Upregulation Of Noxa Research Articles

Gambogenic acid induces apoptosis via upregulation of Noxa in oral squamous cell carcinoma

Gambogenic acid (GNA), a bioactive compound derived from the resin of Garcinia hanburyi, has demonstrated significant antitumor properties. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unclear. This study aimed to elucidate the apoptotic effects of GNA on OSCC cell lines CAL-27 and SCC-15. Our results indicated that GNA induced apoptosis by upregulating the pro-apoptotic protein Noxa. Mechanistic investigations revealed that GNA treatment led to the generation of reactive oxygen species (ROS), which activated endoplasmic reticulum (ER) stress, culminating in cell apoptosis. Inhibition of ROS production and ER stress pathways significantly mitigated GNA-induced Noxa upregulation and subsequent apoptosis. Furthermore, in vivo studies using a murine xenograft model demonstrated that GNA administration effectively inhibited the growth of CAL-27 tumors. Collectively, these findings underscore GNA’s potential as a therapeutic agent for the treatment of OSCC.

ONC212 enhances YM155 cytotoxicity by triggering SLC35F2 expression and NOXA-dependent MCL1 degradation in acute myeloid leukemia cells

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.

Abstract 4579: DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells

Abstract Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) are overexpressed in cancer cells associated with dysregulated gene expression and tumor progression. DNMT1 partnered with Class I HDAC as complex to inhibit gene transcription. Several pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. More selective HDAC inhibitors need to be developed. Psammaplin A (PsA), a symmetrical bromotyrosine-derived disulfide dimer isolated from the Psammaplysilla sponge, is a selective Class I HDAC inhibitor. We tested its cell growth inhibition and apoptosis induction in 12 hematological malignant cell lines with 8 lines sensitive (more than 50% growth inhibition at 1 μM) and 4 lines insensitive (less than 20% growth inhibition at 1 μM). PsA induces acetylation of H3, but not α-tubulin, supporting the Class I HDAC inhibition selection. In the sensitive cell lines, PsA induces apoptosis with down-regulation of c-Flip and upregulation of NOXA as well as DNA damage detected by γ-H2A.X. RNA sequence analysis results confirm the regulation of PsA on NOXA, c-Flip and p21 gene expression. The four insensitive cell lines express high levels of DNMT1 with resistance to apoptosis induction. Silencing DNMT1 with siRNA sensitizes PsA-induced apoptosis with induction of NOXA and γ-H2A.X. Azacytidine, the DNMT1 degrader, in combination with PsA induce synergistic cell growth inhibition and apoptosis in vitro. PsA (20 mg/kg) plus Azacytidine (2.5 mg/kg) synergistically inhibit tumor growth in insensitive mantle cell lymphoma Jeko-1 xenografts, without detected toxic signs in organ pathological analysis. We find a new mechanism of DNMT1 controlling the sensitivity of Class I HDAC inhibitor PsA and provide a rationale of PsA with azacytidine for combination therapy. Citation Format: Yu Bao, Dan Liu, Zi Wu, Yumei He, Ping Gong, Samuel Waxman, Yongkui Jing. DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4579.

Vitamin K2 sensitizes the efficacy of venetoclax in acute myeloid leukemia by targeting the NOXA-MCL-1 pathway.

Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Hydroquinone-selected chronic myelogenous leukemia cells are sensitive to chloroquine-induced cytotoxicity via MCL1 suppression and glycolysis inhibition

Previous studies have provided evidence that repeated exposure to the benzene metabolite hydroquinone (HQ) induces malignant transformation and increases basal autophagy in the chronic myeloid leukemia (CML) cell line K562. This study explored the cytotoxicity of the autophagy inhibitor chloroquine (CQ) on parental and HQ-selected K562 (K562/HQ) cells. CQ triggered apoptosis in these cells independently of inhibiting autophagic flux; however, in K562/HQ cells, CQ-induced cytotoxicity was higher than in K562 cells. Mechanistically, CQ-induced NOXA upregulation led to MCL1 downregulation and mitochondrial depolarization in K562/HQ cells. MCL1 overexpression or NOXA silencing attenuated CQ-mediated cytotoxicity in K562/HQ cells. CQ triggered ERK inactivation to increase Sp1, NFκB, and p300 expression, and co-assembly of Sp1, NFκB, and p300 in the miR-29a promoter region coordinately upregulated miR-29a transcription. CQ-induced miR-29a expression destabilized tristetraprolin (TTP) mRNA, which in turn reduced TTP-mediated NOXA mRNA decay, thereby increasing NOXA protein expression. A similar mechanism explained the CQ-induced downregulation of MCL1 in K562 cells. K562/HQ cells relied more on glycolysis for ATP production than K562 cells, whereas inhibition of glycolysis by CQ was greater in K562/HQ cells than in K562 cells. Likewise, CQ-induced MCL1 suppression and glycolysis inhibition resulted in higher cytotoxicity in CML KU812/HQ cells than in KU812 cells. Taken together, our data confirm that CQ inhibits MCL1 expression through the ERK/miR-29a/TTP/NOXA pathway, and that inhibition of glycolysis is positively correlated to higher cytotoxicity of CQ on HQ-selected CML cells.

