Functional interplay between NF-κB-inducing kinase and c-Abl kinases limits response to Aurora inhibitors in multiple myeloma.

Considering that Aurora kinase inhibitors are currently under clinical investigation in hematologic cancers, the identification of molecular events that limit the response to such agents is essential for enhancing clinical outcomes. Here, we discover a NF-κB-inducing kinase (NIK)-c-Abl-STAT3 signaling-centered feedback loop that restrains the efficacy of Aurora inhibitors in multiple myeloma. Mechanistically, we demonstrate that Aurora inhibition promotes NIK protein stabilization via downregulation of its negative regulator TRAF2. Accumulated NIK converts c-Abl tyrosine kinase from a nuclear proapoptotic into a cytoplasmic antiapoptotic effector by inducing its phosphorylation at Thr735, Tyr245 and Tyr412 residues, and, by entering into a trimeric complex formation with c-Abl and STAT3, increases both the transcriptional activity of STAT3 and expression of the antiapoptotic STAT3 target genes PIM1 and PIM2. This consequently promotes cell survival and limits the response to Aurora inhibition. The functional disruption of any of the components of the trimer NIK-c-Abl-STAT3 or the PIM survival kinases consistently enhances the responsiveness of myeloma cells to Aurora inhibitors. Importantly, concurrent inhibition of NIK or c-Abl disrupts Aurora inhibitor-induced feedback activation of STAT3 and sensitizes myeloma cells to Aurora inhibitors, implicating a combined inhibition of Aurora and NIK or c-Abl kinases as potential therapies for multiple myeloma. Accordingly, pharmacological inhibition of c-Abl together with Aurora resulted in substantial cell death and tumor regression in vivo The findings reveal an important functional interaction between NIK, Abl and Aurora kinases, and identify the NIK, c-Abl and PIM survival kinases as potential pharmacological targets for improving the efficacy of Aurora inhibitors in myeloma.

Aurora and IKK kinases cooperatively interact to protect multiple myeloma cells from Apo2L/TRAIL.

Constitutive activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathways is frequent in multiple myeloma (MM) and can compromise sensitivity to TRAIL. In this study, we demonstrate that Aurora kinases physically and functionally interact with the key regulators of canonical and noncanonical NF-κB pathways IκB kinase ß (IKKß) and IKKα to activate NF-κB in MM, and the pharmacological blockade of Aurora kinase activity induces TRAIL sensitization in MM because it abrogates TRAIL-induced activation of NF-κB. We specifically found that TRAIL induces prosurvival signaling by increasing the phosphorylation state of both Aurora and IKK kinases and their physical interactions, and the blockade of Aurora kinase activity by pan-Aurora kinase inhibitors (pan-AKIs) disrupts TRAIL-induced survival signaling by effectively reducing Aurora-IKK kinase interactions and NF-κB activation. Pan-AKIs consistently blocked TRAIL induction of the antiapoptotic NF-κB target genes A1/Bfl-1 and/or Mcl-1, both important targets for TRAIL sensitization in MM cells. In summary, these results identify a novel interaction between Aurora and IKK kinases and show that these pathways can cooperate to promote TRAIL resistance. Finally, combining pan-AKIs with TRAIL in vivo showed dramatic efficacy in a multidrug-resistant human myeloma xenograft model. These findings suggest that combining Aurora kinase inhibitors with TRAIL may have therapeutic benefit in MM.

MEK1/2 regulate normal BCR and ABL1 tumor-suppressor functions to dictate ATO response in TKI-resistant Ph+ leukemia.

Resistance to tyrosine kinase inhibitors (TKIs) remains a clinical challenge in Ph-positive variants of chronic myeloid leukemia. We provide mechanistic insights into a previously undisclosed MEK1/2/BCR::ABL1/BCR/ABL1-driven signaling loop that may determine the efficacy of arsenic trioxide (ATO) in TKI-resistant leukemic patients. We find that activated MEK1/2 assemble into a pentameric complex with BCR::ABL1, BCR and ABL1 to induce phosphorylation of BCR and BCR::ABL1 at Tyr360 and Tyr177, and ABL1, at Thr735 and Tyr412 residues thus provoking loss of BCR's tumor-suppression functions, enhanced oncogenic activity of BCR::ABL1, cytoplasmic retention of ABL1 and consequently drug resistance. Coherently, pharmacological blockade of MEK1/2 induces dissociation of the pentameric MEK1/2/BCR::ABL1/BCR/ABL1 complex and causes a concurrent BCRY360/Y177, BCR::ABL1Y360/Y177 and cytoplasmic ABL1Y412/T735 dephosphorylation thereby provoking the rescue of the BCR's anti-oncogenic activities, nuclear accumulation of ABL1 with tumor-suppressive functions and consequently, growth inhibition of the leukemic cells and an ATO sensitization via BCR-MYC and ABL1-p73 signaling axes activation. Additionally, the allosteric activation of nuclear ABL1 was consistently found to enhance the anti-leukemic effects of the MEK1/2 inhibitor Mirdametinib, which when combined with ATO, significantly prolonged the survival of mice bearing BCR::ABL1-T315I-induced leukemia. These findings highlight the therapeutic potential of MEK1/2-inhibitors/ATO combination for the treatment of TKI-resistant leukemia.

