Synergistic Induction Of Cell Death Research Articles

Abstract 6484: Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins

Abstract Lung cancer is the second most common cancer worldwide with 1.8 million deaths in 2020 being the leading cause of cancer related death. With 86% of cases, non-small cell lung cancer (NSCLC) is the most prominent subtype. The survival rate improved over the past 20 years by application of targeted therapies. Driver mutations in protein kinases such as the epidermal growth factor receptor (EGFR) are frequent and have evolved as therapeutic targets in anti-cancer therapy. Inhibitors of receptor tyrosine kinase (RTKIs), prominently EGFR-TKIs, are effective in anti-cancer therapy but tumor evolution inevitably generates resistance. Thus, strategies for the treatment of patients with acquired resistance to RTKIs remains an urgent need. Resistance in solid tumors is overcome by a combination of bortezomib with the BH3-mimetic ABT-199 (Muenchow et al. 2020). Also, overcoming ALK-TKI resistance in NSCLC was described by Tanimoto et al. (2021) by proteasome inhibiton that mediates induction of NOXA. Furthermore, the novel described BCL-2 independent activity of ABT-199 to transactivate NOXA expression (Weller et al. 2022) represents a generally active principle by combining drugs with independent mechanism of action. We hypothesize, that combined treatment of EGFR-TKI Osimertinib (OSI) with BH3-mimetics enhances sensitivity to apoptosis induction. To explore this hypothesis, we investigated cell death induction by combined administration of OSI and BH3-mimetics or PIs in NSCLC differing in their mutational status. In addition, we analyzed the expression of BCL-2 family proteins and their relevance and regulation during RTKI-induced cell death by RNA sequencing and Western Blot. These analyses will be performed in cellular model systems of NSCLC with specific resistance mediating mutations of EGFR or activated parallel survival pathways. We found that BCL-2 inhibitors enhance efficacy of OSI in NSCLC, resulting in synergistic cell death induction. Additionally, the sensitivity to BCL-2 inhibitors tends to increase in OSI-resistant NSCLC. Western Blot analysis and RNA sequencing data of OSI-resistant NSCLC identified BCL-2 overexpression and an EMT signature as resistance-mediating mechanisms, which can be overcome by combination of OSI with BCL-2 inhibitors. Indeed, our data suggest that combination of OSI with BCL-2 inhibitors disrupts resistance to OSI in NSCLC. We propose that BCL-2 inhibitors sensitize OSI-resistant NSCLC to effectively induce cell death. Thus, studying the molecular mechanism of synergistic efficacy of RTKI/BCL-2 inhibitor combination therapies, should prompt future efforts in the clinical evaluation of combinatorial regimens. We hypothesize that a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins represents a generally active principle of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Tobias Beigl, Frank Essmann, Annika Harsch, Hans-Georg Kopp, Tom Ekstrom. Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins [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 6484.

Abstract 2910: Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma progression

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. Poor outcomes for traditional treatments demand targeted therapies based on identified mechanisms that drive tumor development and sustain its malignancy. Molecular pathology studies have classified GBM into subtypes differing in treatment responses and survival rates. Among these subtypes, the mesenchymal (MES) group is associated with the worst prognosis. The Hippo pathway transcriptional co-activator with PDZ-binding motif (TAZ) is one of the three transcriptional regulators that drive the GBM MES gene expression program. Studies from our lab and others have previously shown that TAZ activation is able to promote GBM MES differentiation and progression. Therefore, finding vulnerabilities of TAZ-driven tumors may help to develop targeted therapeutic methods for MES GBM. In an orthotopic GBM mouse model, we found that TAZ-driven tumors show increased expression of thioredoxin reductase 1 (TrxR1), which is responsible for catalyzing the reduction of thioredoxin and maintaining the cellular redox balance. In searching for the cause of increased expression of TrxR1, we found that glucose deprivation can induce TrxR1 expression, suggesting TrxR1 may be an adaptive mechanism for tumor cells to survive in glucose deprivation conditions. Indeed, either depleting TrxR1 genetically or inhibiting TrxR1 pharmacologically can promote tumor cell death under glucose deprivation. Furthermore, knockout TrxR1 in a TAZ-driven mouse GBM model significantly decelerated tumor progression. Collectively, our studies revealed that inhibition of the Trx redox system under glucose deprivation conditions leads to a synergistic cell death induction in GBM cells, therefore making targeting TrxR1 a potential therapeutic strategy for TAZ-driven MES GBM. Citation Format: Miaolu Tang, Kaitlyn Dirks, Soo Yeon Kim, Jessica Thorpe, Wei Li. Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma progression [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 2910.

