Abstract
Abstract 1760
In chronic eosinophilic leukemia (CEL), the transforming oncoprotein FIP1L1-PDGFRA (F/P) is a major target of therapy. In most patients, the PDGFRA-targeting tyrosine kinase inhibitor (TKI) imatinib induces complete and durable molecular remissions. For patients who are intolerant or resistant against imatinib, novel TKI may serve as potential alternative therapy. Indeed, several different TKI have been described to act on Ba/F3 cells transfected with F/P, and some even block the activity of imatinib-resistant F/P mutants. However, little is known about the effects of novel TKI on growth and survival of primary neoplastic eosinophils. In the current study, we examined the in vitro effects of 12 kinase blockers on growth and viability as well as cytokine-induced migration of EOL-1 cells, a human F/P+ eosinophil leukemia cell line. In addition, we examined TKI effects on primary human neoplastic eosinophils obtained from a patient with F/P+ CEL, one with aggressive systemic mastocytosis and massive eosinophilia (ASM-eo) and one with reactive hypereosinophilia (HE). In EOL-1 cells, major growth-inhibitory effects were seen with all PDGFRA-blocking agents, with IC50 values in the low nM-range: ponatinib: 0.1–0.2 nM, sorafenib: 0.1–0.2 nM, masitinib: 0.2–0.5 nM, nilotinib: 0.2–2 nM, dasatinib: 0.5–2 nM, sunitinib: 1–2 nM, and midostaurin: 5–10 nM. These drugs were also found to block the activity of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. Targeting of individual downstream molecules with specific inhibitors (PI3-kinase: NVP-BEZ235; mTOR: everolimus; STAT5: pimozide and piceatannol) also induced growth-inhibition in EOL-1 cells, although IC50 values were higher compared to that obtained with PDGFR-blocking TKI. All effective TKI produced dose-dependent apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI staining, and staining for active caspase-3. In a next step, we applied the most effective TKI on primary neoplastic eosinophils. In these experiments, ponatinib, dasatinib, and nilotinib were found to suppress the growth of primary neoplastic eosinophils obtained from a patient with F/P+ CEL and one with ASM-eo, in a dose-dependent manner (IC50 <0.5 μM). In the patient with reactive HE, the TKI also produced growth inhibition, but IC50 values were higher compared to neoplastic eosinophils. We also examined drug effects on growth of Ba/F3 cells expressing the imatinib-resistant F/P mutants T674I and D842V. In these experiments, sunitinib was found to inhibit the growth of Ba/F3 cells expressing the T674I mutant of F/P. By contrast, no substantial effects of masitinib or nilotinib on Ba/F3 cells expressing this mutant were found, and Ba/F3 cells expressing F/P D842V were found to be resistant against sunitinib and masitinib. Strong inhibitory effects on both mutants were only seen with ponatinib. We next examined the effects of various TKI on cytokine-induced migration of neoplastic eosinophils. Unexpectedly, of all cytokines tested including IL-5 and eotaxin, only SDF-1A was found to induce in vitro migration of EOL-1 cells. We found that imatinib, nilotinib, dasatinib, ponatinib, sorafenib, and masitinib inhibit SDF-1A-induced migration of EOL-1 cells in a dose-dependent manner (effective range: 10–100 nM). Finally, we analyzed TKI effects on expression of activation-linked cell surface antigens on EOL-1 cells. In these experiments, we found that ponatinib and sorafenib downregulate expression of CD25 and CD63 in EOL-1 cells, whereas the other TKI tested showed no effects. By contrast, no effects of ponatinib or sorafenib on expression of HLA-DR, CXCR4 and CD95 on EOL-1 cells were seen. We were also unable to detect any significant effects of the other TKI on expression of activation-linked cell surface antigens in EOL-1 cells. In summary, our data show that various novel TKI counteract growth, survival, activation, and migration of neoplastic human eosinophils. The most potent agent that also blocks all known mutant-forms of F/P appears to be ponatinib. Novel PDGFR-targeting TKI, such as ponatinib, may be attractive alternative drugs for the treatment of imatinib-resistant or intolerant CEL.
Disclosures:
Valent: Phadia: Research Funding.
Synthesis and evaluation of some novel derivatives of 2-propoxybenzylidene isonicotinohydrazide for their potential antimicrobial activity
