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1783 Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Combination therapy, Therapies
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Warren C. Fiskus, BSc, PhD1, Prithviraj Bose, MD2, Lucia Masarova, MD3, Christopher Peter Mill, PhD, BA4, Christine Birdwell, PhD4, John Davis4*, Kaberi Das1*, Hanxi Hou2*, Taghi Manshouri, PhD1*, Kevin Philip2*, Noor Alhamadani2*, Alicia Matthews2*, Sanam Loghavi, MD5 and Kapil N. Bhalla, MD3

1UT MD Anderson Cancer Center, Houston, TX
2The University of Texas MD Anderson Cancer Center, Houston, TX
3Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
4MD Anderson Cancer Center, Houston, TX
5Hematopathology, MD Anderson Cancer Center, Houston, TX

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.

Disclosures: Bose: Incyte, BMS, CTI, Morphosys, Blueprint, Cogent, Sumitomo, Ionis: Honoraria, Research Funding; Kartos, Telios, Disc, Janssen, Geron: Research Funding; GSK, Novartis, Karyopharm, AbbVie, Pharma Essentia, Jubilant, Morphic: Honoraria. Masarova: MorphoSys US: Membership on an entity's Board of Directors or advisory committees. Loghavi: QualWorld: Consultancy; Gerson Lehrman Group: Consultancy; Abbvie: Consultancy; Blueprint Medicine: Consultancy; Caris Diagnostics: Consultancy; Astellas: Research Funding; Amgen: Research Funding; Abbvie: Current equity holder in publicly-traded company; Guidepoint: Consultancy; Recordati/ EUSA Pharma: Consultancy; Daiichi Sankyo: Consultancy. Bhalla: Foghorn Therapeutics Inc.: Research Funding.

*signifies non-member of ASH