) cytokines. Small molecules of StemRegeninl (SR1 , 0.5 uM) as well as UM729 (0.5 uM) were supplemented.CD34+ retention: isolated CD34+ HSPCs were cultured at 10,000 cells per well in presence of indicated cytokines over 8 weeks. The percentage of CD34+ cells along with other differentiated markers were determined using flow cytometry with indicated antibodies (Table 2).Table 2: Flow cytometry antibodies used for human cellsInhibitor assay: isolated mononuclear cells were cultured at 20,000-30,000 cells per well in presence of indicated inhibitors at 3 different concentrations. Compound concentrations chosen as not to be toxic for the cells based on initial MTS screenings. Cells were cultured for 7 days with feeding twice in between using IMDM medium supplemented with 20% FBS, 2mM Lglutamine, and 100U / mL penicillin-streptomycin. IL-3 (25 ng / mL), IL-6 (20 ng / mL), SCF (100 ng / mL), FLT3- ligand (50 ng / mL), TPO (25 ng / mL) cytokines. Small molecules of StemRegeninl (SR1 , 0.5 pM), as well as UM729 (0.5 pM), were supplemented. MIF was not supplemented in culture media as MIF is secreted with many cell types in media. Cell counts were obtained to determine and cells maintained >90% viability in all conditions. Differentiation statues of cells were determined using flow cytometry.Lineage differentiation: Purified CD34+ HSPCs were cultured at 2,000 cells per well in 200uL using StemSpan SFEM II media (StemCell, catalog 9655). Myeloid (StemCell, catalog 2694), megakaryocyte (StemCell, catalog 2692), and erythroid (StemCell, catalog 2696) lineage differentiation cytokine cocktails were added at 1x final concentration. Cells were fed accordingly twice a week. The differentiation capacity of cells was determined over 2 weeks using flow cytometry.

[0084] Colony formation assay: Human Samples'. Isolated CD34+ cells from bone marrow samples of FPD and HD primary patients were cultured in methocult H4434 (StemCell) at 1000 cells per well for 14 days at 37°C in 5% CO2 and in presence of different cytokine and inhibitors with mentioned concentrations. Colonies counted and identified according to the company’s characterization. Cells harvested from colonies and re-plated at 100,000 cells per well for 14 days. Small fraction of cells used for flow cytometry analysis as well. Murine Samples’. Lineage negative cells were isolated from wildtype (WT) and Runx1R188Q / +(Het) bone marrow cells via magnetic selection and cultured overnight in DMEM medium supplemented with 15% FBS, 15% WEHI, 2mM L-glutamine, and 100U / mL penicillin-streptomycin. Supplementary cytokines IL-3 (7 ng / mL), IL-6 (12 ng / mL), and SCF (56 ng / mL) were also added. Lineage negative cells either used to flow sort Lineage- Sca1+ cKit+ (LSK) cells or directly seeded in methocult M3434 (StemCell) andcultured for 7 days. For LSK cells, 600 cells per well and for lineage negative cells, 3000 cells per well used for initial plating in presence of indicated cytokines and inhibitors. Cells harvested after counting colonies and re-plated at 100,000 and 200,000 cells per well for lineage negative and LSK cells, respectively.

[0085] Cytokine measurement and analysis: Cytokine concentrations in the plasma of primary FPD and healthy samples were measured using a 65-plex multiplex magnetic beadbased Luminex assay (ThermoFisher Scientific). Additionally, CD34+ progenitors purified from samples were cultured for 48 hrs and conditioned media was evaluated for secreted cytokines and growth factors. Standard curves were evaluated using ProcartaPlex software (ThermoFisher Scientific) and each cytokine concentration was determined in individual samples. Average of healthy samples was used to calculated fold differences in FPD over healthy controls. Bone marrow from tibia of the mice was flushed at once with 200uL media (DMEM+10%FBS). Cells were spun down and media collected to perform cytokine measurement using 36-plex multiplex Luminex assay (ThermoFisher Scientific).

[0086] Phosphoflow and intracellular flow cytometry: Cells were treated with the indicated concentrations of inhibitors for 48 hours and fixed / permeabilized using BD cytofix / cytoperm kit and according to company protocol (BD Bioscience 554714). Cells were stained with extracellular and intracellular antibodies (Table 2) for 1 hour at room temp and analyzed using Cytek™ Aurora flow cytometry machine.

