VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

Background: Chronic Lymphocytic Leukemia (CLL) is a genetically heterogeneous disease characterized by clonal expansion of B-lymphocytes that induce secondary immune suppression. CLL is now treated with inhibitors of Bruton tyrosine kinase (BTK) and BCL2. Virtually all patients respond to therapy, however resistance to these therapies has been described justifying the need for novel CLL therapies. Broad inhibition of cyclin dependent kinases (CDK) and associated alternative target enzymes with agents such as flavopiridol or dinaciclib have demonstrated significant clinical activity in CLL but are hindered by a relatively narrow therapeutic window. VIP152 is a highly specific inhibitor of CDK9 - considered the most important CDK kinase member for CLL clinical activity. VIP152 has favorable pharmacokinetic properties and has demonstrated durable, preliminary single-agent clinical activity in double-hit diffuse large B-cell lymphoma. Herein, we report the efficacy of VIP152 preclinically in CLL. Methods: On-target activity of VIP152 was measured using a KinomeScan from DiscoveryRx at 100nM and 1000nM. Kinase profiling for VIP152 was performed on 6 kinases in a 10-dose assay at ReactionBio. Cell-based viability and proliferation assays (MTS, Annexin-V (AV), and propidium iodine (PI)) were performed in primary CLL cells and the CLL cell lines, HG3 and MEC1. Transcriptional activity after VIP152 exposure was measured via qPCR and limiting-cell RNA sequencing (lcRNAseq). Proteomic and immunoblot studies were performed to measure perturbations in CDK9 binding partners and on-target activity of VIP152. A genome-wide CRISPR/CAS9 knockout screen was performed to identify any synthetically lethal targets and pathways. Results: The KinomeScan identified CDK9 as the kinase with maximal inhibition upon VIP152 treatment and no other CDKs were identified at 100nM. Kinase profiling revealed the IC 50 of VIP152 was lowest for CDK9/Cyclin T1 and CDK9/Cyclin T2 with close similarity to dinaciclib and greater than 1 log superiority over KB-0742. Co-immunoprecipitation and proteomics experiments have identified a CDK9 specific mechanism of action relating to perturbations of CDK9 binding partners. Specifically, we showed that CDK9 nuclear immunoprecipitation resulted in decreased co-immunoprecipitation of 7SK RNA components (HEXIM1 & MEPCE) as well as decreased RNA Polymerase II (RNAP2). The decrease in RNAP2 CoIP was further seen via proteomics. A 2-hour exposure of VIP152 agai