Duloxetine enhances the sensitivity of non-small cell lung cancer cells to EGFR inhibitors by REDD1-induced mTORC1/S6K1 suppression.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been effective targeted therapies for non-small cell lung cancer (NSCLC), most advanced NSCLC inevitably develop resistance to these therapies. Combination therapies emerge as valuable approach to preventing, delaying, or overcoming disease progression. Duloxetine, an antidepressant known as a serotonin-noradrenaline reuptake inhibitor, is commonly prescribed for the treatment of chemotherapy-induced peripheral neuropathy. In the present study, we investigated the combined effects of duloxetine and EGFR-TKIs and their possible mechanism in NSCLC cells. Compared with either monotherapy, the combination of duloxetine and EGFR-TKIs leads to synergistic cell death. Mechanistically, duloxetine suppresses 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) activity through mechanistic target of rapamycin complex 1 (mTORC1), and this effect is associated with the synergistic induction of cell death of duloxetine combined with EGFR-TKIs. More importantly, activating transcription factor 4 (ATF4)-induced regulated in development and DNA damage response 1 (REDD1) is responsible for the suppression of mTORC1/S6K1 activation. Additionally, we found that the combination effect was significantly attenuated in REDD1 knockout NSCLC cells. Taken together, our findings reveal that the ATF4/REDD1/mTORC1/S6K1 signaling axis, as a novel mechanism, is responsible for the synergistic therapeutic effect of duloxetine with EGFR-TKIs. These results suggest that combining EGFR-TKIs with duloxetine appears to be a promising way to improve EGFR-TKI efficacy against NSCLC.

A metabolic shift toward glycolysis enables cancer cells to maintain survival upon concomitant glutamine deprivation and V-ATPase inhibition.

It is widely known that most cancer cells display an increased reliance on glutaminolysis to sustain proliferation and survival. Combining glutamine deprivation with additional anti-cancer therapies is an intensively investigated approach to increase therapeutic effectiveness. In this study, we examined a combination of glutamine deprivation by starvation or pharmacological tools, with the anti-cancer agent archazolid, an inhibitor of the lysosomal V-ATPase. We show that glutamine deprivation leads to lysosomal acidification and induction of pro-survival autophagy, which could be prevented by archazolid. Surprisingly, a combination of glutamine deprivation with archazolid did not lead to synergistic induction of cell death or reduction in proliferation. Investigating the underlying mechanisms revealed elevated expression and activity of amino acid transporters SLC1A5, SLC38A1 upon starvation, whereas archazolid had no additional effect. Furthermore, we found that the export of lysosomal glutamine derived from exogenous sources plays no role in the phenotype as knock-down of SLC38A7, the lysosomal glutamine exporter, could not increase V-ATPase inhibition-induced cell death or reduce proliferation. Analysis of the cellular metabolic phenotype revealed that glutamine deprivation led to a significant increase in glycolytic activity, indicated by an elevated glycolytic capacity and reserve, when V-ATPase function was inhibited concomitantly. This was confirmed by increased glutamine uptake, augmented lactate production, and an increase in hexokinase activity. Our study, therefore, provides evidence, that glutamine deprivation induces autophagy, which can be prevented by simultaneous inhibition of V-ATPase function. However, this does not lead to a therapeutic benefit, as cells are able to circumvent cell death and growth inhibition by a metabolic shift toward glycolysis.

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.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Abstract 1946: Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression

Abstract 50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1946.