[0087] Immunoblotting analysis: RUNX1- mutation positive OCI-AML5 cells were treated with MIF (50ng / mL) for 30 and 60 minutes in MEM-a+0.1%BSA media. Similarly, OCI-AML5 cell line with CD74 knockout were cultured overnight in MEM-a+0.1%BSA and treated with MIF (50ng / mL) and FBS (10%) for 30 minutes and 1 hour. Harvested cells were subject to immunoblotting analysis using antibodies for total and phosphorylation of indicated proteins using actin as a loading control. iPSCs cells derived from FPD patients (JPC192) and CRISPR corrected for RUNX1-mutation (JPC323) and differentiated into CD34+ cells in vitro. Immunoblotting analysis was performed using vinculin as a control. Since CD74 is type II transmembrane receptor, an antibody recognizing the C-terminal (Lysine 190) end of the protein was used.

[0088] Human CD34+ CRISPR editing: Guide RNA (sgRNA) for the human CD74 gene was synthesized. Gene Knockout kit (Synthego) was used to edit purified CD34+ FPD cells according to the company’s protocol. Briefly, 100-150k cells were mixed in nucleofector solution, 60pmol sgRNA and 20pmol Cas9 were added, and the mixture was subjected to electroporation. The cells were recovered overnight in serum-free media (SEM II, StemCell) supplemented with SCF(100ng / mL), TPO (100ng / mL), FLT3-L (WOng / mL), IL-6 (lOOng / mL), LDL (10ug / mL), and UM171 (35nM).

[0089] CD74 Overexpression: CD74 gene (NM_001025158.3) overexpression vector was synthesized commercially in Tet-on inducible lentivirus expressing GFP+ (Vector Builder). Lentivirus was generated by transfecting 293FT cells using VSVG and psPAX2 helper plasmids. Viral particles were collected and concentrated by ultracentrifugation at 28,000 rpm for 2 hours. Primary CD34+were infected with the concentrated virus in the presence of polybrene (5pg / mL) and HEPES (1 mM) and centrifuged at 2,400 rpm for 90 minutes. The cells were treated with doxycycline (1 pg / mL) after 48 hours of transfection and used for colony formation assay in methocult (H4434, StemCell) at 1000 cells / well concentration. The leftover cells were cultured for another 48 hours and analyzed for overexpression of CD74 using flow cytometry. Colonies counted on day 14 post-culture.

[0090] Mouse model and in vivo inhibitor treatments: Gender- and age-matched wild type and Runx1R188Q / +mice (C57BL / 6J-Runx1<tm1Lhc>R188Q) were selected22. Mice were treated with rapamycin (4.8mg / kg, every other day, via intraperitoneal injection), ruxolitinib (36mg / kg, every day, via oral gavage), ISO-1 (5mg / kg, every day, via intraperitoneal injection), and vehicle for 8 weeks. Rapamycin was prepared in 5% Tween-80 and 5% PEG-400, ruxolitinib in 1% (w / v) methylcellulose in saline buffer, and ISO-1 in PBS. Mice were monitored for weight loss and differential blood count data. At the end point, mice were sacrificed. Bone marrow and peripheral blood samples were collected, processed to mononuclear cells and stained with mouse hematopoietic antibodies (Table 3) to be analyzed using flow cytometry.Table 3: Flow cytometry antibodies used for mouse cells

[0091] In vitro murine platelet assay: mouse blood was collected and diluted with 3.8% sodium citrate in 9 to 1 ratio. Platelets in peripheral blood was activated with 0.1U thrombin for 15 minutes at room temperature while staining with flow antibodies (CD41 , CD61 , CD62P, and Terri 19. Table 3). Buffer HEPES / Tyrode (H-T) (129 mM NaCI, 0.34 mM Na2HPO4, 2.9 mM KCI, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCI2; pH 7.3) was used to dilute samples. Samples were fixed and analyzed using flow cytometry. The results were compared between activated and non-activated groups.

[0092] Single-Cell RNA-Sequencing: Freshly purified MNCs were derived from FPD and healthy donors. Each sample was loaded onto a Chromium Controller (10x Genomics) and libraries were generated following manufacture instructions and sequenced using a NovaSeq- 6000. The data analysis methods are described below. Validation of key pathways was performed using GeoMx Digital Spatial Profiling (DSP) and nanostring analyses as described below.

[0093] Nanostring: Magnetically isolated CD34+ cells from FPD bone marrow (n=3) were treated with MIF (10ng / ml_), ISO-1 (100nM) and vehicle for 48 hours. Cells were pelleted and RNA was extracted using Qiagen RNA extraction kit (RNeasy Plus Micro Kit, catalog 74034). RNA (50ng) products were set up for hybridization with capture and reporter probes overnight for 16 hours according to the company’s protocol (Nanostring, catalog MAN10051). The nCounter Pan Cancer Pathways panel was used (Nanostring, catalog 115000092). The hybridization products were loaded on the cartridge (lot 3010272353) and nCounter SPRINT machine was used to run. Data analysis was done using nSolver 4.0 and gene expression was determined after internal data clean up and QC.55

