Session: 503. Clonal Hematopoiesis, Aging, and Inflammation: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, CHIP, Hematopoiesis, Immune mechanism, Immunology, Biological Processes
We sought to characterize novel drivers of clonal expansion by examining recurrent mutations in publicly available sequencing data and newly identified CH individuals. We describe recurrent MYD88 mutations, including the lymphoma-associated mutation L265P, alongside newly discovered mutations at T71I, R140Q, and C203R, identified in the mononuclear cells of newly diagnosed solid tumor cancer patients. MYD88 is a critical adaptor in innate and inflammatory signaling. Upon activation of the Toll-like receptor (TLR) superfamily, MYD88 recruits IRAK family members (IRAK1 and IRAK4), leading to the activation of NF-kB, MAPKs, and other inflammatory pathways. Unlike wild-type MYD88, we found that the expression of the MYD88 CH mutations in myeloid hematopoietic cells results in the activation of NF-kB signaling without TLR stimulation, suggesting that the L265P, T71I, R140Q, and C203R mutations lead to MYD88 gain-of-function activity, similar to the previously reported MYD88 L265P mutation in human lymphoma. Moreover, the expression of Myd88 L252P (equivalent of L265P in humans) in mouse cKit+ bone marrow (BM) cells resulted in elevated NF-kB activation. These findings indicate that MYD88 CH mutations exhibit gain-of-function activity and result in NF-kB signaling in HSPCs. To determine whether the expression of MYD88 CH mutations in HSPCs results in a clonal fitness advantage, we first examined colony-forming potential in methylcellulose using the conditional Myd88 L252P mouse model (under the control of RosaCreERT2 inducible reporter). Expression of Myd88 L252P in mouse cKit+ BM cells resulted in increased colony replating potential compared to WT cKit+ BM cells. Moreover, in competitive BM transplantation assays, the expression of Myd88 L252P conferred a competitive advantage in the PB and BM compared to WT cells. Examination of BM and PB by flow cytometry revealed that Myd88 L262P HSPCs exhibited myeloid-biased hematopoiesis, including increased monocytes and neutrophils. Furthermore, the expression of Myd88 L262P in hematopoietic cells led to a hematologic disease without evidence of lymphoid malignancies. These observations revealed that MYD88 CH mutations lead to a competitive fitness advantage of BM cells, consistent with the more common CH mutations, such as in TET2 and DNMT3A.
Given that active MYD88 mutations are associated with dysregulated NF-κB signaling and inflammatory pathways, we also analyzed inflammatory cytokine profiles and observed a significant increase in pro-inflammatory cytokines in the BM of Myd88 L262P mice compared to WT mice. Through single-cell RNA sequencing of hematopoietic cells (cKit+ enriched BM cells), we found that Myd88 L262P expression alters the expansion of distinct hematopoietic cell clusters, especially immature and mature neutrophil populations, and enrichment of innate immune and inflammatory pathways in HSPCs. We next determined whether the fitness advantage of MYD88 mutant HSPCs is due to NF-kB activation. For this, we performed a competitive BM transplantation using Myd88 L262P BM cells and then treated the mice with a dual IRAK1/4 inhibitor (NCGC-1481) for 6 weeks. Treatment with the IRAK1/4 inhibitor suppressed NF-kB activation in vitro and reversed the competitive fitness advantage of MYD88 L262P BM cells in vivo. In summary, we demonstrate that MYD88 gain-of-function mutations contribute to CH by inducing innate immune pathways in HSPCs and inflammatory cytokine expression. These findings offer new perspectives on the functional impact of genetic variants on HSPC fitness and the relevance of previously under reported CH mutations.
Disclosures: Hertlein: The Ohio State University: Consultancy; Eilean Therapeutics: Current equity holder in private company, Research Funding. Byrd: Vincerx Pharma, Eilean Therapeutics, and Kurome Therapeutics: Current equity holder in private company; Abbvie, AstraZeneca, and Syndax: Consultancy. Starczynowski: Curis: Honoraria; Kurome: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Kymera: Consultancy; Tolero: Research Funding; Treeline Biosciences: Research Funding.
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