Non-canonical role for the ataxia-telangiectasia-Rad3 pathway in STAT3 activation in human multiple myeloma cells.

PURPOSE: The goal of this study was to characterize the relationship between ATR and STAT3 interactions in human multiple myeloma (MM) cells. METHODS: Various MM cell lines, including IL-6-dependent cells were exposed to ATR inhibitors and effects on STAT3 Tyr705 and Ser727 were monitored by WB analysis and ImageStream analysis. Parallel studies examined induction of cell death, STAT3 DNA binding activity, and expression of STAT3 downstream targets (BCL-XL, MCL-1, c-MYC). Validation was obtained in ATR shRNA knock-down cells, and in cells ectopically expressing BCL-XL, MCL-1, or c-MYC. Analogous studies were performed in primary MM cells and in a MM xenograft model. RESULTS: Multiple pharmacologic ATR inhibitors inhibited STAT3 Tyr705 (but not Ser727) phosphorylation at low uM concentrations and down-regulated BCL-XL, MCL-1, c-MYC in association with cell death induction. Compatible results were observed in ATR shRNA knock-down cells. Cell death induced by ATR inhibitors was significantly attenuated in cells ectopically expressing constitutively active STAT3, BCL-XL, MCL-1, or c-MYC. Concordant results were observed in primary human MM cells and in an in vivo MM xenograft model. CONCLUSIONS: Collectively, these findings argue for a non-canonical role for the ATR kinase in STAT3 activation in MM cells, and suggest that STAT3 inactivation contributes to the lethal actions of ATR inhibitors in MM.

Synergistic Interactions between the Hypomethylating Agent Thio-Deoxycytidine and Venetoclax in Myelodysplastic Syndrome Cells.

Interactions between the novel hypomethylating agent (HMA) thio-deoxycytidine (T-dCyd) and the BCL-2 antagonist ABT-199 (venetoclax) have been examined in human myelodysplastic syndrome (MDS) cells. The cells were exposed to agents alone or in combination, after which apoptosis was assessed, and a Western blot analysis was performed. Co-administration of T-dCyd and ABT-199 was associated with the down-regulation of DNA methyltransferase 1 (DNMT1) and synergistic interactions documented by a Median Dose Effect analysis in multiple MDS-derived lines (e.g., MOLM-13, SKM-1, and F-36P). Inducible BCL-2 knock-down significantly increased T-dCyd's lethality in MOLM-13 cells. Similar interactions were observed in the primary MDS cells, but not in the normal cord blood CD34+ cells. Enhanced killing by the T-dCyd/ABT-199 regimen was associated with increased reactive oxygen species (ROS) generation and the down-regulation of the anti-oxidant proteins Nrf2 and HO-1, as well as BCL-2. Moreover, ROS scavengers (e.g., NAC) reduced lethality. Collectively, these data suggest that combining T-dCyd with ABT-199 kills MDS cells through an ROS-dependent mechanism, and we argue that this strategy warrants consideration in MDS therapy.

Dual mTORC1/2 Inhibition Synergistically Enhances AML Cell Death in Combination with the BCL2 Antagonist Venetoclax.

PURPOSE: Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. EXPERIMENTAL DESIGN: Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. RESULTS: Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. CONCLUSIONS: The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.

Mechanisms underlying synergism between circularized tumor necrosis factor-related apoptosis inducing ligand and bortezomib in bortezomib-sensitive or -resistant myeloma cells.

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor-related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib-resistant U266 cells were exposed to low nanomolar concentrations of bortezomib ± CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ-naïve and -resistant cells. The regimen up-regulated DR4 receptor internalization in MM cells, known to modulate both NF-κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non-canonical NF-κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up-regulation, NF-κB inducing kinase down-regulation, diminished p52 and p50 processing, and B-cell lymphoma-extra large (BCL-XL) down-regulation, but failed to inactivate the canonical NF-κB pathway, reflected by unchanged or increased expression of phospho-p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock-down, dominant-negative Fas-associated protein with death domain, knock-down of caspase-8, BCL-2/BCL-XL, or exposure to a caspase-9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib-resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34+ hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient-derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non-canonical NF-κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.

Chk1 Inhibition Potently Blocks STAT3 Tyrosine705 Phosphorylation, DNA-Binding Activity, and Activation of Downstream Targets in Human Multiple Myeloma Cells.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma cells. Gene expression profiling of U266 cells exposed to low (nmol/L) Chk1 inhibitor [PF-477736 (PF)] concentrations revealed STAT3 pathway-related gene downregulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by chromatin immunoprecipitation assay, and downregulation of STAT-3-dependent proteins. Similar findings were obtained in other multiple myeloma cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. Surface plasmon resonance analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or short hairpin RNA knockdown cells also displayed STAT3 inactivation and STAT3-dependent protein downregulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and downregulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (γH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ multiple myeloma cells and tumors extracted from an NSG multiple myeloma xenograft model while inhibiting tumor growth. IMPLICATIONS: These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.

IAP and HDAC inhibitors interact synergistically in myeloma cells through noncanonical NF-κB- and caspase-8-dependent mechanisms.

Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8-related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589-induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell-enriched CD138-/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.

Cotargeting BCL-2 and PI3K Induces BAX-Dependent Mitochondrial Apoptosis in AML Cells.

Inhibitors targeting BCL-2 apoptotic proteins have significant potential for the treatment of acute myeloid leukemia (AML); however, complete responses are observed in only 20% of patients, suggesting that targeting BCL-2 alone is insufficient to yield durable responses. Here, we assessed the efficacy of coadministration of the PI3K/mTOR inhibitor GDC-0980 or the p110ß-sparing PI3K inhibitor taselisib with the selective BCL-2 antagonist venetoclax in AML cells. Tetracycline-inducible downregulation of BCL-2 significantly sensitized MV4-11 and MOLM-13 AML cells to PI3K inhibition. Venetoclax/GDC-0980 coadministration induced rapid and pronounced BAX mitochondrial translocation, cytochrome c release, and apoptosis in various AML cell lines in association with AKT/mTOR inactivation and MCL-1 downregulation; ectopic expression of MCL-1 significantly protected cells from this regimen. Combined treatment was also effective against primary AML blasts from 17 patients, including those bearing various genetic abnormalities. Venetoclax/GDC-0980 markedly induced apoptosis in primitive CD34+/38-/123+ AML cell populations but not in normal hematopoietic progenitor CD34+ cells. The regimen was also active against AML cells displaying intrinsic or acquired venetoclax resistance or tumor microenvironment-associated resistance. Either combinatorial treatment markedly reduced AML growth and prolonged survival in a systemic AML xenograft mouse model and diminished AML growth in two patient-derived xenograft models. Venetoclax/GDC-0980 activity was partially diminished in BAK-/- cells and failed to induce apoptosis in BAX-/- and BAX-/-BAK-/- cells, whereas BIM-/- cells were fully sensitive. Similar results were observed with venetoclax alone in in vitro and in vivo systemic xenograft models. Collectively, these studies demonstrate that venetoclax/GDC-0980 exhibits potent anti-AML activity primarily through BAX and, to a lesser extent, BAK. These findings argue that dual BCL-2 and PI3K inhibition warrants further evaluation in AML.Significance: Combined treatment with clinically relevant PI3K and BCL-2 inhibitors may prove effective in the treatment of acute myeloid leukemia. Cancer Res; 78(11); 3075-86. ©2018 AACR.