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Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, has been implicated in the pathogenesis of many cancers, with involvement in cell-cycle regulation, apoptosis, and immune response. Small-molecule GSK-3β inhibitors are currently undergoing clinical investigation. Tumor sequencing has revealed genomic alterations in GSK-3β, yet an assessment of the genomic landscape in malignancies is lacking. This study assessed >100,000 tumors from two databases to analyze GSK-3β alterations. GSK-3β expression and immune cell infiltrate data were analyzed across cancer types, and programmed death-ligand 1 (PD-L1) expression was compared between GSK-3β–mutated and wild-type tumors. GSK-3β was mutated at a rate of 1%. The majority of mutated residues were in the kinase domain, with frequent mutations occurring in a GSK-3β substrate binding pocket. Uterine endometrioid carcinoma was the most commonly mutated (4%) tumor, and copy-number variations were most commonly observed in squamous histologies. Significant differences across cancer types for GSK-3β–mutated tumors were observed for B cells (P = 0.018), monocytes (P = 0.002), dendritic cells (P = 0.005), neutrophils (P = 0.0003), and endothelial cells (P = 0.014). GSK-3β mRNA expression was highest in melanoma. The frequency of PD-L1 expression was higher among GSK-3β–mutated tumors compared with wild type in colorectal cancer (P = 0.03), endometrial cancer (P = 0.05), melanoma (P = 0.02), ovarian carcinoma (P = 0.0001), and uterine sarcoma (P = 0.002). Overall, GSK-3β molecular alterations were detected in approximately 1% of solid tumors, tumors with GSK-3β mutations displayed a microenvironment with increased infiltration of B cells, and GSK-3β mutations were associated with increased PD-L1 expression in selected histologies. These results advance the understanding of GSK-3β complex signaling network interfacing with key pathways involved in carcinogenesis and immune response.

ONC212 is a fluorinated imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo. Thus, ONC212 is a novel mitocan targeting oxidative metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

Background: 9-ING-41, a small molecule specific GSK-3 inhibitor, demonstrated favorable efficacy and safety in a Phase I clinical trial evaluating 9-ING-41 monotherapy and 8 chemotherapy combinations in 236 patients (pts) that has since been expanded to a Phase II multicenter study (NCT03678883). Interim results documented encouraging clinical activity, especially in patients with advanced pancreatic ductal adenocarcinoma (PDAC) and durable responses in melanoma and ATLL. Potential genomic biomarkers for 9-ING-41 response have not yet been evaluated. Methods: Next-generation sequencing (NGS) of tumor samples was obtained, whenever possible, for those receiving 9-ING-41 alone or in combination with chemotherapy across all sites. Genomic sequencing reports were reviewed for somatic alterations deemed potentially actionable or biologically relevant. For those with PDAC, best response was determined using RECIST 1.1 for those who completed at least two cycles of treatment. Chi-square frequency statistics were used to show the observed versus expected rate of pathogenic variants between patients with disease control (complete response [CR], partial response [PR] or stable disease [SD]) and progressive disease (PD). Results: NGS results were available for 135 pts across 11 sites. Most common histologies were PDAC (n=32), colorectal (n=17), and melanoma (n=9). Among pts with PDAC, NGS results were available for 32 pts (23 tumor samples, 9 ctDNA). Four patients received 9-ING-41 monotherapy; 28 received 9-ING-41 combined with gemcitabine/nab-paclitaxel (n=18), gemcitabine (n=7), or irinotecan (n=3). The most frequently mutated genes among PDAC included KRAS (n=22 pts), TP53 (n=21), CDKN2A (n=12), SMAD4 (n=4), CDKN2B (n=4), MTAP (n=3), ATM (n=3), AKT2 (n=2), and ARID1A (n=2). Of these 32 pts, 25 pts were evaluable for response: 2 pts had CR, 2 PR, 9 SD, and 12 PD. Among the pts with CR, one had tumor without pathogenic variants, the other displayed TP53 and KRAS mutations. Pts with PR: one tumor had no pathogenic mutations, the other tumor harbored mutations in ARID1A, TP53, FGF14, and ROS1. Of the nine patients with SD, 8 had KRAS mutations, 5 had CDKN2A loss of function, and 4 inactivating mutations in SMAD4. Eight out of 12 pts with PD had KRAS mutations. There were no significant differences between pts who had disease control and those with PD in the frequency of KRAS (χ2= 0.0189, P=0.89), TP53 (χ 2= 0.0712, P=0.78), or SMAD4 (χ 2= 1.9631, P= 0.1611). Conclusions: 9-ING-41 has shown clinical benefit in patients with PDAC independent of tumor somatic mutational profile. Preliminary analysis does not reveal pathogenic mutations that are associated with clinical benefit. Additional biomarker studies are ongoing. Citation Format: Brittany A. Borden, Andre De Souza, Devalingam Mahalingam, Steven Powell, Pamela N. Munster, Kelsey Huntington, Andrew Mazar, Ludmila Calvacante, Francis J. Giles, Wafik S. El-Deiry, Howard P. Safran, Benedito A. Carneiro. Genomic biomarkers for response to 9-ING-41, a small molecule selective glycogen synthase kinase-3 (GSK-3) inhibitor, in pancreas cancer: Preliminary results [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P021.

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G1 phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290–8. ©2017 AACR.

Circulating tumor cells (CTC) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of patients with cancer. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared with the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of patients with breast and pancreatic cancer, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. Mol Cancer Ther; 14(3); 835–43. ©2015 AACR.

ONC201, the founding member of the imipridone class of small molecules, has broad spectrum anti-tumor activity and achieved tumor regressions and prolonged disease stability in clinical trials, especially in patients with histone H3 K27M mutated glioma. Levels of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3) are reduced globally in H3 K27M patient samples. EZH2 is the catalytic component of the Polycomb Repressor Complex 2 (PRC2), a conserved multi-subunit that represses gene transcription by methylating lysine 27 on Histone H3 (H3K27). EZH2 is frequently overexpressed or mutated (activating) in numerous human malignancies and EZH2 inhibitors represent a promising therapeutic strategy. We hypothesized that cancer cells could be sensitized to ONC201 by EZH2 inhibition through suppression of H3K27 methylation that may mimic tumors such as DIPG with H3K27M mutations which are known to respond to ONC201 in the clinic and are defective in H3K27 methylation. 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. Cell viability was determined with the Cell Titer-Glo assay. 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. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response and apoptosis in combination with EZH2 inhibitor EPZ-6438. There was greater induction of ATF4 in combination therapy-treated tumor cells versus monotherapy. Intriguingly, U-251 GBM cells that were resistant to ONC201 under hypoxia could be sensitized to ONC201 by EZH2 inhibitor EPZ-6438. Our findings provide a preclinical rationale for developing ONC201 and EZH2 inhibitors as a novel synergistic combinatorial regimen with the potential for clinical translation in the treatment of several human malignancies including GBM, pancreatic cancer, colorectal cancer, and breast cancer. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Robyn Borsuk, Howard P. Safran, Attila A. Seyhan, Wafik S. El-Deiry. Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C10. doi:10.1158/1535-7163.TARG-19-LB-C10

Background: GSK3β, a serine/threonine kinase, has been implicated in the pathogenesis of many cancers. It can act both as a tumor suppressor and a proto-oncogene. Overexpression of GSK3β promotes tumor growth and chemotherapy resistance. Genomic alterations in GSK3β have been identified in many cancer types but no comprehensive analysis is available. Characterization of both the landscape and functional implications of GSK3β alterations will be critical to guide patient selection for treatment with GSK3β inhibitors and related combinations in clinical development, including 9-ING-41, a first-in-class, small molecule potent selective GSK3β inhibitor currently in phase 1/2 clinical trial (NCT03678883). Methods: Publicly-available tumor genomic data was accessed from cBioPortal. Data from 159 studies, curated to exclude duplicate tumor samples, was analyzed. Tumor samples with a GSK3β genomic alteration, including mutations or copy number variations (CNV), were included for analysis. For each tumor, histology, GSK3β protein change or type of CNV, and predicted functional impact of mutation were obtained. Results: 42,199 tumor samples were assessed for GSK3β genomic alterations that were identified in approximately 1% of the specimens (n=414). GSK3β mutations, fusions, frameshift insertions/deletions, and splice site mutations, were present in 211 (0.5%) tumors. Missense mutations were most prevalent (67%). Of 117 unique missense mutations, 68% were predicted to be deleterious by SIFT criteria, while 66% were predicted to be at least possibly damaging by Polyphen-2. Of all mutations, 58% were found in the GSK3β kinase domain. Of all 211 tumors, 10 (5%) had mutations in one of three key GSK3β residues that form a binding pocket for the phosphate group of a primed substrate (R96, R180, and K205). R96 and R180 were found to be of the most commonly mutated residues in this dataset. Cancers most frequently containing a GSK3β mutation were uterine endometrioid carcinoma (n=30, 5% of all cBioPortal uterine endometrioid carcinoma samples included in 42,199 analyzed tumors), melanoma (n=24, 2%) colorectal adenocarcinoma (n=30, 1%), and lung adenocarcinoma (n=19, 1%). GSK3β gene fusions were identified in 17 cases with prostate cancer being the most frequent cancer type (53%). GSK3β CNV, including amplification and homozygous deletion, were found in 217 (0.5%) tumors. Cancers most frequently containing GSK3β CNV were cervical squamous cell carcinoma (n=15, 6%), lung squamous cell carcinoma (n=36, 5%), head and neck squamous cell carcinoma (n=15, 3%), serous ovarian cancer (n=15, 3%), prostate adenocarcinoma (n=40, 2%), and breast invasive ductal carcinoma (n=18, 1%). Conclusions: GSK3β genomic alterations are present in a subset of several malignancies (1-6%). Commonly mutated residues include those that form the binding pocket for the phosphate group of a primed GSK3β substrate, and the majority of GSK3β mutations occur in the kinase domain. The most commonly mutated histologies are distinct from those with GSK3β CNV. Ongoing additional analyses include assessment of the GSK3β genomic landscape in an expanded cohort and functional and pathway analyses to investigate potential biomarkers for treatment response or resistance to selective GSK3β inhibitor 9-ING-41. Citation Format: Brittany A. Borden, Lorin Crawford, Howard P. Safran, Francis J. Giles, Wafik S. El-Deiry, Benedito A. Carneiro. Assessment of glycogen synthase kinase-3 beta (GSK3β) genomic landscape to inform use of cancer therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B092. doi:10.1158/1535-7163.TARG-19-B092