DRP1 Inhibition Enhances Venetoclax-Induced Mitochondrial Apoptosis in TP53-Mutated Acute Myeloid Leukemia Cells through BAX/BAK Activation.

Although TP53 mutations in acute myeloid leukemia (AML) are associated with poor response to venetoclax, the underlying resistance mechanism remains unclear. Herein, we investigated the functional role of dynamin-related protein 1 (DRP1) in venetoclax sensitivity in AML cells with respect to TP53 mutation status. Effects of DRP1 inhibition on venetoclax-induced cell death were compared in TP53-mutated (THP-1 and Kasumi-1) and TP53 wild-type leukemia cell lines (MOLM-13 and MV4-11), as well as in primary AML cells obtained from patients. Venetoclax induced apoptosis in TP53 wild-type AML cells but had limited effects in TP53-mutated AML cells. DRP1 expression was downregulated in MOLM-13 cells after venetoclax treatment but was unaffected in THP-1 cells. Cotreatment of THP-1 cells with venetoclax and a TP53 activator NSC59984 downregulated DRP1 expression and increased apoptosis. Combination treatment with the DRP1 inhibitor Mdivi-1 and venetoclax significantly increased mitochondria-mediated apoptosis in TP53-mutated AML cells. The combination of Mdivi-1 and venetoclax resulted in noticeable downregulation of MCL-1 and BCL-xL, accompanied by the upregulation of NOXA, PUMA, BAK, and BAX. These findings suggest that DRP1 is functionally associated with venetoclax sensitivity in TP53-mutated AML cells. Targeting DRP1 may represent an effective therapeutic strategy for overcoming venetoclax resistance in TP53-mutated AML.

P53 triggers mitochondrial apoptosis following DNA damage-dependent replication stress by the hepatotoxin methyleugenol

Liver cancer is one of the most frequent tumor entities worldwide, which is causally linked to viral infection, fatty liver disease, life-style factors and food-borne carcinogens, particularly aflatoxins. Moreover, genotoxic plant toxins including phenylpropenes are suspected human liver carcinogens. The phenylpropene methyleugenol (ME) is a constituent of essential oils in many plants and occurs in herbal medicines, food, and cosmetics. Following its uptake, ME undergoes Cytochrome P450 (CYP) and sulfotransferase 1A1 (SULT1A1)-dependent metabolic activation, giving rise to DNA damage. However, little is known about the cellular response to the induced DNA adducts. Here, we made use of different SULT1A1-proficient cell models including primary hepatocytes that were treated with 1′-hydroxymethyleugenol (OH-ME) as main phase I metabolite. Firstly, mass spectrometry showed a concentration-dependent formation of N2-MIE-dG as major DNA adduct, strongly correlating with SULT1A1 expression as attested in cells with and without human SULT1A1. ME-derived DNA damage activated mainly the ATR-mediated DNA damage response as shown by phosphorylation of CHK1 and histone 2AX, followed by p53 accumulation and CHK2 phosphorylation. Consistent with these findings, the DNA adducts decreased replication speed and caused replication fork stalling. OH-ME treatment reduced viability particularly in cell lines with wild-type p53 and triggered apoptotic cell death, which was rescued by pan-caspase-inhibition. Further experiments demonstrated mitochondrial apoptosis as major cell death pathway. ME-derived DNA damage caused upregulation of the p53-responsive genes NOXA and PUMA, Bax activation, and cytochrome c release followed by caspase-9 and caspase-3 cleavage. We finally demonstrated the crucial role of p53 for OH-ME triggered cell death as evidenced by reduced pro-apoptotic gene expression, strongly attenuated Bax activation and cell death inhibition upon genetic knockdown or pharmacological inhibition of p53. Taken together, our study demonstrates for the first time that ME-derived DNA damage causes replication stress and triggers mitochondrial apoptosis via the p53-Bax pathway.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

