Detailed results in apoptosis assay of primary AML cells treated with DS-3032b.

Supplemental Methods Table S1. Growth inhibition by DS-3032b and nutlin-3a in tumor cell lines with different p53 status. Table S2. Percent tumor growth inhibition (%TGI ) values at day 20 in SJSA-1 xenograft mice treated with DS-3032a. Table S3. GI50 values of cancer cell lines treated with DS-3032b. Table S4. Clinical information of the 41 samples Table S5. Confusion matrix of predicted and actual sensitivity of primary AML cells to DS-3032b according to TP53 mutation status. Table S6. The cut-off values used for definining sensitivity/resistance to each drug in the different phases of the signature development and validation process. Table S7. Prediction of sensitivity by 175-gene signature scoring and actual results in patient-derived xenograft models of various tumors treated with DS-3032a. Table S8. Confusion matrix of predicted and actual sensitivity of primary AML cells to DS-3032b using previously reported 4-gene signatures. Table S9. The top-ranked p53-inducible genes in the 175-gene signature. Table S10. Cancer type and TP53 mutational frequency. Table S11. Referenced 32 genes. Figure S1. Effects of DS-3032b as an MDM2 inhibitor in vitro and in vivo. Figure S2. Effects of MDM2 inhibitors (DS-3032b, DS-5272 and nutlin-3a) in OncoPanel cell lines. Figure S3. Prediction of sensitivity of tumor cells to MDM2 inhibition using the 175 gene signature.

List of sensitive or resistance signature genes.

Detailed results in viability assay of Oncopanel cell lines treated with MDM2 inhibitors.

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.