[0094] Nanostring GeoMX digital spatial profiling (DSP): bone marrow biopsy slides were prepared at the NIH through fixation and decalcification process via neutral buffered formalin.Tissue slides were stained with multiplex panel of antibodies containing photocleavable indexing oligonucleotides. Between 12-25 regions of interest (ROIs) were selected for each slide randomly based on CD45 staining among with DNA and PanCK staining. DSP prototype instrument was then used to illuminate UV light to localized ROIs to release oligonucleotides and collection into microcapillary tubes. Spatially resolved oligonucleotides are then hybridized to Nanostring fluorescent barcodes and quantified via an nCounter SPRINT machine. The geometric mean of all markers were used to calculate the background noise. Markers of interest were normalized to GAPDH in each ROI and fold changes for FPD samples was calculated over average HD samples.5657

[0095] Single cell sequencing analysis: Data was pre-processed using cellRanger v 3.0.2. All scRNA-seq preprocessing was done using Seruat v3. Cells were filtered based upon the nFeatures_RNA and percent mitochondrial reads. Using Seraut v3 integrateData function, all samples were integrated. Dimensionality reduction was done using UMAP using n.neighbors=40 and min.dist = 0.3 (CITE). Cells were assigned their cell types based upon their expression of cell type specific markers found in Van Galen et al. The population of MKP cells were found looking at the average expression of cell specific markers found in Yi-Chien Lu et al. Differential expression was assessed using the findMarkers function in Seurat using default parameters, as well as logfc.threshold = -Inf, min. pct = -Inf and diff.pct = -Inf so as to include all genes. To control for donor specific effects, each comparison was down sampled to an equal number of cells per donor per comparison.58’59’60’61

[0096] Velocity analysis: RNA velocity of single cells was estimated by distinguishing between unspliced and spliced mRNAs, which is predictive of the rate of transcriptome change over time, using Velocyto [version 0.17.17], 8 samples (v3 10x kit unenriched: 5 FPD and 3 control) were aligned to reference genome GRCh38-3.0.0 with Cellranger (v. 3.0.2). UMI count matrices were masked using the UCSC repeatMasker track. Spliced and unspliced mRNAs were quantified with Velocyto, using the velocyto run 10x command (La Manno et al. 2018). The python program scvelo (https: / / qithub.com / theislab / scvelo) was used to infer future states of single cells. The samples were filtered and normalized with scv.pp.filter_and_normalize(min_shared_counts=20, n_top_genes=None). First and second order moments were calculated with scvelo.pp. moments (n_pcs = 30, n_neighbors = 30). Velocities were fit with the scvelo. tl.velocity() (mode set to ‘dynamical’) and scvelo.tl.velocity_graph() functions. The scvelo.tl.velocity_embedding() function was used to visualize velocities on UMAP coordinates previously assigned.62 63

[0097] Example 2: RUNX1-FPD HSPCs exhibit increased stem cell retention, serial replating, and myeloid differentiation along with defective megakaryopoiesis in vitro.

[0098] Bone marrow cells derived from FPD individuals harboring various RUNX1 mutations (Table 4) were used who have not developed leukemia yet. Most of these patients have bleeding disorders and low platelet counts. It was found that in cell growth assays, FPD HSPCs retained higher CD34 positivity with a higher percentage of myeloid and monocyte progenitors (CD34+ / 33+ / 13+) by week 2 compared to healthy HSPCs (Figure 1A), indicating increased stem cell retention and myeloid differentiation. Accordingly, FPD cells also exhibited an elevated percentage of pro-myeloid / monocyte (CD33+ / 13+) and myeloid (CD13+ / 14+) cell populations in cytokine supplemented and lineage-specific cytokine media. The granulocyte population (CD11b714 ) was similar between FPD and healthy cells. Furthermore, FPD HSPCs had reduced megakaryocytic (CD41761+) differentiation capacity and trended towards slower erythroid differentiation with slightly lower erythroblasts (CD235771+) compared to healthy HSPCs. Consistently, FPD HSPCs showed an increased number of myeloid colonies, particularly granulocyte-macrophage progenitor (CFU-GM) and macrophage (CFU-M) colonies compared to healthy HSPCs (Figure 1B). Additionally, FPD progenitors displayed enhanced serial replating with increased myeloid colonies while healthy progenitors failed to grow at second plating. It was found that 87% of FPD samples have increased myeloid differentiation, 75% have increased myeloid colony formation ability and 92% have a decrease in megakaryopoiesis regardless of mutation type. Overall, the results demonstrated FPD progenitors exhibit increased myeloid bias with defective megakaryocytic differentiation in vitro.Table 4: FPD and HD patient sample data

[0099] Example 3: Single-cell RNA sequencing identified a unique transcriptome profile in FPD HSPCs compared to healthy HSPCs.[000100] To identify the mechanistic basis of the skewed differentiation and growth defects in FPD, single-cell RNA-sequencing (scRNA-seq) analysis was performed using healthy (N=4) and FPD bone marrow cells (N=10). Analysis of 48,781 healthy and 122,021 FPD cells was performed and 18 transcriptionally distinct clusters were identified. FPD samples exhibited altered cluster sizes with higher monocyte and T-cell populations and lower megakaryocyte-erythroid progenitors (MKP) and B-cell populations compared to healthy controls. Accordingly, there was a notable shift in gene expression between FPD versus healthy bone marrow in cluster-definingfeatures between early progenitors (CD34, MSI2, CD38l0W), committed progenitors of MKP and granulocyte-monocyte progenitors (GMP) (LYZ, AZU1, and MPO), and more mature lineages such as pro-monocytic and monocytic clusters LYZ, MPO, ELANE, FCER1G, CD14). Further, pseudotime analysis revealed a higher percentage of FPD cells differentiating towards monocytes, supporting enhanced myeloid differentiation. In contrast, healthy progenitors displayed a higher percentage of cells differentiating to megakaryocytes compared to FPD. RNA velocity analysis showed that FPD HSCs, GMPs, and monocytes have increased RNA velocity compared to healthy cells, indicating significantly increased transcriptional activity in those clusters.[000101] Differentially expressed genes (DEGs) from scRNA-seq showed increased expression of genes associated with myeloid differentiation and cytokine signaling, as well as enrichment of SPI1 and RELA transcription factors that are associated with myeloid differentiation and inflammatory signaling (Table 5). Collectively, these findings demonstrated that the myeloid bias of FPD HSPCs compared to healthy is likely due to the increased activation of the myeloid differentiation program. The simultaneous upregulation of cytokine signaling pathways is also a likely contributor to the observed myeloid skewing.Table 5: Differentially expressed genes[000102] Example 4: FPD stem and progenitor cells show upregulation of inflammatory pathways.[000103] The present disclosure reveals the upregulation of many inflammatory pathways in FPD HSCs as well as GMPs compared to healthy cells, including TNF-O / NF-KB, interferon-gamma response (IFN-y), inflammatory response, and TGF-p signaling (Tables 6 and 7). Accordingly, there is upregulation of many cytokines, chemokines, growth factors, and soluble receptors in the bone marrow extracellular fluid of FPD compared to healthy samples. Specifically, CCL24, CXCL8, GM-CSF, CCL2, IL-1p, TNF-a, and IL-6 were significantly upregulated (> 2-fold) in FPD compared to healthy donors. Similarly, increased cytokine levels were observed in peripheral blood samples, including IL-6, IL-1p, and GM-CSF. Cell type analysis showed that T-cells, myeloid, and stromal cells were the primary sources of upregulated cytokines. Enrichr and STRING-db analyses of upregulated cytokines showed their regulation via RELA, which was alsoenriched in transcription factor enrichment analysis using DEGs (Table 8). The upregulated cytokines are associated with hematopoietic differentiation as well as JAK / STAT and PI3K / AKT signaling. These results confirmed that FPD HSPCs experience high inflammatory stress in their microenvironment.Table 6: Pathway enrichment analysis for upregulated genes in HSC cluster via MSigDB Hallmark2020Table 7: Pathway enrichment analysis for upregulated genes in Progenitor cluster via MSigDBHallmark 2020Table 8: Transcription factor enrichment analysis for upregulated genes in Progenitor cluster viaENCODE and ChEA Consensus TFs from ChlP-X[000104] Example 5: Intrinsic and extrinsic inflammatory stress promotes the expansion and survival of FPD cells.[000105] Recent studies show that extrinsic inflammatory stress using lipopolysaccharide (LPS) promotes the production of cytokines from RLWXI-mutated neutrophils.1923In the present study, it was found that FPD CD34+progenitors and CD14+monocytes also show exaggerated production of cytokines (CXCL8, CCL24, IL-1 p, and CCL2) upon LPS exposure. This is consistent with the upregulated cytokines observed in bone marrow extracellular fluid in FPD. To understand the causal relation of upregulated cytokines, the impact of LPS and upregulated cytokines on myeloid colony formation was tested. FPD HSPCs showed resistance to growth suppression to LPS-mediated inflammatory stress while colony formation by healthy progenitors was significantlyreduced (Figure 2A). In secondary plating, FPD progenitors maintained myeloid colony formation ability while erythroid colonies were suppressed upon LPS exposure. Similar results were obtained upon exposure to individual cytokines including CCL2 and CCL24. CXCL8 and IL-1 p reduced colony formation of FPD in the first and second plating, respectively; but the reduction was not as pronounced as in healthy progenitors (Figures 2B-2D). Inhibition of these cytokines individually showed slight colony reduction (Figure 6) with minimal to no effect on the percentage of CD34+progenitors, monocytes, and megakaryocytes. These results suggest that targeting each cytokine individually is insufficient to reverse the effects. This could be due to compensatory roles played by multiple cytokines and prompted us to evaluate the upstream mechanism regulating cytokine expression. Overall, these results demonstrated that FPD HSPCs have fitness advantages and an increased myeloid bias due to intrinsic and extrinsic inflammatory stress.