Abstract 1398: Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199

Abstract Metastatic Soft-Tissue Sarcomas (STS) are a rare and highly heterogeneous group of mesenchymal malignancies that carry a poor prognosis. Therapy is hampered by a limited number of effective treatments, and the almost non-existing long-term survival rate illustrates the need of effective targeted treatment. As a potential approach, we investigated combination of the clinically approved BH3-mimetic drug ABT-199 with different proteasome inhibitors (PIs): Bortezomib, Carfilzomib and Ixazomib, each with proven efficacy, e.g., in multiple myeloma. ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 while Bortezomib (BOZ) and Ixazomib (IXZ) bind reversible to the β1 and β5 subunits of the 20S proteasome and Carfilzomib (CFZ) irreversibly blocks the subunits β2 and β5. We investigated whether the combination of ABT-199 with CFZ or IXZ shows synergistic activity as recently published for BOZ. SW982 sarcoma cells were cultured with PIs alone or in combination with ABT-199. Cell death was detected by flow cytometric analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate potential differences due to specificity of PIs, we analyzed expression of BCL-2 family proteins by Western Blot and performed analogue experiments in knock-out (BAXKO, BAKKO, BOKKO) cell lines. In combination, ABT-199&BOZ or ABT-199&CFZ showed comparable synergistic cell death induction in SW982 while ABT-199&IXZ less efficiently induced cell death. Engineered SW982 knock-out cell lines suggests specific relevance of BOK in IXZ induced apoptosis, whereas all tested PIs crucially depend on BAX for apoptosis induction in combination with ABT-199. Also, as shown for ABT-199&BOZ, both CFZ and IXZ, alone and in combination with ABT-199, efficiently induced expression of NOXA. Strikingly, and in line with augmented cell death induction, ABT-199&CFZ resulted in highest expression of NOXA as compared to BOZ and IXZ. ABT-199&PIs synergistically induce apoptotic cell death in SW982 and corresponding knock-out cell lines with CFZ showing exacerbated expression of NOXA. ABT-199&CFZ also induced strongest stabilization of p53, a transcriptional key regulator of NOXA. We suggest that simultaneous inhibition of anti-apoptotic BCL-2 by ABT-199 and the enhanced expression of NOXA double-hit on the BCL-2 and the MCL-1 signaling axis force the cells over the threshold of apoptosis. Enhanced efficacy of CFZ is dually caused by reduced degradation of NOXA and enhanced stabilization of p53, which in turn transactivates expression of NOXA. In conclusion, PI specificity and efficacy modulate apoptosis induction by combination with ABT-199. Future efforts, e.g. knock-out of relevant BH3-only proteins, will elucidate the observed variable efficacy of these and other PIs for prospective translation in clinical applications. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias Beigl, Kathrin Böpple, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1398.

Targeting PI3K, mTOR, ERK, and Bcl-2 Signaling Network Shows Superior Antileukemic Activity Against AML

Standard induction therapy for the majority of acute myeloid leukemia (AML) patients has not changed over the past 40 years in spite of unacceptably low overall survival rates and high relapse rates, emphasizing the shortfall of current chemotherapy and stressing the need for new therapies. The PI3K/mTOR pathway, which is involved in proliferation, differentiation, and survival, has been shown to be constitutively active in 50-80% of AML patients. Promising preclinical data of PI3K inhibitors have been overshadowed by disappointing clinical results. PI3K/mTOR inhibition has been shown to cause compensatory ERK activation, resulting in cell survival. Thus, both signaling pathways must be targeted to induce AML cell death. However, other mechanisms of survival can remain intact after PI3K/mTOR and ERK inhibition. AML cells can escape cell death through upregulation of the anti-apoptotic Bcl-2 family proteins. Although results from a Phase II AML clinical trial using the Bcl-2-selective inhibitor ABT-199 (Venetoclax) showed promising results, all the ABT-199 treated patients relapsed in a short period of time (median time to relapse was 2.5 months). Therefore, we hypothesize that targeting the PI3K, mTOR, ERK, and Bcl-2 signaling network would result in superior antileukemic activity against AML. To begin our investigation, we first considered inhibition of PI3K and mTOR in AML cells using a novel dual PI3K/mTOR inhibitor VS-5584. VS-5584 sensitivity, as measured by MTT assays, varied among the AML cell lines (n=7) and primary AML patient samples (n=43), with IC50s ranging from 303 nM to 1.4 µM in the AML cell lines and 7 nM to 5.3 µM in the primary AML patient samples (median IC50 was 1.1 µM). Annexin V/propidium iodide staining and flow cytometry analysis revealed that VS-5584 induced cell death including caspase-independent apoptosis due to the lack of PARP and caspase-3 cleavage and change on the mitochondrial outer membrane potential, and the inability of the pan-caspase inhibitor Z-VAD-FMK to prevent cell death. Western blot analysis showed that VS-5584 treatment resulted in decreased phosphorylation of AKT (S473 and T308) and S6 (S240/244), indicating inhibition of both PI3K and mTOR. However, increased phosphorylation of ERK (T202/204) was detected, which could be abolished by the ERK-selective inhibitor SCH772984. Although the combination of VS-5584 and SCH772984 indeed resulted in synergistic induction of cell death, the levels of cell death were modest in some of the AML cell lines and primary patient samples tested. Although Z-VAD-FMK could partially prevent cell death of AML cells exposed to VS-5584 and SCH772984 for a short time (12 h), it could not save the cells post longer drug exposure (48 h). Western blot analysis showed downregulation of Mcl-1 and upregulation of Bim following inhibition of PI3K/mTOR, which were further enhanced by the combined inhibition of PI3K/mTOR and ERK. shRNA knockdown of Bim and overexpression of Mcl-1 at least partially prevented the combination of VS-5584 and SCH772984 induced cell death at an early time point, showing that Bim and Mcl-1 play an important role in cell death induced by the combination. Interestingly, immunoprecipitation of Bcl-2 revealed increased binding of Bim to Bcl-2 in AML cells post combined VS-5584 and SCH772984 treatment, which was abolished by the addition of ABT-199, suggesting that the increased Bim was bound to Bcl-2 which prevented its enhancement of cell death. The three drug combination induced significantly more cell death compared to control, single drug treatment or any of the two drug combinations, and was at least partially mediated through the intrinsic apoptotic pathway, as demonstrated by the ability of Z-VAD-FMK to partially prevent cell death induced by the three drug combination. Treatment of normal human CD34+ cells with the VS-5584, SCH772984, and ABT-199 combination showed no significant effect on colony formation, suggesting that the three drug combination spares normal hematopoietic stem/progenitor cells. Taken together, our findings show that inhibition of PI3K/mTOR and ERK synergistically induce cell death in AML cells, and addition of ABT-199 can improve the efficacy. Thus, our data support targeting the PI3K, mTOR, ERK, and Bcl-2 signaling network for the treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Phosphoproteomics Uncovers Synergy between DNA-PK and FLT3 Inhibitors in Acute Myeloid Leukaemia