Cellular stress response has dual aspects; cell-protective or lethal. Mitochondria have their unique organellar response termed "mitochondrial unfolded protein response (UPRmt)" induced by damaged mitochondrial (mt) matrix proteins. While recent discoveries have successfully targeted BCL2, a regulator of mt integrity in acute myeloid leukemia (AML), the significance of UPRmt is unknown. We hypothesized that priming UPRmt towards cell death would be a novel therapeutic strategy for AML. UPRmt is generally induced by dysregulation of mt protein pools. Therefore, to test if UPRmt signaling is also operational in AML cells, we selected classical or putative UPRmt inducers; the mt translation inhibitors tetracycline and tigecycline, the mt protein transport inhibitor MitoBlock6, and the mtDNA damaging agent ethidium bromide. In OCI-AML3 and HL60 cells, these agents indeed induced the transcription factor ATF5, which was reported as a central inducer of UPRmt, and its targets (e.g., LonP, HSPA9), triggering apoptosis in AML cells. In addition, we here report imipridones (ONC201 and ONC212), the activators of mt protein degradation, as novel UPRmt inducers. We recently reported that imipridones non-covalently bind the mt protease ClpP and allosterically activate it. They induced prominent apoptosis in primary AML progenitor and leukemia initiating cells (LICs) in vitro and in vivo, but not in normal bone marrow cells, following "mitochondrial proteolysis" with reduction of selective mt matrix proteins (e.g., SDHB, NDUFA12) and resultant inhibition of oxidative phosphorylation (Oxphos) (Ishizawa, Zarabi et al, Cancer Cell 2019). We then postulated that dysregulation of mt protein pools by mitochondrial proteolysis can also induce UPRmt. Indeed, our gene expression profiles of ONC201-treated Z138 and Jeko-1 cells were highly enriched for previously published UPRmt gene signatures, and UPRmt effectors were induced also in AML cells. Of potentially high clinical significance is the finding of synergistic anti-leukemia effects of imipridones when combined with the selective BCL2 inhibitor venetoclax, in vitro and in vivo (Ishizawa et al. Science Signaling 2016, and Nii et al. Blood 2019). However, its underlying molecular mechanism is unclear. Since BCL2 is reported to be induced by UPRmt, we hypothesized that BCL2 is critical for the ClpP-mediated UPRmt to have the cell protective effects, contrary to lethal effects, as dual aspects of stress response. We utilized the tetracycline-inducible system of an activated mutant (Y118A) form of ClpP in OCI-AML3 cells, and demonstrated that venetoclax treatment sensitizes OCI-AML3 cells to genetic activation of ClpP towards apoptosis. Furthermore, other UPRmt inducers (tetracycline, tigecycline, and MitoBlock6) in combination with venetoclax also synergistically induced apoptosis in AML cells, suggesting that BCL2 inhibition and UPRmt induction generally exerts synergistic anti-leukemia effects. We next focused on the enhanced effect observed for the combination of imipridones with venetoclax as compared to other UPRmt inducers, searching for other targets that could further enhance the synergy. We then hypothesized that the synergism between ClpP activation and BCL2 inhibition involves SDHB, a respiratory chain complex II subunit degraded by activated ClpP but not targeted by any of other UPRmt inducers. Consistently, SDHB knockdown sensitized OCI-AML3 cells to venetoclax-induced apoptosis, indicating that SDHB reduction and UPRmt by ClpP activation concomitantly enhance the cell lethality by BCL2 inhibition. Collectively, UPRmt is a new potential therapeutic target for AML, which significantly enhances the cell death effects of BCL2 inhibition on AML cells. In particular, ClpP activation induces UPRmt and, concomitantly, downregulates SDHB, thus targeting the respiratory chain complex II, which results in improved synergistic leukemia cell apoptosis when combined with BCL2 inhibition. Oxphos is also a hallmark of drug resistant AML stem cells, which supports the notion that Oxphos inhibition by this combination targets LICs. Based on promising preclinical anti-tumor efficacy, ONC201 as a single agent is being evaluated in early phase clinical trials, showing clinical responses in AML and midline gliomas. A clinical trial testing the combinatorial strategy of targeting ClpP and Bcl-2 is under development. Disclosures Borthakur: Novartis: Research Funding; NKarta: Consultancy; Eisai: Research Funding; Oncoceutics: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel: Research Funding; Strategia Therapeutics: Research Funding; Eli Lilly and Co.: Research Funding; Arvinas: Research Funding; Merck: Research Funding; AstraZeneca: Research Funding; PTC Therapeutics: Consultancy; Agensys: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; Incyte: Research Funding; Janssen: Research Funding; AbbVie: Research Funding; BMS: Research Funding; Oncoceutics, Inc.: Research Funding; Bayer Healthcare AG: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Tetralogic Pharmaceuticals: Research Funding; Cantargia AB: Research Funding; Polaris: Research Funding; Xbiotech USA: Research Funding. Stogniew:Oncoceutics, Inc.: Employment. Oster:Oncoceutics, Inc.: Employment. Kantarjian:BMS: Research Funding; AbbVie: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Agios: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Takeda: Honoraria; Astex: Research Funding; Daiichi-Sankyo: Research Funding. Schimmer:Novartis Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Andreeff:Eutropics: Equity Ownership; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.

Imipridones constitute a novel class of antitumor agents. Here, we report that a second-generation imipridone, ONC212, possesses highly increased antitumor activity compared to the first-generation compound ONC201. In vitro studies using human acute myeloid leukemia (AML) cell lines, primary AML, and normal bone marrow (BM) samples demonstrate that ONC212 exerts prominent apoptogenic effects in AML, but not in normal BM cells, suggesting potential clinical utility. Imipridones putatively engage G protein-coupled receptors (GPCRs) and/or trigger an integrated stress response in hematopoietic tumor cells. Comprehensive GPCR screening identified ONC212 as activator of an orphan GPCR GPR132 and Gαq signaling, which functions as a tumor suppressor. Heterozygous knock-out of GPR132 decreased the antileukemic effects of ONC212. ONC212 induced apoptogenic effects through the induction of an integrated stress response, and reduced MCL-1 expression, a known resistance factor for BCL-2 inhibition by ABT-199. Oral administration of ONC212 inhibited AML growth in vivo and improved overall survival in xenografted mice. Moreover, ONC212 abrogated the engraftment capacity of patient-derived AML cells in an NSG PDX model, suggesting potential eradication of AML initiating cells, and was highly synergistic in combination with ABT-199. Collectively, our results suggest ONC212 as a novel therapeutic agent for AML.