Shikonin derivatives cause apoptosis and cell cycle arrest in human chondrosarcoma cells via death receptors and MAPK regulation

BackgroundAlthough chondrosarcoma is the second most common primary malignant bone tumor, treatment options are limited due to its extensive resistance to a chemo- and radiation therapy. Since shikonin has shown potent anticancer activity in various types of cancer cells, it represents a promising compound for the development of a new therapeutic approach.MethodsThe dose-relationships of shikonin and its derivatives acetylshikonin and cyclopropylshikonin on two human chondrosarcoma cell lines were measured using the CellTiter-Glo®. The changes in the cell cycle were presented by flow cytometry. Protein phosphorylation and expression apoptotic markers, MAPKs and their downstream targets were analyzed using western blotting and gene expression were evaluated using RT-qPCR.ResultsChondrosarcoma cells showed a dose-dependent inhibition of cell viability after treatment with shikonin and its derivatives, with the strongest effect for shikonin and IC50 values of 1.3 ± 0.2 µM. Flow cytometric measurements revealed a G2/M arrest of the cells after treatment. Protein and gene expression analysis demonstrated a dose-dependent downregulation of survivin and XIAP, and an upregulation of Noxa, γH2AX, cleaved caspase-8, -9, -3, and -PARP. Furthermore, the expression of various death receptors was modulated. As MAPK signaling pathways play a key role in tumor biology, their phosphorylation pattern and their corresponding downstream gene regulation were analyzed. Treatment with shikonin derivatives caused an inhibition of pSTAT3 and an increase of pAKT and the MAPKs pERK, pJNK, and pp38 in a dose-dependent manner.ConclusionsThese data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

High-throughput screening identifies stevioside as a potent agent to induce apoptosis in bladder cancer cells

BackgroundBladder cancer (BC) is a global health issue that lacks effective treatment strategies. Growing evidence suggests that various natural products possess anti-tumour effects. This study aims to identify a novel agent that can be used in the treatment of BC. MethodsHigh-throughput screening was conducted to search for potential anti-BC natural agents. Cell viabilities were measured by the CCK-8 assay. Cell death, cellular reactive oxygen species (ROS), and mitochondrial outer membrane potential (MOMP) were measured by flow cytometry. RNA sequencing was conducted to identify the affected signalling pathways. Western blots were used to measure the change of proteins. Xenografts models were used to assess the anti-tumour effects in vivo. ResultsThrough high-throughput screening, we identified stevioside, a diterpenoid glycoside isolated from Stevia rebaudiana, which selectively inhibited the viability of BC cells and induced their intrinsic apoptosis sparing normal cells. Stevioside also induced mitochondrial stress in BC cells, and activated Bax by downregulating Mcl-1 and upregulating Noxa. RNA sequencing revealed that stevioside treatment caused activation of GSK-3β and endoplasmic reticulum (ER) stress signalling pathways. Activation of GSK-3β induced upregulation of FBXW7, which effectuated the downregulation of Mcl-1. In addition, activation of GSK-3β triggered ER stress, leading to the upregulation of Noxa. Further investigations revealed that the accumulation of ROS was responsible for the activation of the GSK-3β signalling pathway in BC cells. Moreover, we also found that stevioside inhibited the growth of BC cells in vivo. ConclusionsCollectively, our data suggest that stevioside can be a potential agent for the treatment of BC.

Abstract 3709: Synergistic activity of ABT-263 and ONC201 against solid tumor cell lines is associated with suppression of BAG3, Mcl-1, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis

Abstract There is an urgent need for the development of novel therapeutic approaches that can address the complex nature of cancer, including resistance to therapy. A major underlying cause of the resistance is the evasion of cell death following anti-cancer drug treatment. A great deal of progress has been made in the discovery and early development of cancer therapeutics that target cell death via the intrinsic or extrinsic cell death pathways. Two such drugs are ABT-263 and ONC201. ABT-263 is a small molecule that targets the pro-apoptotic cell death of tumor cells by blocking the anti-apoptotic function of BCL-XL and BCL-2; ONC201/TIC10 is a first-in-class small molecule originally discovered as a TRAIL-inducing compound. Previous studies have found that a mechanism of resistance to ABT-263 involves upregulation of anti-apoptotic Mcl-1 in ABT-263-treated tumors, while the efficacy of ONC201 is limited by BCL-2 overexpression. Given that ONC201 is known to downregulate Mcl-1 expression and that BCL-2 is a target of ABT-263, the combination of the two drugs has great synergistic potential. ONC201 and ABT-263 been tested in glioblastoma; however, ABT-263 is not able to cross the blood-brain barrier, and the combination of ONC201 and ABT-263 has not been tested in other solid tumors. It remains a gap in the field to test this combination therapy in tissues to which ONC201 and ABT-263 are bioavailable. We explored the combination of TRAIL-inducing compound ONC201/TIC10 and BCL-xl/BCL-2 inhibitor ABT-263 to target the extrinsic and intrinsic apoptotic pathways, respectively, in solid tumor cell lines (N=13) derived from different tissues (colon, prostate, lung, breast, ovary, bladder). We found an IC50 range of 0.83-20.10 μM for ONC201 and 0.06-14.75 μM for ABT-263 among the 13 cell lines. We show that combination of ONC201 and ABT-263 produces a strong synergistic effect leading to tumor cell death across the 1cell line panel, with maximum synergy scores ranging from 13.56-52.23 (scores were calculated using SynergyFinder; scores 0-10 indicate additive effects, while scores >10 indicate synergism), and that the combination is not toxic to human fibroblast cells. We also found that the combination of these two agents resulted in caspase activation and PARP cleavage consistent with induction of apoptosis, and that these effects where over and above individual drug effects. Combination therapy-induced cell death correlated with decreased levels of Mcl-1, BAG3, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis. Our data provide evidence for synergy from the combination of ONC201 and ABT-263 against human solid tumor cell lines associated with alterations in cell death and pro-survival mediators. The combination of ONC201 and ABT-263 merits further exploration in vivo in a variety of solid malignancies. Citation Format: Francesca R. Di Cristofano, Mara Fong, Lanlan Zhou, Wafik S. El-Deiry. Synergistic activity of ABT-263 and ONC201 against solid tumor cell lines is associated with suppression of BAG3, Mcl-1, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis [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 3709.

Abstract 5854: Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells

Abstract The tumor suppressor p73, a member of the p53 family, transcriptionally activates numerous p53-target genes in cell cycle regulation and apoptosis. Therapeutic activation of p73 can restore p53-signaling in mutant p53-expressing cancer cells effectively bypassing the p53 deficiency. In addition to pro-apoptotic signaling, outcomes of p73 activation contain survival signals and this may provide insights in cell fate outcomes between cell survival and apoptosis after cellular stress. We report that cellular FLICE inhibitory protein (c-FLIP), a master antiapoptotic factor, is a novel transcriptional target of p73. To test if p73 regulates c-FLIP expression in p53-deficient cancer cells, we overexpressed p73 in p53-deficient cancer cells using p73-adenovirus. Overexpression of p73 dramatically increased c-FLIP at the protein and RNA levels. The effect of p73 on c-FLIP upregulation was blocked by knock-down of p73. ChIP assay showed that p73 binds to the c-FLIP promoter in a p73-level dependent manner. Furthermore, a luciferase reporter assay showed that p73 increased the c-FLIP-L-promoter-mediated luciferase reporter expression in p53-deficient cancer cells. To test the effect of the outcomes of p73 signaling on cell survival or death, we performed cell viability assay and found that p73 reduces cell viability in a p73-level dependent manner. We found an increase of both p21 and c-FLIP at the protein levels in response to overexpression of p73 in p53-deficient cancer cells, but no cleaved-PARP was detected and flow cytometric analysis showed the cells were arrested in G1 phase. These results suggest that p73 arrests the cell cycle and prevents apoptosis at the tested levels of p73. We knocked down c-FLIP using siRNA in p53-deficient cancer cells, and found that knockdown of c-FLIP enhanced cleaved-PARP, cleaved caspase- 3 and -8 correlated to high level of apoptotic signaling with Noxa and puma upregulation in response to the overexpression of p73. Our results suggest that the depletion of c-FLIP primes cellular apoptosis switching from p73-induced cell cycle arrest (cell survival) and sensitizes cells to p73-induced apoptotic signals in p53-deficient cells. The cell fate decision following p73 activation is determined by the adjustment of the balance of outcomes of p73 activation between p73-induced pro-apoptotic signaling and c-FLIP expression in p53-deficent cancer cells. Our discovery of the new function of p73 to upregulate c-FLIP provides a rationale for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells [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 5854.

