Mechanisms of chemotherapy resistance driven by Mecom overexpression

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that causes >11,000 deaths per year in the United States. Front-line therapy for young, medically fit patients involves intensive induction chemotherapy with an anthracycline and cytarabine (AraC), referred to as “7+3”, followed by consolidation with high-dose AraC or an allogeneic hematopoietic cell transplant. Approximately 25% of patients are refractory to induction chemotherapy, and these patients have limited treatment options and poor outcomes. Prior studies have shown that 5-10% of AMLs exhibit MECOM overexpression, which is associated with higher rates of induction failure and poor overall survival. However, the mechanism of chemotherapy resistance in AMLs with MECOM overexpression is poorly understood, and there are no FDA approved therapies targeting MECOM. To evaluate the impact of MECOM overexpression on chemoresistance, we utilized a previously established mouse model (Ayoub et al. Nature Communications. 2018) with dose-dependent doxycycline-inducible Mecom overexpression (“Mecom-TO”). We used single cell RNA-seq to show that in vitro doxycycline treatment leads to Mecom overexpression in both immature and mature bone marrow (BM) cell populations. Mecom overexpression in lineage-depleted pre-leukemic BM cells for only 24 hours directly induced resistance to both AraC and doxorubicin in vitro. Additionally, mixed BM chimeras composed of WT (Ly5.1+) and Mecom-TO (Ly5.2+) cells treated with doxycycline chow demonstrated that Mecom overexpression in vivo induces striking AraC resistance in pre-leukemic mice (5.3-fold, p=0.0009). Cell cycle analysis showed that Mecom overexpression blocked cell cycle progression in lineage-depleted pre-leukemic BM cells, which may contribute to resistance to S-phase specific chemotherapeutic agents. To characterize the molecular mechanisms through which Mecom overexpression impacts cell cycle progression and chemoresistance, RNA-seq, proteomics, metabolomics, and lipidomics were performed in pre-leukemic Mecom-TO BM cells after doxycycline treatment in vitro for 18 hours. These datasets identified dysregulated choline metabolism (5.9-fold increase in CDP-choline; 2-fold increase in RNA levels of the rate-limiting enzyme Pcyt1a) after short-term Mecom overexpression, which has previously been associated with chemoresistance. To extend these findings to AML cells, we have generated 3 independent spontaneous AMLs from Mecom-TO mice. Whole genome sequencing of Mecom-TO AMLs demonstrated that each AML acquired distinct clonal genetic alterations, including mutations in AML-associated epigenetic modifier genes (Bcor and Arid2) and a focal 51 Mb amplification of Chromosome 11, that contains 959 genes, including Ncor1. Notably, prior studies have identified enrichment of BCOR mutations in human AML with MECOM rearrangements. Additionally, NCOR1 is overexpressed broadly in AML compared to healthy donor CD34 cells in the TCGA dataset, and ARID2 mutations are rare, but recurrent, in AML. Overall, these results suggest that Mecom overexpression directly induces chemotherapy resistance in the absence of transformation, and suggest that mutations in epigenetic modifiers can potentially cooperate with Mecom overexpression to drive leukemic transformation. Studies are ongoing to characterize mechanisms of Mecom-induced chemoresistance in AMLs from Mecom-TO mice.