Effects Of ONC201 Research Articles

Abstract 1856: Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206

Abstract ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving agonist activity against mitochondrial ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may compete with ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, lung cancer, GBM, and DIPG with dopamine for 12 hours, 24 hours or 48 hours, respectively, followed by treatment of ONC201 or ONC206 for 72hours. Cell viability was determined by the CellTiter Glo assay. Single treatment with dopamine did not increase cell viability. We observed that 48 hours of pre-treatment with dopamine at 50 μM, 100 μM or 200 μM impaired the cell viability suppression effect of ONC201 at 4 μM and 8 μM, while ONC206 at 0.5 μM, 1 μM and 2 μM led to similar protection for loss of cell viability in pancreatic cancer cells. The impairment of the anti-cancer effect by dopamine was also observed in colorectal cancer cells with 48 hours pre-treatment with dopamine at 50 μM, 100 μM, 200 μM or 400 μM and treatment with ONC201 at 4 μM and 8 μM, or ONC206 at 0.5 μM, 1 μM, or 2 μM. In addition, we treated pancreatic cancer and colorectal cancer cells with ONC212 after 48 hours pre-treatment with dopamine and observed a similar impairment of the anti-cancer effect, even though ONC212 is not a DRD2 antagonist. We pre-treated pancreatic cancer and colorectal cancer cells with specific DRD2 agonist, sumanirole, for 48 hours followed by treatment of ONC201, ONC206, or ONC212 for 72 hours, and no impairment was seen. We pre-treated multiple cancer cell lines with 2 μM or 10 μM dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine at 10 μM impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer. Pre-treatment with dopamine at 2 μM or 10 μM impaired the anti-tumor effect of ONC201 in GBM cells. By contrast to the cell viability result, no impairment of ONC212 effect from dopamine was seen in pancreatic cancer cells. Western blotting was conducted to investigate whether dopamine pre-treatment impacted signaling pathways reported to be affected by ONC201. Dopamine pre-treatment did not impact changes induced by ONC201 in ATF4, CHOP, DR5, ClpX, clpP, pERK, pAkt and pS6. We are pursuing whether the protective effect of dopamine is due to the agonism of DRD2 or alternative pro-survival mechanisms. Citation Format: Yiqun Zhang, Wafik S. El-Deiry. Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206 [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 1856.

Abstract 5380: Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death

Abstract Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [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 5380.

Caseinolytic protease P (CLPP) activated by ONC201 inhibits proliferation and promotes apoptosis in human epithelial ovarian cancer cells by inducing mitochondrial dysfunction.

BackgroundCaseinolytic protease P (CLPP) is a mitochondrial specific protein which has been reported to be related to tumor cell apoptosis. This study aims to explore the roles of CLPP in human epithelial ovarian cancer (EOC).MethodsWe determined CLPP expression in paracancerous tissues and cancer tissues obtained from 20 EOC patients, and also in 4 EOC cell lines, and one normal ovarian cell line (IOSE-80). We knocked CLPP expression down in SK-OV-3 and A2780 cells and overexpressed it in SW626 and OVcar3 cells. The effect of CLPP expression on cell proliferation, mitochondrial membrane potential, and apoptosis was then assessed by flow cytometry assay. Furthermore, the effect of ONC201 (agonist of CLPP) on the EOC cell lines was also investigated.ResultsThe CLPP expression was markedly down-regulated in EOC cancer tissues, and the Kaplan-Meier Plotter database revealed its low expression was linked to poor prognosis in EOC patients. Low expression of CLPP up-regulated the expression of NADH: ubiquinone oxidoreductase subunit A12 (NDUFA12), succinate dehydrogenase complex flavoprotein subunit A (SDHA), and succinate dehydrogenase complex iron sulfur subunit B (SDHB), which are key members of the mitochondrial respiratory chain, and these up-regulated proteins further led to the increase of mitochondrial membrane potential, cell proliferation promotion and neoplasm metastasis. Conversely, while overexpression of CLPP led to the opposite results, including inducing the decrease of mitochondrial membrane potential and apoptosis. In addition, stimulation with ONC201 enhanced the function of CLPP in SW626 and OVcar3 cells, and silencing of CLPP could neutralize the effect of ONC201.ConclusionsOur findings suggest that CLPP mediated mitochondrial dysfunction inhibits the proliferation and migration of EOC cells.

Abstract 2398: Disruption of mitochondrial metabolism by ClpP activation in triple negative breast cancer

