Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood.

We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 ⁺ leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression.

In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia.