Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad

ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can overcome such resistance to restore the sensitivity. In the present study, a second-generation selenium compound methylseleninic acid (MSeA) synergistically sensitized MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells and DU145 human prostate cancer cells to apoptosis induction by ABT-737, as evidenced by greater than additive enhancement of Annexin V/FITC positive (apoptotic) cells and activation of multiple caspases and PARP cleavage. Mechanistic investigation demonstrated that MSeA significantly decreased basal Mcl-1 expression and ABT-737-induced Mcl-1 expression. Knocking down of Mcl-1 with RNAi approach supported the functional significance of this molecular target. More importantly, we identified inactivation of Bad by phosphorylation on ser-136 and ser-112 as a novel mechanism involved in ABT-737 resistance, which can be overcome by combining with MSeA. In addition, we found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737.     

Platinum compounds sensitize ovarian carcinoma cells to ABT-737 by modulation of the Mcl-1/Noxa axis

Ovarian cancer is the leading cause of death from gynecological cancer. The anti-apoptotic protein Bcl-x_L is frequently overexpressed in ovarian carcinoma which correlates with chemotherapy resistance. It has been demonstrated that Bcl-x_L cooperates with another anti-apoptotic protein, Mcl-1, to protect ovarian cancer cells against apoptosis, and that their concomitant inhibition induces massive cell death. Here, we examined the interest of ABT-737, a potent BH3-mimetic molecule targeting Bcl-x_L, both alone and in combination with Mcl-1 modulators, in ovarian cancer cell lines. As a single agent, ABT-737 was ineffective at promoting cell death in the four cell lines we tested in vitro. However, the specific inhibition of Mcl-1 by siRNA dramatically increased the sensitivity of chemoresistant cells to ABT-737. Platinum compounds also sensitize to ABT-737 by dose-dependently decreasing Mcl-1 expression or by increasing the expression of pro-apoptotic BH3-only proteins Noxa and, to a lower extent, Bim. Furthermore, we demonstrated that Noxa accumulation was involved in apoptosis occurring in response to the combination of ABT-737 and platinum compounds, since cells were protected from apoptosis by its silencing. Moreover, the combination was also highly cytotoxic ex vivo in sliced SKOV3 tumor nodes. However we observed in these slices a strong basal expression of Noxa and apoptotic cell death in response to ABT-737 alone. Therefore, we have revealed that the modulation of the Mcl-1/Noxa axis by platinum compounds results in a strong sensitization of chemoresistant ovarian carcinoma cells to ABT-737, which could constitute a promising therapeutic in these cancers.     

Inhibition of PIM1 blocks the autophagic flux to sensitize glioblastoma cells to ABT-737-induced apoptosis

Overcoming apoptosis resistance is one major issue in glioblastoma (GB) therapies. Accumulating evidence indicates that resistance to apoptosis in GB is mediated via upregulation of pro-survival BCL2-family members. The synthetic BH3-mimetic ABT-737 effectively targets BCL2, BCL2 like 1 and BCL2 like 2 but still barely affects cell survival which is presumably due to its inability to inhibit myeloid cell leukemia 1 (MCL1). The constitutively active serine/threonine kinase proviral integration site for moloney murine leukemia virus 1 (PIM1) was recently found to be overexpressed in GB patient samples and to maintain cell survival in these tumors. For different GB cell lines, Western Blot, mitochondrial fractionation, fluorescence microscopy, effector caspase assays, flow cytometry, and an adult organotypic brain slice transplantation model were used to investigate the putative PIM1/MCL1 signaling axis regarding potential synergistic effects with ABT-737. We demonstrate that combination of the PIM1 inhibitor SGI-1776 or the pan-PIM kinase inhibitor AZD1208 with ABT-737 strongly sensitizes GB cells to apoptosis. Unexpectedly, this effect was found to be MCL1-independent, but could be partially blocked by caspase 8 (CASP8) inhibition. Remarkably, the analysis of autophagy markers in combination with the observation of massive accumulation and hampered degradation of autophagosomes suggests a completely novel function of PIM1 as a late stage autophagy regulator, maintaining the autophagic flux at the level of autophagosome/lysosome fusion. Our data indicate that PIM1 inhibition and ABT-737 synergistically induce apoptosis in an MCL1-independent but CASP8-dependent manner in GB. They also identify PIM1 as a suitable target for overcoming apoptosis resistance in GB.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.     

Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

Background and purpose

Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.

Experimental approach

For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.

Key results

The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC₅₀ of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.

Conclusions and implications

Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.     

Targeting A20 enhances TRAIL-induced apoptosis in hepatocellular carcinoma cells

A20 was initially identified as a primary gene product following TNF α treatment in human umbilical vein endothelial cells. Increased A20 expression is associated with tumorigenesis in many cancers, whereas the loss of A20 function is linked to lymphoma. It has been reported that A20 protects cells from TRAIL-induced apoptosis; however, the mechanism by which A20 is involved is still largely unknown. Our results indicate that TRAIL induces the hepatocellular carcinoma apoptosis associated with A20 knockdown in a concentration-dependent manner. TRAIL-induced apoptosis requires p18 caspase-8 activation, and, the activation of caspase-8 is at least in part, due to the direct cleavage of RIP1 by A20 knockdown. These findings suggest that A20 modulates the sensitivity to TRAIL by RIP1 ubiquitination, thereby repressing the recruitment and activation of pro-caspase-8 into the active form caspase-8. Thus, our study suggests that A20 protects against TRAIL-induced apoptosis through the regulation of RIP1 ubiquitination.     

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.     

Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ～60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.     

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.     

Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma’s well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.     

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

Background

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma''s striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance.

Methodology/Principal Findings

Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity.

Conclusions/Significance

Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.