Abstract Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research
Background: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by a hyperactive JAK-STAT pathway, bone marrow fibrosis, extramedullary hematopoiesis, and splenomegaly. Ruxolitinib (RUX) and fedratinib (FEDR) are JAK inhibitors used for the treatment of patients (pts) with higher-risk MF. Furthermore, FEDR is clinically efficacious in pts previously exposed to RUX (Gupta, et al., Blood 2022: abstract 1711). The cellular and molecular mechanisms of FEDR efficacy in RUX-exposed/resistant pts are not fully understood.
Aims: To explore potential mechanisms of FEDR efficacy after RUX exposure using clinical and preclinical data.
Methods: In the FREEDOM study (NCT03755518), pts with MF (N=38) received FEDR 400 mg once daily in continuous 28-day cycles until lack of efficacy, intolerance, disease progression, or consent withdrawal. The primary endpoint was the percentage of pts with ≥35% spleen volume reduction at the end of cycle 6 (SVR35 at EOC6). In the FREEDOM biomarker cohort (pts with paired samples at cycle 1 day 1 (C1D1) and EOC6 [n=19]), levels of 85 serum cytokine levels were measured using the RBM HumanMAP v2.0 panel. BaF3 cell lines overexpressing JAK2 V617F were incrementally exposed to RUX over 3 weeks (up to IC90) to induce RUX-resistance. Cell proliferation and phosphorylation of STAT5 in FEDR- and RUX-treated cells was measured using CellTiter-Glo and immunoblotting, respectively. Kinome screening was performed using the ThermoFisher LanthaScreen Eu kinase binding assay and the reaction biology kinome assay.
Results: Paired analysis between C1D1 and EOC6 revealed increases in cytokines including adiponectin, carcinoembryonic antigen (CEA), erythropoietin (EPO), and ferritin and decreases in pro-inflammatory EN-RAGE, IL-16, IL-18, IL1-RA, TIMP-1, TNFR2, MPO, VCAM1, and VEGF (P<0.001). Increases in adiponectin, CEA, ferritin, haptoglobin, EPO, and CCL18 also significantly correlated with SVR (P<0.05) (Jeyaraju, et al., Blood 2022: abstract 1682). The cytokine profile of RUX-exposed pts in FREEDOM was similar to cytokine changes previously measured in RUX-naive pts (Pardanani et al., Blood Cancer J 2015), suggesting that prior RUX exposure/resistance does not interfere with FEDR efficacy or mechanism of action.
RUX-resistant JAK2 V617F BaF3 cell lines showed <10% loss of proliferation when treated with 4mM RUX for 24 hours, while control cells remained sensitive to RUX (IC50 120nM). In contrast, both RUX-resistant and control cells showed loss of proliferation when treated with FEDR (IC50 650nM vs 1552nM, respectively) (Figure). Immunoblotting showed that RUX-resistant cells treated with 2uM RUX maintained high levels of STAT5 phosphorylation, while in RUX-resistant cells treated with 2uM FEDR, STAT5 phosphorylation was inhibited. FEDR also had a 2–3-fold higher kinase inhibitory profile compared with RUX in an in-vitro kinome screen.
Summary/Conclusion: In the FREEDOM study, FEDR treatment resulted in cytokine changes, some of which suggest an anti-inflammatory and anti-fibrotic effect. Changes to cytokine profile, specifically anti-inflammatory effects, are a potential disease-modifying effect in MF therapies. Preclinical studies at clinically relevant exposures further demonstrated that FEDR inhibited proliferation and STAT phosphorylation in RUX-resistant cell lines, and had a broader kinase inhibition profile compared with RUX. These results indicate additional effects of FEDR which allow efficacy in previously RUX-exposed pts.
Keywords: Myeloproliferative disorder, Janus Kinase inhibitor