The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.

Background: Standard treatment for ovarian cancer with a platinum and taxane is effective initially but associated with significant relapse. Our group was first to demonstrate genetically modified MSC preferentially engraft and secrete gene products such as interferon beta (IFNB) at tumor sites, resulting in complete responses in animal models (Studeny M et al. Cancer Res 2002;62:3603; JNCI 2004;96:1593). Methods: We conducted a single-center phase 1 trial to evaluate the safety, feasibility tumor response of mesenchymal stem cells transfected with plasmid secreting IFNB administered via intraperitoneal infusion (IPI) for women with advanced refractory epithelial ovarian cancer. We electroporated MSC derived from male donors with a plasmid vector containing the IFNB gene (MSC-IFNB). MSC-IFNB were then administered on an outpatient basis via IPI. Pretreatment biopsy, peritoneal fluid sample, blood draw, and a CT were performed at enrollment. Results: Three patients were enrolled at dose level 1 (105 MSC/kg) and received 4 weekly IPI of MSC-IFNB between 6-10/2016. After the 4th dose we performed a restaging CT and a biopsy, which was examined by FISH for the presence of male Y-chromosome positive MSCs, for production of IFNB assessed by multispectral multiplexed IHC, presence of injected MSCs, changes in immune cell phenotypes, and tumor cell death. We demonstrated detectable production of IFNB in the serum of patient 1, and at week 4 detection of IFNB in the peritoneal fluid, suggesting injected MSC-IFNB produce IFNB locally. We performed multispectral analysis to identify and quantitate the constituents of the ovarian tumor microenvironment in pre- and post-treatment biopsy specimens. We focused on ovarian tumor cells (CK3/5+,AE1+), as well as stromal cells (αSMA+), and IFNB. We observed in all 3 patients colocalization of IFNB (red) with αSMA-(green,) suggesting MSC homing and production of IFNB at tumor stroma interface. To confirm the presence of injected MSC (derived from male donors), we then analyzed the pre- and post-treatment tumor biopsies for presence of the “XY” chromosome by FISH. We identified “XY” chromosomes MSC only in the post-tumor biopsy, suggesting incorporation of injected MSC at the tumor site. There were no infusion- or MSC-related adverse events and 1 patient had stable disease on CT scan at 4 months after treatment. Conclusion: We demonstrate, for the first time, proof of principle that MSC-IFNB home to and are incorporated into tumors and locally produce beta-interferon after IP injection in patients with ovarian cancer. We anticipate increased responses at higher MSC dose levels and envision MSC-IFNB may be used in combination with immunotherapies and chemotherapy, as we have shown in animal models that MSC-IFNB modulate the immune phenoptype of the tumor, recruit FOXp3 immune cells, and facilitate long-term control of tumor growth and metastases. Citation Format: Michael Andreeff, Frank C. Marini, Shannon N. Westin, Robert L. Coleman, Peter F. Thall, Vivian Aljahdami, Muzaffar H. Qazilbash, Katy Rezvani, Melissa Timmons, Lauren Heese, Rui-Yu Wang, Richard E. Champlin, Elizabeth J. Shpall, Amanda Olson. A phase I trial of mesenchymal stem cells transfected with a plasmid secreting interferon beta in advanced ovarian 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 75.