Endoplasmic Reticulum Stress Contributed to Dipyridamole-Induced Impaired Autophagic Flux and Glioma Apoptosis.

Elevation of intracellular cAMP levels has been implicated in glioma cell proliferation inhibition, differentiation, and apoptosis. Inhibition of phosphodiesterase is a way to elevate intracellular cAMP levels. The present study aimed to investigate the anti-glioma potential of dipyridamole, an inhibitor of phosphodiesterase. Upon treatment with dipyridamole, human U87 glioma cells decreased cell viability, clonogenic colonization, migration, and invasion, along with Noxa upregulation, Endoplasmic Reticulum (ER) stress, impaired autophagic flux, Yes-associated Protein 1 (YAP1) phosphorylation, and YAP1 reduction. Pharmacological and genetic studies revealed the ability of dipyridamole to initiate Noxa-guided apoptosis through ER stress. Additionally, the current study further identified the biochemical role of YAP1 in communicating with ER stress and autophagy under situations of dipyridamole treatment. YAP1 promoted autophagy and protected glioma cells from dipyridamole-induced apoptotic cell death. Dipyridamole impaired autophagic flux and rendered glioma cells more vulnerable to apoptotic cell death through ER stress-inhibitable YAP1/autophagy axis. The overall cellular changes caused by dipyridamole appeared to ensure a successful completion of apoptosis. Dipyridamole also duplicated the biochemical changes and apoptosis in glioma T98G cells. Since dipyridamole has additional biochemical and pharmacological properties, further research centered on the anti-glioma mechanisms of dipyridamole is still needed.

APR-246 induces apoptosis and enhances chemo-sensitivity via activation of ROS and TAp73-Noxa signal in oesophageal squamous cell cancer with TP53 missense mutation.

Mutations in p53, identified in 90% of oesophageal squamous cell carcinoma (ESCC), are associated with unfavourable prognosis and chemo-resistance. APR-246 induces apoptosis by restoring transcriptional ability of mutant p53, and may be a promising therapeutic agent to overcome chemo-resistance in ESCC. In ESCC cell lines differing in p53 status, we performed in vitro cell viability and apoptosis assays, evaluated reactive oxygen species (ROS) generation, and assessed signal changes by western blot after APR-246 administration with/without chemo-agent. Antitumour effects and signal changes were evaluated in in vivo experiments using xenograft and patient-derived xenograft (PDX) mouse models. APR-246 administration induced significant apoptosis by upregulating p73 and Noxa via ROS induction in ESCC cell lines harbouring p53 missense mutations. Moreover, APR-246 plus chemotherapy exerted combined antitumour effects in ESCC with p53 missense mutations. This effect was also mediated through enhanced ROS activity, leading to massive apoptosis via upregulation of p73 and Noxa. These findings were confirmed by xenograft and PDX models with p53 mutant ESCC. APR-246 strongly induced apoptosis by inducing ROS activity and p73-Noxa signalling, specifically in ESCC with p53 missense mutation. This antitumour effect was further enhanced by combination with 5-FU, which we first confirmed in ESCC preclinical model.

Dual Sphingosine Kinase and Bcl-2 Inhibition Exhibits Synergistic Cell Death in Acute Myeloid Leukemia