*These authors contributed equally to this work Background:Acute Myeloid Leukaemia (AML) is the most common and aggressive form of acute leukaemia, with a 5-year survival rate of just 24%. Activating mutations in the receptor tyrosine kinase FLT3 are the most common driver mutations in AML (25-30% of patients). Inhibiting the FLT3 receptor as a mono-therapeutic strategy in AML has proven difficult however, due to the development of treatment resistance and relapse. In order to identify improved therapeutic targets, the oncogenic signalling pathways downstream of mutant FLT3 require characterisation. Methods:Quantitative, label-based phosphoproteomics was performed on primary blasts from 7 AML patients (4 mutant-FLT3, 3 wildtype-FLT3). Differentially phosphorylated pathways were identified using Ingenuity Pathway Analysis, and kinase activation was assessed by kinase substrate enrichment analysis. Validation of results was performed using targeted mass spectrometry. Proliferation, apoptosis, and cell cycle assays were used to assess drug toxicity; drug synergy was evaluated using Chou-Talalay and Webb analyses. Results:Analysis of differentially expressed phosphoproteins in mutant-FLT3 compared to wildtype-FLT3 AML patient blasts revealed dysregulation of DNA repair pathways. Specifically, mutant-FLT3 samples displayed increased phosphorylation of proteins within the error-prone Non-Homologous End Joining (NHEJ) repair pathway, indicating NHEJ pathway activation. Kinase enrichment analysis predicted increased activity of the NHEJ core kinase, DNA-dependent protein kinase (DNA-PK), in mutant-FLT3 samples. Accordingly, proliferation assays revealed that mutant-FLT3 cell lines were sensitive to inhibition of DNA-PK. FLT3-inhibitor treatment reduced DNA-PK phosphorylation in mutant-FLT3 cells, suggesting that activation of DNA-PK is downstream of FLT3 activation. Inhibition of DNA-PK kinase activity combined with FLT3 inhibitors led to synergistic induction of cell death, selectively in mutant-FLT3 cell lines. DNA-PK inhibitors combined with FLT3 inhibitors also co-operatively induced cell death in mutant-FLT3 primary AML patient samplesex vivo, and significantly prolonged survival compared to either monotherapy in a human AML xenograft mouse model. Conclusions:Mutant-FLT3 AML is associated with activation of the error-prone NHEJ repair pathway, which may contribute to genomic instability. Targeting the NHEJ kinase, DNA-PK, in combination with FLT3 inhibitors has the potential to improve outcomes for this poor-prognosis AML subtype. Disclosures Enjeti: Bayer:Speakers Bureau;AbbVie:Membership on an entity's Board of Directors or advisory committees;Alexion:Speakers Bureau;Novartis:Membership on an entity's Board of Directors or advisory committees;Astellas:Membership on an entity's Board of Directors or advisory committees;Sanofi:Speakers Bureau.

Menin‑MLL inhibitors induce ferroptosis and enhance the anti‑proliferative activity of auranofin in several types of cancer cells.

Menin‑mixed‑lineage leukemia (MLL) inhibitors have potential for use as therapeutic agents for MLL‑rearranged leukemia. They are also effective against solid cancers, such as breast cancer. The present study demonstrated that menin‑MLL inhibitors, such as MI‑463, unexpectedly induced the ferroptotic cell death of several cancer cell lines. MI‑463 at a double‑digit nM concentration markedly decreased the viable number of OVCAR‑8 ovarian cancer cells for 3days. Ferrostatin‑1 (a ferroptosis inhibitor) almost completely abrogated the MI‑463‑induced decrease in viable cell numbers. Furthermore, the cancer cell‑killing activity was inhibited by N‑acetylcysteine [a scavenger of reactive oxygen species (ROS)], deferoxamine (DFO, an iron chelator), PD146176 (a specific inhibitor of arachidonate 15‑lipoxygenase), idebenone (a membrane‑permeable analog of CoQ10) and oleic acid [a monounsaturated fatty acid and one of the end products of stearoyl‑CoA desaturase 1 (SCD1)], whereas Z‑VAD‑FMK (an apoptosis inhibitor) had a negligible effect on cell death. It was also found that MI‑463 in combination with auranofin (a thioredoxin reductase inhibitor) synergistically increased cancer the death of breast, ovarian, pancreatic and lung cancer cell lines (88%, 14/16 cell lines). The synergistic induction of cell death was abrogated by ferroptosis inhibitor and DFO. Inhibitors of SCD1, similar to MI‑463, also enhanced cancer cell death synergistically with auranofin, while inhibitors of SCD1 and MI‑463 did not additively induce cell death. Treatment with zinc protoporphyrin‑9, a specific inhibitor of heme oxygenase‑1 (HO‑1), markedly attenuated the cell death induced by MI‑463 plus auranofin. On the whole, these results suggest that the MI‑463‑induced decrease in cell viability may be at least partly associated with the inhibition of SCD1 activity. In addition, the potent induction of HO‑1 contributed to the synergistic effects of MI‑463 plus auranofin. Therefore, menin‑MLL inhibitors, such as MI‑463, in combination with auranofin represent an effective therapeutic approach for several types of cancer via the induction of ferroptosis.

Abstract 6388: MERTK and ROCK inhibitors mediate synergistic AML cell death and enhance therapeutic activity in combination with cytotoxic chemotherapy

Abstract Survival rates are suboptimal for adult and pediatric patients with acute myeloid leukemia (AML). Cytotoxic chemotherapies have short and long-term side effects and are often contraindicated for elderly patients. Targeted agents can reduce toxicity compared to chemotherapy; however, resistance to single-agents often develops, thus combination therapies may provide more durable responses. To increase efficacy and reduce exposure to cytotoxic agents, a targeted combination therapy (MERTK/ROCKi) was utilized alone and with doxorubicin or etoposide, frontline cytotoxic chemotherapeutics routinely used for AML treatment. MERTK is aberrantly expressed in >80% of AML patient samples and MERTK inhibitors are in clinical development. Both MERTK and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) regulate actin/microtubule dynamics and cell cycle progression and shMERTK or siROCK1 knockdown induces apoptosis in AML cells. Here, analysis of The Cancer Genome Atlas database revealed poorer overall survival in patients with higher levels of ROCK1 mRNA (p<0.01, n=172). MERTK inhibitors (MRX-2843 or UNC3997) and ROCK1/2 inhibitors (RKI-1447 or GSK269962A) synergized to reduce cell density in 4 of 6 AML cell lines tested and had an additive effect in the remaining cell lines. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in KG-1 (p<0.05), OCI-AML5 (p<0.05), and NB4 (p<0.1) cell cultures and an additive effect in Kasumi-1 cultures. Cells treated with the combination therapy failed to expand, even when cultured in the absence of inhibitor(s), and cell density was reduced in response to treatment with the combination therapy compared to single agents (p<0.01). Flow cytometric analysis of DNA content revealed an increased fraction of cells with G2/M DNA content in cultures treated with MRX-2843 (p<0.01). This effect was more pronounced in cultures treated with the combination therapy (p<0.05-p<0.0001) and was accompanied by an accumulation of cells with slightly reduced PI staining relative to untreated G2 cells. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. Addition of MERTK/ROCKi therapy to doxorubicin or etoposide additively decreased cell density relative to chemotherapy alone in OCI-AML5 and KG-1 cultures (p<0.0001). Treatment with doxorubicin and MERTK/ROCKi therapy also decreased OCI-AML5 cell density compared to MERTK/ROCKi therapy alone (p<0.01). These data indicate that combination therapies targeting MERTK and ROCK1/2, alone or in conjunction with chemotherapy, may be particularly effective for treatment of AML and support further studies in animal models. Citation Format: Dawn E. Barnes, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Barnes. MERTK and ROCK inhibitors mediate synergistic AML cell death and enhance therapeutic activity in combination with cytotoxic chemotherapy [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 6388.

