Knockdown Of Noxa Research Articles

The chromatin remodeling protein BRG1 contributes to liver ischemia-reperfusion injury by regulating NOXA expression

AimsHepatic ischemia-reperfusion injury (IRI) is a common complication secondary to liver transplantation. Extensive death of hepatocytes, typically in the form of apoptosis, is observed in and contributes to IRI. In the present study we investigated the role of BRG1 (encoded by Smarca4), a chromatin remodeling protein, in the pathogenesis of liver IRI focusing on the transcriptional mechanism and translational potential. MethodsSmarca4f/f mice were crossed to Alb-Cre mice to generate hepatocytes-specific BRG1 knockout mice (CKO). Alterations in cellular transcriptome were evaluated by RNA-seq. ResultsBRG1 expression was up-regulated in liver tissues of mice subjected to I/R and in hepatocytes exposed to hypoxia-reoxygenation (H/R). Compared to wild type (WT) littermates, the BRG1 CKO mice displayed significant amelioration of liver injury following ischemia-reperfusion as evidenced by decreased ALT/AST levels and cell apoptosis. Primary hepatocytes isolated from the CKO mice were protected from H/R-induced apoptosis compared to those from the WT mice. RNA-seq analysis revealed phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA) as a novel target for BRG1. Consistently, NOXA knockdown attenuated liver IRI in mice. More importantly, administration of a small-molecule BRG1 inhibitor (PFI-3) protected the mice from liver IRI. ConclusionsOur data uncover a pivotal role for BRG1 in liver IRI and suggest that targeting BRG1 with small-molecule inhibitors can be considered as a reasonable therapeutic strategy.

Abstract 3710: Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA

Abstract Ovarian cancer is among the top ten leading causes of cancer death in women worldwide with a mortality rate of roughly 5% among all cancer deaths. So far, there are no recommended screening tests for the early detection of ovarian cancer. Therefore, patients are frequently diagnosed at late stages of ovarian cancer, which is associated with unfavorable 5-year survival. Patients suffering from ovarian cancer often show a good response to initial therapy with platinum-based compounds and/or taxanes subsequent to resection of the affected tissue. Unfortunately, ovarian cancer shows a high frequency of relapse and after chemotherapy persistent tumor cells re-expand and successively evolve into therapy-resistant tumors. We recently reported that cisplatin (cisPt) treatment of ovarian cancer cells enhances mitochondrial content and mitochondrial reactive oxygen species (Kleih et al. 2019). We therefore speculated that the enhanced mitochondrial content primes cisPt-treated cells for apoptosis induced by BH3 mimetic drugs, such as Venetoclax (ABT-199), that act on anti-apoptotic Bcl-2 proteins at the outer mitochondrial membrane. To explore this hypothesis, we investigated whether the combined administration of cisPt and ABT-199 leads to a synergistic cell death induction in ovarian cancer cells with different cisPt resistance and mitochondrial content. Detection of cell death by flow cytometric measurements of the mitochondrial membrane potential and exposure of phosphatidylserine demonstrated a synergistic cell death induction by cisPt and ABT-199 in OVCAR4 and OVCAR8 cells, irrespective of the cisPt resistance. Interestingly, Western blot analysis revealed that ABT-199 induced the accumulation of the BH3-only protein NOXA in both cancer cell lines. Accumulation of NOXA was dependent on ABT-199-mediated activation of the stress-responsive transcription factor ATF4. Knock-down experiments showed that apoptosis of cisPt-resistant OVCAR8 cells by the combined treatment with ABT-199 and cisPt was entirely dependent on NOXA. Moreover, also cisPt-sensitive OVCAR4 cells revealed an initially reduced apoptosis induction by the NOXA knock-down. Our data therefore elucidate the molecular mechanism of the synergistic efficacy of cisPt/ABT-199 combination therapies, which should be promising especially for the treatment of recurrent tumors developing reduced sensitivity to cisPt-based treatments. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias B. Beigl, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3710.

