Transcription Regulation Targeting in Peripheral T Cell Lymphomas Induces Apoptosis and Sensitization to BCL2 Inhibitors (2014)

Abstract Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival. To identify active drugs for PTCL patients, we performed a cell-based progressive screen from a library of 105 anti-neoplastic drugs in clinical use. Primary screening was done in the PTCL-NOS cell line (OCI-Ly12), using three drug concentrations. We identified 3 active drug groups within the clinical-range limit: HDAC inhibitors (HDI) (romidepsin), proteasome inhibitors (bortezomib, carfilzomib) and transcription inhibitors (dactinomycin). Secondary screening was conducted for the active drug groups with 6 drug concentrations, in an extended panel of 9 TCL cell lines. We also expanded the drugs in each group by adding vorinostat, panobinostat and valproic acid for HDI and SNS032 (CDK9&gt;2&gt;&gt;7 inhibitor) and THZ1 (CDK7&gt;12 inhibitor) for transcription inhibitors. We demonstrated that the most active drugs (within serum achievable concentrations) were bortezomib, carfilzomib, romidepsin, dactinomycin and THZ1. Since proteasome inhibitors and romidepsin are being currently tested in PTCL clinical trials, we focused on the transcriptional inhibitors. Actinomycin inhibits the transcription initiation complex and prevents RNA elongation by RNA-POL2; given this broad mechanism of action, it is associated with serious side effects that limit its clinical use either alone or in combination. On the other hand, a recently discovered CDK7&gt;12 inhibitor, THZ1, showed minimal side effects in vivo (Kwiatkowski N, et al. Nature 2014 Jun22), and high potency in our secondary screening yielding IC50s in TCL of ~200nM. We therefore used THZ1 to investigate the functional relevance of CDK7/12 targeting in PTCL. CDK7 is a critical component of both CDK-activating kinase (CAK) and transcription factor II human (TFIIH) complexes that phosphorylates cell cycle CDKs and C-terminal domain (CTD) of RNA-POL2, respectively. CDK7 inhibition decreases the phosphorylation of RNA-POL2 at Ser5 and Ser2, leading to transcriptional inhibition of susceptible loci. CDK12 in complex with CCNK displays a CTD kinase activity that is required for RNA splicing. To better understand the sensitivity of TCL to THZ1, we first determined the expression of all CAK components (CDK7, CCNH, and MAT1A) and CDK12/CCNK in 9 TCL cell lines that were found overexpressed compared to normal T cells from tonsils. Treatment of OCI-Ly12 and OCI-Ly13.2 (ALCL-ALK negative) cell lines with THZ1 for up to 48 h induced dose- and time-dependent decrease of phospho-POL2-ser5 and phospho-POL2-ser2, followed by PARP cleavage, caspase 7/3 activation and apoptosis. Genes with super-enhancers were found to be more susceptible to THZ1, and we also found these enhancers in genes associated with PTCL prognosis such as MCL1, JAK1 and MYC. Accordingly, THZ1 decreased mRNA and protein levels of MCL1, JAK1 and MYC as early as 3 hours after treatment. This was followed by decreasing levels of BCL2, BCL-XL, JUND and NFkB, and increased expression of pro-apoptotic proteins such as BAX at later time points. The decrease in JAK1 abundance led up to 70% reduction of phospho-STAT3 activity (as determined by immunoblotting and EMSA-like assays). Moreover, induction of STAT3 phosphorylation by IL-7/IL-15 treatment partially rescued the effect of THZ1, suggesting that JAK1 is a relevant target of CDK7 in PTCLs. The THZ1-dependent decrease in anti-apoptotic BCL2-family members prompted us to determine whether CDK7/12 inhibition can re-sensitize TCL to these drugs. We found either synergistic or re-sensitization effects on combination of THZ1 with BH3-family inhibitors, ABT-737 and obatoclax, in both OCI-LY12 and OCI-Ly13.2 cells. In sum, we identified a mechanism by which CDK7/12 inhibition, with the irreversible clinical candidate compound THZ1, simultaneously inhibits prominent PTCL survival pathways (JAK/STAT3, MYC and BCL2) causing apoptosis and re-sensitization to BCL2-family inhibitors. Disclosures Off Label Use: THZ1 (CDK7 and CDK12 inhibitor) for inducing apoptosis and sensitization to BCL2 inhibitors in peripheral T cell lymphomas.. Kwiatkowski:Syros Pharmaceuticals: One of the inventors Patents &amp; Royalties. Zhang:Syros Pharmaceuticals: One of the inventors Patents &amp; Royalties. Gray:Syros Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.