Abstract Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type TP53 tumors are sensitive to MDM2 inhibition. Therefore, more potent inhibitors and biomarkers predictive of tumor sensitivity are needed. The novel MDM2 inhibitor DS-3032b is 10-fold more potent than the first-generation inhibitor nutlin-3a. TP53 mutations were predictive of resistance to DS-3032b, and allele frequencies of TP53 mutations were negatively correlated with sensitivity to DS-3032b. However, sensitivity to DS-3032b of TP53 wild-type tumors varied greatly. We thus used two methods to create predictive gene signatures. First, by comparing sensitivity to MDM2 inhibition with basal mRNA expression profiles in 240 cancer cell lines, a 175-gene signature was defined and validated in patient-derived tumor xenograft models and ex vivo human acute myeloid leukemia (AML) cells. Second, an AML-specific 1,532-gene signature was defined by performing random forest analysis with cross-validation using gene expression profiles of 41 primary AML samples. The combination of TP53 mutation status with the two gene signatures provided the best positive predictive values (81% and 82%, compared with 62% for TP53 mutation status alone). In addition, the top-ranked 50 genes selected from the AML-specific 1,532-gene signature conserved high predictive performance, suggesting that a more feasible size of gene signature can be generated through this method for clinical implementation. Our model is being tested in ongoing clinical trials of MDM2 inhibitors. Significance: This study demonstrates that gene expression profiling combined with TP53 mutational status predicts antitumor effects of MDM2 inhibitors in vitro and in vivo. Cancer Res; 78(10); 2721–31. ©2018 AACR.

Abstract Imipridones are first-in-class anti-tumor compounds including ONC201, which has shown promising clinical activity. ONC212 was designed as a second-generation imipridone. We first confirmed the ONC212 effects in a collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project; leukemia was identified as the most sensitive tumor type. In fact, ONC212 exerted prominent apoptogenic effects in acute myeloid leukemia (AML) cell lines and primary AML, but not normal bone marrow (BM) cells. We investigated the effects of ONC212 in vivo in an aggressive systemic AML xenograft model using OCI-AML3 cells. ONC212 markedly inhibited AML expansion and prolonged median survival (controls: 43 d, ONC212: 49 d; p = 0.0003). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells (LSPCs), we treated patient-derived xenograft (PDX) cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected into recipient NSG mice. After one month, the human leukemic CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212 treated group. The median survival was remarkably prolonged (controls: 36 d, ONC212: 82 d; p < 0.0001). These results indicate that ONC212 has anti-LSPC effects to reduce the engraftment potential. We previously demonstrated that the prototype it compound ONC201 induces apoptosis via an atypical integrated stress response (ISR; Ishizawa et al., Sci Signal, 2016). As expected, ONC212 induced the transcription factor ATF4, a key effector of ISR. Because BCL-2 is generally considered to be protective against ISR-mediated apoptosis, we hypothesized that the BCL-2 inhibitor ABT-199 could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 synergistically induced apoptosis in AML cells. Furthermore, the combination showed highly significant synergistic anti-leukemia effects in vivo. The combinatorial treatment prolonged overall median survival (controls: 20 d, each agent: 21 d, the combination 30 d; p < 0.0001). Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of ONC201, we hypothesized that ONC212 also targets GPCRs. The PathHunter β-arrestin screening discovered that ONC212 specifically activated the orphan GPCR GPR132. Consistently, the GPR132 mRNA expression was correlated with ONC212 sensitivity. On the other hand, GPR132 overexpression induced cell death in AML cells, which is consistent with previous reports implicating GPR132 as a tumor suppressor. Furthermore, ONC212 increased GPR132 mRNA expression. These results suggest that GPR132 could be a potential therapeutic target in AML. Taken together, ONC212 has potential as a novel agent for AML therapy. This study provides the first reported opportunity to therapeutically target GPR132 in oncology. Citation Format: Takenobu Nii, Jo Ishizawa, Varun V. Prabhu, Vivian Ruvolo, Neel Madhukar, Ran Zhao, Hong Mu, Lauren Heese, Kensuke Kojima, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Olivier Elemento, Joshua Allen, Wolfgang Oster, Martin Stogniew, Michael Andreeff. The novel imipridone ONC212 highly synergizes with the BCL-2 inhibitor ABT-199 in AML and activates orphan receptor GPR132 [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 4957.

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).