AbstractConsidering 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.
Despite encouraging advances in therapy, multiple myeloma (MM) remains an incurable disease due to complex genomic alterations, lower sensitivity to chemotherapy of MM cells in the bone marrow microenvironment, and the emergence of drug resistance.1
Recent genetic evidence has established a pathogenetic role for NF-κB signaling in MM.42 In particular, at various frequencies, MM cells harbor gain-of-function mutations as well as loss-of-function mutations in genes encoding components of the classical and the alternative NF-κB pathways.42 Among these, mutations in the genes encoding NF-κB-inducing kinase (NIK) or its negative regulators TRAF2, TRAF3, cIAP1, and cIAP2 lead to increased stability of NIK and subsequent aberrant activation of the non-canonical and canonical NF-κB pathways.72
In addition to regulating NF-κB pathways, the NIK signaling pathway has been demonstrated to crosstalk with and activate other critical cancer-associated pathways including the MAPK-ERK98 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, including myeloma.1211
NF-κB and STAT3 signaling can also be regulated by c-Abl,1413 a ubiquitously expressed non-receptor tyrosine kinase that plays an important role in regulating critical cellular processes, including proliferation, survival, apoptosis, differentiation, invasion, adhesion, migration, and stress responses.1615
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,1817 whereas activation of nuclear c-Abl in response to DNA damage can negatively regulate cell proliferation and mediate apoptosis/necrosis.15
The subcellular localization of c-Abl is critically controlled 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 activities 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.2221 However, most of its nuclear tumor suppressor functions are compromised because of the disruption of the ABL-YAP1-p73 axis.21
In MM and other hematologic and solid malignancies, genomic instability, centrosome amplification and aneuploidy 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 pathways.2724 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.312825 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 combination with other targeted therapy agents.3129 The poor efficacy of AKI therapies in MM may, in part, be related to the still undetermined drug-induced compensatory mechanisms occurring in both the MM cells and their microenvironment, and, consequently, to the lack of appropriate mechanism-based combination therapies.
In this study, we demonstrate that pan-AKI generate pro-survival signals in MM cells by inducing the expression/activation of the pro-survival serine/threonine kinas-es PIM1 and PIM232 through a NIK/c-Abl-mediated activation of STAT3, a cascade of molecular events that consequently 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.
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]-methylene]-2,4-thiazolidinedione (Sigma-Aldrich, St. Louis, MO, USA).
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 previously 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” (identification 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 immunofluorescent studies are described in the Online Supplementary Methods.
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, we formulated the hypothesis that NIK, a kinase capable of activating both the alternative and classical NF-κB pathways through IKKα/β phosphorylation,42 could interfere with the inhibitory effects of pan-AKI on NF-κB signaling.2524
To investigate this hypothesis, we blocked Aurora kinase activity with the pan-AKI MK-0457 or PHA-680632,2925 and monitored the impact on NIK levels in HMCL with barely detectable (OPM-2), very low (U266), low (8226 and R5), or high (JJN3) NIK expression.42 Interestingly, pan-AKI significantly increased NIK protein levels in all the tested HMCL, although to varying degrees depending on the cell line examined, with an average fold increase ranging from 1.3 (U266) to 7.8 (OPM-2) (Figure 1A). Furthermore, consistent with previous studies demonstrating that MM-microenvironmental interactions induce reciprocal activation of NF-κB in both cellular compartments,35 together with the fact that NIK stabilization is a critical step for NF-κB activation in MM cells,42 we found that adherence of MM cells to HS-5 stromal cells caused a significant accumulation of NIK protein in 4 of 5 HMCL tested (except JJN3) and also in the HS-5 stromal cells, and that this increment was further enhanced by pan-AKI treatment in both the co-cultured cell populations, MM and stromal cells (Figure 1B). Notably, pan-AKI did not significantly affect NIK mRNA levels in MM cells (Figure 1C), thereby suggesting that pan-AKI-induced NIK protein accumulation in MM cells is mainly due to post-translational rather than transcriptional regulation.
