Mecom gene network drives chemotherapy resistance in high-risk acute myeloid leukemia Free

Despite recent advances in targeted chemotherapies for acute myeloid leukemia (AML), treatment of relapsed and refractory AML remains a significant challenge. Overexpression or rearrangement of the Myelodysplasia Syndrome (MDS) and Ecotropic Virus Integration Site 1 (EVI1) Complex locus (MECOM) is associated with particularly poor prognoses. High MECOM expression establishes a population of leukemia stem cells (LSCs) that have increased resistance to chemotherapy. Prior data from our group revealed a MECOM associated gene regulatory network that is essential in hematopoietic stem cell self-renewal and function, which may represent a therapeutic vulnerability in high-risk AML. Here, we investigated the association of the MECOM transcriptional network with chemoresistance in multiple AML models and found that perturbation of MECOM increased sensitivity to standard chemotherapies independent of cell state changes.

To probe the interaction of MECOM with chemosensitivity, we engineered multiple high MECOM expressing human AML cell lines by targeting the endogenous MECOM locus with a FKBP12^F36V degron that enabled rapid and complete MECOM protein degradation with exposure to the small molecule dTAG. MUTZ-3, an inv(3) AML cell line, was the primary system used as it can expand indefinitely and has a subpopulation of CD34+ progenitors that either self-renew or differentiate into CD14+ monocytes. Findings were later verified in the UCSD-AML-1 and HNT-34 cell lines. Using these models, we determined differential chemosensitivity of AML cells following MECOM protein degradation compared to control cells by characterizing phenotypic properties and differential gene expression profiles with etoposide and doxorubicin treatment, common backbones in current AML treatment regimens.

In MUTZ-3 cells, etoposide treatment increased the CD34+ stem cell progenitor population from 59.5% to 67% compared to the control (p<0.05, n=3). CD34+ MUTZ-3 LSCs are less likely to undergo apoptosis following etoposide treatment (5.5%, n=3) compared to more differentiated CD14+ cells (60.6%, n=3), demonstrating that etoposide selects for LSCs. Hypothesizing that some of this chemoresistance of LSCs was driven by MECOM, we determined IC₅₀ values of etoposide and doxorubicin after MECOM degradation. MECOM degradation led to a 2.2-fold, 1.6-fold, and 1.7-fold decrease in etoposide IC₅₀ respectively in MUTZ-3, UCSD-AML-1, and HNT-34 cells (p<0.05, n=3). Similarly, MECOM degradation led to a 1.5-fold, 1.8-fold, and 1.5-fold decrease in doxorubicin IC₅₀ in MUTZ-3, UCSD-AML-1, and HNT-34 cell lines (p<0.05, n=3), which indicates that MECOM degradation sensitizes AML cells to etoposide and doxorubicin.

To examine the transcriptional signatures associated with chemotherapy treatment and MECOM degradation, we performed bulk RNA sequencing with the CD34+ subpopulation of MUTZ-3 cells. We observed significant enrichment of the MECOM gene network following treatment with etoposide (NES =1.81, p <0.001) and doxorubicin (NES=1.91, p <0.001) that is reversed with degradation of MECOM with both etoposide (NES = -2.610, p<0.001) and doxorubicin treatment (NES= -2.634, p<0.001).

To determine the relevance of this MECOM gene set in primary AMLs, we evaluated RNA sequencing data from adult patients in the BEAT-AML data set. We compared samples at diagnosis (n=450) and relapse (n=37) and found enrichment of the MECOM gene network in relapsed samples compared to diagnostic samples (NES = 1.9, p <0.001). We additionally examined diagnostic samples from patients that were later found to have a complete (n=220) or refractory (n=103) response to first-line chemotherapy and found an enrichment of the MECOM gene network in patients with a refractory response (NES = 1.7, p <0.002).

In summary, the MECOM gene network is associated with etoposide and doxorubicin resistance in AML human cell models, suggesting that chemotherapy resistance is not only mediated by cell state changes, but is also modified by transcriptional changes in LSCs. Additionally, this gene set is enriched in relapsed and refractory primary AML patient samples. Further study of the mechanisms underlying the observed chemotherapy resistance and ways to overcome it are desperately needed for the development of newer and more effective therapies for high-risk AML.