P479: treatment is shaping clonal evolution and resistance patterns in murine kmt2a-rearranged leukemia with subclonal flt3n676k

Background: Our understanding of how individual mutations, whether present in all or just a fraction of the leukemia cells, affect cellular response to therapy is limited. Leukemia mouse models provide a unique possibility to explore how therapy affects evolution of genetically distinct clones and identify mechanisms of resistance allowing transfer to human disease. Aims: Herein, we investigated the impact of different therapies on survival, clonal evolution, and resistance patterns in a leukemia mouse model of KMT2A::MLLT3 with subclonal FLT3N676K. Methods: Bone marrow (BM) from a leukemia expressing KMT2A::MLLT3-mCherry in all cells and FLT3N676K-GFP in 40% of cells were re-transplanted to sublethally irradiated recipients. Upon engraftment, treatment was started with either chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was given for 28 days; controls received vehicle (Fig 1a). The leukemias were analyzed by flow-cytometry, bulk- and single cell RNA-sequencing and targeted gene re-sequencing. Results: Each treatment prolonged survival with a median latency of 30 days for chemotherapy, 37.5 days for AC220, and for the chemotherapy+AC220 and AC220+Trametenib groups, the median survival was 42 days and 45 days, respectively. The control mice that did not receive any treatment succumbed to disease with a median latency of 25.5 days. At sacrifice, all mice displayed splenomegaly and leukocytosis. Treatment impacted evolution of the KMT2A::MLLT3+FLT3N676K cells and they constituted all cells in control and chemotherapy-treated mice but had 3 distinct patterns of evolution in AC220-treated mice with either >80% of KMT2A::MLLT3+FLT3N676K cells, >80% of cells expressing KMT2A::MLLT3-alone, or dual similar sized clones expressing KMT2A::MLLT3 alone or KMT2A::MLLT3+FLT3N676K (Fig1 b). Eradication of the FLT3-leukemia cells was rare, but most common in mice receiving AC220+Trametinib and the frequency of dual clones increased when mice received chemotherapy followed by AC220, in line with treatment selectively affecting evolution. Next, RNA-sequencing unveiled three major clusters including 1) mice expressing KMT2A::MLLT3-alone, 2) control and chemotherapy-treated mice or 3)AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting resistance through activation of the ERK-pathway. Control/chemotherapy-treated leukemias exhibited a myeloid phenotype, with an aberrant expression of B220 and enrichment of gene sets correlating with stemness and oxidative phosphorylation, suggested a switch in cellular phenotype and metabolic state. By contrast, AC220-treated leukemias showed enrichment of gene sets correlating with granulocyte-monocyte progenitor (GMP) and immune regulatory pathways, indicating selective dependence of distinct cellular pathways upon resistance (Fig1 c). Acquisition of AC220-resistance mutations was rare, seen in only 2 mice. Moreover, single-cell RNA-sequencing revealed the emergence of a distinct cellular subpopulation in AC220-treated samples upon resistance with a strong GMP signature and upregulation of Six1 (Fig 1d). Summary/Conclusion: The specific treatment given affected survival and impacted the evolution of genetically distinct cells. The general lack of acquired mutations upon targeted treatment suggests that target-independent mechanisms that result in alternate activation of survival/proliferation explains acquired resistance in a majority of mice.Keywords: flt3 inhibitor, AML, KMT2A