Combination of Bortezomib and Venetoclax Induces Synergistic Killing of Epstein-Barr Virus-Driven Lymphoproliferative Diseases By Targeting the Pro-Survival Function of Latent Membrane Protein-1 and Epstein-Barr Nuclear Antigen-3C

Epstein-Barr virus (EBV) is strongly associated with lymphoproliferative diseases (LPDs), in particular B cell, in both immunocompetent and immunocompromised hosts. The virus is able to manipulate host cell machineries such as the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and the survival of the host cells through expression of various viral proteins in distinct latency patterns. Latent membrane protein-1 (LMP-1) is a constitutively active CD40 receptor homolog and activates NF-κB signaling pathways to induce Bcl-2 expression whilst Epstein-Barr nuclear antigen-3C (EBNA-3C) interacts with and mediates proteasomal degradation of Bcl-6 protein which, in turn, increases Bcl-2 expression. We hypothesize that combining a proteasome inhibitor, bortezomib, with a Bcl-2 inhibitor, venetoclax, will induce synergistic killing of EBV-driven LPDs expressing both LMP-1 and EBNA-3C in the latency III pattern. Isobologram showed that combination of bortezomib and venetoclax could synergistically induce potent apoptosis of spontaneous lymphoblastoid cell lines (sLCL), derived from patients with post-transplant lymphoproliferative disorder (PTLD), expressing LMP-1 and EBNA-3C proteins. The mechanism of killing was related to the suppression of NF-κB signaling pathway induced by LMP-1. The phosphorylation of serine 70 of Bcl-2, which enhances the anti-apoptotic activity of Bcl-2 through stabilization of its interaction with other pro-apoptotic proteins such as Bak and Bim, was decreased in the sLCL, but not in the LMP-1 or EBNA-3C knockdown LCL, upon treatment with the drug combination. Activation of DNA damage response and production of reactive oxygen species were observed in the sLCL, contributing to the synergistic cell death. Bortezomib induced the expression of pro-apoptotic initiator, NOXA, to enhance the susceptibility of the sLCL to apoptosis upon treatment with venetoclax whilst knockdown of NOXA in the sLCL led to the resistance of the cells to apoptosis induced by the drug combination. In-vivo study demonstrated that the drug combination significantly inhibited the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, we conclude that the combination of bortezomib and venetoclax induces synergistic killing of EBV-driven LPDs such as PTLD by targeting the pro-survival function of LMP-1 and EBNA-3C. Disclosures No relevant conflicts of interest to declare.

Abstract 6227: ABT-199 and Bortezomib synergistically induce apoptosis in soft-tissue sarcomas

Abstract Soft-Tissue Sarcomas (STS) are mesenchymal malignancies with high heterogeneity that predominantly affect children and young adults. Despite routinely applied therapy strategies including radiotherapy, surgery and chemotherapy, the five-year survival rate of metastatic STS is only 50 %. Therefore, it is of high importance to focus on possible combinational therapies for the effective treatment of all kinds of STS. For such a therapy we combined the clinically approved BH3-mimetic drug ABT-199 (Venetoclax) with the proteasome inhibitor Bortezomib (Velcade). ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 whereas the proteasome inhibitor Bortezomib is effective, e.g., in multiple myeloma. Sarcoma cell lines were incubated with ABT-199 and Bortezomib alone or in combination and apoptotic cell death was flow cytometric detected by analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate an underlying mechanism for apoptosis induction, we analyzed expression of members of the Bcl-2 family involved in the apoptosis pathway by Western Blotting and performed analogue experiments in knock-out cell lines. Combined treatment with ABT-199 and Bortezomib showed synergistic cell death induction in several sarcoma cell lines including Rhabdomyosarcoma, Leiomyosarcoma and Synovial sarcoma. Loss of mitochondrial membrane potential and Annexin V staining revealed apoptosis as the underlying cell death mechanism. Interestingly, expression of Bok, a homologue of the pore-forming effector proteins Bax and Bak, was increased in response to ABT-199 and Bortezomib. Also, expression was increased for the BH3-only protein Noxa and its anti-apoptotic interaction partner Mcl-1. Knock-out (KO) of effector proteins Bax, Bak or Bok in the sarcoma cell line SW982 reduced apoptosis induction by ABT-199/Bortezomib with the most pronounced reduction in SW982/Bax-KO. Additional shRNA mediated knock-down of Noxa in Bax-KO, Bak-KO or Bok-KO SW982 cells further reduced apoptosis compared to Noxa knock-down alone. ABT-199 and Bortezomib synergistically induce apoptotic cell death in various sarcoma cell lines concomitant with enhanced expression of the Bcl-2 proteins Bok, Noxa and Mcl-1. Hence, we suggest a mechanism in which the simultaneous inhibition of anti-apoptotic Bcl-2 by ABT-199 and the stabilization of pro-apoptotic proteins Bok and/or Noxa shift the equilibrium of BCL-2-proteins towards apoptosis. Experiments in Bax, Bak, Bok, and Noxa deficient SW982 cells indicate that Bax and Noxa are crucial for the observed synergistic effect. based on these results we propose the combined treatment with ABT-199 and Bortezomib as a new and highly promising therapy option for advanced STS. Future efforts, e.g. simultaneous knock-out of relevant Bcl-2 proteins, will unravel the detailed underlying mechanism of the observed synergistic cell death induction by ABT-199 and Bortezomib. Citation Format: Sandra Weller, Alina Muenchow, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. ABT-199 and Bortezomib synergistically induce apoptosis in soft-tissue sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6227.

A novel derivative of valepotriate inhibits the PI3K/AKT pathway and causes Noxa-dependent apoptosis in human pancreatic cancer cells.

Natural compound valepotriate exhibits inhibitory activity against a number of cancers, but the effect of valepotriate against pancreatic cancer is unclear, and the structure–activity relationship of valepotriate has not been characterized. In this study, we performed a structure-based similarity search and found 16 hit compounds. Among the 16 hits, (1S,6S,7R)-6-(acetyloxy)-1-[(3-methylbutanoyl)oxy]-4a,5,6,7a-tetrahydro-1H-spiro[cyclopenta[c]pyran-7,2’-oxiran]-4-ylmethyl 3-methylbutanoate (denoted as Amcp) exhibited superior anticancer activity against human pancreatic cancer BxPC-3 and SW1990 cells. The anti-proliferation activity of Amcp was validated in human pancreatic cancer BxPC-3 and SW1990 cells in vitro. Amcp more effectively induced apoptosis in BxPC-3 and SW1990 cells than gemcitabine. At a concentration of 15 μM, Amcp significantly suppressed the PI3K/AKT pathway and disrupted the mitochondrial membrane equilibrium through modulation of Noxa and Mcl-1 balance in both cell lines. Meanwhile, knockdown of Noxa substantially attenuated Amcp-induced reduction of cell viability and anti-apoptotic protein Mcl-1 level in BxPC-3 cells. In addition, Amcp showed synergistic anticancer effects when combined with gemcitabine in BxPC-3 cells. To conclude, this work not only suggests that Amcp possesses a dual-inhibitory activity towards PI3K/AKT pathway and Mcl-1, but also enlightens further development of bioactive valepotriate derivatives.

β,β-Dimethylacrylshikonin Induces Apoptosis in Melanoma Cell Lines by NOXA Upregulation.

Melanoma is the most aggressive form of skin cancer, with high metastasis rates and poor prognosis. Survival rates and possible therapies depend on the state of the tumor and its mutational profile. BRAF and NRAS are the most frequent driver mutations. Currently, there is no efficient therapy for NRAS-mutated or late-stage melanoma. In this study, the therapeutic potential of β,β-dimethylacrylshikonin (DMAS) was investigated on melanoma. The influence of DMAS was determined in five different melanoma cell lines with different mutational profiles. The effects of this compound on cell viability, apoptosis, and gene and protein expression were examined. The results obtained were validated in vivo. DMAS significantly reduced the viability of several melanoma cell lines in a concentration- and time-dependent manner. Furthermore, DMAS induced caspase-3-dependent apoptosis via NOXA upregulation, as confirmed by NOXA knockdown experiments. This is the first time that NOXA-dependent apoptosis was shown with respect to a shikonin derivative and melanoma. Additionally, tumor regression and necrosis under DMAS treatment were demonstrated in vivo. Importantly, BRAF as well as NRAS-mutated metastatic human melanoma cell lines were treated successfully in vitro and in vivo. Taken together, DMAS showed promising results and is worthy of further study.

Homoharringtonine interacts synergistically with bortezomib in NHL cells through MCL-1 and NOXA-dependent mechanisms

BackgroundInteractions between the protein synthesis inhibitor homoharringtonine (HHT) and the proteasome inhibitor bortezomib were investigated in DLBCL and mantle cell lymphoma cells (MCL).MethodsVarious DLBCL and MCL cells were exposed to HHT and bortezomib alone or together after which apoptosis and signaling pathway perturbations were monitored by flow cytometry and Western blot analysis. Xenograft mouse models were used to assess tumor growth and animal survival.ResultsHHT and bortezomib co-administration synergistically induced apoptosis in GC-, ABC- and double-hit DLBCL cells. Similar interactions were observed in MCL cells and in primary lymphoma cells. HHT/bortezomib co-administration diminished binding of MCL-1 to both BAK and NOXA. Knock-down of NOXA significantly diminished lethality whereas MCL-1 knock-down or ectopic NOXA expression increased cell death. Notably, HHT/bortezomib lethality was dramatically reduced in BAK knockout or knockdown cells. Finally, HHT/bortezomib co-administration significantly improved survival compared to single agents in GC- and ABC- xenograft models while exhibiting little toxicity.ConclusionsThese findings indicate that HHT and bortezomib cooperate to kill DLBCL and MCL cells through a process involving MCL-1 down-regulation, NOXA up-regulation, and BAK activation. They also suggest that a strategy combining HHT with bortezomib warrants attention in DLBCL and MCL.

PS02.024: PRIMA-1 INDUCES P53-MEDIATED APOPTOSIS BY UPREGULATING NOXA IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA WITH TP53 MISSENSE MUTATION

Abstract Background TP53 is associated with the resistance of cytotoxic treatment and patient prognosis, and the mutation rate of TP53 in esophageal squamous cell carcinoma (ESCC) is extraordinarily high, at over 90%. PRIMA-1 (p53 re-activation and induction of massive apoptosis) has recently been reported to restore wild type activity to mutant p53 and induce massive p53-dependent apoptosis. APR-246 (methylated PRIMA-1) has been tested in a phase I/II clinical trial with promising results; however, the effects and mechanism in ESCC remain unknown. This study was designed to assess the antitumor effect of PRIMA-1 treatment in both ESCC cell lines with different TP53 status and an ESCC xenograft model and uncover the molecular mechanism of PRIMA-1. Methods After evaluating the TP53 mutation status of a panel of eleven ESCC cell lines by Sanger sequencing, we assessed the in vitro effect of PRIMA-1 administration on cells with different p53 status by conducting cell viability and apoptosis assays. The expression levels of proteins in TP53-related pathways were examined by Western blotting, while knockdown studies were conducted to investigate the mechanism underlying PRIMA-1’s function. An ESCC xenograft model was further used to evaluate the therapeutic effect of PRIMA-1 in vivo. Results PRIMA-1 markedly inhibited cell growth and induced apoptosis by upregulating Noxa expression in ESCC cell lines with a TP53 missense mutation, whereas no apoptosis was induced in ESCC with wild type TP53 and with TP53 frameshift and nonsense mutations. Importantly, the knockdown of Noxa cancelled the apoptosis induced by PRIMA treatment in ESCC cell lines with a TP53 missense mutation. PRIMA-1 administration, compared with placebo, showed a significant antitumor effect by inducing Noxa in the xenograft model of an ESCC cell line with a TP53 missense mutation. Conclusion PRIMA-1 exhibits a significant antitumor effect, inducing massive apoptosis through the upregulation of Noxa in ESCC with a TP53 missense mutation. Disclosure All authors have declared no conflicts of interest.

Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL

The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression increased in cells exposed to UVB and the TOR kinase inhibitor Torin 2. Similar to knockdown of FOXO3a, NOXA knockdown reversed the sensitization to UVB-induced apoptosis caused by mTORC2 inhibition. In contrast, loss of TRAIL by either knockdown or knockout actually enhanced expression of nuclear FOXO3a, which maintained apoptosis. These surprising results are not due to faulty death receptor signaling in HaCaT cells, as we found that the cells undergo extrinsic apoptosis in response to treatment with recombinant TRAIL. Even more striking, TRAIL knockout cells were sensitized to recombinant TRAIL-induced apoptosis compared to wild-type HaCaT cells, with the largest increase occurring in the presence of mTORC2 inhibition. Taken together, these studies provide strong evidence that mTORC2 controls UVB-induced apoptosis by regulating NOXA expression downstream of FOXO3a. Moreover, FOXO3a transcriptional activation by mTORC2 inhibitors may be a valuable target for prevention or therapy of NMSC, especially in cases with low endogenous TRAIL.

Abstract 4828: Newly identified p53 pathway-restoring compound CB002 and its derivatives sensitize colorectal and multiple myeloma cancer cell lines to front-line cancer therapies

Abstract Proapoptotic NOXA protein is a p53 transcriptional target that has been shown to be a crucial mediator of cell death in response to chemotherapy agents. Given the fact that NOXA is downregulated in multiple cancers, induction of this protein by small-molecule exposure makes it an attractive therapeutic strategy. Although cancer patients often respond to initial therapy, later on therapy resistance develops, leaving them with limited therapy options. Therefore, new therapeutic regimens that overcome resistance mechanisms are of high importance. The purpose of this study was to identify new small molecules that restore the p53 pathway and, in combination with chemotherapy agents, could result in therapy sensitization. We have discovered a family of small molecules (CB002 and 12 structural analogs) that restore the p53 pathway through NOXA in various cancer cell lines including SW480, DLD-1 and HCT116 colorectal cancer (CRC) cells. In order to test if CB002 and its derivatives sensitize cells to first-line CRC therapies, we treated SW480 and DLD-1 cells in combination with irinotecan, fluorouracil (5-FU), doxorubicin, or oxaliplatin. Our data suggest that treatment combinations of CB002 with first-line CRC therapies result in enhanced cell death and decreased IC50. Particularly, sensitization to doxorubicin and irinotecan was dependent on NOXA expression as stable NOXA knockdown abolished therapy sensitization. NOXA induction has been shown to overcome bortezomib resistance in multiple myeloma (MM) cells. Thus, we investigated if CB002 and more potent derivatives could resensitize bortezomib-resistant cells. We treated MM1S (bortezomib-sensitive) and 33X (bortezomib-resistant) cells with bortezomib along with our CB002 structural analogs. Our data indicate that combination treatment with our structural analogs sensitizes 33X cells to bortezomib treatment. Taken together, our evidence supports that our novel identified structural analogs of CB002 can be further tested in vivo in combination with front-line therapies to improve therapeutic modalities and overcome resistance in CRC and MM. Our experiments will further assess toxicity to normal cells and tissues. Current experiments are focused on using NOXA as a pharmacodynamic biomarker to predict therapeutic efficacy. Our data argue that p53 pathway restoring compounds are worth pursuing further to determine if they can provide a therapeutic advantage to patients who are not responsive to front-line cancer therapies. Citation Format: Liz J. Hernandez Borrero, David T. Dicker, Wafik S. El-Deiry. Newly identified p53 pathway-restoring compound CB002 and its derivatives sensitize colorectal and multiple myeloma cancer cell lines to front-line cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4828.

Prox1 is essential for oligodendrocyte survival and regulates oligodendrocyte apoptosis via the regulation of NOXA

Demyelinating diseases, such as multiple sclerosis, are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. Its underlying molecular mechanisms, however, remain unclear. The transcription factor prospero homeobox 1 (Prox1) plays an essential role during embryonic development of the central nervous system and cell differentiation. Thus, we aimed to investigate the role of Prox1 in the survival and differentiation of oligodendrocytes. Cell viability was measured by MTT assay. Flow cytometry was conducted to analyze cell apoptosis. Ectopic-Prox1 and shProx1 were used for the overexpression and knockdown respectively of Prox1 in FBD-102b cells. Real-time reverse transcriptase polymerase chain reaction and western blot analysis were used to assess the alterations of signaling pathway-related mRNAs and proteins, respectively. Results showed that Prox1 was upregulated in differentiating oligodendrocytes, and Prox1 knockdown inhibited the differentiation of oligodendrocytes. In addition, overexpression of Prox1 promoted oligodendrocyte differentiation, as shown by the change in myelin basic protein expression. The overexpression of Prox1 had no effect on oligodendrocyte survival, while Prox1 knockdown impaired cell survival. Further study demonstrated that Prox1 knockdown promoted oligodendrocyte apoptosis and activated NOXA, a pro-apoptotic member of the Bcl-2 protein family. Knockdown of NOXA by siRNA abrogated Prox1 knockdown-induced apoptosis. Our findings indicated that Prox1 regulated the differentiation of oligodendrocyte precursor cells via the regulation of NOXA. Therefore, Prox1 could be a potential modulator of demyelinating diseases in clinical settings.

BCL-2 selective inhibitor ABT-199 primes rhabdomyosarcoma cells to histone deacetylase inhibitor-induced apoptosis.

BH3 mimetics are emerging novel anticancer therapeutics that potently and specifically inhibit antiapoptotic BCL-2 proteins and thereby induce cell death in many cancer entities. Previously, we demonstrated that JNJ-26481585 (JNJ), a second-generation histone deacetylase inhibitor (HDACI), engages mitochondrial apoptosis via upregulation of several BH3-only proteins. In the present study, we describe synergistic interactions of JNJ with BH3 mimetics (i.e. ABT-737, ABT-199) in rhabdomyosarcoma (RMS) cells. Importantly, JNJ synergizes with ABT-199 to trigger apoptosis in primary-derived RMS cells isolated from tumor samples, underlining the translational importance of combining these compounds and their potential to improve cancer therapy. Importantly, JNJ/ABT-199 cotreatment also significantly inhibits long-term survival of RMS cells. Mechanistically, JNJ increases expression levels of the BH3-only protein BIM, while exposure to ABT-199 displaces BIM from BCL-2 and shuttles BIM to MCL-1, which also constitutively sequesters NOXA. Both BIM and NOXA contribute to JNJ/ABT-199-mediated cell death, as individual knockdown of NOXA or BIM significantly prevents cell death. Further, JNJ and ABT-199 act in concert to activate BAK and BAX, resulting in loss of the mitochondrial membrane potential (MMP) and caspase activation. These events are required for JNJ/ABT-199-mediated apoptosis, since BAK or BAX silencing or inhibition of caspases significantly protects from JNJ/ABT-199-induced cell death. Rescue experiments demonstrate that overexpression of MCL-1, but not overexpression of BCL-2, blocks JNJ/ABT-199-induced apoptosis. In conclusion, this study provides the first demonstration of ABT-199-induced priming, which sensitizes RMS cells to HDACI, such as JNJ, by engaging mitochondrial apoptosis, highlighting that BH3 mimetics show great promise for the treatment of RMS.