Given the critical role of TRAF2 and TRAF3 in regulating cIAP1/2-mediated NIK proteasomal degradation,72 we investigated the effects of pan-AKI on the protein expression of these NIK negative regulators. We found that pan-AKI treatment induced a significant reduction in the protein levels of TRAF2 but not TRAF3 in all the tested HMCL, either cultured alone (Figure 1A) or co-cultured with HS-5 stromal cells (Figure 1B). Furthermore, pan-AKI treatment did not modulate TRAF2 mRNA levels in MM cells (Figure 1C), thereby indicating that its protein expression is not regulated at transcriptional levels by these inhibitors.
Furthermore, small interfering RNA (siRNA)-mediated knockdown of Aurora-A and -B recapitulated the ability of pan-AKI to down-regulate the negative regulator of NIK, TRAF2, and increase NIK protein levels (Figure 1D), thereby confirming the significant role of Aurora kinases in modulating NIK stability through TRAF2 in MM cells. On the other hand, siRNA-mediated knockdown of TRAF2 led to NIK accumulation in all the HMCL studied (Figure 1E and Online Supplementary Figure S1), including those with deletion or inactivating mutations of TRAF3 (U266, 8226 and 8226/R5) or bearing alterations in the TRAF3-binding domain of NIK (JJN3),32 thereby confirming the important role of TRAF2 in regulating NIK degradation in MM.42
NF-κB-inducing kinase attenuates the anti-tumor activity of pan-AKI in multiple myeloma cells
We found that NIK inhibition by either the NIK small-molecule inhibitor 4H-isoquinoline-1,3-dione (NIK-in)36 or the NIK-specific siRNA significantly enhanced pan-AKI-induced cell death in all the HMCL tested, either cultured alone or in co-culture with HS-5 stromal cells (Figure 2A). Importantly, NIK-in synergized with pan-AKI to kill MM cells (Figure 2B and Online Supplementary Table S1). Furthermore, adherence of MM cells to HS-5 stromal cells conferred significant protection against pan-AKI-induced cell death in the majority of the HMCL analyzed. However, this protective effect was significantly reduced by NIK inhibition (Figure 2A), thus confirming the important role of NIK in the stroma-mediated pan-AKI protection. Finally, NIK-in significantly (P<0.005; n=10) increased the cytotoxicity of pan-AKI in patient-derived primary MM cells (Figure 2C and Online Supplementary Figure S2A), with no significant differences in the response rates between newly diagnosed (n=3) and relapsed (n=7) patients (Online Supplementary Figure S3) but not on PBMC from healthy individuals (Figure 2C and Online Supplementary Figure S2B). These observations thereby indicate that NIK plays an important role in the responsiveness of MM cells to pan-AKI. (Patients’ demographic and clinical characteristics are summarized in Online Supplementary Table S2).
It is also important to highlight the fact that treatment of MM cells with the proteasome inhibitor bortezomib (currently the standard of care for MM) caused a strong accumulation of the NIK protein in the majority of the HMCL analyzed (Online Supplementary Figure S4A) and its chemical inhibition significantly enhanced the anti-myeloma effects of bortezomib, thereby indicating that NIK can influence the sensitivity of MM cells to this drug (Online Supplementary Figure S4B).
NF-κB-inducing kinase interferes with the inhibitory activity of pan-AKI on NF-κB-inducing kinase
To examine whether NIK accumulation induced by pan-AKI counteracts their ability to inhibit NF-κB pathways in MM cells, we blocked its function with a NIK-specific siRNA and monitored NF-κB activity in response to pan-AKI. We found that in 4 of 5 HMCL tested (except OPM-2 cells that have low NF-κB index2543), NIK knockdown reduced the basal phosphorylation/activation status of IKKα and IKKβ (p-IKKα/β) and their respective downstream direct targets NF-κB2/p100 and IκB-α (Figure 3A), thus confirming that NIK affects not only the non-canonical but also the canonical NF-κB pathway in MM cells.42 Notably, pan-AKI were ineffective (OPM-2) or only partially effective (all the other HMCL analyzed) in attenuating NF-κB signaling25 (Figure 3A), and their reduced inhibitory activity on NF-κB signaling was closely linked to NIK induction because its knockdown by siRNA completely abrogated the pan-AKI-induced NF-κB activation in OPM-2 as well as greatly enhanced the pan-AKI-induced NF-κB inhibition in all the other HMCL analyzed (Figure 3A).