Abstract ONC201 is a novel anticancer compound currently in over 20 clinical trials for multiple different cancers, including triple negative breast cancer (TNBC). We recently demonstrated that ONC201 and the more potent TR compounds selectively activate the mitochondrial protease ClpP. While previous studies demonstrated substantial effects on mitochondrial metabolism, the consequences of ClpP activation on cancer cell metabolism are yet to be defined. To investigate this, we performed an unbiased metabolomics and proteomics analysis comparing the effects of ONC201 and TR-57 on the TNBC metabolome and proteome. Consistent with these compounds targeting the mitochondria, our studies identified multiple mitochondrial metabolites and enzymes that were impacted by ClpP activation, such as TCA cycle intermediates and enzymes, heat shock proteins and mitochondrial ribosomal proteins. In addition to previously reported elements of the integrated stress response (ATF4, CHOP), we identified several proteins that were upregulated as part of the mitochondrial-nuclear signaling process. qRT-PCR was used to determine the impact of ONC201 or TR-57 treatment on transcript levels of proteomic hits. Importantly, the effects of ONC201 and the TR compounds on these proteins and cell growth were abolished in ClpP null cells. In summary, our studies further demonstrate that loss of metabolic function in mitochondria contributes to the anticancer activity of these ClpP activator compounds. Citation Format: Emily M. Fennell, Lucas J. Aponte-Collazo, Blake R. Rushing, Yuan-Yuan Li, Wimal Pathmasiri, Joshua D. Wynn, Paul R. Graves, Ekhson L. Holmuhamedov, Laura E. Herring, Edwin J. Iwanowicz, Lee M. Graves. Disruption of mitochondrial metabolism by ClpP activation in triple negative breast cancer [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 2398.

Abstract 2017: Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML)

Abstract Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment. Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [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 2017.

DIPG-52. PHASE I CLINICAL TRIAL OF ONC201 IN PEDIATRIC H3 K27M-MUTANT GLIOMA OR NEWLY DIAGNOSED DIPG

H3 K27M-mutant gliomas often manifest as midline gliomas, have a dismal prognosis, and have no effective treatments. ONC201 efficacy has been shown in high-grade glioma preclinical models and durable responses with single agent ONC201 have been reported in adults with recurrent H3 K27M-mutant gliomas. These observations led to a Phase I pediatric clinical trial of ONC201 dosed by body weight. This multi-center, open-label, 3 + 3 dose-escalation and dose-expansion clinical trial (NCT03416530) for H3 K27M-mutant glioma or non-biopsied DIPG has 6 arms: arms A and E determine the RP2D in pediatric post-radiation (recurrent or not-recurrent) H3 K27M-mutant glioma patients with ONC201 administered as an oral capsule as well as a liquid formulation, respectively. Both arms have completed accrual. The study is currently enrolling newly diagnosed DIPG patients to determine the RP2D for ONC201 in combination with radiation (arm B). Dedicated assessment of intratumoral ONC201 concentrations in midline gliomas patients (arm C) and the effects of ONC201 in H3K27M DNA levels in circulating CSF (arm D) are currently enrolling patients. ONC201 as a single agent in patients with progressive H3K27M mutant tumors following irradiation (excluding DIPG/spinal cord tumors) was recently opened (arm F). Once the RP2D is confirmed, there is a dose-expansion cohort to confirm the safety, radiographic efficacy and survival with ONC201. The primary endpoints of arms A, B, and E have been established with the RP2D of 625mg scaled by body weight as a capsule or liquid formulation administered alone or in combination with radiation without incidence of dose-limiting toxicity.

Abstract 4794: Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells

Abstract Background: We previously reported that ONC201, a novel anti-tumor drug currently being tested in early phase clinical trials, impairs mitochondrial structure and function in breast cancer cells (Greer et al., 2018). Recent studies demonstrated that ONC201 and the related TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein to mitochondrial protein homeostasis (Graves et al. 2019). Cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we tested the impact of ClpP agonists-mediated mitochondrial dysregulation on breast CSCs. Methods: Mitochondrial DNA (mtDNA)-depleted (rho0) MDA-MB231 (MB231) cells were generated by ethidium bromide treatment. MtDNA copy numbers were measured by qPCR. Mitochondrial respiration was monitored by Seahorse XF analyzer. Cell viability was analyzed by CellTiter-Glo 2.0 and RealTime-Glo assay. Protein expression was examined by Western blotting. ClpP knockout (KO) cells were generated by CRISPR/Cas9 technology. Mammosphere formation assay was performed to evaluate CSC function. MB231 cells stably expressing SORE6 (OCT4/SOX2 response element)-mCherry reporter gene were used to monitor CSC numbers. In vivo limiting dilution analysis was performed to examine the effect of ClpP agonists on MB231 mammary fat pad tumor initiation capability in athymic nude female mice. Results: We observed that mammospheres formed by MB231were enriched in mtDNA compared to cells grown in 2D culture while rho0 cells lacking functional mitochondria failed to form mammospheres. This suggested that mitochondria are required for CSC function. The effects of ONC201 and TR compounds in breast cancer cells were tested. First, we confirmed that TR compounds (TR-65, TR-57) had ~60-250 fold higher potency compared to ONC201 in cell viability assays. Both ONC201 and TR-57 impaired mitochondrial structure, function, inhibited cell growth in 2D, and significantly inhibited mammosphere formation in multiple breast cancer cell lines. Importantly, ONC201 and TR-57 did not affect mitochondrial function, cell growth in 2D, nor mammosphere formation in CLPP KO cell lines, confirming ClpP is the target of these drugs. Flow cytometry analysis of MB231-SORE6-mCherry expressing cells demonstrated that ONC201 treatment significantly reduced the fraction of cells that were positive for the SORE6-mCherry activity, consistent with loss of CSCs. Finally, using in vivo limiting dilution experiments, CSC frequency was significantly lower (p<0.05) in the ONC201-treated group compared with the control group, indicating that ONC201 inhibits tumor initiation capability in breast cancers. Conclusion: Our studies suggest that targeting mitochondria by ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Voeller Donna, Raj Chari, Sam Gilbert, Binwu Tang, Christina Annunziata, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells [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 4794.

