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L. Mazzera et al.
TRAF3, cIAP1, and cIAP2 lead to increased stability of NIK and subsequent aberrant activation of the non-canonical and canonical NF-κB pathways.2-7
In this study, we demonstrate that pan-AKI generate pro-survival signals in MM cells by inducing the expres- sion/activation of the pro-survival serine/threonine kinas- es PIM1 and PIM232 through a NIK/c-Abl-mediated activa- tion of STAT3, a cascade of molecular events that conse- quently limit the response to pan-AKI. Our findings reveal a novel functional interplay between NIK and c-Abl with implications for treatment of MM. They therefore provide the rationale for targeting c-Abl as a novel strategy to enhance activity of Pan-AKI.
Methods
Reagents
Pan-AKI MK-0457 (Merck & Co. Rahway, NJ, USA); pan-AKI PHA-680632 (Pfizer/Nerviano, Italy); pan-AKI AMG-900 (Cayman Chemical Company; Ann Arbor, MI, USA); NIK inhibitor isoquinoline-1,3(2H,4H)-dione (Santa Cruz Biotechnology, Santa Cruz, CA, USA); proteasome inhibitor bortezomib (PS-341) from Janssen-Cilag (Milan, Italy); c-Abl inhibitors imatinib mesylate and nilotinib (Novartis Pharmaceuticals, Basel, Switzerland). STAT3 inhibitor Stattic (6- Nitrobenzo [b]thiophene-1,1-dioxide) and pan-PIM kinase inhibitor SMI-4A (5Z)-5-[[3-(Trifluoromethyl)-phenyl]-methyl- ene]-2,4-thiazolidinedione (Sigma-Aldrich, St. Louis, MO, USA).
Cell cultures
Cell cultures were: human myeloma cell lines (HMCL) OPM-2, U266, RPMI-8226 and JJN3 (DSMZ, Braunschweig, Germany); multidrug-resistant RPMI-8226/R5 HMCL was established as pre- viously described;33 human bone marrow-derived stromal cell line HS-5 (ATCC, Manassas, VA, USA). Primary MM cells from MM patients and peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated and treated as described in the Online Supplementary Methods. The study was approved by the Ethics Committee of the University of Bari “Aldo Moro” (identifi- cation n. 5143/2017), and all patients and healthy donors provided informed consent in accordance with the Declaration of Helsinki.
Apoptosis assays, siRNA and plasmid transfections, molecular and statistical analysis
These methods have been previously published34 and are described in the Online Supplementary Methods.
Animal studies, histology, immunohistochemistry and immunofluorescence
The animal study was approved by the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna review board (n. PRC 2009018). Five-week old non-obese diabetic (NOD) severe combined immunodeficiency (SCID) NOD.CB17- Prkdcscid/J (NOD-SCID) mice (Jackson Laboratory, Bar Harbour, ME, USA) were maintained under the same specific pathogen-free conditions. Histological, immunohistochemical and immunofluo- rescent studies are described in the Online Supplementary Methods.
Results
Pharmacological blockade of Aurora kinases elevates NF-κB-inducing kinase protein levels through TRAF2 degradation
Although pan-AKI were able to prevent TRAIL-induced canonical and non-canonical NF-κB activation, they proved to be only partially effective in reducing the basal NF-κB activity of MM cells.25 Based on these observations,
In addition to regulating NF-κB pathways, the NIK sig- naling pathway has been demonstrated to crosstalk with and activate other critical cancer-associated pathways including the MAPK-ERK8,9 and JAK/STAT3.10 Moreover, these pathways are highly interconnected at many levels, and have been demonstrated to be often persistently and simultaneously activated in many human cancers, includ- ing myeloma.11,12
NF-κB and STAT3 signaling can also be regulated by c- Abl,13,14 a ubiquitously expressed non-receptor tyrosine kinase that plays an important role in regulating critical cellular processes, including proliferation, survival, apop- tosis, differentiation, invasion, adhesion, migration, and stress responses.15,16
The tyrosine kinase c-Abl has been reported to have opposing and antagonistic functions in the regulation of cell proliferation and survival depending on its subcellular localization, phosphorylation state, and cellular context.17 In particular, activation of cytoplasmic c-Abl in response to growth factors, cytokines and Src tyrosine kinases, can promote mitogenic and survival signals,17,18 whereas acti- vation of nuclear c-Abl in response to DNA damage can negatively regulate cell proliferation and mediate apopto- sis/necrosis.15
The subcellular localization of c-Abl is critically con- trolled by binding with the 14-3-3 protein, which requires the phosphorylation of c-Abl at an amino acid residue Thr735.19
Wild-type c-Abl is localized both in the nucleus and cytoplasm, in contrast to its oncogenic forms that are localized exclusively in the cytoplasm. Oncogenic forms of c-Abl exhibit enhanced kinase and transforming activi- ties and play a critical role in the pathogenesis of chronic and acute leukemias.20 MM cells display high levels of nuclear c-Abl in response to ongoing DNA damage and genomic instability.21,22 However, most of its nuclear tumor suppressor functions are compromised because of the dis- ruption of the ABL-YAP1-p73 axis.21
In MM and other hematologic and solid malignancies, genomic instability, centrosome amplification and aneu- ploidy have been associated with the overexpression of Aurora kinases, a family of serine/threonine kinases that play essential and distinct roles in mitosis.23
In addition to their mitosis specific substrates, Aurora kinases have also been found to functionally interact with proteins involved in critical cancer-associated pathways including NF-κB, STAT3 and DNA-damage response path- ways.24-27 On the basis of these findings, Aurora kinases have been considered as therapeutic targets for cancer and Aurora kinases inhibitors (AKI) have been extensively explored. These have shown encouraging pre-clinical and early clinical activity in different cancer types either alone or in combination with other agents.25,28-31 Unfortunately, AKI have not proved to be sufficiently effective and/or caused too many adverse side-effects in myeloma patients, both when used as monotherapy or in combina-
29-31
tion with other targeted therapy agents. The poor effi-
cacy of AKI therapies in MM may, in part, be related to the still undetermined drug-induced compensatory mech- anisms occurring in both the MM cells and their microen- vironment, and, consequently, to the lack of appropriate mechanism-based combination therapies.
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haematologica | 2019; 104(12)