In support of these data, we found that experimental overexpression of NIK in MM cells (Figure 3B) caused enhanced phosphorylation of IKKα/β, NF-κB2/p100 and IκB-α, and increased nuclear localization and DNA binding activities of the NF-κB p65, p50, p52, and RelB subunits (Figure 3C). In contrast, NIK knockdown in these NIK-over-expressing MM cells consistently and significantly decreased their basal NF-κB activity (Figure 3D), thus confirming the important role of NIK in controlling NF-κB signaling in MM.43
NF-κB-inducing kinase induction by pan-AKI activates the STAT3 signaling pathway in multiple myeloma cells
Because NIK induction by pan-AKI was not associated with an increased activation of NF-κB pathways in 4 of 5 HMCL tested (except OPM-2), and yet NIK signaling has been demonstrated to crosstalk at different levels with other important prosurvival signaling pathways including MEK-ERK and STAT3 pathways,108 we explored whether NIK induction by pan-AKI affected these pathways in MM cells.
Because NIK can phosphorylate MEK1 and thereby cause activation of downstream MAPK ERK,9 we investigated whether NIK induction by pan-AKIs is associated with increased phosphorylation/activation of ERK in MM cells.
We found no significant change (U266, R5) or even a decrease (OPM-2, JJN3) in ERK activity (p-ERK1/2) in the pan-AKIs-treated HMCL (Figure 4A), thereby indicating that NIK, stabilized by pan-AKI, does not act through this pathway. Because STAT3 activity is regulated by two independent phosphorylations, one occurring at Tyr705 and one at Ser727, which are both required for it to be fully functional,37 we specifically analyzed the STAT3 (Tyr705) and STAT3 (Ser727) phosphorylation patterns alongside with the overall protein expression levels. We found that treatment with pan-AKI significantly increased both Ser727 and Tyr705 STAT3 phosphorylation in OPM-2, RPMI-8226 and 8226/R5, but not in U266 and JJN3 HMCL where no significant changes in p-Ser-STAT3 or a decrease in p-Tyr-STAT3 phosphorylation were observed (Figure 4A).
Notably, NIK knockdown in MM cells completely abrogated both Ser727 and Tyr705 STAT3 phosphorylation induced by pan-AKI (Figure 4B), which would suggest that NIK is involved in the pan-AKI-mediated STAT3 activation. Confirming these data, we found that ectopic expression of NIK in MM cells caused enhanced phosphorylation of STAT3 in both serine and tyrosine residues (Figure 4C), whereas its depletion in these NIK-over-expressing MM cells consistently and significantly (P<0.001) decreased their basal STAT3 activity levels (Figure 4D).
In the light of evidence supporting reciprocal regulatory mechanisms and crosstalk between the NIK and STAT3 proteins,10 we examined whether NIK exists in a complex with STAT3 in MM cells. Co-immunoprecipitation showed that STAT3 was associated with NIK and that this association was significantly enhanced by pan-AKI treatment of the cells (Figure 4E).
We also examined the Ser727 and Tyr705 phosphorylation state of STAT3 that co-immunoprecipitated with NIK and found that treatment with pan-AKI promoted a strong increase in the phosphorylation of NIK-associated STAT3 in both serine and tyrosine residues (Figure 4E and F), stressing the putative function of NIK in controlling STAT3 activation.
Aurora kinases inhibitors induce a NF-κB-inducing kinase dependent cytoplasmic relocalization and activation of c-Abl and promote the formation of the NIK-c-Abl-STAT3 ternary complex in multiple myeloma
Given the high levels of tyrosine-phosphorylated STAT3 that co-immunoprecipitates with the serine/threo-nine kinase NIK in response to pan-AKI treatment, we explored whether pan-AKI affect the Stat3 upstream tyrosine kinases JAK2, Src and/or c-Abl38 activity/expression.
We found that, depending on the HMCL examined, pan-AKI caused a decrease or no significant changes in the Tyr-phosphorylation/activity of JAK2 (p-JAK2) and Src (p-SRC) kinases (Figure 5A), whereas they were able to significantly activate c-Abl in 4 of 6 HMCL tested (except U266 and JJN3 in which no significant changes or a decrease in c-Abl tyrosine-phosphorylation levels were observed, respectively) (Figure 5A). A significant increase (>3-fold) in p-c-Abl, but not in p-JAK2 and p-Src, was also observed in untreated 8226-NIK as compared to untreated 8226 HMCL (Figure 5A, lane 5 vs. lane 3). This finding links NIK to c-Abl signaling and, indeed, experimental overexpression of NIK in MM cells causes enhanced phosphorylation of endogenous c-Abl on Tyr245 and Tyr412 residues (both commonly used as Abl activation markers),1716 as well as Tyr705 phosphorylation of STAT3. Conversely, knockdown of NIK in these NIK-over-expressing MM cells consistently and significantly decreased their basal tyrosine-phosphorylation levels (Figure 5B). Accordingly, abrogation of Aurora-A and -B induced c-Abl phosphorylation at both Tyr245 and Tyr412 residues in MM cells (Figure 5C).