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.

Redox Modulation and Induction of Ferroptosis as a New Therapeutic Strategy in Hepatocellular Carcinoma

Ferroptosis, a newly discovered form of cell death mediated by reactive oxygen species (ROS) and lipid peroxidation, has recently been shown to have an impact on various cancer types; however, so far there are only few studies about its role in hepatocellular carcinoma (HCC). The delicate equilibrium of ROS in cancer cells has found to be crucial for cell survival, thus increased levels may trigger ferroptosis in HCC.In our study, we investigated the effect of different ROS modulators and ferroptosis inducers on a human HCC cell line and a human hepatoblastoma cell line. We identified a novel synergistic cell death induction by the combination of Auranofin and buthionine sulfoxime (BSO) or by Erastin and BSO at subtoxic concentrations. We found a caspase-independent, redox-regulated cell death, which could be rescued by different inhibitors of ferroptosis. Both cotreatments stimulated lipid peroxidation. All these findings indicated ferroptotic cell death. Both cotreatments affected the canonical ferroptosis pathway through GPX4 downregulation. We also found an accumulation of Nrf2 and HO-1, indicating an additional effect on the non-canonical pathway. Our results implicate that targeting these two main ferroptotic pathways simultaneously can overcome chemotherapy resistance in HCC.

Abstract 2192A: Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML

Abstract Acute myeloid leukemia (AML) has a 5-year overall survival rate under 26% in adults and current cytotoxic chemotherapies are toxic, can cause long-term side effects and are often contraindicated for elderly patients. Targeted therapies may have reduced toxicity compared to chemotherapy; however, resistance to single-agents often develops and combination therapies may provide more durable responses. MERTK is aberrantly expressed in >80% of AML patient samples and is a promising therapeutic target. MRX-2843 is a novel small molecule dual MERTK and FMS-like tyrosine kinase 3 (FLT3) inhibitor that is currently in clinical trials. Here, we report synergistic anti-leukemia activity mediated by MRX-2843 and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) inhibitors. Both MERTK and ROCK1/2 have been implicated in actin/microtubule dynamics and cell cycle progression and previous publications demonstrated induction of apoptosis in AML cells in response to RNAi-mediated MERTK or ROCK1 inhibition. ROCK1/2 inhibitors RKI-1447 or GSK269962A synergized with sub-therapeutic doses of MRX-2843 to reduce cell density as indicated by decreased Presto Blue staining in cultures of 4 of 6 AML cell lines tested and had an additive effect in the remaining 2 cell lines. Similar interactions were observed between RKI-1447 and UNC3997, a MERTK-selective TKI with 15-fold weaker FLT3 activity. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in the KG-1, OCI-AML5, and NB4 cell lines and an additive effect in Kasumi-1 cultures. In addition, cells that survived treatment exhibited subsequent defects in expansion, even when they were cultured without inhibitor(s), and these effects were significantly more pronounced in cells treated with the combination therapy compared to single agents. Flow cytometric analysis of PI-stained permeabilized cells revealed an increased fraction of cells in G2/M phase in cultures treated with MRX-2843. This effect was more pronounced in cultures treated with the combination therapy and was accompanied by accumulation of a population with slightly reduced DNA content relative to untreated G2 cells, suggestive of a defect in late S-phase. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. In addition, a preliminary analysis of The Cancer Genome Atlas database revealed significantly poorer overall survival in patients with higher levels of ROCK1 expression (p=0.00147, n=172). Together, these data suggest that combination therapies targeting MERTK and ROCK1/2 may be particularly effective for treatment of AML and support further studies to test the effects of this novel strategy in animal models. Citation Format: Dawn E. Barnes, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2192A.

