Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34⁺/CD38⁻ MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34⁺/CD38⁻ MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V⁺ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.

Advanced systemic mastocytosis (advSM), including aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL), are rare, mostly KIT D816V-driven malignancies with a poor prognosis. KIT D816V-targeting drugs, such as midostaurin or avapritinib, can induce hematologic responses in most patients with advSM. However, relapses may occur. Therefore, current research focuses on novel drugs and drug combinations to improve outcomes in advSM. We here describe that the synthetic triterpenoids bardoxolone methyl and omaveloxolone suppress proliferation in various human MCL-like cell lines expressing or lacking KIT D816V, with IC₅₀ values of 0.2–0.4 µM. Growth-inhibitory effects were accompanied by induction of apoptosis, decreased surface expression of CD44, CD71 and KIT, and downregulation of cytoplasmic AKT. Antiproliferative effects of bardoxolone methyl and omaveloxolone were also observed in primary neoplastic cells obtained from patients with advSM. Moreover, both synthetic triterpenoids were found to cooperate with midostaurin and avapritinib in suppressing the proliferation of HMC-1 and MCPV-1 cells. However, bardoxolone methyl and omaveloxolone also augmented expression of heme oxygenase-1 (HO-1), a mediator of cell survival and drug resistance in these cells. The HO-1 inhibitor SMA-ZnPP was subsequently applied and found to sensitize neoplastic mast cells against synthetic triterpenoids and KIT D816V-targeting drugs. Together, application of bardoxolone methyl and omaveloxolone, as single agents or in drug combinations involving HO-1 blockers and/or KIT D816V-targeting drugs, may be a suitable strategy to improve therapy and to overcome resistance in advSM. The clinical value of this combination approach remains to be assessed in clinical trials.