Codon-Specific Differences in RAS Mutations in AML and Differential Response to Therapeutic Agents

Background

RAS pathway mutations are a key component of the proliferative drive in a subset of acute myeloid leukemia (AML), present in 10-20% of cases at diagnosis. In addition, they are associated with relapse after azacitidine/venetoclax and targeted therapy agents including FLT3 and IDH1/2 inhibitors. Bulk sequencing studies have shown heterogeneity in RAS mutations in AML, with NRAS mutations occurring more frequently than KRAS, and G12/G13 codon mutations predominating, followed by Q61 codon alterations. NRAS mutations frequently co-occur with NPM1 mutations, however there is limited data assessing codon-specific differences in co-mutation patterns, disease characteristics and responsiveness to therapy.

Methods

To compare disease characteristics of AML with different RAS codon mutations, and cooperativity between NPM1 and NRAS mutations, we characterized tamoxifen inducible, Cre-recombinant single- and double-mutant mouse models harboring Npm1c, NrasG12D, NrasG13D, and NrasQ61R mutant alleles. Mice were regularly monitored via peripheral blood draws and flow cytometric analysis of whole bone marrow and spleen cells at endpoints.

Results

Using single- and double-mutant Cre-inducible mouse models, we assessed disease symptoms and complete blood counts at 12 weeks post activation of mutant allele(s). We found that double mutant Npm1c/NrasG12D (NRG12) and Npm1c/NrasQ61R (NRQ61) models exhibited significantly higher WBC counts and spleen weights, relative to single-mutant models, with most significant increases observed in NRQ61 model. Flow cytometric analysis of stem/progenitor cells and mature lineage cells harvested from 12-week cohorts revealed significant defects in normal hematopoiesis in double-mutant models compared to their respective single-mutant cohorts. Additionally, we observed codon specific differences in hematopoietic compartments between Nras G12/G13 and Nras Q61 mutant cohorts.

To assess clonal fitness differences, we performed competitive transplants of CD45.1 WT, Npm1c single-mutant, and NR double-mutant cells mixed at a 50:40:10 ratio. We found that all double-mutant NR cells outcompeted WT and Npm1c single-mutant cells. However, NRQ61 double-mutant cells outcompeted WT/Npm1c cells faster than NRG12 or NRG13 cells. These findings suggest that, at least in the short-term, NR double mutant cells have increased fitness over WT and Npm1c single-mutant cells.

We next compared overall survival of the single- and double-mutant mouse models. We observed that single-mutant Npm1c, NrasG12D and NrasG13D have the least aggressive disease phenotypes. Interestingly, single-mutant NrasQ61R mice exhibited significantly faster disease development compared to other single mutants, with an overall survival similar to double-mutant cohorts. All double-mutant cohorts showed decreased overall survival compared to their respective single-mutant cohorts. These findings suggest that co-occurring Npm1c and Nras mutations can synergize to drive rapid disease development, albeit with different latencies. However, analysis of double-mutant moribund mice suggest there are significant codon specific differences in disease phenotype and ability to reconstitute leukemia.

Gene expression analysis of single- and double-mutant HSPCs also revealed codon-specific transcriptional regulation, underscoring the divergent synergies between Npm1c and different Ras codons. Ongoing studies are comparing the effect of targeted therapeutic agents, including novel RAS inhibitors, on the activity of distinct Nras codon mutations in AML, to further delineate codon-specific differences in responsiveness to therapy.

Conclusions

Overall, our findings suggest that there are codon-specific differences in disease latency and overall survival in models of AML with distinct Nras codon mutations, with Npm1c/NrasQ61 co-mutation models demonstrating more aggressive phenotype than G12/G13 codon alterations.

Starczynowski:Treeline Biosciences: Research Funding; Tolero: Research Funding; Kymera: Consultancy; 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.