Background:Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their ability to suppress the apoptotic potential of pro-apoptotic Bax and Bak. In response to cell stress, BH3-only proteins sequester Bcl-2 family members, promoting apoptosis. Small molecule inhibitors such as the Bcl-2 inhibitor Venetoclax have demonstrated poor single agent efficacy in AML. This poor efficacy is a result of AML cells being highly dependent on the pro-survival Bcl-2 family member Mcl-1. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. SK1 promotes oncogenic signalling through the production of sphingosine-1-phopshate and subsequent activation of G-protein coupled receptors. In addition, SK1 has been reported to modulate Mcl-1 expression in a number of haematological malignancies including AML. Thus we sought to investigate the efficacy of targeting SK1 to sensitise AML cells to Venetoclax.Results:To determine how SK1 inhibition influences Mcl-1 levels we utilised a short hairpin RNA (shRNA) approach targeting members of the BH3 only protein family (Bid, Bim, Bad, Puma and Noxa). Targeting of Noxa significantly attenuated the cytotoxic effects of the SK1 inhibitor, MP-A08. Immunoprecipitation of Mcl-1 in response to MP-A08 treatment revealed increased association of BH3 only protein, Noxa and ubiquitin-ligase FBW7 suggesting this triggers Mcl-1 degradation in agreeance with previous studies. No interaction was observed between Mcl-1 and Bim further supporting the importance of Noxa in MP-A08 induced Mcl-1 degradation.Canonical pathway analysis of MP-A08 treated cells has previously shown to enrich for genes associated with the unfolded protein response (UPR) (Powell et al. Blood 2017). Western blot analysis recapitulated these findings with activation of the PERK arm, typified by eIF2a phosphorylation and ATF4 upregulation. ATF4 has previously been shown to bind within the Noxa promoter and drive transcription in response to ER stress (Wang et al PNAS 2009). Maintaining global translation with eIF2B agonist, ISRIB blocked Noxa transcription in response to MP-A08 suggesting ATF4 drives Noxa mediated Mcl-1 degradation.Given the Noxa dependent Mcl-1 degradation in response to MP-A08 treatment, we sought to assess the efficacy of combined treatment with the Bcl-2 inhibitor, Venetoclax. Combinational therapies with sub-cytotoxic doses of MP-A08 and Venetoclax induced synergistic cell death in AML cell lines, primary patient AML blasts and leukemic stem and progenitor cells. Synergy could be observed as early as two hours, in agreeance with Noxa upregulation and binding Mcl-1, implicating this as key for synergistic cell death.Conclusions:We have demonstrated SK1 inhibition invokes a pro-apoptotic UPR response via ATF4 driven Noxa upregulation and Mcl-1 degradation. Nullification of the pro-survival function of Mcl-1 by Noxa renders AML cells vulnerable to Bcl-2 targeting strategies such as Venetoclax providing a novel therapeutic angle to enhance patient response to Venetoclax. Current work is underway assessing MP-A08 and Venetoclax using patient-derived xenograft models. DisclosuresNo relevant conflicts of interest to declare.

Targeting Mcl-1 By Proteasome Inhibitors Enhances the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphomas and is a therapeutic challenge. At the molecular level, MCL is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) altering apoptosis, as well as, cell cycle (cyclin D1) partially regulated by the ubiquitin-proteasome system (UPS). Inhibitors of the UPS, also known as proteasome inhibitors (PI), have been tested against MCL. The lethality by PIs has been associated with pronounced up-regulation of the pro-apoptotic protein Noxa, which prevented the upregulation of Mcl-1. Changes in the ratio of Mcl-1 and Bak/Bax result in the release of Bak triggering apoptosis particularly in cells with functional p53. Venetoclax is a highly selective Bcl-2 inhibitor, BH-3 mimetic with potent activity against MCL, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in a t(14;18)-carrying xenograft model with an established tolerability and safety in a recent phase I trial. We sought to look at further enhancing the pre-clinical activity of venetoclax using PIs in MCL models. We hypothesized that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 could result in synergistic activity that could be translated clinically. Materials and Methods: We performed in vitro studies on a panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell line clones (Jeko araC, HBL-2 araC, and Mino araC). MCL cell lines were exposed to venetoclax, carfilzomib, ixazomib and bortezomib for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of venetoclax (0.001uM-5uM) and carfilzomib, an irreversible PI (1.5625nM-50nM); or two clinically available PI: bortezomib (3.125nM-100nM) or ixazomib (3.125nM-100nM). Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including MCL were exposed ex vivo to venetoclax +/- carfilzomib or bortezomib. Results: In vitro exposure of MCL cell lines to either venetoclax, carfilzomib, bortezomib and ixazomib induced cell death in a dose- and time-dependent manner. Anti-tumor activity was observed in cytarabine sensitive or resistant cell lines. Significant synergistic activity was observed by combining both venetoclax with carfilzomib or ixazomib at known sub-therapeutic and therapeutic doses of individual agents. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Conclusion: Our data suggests that venetoclax exhibits strong synergistic activity with carfilzomib and ixazomib in vitro and ex vivo MCL models. This pharmacological interaction could be related to the effects of proteasome inhibition on Mcl-1 in MCL cells, further enhancing the activity of venetoclax. In vivo experiments will further confirm the synergistic activity observed between BH3 mimetics and PIs. (Supported by a NCI SPORE Lymphoma grant: 5 P50 CA130805-04, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) DisclosuresNo relevant conflicts of interest to declare.