As c-Abl may exhibit both pro- and antiapoptotic functions depending on the subcellular localization (nuclear or cytoplasmic),1815 and its intracellular localization is regulated by phosphorylation of its Thr735 residue promoting cytoplasmic sequestration by the 14-3-3 protein,19 we explored whether pan-AKI affect Thr735 phosphorylation and/or subcellular localization of c-Abl in MM cells in which the pervasive DNA damage leads to a predominantly nuclear localization of c-Abl.2221 As shown in Figure 6 and Online Supplementary Figure S5, endogenous c-Abl was predominantly accumulated in the nucleus of the MM cells,21 while pan-AKI were able to cause a significant translocation of c-Abl from the nucleus to cytoplasm, thus elevating its cytoplasm/nucleus ratio in 4 of 5 HMCL tested (except JJN3) (Figure 6). Notably, the pan-AKI-induced cytoplasmic accumulation of c-Abl was associated with increased Thr735 phosphorylation of the cytoplasmic fraction of c-Abl (Figures 6 and 7A).
Both processes, Thr735 phosphorylation and concomitant cytoplasmic accumulation of c-Abl, were closely linked to NIK induction since its overexpression in MM cells increased Thr735 phosphorylation of cytoplasmic c-Abl (Figure 7A) and caused c-Abl to translocate from the nuclear to the cytoplasmic compartment, whereas its inhibition in these NIK-over-expressing cells reversed this shuttling (Figure 7A). These data were further confirmed by immunofluorescence analysis (Online Supplementary Figure S6).
A closer examination revealed that NIK was diffused in the cytoplasm with an accumulation around the nucleus of the tumor cells treated with pan-AKI (Figure 7B), and its overexpression also caused enhanced tyrosine phosphorylation of cytoplasmic c-Abl (Figure 7A), to elicit its anti-apoptotic functions.1815
Together with these results, siRNA-mediated knockdown of NIK completely abrogated the pan-AKI-induced Thr735 phosphorylation of c-Abl in OPM-2 and greatly decreased the high basal c-Abl Thr735 phosphorylation in the high NIK expressing JJN3 HMCL (Figure 7C).
Because pan-AKI can induce NIK accumulation and concomitant c-Abl activation, and both these kinases converge on and activate the STAT3 pathway,1710 we next investigated whether c-Abl can form a heterotrimeric complex with NIK and STAT3 in MM cells. As indicated in Figure 8A and B, there was little if any detectable interaction of c-Abl and NIK in untreated MM cells. However, exposure of MM cells to Pan-AKI led to an increase in the association of c-Abl with NIK kinases that was at least a 3-fold higher than in untreated control cells (Figure 8C).
The interaction between NIK and c-Abl, and that previously shown between NIK and STAT3 (Figure 4E), together with the fact that c-Abl can regulate the activation of STAT3 in cancer cells,17 indicated that these three proteins may form a trimeric complexes in pan-AKI-treated MM cells. Accordingly, as shown in Figure 8B, immunoprecipitation of endogenous c-Abl from lysates of untreated or pan-AKI-treated MM cells followed by STAT3 immunoblotting revealed that the pharmacological blockade of Aurora kinases induced a physical interaction of c-Abl with STAT3, thus confirming that, in MM cells, pan-AKI can promote the formation of the ternary complex NIK-c-Abl-STAT3.