[000106] Example 6: CD74 receptor drives the activation of many prosurvival pathways and increases the secretion of inflammatory cytokines in FPD.[000107] The upstream regulator of elevated cytokines and prosurvival pathways in FPD HSPCs were investigated, in the present study, by evaluating receptor expression using scRNA-seq analysis data. The study revealed significant upregulation of CD74 expression in FPD HSC, progenitor, GMP, and monocyte clusters. Additional genes in the CD74 network such as CD44, CXCL8, and CXC1 are also upregulated in HSCs. It was confirmed that expression of CD74 in FPD CD34+HSPCs is high compared to healthy cells (Figure 3A). CD74, a type II transmembrane and a MIF receptor, is known to be expressed in antigen-presenting cells to regulate cell survival.24Further, immunohistochemistry of the bone marrow biopsies verified that CD74 levels are upregulated in FPD patients and remained upregulated in transformed germline RUNX1 MDS and CMML patients (Figure 3B). Primary AML samples harboring RUNX1 mutations have higher expression of CD74. Next, the impact of MIF / CD74 signaling on the growth and differentiation of FPD HSPCs was tested. Knocking out CD74 in FPD HSPCs caused the reduction of colony formation, while HD HSPCs showed no change (Figure 3C). CD74 has two ligands, MIF and MIF- 2 (D-DT). In our scRNA-seq analysis, MIF is overexpressed in FPD compared to healthy but not MIF-2. We confirmed the upregulation of MIF in cultured media of FPD cells and compared it to healthy cells. Further, MIF levels are significantly higher in FPD CD34+progenitors and stromal cells than in healthy controls. In AML patients, higher MIF expression is also associated with a slightly lower survival rate. FPD HSPCs showed resistance to MIF-induced growth suppression as seen in healthy cells with increased myeloid and granulocyte colonies (CFU-G and CFU-M) upon secondary plating (Figure 3D). Overexpression of CD74 in healthy CD34+ progenitorsshowed increased colony formation and showed similar resistance to MIF-induced colony formation ability reduction compared to healthy HSPCs without CD74 over expression as seen in FPD. Inhibition of MIF using ISO-1 , a MIF antagonist, suppressed myeloid colonies (CFU-GM) in FPD, with no effect on healthy progenitors (Figure 3E), suggesting healthy progenitors are protected from CD74 target inhibition. ISO-1 treatment in FPD also resulted in rescuing megakaryopoiesis in vitro (Figure 3F). Further, ISO-1 treatment of FPD CD34+cells resulted in decreased levels of multiple cytokines such as CCL24, IL-1 |3, TNF-a, and CXCL8 which were found to be upregulated in FPD bone marrow extracellular fluid and peripheral blood. Stimulation of FPD CD34+cells with MIF resulted in upregulation of PI3K, JAK / STAT, and MAPK pathways as measured via nanostring and GSEA analysis and consistent with scRNA-seq analysis. Immunoblot analysis using a RUNX1-mutated AML cell line (OCI-AML5) also confirmed that MIF stimulation caused upregulation of phosphorylated mTOR (mTOR, 4EBP1 , and S6), PI3K (p85), JAK / STAT (STAT-3 / 5), and MAPK (ERK1 / 2, and MEK) pathways members. Inhibition of MIF via ISO-1 decreased phosphorylation of ERK1 / 2, P65, STAT3, S6, and SRC in FPD CD34+cells by nanostring and phosphoflow analyses (Figure 3G). Similar results were obtained upon knocking out CD74 in FPD CD34+cells (Figure 3H) or in MIF-stimulated OCI-AML5 cells compared to nontargeting control. These results demonstrated that MIF / CD74 signaling drives increased expression of pro-survival pathways such as PI3K / mT0R and JAK / STAT as well as cytokines in FPD. Targeting MIF / CD74 rescued megakaryocytic and myeloid differentiation skewing in FPD HSPCs.[000108] Example 7: Targeting PI3K / mTOR or JAK pathways rescued FPD differentiation defects and suppressed the inflammatory stress.[000109] Single-cell analysis of FPD progenitors revealed upregulation of prosurvival pathways including mTORCI and PI3K / AKT, as well as inflammatory pathways such as JAK / STAT signaling. Digital spatial profiling (GeoMx DSP) confirmed the upregulation of PI3K / AKT / mTOR pathway member proteins (phosphorylated AKT1, S6, and 4EBP1) in FPD bone marrow biopsies compared to healthy controls and which remained upregulated in transformed MDS and CMML patients (Figure 4A). These pathways are also downstream of CD74 signaling (Figures 3A-3H). Therefore, to determine the significance of these pathways in rescuing the function of FPD progenitors, an in vitro small molecule inhibitors screen was performed using a concentration gradient. The least cytotoxic concentrations for each inhibitor were selected by comparing cell viability to identify therapeutic windows for RUNX-mutated FPD or AML cells compared to healthy or non-mutated AML cells, respectively (Figures 7A-7D). Specifically, inhibition of mTOR viaAZD2014 or rapamycin (sirolimus), PI3K / AKT pathway via idelalisib, or JAK kinase via ruxolitinib all significantly increased megakaryocytes while decreasing monocytes in FPD with no change in frequency of CD34+HSPCs (Figure 4B). These inhibitors also showed a significant reduction in myeloid colonies of FPD with minimal impact on healthy controls (Figure 4C). Interestingly, RUNX1-wi\d type and -mutated primary AML samples showed no difference in sensitivity to mT0R / PI3K (idelalisib, sirolimus) and JAK / STAT (ruxolitinib) inhibition (Figure 8A). However, the AML samples with higher CD74 expression showed significant sensitivity to these inhibitors (Figure 8B), suggesting CD74 expression can be used to stratify AML patients for the treatments. Further, it was observed that high CD74 expression and sensitivity to these pathways were regardless of RUNX1 mutation status in AML samples, suggesting that other mechanisms also regulate the CD74 expression and mT0R / PI3K and JAK / STAT signaling in AML. A decrease in cytokine levels was also observed upon JAK inhibition, including CCL24, CCL2, CXCL8, IL-1 , and MIF (Figures 8C-8F). Inhibition of mTOR, PI3K, and JAK pathways also reduced phosphorylation of 4eBP1 , S6, AKT, P65, STAT5, and SRC in FPD. These results suggest that the FPD inflammatory-rich microenvironment is due to the activation of PI3K / mT0R and JAK1 / 2 pathways which are downstream of CD74 signaling. Targeting these pathways reversed FPD phenotype and decreased inflammatory cytokines and has much more pronounced effects compared to single cytokine inhibition.