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.

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.

The p53 family protein p73 provides new insights into cancer chemosensitivity and targeting.

The p53 tumor suppressor is part of a small family of related proteins that includes two other members, p73 and p63. Interest in the p53 family members, their functions and their complex interactions and regulation, has steadily grown over recent years and does not show signs of waning. p73 is a major determinant of chemosensitivity in humans, and mutant p53 proteins carrying specific polymorphisms can induce drug resistance by inhibiting TAp73. Cooperation between TA (transactivating, proapoptotic, antiproliferative) and Delta N (truncated, antiapoptotic, pro-proliferative) p73 isoforms and among the three family members guarantees equilibrium between proliferation, differentiation, and cell death, thus creating a harmony that is lost in several human cancers. In this article, we review our current knowledge of the role of p73 in cancer chemosensitivity and the real prospect of therapy targeting this molecule. We also draw attention to the crucial role of specific phosphorylation and acetylation events for p73-induced apoptosis and drug chemosensitivity.

Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways.

We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by myeloma cells and suppresses hypoxia-inducible factor-1 alpha (HIF-1alpha) activity: involvement in myeloma-induced angiogenesis.

Angiogenesis has a critical role in the pathophysiology of multiple myeloma (MM); however, the molecular mechanisms underlying this process are not completely elucidated. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of angiogenesis. In this study, we found that ING4 expression in MM cells was correlated with the expression of the proangiogenic molecules interleukin-8 (IL-8) and osteopontin (OPN). Moreover, we demonstrate that ING4 suppression in MM cells up-regulated IL-8 and OPN, increasing the hypoxia inducible factor-1alpha (HIF-1alpha) activity and its target gene NIP-3 expression in hypoxic condition. In turn, we show that the inhibition of HIF-1alpha by siRNA suppressed IL-8 and OPN production by MM cells under hypoxia. A direct interaction between ING4 and the HIF prolyl hydroxylase 2 (HPH-2) was also demonstrated. Finally, we show that ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN. These in vitro observations were confirmed in vivo by finding that MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD. Our data indicate that ING4 exerts an inhibitory effect on the production of proangiogenic molecules and consequently on MM-induced angiogenesis.

MEK1 inhibition sensitizes primary acute myelogenous leukemia to arsenic trioxide-induced apoptosis.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative DeltaNp73 isoforms, thereby failing to elevate the TA/DeltaNp73 ratio. Conversely, treatment with PD184352 reduces the level of DeltaNp73 and blunts the arsenic-mediated up-regulation of DeltaNp73, thus causing an increase in the TA/DeltaNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.