Abstract 3173: Predictive biomarker evaluation for the anti-cancer imipridone ONC201

Abstract Small molecule imipridone ONC201 is a selective, mixed competitive and non-competitive antagonist of dopamine receptor D2 (DRD2), a GPCR that is overexpressed in a broad range of malignancies (Madhukar et al, Nature Communications, 2019). ONC201 was recently found to also act as an allosteric agonist of the mitochondrial protease ClpP that degrades members of the electron transport chain and other mitochondrial substrates (Ishizawa et al, Cancer Cell 2019). ONC201 is currently being tested in several Phase II clinical trials with evidence of clinical activity in DRD2-dysregulated tumor types, including high-grade glioma, endometrial cancer, mantle cell lymphoma and adrenal tumors. Previously, we reported that DRD2 and DRD5 expression correlates with ONC201 efficacy in the NCI60 and GDSC panels (>1000 cancer cell lines) (Prabhu et al, Clinical Cancer Research, 2019). In this study, we explored additional predictive biomarkers of response for ONC201, including ClpP and known downstream effects of ONC201 across the NCI60 and GDSC cancer cell line panels. Elevated ClpP expression emerged as a top predictive biomarker for ONC201 anti-cancer efficacy (low IC50) in the NCI60 and GDSC (p = 0.0066 and p = 0.00071, respectively) panels. Upregulation of HIF1 expression is known to drive cancer cells toward glycolysis; conversely, its loss shunts cellular energetics toward oxidative phosphorylation as the primary mechanism of ATP generation (Clark et al, Cell, 2019) that is inhibited by ONC201 via ClpP activation. Accordingly, decreased HIF1a expression correlated with increased anti-cancer efficacy in the NCI60 (p = 0.058) and GDSC (p = 0.0021) panels. ONC201 is known to cause downstream activation of the integrated stress response pathway, triggering induction of ATF4 (Ishizawa et al, Science signaling, 2016). High baseline ATF4 expression correlated with lower ONC201 IC50s in the GDSC panel (p = 0.027). Proteasomal degradation of c-myc by ONC201 has been reported in association with DRD2 antagonism and inhibition of oxidative phosphorylation in GBM (Ishida et al Clinical Cancer Research, 2018). High c-myc expression correlated with lower ONC201 IC50s in the GDSC panel (p = 2.5e-6). Ongoing studies are investigating biomarker combinations and identifying tumor types that are enriched for these predictive biomarkers. In conclusion, a series of target and downstream signaling biomarkers can be used to predict innate ONC201 tumor cell sensitivity that may provide guidance for patient and tumor type selection in future ONC201 clinical trials. Citation Format: Sara Morrow, Varun Vijay Prabhu, Joshua E. Allen, Wolfgang Oster. Predictive biomarker evaluation for the anti-cancer imipridone ONC201 [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 3173.

Abstract 3797: Anti-cancer effects of novel imipridone DRD2 antagonists in a panel of human cancer cell lines

Abstract Purpose: The dopamine D2-like receptors (DRD2) belong to the family of G-protein coupled receptors, which play crucial roles in various neurophysiological processes and in tumorigenesis. Several reports have described the role of DRD2 in cancer progression by influencing cellular invasion, migration, apoptosis and proliferation. Thereby, the pharmacological targeting of the DRD2 may serve as a potential therapeutic approach for cancer treatment. The imipridones ONC201 and ONC206 (Oncoceutics) are selective small molecule antagonists of DRD2 that inhibit Akt/Erk signaling pathway, and thus serve as promising anti-cancer agents for many tumors. Methods and Results: A MTT ([3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]) cell growth assay was used to measure the in vitro anti-proliferative effects of ONC201 and ONC206 on different types of human cancer cells, namely HCT116 (colon), MCF7 (breast), UC6 (bladder), PC3 and DU145 (prostate). Cells were seeded 24 hours (h) before drug treatment, and then treated with either ONC201 or ONC206 at different concentrations ranging from 1 to 100 µM. Cell proliferation was measured at 24, 48 and 72 h post drug treatment. Our results show that both ONC201 and ONC206 had considerable, dose-dependent anti-proliferative effects on all the tested cell lines after three consecutive days of treatment. Furthermore, we assessed the effects of the two drugs on nervous system tumors, namely neuroblastoma (NB) and medulloblastoma (MB). Pediatric human NB cell lines (MYCN-amplified IMR-32 and the non MYCN-amplified SKNSH) exhibited a dose-dependent inhibition on cellular proliferation and increased apoptosis at 48 and 72 h after drug treatment. ONC206 was more potent than ONC201 at inhibiting cell proliferation in both cell lines. Cell migration was also inhibited with both drugs in the IMR-32 and SKNSH NB cell lines. Cell viability was assessed in human MB D556 and D283 cell lines, which was reduced at 48 and 74 h post treatment with both ONC201 and ONC206 compared to vehicle treated cells. Tumor-sphere formation efficiency was inhibited in a dose-dependent manner in both human NB (IMR-32 and SKNSH) and MB (D556 and D283) cells after treatment with either drug. Western blot analysis revealed differential protein expression of stem cell maintenance and tumorigenic proteins, including OCT-4, MYCN, L1-CAM, HSP90, FABP5, VEGFR2 and RAB5C after treatment with either ONC201 or ONC206. Conclusion: Our data show potent anti-stem cell maintenance as well as anti-proliferative, anti-migratory, anti-viability and pro-apoptotic activity of both ONC201 and ONC206 on cancer cell lines, with ONC206 showing greater potency than ONC201. Our future directions include utilizing a combinatorial multi-modality therapy to treat a panel of pediatric and adult neurological tumors with ONC 201/206 and other selective inhibitors of tumorigenic pathways to completely eliminate the highly resistant sub-population of cancer stem cells, and delay malignant recurrence. Citation Format: Reine Hanna, Reem Daouk, Farah Ballout, Sarra El-Soussie, Jad Abdallah, Wassim Abou-Kheir, Tamara Mary Abou-Antoun. Anti-cancer effects of novel imipridone DRD2 antagonists in a panel of human cancer cell lines [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 3797.

Abstract 14: TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model

Abstract Colorectal cancer (CRC) is a major health problem globally. Preventing CRC in the general population, and especially in high-risk cohorts with colonic polyps, or its recurrence, is a high priority. ONC201, an orally active small molecule with robust antitumor activity, is currently in clinical trials for advanced cancers. The aim of this study was to determine the CRC preventive potential of ONC201 using the Apcmin/+ mouse model for CRC in familial adenomatous polyposis. First, the optimal and non-toxic doses of ONC201 were determined. C57BL/6J mice (n=6/group) were randomized, and five different doses of ONC201 (0-100mg/kg BW) were administered orally twice a week by gavage. After six weeks of treatment, all mice were euthanized, and tissues were analyzed for optimal dose and toxicity. Based on the body weight gain, gross organ weights at termination, blood profiling, plasma liver enzyme profile, and histopathological analysis, it was concluded that there were no toxicities associated with ONC201 within the tested dose range. For efficacy evaluation, Apcmin/+ male and female mice (n≥20) were grouped and ONC201 (0, 25 and 50 mg/kg BW) was administered by gavage, twice weekly for 14 weeks. At termination, intestinal tumors were evaluated for incidence and multiplicity to determine the efficacy of ONC201. Results indicate a strong suppressive effect of ONC201 against colonic and small intestinal (SI) tumor multiplicity in mice of both genders. Colonic tumor incidence was significantly reduced by >50% in ONC201-treated (50 mg/kg BW) male (40% in treated vs. 92% in control; p<0.0002) and female mice (30% in treated vs. 81% in control; p<0.001) when compared with placebo. Colonic tumor multiplicity was reduced by 68% in ONC201-treated (50 mg/kg BW) male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in control; p<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in control; p<0.0003). SI polyps were suppressed by up to 68% in both male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in control; p<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in control; p<0.0001). Snap-frozen colon tumor samples were analyzed for various biomarkers. Western blot protein analysis of the tumors suggested a dose-dependent increase in TRAIL expression upon treatment with ONC201. Similar increases were observed for DR5 and cleaved caspase 8 protein expression. Cleaved caspase 7, caspase 3, FADD and p21 were found to be increased in the treated tumor samples when compared with control. Immunohistochemical analysis indicated a significant decrease in PCNA index in the ONC201-treated tumors compared with controls. Serum analysis indicated a decrease in pro-inflammatory biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated samples compared to controls. Overall, chronic oral administration of ONC201 demonstrated strong chemopreventive efficacy against intestinal tumorigenesis in the Apcmin/+ mouse model. (Supported by NCI HHSN 261201500038I) Citation Format: Chinthalapally V. Rao, Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Niklas K. Finnberg, Howard P. Safran, Jennifer Fox, Shizuko Sei, Elizabeth R. Glaze, Robert Shoemaker, Wafik S. El-Deiry. TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model [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 14.