[000110] Example 8: Targeting CD74-driven signaling shows a reversal in the megakaryocytic defect and suppression of myeloid differentiation in Runxl- mutated mice in vivo.[000111] The significance of targeting CD74 as well as downstream PI3K / mTOR and JAK1 / 2 signaling was validated using Runxl-mutated mice Runx1R188Q / +mice, equivalent to human R201Q). Heterozygous Runx1R188Q / +(Het) mice have an inflammatory-rich bone marrow microenvironment with cytokines such as CCL2, GM-CSF, and TNF-a upregulated. Heterozygous Runx1R188Q / +(Het) mice showed increased expression of CD74 in lineage-negative bone marrow cells (Figure 9A). In addition, Runx1R188Q / +bone marrow progenitors show increased colony number, and resistance to inflammatory stress compared to wild type (WT). MIF exposure resulted in a significant increase in Het granulocyte-macrophage (CFU-GM) and granulocyte (CFU-G) colonies compared to WT upon serial replating (Figure 5A). Importantly, inhibition of MIF via ISO-1 reduced the colony formation ability of Het cells. In an in vivo experiment, WT and Het mice were treated with ruxolitinib (JAK1 / 2i, 36mg / kg, every day, via oral gavage), rapamycin / sirolimus (mTORCIi, 4.8mg / kg, every other day, via intraperitoneal injection), andISO-1 (MIF-antagonist, 5mg / kg, every day, via intraperitoneal injection) for 8 weeks. A significant decrease (1.4-fold) was observed in CD62P+expression of megakaryocytes (CD41+ / 61+) upon platelet activation via thrombin stimulation in Het compared to WT mice. Het mice treated with rapamycin and ISO-1 showed significantly increased platelet activation, rescued to the level of WT, compared to the vehicle-treated control. This effect is specific to Het mice as treatments caused no significant changes in WT controls (Figure 5B). The same effect was not observed on platelet activation in ruxolitinib-treated Het mice; however, immunophenotyping of the bone marrow at the end point of the experiment showed a reduction in CD11 b+monocytes, neutrophils, and macrophages with ruxolitinib and ISO-1 treatments but not rapamycin / sirolimus (Figure 5C). Differential blood count analysis showed a slight decrease in white and red blood cell counts of Het mice with all the treatments (Figure 9B). No significant changes in platelet counts for all the treatments were observed. This is consistent with previous studies showing that the Runx1R188Q / +does not show differences in platelet counts at steady state, but reduced platelet activation was observed compared to wild-type mice (Figure 9C). Targeting CD74 signaling is able to rescue both myeloid and megakaryocytic differentiation defects more effectively compared to mTOR or JAK inhibition, being an upstream target. Taken together, these data showed that targeting CD74 and its downstream signaling results in the reversal of F?unx7-mutated murine cells, a phenotype similar to human FPD samples, and could serve as an early intervention strategy in FPD.(V) References1. Jongmans MC, Kuiper RP, Carmichael CL, et al. Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD / AML syndrome. Leukemia 2010;24:242-6.2. Owen CJ, Toze CL, Koochin A, et al. 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Enhancing autophagy protects platelets in immune thrombocytopenia patients. Ann Transl Med 2019;7:134.51. Reda G, Cassin R, Artoni A, et al. Idelalisib rapidly improves platelet function tests in patients with chronic lymphocytic leukaemia. Br J Haematol 2018;183:825-8.52. Rojek K, Nickels E, Neistadt B, et al. Identifying Inherited and Acquired Genetic Factors Involved in Poor Stem Cell Mobilization and Donor-Derived Malignancy. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 2016;22:2100-3.53. Xiao H, Shi J, Luo Y, et al. First report of multiple CEBPA mutations contributing to donor origin of leukemia relapse after allogeneic hematopoietic stem cell transplantation. Blood 2011 ;117:5257-60.54. Jaatinen T, Laine J. Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient. Curr Protoc Stem Cell Biol 2007;Chapter 2:Unit 2A.1.55. Malkov VA, Serikawa KA, Balantac N, et al. 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U.S. Patent No. 9,133,164 (Sep 2015).[000112] Each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.[000113] Unless otherwise indicated, all numbers expressing quantities of ingredients, agent / drug / inhibitor concentrations, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.[000114] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in their respective testing measurements.[000115] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.[000116] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.[000117] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.[000118] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.[000119] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.[000120] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.[000121] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006).

