Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML

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 TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated epigenome/transcriptome/proteome that inhibits differentiation and induces leukemia transformation (sAML) and therapy-refractoriness in MPN with excess blasts (> 5% in PB) or MPN transformed to AML (sAML). Treatment with JAK inhibitor (JAKi), e.g., ruxolitinib, venetoclax or hypomethylating agents alone or in combination are ineffective in improving the poor survival in MPN-sAML. CDK7 is the catalytic component of the general transcription factor TFIIH recruited with RNA-pol II (RNAP2) to transcription start-sites to initiate mRNA transcription. By phosphorylating CDK9 and negative transcript elongation factors, CDK7 also enables productive transcript elongation of oncogenes involved in growth and survival. In MPN-sAML, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1, and MYC. By phosphorylating CDK1/2/4/6, CDK7 also induces cell cycle. RNA-Seq analysis in BEAT AML study showed significantly higher CDK7 expression in MPN-sAML compared to normal progenitor cells. DepMap data involving CRISPR-mediated gene-knockouts show CDK7 as a dependency in the MPN-sAML SET2 and HEL cell lines. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and clinical grade SY-5609, dose-dependently (20 to 250 nM) increased % G1 while reducing the % of cell-cycle S phase SET2 and HEL cells. CDK7i treatment induced in vitro loss of viability in these cells, as well as in patient-derived (PD), CD34+ MPN-sAML but not in normal CD34+ progenitor cells. CRISPR-mediated CDK7 depletion (over 85%) in HEL cells significantly reduced SY-5609-mediated loss of viability. Importantly, pre-treatment with SY-5609 significantly increased sensitivity to ruxolitinib-induced loss of viability in JAKi-resistant SET2 and HEL cells, suggesting that CDK7 inhibition could overcome resistance to JAKi in MPN-sAML cells. RNA-Seq analysis conducted after SY-5609 treatment (100 nM for 16 hours) showed significant negative-enrichment of the gene-sets of MYC and E2F targets, cell-cycle checkpoints and of protein translation initiation/elongation, with log2 fold-decline in the mRNA levels of MYC, MYB, PLK1/4, KIT, CDK6, AURKA, PIM1 and CCND1, but upregulation of CDKN1A and HEXIM1. Following SY-5609 treatment of HEL cells, mass spectrometry demonstrated significant log2 fold-decline in c-Myc, c-Myb, RUNX1, PLK1, PIM1, but increase in p21, CASP9, BAD, DAP and TGFβ1 protein levels. CyTOF analysis demonstrated that SY-5609 treatment markedly reduced c-Myc, RUNX1 and MCL1, while increasing TP53, p21, and cleaved PARP proteins levels in MPN-sAML stem-progenitor cells defined by high expression of CD34, CLEC12A, CD123, CD99 but low expression of CD11b. Co-treatment with SY-5609 and ruxolitinib induced synergistic loss of viability in HEL, SET2 and PD MPN-sAML cells (n = 5) (delta synergy scores > 1.0 by the ZIP method). A CRISPR screen in SET2 and HEL cells targeting epigenetic regulators revealed significant log2 fold-decline in gRNA targeting BRD4, CBP and p300, highlighting them as dependencies. Consistent with this, co-treatment with SY-5609 and the BETi OTX015 was synergistically lethal in SET2, HEL, mouse MPN (JAK2-V617F plus TP53 loss) cells and PD MPN-sAML cells (n=5). SY-5609 treatment also exerted synergistic lethality with the BETi pelabresib or BD2-selective BETi ABBV-744 or the CBP/p300 inhibitor GNE-049 in MPN-sAML cells. In the xenograft model of HEL-Luc/GFP cells in NSG mice, monotherapy with SY-5609 (1.5 or 3.0 mg/kg/day, by oral gavage), vs vehicle control, significantly reduced the MPN-sAML burden and improved survival, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with SY-5609 and OTX015 (30 mg/kg/day by oral gavage) reduced more MPN-sAML burden and significantly improved survival in a HEL-Luc/GFP xenograft model without inducing toxicity. These findings demonstrate promising preclinical activity of CDK7 inhibition against the cellular models of MPN-sAML, supporting the rationale to further evaluate in vivo efficacy of CDK7i-based combinations against advanced MPN with excess blasts or MPN-sAML.