Abstract LB-315: Cyclin dependent kinase 7 represents a promising therapeutic target in glioma

Cyclin dependent kinase 7 (CDK7), the enzyme which facilitates cell cycle progression and stabilizes RNA polymerase II-based transcription, is central to two critical instigators of high grade glioma (HGG). Yet this target has not been identified or evaluated for HGG therapy. CDK7 can be inhibited using THZ1, a novel irreversible inhibitor of CDK7 whose mechanism(s) of action are largely unknown. We hypothesized that, due to its crucial role in activating the central drivers of HGG, CDK7 inhibition would offer a therapy which is broad in its efficacy across multiple HGG subtypes. In this study, we sought to prove this by determining the efficacy of THZ1 against HGG and to elucidate the biological processes disrupted by the drug. We used microarray and RT-qPCR to monitor transcript expression and splicing, multiplex immunofluorescence to monitor subnuclear compartment integrity, western blotting for protein expression, cell viability assays, flow cytometry to measure cell cycle, DNA damage and mitosis and high resolution respirometry to monitor mitochondrial health. We show that low nanomolar THZ1 has profound antitumor activity against 14 HGG patient-derived primary cell lines and ex vivo HGG patient tissue slices. CDK7 appears indispensable for gliomagenesis as THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest, nuclear DNA damage and caspase-independent cell death. THZ1 also disabled mitochondrial function by halting transcription of the nuclear-encoded mitochondrial ribosomal genes, subsequently reducing mitochondrial translation of the electron transport chain which, in turn, hampered oxidative respiration. This also resulted in the release of the mitochondrial located nuclease, apoptosis inducing factor, to translocate to the nucleus to augment DNA damage. THZ1 also inhibited the expression of multiple RTKs such as EGFR, PDGFR-α and MET, reducing signaling flux through downstream pathways such as AKT, ERK1/2 and STAT3. Finally, THZ1 disrupted nucleolar, Cajal-body and nuclear speckle formation resulting in reduced cytosolic translation and aberrant mRNA splicing. Hence, THZ1 sets in motion a convergence of multiple cellular insults to potently kill cells in a caspase-independent manner, providing the first description of the biological processes affected by THZ1. We also generated a THZ1 resistant patient-derived HGG cell line following six months of selection which possessed an IC50 40-fold greater than parental cells. Further analysis demonstrated a 30,000-fold upregulation of the ABCB1 (P-glycoprotein) transporter but not the ABCC1 or ABCG2 transporters. Blocking ABCB1 activity using Tariquidar or Zoriquidar fully reversed resistance to THZ1, demonstrating that ABCB1 mediated efflux of THZ1 is the main resistance mechanism to the drug. These studies validate CDK7 as a therapeutic target in HGG and provide new insights into the anti-tumor activity of THZ1 and the resistance mechanisms employed by HGG to counteract THZ1 therapy. Citation Format: Sameer A. Greenall, Terrance G. Johns. Cyclin dependent kinase 7 represents a promising therapeutic target in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-315. doi:10.1158/1538-7445.AM2017-LB-315