CDK8 Inhibitors Induce Transcriptional Reprogramming of AML Cells Associated with Differentiation

Introduction

Targeting Cyclin Dependent Kinase 8 (CDK8) is a novel therapeutic strategy for Acute Myeloid Leukemia (AML), a disease associated with poor outcome. CDK8 has been implicated in the regulation of transcription, however antileukemic mechanism of CDK8 inhibitors (CDK8i) are not fully elucidated.

SEL120 is an orally available, selective inhibitor of CDK8 and its paralog CDK19. Preclinical characterization of SEL120 indicated a potent inhibition of CDK8, accompanied by transcriptional changes. A first-in-human phase Ib clinical trial with SEL120 in patients with AML or higher-risk myelodysplastic syndrome (HR-MDS) was initiated in June 2019. Inclusion criteria for patients include relapsed or refractory disease and no more than 3 prior lines of standard therapy. To clarify the role of CDK8 in AML, we have used SEL120 and other chemically- unrelated CDK8i to characterize transcriptional and epigenetic changes associated with the antileukemic activity.

Material and methods

A panel of CDK8i- resistant and sensitive AML cell lines was treated with SEL120 and other CDK8i. Global transcriptional changes were assessed by RNAseq at different timepoints in order to capture early and long-term effects of CDK8i. Transcriptome- wide kinetics of RNA synthesis and turnover in AML cells was captured by Thiol (SH)- Linked Alkylation for the Metabolic sequencing of RNA (SLAMseq). Transcriptional changes and promoter- enhancer interaction analysis data were correlated with chromatin immunoprecipitation results using antibodies against subunits of Polycomb repressive complex 2 (PRC2), trimethylation of histone H3 at lysine 27 (H3K27me3), CDK8 and RNA polymerase II (RNAPII).

Results

Viability screening with three structurally- unrelated CDK8 inhibitors identified CDK8i- responsive AML cell lines. Transcriptional profiling after CDK8i treatment revealed profound activation of SE- associated genes in both responder and non- responder cell lines, at least partially disconnecting these effects from the anticancer activity of CDK8i. Although SEL120 significantly repressed many genes, including oncogenic MYC-dependent signatures, broad transcriptional profiling indicated rather stimulatory effects on gene expression in AML cells. Detailed transcriptomic analysis of AML cells revealed that SEL120 induced expression of many genes involved in lineage controlling functions. Derepression of genes marked with H3K27me3, including homebox family of transcriptional factors, was one of the earliest transcriptional events in sensitive AML cells treated with SEL120. Morphological changes in AML blasts required prolonged exposure to SEL120 and were correlated with increased expression of lineage markers. A particularly high increase was observed for CD38 and erythroid markers, including erythroid transcription factors - GATA-binding factor 1 and 2 (GATA1/2), and hemoglobin genes. Forced differentiation and reduced viability were also confirmed in patient- derived cells treated with SEL120.

Conclusions

CDK8 inhibitors act both by inducing expression and repression of distinct genes. Our study identifies that CDK8i cause epigenetic reprogramming that results in antileukemic efficacy by activation of H3K27me3 differentiation programs and inhibition of MYC-dependent genes. Because PRC2- dependent H3K27me3 maintains the stemness by repression of lineage commitment genes, our data suggest that CDK8i may negatively affect core properties of LSCs. Collectively, our studies demonstrated underlying mechanisms of anticancer activity of SEL120 in AML cells, currently investigated in clinical studies.