Delineating CDK9- Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Abstract

Abstract Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, and RNA Pol II, but not other genes (e.g., Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, and RNA Pol II, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 reduced mRNA/ protein levels of CDK9 as well as of Mcl-1, cMyc, RNA Pol II, and Mdm2, and increased p53 levels in a dose- and time-dependent manner, both in MM cell line and primary cells. In contrast, Thal-sns-032 did not inhibit survival of peripheral blood mononuclear cells (PBMCs) from healthy donors. Furthermore, Thal-sns-032 induced downregulation of CDK9, Mcl-1 and cMyc in MM.1S co-cultured with BMSCs cells. Of note, Thal-sns-032- mediated anti-MM effects were abrogated upon its wash-out. Moreover, knockdown CDK9 by shRNA inhibited proliferation and survival, both in MM tumor cell- and tumor cell/BMSC co-cultures. Rationally derived combination strategies of Thal-sns-032 with venetoclax, navitoclax, Selinexor or Carfilzomib as well as other investigational and established MM therapies induced synergistic anti-MM effects in MM cells or BMSC co-cultures. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derived MM combination treatment strategies.