Cotreatment with sorafenib and oleanolic acid induces reactive oxygen species-dependent and mitochondrial-mediated apoptotic cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most common liver malignancy, and the lack of effective chemotherapies underlines the need for novel therapeutic approaches for this disease. Recently, we discovered a novel synergistic induction of cell death by combining sorafenib, the only routinely used palliative chemotherapeutic agent, and the triterpenoid oleanolic acid (OA). However, the underlying mechanisms of action have remained obscure. Here, we report that sorafenib and OA acted in concert to trigger mitochondria-mediated apoptotic cell death, which is dependent on reactive oxygen species (ROS). Sorafenib/OA cotreatment significantly increased ROS production, which was prevented by the ROS scavengers α-tocopherol and MnTBAP. Importantly, rescue experiments showed that ROS were required for sorafenib/OA-induced apoptosis as ROS scavengers protected HCC cells against cell death. In addition, sorafenib and OA cotreatment cooperated to decrease myeloid cell leukaemia-1 expression and to activate Bak, two events that were prevented by ROS scavengers. Bak activation was accompanied by the loss of mitochondrial membrane potential, followed by PARP cleavage, DNA fragmentation and, finally, apoptotic cell death in HCC cells. By providing new insights into the molecular regulation of sorafenib/OA-mediated and ROS-dependent cell death, our study contributes toward the development of novel treatment strategies to overcome sorafenib resistance in HCC.

Metabolic Reprogramming by Dual AKT/ERK Inhibition through Imipridones Elicits Unique Vulnerabilities in Glioblastoma.

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM).Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency.Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3β at serine 9. GSK3β promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392-406. ©2018 AACR.

PO-091 Induction of anti-Fas regulates PUMA and MYC: P53-dependent apoptosis pathway in hepatocellular carcinoma cells

IntroductionIncreased oxidative stress is a hallmark of cancer cells, which makes them more vulnerable to induction of reactive oxygen species (ROS). P53 plays a crucial role in sensing and removing oxidative damage to DNA, and inactivating mutations in the TP53 gene attenuate this function. In addition, it was shown that mutant p53 is able to suppress the function of major antioxidant factors. Therefore, mutant p53 renders cancer cells even more susceptible to the induction of oxidative stress. Besides p53, the poly (ADP-ribose) polymerase 1 (PARP-1) protein plays an important role in the repair of ROS-induced DNA-damage. This led us to explore the potential of combining oxidative stress induction with the targeted inhibition of the PARP-1 protein to selectively target mutant p53 NSCLC cancer cells.Material and methodsAPR-246 and Auranofin (inhibition glutathione (GSH) and/or thioredoxin reductase 1 (TrxR1)) and Olaparib (PARP-1 inhibitor) were used. The cytotoxicity (SRB-assay) of these compounds was determined in a panel of NSCLC cell lines with different p53 status, including isogenic cell lines (p53 shRNA-knockdown, p53 knock-in). Total GSH content (GSH/GSSG-GloTM) and ROS content (CellROX) were determined. N-acetyl-l-cysteine (NAC) was used as a potent ROS-scavenger. Induction of apoptosis/cell death was determined by the AnnV/PI assay (FC) or the Cytotox Reagent (IncuCyte). DNA-damage was assessed by g-H2AX foci (IF) and the Comet Assay. Synergism was determined using the Additive model.Results and discussionsP53Mut knock-down reduces the cytotoxic effect of APR-246, Auranofin and Olaparib, while p53Mut knock-in sensitised cells for all three compounds. APR-246/Olaparib treatment reduced GSH levels and increased ROS content, resulting in a strong accumulation of DNA-damage and synergistic induction of cell death. Co-treatment with NAC or p53-knockdown significantly reduced this cytotoxic response. Similar synergistic effects were observed for Auranofin/Olaparib treatment in several cell lines with clinically relevant p53 mutations.ConclusionMutant p53 protein expression renders NSCLC cells more susceptible to APR-246, Auranofin and Olaparib treatment. In addition, the combination of oxidative stress induction (APR-246, Auranofin) with PARP-1 inhibition (Olaparib) results in remarkable synergistic effects in the presence of mutant p53. Therefore, this combination strategy could be a promising and selective treatment option for mutant p53 NSCLC patients in which resistance to standard therapies often occurs.

