CD74 Signaling Regulates Inflammatory Stress to Drive Defective Hematopoiesis and Fitness Advantage of Progenitors in RUNX1-Familial Platelet Disorder

Background: Familial platelet disorder (FPD) caused by RUNX1 germline mutations is one of the most common pre-disposition syndromes to myeloid malignancies. FPD is characterized by platelet maturation defects, thrombocytopenia, and increased bleeding. FPD individuals have 40% increased life-long risk of developing hematopoietic malignancies, primarily MDS and AML, at the median age of 29 years. Though the mechanism of disease progression in FPD is unknown, patients suffer from autoimmune diseases and inflammatory disorders. We have shown increased levels of cytokines in bone marrow microenvironment of FPD, and upregulation of inflammatory and prosurvival pathways driving hematopoietic defects. Here, we sought to identify a master regulator of these pathways and phenotypes in FPD.

Methods: We performed 10x single-cell RNA sequencing (scRNA-seq, 70k cells), digital spatial profiling (N=3), and cytokine analyses (N=40) on FPD and healthy bone marrow cells. Using primary FPD and healthy samples, we evaluated the expression of various cytokine receptors using scRNA-seq and confirmed their expression via flow cytometry and immunohistochemistry. We tested the impact of the MIF/CD74 pathway and its blockade on FPD hematopoiesis using in vitro differentiation, colony formation, and multiplex cytokine profiling. The results were validated using Runx1-mutated murine model in vitro and in vivo.

Results: We found that among various cytokine receptors, CD74 is significantly overexpressed in HSC, progenitors, GMP, and monocyte populations in FPD compared to healthy. CD74 is a class II transmembrane protein, and its role in FPD hematopoiesis has not been evaluated. We confirmed that FPD CD34⁺ progenitors have a higher surface expression of CD74 (>2.0-fold, p<0.05) compared to healthy using flow cytometry. Accordingly, immunohistochemistry of FPD bone marrow biopsies at pre- and post-MDS/CMML transformation showed high levels of CD74. MIF, CD74 ligand, was also increased in the culture media of FPD CD34⁺ progenitors and stromal cells. MIF stimulation led to exhaustion of healthy progenitors in colony formation assay. However, FPD human and murine progenitors were resistance to stress-induced cell growth inhibition and showed increased colony formation ability upon MIF stimulation. We found that upregulated PI3K/mTOR and JAK signaling are also downstream of CD74 in FPD HSPCs leading to increased cytokine levels in the bone marrow microenvironment. Digital spatial profiling of the bone marrow biopsies from FPD and FPD-MDS/CMML confirmed upregulation of PI3K/AKT and mTOR pathway members such as pAKT1, pS6, and p4EBP1 compared to healthy. Consistently, there was a positive correlation between these pathway members and CD74 levels (R²>0.5, p<0.05). ChIP-qPCR analysis showed increased enrichment of RUNX1, p65, and PU.1 transcription factors at CD74 transcription start site in RUNX1-mutated compared to non-mutated cells. Genetic knockout of CD74 in FPD progenitors reduced myeloid colony formation (<0.5-fold, p<0.05). Pharmacological inhibition of MIF via ISO-1 resulted in increased megakaryocytic differentiation (>1.5-fold, p<0.05) of FPD, decreased cytokine levels (CCL24, IL-1β, TNF-α, CXCL8, etc), and decreased phosphorylation of PI3K/AKT, mTOR, and JAK/STAT pathway members. ISO-1 treatment also reduced colony formation ability of human and murine FPD progenitors. The in vivo treatment of Runx1^R188Q/+ mouse with ISO-1 rescued platelet activation defects (>2-fold, p<0.05) while suppressing myeloid differentiation to the levels of wild type mice (up to 0.5-fold change, p<0.05).

Conclusion: Skewed hematopoietic differentiation of FPD HSPCs is the result of RUNX1-mutation mediated transcriptomic changes leading to upregulation of MIF/CD74. The inflammatory MIF/CD74 regulates downstream PI3K/mTOR and JAK signaling leading to an increased aberrant inflammatory signaling, providing a fitness advantage and myeloid bias to the FPD HSPCs. The MIF/CD74 pathway is upregulated at early FPD and transformed leukemia stages, suggesting an early event in disease evolution. Genetic and pharmacological inhibition of MIF/CD74 rescued hematopoietic defects in RUNX1-mutated progenitors. To our knowledge, this is the first study that identified a novel role of MIF/CD74 signaling in FPD and its feasibility to serve as a new early intervention strategy to prevent or delay FPD evolution.

Keel:Disc Medicine: Consultancy, Other: Principal Investigator on clinical trial.