CDK7 Inhibitor THZ1 Inhibits Glycolytic Metabolism By Suppressing HIF1α Activity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western adults, is characterized by the progressive accumulation of CD19+ malignant B cells in blood, secondary lymphoid tissues and bone marrow. Currently, CLL remains incurable in spite of the explosive development of novel therapeutics in the past decade. Cyclin-dependent kinases (CDKs) have been suggested as potential therapeutic targets for CLL. Flavopiridol, a pan-CDK inhibitor, has demonstrated safety and efficacy in patients with relapsed CLL, including patients with high-risk cytogenetic features. In this study, we assessed the therapeutic potential of a covalent CDK7 inhibitor, THZ1, in preclinical CLL models. We observed that THZ1 had a significantly lower IC₅₀ in CLL cell lines MEC1 and MEC2 (7.23nM and 7.35nM, respectively), which was 10-fold more potent than flavopiridol and CR8, two other CDK inhibitors that were previously studied in CLL. THZ1 inhibited cell growth and induced apoptosis in MEC1, MEC2 and primary CLL cells in both a dose-dependent and time-dependent manner. The induction of apoptosis by THZ1 in MEC1, MEC2 and primary CLL cells was associated with PARP1 cleavage and down-regulation of anti-apoptotic protein MCL-1. Particularly, THZ1 was able to overcome protection signals provided by the tumor microenvironments and induce apoptosis in primary CLL cells in stromal-coculture conditions. To elucidate the mechanism of action of THZ1, we performed a time course RNA-seq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of transcript levels over a 12-hour period, we identified 728 and 647 THZ1-sensitive transcripts (~5.2% and 4.5% of all active transcripts) in MEC1 and MEC2 cell lines, respectively. Pathway analysis demonstrated that THZ1 down-regulated transcripts are significantly enriched in regulation of cellular metabolism including hypoxia, mTORC1 signaling, and glycolysis pathways. In particular, when analyzing the entire THZ1 down-regulated expression signature for both MEC1 and MEC2 at 8 hours using Gene Set Enrichment Analysis (GSEA), two of the most significantly enriched gene sets contain gene expression signatures from hypoxia induction and HIF1α target genes. Several of the metabolic transcripts sensitive to THZ1 treatment including SLC2A1 , SLC2A3 , HK2 , ENO2 , and PKFKB4 were validated using quantitative RT-PCR. Immunoblot analysis demonstrated that mTORC1 substrates 4E-BP1 and p70S6K were hypophosphorylated in MEC1 and MEC2 when treated with increasing concentrations of THZ1 over time, indicating the inhibition of mTORC1 activity by THZ1. We also observed that the metabolically sensitive AKT Ser473 phosphorylation had essentially been fully repressed by THZ1 treatment in both MEC1 and MEC2. Furthermore, THZ1 was able to mitigate glucose uptake and reduce lactate production in MEC1 and MEC2 cells. THZ1 also prevented the induction of glycolytic transcripts by hypoxia and hypoxia-mimetic CoCl2 (200μM) treatment in MEC1, MEC2 and primary CLL cells. Analysis of CDK7 ChIP-seq data in Jurkat cells revealed that CDK7 directly binds to the promoter and -24kb, -35kb distal enhancers of SLC2A3 that are known to directly interact with HIF1α, suggesting that CDK7 is likely involved in transcriptional regulation of HIF1α target genes via direct enhancer binding. Experiments are currently underway to determine if CDK7 physically interacts with HIF1α under hypoxia. Overall, we observed that THZ1 could effectively disrupt CLL cell viability and overcome the protective signals present in the leukemia microenvironment by inhibiting the transcriptional activity of HIF1α, which is a key regulator of the interaction of CLL cells with the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of the CDK7 inhibitor as novel therapies for CLL.