Human HSPCs CRISPR-engineered to endogenously express oncogenic NRAS generate a transplantable lethal myeloid malignancy targetable with novel RAS therapeutics Free

Introduction: NRAS mutations are frequently found in human leukemias, including 20-30% of acute myeloid leukemia (AML) patients and 50-70% of chronic myelomonocytic leukemia (CMML) patients. Preclinical models of oncogenic NRAS are critical for understanding their biology and for evaluating novel RAS therapeutics. Recently, it has been reported that oncogenic NRAS expression alone in human hematopoietic stem and progenitor cells (HSPCs) fails to engraft or cause a myeloid malignancy upon xenotransplantation. Notably, our group has demonstrated that introduction of NRAS^G12D into the AAVS1 safe-harbor locus in human HSPCs results in a rapidly fatal, high-grade myeloid neoplasm in xenotransplanted mice. However, it is unknown whether this model of exogenous NRAS^G12D accurately reflects physiological expression and human disease. Here, we generate a model of oncogenic NRAS expression from the endogenous locus in human HSPCs and compare it to our previous model of exogenous NRAS^G12D. We then use our endogenous NRAS model to characterize a panel of oncogenic NRAS mutations and evaluate their response to the novel multi-selective RAS inhibitor RMC-7977.

Methods: We used CRISPR/Cas9-mediated gene editing and AAV6-mediated homology directed repair to introduce NRAS^G12D into either the endogenous locus (driven by the endogenous promoter, denoted endoNRAS^G12D) or the AAVS1 locus (driven by the UBC promoter, denoted UBC-NRAS^G12D) in human cord blood CD34+ HSPCs. Successfully engineered cells also express fluorescent proteins which were used for purification by FACS, followed by intrafemoral transplantation into immunocompromised mice. Additionally, we introduced the G12C, G13D, and Q61R mutations into the endogenous NRAS locus, compared their functional effects to endoNRAS^G12D, and characterized their response to RMC-7977 treatment.

Results: Consistent with our previous findings, mice engrafted with human HSPCs expressing UBC-NRAS^G12D developed a lethal myeloid malignancy resulting in cytopenias, enlarged spleens, and rapid morbidity (median survival = 31 days). Interestingly, endoNRAS^G12D mice also developed cytopenias and enlarged spleens but with a prolonged latency (median survival = 99 days). Analysis of bone marrow cells showed similarly high levels of human engraftment (UBC-NRAS^G12D mean = 58.4%, endoNRAS^G12D mean = 40.4%) and a predominance of CD33+ myeloid cells (UBC-NRAS^G12D mean = 97.3%, endoNRAS^G12D mean = 87.7%). Notably, the spleens of endoNRAS^G12D mice had significantly lower human engraftment, lower CD33+ fraction, and higher CD19+ fraction compared to UBC-NRAS^G12D. Further characterization of these models is ongoing to uncover mechanisms underlying the differences in their disease kinetics and phenotype.

Given that our endoNRAS^G12D-expressing HSPCs demonstrated transforming ability in vivo, we aimed to explore whether introducing other leukemia-associated NRAS mutations in human HSPCs also affected their function, as assessed with in vitro colony forming assays. G12C, G12D, G13D, and Q61R mutant HSPCs all showed significantly increased colony formation after replating compared to NRAS-wildtype HSPCs. We then evaluated the effects of RMC-7977 treatment in NRAS-mutant HSPCs. Treatment of cells with 10 nM RMC-7977 significantly decreased colony formation upon serial replating. Furthermore, treatment with 10 nM RMC-7977 inhibited cell proliferation of mutant HSPCs but not NRAS-wildtype HSPCs. Ongoing efforts are aimed at characterizing the transformation potential of the additional mutants in vivo and validating the in vivo efficacy of RMC-7977.

Conclusion: Here, we established a model of endogenous NRAS^G12D in human HSPCs and showed that transplantation of such engineered cells into mice leads to a lethal myeloid neoplasm with prolonged latency compared to our UBC-NRAS^G12D model. We extended this model to other oncogenic NRAS mutations and demonstrated that the pan-RAS inhibitor RMC-7977 treatment reduces their proliferation and replating potential, while having little effect on NRAS-wildtype cells. Thus, both exogenous and endogenous oncogenic NRAS expression in human HSPCs leads to engraftment and development of myeloid malignancy with features of primary human disease. These findings highlight the critical role of oncogenic NRAS in myeloid malignancies and provide a valuable platform for testing targeted therapies, paving the way for future advancements in the treatment of NRAS-driven leukemias.