Abstract 4042: Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors

Abstract ONC201 is a promising anti-cancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. It has broad spectrum anti-tumor activity and achieved tumor regression and prolonged disease stabilization in clinical trials, especially in patients with H3K27M mutant gliomas. Based on the fact that the H3K27M mutation reduces the level of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that cancer cells could be sensitized to ONC201 by epigenetic modulators that impact on histone methylation or acetylation. We tested the synergy between epigenetic modulators and ONC201 in tumor cell lines. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. We also treated cancer cell lines originating from colorectal cancer, breast cancer, gastric cancer, prostate cancer, GBM and DIPG with combination of histone deacetylase inhibitor (HADCi), vorinostat, and ONC201. Cell viability was determined with the Cell Titer-Glo assay. Apoptosis was evaluated through flow cytometry analysis of cell distribution. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Cytotoxic TRAIL pathway signaling was evaluated using Western blot analysis of DR5. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response, cytotoxic TRAIL pathway signaling and apoptosis in combination with epigenetic modulators. There was greater induction of ATF4 and DR5 in combination therapy-treated tumor cells versus monotherapy. Immunoblotting analysis showed that combination of ONC201 and EZH2 inhibitor EPZ-6438 reduced H3K27me3 and EZH2. ONC201 is an antagonist of dopamine receptor D2 (DRD2). Dopamine receptor D5 (DRD5) is a dopamine receptor family member that opposes DRD2 signaling. The DRD2+DRD5- biomarker signature is associated with enhanced ONC201 tumor cell sensitivity. In order to investigate the role of dopamine receptors in anti-tumor effect of ONC201, we also tested the mRNA level of dopamine receptors by RT-PCR and observed that both vorinostat and EPZ-6438 upregulated DRD5 more than DRD2. Thus, we suspect other target genes may be involved in sensitization to ONC201 due to epigenetic modulation. We are currently investigating the interplay between H3K27 methylation versus acetylation in ONC201 sensitivity with or without additional therapeutic modulation by epigenetic drugs. Our results unravel potent synergy between ONC201 and EZH2 inhibitors or HDAC inhibitors and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Michael Glantz, Howard Safran, Attila Seyhan, Wafik S. El-Deiry. Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors [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 4042.

Anti-Tumor and Anti-Invasive Effects of ONC201 on Ovarian Cancer Cells and a Transgenic Mouse Model of Serous Ovarian Cancer

Anti-Tumor and Anti-Invasive Effects of ONC201 on Ovarian Cancer Cells and a Transgenic Mouse Model of Serous Ovarian Cancer

Abstract 364: The dopamine receptor D2 antagonist ONC201 has anti-tumorigenic activity in obesity-driven epithelial ovarian cancer

Abstract Objectives: ONC201 is an orally bioavailable dopamine receptor D2 antagonist that induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and TRAIL has been implicated in both the physiopathology of ovarian cancer (OC) and obesity. In addition, DRD2 is a G protein-coupled receptor that activates the trimeric G-protein complex Ras, modulating downstream metabolic pathways such as Akt-signaling which is highly dysregulated in both obesity and OC. ONC201 has demonstrated anti-tumorigenic activity in solid tumors, including uterine serous carcinoma, without significant toxicity in phase 1/2 trials. Thus, we sought to evaluate the anti-tumorigenic effects of ONC201 in human ovarian cancer (OC) cell lines and a high grade serous OC mouse model (K18-gT121+/-;p53fl/fl;Brca1fl/fl; KpB), using both obese and lean mice. Methods: The human epithelial OC cell lines (OVCAR3, SKOV3, IGROV, OVCAR433, and OVCAR5) were treated with ONC201 at doses of 0.01 to 250 μM. Cell proliferation and apoptosis were assessed by MTT and Annexin V assays, respectively. Adhesion and invasion were assessed by laminin and wound healing assays, respectively. For our animal study, we utilized the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) high grade serous OC mouse model. KpB mice were fed either a low fat diet (LFD, lean) with 10% calories from fat, or a high fat diet (HFD, obese) with 60% calories from fat in order to mimic diet-induced obesity. Following tumor onset, obese and lean mice were treated with vehicle or ONC201 (130 mg once a week, oral gavage) for 4 weeks (n = 8-10 mice/group). Results: ONC201 significantly decreased cell proliferation in a dose-dependent manner in all five cell lines, with a range of IC50s between 1 and 10μM. Moreover, ONC201 increased Annexin V expression in a dose-dependent manner in both the SKOV3 and OVCAR5 cell lines (p<0.05). Treatment with ONC201 reduced cell adhesion by 25-30% as well as invasion by 20-25% in both OC cell lines when compared to controls (p<0.05). HFD-fed mice (obese) had tumors that were significantly greater in size as compared to LFD-fed mice (lean) (p<0.05). ONC201 decreased tumor weight/size by 65% in the obese mice and 61% in the lean mice (p<0.01). Conclusions: ONC201 inhibited cell growth, induced apoptosis and decreased cellular adhesion and invasion in human OC cells. ONC201 was efficacious in reducing ovarian tumor growth in both obese and non-obese KpB mice. These results suggest that ONC201 holds promise as a novel targeted therapy in the treatment of OC. Citation Format: Allison Staley, Katherine Tucker, Ziwei Fang, Yali Fan, Wenchuan Sun, Yajie Yin, Yingao zhang, Varun Prabhu, Chunxiao Zhou, Victoria Bae-Jump. The dopamine receptor D2 antagonist ONC201 has anti-tumorigenic activity in obesity-driven epithelial ovarian cancer [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 364.

