Modeling KRAS mutations in KMT2A::MLLT3 acute myeloid leukemia promotes leukemic progression and transcriptional rewiring

Acute myeloid leukemia (AML) is a biologically heterogeneous disease where the mutational profile of the leukemic cells is a key determinant of prognosis, including disease-free survival and overall survival (OS). Rearrangements of the KMT2A gene occur in about 10% of AML cases, with MLLT3 being the most common fusion partner [ 1 ]. Although KMT2A fusions can initiate leukemogenesis, most KMT2A -rearranged ( KMT2A -r) leukemias acquire additional mutations that provide a selective advantage to subclones, allowing them to expand and eventually dominate the leukemic population. Despite advances in next-generation sequencing that have identified these secondary alterations, their functional roles remain poorly understood. Among these events, activating mutations in the RAS pathway, most frequently involving NRAS, KRAS and PTPN11, are detected in 10–25% of AML cases [ 2, 3 ] and in up to 32% of KMT2A -r AML [ 4 ]. Their typically low variant allele frequency (VAF) suggests that they arise later during leukemogenesis as subclonal events [ 5 ]. Their clinical significance remains debated, with some studies linking KRAS pathogenic variants to lower remission rates and poorer survival [ 6 ]. Relapses remain common due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs) [ 7 ]. To better understand the functional contribution of KRAS pathogenic variants in this context, we generated a human KMT2A::MLLT3 ( KM3 ) AML in vivo model to investigate their role in disease progression. We have previously shown that human CD34+ cells transduced with KM3 are sufficient to induce AML in vivo when injected into NSG-SGM3 immunodeficient mice [ 8 ]. To evaluate the impact of RAS activation, we introduced a KRAS G13D point mutation using CRISPR-Cas9 in KM3 -transduced human CD34+ cells before transplantation, while control mice received KM3 cells without the KRAS pathogenic variant (Fig. 1A ). KM3 + KRAS G13D mice developed leukemia with a significantly shorter latency, on average 2.16 ± 0.65-fold earlier than KM3 control mice across four independent experiments (Fig. 1B ). PCR and Sanger sequencing confirmed the presence of the KRAS pathogenic variant in leukemic cells, with VAFs ranging from 6.8% to 48.9%, comparable to those observed in patients [ 4 ]. The cell surface phenotype and morphology were similar between groups, expressing CD45, CD33, CD11b, HLA-DR and CD38, indicating that the shorter latency was not associated with phenotypic changes. All experiments were reproduced with the KRAS G12C pathogenic variant and showed similar results, except for the VAF, which was on average ~5%. KRAS pathogenic variants (G13D or G12C) slightly increase proliferation in KM3 cells, leading to a higher proportion of AML cells in the G2/M phase (Fig. 1C ). We next performed limiting dilution analysis (LDA) in secondary transplantation to quantify LSC frequency in KM3 control and KM3 + KRAS G13D leukemias. Secondary KM3 control mice remained healthy for >100 days with low engraftment (1–32%), whereas KM3 + KRAS G13D mice developed disease rapidly with high engraftment (Fig. 1D ). LSC frequency was estimated at >1 in 17 000 in KM3 + KRAS G13D AMLs versus 1 in 360 000 in KM3 controls ( p = 0.0069), indicating a > 20-fold increase in LSC frequency in our KMT2A -r AML model. PCR and sequencing confirmed the maintenance of the KRAS G13D pathogenic variant in secondary mice with VAFs between 16.8% and 68.8%. Fig. 1: KRAS pathogenic variants shorten KM3 AML latency, increase LSC frequency and induce gene expression changes. Full size image A Schematic representation of the in vivo experiments. B Representative Kaplan-Meier survival curves of mice KM3 control or KM3 + KRAS G13D injected with ~400 000 cells per mouse. C Cell-cycle analysis of KM3 control ( n = 6) and KM3 + KRAS -mutated ( n = 10) cells from bone marrow (BM) determined by flow cytometry. P value was determined by Student t test. * P < 0.05. D Kaplan-Meier survival curves of secondary mice ( KM3 control or KM3 + KRAS G13D) injected with 500,000, 50,000 or 5000 leukemic cells for limiting dilution experiment assay (LDA). ELDA software was used to calculate LSC frequencies. E OS of KM3 AML in the Harmony cohort of patients ≤ 60 years. F Principal Component Analysis (PCA). Principal components were calculated and the first two ranked by proportion of variation explained are shown (PC1 and PC2). CD34+ cells are represented in blue, KM3 control cells in purple (WT), KM3 + KRAS G13D in green and KM3 + KRAS G12C in red. G Volcano plot showing log2 fold-change (x-axis) and their −log10 FDR values (y-axis) for the DEGs detected between KM3 control vs. KM3 + KRAS pathogenic variants (G13D + G12C) cells. AML acute myeloid leukemia, BM bone marrow, DEGs differentially expressed genes, ELDA extreme limiting dilution analysis, KM3 KMT2A::MLLT3; LDA, limiting dilution analysis, LSC leukemic stem cell, OS overall survival, PCA principal component analysis, WT wild type. Taken together, KRAS G13D accelerates disease onset without altering cellular phenotype and markedly increases LSC frequency in secondary recipients. KRAS VAFs closely mirror those observed in patients and remain stable upon serial transplantation, supporting the clinical relevance of our model. Consistent with these findings, analysis of the HARMONY KMT2A::MLLT3 adult AML cohort showed that KRAS pathogenic variants ( n = 22) were associated with significantly reduced OS (median OS: 9.6 vs. 38.1 months in KRAS -mutated vs. wild-type (WT) patients; p = 0.006 in patients younger than 60 years (Fig. 1E )), a trend that remained evident across the entire cohort regardless of KMT2A fusion partner (data not shown). RNA sequencing (RNA-seq) was conducted on leukemic cells isolated from mice to assess transcriptional changes induced by KRAS pathogenic variants (G13D and G12C). Principal component analysis (PCA) showed clear separation between healthy cord blood CD34+ cells (blue, CD34 + ), KM3 control ce