The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212...Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.

We demonstrate that GPX4 inhibition with doxycycline-inducible shRNA or the specific inhibitor ML210 induces ferroptosis in leukemia cells, evidenced by iron-dependent lipid peroxidation and blockade of cell death by the iron chelator deferoxamine. Of note, the classical ferroptosis inducers (xCT inhibitors) sulfasalazine and sorafenib did not induce ferroptosis in AML cells, suggesting a specific relevance of GPX4 in AML ferroptosis...Consistently, degradation of the respiratory complex with hyperactivation of mito-protease ClpP by imipridone ONC212 sensitized cells to GPX4 inhibition in HL60 parental cells but not in rho0 cells, further supporting the role of mito-respiration in the protection of cells against ferroptosis. As venetoclax (Ven) is one of the most widely used mitochondria-targeting agents in AML that also inhibits mito-respiration, we tested the combination of GPX4 inhibitor ML210 and Ven...Unexpectedly, and as a novel mechanism of GPX4 inhibition-mediated ferroptosis, we found that ML210 induces BAX/BAK-independent cytochrome C release from mitochondria, which is blocked by MitoQ... Our data suggest therapeutic potential of ferroptosis induction in AML by targeting GPX4 with the mechanistic involvement of mito-respiration. The combination of GPX4 and BCL-2 inhibition is synergistic and capable of overcoming Ven resistance. Studies are in progress to elucidate the molecular link between mito-respiration/redox and cytochrome C release and to investigate ferroptosis induction in vivo.

TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models...The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212...ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

We showed that activation of the mitochondrial ClpP protease by mutant ClpP (Y118A) or through utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (panobinostat) or selective (romidepsin) HDAC inhibitors caused synergistic reduction of viability in GBM model systems, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC inhibitors and imipridones prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

We investigated the anti-tumor effect of three imipridones (ONC201, ONC206, and ONC212), a promising new class of small molecules, in the treatment of neuroblastoma...Cell viability assays with HDAC inhibitors Vorinostat and Panobinostat demonstrated single-agent efficacy in vitro...However, further investigation is needed to determine synergy and mechanisms of synergy when histone deacetylase (HDAC) inhibitors are used in novel combinations with imipridones. Overall, our data reveals promise in imipridone therapy for neuroblastoma, and future studies are proposed to explore potential novel therapeutic combinations in this difficult-to-treat pediatric cancer.

Further investigation has revealed that ONC212 also appears to synergize with various autophagy inhibitors including hydroxychloroquine and chloroquine. Further in vitro experiments will be conducted to evaluate the effect of ONC212, trametinib, and other autophagy inhibitors on the PDAC tumor microenvironment using a T cell co-culture system. Similarly, in vivo murine studies will also be performed to assess the translational potential of this combination.

By integration of a transcriptome, metabolite and U-13C-glucose tracing analyses, we showed that activation of the mitochondrial ClpP protease through constitutively active ClpP (Y118A) or utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (Panobinostat) and selective (Romidepsin) HDAC inhibitors caused synergistic reduction of viability in established, neuro-sphere and patient-derived xenograft (PDX) cultures of human GBM, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC-inhibitors and imipridones reduced tumor growth and prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Background: Targeting apoptosis pathways in cancer has been extensively studied including the recent breakthrough therapy using venetoclax in acute myeloid leukemia (AML)...The anti-leukemia effects were associated with lipid peroxidation and were almost completely abrogated by a lipophilic antioxidant Liproxstatin-1 (Lip1). The effect of ML210 was also blocked by the iron chelator deferoxamine, indicating ferroptosis...Meanwhile, GPX4 is among the top 15 sensitization hits in a previously published genome-wide CRISPR screening of leukemia cells treated with potent ClpP agonists ONC201 and ONC212 (Jacques et al... Our data suggests the potential involvement of mitochondrial lipid peroxidation in the anti-leukemia effects of GPX4 inhibition, along with its therapeutic potential in conjunction with mito-oxidative stress induction though instability in mito-proteome. Further investigations are in progress to assess the molecular mechanisms and the in vivo efficacy of the combinatorial treatment.