Direct-Acting Small Molecule Activators of PP2A (SMAPs) in Myeloid Malignancies: Understanding Mechanisms of Cytotoxicity to Inform Rational Combinatorial Therapeutic Strategies

The protein phosphatase 2A (PP2A) functions as a tumor suppressor by counteracting oncogenic kinase signaling. Suppression of PP2A activity represents a critical process in the development and maintenance of many neoplasms, including myeloid malignancies; PP2A inhibition de-represses numerous proliferative and survival signaling pathways. However, complete loss of PP2A is lethal to cells. As such, indirect reactivation of PP2A has emerged as a promising therapeutic strategy for myeloid leukemias. Yet the broad applicability of such a strategy remains uncertain, as it relies on targeting endogenous PP2A inhibitors. In the present work we investigated the effects of direct acting small molecule activators of PP2A (SMAPs) (Sandokar et al. JCI 2017) on myeloid leukemia cells and uncovered a novel link between PP2A reactivation and NOXA-mediated apoptosis. This mechanism explains our observation of cooperativity between SMAPs and the BH3 mimetic ABT-199 (venetoclax). Additionally, we found that SMAPs elicit a protective stress response that can be overcome by inhibition ofprotein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).Given the known role of PP2A inactivation in both acute and chronic myeloid leukemia (AML and CML), we first tested the activity of SMAPs against cell lines derived from both diseases. Across all cell lines tested (CML- K562, Meg-01; AML- MOLM-14, HL-60, OCI-AML3, THP-1) SMAPs caused a dose-dependent decrease in cell viability. It is known that reactivation of PP2A induces apoptosis in myeloid leukemia cells, though this area has yet to be thoroughly investigated. Therefore, we first sought to clarify the mode of cell death in response to PP2A reactivation. Both by biochemical and flow cytometric analyses, SMAPs showed activation of the intrinsic apoptotic cascade. Furthermore, SMAP-mediated cell death was blocked by co-treatment with the caspase inhibitor z-VAD-FMK (50 μM). We previously demonstrated the target specificity of SMAPs for PP2A with methods including expression of SV40 small T antigen and PP2A alpha subunit drug-binding mutants (Sandokar et al. JCI 2017). Here, we confirmed that pre-treatment with the PP2A inhibitor okadaic acid at a dose that retains selectivity for PP2A (10 nM) attenuated SMAP-induced cell death and cleavage of PARP and caspase-3.Next, we investigated the effectors of SMAP-mediated apoptosis. To this end, we performed Western blots to detect changes in abundance and/or phosphorylation of a panel of Bcl-2 family member proteins. Interestingly, a strong upregulation of the pro-apoptotic protein NOXA occurred consistently across AML and CML cell lines. Further RT-PCR studies showed this increase to occur at the transcript level. Knockdown studies confirmed the importance of NOXA for SMAP-mediated apoptosis. These data represent the first reported link between PP2A and NOXA regulation. Since SMAPs target the NOXA-MCL1 axis of the Bcl-2 family, we next tested their efficacy in combination with the Bcl-2-selective BH3 mimetic ABT-199. We found co-treatment with SMAPs and ABT-199 to exhibit enhanced cell death compared to either agent alone.Concurrent with NOXA elevation, SMAP treatment modulated protein expression and phosphorylation levels for several known pathways downstream of PP2A. Interestingly, we additionally observed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) in SMAP-treated cells which correlated with upregulation of mRNA and protein for the downstream transcription factor ATF4. Blockade of eIF2α phosphorylation with PERK inhibitor GSK2656157 (350 nM) prevented ATF4 elevation and enhanced SMAP-induced apoptosis. This result supports the idea that the integrated stress response (ISR) via PERK-eIF2α-ATF4 acts to counter apoptosis in the context of PP2A reactivation in myeloid leukemia cells. Thus, the efficacy of SMAPs and other PP2A-activating therapeutics may be enhanced by combination with both BH3 mimetics and inhibitors of the ISR. DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

Edo-S101, a Bendamustine (BDM)/ Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule, Demonstrates Potent Preclinical Activity Against T-Cell Malignancies and Overcomes BDM-Resistance

