CDK8 Inhibition Represses Monocyte-like Gene Expression in Acute Myeloid Leukemia Cells and Antagonizes In Vivo Resistance to FLT3 Inhibition

Clinical resistance shortens survival for acute myeloid leukemia (AML) patients treated with targeted inhibitors. Resistance mechanisms to AML targeted therapies, including inhibitors of FLT3, BCL2 and IDH1/2, often converge upon the RAS/MAPK signaling pathway. Monocytic differentiation of leukemic cells also imparts resistance to venetoclax-based therapies, potentially due, in part, to upregulation of anti-apoptotic proteins such as MCL1 and BCL2A1. Others have linked RAS and monocytic drug resistance by showing that RAS mutations in committed GMPs generate venetoclax-resistant LSCs. Monocytic differentiation also confers relative in vitro resistance to FLT3 TKIs.

We performed two genome-wide CRISPRi apoptosis screens to identify gene targets that overcome such resistance by re-sensitizing AML cells to FLT3 inhibition in TKI-resistant conditions. Specifically, we performed screens in gilteritinib-treated MOLM-14 FLT3-ITD+ AML cells stimulated to hyperactivate MAPK signaling via either co-culture in cytokine-enriched conditioned media or by secondary expression of mutant NRAS G12C. We used a published monocyte-like AML gene expression set derived from single-cell transcriptomics (van Galen, et al, Cell 2019) and applied RNA-seq and GSEA to demonstrate that both screening conditions upregulated a monocyte-like gene signature, consistent with reports linking RAS/MAPK signaling to monocytic differentiation.

Both screens identified the Mediator complex/CDK8 as regulators of gilteritinib resistance that can be co-targeted by SEL120, a small-molecule CDK8 inhibitor. To define mechanisms of combinatorial inhibitor activity, we again used GSEA and discovered that FLT3-mutant AML cells treated with gilteritinib activated an inflammatory/interferon transcriptional response between early (4h) and late (16h) treatment timepoints. Additionally, the monocyte-like AML gene signature also became upregulated at the late timepoint relative to early treatment, suggesting apoptotic resistance may arise from these adaptive changes. Co-treatment with SEL120 blocked induction of these transcriptional programs and repressed SPI1-associated gene expression, a driver of monocyte development. In concordance, SPI1 knockdown strongly sensitized multiple FLT3-mutant AML cell lines to gilteritinib-induced apoptosis, including MOLM-14, MV4-11 and MOLM-14 NRAS-G12C cells. Of individual genes from the monocytic AML gene set, SEL120 strongly repressed expression of CD68, TLR4, and BCL2A1 alone and when combined with gilteritinib. SEL120 treatment also was associated with downregulation of MCL1 and ITGAM (CD11b), the latter a marker used to identify leukemic blasts of monocytic differentiation.

We then assessed the gilteritinib/SEL120 combination in a patient-derived xenograft (PDX) murine AML model. NSGS mice expressing human IL3, GM-CSF, and SCF were xenografted with a FLT3-ITD+ AML patient sample. Mice were treated for 6-weeks with vehicle, gilteritinib, SEL120, or the drug combination and percentage of human CD45+ cells were measured across different organ tissues. When compared to vehicle, gilteritinib significantly reduced leukemic burden in cardiac blood but had significantly less anti-leukemic activity in spleen and bone marrow. These findings recapitulated early human FLT3 TKI clinical trials in which circulating AML blasts often rapidly cleared but few patients exhibited bone marrow responses, likely reflecting a cytoprotective tumor microenvironment. SEL120 alone exhibited no anti-leukemic activity, but gilteritinib/SEL120 significantly reduced hCD45+ AML cells in spleen and bone marrow compared to gilteritinib alone. Moreover, monocytic CD11b expression within the hCD45+ population was increased in the marrow and spleens of gilteritinib-treated mice, suggesting selection for monocytic differentiation. This upregulation was mitigated by SEL120 co-treatment.

In sum, CDK8 inhibition appears to effectively overcome in vivo FLT3 TKI resistance driven by RAS/MAPK and monocytic differentiation. This activity may stem in part from preventing AML cell activation of inflammatory and monocyte-like gene expression that may otherwise promote cell survival. Further exploration is warranted to determine whether repression of this gene signature by CDK8 inhibition could also reduce resistance to venetoclax and other targeted AML therapies.