Pharmacological blockade of c-Abl sensitizes multiple myeloma cells to pan-AKI
To examine the functional significance of the pan-AKI-induced activation of c-Abl in MM cells we blocked its function using the Abl kinase inhibitors imatinib or nilotinib20 and monitored cell death in response to pan-AKI treatment. Both imatinib or nilotinib significantly increased the pan-AKI-induced cell death in the majority of the HMCL as well as in patient-derived primary MM cells, (P<0.005; n=9) (Figure 9A and B and Online Supplementary Figure S7A and B), with no significant differences observed in the response rates of newly diagnosed (n=4) versus relapsed (n=5) patients (Online Supplementary Figure S8) and no effects seen in normal PBMC (Figure 9B and Online Supplementary Figure S7C).
In agreement with these results, Aurora-A and -B inhibition by either Aurora A/B-specific siRNA or AMG-900,2923 a potent and highly selective pan-AKI, significantly enhanced the sensitivity of MM cells to c-Abl inhibitors (Figure 9C, Online Supplementary Figures S9 and S10A and B). Furthermore, c-Abl kinase inhibitors consistently synergized with pan-AKI to induce cell death in MM cells (Online Supplementary Figure S11 and Online Supplementary Table S3).
Remarkably, as observed in the majority of the HMCL analyzed, treatment of cells isolated from MM patients with Pan-AKI induced NIK accumulation, increased Thr735, Tyr245 and Tyr412 phosphorylation of c-Abl and Ser727 and Tyr705 phosphorylation of STAT3 (Figure 9D). None of these conditions was observed in similarly treated PBMC from healthy donors.
To verify that the anti-tumor activity of pan-AKI and the synergizing effects of c-Abl inhibitors observed on cultured/isolated MM cells could be reproduced in vivo, we set up a multidrug-resistant xenograft mouse model of human MM. Consistent with our in vitro results, imatinib significantly potentiated the anti-tumor activity induced by pan-AKI in this in vivo setting, while having no effect as a single agent in vivo in a multidrug-resistant xenograft mouse model of human MM (Figure 10A). Animal survival was also significantly improved in mice treated with the combination imatinib/pan-AKI versus those that received monotherapies or vehicle alone (P<0.0015) (Figure 10A and Online Supplementary Table S4).
Immunobloting analyses on tumor masses harvested after five days post treatment confirmed decreases in the phosphorylation levels of Aurora kinases, enhancement of NIK protein, and increases in the Thr735, Tyr245 and Tyr412 phosphorylation of c-Abl and Tyr705 phosphorylation of STAT3 in the case of xenografted animals treated with pan-AKI when compared to vehicle-treated controls (Figure 10B). In addition, immunohistochemical staining of tumor lesions for NIK and c-Abl revealed that also in vivo pan-AKI were capable of causing cytoplasmic NIK accumulation, which was most prominent around the nucleus of the tumor cells (Figure 10C and Online Supplementary Figure S12), whereas c-Abl was observed to have been extensively translocated from the nucleus to the cytoplasm (Figure 10C).
Finally, immunohistochemical analysis of tumor lesions isolated from pan-AKI-treated animals consistently revealed a significant reduction in the phosphorylation of Histone H3 on Ser10 (Figure 10D), a protein known to be a physiological substrate of Aurora kinases and a cellular proliferation marker.39 This result would be consistent with the retardation of tumor growth observed in pan-AKI-treated versus vehicle-treated mice (Figure 10A).
Notably, combined imatinib and pan-AKI treatment blunted the pan-AKI-induced tyrosine (but not threonine) phosphorylation of c-Abl (Figure 10B) and increased the levels of apoptosis (cleaved-PARP and -caspase-3 staining), relative to that seen with monotherapies and vehicle alone (Figure 10D); a result that agreed with the tumor regression and the improved survival rate observed in mice treated with the imatinib-Pan-AKI combination therapy (Figure 10A).
Pan-AKI-induced NF-κB-inducing kinase accumulation promotes survival signaling through PIM kinases activation
Consistent with the fact that NIK can elicit pro-survival signals in MM cells through activation of NF-κB and STAT3 pathways, we found that experimental overexpression of NIK in MM cells caused the induction of the antiapoptotic NF-κB/STAT3 regulated genes Bcl-xL, A1/Bfl-1, Mcl-1 and XIAP40 (Figure 11A), all of which represent important targets for sensitizing MM cells to anticancer agents,1 including pan-AKI.25 NIK overexpression was also associated with upregulation of PIM1 and PIM2 (Figure 11A), both oncogenic, constitutively active serine/threonine kinases transcriptionally regulated either by NF-κB or STAT3, that mediate survival signaling through the phosphorylation and inactivation of Bad4132 (Figure 11A). In accordance with its role in controlling anti-apoptotic signal transduction events, NIK overexpression protected MM cells from pan-AKI-induced cell death, which was reversed by the chemical or genetic disruption of NIK functions (Figure 11B).
We further found that in 5 of 7 HMCL tested (except U266 and JJN3 cells), the pan-AKI-induced NIK-stabilization was associated with enhanced levels of PIM1 and PIM2 proteins, and phosphorylation of their direct downstream target Bad (Figure 11C and Online Supplementary Figure S13); RNA interference-mediated knockdown of NIK or the use of a NIK-inhibitor (NIK-in) prevented these increments (Figure 11C), thus confirming the role of NIK in PIM kinases induction in MM cells. Taken together with our previous findings (Figures 4–8), the observations also supported the existence of a NIK/c-Abl /STAT3 /PIM /Bad signaling axis in pan-AKI-treated MM cells.
Consistent with the fact that STAT3 can regulate the expression of PIM kinases,4132 we found that its inhibition by siRNA completely abrogated the pan-AKI-induced PIM1 and PIM2 upregulation in OPM-2, RPMI-8226 and RPMI-8226-NIK HMCL, and greatly decreased their basal levels in JJN3 cells (Figure 11D).
Loss-of-function of STAT3 by either siRNA or the small-molecule inhibitor STATTIC42 significantly enhanced the pan-AKI sensitivity of MM cells (Figure 11D and Online Supplementary Figure S14), thereby indicating that STAT3 activated by pan-AKI acted as a prosurvival, antiapoptotic transcription factor in MM.
PIM kinases have been implicated in the regulation of MM cell proliferation, survival, and drug resistance.43 Given this, we examined whether their inhibition affected the responses of MM cells to pan-AKI. PIM1/2 inhibition, by either the specific small-molecule inhibitor SMI-4a44 or by PIM1/2-specific siRNA significantly increased the pan-AKI-induced cell death in all the HMCL tested either cultured alone or together with HS-5 cells, except for U266 and JJN3 (Figure 12A and B, and Online Supplementary Figure S15), in which pan-AKI failed to increase PIM kinases levels (Supplementary Figure S14).
Furthermore, treatment of patient-derived MM cells, but not normal PBMC, with Pan-AKI led to an increment of PIM1/2 protein levels (Figure 12C), that significantly (P<0.005; n=10) influenced the responsiveness of the cells to pan-AKI, with similar response rates between newly diagnosed (n=3) and relapsed (n=7) patients (Figure 12D and Online Supplementary Figures S2A and S3), thereby indicating that these kinases may significantly impact on the susceptibility of MM cells to pan-AKI exposure.
The critical role of NIK in regulating non-canonical and canonical NF-κB pathways in MM,64 together with the fact that NIK and Aurora kinases can converge on common targets,2624 prompted us to hypothesize that NIK might interfere with and reduce or bypass the NF-κB inhibitory effects exerted by pan-AKI on MM cells. In support of this hypothesis, we found that pan-AKI induce NIK protein stabilization and that this depended on the downregulation of the TRAF2 protein, one of the critical NF-κB negative regulators that, together with TRAF3, form a molecular bridge that couples NIK to the NIK K48-ubiquitin ligase cIAP1/2.76 We also found that TRAF2 reduction was sufficient to elevate NIK protein levels in MM cells harboring alterations in the TRAF3-binding domain of NIK or in TRAF3 itself, thus confirming that TRAF2 can regulate NIK stabilization independent of TRAF3.454
Although experimental overexpression of NIK led to a marked activation of both NF-κB and STAT3 pathways, its induction by pan-AKI resulted in the activation of only the STAT3 pathway, thereby suggesting that Aurora kinases can significantly contribute to the basal NF-κB activity of MM cells and that their inhibition can partially compensate for the NIK-induced activation of NF-κB pathways.