Abstract 2664: Differential activation of the integrated stress response correlates with anti-tumor activity of imipridones ONC201 and ONC206 in pediatric sarcomas

Abstract Cure rates for pediatric sarcomas have minimally improved with intensification of genotoxic chemotherapy. We have previously shown that activation of the Integrated Stress Response (ISR), a cellular stress response that can lead to apoptosis, is a novel, effective means of selectively inducing the death of rhabdomyosarcoma (RMS) cells. However, context-specific wiring of homeostatic components such as protein chaperones dictates unique barriers to ISR activation. We hypothesized that just as HSP70 inhibition can preferentially activate the ISR in RMS, but not Ewing sarcoma, alternative small molecules might highlight dependencies unique to other sarcoma subtypes. ONC201 is a selective dopamine receptor (DRD2/3) antagonist that has previously shown antitumor activity through ISR activation in solid tumor models. ONC206 is a more potent analogue of ONC201 that was shown to be effective in Ewing sarcoma cell lines with sub-micromolar IC50. We used these molecules to test the hypothesis that DRD2/3 antagonism can activate the ISR in pediatric sarcomas, identifying a new therapeutic strategy. We measured the growth inhibitory effects of ONC201 and ONC206 across a panel of four rhabdomyosarcoma (RMS) and three Ewing sarcoma (ES) patient-derived cell lines. Across all sarcoma cell lines tested, ONC201 and ONC206 inhibited growth with low micromolar and sub-micromolar IC50 values respectively. ES cell lines were more sensitive than RMS lines, with mean ONC201 IC50 of 7.7 micromolar vs 3.2 micromolar (p = 0.0016). To explore this difference, we measured ISR activation by immunoblot. The ISR initiates with phosphorylation of eIF2α, leading to a halt in protein translation. RMS cells showed a strong increase in phosphorylated eIF2α one hour after treatment with either drug that was sustained through 24 hours, but attenuated by 48 hours of treatment. ES cells, however, did not show increased eIF2α phosphorylation until 48 hours of treatment, at which point levels of phosphorylation were much higher than in RMS cells. Correspondingly, PARP cleavage was relatively modest in RMS cells, but robust in ES cells at 48 hours of treatment. We conclude that two phenotypes of ISR activation – early and attenuated, versus late and sustained – correlate with the therapeutic efficacy of these agents. Our data suggest that regulation of the ISR downstream of DRD2/3 antagonism underlies differential response to two clinically relevant agents. The development of biomarkers of the strength and duration of ISR activity may thus guide the clinical application of ONC201, ONC206, or other ISR-activating therapies in pediatric sarcomas. Ongoing work will test the expression and activity of eIF2α phosphatases and the necessity and sufficiency of terminal ISR effectors in controlling this differential response, as well as the efficacy of these agents in relevant in vivo models of disease. Citation Format: David V. Allegakoen, Rohinton S. Tarapore, Joshua E. Allen, Amit J. Sabnis, Trever G. Bivona. Differential activation of the integrated stress response correlates with anti-tumor activity of imipridones ONC201 and ONC206 in pediatric sarcomas [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 2664.

Small molecule ONC201 inhibits HIV-1 replication in macrophages via FOXO3a and TRAIL

Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.

Abstract LB-082: ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation

Abstract ONC201 is a potent inducer of cancer cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. ONC201 and its structural analogs comprise a novel class known as the imipridones. The compound is being tested clinically against a range of solid tumors and hematological malignancies. We found that both triple negative and non-triple negative breast cancer cells are sensitive to ONC201. This was initially surprising, as the majority of non-triple negative breast cancer cells are resistant to TRAIL. In TRAIL-sensitive cells, ONC201 induced cell death in a TRAIL-dependent manner. In TRAIL-resistant cells, the effects of ONC201 are not pro-apoptotic but are rather anti-proliferative. Here we further investigate the effects of ONC201 in TRAIL-resistant breast cancers from multiple molecular subtypes. ONC201 has been previously shown to induce transcriptional upregulation of TRAIL receptor death receptor 5 (DR5). A known mechanism of TRAIL resistance in breast cancer is low cell surface DR5 expression. We hypothesized that pre-treatment with ONC201 would upregulate DR5 and lead to TRAIL sensitization in resistant cells. Our results confirm our hypothesis and show that treatment with ONC201 transcriptionally upregulates DR5 and increases its expression at the cell surface. In addition, pre-treatment with ONC201 sensitizes TRAIL-resistant breast cancer cells to the pro-apoptotic effects of TRAIL. Caspase-8 and PARP are cleaved when cells are treated with the combination of ONC201 and TRAIL, but not when treated with either compound alone. Annexin-V PI staining and quantitation of subG1 DNA content via flow cytometry further confirmed these results. Knockdown of DR5 abrogated TRAIL sensitization by ONC201. Natural killer (NK) cells are known to use TRAIL to kill targets like tumor cells. We are exploring the hypothesis that pre-treatment of tumor cells with ONC201 would increase their killing by NK cells. We are currently investigating the importance of the TRAIL pathway and DR5 upregulation by imipridone compounds in this sensitization to NK cell induced apoptosis. In summary, these results describe a novel mechanism by which ONC201 sensitizes breast cancer cells to the effects of apoptosis inducing ligand TRAIL. They also provide preclinical rationale for the testing of the compound against breast cancers in the clinic. Citation Format: Marie D. Ralff, Jessica Wagner, Wafik S. El-Deiry. ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation [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 LB-082.