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing familial platelet disorder in a subject, the method comprising the steps of:(a) obtaining a biological sample from the subject;(b) isolating CD34+ mononuclear cells from the biological sample obtained from the subject;(c) measuring in the isolated CD34+ mononuclear cells an expression level of CD74 receptor;(d) diagnosing the subject based on the measured expression level of CD74 receptor in the CD34+ mononuclear cells; and(e) managing familial platelet disorder associated with the subject.

2. A method of diagnosing familial platelet disorder in a subject, the method comprising the steps of:(a) isolating CD34+ mononuclear cells from a biological sample obtained from the subject;(b) measuring in the isolated CD34+ mononuclear cells an expression level of CD74 receptor;(c) diagnosing the subject based on the measured expression level of CD74 receptor in the CD34+ mononuclear cells; and(d) managing familial platelet disorder associated with the subject.

3. The method of claim 1 , wherein the biological sample comprises peripheral blood or bone marrow aspirate.

4. The method of claim 1 , wherein the CD34+ mononuclear cells comprise CD34+ progenitor cells, and wherein measuring the expression level of CD74 receptor comprises performing flow cytometry on the CD34+ mononuclear cells.

5. The method of claim 4, wherein diagnosing the subject as having familial platelet disorder includes detecting an upregulation in the expression level of CD74 receptor in the CD34+ progenitor cells.

6. The method of claim 1 , wherein managing familial platelet disorder in the subject comprises administering to the subject in need thereof a therapeutically effective amount of one or more therapeutic agents selected from the group of a CD74 expression inhibitor and an antagonist of CD74 function.

7. The method of claim 6, wherein the one or more therapeutic agents are selected from the group of (S,R)-3-(4 hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) and an anti-CD74 monoclonal antibody.

8. The method of claim 6, further comprising administering to the subject in need thereof a therapeutically effective amount of one or more drugs that inhibit the PI3K / mT0R pathway or inhibit the JAK / STAT pathway downstream of CD74 signaling.

9. The method of claim 8, wherein the one or more drugs are selected from the group of Rapamycin, Vistusertib (AZD2014), Idelalisib, and Ruxolitinib.

10. A method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ mononuclear cells, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits or reduces expression of CD74 receptor or comprises an antagonist of CD74 function.

11. The method of claim 10, wherein the CD34+ mononuclear cells comprise CD34+ progenitor cells.

12. The method of claim 10, wherein the agent comprises (S,R)-3-(4 hydroxyphenyl)-4,5- dihydro-5-isoxazole acetic acid methyl ester (ISO-1).