In MM, the pervasive DNA damage triggers constitutive activation of the ATR/ATM-regulated DNA damage response proapoptotic network which in turn leads to a prominent and preferential nuclear localization of c-Abl. Here, however, it is unable to induce apoptosis because of disruption of the ABL-YAP1-p73 axis.21 The nuclear accumulation of c-Abl in MM21 may explain its marginal role in MM pathogenesis46 and the therapeutic inefficacy of c-Abl inhibitors in monotherapy regimens or when used in combination with other agents for the treatment of MM.47 Instead, by inducing a NIK-dependent cytoplasmic relocalization and activation of c-Abl, pan-AKI switch it from a pro-apoptotic to a pro-survival factor, thereby turning it into a potentially effective target for MM. In accordance with this, we demonstrate here that c-Abl inhibitors consistently increase the sensitivity of MM cells to pan-AKI in different experimental settings and in patient-derived cells.
Our data identify NIK as a kinase responsible for phosphorylation of c-Abl at Thr735, which is critical for its cytoplasmic retention, thereby indicating that NIK influences the subcellular localization of c-Abl in MM cells. NIK, stabilized by pan-AKI, forms a trimeric complex with c-Abl and STAT3 and, together with c-Abl, contributes to the serine 727 and tyrosine 705 phosphorylation of STAT3. NIK is also involved in the tyrosine-phosphorylation/activation of c-Abl observed after pan-AKI treatment, as supported by our genetic perturbation experiments of NIK in MM cells. This recalls the fact that also serine/threonine kinases, in addition to SRC family kinas-es, may regulate the catalytic activity of c-Abl via direct protein-protein interactions and/or by promoting phosphorylation of c-Abl on serine and/or threonine residues.1716 Moreover, pan-AKI failed to induce c-Abl and STAT3 tyrosine phosphorylation in those HMCL (U266 and JJN3) in which the high basal activity of Src kinase was significantly inhibited by pan-AKI, thereby indicating that Src kinase, of which c-Abl and STAT3 are direct downstream substrates and effectors,1816 may compensate for or obscure the pan-AKI-induced NIK-dependent c-Abl activation.
Based on its off-target activity against wild-type and mutated BCR-ABL including the imatinib-/nilotinib-/dasatinib-resistant T315I-BCR-ABL, MK-0457 has shown clinical efficacy in chronic myelogenous leukemia patients bearing T315I mutated BCR-ABL.5048 However, it has been demonstrated that MK-0457 is able to inhibit the autophosphorylation of T315I mutant BCR-ABL at concentrations (IC50 values ranging from 5 to 10 μM)525149 which are significantly higher than those clinically achievable (plasma/serum concentrations 1-3 μM)535048 and 12.5-100-fold greater than those required to inhibit Aurora kinases and exert its anti-tumor activities.5149 Particular attention was, therefore, given here to assay the effects of MK-0457 at submolar concentrations (0.1-0.4 μM) in all of our in vitro experiments.
Interestingly, MK-0457, in its cytostatic and/or cytotoxic potential, did not discriminate between parental and wild-type or mutants BCR-ABL-transformed Ba/F3 cells.5249 However, hematologic responses were also observed in patients treated with MK-0457 at clinical doses that had not been reported to affect BCR-ABL kinase activity.50 This would suggest that the activity of the drug is mainly exerted through inhibition of Aurora kinases rather than by interference with BCR-ABL function.
Finally, a role for c-Abl in the activation of STAT3 has been reported,3813 as well as a connection between c-Abl and Mcl-1 in chronic myelogenous and lymphocytic leukemias.5413 Indeed, we have previously showed that pan-AKIs were capable of up-regulating Mcl-1 in MM cells.25
Thus, cytoplasmic relocalization and activation of c-Abl secondary to NIK cytoplasmic accumulation, together with the formation of NIK/c-Abl/STAT3 trimeric complexes, emerge as novel survival mechanisms that significantly impair the antitumor efficacy of pan-AKI and identify potential translational approaches for targeting these mechanisms by using pan-AKI in combination with NIK, c-Abl, STAT3 and/or PIM inhibitors (Figure 13).
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/12/2465
- Funding This work was supported by grants from “Chiara Tassoni” Onlus Association, Parma, Italy (LM), Associazione Italiana per la Ricerca sul Cancro (IG, Rif 10670, Italian Association for Cancer Research, Milan, Italy; AB), and Fondazione Cassa di Risparmio di Parma (Cariparma, Parma, Italy; AB). This work was supported by the Italian Ministry of Health (GR-2016-02363646 to LM) and by Regione Emilia Romagna L. 20/2002 (GPG/2018/918 to PL).
- Received October 17, 2018.
- Accepted April 3, 2019.
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