PF396 HISTONE DEACETYLASE INHIBITOR PANOBINOSTAT TARGETS CALCINEURIN/NFATC1 SIGNALING AND ABROGATES THE RESISTANCE OF PH+ LEUKEMIA CELLS TO DASATINIB

Abstract

Background:BCR‐ABL‐targeted tyrosine kinase inhibitors (TKI) revolutionized the outcome of patients inflicted with CML and Ph+ B‐ALL. However, TKI resistance continues to pose challenges for both CML and ALL.Aims:We hypothesized that overcoming the BM microenvironment‐mediated protection of Ph+ leukemic cells from TKI‐mediated apoptosis may further enhance the responsiveness to TKI therapy.Methods:In vitro experiments using BCR‐ABL‐positive ALL (BV‐173, NALM1 and NALM20) and CML (K562 and LAMA84) cell lines.Results:Dasatinib treatment significantly suppressed the growth of BCR‐ABL‐positive ALL and CML cell lines (p < 0.01). However, co‐culture with bone marrow stromal cells (BMSCs) protected both ALL and CML cells from dasatinib‐mediated toxicity. Furthermore, dasatinib treatment promoted significant upregulation of the chemokine receptor CXCR4, on both mRNA and cell surface levels. Elevated CXCR4 expression was accompanied by increased responsiveness of ALL and CML cells to CXCL12 stimulation, resulting in strong and sustained phosphorylation of Erk1/2 and Akt and increased adhesion capacity to BMSCs. Consequently, dasatinib‐induced upregulation of CXCR4 promotes the stroma‐mediated survival advantage of Ph+ leukemia cells upon TKI therapy.Next, in order to overcome the CXCR4‐mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete CXCR4 in AML cells. Single‐agent treatment with panobinostat demonstrated significant inhibition of Ph+ ALL and CML cell growth at low nanomolar concentrations (p < 0.01). Importantly, combination of panobinostat with dasatinib synergized (CI<0.5), effectively overcoming the protection provided by BMSCs and inducing apoptosis of Ph+ leukemia cells, as demonstrated by phosphatidylserine externalization and mitochondrial depolarization. Moreover, panobinostat/dasatinib treatment suppressed cell cycle progression, inducing subG0/G1 DNA fragmentation. Furthermore, combining panobinostat with dasatinib significantly reduced CXCR4 surface levels and CXCL12‐mediated responses, including Erk1/2 and Akt activation in Ph+ leukemic cells. Notably, panobinostat/dasatininb combination significantly inhibited soft agar colony formation of K562 cells.To elucidate the molecular mechanisms, we performed gene and protein expression analysis. Panobinostat, alone or in combination with dasatinib, significantly down‐regulated the protein levels of calcineurin, a serine–threonine protein phosphatase previously implicated in ALL and CML pathogenesis, as well as of NFATc1, a critical effector of the calcineurin signaling cascade, and NFATc1‐regulated target genes. Additionally, inhibiting calcineurin activity with cyclosporin A (CsA) decreased both basal and dasatinib‐induced CXCR4 surface levels and sensitized leukemic cells to dasatinib, overcoming the protection of the BMSCs. In addition, combining CsA with panobinostat demonstrated synergistic (CI<0.5) anti‐leukemic effect that results in deeper suppression of NFATc1‐regulated target genes. We thus link the effect of panobinostat with calcineurin‐dependent downregulation of CXCR4 and NFTAc1 signaling, blocking the ability of the leukemic cells to respond to CXCL12‐mediated stromal support and enhancing their response to BCR‐ABL inhibition.Summary/Conclusion:Taken together, our results identify calcineurin/NFATc1 signaling pathway as a novel target of panobinostat in Ph+ leukemia cells. Based on these findings, HDAC inhibition with panobinostat may be effective strategy for facilitating the anti‐leukemic activity of TKI therapy.