PRIMA-1 induces p53-mediated apoptosis by upregulating Noxa in esophageal squamous cell carcinoma with TP53 missense mutation.

TP53 is associated with the resistance of cytotoxic treatment and patient prognosis, and the mutation rate of TP53 in esophageal squamous cell carcinoma (ESCC) is extraordinarily high, at over 90%. PRIMA‐1 (p53 re‐activation and induction of massive apoptosis) has recently been reported to restore the function of mutant TP53; however, its antitumor effect and mechanism in ESCC remain unclear. After evaluating the TP53 mutation status of a panel of 11 ESCC cell lines by Sanger sequencing, we assessed the in vitro effect of PRIMA‐1 administration on cells with different TP53 status by conducting cell viability and apoptosis assays. The expression levels of proteins in p53‐related pathways were examined by Western blotting, while knockdown studies were conducted to investigate the mechanism underlying PRIMA‐1's function. An ESCC xenograft model was further used to evaluate the therapeutic effect of PRIMA‐1 in vivo. PRIMA‐1 markedly inhibited cell growth and induced apoptosis by upregulating Noxa expression in ESCC cell lines with TP53 missense mutations, whereas no apoptosis was induced in ESCC with wild‐type TP53 and TP53 with frameshift and nonsense mutations. Importantly, the knockdown of Noxa canceled the apoptosis induced by PRIMA treatment in ESCC cell lines with TP53 missense mutations. PRIMA‐1 administration, compared with placebo, showed a significant antitumor effect by inducing Noxa in the xenograft model of an ESCC cell line with a TP53 missense mutation. PRIMA‐1 exhibits a significant antitumor effect, inducing massive apoptosis through the upregulation of Noxa in ESCC with TP53 missense mutations.

Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death

Rhabdomyosarcoma (RMS) cells have recently been reported to be sensitive to oxidative stress. Therefore, we investigated whether concomitant inhibition of the two main antioxidant defense pathways, that is, the thioredoxin (TRX) and the glutathione (GSH) systems, presents a new strategy to trigger cell death in RMS. In this study, we discover that GSH-depleting agents, i.e. γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine (BSO) or the cystine/glutamate antiporter inhibitor erastin (ERA), synergize with thioredoxin reductase (TrxR) inhibitor auranofin (AUR) to induce cell death in RMS cells. Interestingly, AUR causes accumulation of ubiquitinated proteins when combined with BSO or ERA, in line with recent reports showing that AUR inhibits the proteasome besides TrxR. Consistently, AUR/BSO or AUR/ERA cotreatment increases ubiquitination and expression of the short-lived proteins NOXA and MCL-1, accompanied by increased binding of NOXA to MCL-1. Notably, NOXA knockdown significantly rescues RMS cells from AUR/BSO- or AUR/ERA-induced cell death. In addition, AUR acts together with BSO or ERA to stimulate BAX/BAK and caspase activation. Of note, BSO or ERA abolish the AUR-stimulated increase in GSH levels, leading to reduced GSH levels upon cotreatment. Although AUR/BSO or AUR/ERA cotreatment enhances reactive oxygen species (ROS) production, only thiol-containing antioxidants (i.e., N-acetylcysteine (NAC), GSH), but not the non-thiol-containing ROS scavenger α-Tocopherol consistently suppress AUR/BSO- and AUR/ERA-stimulated cell death in both cell lines. Importantly, re-supply of GSH or its precursor NAC completely prevents AUR/ERA- and AUR/BSO-induced accumulation of ubiquitinated proteins, NOXA upregulation and cell death, indicating that GSH depletion rather than ROS production is critical for AUR/BSO- or AUR/ERA-mediated cell death. Thus, by demonstrating that GSH-depleting agents enhance the antitumor activity of AUR, we highlight new treatment options for RMS by targeting the redox homeostasis.