13. The method of claim 12, wherein the therapeutically effective amount of ISO-1 ranges from about 1 mg / kg to about 10 mg / kg.

14. The method of claim 13, wherein the therapeutically effective amount of ISO-1 administered to the subject is 5 mg / kg.

15. The method of claim 10, wherein the agent comprises a monoclonal antibody, or binding fragment thereof that binds to CD74 receptor.

16. A method of treating familial platelet disorder (FPD) in a subject in need thereof, the method comprising:(a) obtaining a biological sample from the subject, wherein the biological sample comprises peripheral blood or bone marrow aspirate;(b) isolating CD34+ mononuclear cells from the biological sample obtained from the subject;(c) detecting an upregulation of CD74 signaling in the isolated CD34+ mononuclear cells; and(d) administering to the subject a therapeutically effective amount of an agent that inhibits CD74 expression or comprises an antagonist of CD74 signaling.

17. A method of treating familial platelet disorder (FPD) in a subject in need thereof, the method comprising: a) isolating CD34+ mononuclear cells from a biological sample obtained from the subject, wherein the biological sample comprises peripheral blood or bone marrow aspirate; b) detecting an upregulation of CD74 signaling in the isolated CD34+ mononuclear cells; and c) administering to the subject a therapeutically effective amount of an agent that inhibits CD74 expression or comprises an antagonist of CD74 signaling.

18. The method of claim 16, wherein the CD34+ mononuclear cells comprise CD34+ progenitor cells.

19. The method of claim 16, wherein the agent comprises (S,R)-3-(4 hydroxyphenyl)-4,5- dihydro-5-isoxazole acetic acid methyl ester (ISO-1) or an anti-CD74 monoclonal antibody.

20. The method of claim 19, wherein the agent comprises ISO-1 , and wherein the therapeutically effective amount of ISO-1 ranges from about 1 mg / kg to about 10 mg / kg.

21. The method of claim 20, wherein the therapeutically effective amount of ISO-1 administered to the subject is 5 mg / kg.

22. The method of claim 16, further comprising administering to the subject a therapeutically effective amount of an agent that inhibits PI3K / mTOR pathway downstream of CD74 signaling.

23. The method of claim 22, wherein the agent is selected from the group of Rapamycin, Vistusertib (AZD2014), and Idelalisib.

24. The method of claim 23, wherein the agent comprises rapamycin, and wherein the therapeutically effective amount of rapamycin ranges from about 0.1 mg to about 10 mg per day.

25. The method of claim 24, wherein the therapeutically effective amount of rapamycin administered to the subject ranges from about 1 mg to about 5 mg per day.

26. The method of claim 16, further comprising administering to the subject a therapeutically effective amount of an agent that inhibits JAK / STAT pathway downstream of CD74 signaling.

27. The method of claim 26, wherein the agent comprises ruxolitinib, and wherein the therapeutically effective amount of ruxolitinib ranges from about 1 mg to about 150 mg.

28. The method of claim 27, wherein the therapeutically effective amount of ruxolitinib administered to the subject ranges from about 1 mg to about 100 mg.

29. A method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ progenitor cells, the method comprising administering to the subject a therapeutically effective amount of a first agent that reduces expression of CD74 receptor or comprises an antagonist of CD74 signaling, and a therapeutically effective amount of a second agent that inhibits PI3K / mTOR pathway downstream of CD74 signaling.

30. The method of claim 29, wherein the first agent and the second agent are administered to the subject simultaneously or separately.

31. The method of claim 29, wherein the first agent comprises ISO-1 or an anti-CD74 monoclonal antibody, and wherein the second agent comprises Rapamycin, Vistusertib (AZD2014), or Idelalisib.

32. A method of treating familial platelet disorder in a subject, wherein an upregulation of CD74 signaling is present in CD34+ progenitor cells, the method comprising administering to the subject a therapeutically effective amount of a first agent that reduces expression of CD74 receptor or comprises an antagonist of CD74 signaling, and a therapeutically effective amount of a second agent that inhibits JAK / STAT pathway downstream of CD74 signaling.

33. The method of claim 32, wherein the first agent and the second agent are administered to the subject simultaneously or separately.

34. The method of claim 32, wherein the first agent comprises ISO-1 or an anti-CD74 monoclonal antibody, and wherein the second agent comprises ruxolitinib.

35. The method of claim 18, wherein the upregulation of CD74 signaling is determined by comparing percentage positivity of the CD34+ progenitor cells from the biological sample of the FPD subject with a biological sample from a healthy subject.

36. The method of claim 35, wherein the biological sample is determined to have CD74 signaling upregulated if the percentage positivity of the CD34+ progenitor cells exceeds a threshold of 1 .5 fold compared to a threshold obtained from the healthy subject.

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