The small GTPase RhoU lays downstream of JAK/STAT signaling and mediates cell migration in multiple myeloma

Multiple myeloma is a post-germinal center B-cell neoplasm, characterized by the proliferation of malignant bone marrow plasma cells, whose survival and proliferation is sustained by growth factors and cytokines present in the bone marrow microenvironment. Among them, IL-6 triggers the signal downstream of its receptor, leading to the activation of the JAK/STAT pathway. The atypical GTPase RhoU lays downstream of STAT3 transcription factor and could be responsible for mediating its effects on cytoskeleton dynamics. Here we demonstrate that RHOU is heterogeneously expressed in primary multiple myeloma cells and significantly modulated with disease progression. At the mRNA level, RHOU expression in myeloma patients correlated with the expression of STAT3 and its targets MIR21 and SOCS3. Also, IL-6 stimulation of human myeloma cell lines up-regulated RHOU through STAT3 activation. On the other hand, RhoU silencing led to a decrease in cell migration with the accumulation of actin stress fibers, together with a decrease in cyclin D2 expression and in cell cycle progression. Furthermore, we found that even though lenalidomide positively regulated RhoU expression leading to higher cell migration rates, it actually led to cell cycle arrest probably through a p21 dependent mechanism. Lenalidomide treatment in combination with RhoU silencing determined a loss of cytoskeletal organization inhibiting cell migration, and a further increase in the percentage of cells in a resting phase. These results unravel a role for RhoU not only in regulating the migratory features of malignant plasma cells, but also in controlling cell cycle progression

Therapeutic targeting of CK2 in acute and chronic leukemias

Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes. Thus, proper regulation of phosphorylation events is critical to the homeostatic functions of cell signaling. Indeed, deregulation of signaling pathways underlies many human diseases, including cancer.[1] The importance of phosphorylation makes protein kinases and phosphatases promising therapeutic targets for a wide variety of disorders.[2] CK2, formerly known as casein kinase II, was discovered in 1954, [3] although only recently, and especially over the last two decades, it has become one of the most studied protein kinases, due to its ubiquity, pleiotropy and constitutive activity. In particular, appreciation of its pleiotropy has completely changed our vision of CK2 biology, from an ordinary cell homeostasis-maintaining enzyme to a master kinase potentially implicated in many human physiological and pathological events. CK2 is responsible for about 25% of the phosphoproteome,[4] as it catalyzes the phosphorylation of >300 substrates.[5] This partly explains the CK2 interconnected roles that underlie its involvement in many signaling pathways. However, CK2 prevalent roles are promotion of cell growth and suppression of apoptosis. Accordingly, several lines of evidence support the notion that CK2 is a key player in the pathogenesis of cancer. High levels of CK2 transcript and protein expression, as well as increased kinase activity are associated with the pathological functions of CK2 in a number of neoplasias.[6] It was only over the last decade, after extensive analyses in solid tumors, that basic and translational studies have provided evidence for a pivotal role of CK2 in driving the growth of different blood cancers as well, although the first report demonstrating increased CK2 expression in acute myelogenous leukemia (AML) dates back to 1985.[7] Since then, CK2 overexpression/activity has been demonstrated in other hematological malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML). [8] With the notable exceptions of CML and pediatric ALL, many patients with leukemias still have a poor outcome, despite the development of protocols with optimized chemotherapy combinations. Insufficient response to first-line therapy and unsalvageable relapses present major therapeutic challenges. Moreover, chemotherapy, even if successful, could have deleterious long-term biological and psychological effects, especially in children.[9] Furthermore, CML patients can develop resistance to tyrosine kinase inhibitors (TKIs), while both primary chemoresistant and relapsed pediatric ALL cases still remain an unresolved issue.[9

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BACKGROUND: Glycogen Synthase Kinase-3 (GSK-3) \u3b1 and \u3b2 are two serine-threonine kinases controlling insulin, Wnt/\u3b2-catenin, NF-\u3baB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3\u3b1 and GSK-3\u3b2 function in multiple myeloma (MM). METHODS: GSK-3 \u3b1 and \u3b2 expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 \u3b1 and \u3b2 isoforms. Survival signaling pathways were studied with WB analysis. RESULTS: GSK-3\u3b1 and GSK-3\u3b2 were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3\u3b2 knock down decreased MM cell viability, while GSK-3\u3b1 knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of \u3b2-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3\u3b1 knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. CONCLUSIONS: These data suggest that in MM cells GSK-3\u3b1 and \u3b2 i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

Protein kinase CK2 regulates AKT, NF-\u3baB and STAT3 activation, stem cell viability and proliferation in acute myeloid leukemia

Protein kinase CK2 sustains acute myeloid leukemia cell growth, however, its role in leukemia stem cells is largely unknown. Here, we discovered that the CK2 catalytic \uf061 and regulatory \u3b2 subunits are consistently expressed in leukemia stem cells isolated from acute myeloid leukemia patients and cell lines. CK2 inactivation with the selective inhibitor CX-4945 or RNA interference induced an accumulation of leukemia stem cells in the late S-G2-M phases of the cell cycle and triggered late-onset apoptosis. As a result leukemia stem cells displayed an increased sensitivity to the chemotherapeutic agent doxorubicin. From a molecular standpoint, CK2 blockade was associated to a down-modulation of the stem cell-regulating protein BMI-1 and a marked impairment of AKT, NF-\u3baB and STAT3 activation, while FOXO3a nuclear activity was induced. Notably, combined CK2 and either NF-\u3baB or STAT3 inhibition resulted in a superior cytotoxic effect on leukemia stem cells. This study suggests that CK2 blockade could be a rational approach to minimize the persistence of residual leukemia cells.Leukemia accepted article preview online, 01 August 2016. doi:10.1038/leu.2016.209

Protein Kinase CK1α Sustains B-Cell Receptor Signaling in Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is still an incurable B-cell malignancy characterized by poor prognosis and frequent relapses. B Cell Receptor (BCR) signaling inhibitors, in particular of the kinases BTK and PI3Kγ/δ, have demonstrated clinically meaningful anti-proliferative effects in B cell tumors. However, refractoriness to these drugs may develop, portending a dismal prognosis. Protein kinase CK1α is an emerging pro-growth enzyme in B cell malignancies. In multiple myeloma, this kinase sustains β-catenin and AKT-dependent survival and is involved in the activation of NF-κB in B cells. In this study, we analyzed the role of CK1α on MCL cell survival and proliferation, on the regulation of BCR-related BTK, NF-κB, PI3K/AKT signaling cascades and the effects of CK1α chemical inhibition or gene silencing in association with the BTK inhibitor Ibrutinib or the PI3Kγ/δ inhibitor Duvelisib. CK1α was found highly expressed in MCL cells as compared to normal B cells. The inactivation/loss of CK1α caused MCL cell apoptosis and proliferation arrest. CK1α sustained BCR signaling, in particular the NF-κB, AKT and BTK pathways by modulating the phosphorylation of Ser 652 on CARD11, Ser 536 p65 on NF-κB, Ser 473 on AKT, Tyr 223 on BTK, as well as the protein levels. We also provided evidence that CK1α-mediated regulation of CARD11 and BTK likely implicates a physical interaction. The combination of CK1α inhibition with Ibrutinib or Duvelisib synergistically increased cytotoxicity, leading to a further decrease of the activation of BCR signaling pathways. Therefore, CK1α sustains MCL growth through the regulation of BCR-linked survival signaling cascades and protects from Ibrutinib/Duvelisib-induced apoptosis. Thus, CK1α could be considered as a rational molecular target for the treatment of MCL, in association with novel agents