Identification of basophils as a major source of hepatocyte growth factor in chronic myeloid leukemia: a novel mechanism of BCR-ABL1-independent disease progression. Neoplasia (New York, N.Y.) Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML. 10.1593/neo.12724

JAK-STAT pathway activation in malignant and nonmalignant cells contributes to MPN pathogenesis and therapeutic response. Kleppe Maria,Kwak Minsuk,Koppikar Priya,Riester Markus,Keller Matthew,Bastian Lennart,Hricik Todd,Bhagwat Neha,McKenney Anna Sophia,Papalexi Efthymia,Abdel-Wahab Omar,Rampal Raajit,Marubayashi Sachie,Chen Jonathan J,Romanet Vincent,Fridman Jordan S,Bromberg Jacqueline,Teruya-Feldstein Julie,Murakami Masato,Radimerski Thomas,Michor Franziska,Fan Rong,Levine Ross L Cancer discovery UNLABELLED:The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. SIGNIFICANCE:Our results demonstrate that JAK-STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs. 10.1158/2159-8290.CD-14-0736

Mechanisms for mTORC1 activation and synergistic induction of apoptosis by ruxolitinib and BH3 mimetics or autophagy inhibitors in JAK2-V617F-expressing leukemic cells including newly established PVTL-2. Ishida Shinya,Akiyama Hiroki,Umezawa Yoshihiro,Okada Keigo,Nogami Ayako,Oshikawa Gaku,Nagao Toshikage,Miura Osamu Oncotarget 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. 10.18632/oncotarget.25515

AZD1208, a Pan-Pim Kinase Inhibitor, Has Anti-Growth Effect on 93T449 Human Liposarcoma Cells via Control of the Expression and Phosphorylation of Pim-3, mTOR, 4EBP-1, S6, STAT-3 and AMPK. Yadav Anil Kumar,Kumar Vinoth,Bailey David Bishop,Jang Byeong-Churl International journal of molecular sciences Overexpression of Pim kinases has an oncogenic/pro-survival role in many hematological and solid cancers. AZD1208 is a pan-Pim kinase inhibitor that has anti-cancer and anti-adipogenic actions. Here, we investigated the effects of AZD1208 on the growth of 93T449 cells, a differentiated human liposarcoma cell line. At 20 µM, AZD1208 was cytotoxic (cytostatic) but not apoptotic, reducing cell survival without DNA fragmentation, caspase activation or increasing cells in the sub G1 phase; known apoptotic parameters. Notably, AZD1208 reduced phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in 93T449 cells. STAT-3 inhibition by AG490, a JAK2/STAT-3 inhibitor similarly reduced cell survival. AZD1208 down-regulated phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal S6 while up-regulated eukaryotic initiation factor-2α (eIF-2α). In addition, AZD1208 induced a LKB-1-independent AMPK activation, which was crucial for its cytostatic effect, as knock-down of AMPK greatly blocked AZD1208s ability to reduce cell survival. AZD1208 had no effect on expression of two members of Pim kinase family (Pim-1 and Pim-3) but inhibited phosphorylation of 4EBP-1, a downstream effector of Pim kinases. Importantly, a central role for Pim-3 in the actions of AZD1208 was confirmed by knock-down, which not only reduced 93T449 cell survival but also led to the inhibition of 4EBP-1, mTOR, eIF-2α and STAT-3, along with the activation of AMPK. In summary, this is the first report demonstrating that AZD1208 inhibits growth of liposarcoma cells and that this activity is mediated through Pim-3 kinase, STAT-3, mTOR, S6 and AMPK expression and phosphorylation pathways. 10.3390/ijms20020363

Activated Jak2 with the V617F point mutation promotes G1/S phase transition. Walz Christoph,Crowley Brian J,Hudon Heidi E,Gramlich Jessica L,Neuberg Donna S,Podar Klaus,Griffin James D,Sattler Martin The Journal of biological chemistry Hematopoietic stem cells in myeloproliferative diseases mostly retain the potential to differentiate but are characterized by hyper-responsiveness to growth factors, as well as partial factor-independent growth. The V617F activating point mutation in Jak2 has recently been associated with myeloproliferative disorders. Using various cell line models, mechanisms that contribute to Jak2V617-mediated signaling were investigated. Treatment of the Jak2V617F mutant-expressing erythroid leukemia cell line HEL with a small molecule Jak2 inhibitor was associated with a dose-dependent G(1) cell cycle arrest. This inhibition correlated with decreased expression of cyclin D2 and increased expression of the cell cycle inhibitor p27(Kip). Inhibition of Jak2V617F with a Jak2-targeted small interfering RNA approach resulted in a similar phenotype. Mechanisms leading to altered p27(Kip) and cyclin D2 likely involve inhibition of STAT5, a major target of Jak2 in hematopoietic cells, because a constitutively active form of STAT5 reduced p27(Kip) and increased cyclin D2 expression. Jak2V617F and constitutively active STAT5 also induced high levels of reactive oxygen species, which are sufficient to promote G(1)/S phase transition. In contrast, treatment of HEL cells with the antioxidant N-acetylcysteine decreased cell growth or expression of cyclin D2 and increased expression of p27(Kip). Similar results were obtained in BaF3 cells transfected with Jak2V617F, but these cells required coexpression of the erythropoietin receptor for optimal signaling. These results suggest that regulation of cyclin D2 and p27(Kip) in combination with redox-dependent processes promotes G(1)/S phase transition downstream of Jak2V617F/STAT5 and therefore hint at potential novel targets for drug development that may aid traditional therapy. 10.1074/jbc.M600064200