Crebbp Inhibition Potentiates JAK2 Inhibition in Post-MPN Acute Myeloid Leukemia

Post-MyeloProliferative Neoplasm Acute Myeloid Leukemia (Post-MPN AML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms. Currently, there is no standard of care treatment for post-MPN AML patients, with allogeneic stem cell transplantation being the only curative option. However, the advanced age and comorbidities of post-MPN AML patients render them unfit for allo-SCT, emphasizing the urgent unmet need to find novel treatment for this disease. Hyperactivation of JAK/STAT signaling is highly prevalent in post-MPN AML pathogenesis, with as much as 50-90% of MPN cases harboring driver mutations in JAK2, CALR or MPL. The JAK2 inhibitor ruxolitinib has been tested in clinical trials for post-MPN AML and was reported to improve the patient quality of life, however, it was not effective in modifying the course of the disease. Given the limitations of targeting the JAK/STAT pathway, we aimed to identify genes and pathways synergistic with JAK2 inhibitors in post-MPN AML.

Here, we demonstrate through genome-wide CRISPR screens in post-MPN AML line HEL treated with four different JAK2 inhibitors (i.e. ruxolitinib, momelotinib, pacritinib and fedratinib) that depletion of CREBBP sensitizes cells to JAK2 inhibition. CREBBP is a histone acetyltransferase which also acts as a coactivator of essential transcription factors in diverse hematopoietic lineages. CREBBP is overexpressed in AML and is ubiquitously expressed in blood cancer cell lines, underscoring its potential as a therapeutic target. Genetic depletion of CREBBP via CRISPR/Cas9 editing showed that CREBBP, but not its paralog EP300, sensitizes HEL to JAK2 inhibition. In addition, pharmacological approaches showed that JAK2 inhibitors synergize with CREBBP/EP300 inhibitors to effectively kill post-MPN AML lines. In both human and murine models of post-MPN AML, the combination treatment of ruxolitinib plus CREBBP/EP300 inhibitor SGC-CBP30 or CCS1477 substantially induced apoptosis and cell cycle arrest at G1. Moreover, bioluminescence imaging of HEL luciferase mouse xenografts treated with vehicle, ruxolitinib (80 mg/kg), CCS1477 (20mg/kg) and combination showed significant decrease in leukemia burden in the combination-treated animals after 3 weeks of treatment.

Integration of the CRISPR viability screen and transcriptome profiles of HEL cells treated with JAK and CREBBP inhibitors identified 118 genes that are both essential for the proliferation and are downregulated with the combination treatments. GSEA showed enrichment of MYC targets in the said genes with E2F4, MYC and HSF1 being the top transcription factors associated with them. Notably, 21 of the 118 genes identified are part of the epichaperome network which is regulated by MYC. Accordingly, western blot analysis showed decreased MYC, E2Fs, STAT3 and STAT5 protein levels in HEL cells treated with the combinations. In addition, combination treatments also reversed the accumulation of JAK2 protein levels induced by ruxolitinib, which is a well-studied mechanism driving disease persistence. We hypothesize that this is due to the downregulation of MYC-regulated epichaperome genes which are known to regulate JAK/STAT signaling by physically interacting and stabilizing JAK2. Further investigation on the epigenetic changes induced by the ruxolitinib/CREBBP inhibitors is underway to identify their modulatory role of MYC and STAT3/5 regulatory regions.

Overall, our results demonstrate that CREBBP/EP300 inhibition potentiates JAK2 inhibition in post-MPN AML by further attenuating MYC expression and activity, and repressing JAK/STAT and other pathways associated with JAK2 inhibitor persistence. Therefore, we propose CREBBP inhibition as a potential therapeutic strategy to potentiate JAK2 inhibition in post-MPN AML.

Rampal:Incyte Corporation: Consultancy, Research Funding; Celgene/BMS: Consultancy; Blueprint: Consultancy; AbbVie: Consultancy; CTI BioPharma: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Galecto: Consultancy; PharmaEssentia: Consultancy; Jubilant: Consultancy; Constellation/MorphoSys: Consultancy, Research Funding; Sierra Oncology/GSK: Consultancy; Protagonist: Consultancy; Cogent: Consultancy; Sumitomo Dainippon: Consultancy; Kartos: Consultancy; Servier: Consultancy; Zentalis: Consultancy, Research Funding; Karyopharm: Consultancy; Ryvu: Research Funding; Disc Medicine: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Promedior: Consultancy. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Mana: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Kurome: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Scorpion: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Anovia: Consultancy; Bakx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Epiphanes: Membership on an entity's Board of Directors or advisory committees; Jubilant: Membership on an entity's Board of Directors or advisory committees; Bridge Bio: Consultancy; Bridge Medicines: Consultancy. Ferrando:Regeneron Pharmaceuticals: Current Employment.