Arenobufagin Induces Apoptotic Cell Death in Human Non-Small-Cell Lung Cancer Cells via the Noxa-Related Pathway.

Arenobufagin, an active component isolated from the traditional Chinese medicine Chan Su, exhibits anticancer influences in several human malignancies. However, the effects and action mechanisms of arenobufagin on non-small-cell lung cancer (NSCLC) are still unknown. In this study, we reported that arenobufagin acted through activation of Noxa-related pathways and promoted apoptotic cell death in human NSCLC cells. Our results revealed that arenobufagin-induced apoptosis was caspase-dependent, as evidenced by the fact that caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) were cleaved, and pretreatment with a pan-caspase inhibitor Z-VAD-FMK inhibited the pro-apoptosis effect of arenobufagin. Mechanistically, we further found that arenobufagin rapidly upregulated the expression of the pro-apoptosis protein Noxa, and abrogated the anti-apoptosis protein Mcl-1, a major binding partner of Noxa in the cell. More importantly, the knockdown of Noxa greatly blocked arenobufagin-induced cell death, highlighting the contribution of this protein in the anti-NSCLC effects of arenobufagin. Interestingly, arenobufagin also increased the expression of p53, a direct transcriptional activator for the upregulation of the Noxa protein. Taken together, our results suggest that arenobufagin is a potential anti-NSCLC agent that triggers apoptotic cell death in NSCLC cells through interfering with the Noxa-related pathway.

Abstract 5178: CB002, a novel p53 pathway-restoring compound that induces apoptosis through the pro-apototic protein NOXA

Abstract P53 is a transcription factor that becomes stimulated upon genotoxic and cellular stress signals ultimately leading to the transcriptional activation of genes involved in processes such as DNA repair, cell cycle arrest, and apoptosis etc. TP53 is the most commonly mutated tumor suppressor and this is associated with cancer initiation, cancer progression and therapy resistance. P53-targeted therapy has proven to be challenging because direct functional restoration of p53 as a transcription factor has been difficult to achieve. We haven taken a different approach by investigating small molecules that functionally restore the p53-signaling pathway instead of direct p53 targeting. To date there is no approved drug that restores the p53 pathway in cancer cells with mutant p53. Therefore, we performed a high-throughput screen to identify such molecules. We identified a family of small molecules, CB002 and its structural analogs, as candidates for restoration of the p53 pathway in p53-null and mutant p53-expressing cancer cells. CB002 was specific in causing apoptosis in colorectal cancer cells and not in normal human fibroblasts cells as indicated by a Sub-G1 assay. Utilizing the xCELLigence RTCA DP system and CIM-Plate we show that CB002 treatment decreases the ability of colorectal cancer cells to migrate. The ability of CB002 to restore the p53 pathway was validated by studying the activation of p53 target genes p21, DR5, and NOXA in various cancer cell lines and expression of well-established apoptotic markers. Stable knockdown of NOXA completely abrogated the expression of cleaved PARP and reduced Sub-G1 cells, implicating the relevance of NOXA as a key mediator of cell death induction by CB002. We are currently exploring the mechanism by which NOXA induces apoptosis, as preliminary results suggest that degradation of Mcl-1 is not required for CB002 mediated cell death. In addition, we are evaluating the ability of CB002 structural analogs in decreasing the stability of mutant p53 using a larger panel of p53 mutants, as CB002 appears to target R175H p53 mutants. Our data supports the continued investigation of CB002 as a p53 pathway-restoring compound that may be further developed as a cancer therapeutic for p53-null tumors as well as tumors with p53 mutations. Citation Format: Liz J. Hernandez Borrero, Shengliang Zhang, David T. Dicker, Wafik S. El-Deiry. CB002, a novel p53 pathway-restoring compound that induces apoptosis through the pro-apototic protein NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2017-5178

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477-induced death and is regulated by the BH3-only protein PMAIP1/NOXA which sequesters the anti-apoptotic BCL2-protein BCLXL and thereby displaces and activates the autophagy-regulator BECN1/beclin1. Knockdown of NOXA or overexpression of its pro-survival binding partners MCL1 and BCLXL counteracts MG-2477-induced cell death. MG-2477 also rapidly induces the repression of the anti-apoptotic protein Survivin, which promotes autophagy and cell death. We further observed the accumulation of reactive oxygen species (ROS) that triggers autophagy induction suggesting a change of the PI3 kinase-III/BECN1 complex and activates the transcription factor FOXO3, which contributes to final cell death induction. The combined data suggest that MG-2477 induces a sequential process of ROS-accumulation, autophagy and FOXO3-activation that leads to cell death in neuroblastoma cells.

