Abstract 6285: Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN)
Authors: Mill Christopher P. , Fiskus Warren C. , Birdwell Christine E. , Davis John A. , Das Kaberi , Bhalla Kapil N. 
Published: June 2023

Abstract
Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including DNMT3A, TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated transcriptome, which is responsible for the increased growth, survival and therapy-refractoriness in MPN blastic phase (BP). Treatment with JAK inhibitors (JAKis), e.g., ruxolitinib, venetoclax and hypomethylating agents are ineffective in improving survival in MPN-BP. CDK7 is the crucial catalytic component of the general transcript initiation factors TFIIH, which is recruited to the transcription start-sites along with RNA-pol II (RNAP2) to initiate transcription. Complexed with cyclin H, CDK7 phosphorylates CDK1, 2, 4 and 6, as well as serine 5 and 7 in the C terminal domain (CTD) of RNAP2, thus facilitating transcript initiation. CDK7 also phosphorylates CDK9, which in turn phosphorylates serine 2 of the CTD of RNAP2, and phosphorylates and inactivates the negative elongation factors, thus enabling productive mRNA transcript elongation of growth and survival promoting oncogenes. In MPN-BP, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1 and MYC. CRISPR-gRNA dependency-screen (DepMap) demonstrated that CDK7 is a dependency in the Advanced-AML cell lines, SET2 and HEL cells. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and its clinical grade counterpart SY-5609, dose-dependently inhibits cell cycle, growth and induces lethality in SET2 and HEL as well as patient-derived (PD), CD34+ MPN-sAML cells. ChIP-Seq analysis showed that SY-1365 treatment caused log2 fold-reduction in p-Ser2-RNAP2 occupancy on the DNA of MYC, PIM1 and MCL1 genes in HEL cells. RNA-Seq analysis showed that SY1365 treatment caused negative enrichment of gene-sets of MYC and E2F targets, as well as inflammatory response, IL6-JAK-STAT3 and mTORC1 signaling. SY-1365 treatment also dose-dependently depleted protein expressions of p-Ser5 RNAP2, p-Ser2 RNAP2, c-Myc, c-Myb, RUNX1, PU.1, MCL1 and Bcl-xL, while inducing cleavage of PARP protein. As compared to the parental JAKi-sensitive, in vitro generated JAKi-persister/resistant (P/R) SET2/RuxP cells were significantly more sensitive to SY-1365-induced apoptosis. Co-treatment with CDK7i and ruxolitinib or the BET inhibitor OTX015 was synergistically lethal in PD MPN-BP cells (n=7). In an aggressive xenograft model of HEL-GFP/Luc cells in NSG mice, compared to the vehicle control, monotherapy with SY5609 significantly reduced the MPN sAML burden and improved survival of the NSG mice without causing host toxicity. These findings demonstrate promising activity and support the rationale to further evaluate the in vivo efficacy of CDK7i-based combinations against advanced MPN.
Citation Format: Christopher P. Mill, Warren C. Fiskus, Christine E. Birdwell, John A. Davis, Kaberi Das, Kapil N. Bhalla. Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6285.