Abstract 2933: Synergistic antitumor effect of ONC201 in combination with radiation therapy

Abstract Radiation therapy is used as the main treatment when surgery is not feasible, before/after surgery, and to palliate symptoms in advanced stage. About 50% of cancer patients receive radiation therapy. 40% of the cure is achieved by radiation therapy. First-in-class small-molecule imipridone ONC201 can cause dual inactivation of ERK and AKT and induce an integrated stress response, proapoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and cell cycle arrest. ONC201 could synergize with radiation therapy because of its proapoptotic and antimetastasis effects. We tested this hypothesis in colorectal cancer, non-small cell lung cancer and glioblastoma cell lines (HCT116, RKO, LS513, H460, A549 and SNB19). Firstly, CellTiter-Glo® luminescent cell viability assays were performed with single treatment or combination of radiation therapy (0 to 4 Gy) and ONC201 (0 to 20 μM). For combination treatment, cancer cells were pretreated with ONC201 for 24 hours followed by radiation therapy. Next, colony formation assays with single treatment or combination were used to evaluate the effective dose range from cell viability assays. Flow cytometry and Western blot analysis were used for determination of apoptosis. We observed synergism between ONC201 and radiation therapy in several colorectal cancer, non-small cell lung cancer and glioblastoma cell lines. Combination indices are 0.53, 0.59 and 0.68 when non-small cell lung cancer cell line H460 was treated with ONC201 at 5 μM and radiation therapy at 1, 2 and 4 Gy. We are currently pursuing this promising combinational therapy in vivo, using the H460 xenograft model and potential syngeneic lung tumor models with wild-type p53. We plan to pursue the mechanism of this combination from in vivo samples. Our results indicate that the combination of radiation therapy and ONC201 could be an important approach in the application of ONC201 in clinical practice. Citation Format: Lanlan Zhou, Jessica Wagner, Wafik S. El-Deiry. Synergistic antitumor effect of ONC201 in combination with radiation therapy [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 2933.

Abstract 2765: Combination ONC201 and radiation therapy in the treatment of breast cancer

Abstract Purpose: Metastatic breast cancer patients continue to have poor outcomes on current treatments, and thus, novel therapies in combination with standard treatments are needed. Towards this we studied the pre-clinical effects of combining ONC201, a first-in-class imipridone (which works via activation of ER stress, upregulation of TRAIL, and activation of death receptor 5 [DR5]) with radiation therapy (RT). Since the DR5 pathway is also required for RT-induced apoptosis, we hypothesized that addition of ONC201 could increase the effectiveness of RT. Pertinent Experimental Procedures: Human and mouse breast cancer cell lines were used to study in vitro cancer cell death (by AlamarBlue and clonogenic assays). Patient-derived breast cancer tissue and mouse cell lines were used to study in vivo tumor growth kinetics. Cell lines and mice were administered radiation (0-8 Gy) delivered via a Gammacell 40 exactor. ONC201 was given at does 0-10 µM. Mechanisms of tumor cell death were elucidated using western blots of cleaved PARP (cPARP) and caspase 3 (cC3). Immune response mechanisms were elucidated using in vivo cell-specific depletion. Summary of Data: Multiple breast cancer lines (MB231, MB468, and 4T1) exhibited decreased cell viability after combination therapy compared to either single therapy. In particular, ON201 (1 µM) followed by RT (8 Gy) resulted in a decreased MB468 breast cancer cell viability compared to no treatment (36.9%, P<0.0001), RT alone (30.5%, p<0.0001) and ONC201 alone (26.9%, p<0.0001). A clonogenic assay using MB468 revealed a decreased surviving fraction at every RT dose (2-8 Gy). Western blot analysis revealed increasing cPARP and cC3 with increasing ONC201 (2-8 Gy). In vivo studies utilizing 4T1 cells demonstrated that combination RT (6 Gy) and ONC201 (48 mg/kg given 3x weekly) decreases tumor growth and prolongs host survival compared to either treatment alone (P<0.01). Depletion of NK or CD8 T cells (but not CD4 or B cells) abrogated the positive effects of ONC201 + RT. Similar efficacy of ONC201 + RT was achieved in NSG mice surgically transplanted breast cancer patient tumor sections and immune reconstituted with autologous patient immune cells. Conclusions: Treatment with combination ONC201 + RT results in decreased in vitro breast cancer cell viability and increased in vivo cancer control. Future studies are aimed at translating this combination therapy for clinical use towards improving and saving cancer patient lives. Citation Format: Sachin R. Jhawar, Devora Schiff, Hao Wu, Aditya Thandoni, Suemair Hassan, Joshua Allen, Martin Stogniew, Rohinton Tarapore, Wolfgang Oster, Mark Stein, Ann W. Silk, Sharad Goyal, Bruce G. Haffty, Andrew Zloza. Combination ONC201 and radiation therapy in the treatment of breast 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 2765.