Mitochondrial matrix chaperone and c-myc inhibition causes enhanced lethality in glioblastoma.

Malignant gliomas display high levels of the transcription factor c-myc and organize a tumor specific chaperone network within mitochondria. Here, we show that c-myc along with mitochondrial chaperone inhibition displays massive tumor cell death. Inhibition of mitochondrial matrix chaperones and c-myc was established by utilizing genetic as well as pharmacological approaches. Bromodomain and extraterminal (BET) family protein inhibitors, JQ1 and OTX015, were used for c-myc inhibition. Gamitrinib was applied to interfere with mitochondrial matrix chaperones. A xenograft model was used to determine the in vivo efficacy. Combined inhibition of c-myc and mitochondrial matrix chaperones led to a synergistic reduction of cellular proliferation (CI values less than 1) in established glioblastoma, patient-derived xenograft and stem cell-like glioma cultures. The combinatorial treatment of BET inhibitors and Gamitrinib elicited massive apoptosis induction with dissipation of mitochondrial membrane potential and activation of caspases. Mechanistically, BET-inhibitors and Gamitrinib mediated a pronounced integrated stress response with a PERK-dependent up regulation of ATF4 and subsequent modulation of Bcl-2 family of proteins with down-regulation of Mcl-1 and its interacting partner, Usp9X, and an increase in pro-apoptotic Noxa. Blocking ATF4 by siRNA attenuated Gamitrinib/BET inhibitor mediated increase of Noxa. Knockdown of Noxa and Bak protected from the combinatorial treatment. Finally, the combination treatment of Gamitrinib and OTX015 led to a significantly stronger reduction of tumor growth as compared to single treatments in a xenograft model of human glioma without induction of toxicity. Thus, Gamitrinib in combination with BET-inhibitors should be considered for the development for clinical application.

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage.

Purpose: Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a proapoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage.Experimental Design: Methods of immunohistochemistry and immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by a pulldown assay. Polyubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays.Results: UBE2F is overexpressed in non-small cell lung cancer (NSCLC) and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with a low level of NOXA and poor patient survival.Conclusions: By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target. Clin Cancer Res; 23(4); 1104-16. ©2016 AACR.

Upregulation of NOXA by 10-Hydroxycamptothecin plays a key role in inducing fibroblasts apoptosis and reducing epidural fibrosis.

The fibrosis that develops following laminectomy or discectomy often causes serious complications, and the proliferation of fibroblasts is thought to be the major cause of epidural fibrosis. 10-Hydroxycamptothecin (HCPT) has been proven to be efficient in preventing epidural fibrosis, but the exact mechanism is still unclear. NOXA is a significant regulator of cell apoptosis, which has been reported to be beneficial in the treatment of fibrosis. We performed a series of experiments, both in vitro and in vivo, to explore the intrinsic mechanism of HCPT that underlies the induction of apoptosis in fibroblasts, and also to investigate whether HCPT has positive effects on epidural fibrosis following laminectomy in rats. Fibroblasts were cultured in vitro and stimulated by varying concentrations of HCPT (0, 1, 2, 4 µg/ml) for various durations (0, 24, 48, 72 h); the effect of HCPT in inducing the apoptosis of fibroblasts was investigated via Western blots and TUNEL assay. Our results showed that HCPT could induce apoptosis in fibroblasts and up-regulate the expression of NOXA. Following the knockdown of NOXA in fibroblasts, the results of Western blot analysis showed that the level of apoptotic markers, such as cleaved-PARP and Bax, was decreased. The results from the TUNEL assay also showed a decreased rate of apoptosis in NOXA-knocked down fibroblasts. For the in vivo studies, we performed a laminectomy at the L1-L2 levels in rats and applied HCPT of different concentrations (0.2, 0.1, 0.05 mg/ml and saline) locally; the macroscopic histological assessment, hydroxyproline content analysis and histological staining were performed to evaluate the effect of HCPT on reducing epidural fibrosis. The TUNEL assay in epidural tissues showed that HCPT could obviously induce apoptosis in fibroblasts in a dose-dependent manner. Also, immunohistochemical staining showed that the expression of NOXA increased as the concentrations of HCPT increased. Our findings are the first to demonstrate that upregulation of NOXA by HCPT plays a key role in inducing fibroblast apoptosis and in reducing epidural fibrosis. These findings might provide a potential therapeutic target for preventing epidural fibrosis following laminectomy.