Abstract
The activated JAK2-V617F mutant is very frequently found in myeloproliferative neoplasms (MPNs), and its inhibitor ruxolitinib has been in clinical use, albeit with limited efficacies. Here, we examine the signaling mechanisms from JAK2-V617F and responses to ruxolitinib in JAK2-V617F-positive leukemic cell lines, including PVTL-2, newly established from a patient with post-MPN secondary acute myeloid leukemia, and the widely used model cell line HEL. We have found that ruxolitinib downregulated the mTORC1/S6K/4EBP1 pathway at least partly through inhibition of the STAT5/Pim-2 pathway with concomitant downregulation of c-Myc, MCL-1, and BCL-xL as well as induction of autophagy in these cells. Ruxolitinib very efficiently inhibited proliferation but only modestly induced apoptosis. However, inhibition of BCL-xL/BCL-2 by the BH3 mimetics ABT-737 and navitoclax or BCL-xL by A-1331852 induced caspase-dependent apoptosis involving activation of Bak and Bax synergistically with ruxolitinib in HEL cells. On the other hand, the putative pan-BH3 mimetic obatoclax as well as chloroquine and bafilomycin A1 inhibited autophagy at its late stage and induced apoptosis in PVTL-2 cells synergistically with ruxolitinib. The present study suggests that autophagy as well as the anti-apoptotic BCL-2 family members, regulated at least partly by the mTORC1 pathway downstream of STAT5/Pim-2, protects JAK2-V617F-positive leukemic cells from ruxolitinib-induced apoptosis depending on cell types and may contribute to development of new strategies against JAK2-V617F-positive neoplasms.
Highlights
The Janus kinase (JAK) family of cytoplasmic tyrosine kinases, comprised of JAK1, JAK2, JAK3, and TYK2, couples with cytokine receptors upon ligand binding and plays essential roles in transduction of intracellular signaling from these receptors lacking the tyrosine kinase domain [1]
The present study suggests that autophagy as well as the anti-apoptotic BCL-2 family members, regulated at least partly by the mTORC1 pathway downstream of STAT5/Pim-2, protects JAK2-V617F-positive leukemic cells from ruxolitinibinduced apoptosis depending on cell types and may contribute to development of new strategies against JAK2-V617F-positive neoplasms
PVTL-2 cells displayed a near-tetraploid consensus karyotype, 83,XX,X,X,2,5,der(5;7)(p10;q10),7,8,del(12)(q?)x2,13,14,15,16,16,18,20,20,22,add(22)(q11.2),+7mar (Figure 1B), which was quite different from that of PVTL-1 but included some of the common chromosomal abnormalities observed in primary AML cells, such as der(5;7)(p10;q10) and add(22)(q11.2), indicating that this cell line was derived from a different subclone of leukemic cells evolved at the transformation to AML
Summary
The Janus kinase (JAK) family of cytoplasmic tyrosine kinases, comprised of JAK1, JAK2, JAK3, and TYK2, couples with cytokine receptors upon ligand binding and plays essential roles in transduction of intracellular signaling from these receptors lacking the tyrosine kinase domain [1] Among these kinases, JAK2 plays a crucial role in regulation of proliferation and apoptosis of hematopoietic cells by activating various signaling pathways including the STAT5, Ras/Raf-1/. Ruxolitinib has shown only transient and limited effects against post-MPN sAML, which bears the uniformly dismal prognosis with median survival of less than 6 months [7, 8] In this regard, it has been reported that JAK2-V617F-positive cell lines readily gain resistance to JAK inhibitors after a long-term exposure to gradually increasing concentrations of these inhibitors [9,10,11,12]. Development of newer therapeutic strategies for MPNs and, post-MPN sAML is urgently needed
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