The proapoptotic effect of zoledronic acid is independent of either the bone microenvironment or the intrinsic resistance to bortezomib of myeloma cells and is enhanced by the combination with arsenic trioxide.

OBJECTIVE: Multiple myeloma (MM) cells are extremely resistant to drug-induced apoptosis due to both intrinsic- and bone marrow (BM) microenvironment-dependent drug resistance particularly supported by bone cells. Growing evidence suggest that the osteoclast inhibitor zoledronic acid (ZOL) exerts both indirect and direct anti-tumoral effects, including an in vitro proapoptotic effect on MM cells, although this property has not yet been clearly observed in MM patients. MATERIALS AND METHODS: In this study, we attempt to better define the cytotoxic effect of ZOL on MM cells in order to identify novel drug combinations able to potentiate its proapoptotic effect. RESULTS: Our data shows that ZOL at concentrations ranging from 10 to 100 µM was able to induce MM cell apoptosis overcoming the prosurvival effect of both stromal cells and osteoclasts and independent of the intrinsic bortezomib resistance of MM cells. Interestingly, we found that the capacity of ZOL to induce apoptosis in bortezomib-resistant cells was associated with a downregulation of the proapoptotic molecule myeloid cell leukemia-1. A transcriptional analysis by microarray was also performed to identify genes specifically modulated by ZOL in bortezomib-resistant MM cells. Finally, we show an additive effect of arsenic trioxide on apoptosis when used in combination with ZOL. CONCLUSIONS: Our in vitro data suggest that the use of ZOL at appropriate doses could be explored clinically in bortezomib-resistant MM patients and combined with arsenic trioxide to increase its proapoptotic effect.

Interleukin-27 acts as multifunctional antitumor agent in multiple myeloma.

PURPOSE: Multiple myeloma (MM) derives from plasmablast/plasma cells that accumulate in the bone marrow. Different microenvironmental factors may promote metastatic dissemination especially to the skeleton, causing bone destruction. The balance between osteoclast and osteoblast activity represents a critical issue in bone remodeling. Thus, we investigated whether interluekin-27 (IL-27) may function as an antitumor agent by acting directly on MM cells and/or on osteoclasts/osteoblasts. EXPERIMENTAL DESIGN: The IL-27 direct antitumor activity on MM cells was investigated in terms of angiogenesis, proliferation, apoptosis, and chemotaxis. The IL-27 activity on osteoclast/osteoblast differentiation and function was also tested. In vivo studies were done using severe combined immunodeficient/nonobese diabetic mice injected with MM cell lines. Tumors from IL-27- and PBS-treated mice were analyzed by immunohistochemistry and PCR array. RESULTS: We showed that IL-27 (a) strongly inhibited tumor growth of primary MM cells and MM cell lines through inhibition of angiogenesis, (b) inhibited osteoclast differentiation and activity and induced osteoblast proliferation, and (c) damped in vivo tumorigenicity of human MM cell lines through inhibition of angiogenesis. CONCLUSIONS: These findings show that IL-27 may represent a novel therapeutic agent capable of inhibiting directly MM cell growth as well as osteoclast differentiation and activity.

Distinct transcriptional profiles characterize bone microenvironment mesenchymal cells rather than osteoblasts in relationship with multiple myeloma bone disease.

OBJECTIVE: Multiple myeloma (MM) is characterized by a high incidence of osteolytic bone lesions, which have been previously correlated with the gene expression profiles of MM cells. The aim of this study was to investigate the transcriptional patterns of cells in the bone microenvironment and their relationships with the presence of osteolysis in MM patients. MATERIALS AND METHODS: Both mesenchymal (MSC) and osteoblastic (OB) cells were isolated directly from bone biopsies of MM patients and controls to perform gene expression profiling by microarrays and real-time polymerase chain reaction on selected bone-related genes. RESULTS: We identified a series of upregulated and downregulated genes that were differentially expressed in the MSC cells of osteolytic and nonosteolytic patients. Comparison of the osteolytic and nonosteolytic samples also showed that the MSC cells and OB had distinct transcriptional patterns. No significantly modulated genes were found in the OBs of the osteolytic and nonosteolytic patients. CONCLUSIONS: Our data suggest that the gene expression profiles of cells of the bone microenvironment are different in MM patients and controls, and that MSC cells, but not OBs, have a distinct transcriptional pattern associated with the occurrence of bone lesions in MM patients. These data support the idea that alterations in MSC cells may be involved in MM bone disease.