Abstract 5425: Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer

Abstract Background Prostate cancer (PCa) is now the most commonly diagnosed malignancy in men worldwide. SOC combines radiotherapy with ADT, however recurrence rate remains unacceptably high and many men present with late stage aggressive and metastatic disease, with poor prognosis and limited treatment options. Despite tumor suppressor p53 being mutated in approximately 50% of human tumors, it has been reported that p53 alteration occurs in only 12% of primary prostate tumors. Emerging data links epigenetic-mediated gene silencing as a major differentiator between aggressive and indolent disease that is associated with up-regulation of key cell cycle regulator FOXM1, which is known to be suppressed by p53. HDAC inhibitors (HDACi) are an emerging class of epigenetic modifying anti-cancer agents, which we hypothesises will enhance p53 activity, cell death and alter patterns of gene expression to targeting p53 dependent and independent vulnerabilities in PCa. Methods We used basally p53 deficient/proficient PCa models (PC3/LNCaP) and novel C4-2B and LNCap p53 CRISPR-Cas9 knockout models to investigate the combination effects of Nutlin 3A or radiation (IR) with class 1 HDACi Entinostat by Annexin V/PI Flow Cytometry and Western Blot. Transcriptional effects were evaluated by RT-PCR and RNA-seq analysis. TCGA mutational and RNA-seq data were used to link p53 mutation with altered gene expression and outcome. Results We have demonstrated that there is a synergistic induction of cell death when we combine N3A/IR with Entinostat that is p53-dependent. In addition, we see that p53 acts as a barrier to genes associated with poor prognosis including FOXM1 which is phenocopied by single-agent Entinostat independent of WT-p53. This suppression may potentially be downstream of p21 via the DREAM complex. Importantly p53 mutation in TCGA primary PCa patients is associated with poor outcome and elevated FOXM1 expression. Conclusion and Future Work Results to date suggest that Entinostat enhances p53 dependent cell death but also has p53 dependent and independent mechanisms to suppress genes associated with poor prognosis PCa. The mechanism looks to be dependent of p21 and may involve DREAM complex. Citation Format: Gemma M. Gregg, Fiammetta Falcone, Andrea Lees, Gerard Quinn, Peter Gallagher, Richard Kennedy, Ian I. Mills, Simon S. McDade. Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer [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 5425.

Piperlongumine rapidly induces the death of human pancreatic cancer cells mainly through the induction of ferroptosis.

Pancreatic cancer is one of the most lethal types of cancer with a mortality rate of almost 95%. Treatment with current chemotherapeutic drugs has limited success due to poor responses. Therefore, the development of novel drugs or effective combination therapies is urgently required. Piperlongumine(PL) is a natural product with cytotoxic properties restricted to cancer cells by significantly increasing intracellular reactive oxygen species (ROS) levels. In the present study, we demonstrated that PL induced cancer cell death through, at least in part, the induction of ferroptosis, as the cancer cell-killing activity was inhibited by the antioxidant, N‑acetylcysteine, ferroptosis inhibitors (ferrostatin‑1 and liproxstatin‑1) and the iron chelator, deferoxamine (DFO), but not by the apoptosis inhibitor, Z-VAD-FMK, or the necrosis inhibitor, necrostatin‑1. Cotylenin A (CN‑A; a plant growth regulator) exhibits potent antitumor activities in several cancer cell lines, including pancreatic cancer cell lines. We found that CN‑A and PL synergistically induced the death of pancreatic cancer MIAPaCa‑2 and PANC‑1 cells for 16h. CN‑A enhanced the induction of ROS by PL for 4h. The synergistic induction of cell death was also abrogated by the ferroptosis inhibitors and DFO. The present results revealed that clinically approved sulfasalazine (SSZ), a ferroptosis inducer, enhanced the death of pancreatic cancer cells induced by PL and the combined effects were abrogated by the ferroptosis inhibitors and DFO. SSZ further enhanced the cancer cell-killing activities induced by combined treatment with PL plus CN‑A. On the other hand, the synergistic induction of cell death by PL and CN‑A was not observed in mouse embryonic fibroblasts (MEFs), and SSZ did not enhance the death of MEFs induced by PL plus CN‑A. These results suggest that the triple combined treatment with PL, CN‑A and SSZ is highly effective against pancreatic cancer.