Introduction: T-cell and natural killer (NK)-cell lymphomas encompass a heterogeneous spectrum of malignancies with variable degrees of biological and clinical aggressiveness and different patterns of nodal/extra nodal dissemination. Despite advances in understanding of their genetic landscape, treatment options for these tumors are limited and survival remains dismal especially in patients (pts.) who relapse/progress after frontline treatment. Therefore, development of new active agents is a pressing medical need in this setting. The unconventional alkylator BDM and HDACi are clinically effective in pts. with recurring disease and the combination of these agents was suggested as a way to overcome early resistance and improve response and survival rates in T-cell tumors. Since EDO-S101 is a fusion molecule that combines the active structures of BDM and the pan-HDACi vorinostat to simultaneously damage DNA and block damage repair, we investigated its antitumor activity in preclinical models of human T- and NK- lymphomas.Methods: We assessed the patterns of EDO-S101 cytotoxicity and its regulatory effects on genes involved in DNA-damage/repair, apoptosis and drug resistance on a large panel of cell lines representative of most T-lymphoma subtypes, including precursor T-cell lymphoma/leukemia (pTCL; MOLT-4, KE-37, Jurkat, CCRF-CEM), anaplastic large T-cell lymphoma (ALCL; SR-786, SUDHL1) and cutaneous T-cell lymphoma (CTCL; HUT78, MYLA, HH, PB2B, MAC-2A). As a further model, we exploited an ALCL cell line (SUDHL1-R100) selected for resistance to BDM. SUDHL1-R100 display a growth pattern indistinguishable from parental cells in the presence of BDM (100 μmol/L).Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all T-cell lines, with a IC50 at 48 hrs (0.33 to 0.68 μmol/L), that was at least 90% and 50% lower than IC50 of BDM (10.5 to 23.67 μmol/L) and vorinostat (0.42 to 1.17 μmol/L) in the same cell lines. No significant differences emerged across various cell lines as to sensitivity to EDO-S101. Notably, growth inhibitory effects of EDO-S101 were 35% to 40% higher than those achieved by the concomitant exposure of tumor cells to BDM and vorinostat at equimolar concentrations. A significant induction of the DNA double strand breaks marker pγH2AX, along with decreased levels of Thr-1989 pATR and Polo-like kinase 1 (PLK1) were consistently observed in pTCL, ALCL and CTCL cell lines, indicating that EDO-S101 triggered inappropriate mitotic entry with unrepaired DNA damage. In addition, qRT-PCR results showed upregulation of the proapototic genes NOXA, p21 and/or p27 in pTCL, CTCL and ALCL cells. Among DNA repair genes, EDO-S101-induced downregulation of ATM, ATR and EXO1 was paralleled by an increase of MGMT transcripts. Most intriguingly, BDM-resistant ALCL cells (SUDHL1-R100) were highly sensitive to EDO-S101, with an IC50 of only 0.72 μmol/L, but less responsive to vorinostat (IC50: 2.17 μmol/L) than parental cells (IC50: 1.46 μmol/L). Exposure to EDO-S101 also resulted in transcriptional regulation of components of the PI3K pathway, mainly the PI3Kα and δ isoforms and mTOR. These findings, prompted experiments showing that EDO-S101 at sublethal concentrations was highly synergic with the predominant PI3Kα/δ inhibitor copanlisib in inducing cell death of tumor T- and NK-cells. Finally, exposure to EDO-S101 (1.0 μmol/L) for 48 hrs resulted in a sustained upregulation of surface PD1 and PD-L1 in most tumor T- and NK-cell lines, as a possible result of its HDACi component.Conclusions: We have shown for the first time that the BDM/vorinostat fusion molecule EDO-S101 is very active in preclinical models of T/NK cell lymphomas. It shows a superior activity towards different types of tumor T- and NK-cells than its single components administered either individually or in combination by also overcoming acquired resistance to BDM. Data indicate that EDO-S101 simultaneously damages DNA and interferes on its repair pathways in tumor T-cells. We also showed synergy between EDO-S101 and PI3Kα/δ inhibitors possibly due to regulatory effects on specific components of the PI3K pathway as well as a potential for this agent to enhance PD1-blockade strategies. A clinical trial is ongoing to evaluate efficacy and safety of EDO-S101 in pts. with relapsed/refractory hemopoietic malignancies including T-cell lymphomas. DisclosuresMehrling:Mundipharma-EDO Gmbh, Basel, CH: Employment. Pinto:Celgene: Honoraria; Merck Sharp Dome: Honoraria; Millenium Takeda: Research Funding; Roche: Honoraria; Bristol Myers Squibb: Honoraria; Gilead: Honoraria; Helssin: Honoraria; Mundipharma EDO: Speakers Bureau.