Abstract P3-06-08: Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2

Abstract Breast cancer is a major cause of cancer-related death and there is a need for novel therapies with increased efficacy and decreased toxicity. The small molecule ONC201 was initially identified as a TRAIL pathway inducer. The compound has entered early phase clinical trials and is being tested in a range of solid tumors and hematological malignancies. Our previously published data demonstrate that ONC201 has potent anti-proliferative and pro-apoptotic effects in a broad range of breast cancer subtypes through TRAIL-dependent and TRAIL-independent mechanisms. Analogs of ONC201 with a shared pharmacophore have been developed and form the novel “imipridone” class. When compared with ONC201, imipridones ONC206, ONC212, and ONC213 showed increased potency of anti-proliferative or pro-apoptotic effects in breast cancer cells. We were interested in further defining the previously unstudied anti-proliferative effects of the imipridone compounds. Single agent efficacy of potent imipridone ONC212 in a xenograft model of TNBC was observed in the absence of apoptosis induction. This indicates that the anti-proliferative actions of the compound are sufficient for an in vivo anti-tumor effect. Our lab has previously shown that ONC201 activates an ATF4-dependent integrated stress response (ISR), essential for apoptosis induction in colon cancer cells. In contrast, in breast cancer cells, although ATF4 knockdown did not block cell death induced by ONC201 it did partially abrogate the anti-proliferative effects of the compound. The mammalian target of rapamycin complex 1 (mTORC1) is a well-known regulator of cellular growth and proliferation. mTORC1 signaling is inactivated in breast cancer cells following treatment with ONC201 and its analogs ONC212 and ONC213, regardless of whether the cells undergo apoptosis. Knockdown of ATF4 abrogated ONC201-mediated inhibition of p70 S6 kinase and ribosomal protein S6 phosphorylation, linking ISR induction to mTORC1 inhibition. We hypothesized that sestrin2, an endogenous mTORC1 inhibitor known to be upregulated following cellular stress, might represent a link between induction of ATF4 and inhibition of mTORC1. Treatment with ONC201 and its analogs ONC212 and ONC213 in multiple breast cancer cell lines resulted in sestrin2 upregulation. This was blocked by ATF4 knockdown. Furthermore, knockdown of sestrin2 abrogated the effects of ONC201 on mTORC1 signaling in breast cancers from multiple molecular subtypes. Previous mechanistic studies have focused exclusively on the relevance of ATF4 in the pro-apoptotic effects of ONC201. The novel findings described here help to elucidate the mechanism behind the potent and understudied anti-proliferative effects of the imipridones. Our findings also strengthen the preclinical rationale for testing of imipridone compounds against breast cancers regardless of molecular subtype. Citation Format: Ralff MD, Kline CLB, El-Deiry WS. Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-08.

Abstract B023: ONC201 kills breast cancer cells by targeting mitochondria

Abstract ONC201 is a small molecule originally identified as a TRAIL (TNF-related apoptosis-inducing ligand) inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. In this study, we tested ONC201 toxicity in breast cancer cell lines. ONC201 was obtained from Oncoceutics, Inc. Cell viability was tested with MTS assay and CytoTox-Glo assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNA-seq and Western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal and electron microscopy analysis were performed to study mitochondrial morphology. Mitochondrial DNA (mtDNA) copy number was analyzed by Quantitative PCR (qPCR). Mitochondrial defective (rho0) cell lines were generated by ethidium bromide treatment. We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2-amplified, basal A triple-negative (TNBC), and basal B TNBC breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases. Live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggested that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism. Western blots confimed that ONC201 induces ATF4 and CHOP, consistent with reported observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Cytotoxicity and ATP depletion induced by ONC201 were significantly enhanced in nonglucose (galactose) medium compared with glucose-containing medium, suggesting that ONC201 targets mitochondrial respiration. Supplementing glucose to cells grown in galactose medium rescued ONC201-dependent ATP depletion, cytotoxicity, and induction of p-AMPK, ATF4, and CHOP. Seahorse XF analyzer indicated that ONC201 inhibits mitochondrial respiration via an indirect mechanism. Confocal imaging revealed that ONC201 induces mitochondrial fission and reduces membrane potential. Electron microscopic analysis revealed that ONC201 induces mitochondrial structural damages, such as mitochondrial swelling, matrix lysis, and fragmentation. Confocal imaging and qPCR revealed that ONC201 decreases mtDNA. Importantly, ONC201 induced mitochondrial damage and mtDNA depletion even in ONC201-resistant cells, implying that cells that are not dependent on mitochondrial respiration are ONC201 resistant. Indeed, multiple rho0 cell lines were ONC201-resistant. RNAseq analysis confirmed that ONC201 inhibits expression of multiple mtDNA-encoded genes and nuclear encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions, and Western blot reinforced those findings. In summary, our data demonstrate that ONC201 kills breast cancer cells by targeting mitochondria. Citation Format: Yoshimi Endo Greer, Samuel Gilbert, Celia Islam, Ashley Ubaldini, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Yun Ji, Luca Gattinoni, Ferri Soheilian, Kunio Nagashima, Xiantao Wang, Markus Hafner, Jyoti Shetty, Bao Tran, Vishal Koparde, Parthav Jailwala, Maggie Cam, Dan Crooks, W. Marston Linehan, Donna Voeller, William Reinhold, Vinodh Rajapakse, Yves Pommier, Stanley Lipkowitz. ONC201 kills breast cancer cells by targeting mitochondria [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B023.