P986: combination jak1/2 and cdk8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Myeloproliferative neoplasms (MPNs) are characterized by aberrant activation of the JAK-STAT pathway. Ruxolitinib (RUX), is a JAK1/2 inhibitor used to treat myelofibrosis (MF) and hydroxyurea-resistant/intolerant polycythemia vera. While RUX has demonstrated important clinical benefits, a large proportion of patients have persistent disease manifestations despite therapy. CDK8 regulates phosphorylation of STAT proteins in a RUX-independent manner; thus, CDK8 inhibition may alter downstream STAT target gene expression. RVU120 is a highly selective and potent CDK8/19 inhibitor which inhibits STAT1 and STAT5 phosphorylation. Aims: We hypothesized that the combination of RUX and RVU120 would act in a cooperative manner to attenuate JAK-STAT signaling and reduce an MPN phenotype in vivo. Methods: Using the JAK2V617F (JVF) mutant leukemic cell lines: HEL92.1.7, UKE-1, MUTZ-8, and SET-2, we tested the viability of JVF cell lines with increasing concentrations of RVU120. Pharmacodynamic and synergy data were generated by treating JVF cell lines with RUX, RVU120 and combination RUX+RVU120. Pharmacodynamic analysis was conducted via Western blot; synergy data was generated via Loewe additivity model. For in vivo studies, we used a previously-described retroviral transplant murine model of myelofibrosis. Briefly, retrovirally-transfected (MSCV-MPLW515L-IRES-GFP) murine bone marrow (BM) was intravenously injected with wildtype support into lethally-irradiated, congenic recipients. Mice were randomized, treated with vehicle (VEH), single-agent RUX or RVU with one of two dosing regimens: daily (QD) or twice daily (BID), or RUX+RVU120 (RVU QD/BID) arms, and dosed for 3-4wk PO with biweekly bleeds for CBCs and disease progression. Results: We identified dose-responsive changes in viability in all cell lines, particularly in SET-2 and UKE-1 cells. Pharmacodynamic analysis validated that STAT5 protein phosphorylation was decreased by RUX and RVU120, although at different phosphorylation sites by RUX (pTYR694) and RVU120 (pSER726/731). Exposure to both RUX and RVU120 inhibited phosphorylation at both sites. Analysis of combinatorial effect of RUX and RVU120 on viability demonstrated synergy between the drugs (combinatorial index <1) in RUX-naïve and RUX-resistant SET-2 cells. WBCs were significantly decreased in mice treated with RUX+RVU120 versus RUX alone (in K/μL: RUX 132.6, RVU QD/BID ≤61.8, 4wks post-dosing; p<0.002, one-way ANOVA). The proportion of GFP+ cells in the peripheral blood was also significantly reduced in RUX+RVU120 treated mice compared with RUX monotherapy treated mice (RUX 80.2%, RUX+RVU QD/BID 67.1%, 2 weeks post-dosing; p<0.001 QD). Comparison of spleen weight at the time of terminal sacrifice demonstrated a significant reduction in spleen weights comparing RUX and RUX+RVU120 treated mice (average RUX 360mg, RUX+RVU QD 183mg, p=0.009) Flow cytometry on BM revealed reductions in megakaryocyte-progenitor proportions between VEH, RUX, and combination therapy (28.6% CD41+ of Lin-cKit+ cells RUX, 20.8% QD; p=0.033 QD; VEH 38.0%). Histopathologic data will be presented at the meeting. Summary/Conclusion: The combination of RVU120 and RUX demonstrated biochemical synergy and differential inhibition of STAT5 phosphorylation in vitro. Further, in vivo treatment with the combination of RUX and RVU120 resulted in significant reductions of disease manifestation (GFP+ cell fractions, WBC, splenomegaly, and megakaryocyte differentiation) when compared to RUX alone. These data nominate CDK8/19 inhibition in combination with JAK1/2 inhibition as a potential novel therapeutic strategy in MPNs.Keywords: Transcription, Myeloproliferative disorder, Myelofibrosis, Janus Kinase inhibitor