N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells promotes differentiation coupled with reduced proliferation. Conversely, overexpression of wild-type METTL3, but not the catalytic-dead form of METTL3, inhibits differentiation and increases cell growth. METTL3 mRNA and protein is expressed more abundantly in acute myeloid leukemia (AML) cells compared to healthy hematopoietic stem/progenitor cells and other types of tumors. Furthermore, METTL3 depletion in humanmyeloid leukemia cell lines induces differentiation and apoptosis and delays leukemia in recipient mice in vivo. Single-nucleotide resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in human myeloid leukemia MOLM13 cells. Moreover, loss of METTL3 leads to increased levels of pAKT, which contributes to the differentiation effects of METTL3 depletion. Overall these results provide a rationale for therapeutic targeting of METTL3 in myeloid leukemia.

Acquired mutations are pervasive across normal tissues. However, our understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMN). We find mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response (DDR) genes ( TP53, PPM1D, CHEK2 ). Sequential sampling provides definitive evidence that DDR clones outcompete other clones when exposed to certain therapies. Among cases where CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies.

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08–2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

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Clonal hematopoiesis (CH) is frequent in cancer patients and associated with increased risk of therapy related myeloid neoplasms (tMN). To define the relationship between CH, oncologic 90 therapy, and tMN progression, we studied 24,439 cancer patients. We show that previously treated patients have increased rates of CH, with enrichment of mutations in DNA Damage Response (DDR) genes (TP53, PPM1D, CHEK2). Exposure to radiation, platinum and topoisomerase II inhibitors have the strongest association with CH with evidence of dose-dependence and genetreatment interactions. We validate these associations in serial sampling from 525 patients and 95show that exposure to cytotoxic and radiation therapy imparts a selective advantage specifically in hematopoietic cells with DDR mutations. In patients who progressed to tMN, the clone at CH demarcated the dominant clone at tMN diagnosis. CH mutational features predict risk of therapyrelated myeloid neoplasm in solid tumor patients with clinical implications for early detection and treatment decisions. 100