We demonstrate synergy between ONC201, ONC206 and ONC212, and targeted therapies with known preclinical activity against DIPG...Six patient-derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36) were exposed to imipridones alone or combinations with histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide...Our results suggest increased sensitivity of H3K27M-mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is synergistic cell death with combination of imipridones and panobinostat, romidepsin, or marizomib, which may be further tested in vivo and in clinical trials.

Introduction: The combination of the autophagy inhibitor chloroquine and the MEK inhibitor trametinib has been shown to exhibit synergy in KRAS-mutated pancreatic cancer cell lines and mouse models. The combination of trametinib with ONC201 or ONC212 exerts a synergistic cytotoxic effect on tumor cells at doses that are non-toxic to control cells. In a KRAS-mutated GA cell line, this is likely related to reduction of autophagic flux. Similar synergistic effects were noted in a KRAS-wt GA cell line but with down-regulation of autophagy markers, suggesting an autophagy-independent mechanism.

Liver cancer cell lines Hep3B and HepG2 were treated with ONC201, ONC206, and ONC212 to determine dose-response effect...Combination treatment effect was also tested between ONC201 and the HDACi vorinostat, PARPi nariparib, and the proteasome inhibitor marizomib...These results suggest that the imipridone family of small molecules is a novel therapy for liver cancer with efficacy as a single agent and in combination with existing targeted agents. Future studies will clarify the mechanisms of synergy and expand the findings into small animal models.

We performed combinatorial drug treatment on Ewing sarcoma cell lines SK-N-MC and RD-ES with 3 imipridones (ONC201, ONC206, and ONC212) and 3 HDAC inhibitors (vorinostat, entinostat, and panobinostat). Cell viability studies and analysis with Compusyn demonstrate potent synergistic effects causing tumor cell death when imipridones are combined with HDAC inhibitors. Our work presents a novel therapeutic combination for the treatment of Ewing sarcoma.

Autophagy, a catabolic process used by many tumors to maintain viability, can be targeted with various agents such has hydroxychloroquine and chloroquine, and recent preclinical data has shown that the combination of these agents with inhibitors of MEK/ERK can synergistically induce cell death...In conclusion, the combination of trametinib and ONC212 produces a synergistic cytotoxic effect on tumor cells at doses that are non-toxic to normal cells...Similar synergistic effects were noted in a KRAS wild-type pancreatic cancer cell lines, suggesting an autophagy-independent mechanism. Thus, this combination may represent a potential therapeutic modality for PDAC.

Here, we showed that pharmacological activation of the mitochondrial ClpP protease through utilization of the novel imipridone compounds (ONC206 and ONC212) in combination with global (Panobinostat) and selective (romidepsin) HDAC – inhibitors caused synergistic reduction of viability in established and patient-derived xenograft (PDX) cultures of human GBM. Finally, using a PDX model, we demonstrated that the combination treatment of romidepsin and ONC206 reduced tumor growth more potently than single treatments or vehicle by enhanced reduction of cellular proliferation and pronounced induction of cell death in vivo. Collectively, these observations suggest that the efficacy of HDAC-inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Enhancement of glycolytic flux may represent an escape mechanism active in most PDAC cells. Switching metabolic dependence towards mitochondria or combining ONC212 with a glycolytic inhibitor may significantly increase its antineoplastic potential in this highly aggressive malignancy.

Future experiments aim to examine G1 arrest after treatment with ONC201 and ONC212 via western blot and flow cytometry, to confirm surface expression of TRAIL and DR5 via flow cytometry, and to determine efficacy in pancreatic cancer xenografts. Taken together, these data indicate a novel therapeutic strategy for treatment of patients with ONC201 and ONC212 resistant pancreatic tumors.