Pancreatic cancer is almost invariably fatal, often diagnosed in advanced stage and carrying a five-year survival rate of less than 5%. Cell-intrinsic oncogenic signaling in a supportive microenvironment renders neoplastic cells resistant to standard chemotherapy or immunotherapy, raising the urgent need for novel agents. ONC212, a fluorinated imipridone, is cytotoxic at nM doses against a variety of cancer preclinical models. ONC212 engages the seven-passage transmembrane receptor GPR132, which is considered a pH sensor and one of the lactic acid receptors. We previously demonstrated that ONC212 inhibits growth of several pancreatic cancer xenografts, some of which underwent apoptosis. We hypothesized that GPR132 modulates ONC212 anti-tumor response in the acidic tumor microenvironment in pancreatic cancer. We studied the growth inhibitory effects of ONC212 in the presence of lactic acid, an essential metabolite within the pancreatic tumor microenvironment resulting from the Warburg effect. We investigated a panel of human pancreatic cancer cell lines (AsPC1, HPAFII, BxPC3, CAPAN2 and PANC1) and two normal fibroblast cell lines (MRC-5 and WI-38) in vitro by CellTiter-Glo assays to assess cell viability and western blots to assess intracellular signaling upon ONC212 treatment. ONC212 inhibited cancer cell growth with a GI50 ranging from 0.09 to 0.47 μM, whereas fibroblasts were resistant. PKC became transiently phosphorylated, as would be expected in response to GPR132 engagement, by 15-30 minutes after ONC212 exposure of AsPC1 cells. Pro-growth and survival Akt and ERK phosphorylation, constitutively detected in most pancreatic cancer cells, was suppressed by 1 hour after ONC212 treatment and this was also observed at 48 hours. After 48 hours, we observed PARP cleavage in the most sensitive pancreatic cancer cell lines such as AsPC1 and HPAFII, whereas the anti-apoptotic protein Mcl-1 decreased in all the cell lines treated with ONC212. Interestingly, LC3II and Beclin 1, two well-recognized autophagic markers, were suppressed by ONC212 in cell lines undergoing apoptosis but not in the others, pointing to autophagy as a possible escape mechanism from ONC212. GPR132 was constitutively expressed in pancreatic cancer cells and decreased upon 48-hour ONC212 treatment. Importantly, GPR132 knockdown reduced cell survival while synergizing with ONC212 in pancreatic cancer cells, suggesting its cytotoxic effect may be mediated by engagement and subsequent abrogation of pro-survival GPR132 signaling. The combination of ONC212 and lactic acid, sharing GPR132 as surface receptor, resulted in strong synergism in inhibiting cancer cell growth. This was evident in fibroblast cell lines too, hinting that stromal tumor-supportive cells residing in cancer acidic microenvironment may be inhibited by treatment. ONC212 shows pre-clinical activity against pancreatic cancer through apoptosis induction and autophagy blockade. Suppression of GPR132 may contribute to ONC212 cytotoxicity. ONC212 is a novel therapeutic active against the acidic tumor microenvironment of pancreatic cancer through suppression of pH sensor and pro-survival GPR132. Citation Format: Isacco Ferrarini, Aakash Jhaveri, Young Lee, Howard Safran, Lanlan Zhou, Wafik S El-Deiry. Imipridone ONC212 induces apoptosis, suppresses autophagy and synergizes upon GRP132 knockdown or exposure to lactic acid in preclinical studies of the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C08. doi:10.1158/1535-7163.TARG-19-LB-C08

Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLM). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or nonmalignant cell lines at submicromolar concentrations. SLM6 was the most active compound in vivo, where it was well tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (MAF, CCND1, MYC, and others). Furthermore, SLM6 showed superior in vivo anti-MM activity more than the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings show that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent. Mol Cancer Ther; 11(11); 2321–30. ©2012 AACR.

Background: ONC201, an imipridone that is a selective antagonist of the G protein-coupled receptors dopamine receptor D2 (DRD2) and D3 (DRD3), has exhibited biologic activity and an exceptional safety profile in a phase II study in bevacizumab-naïve recurrent glioblastoma (Arrillaga et a.l, 2017). Single-agent ONC201 efficacy has been observed in preclinical glioblastoma models in addition to robust penetrance of the blood-brain barrier (Allen et al., 2013). DRD2 antagonism induces tumor cell apoptosis via the same signaling pathways affected by ONC201. In addition, DRD2 is expressed on NK and other immune cells and DRD2 antagonism can induce their activation. Methods: Cell viability assays were performed with ONC201 in >1000 Genomic of Drug Sensitivity in Cancer (GDSC) cell lines and NCI60. Immunohistochemistry staining of DRD2/DRD5 was performed in glioblastoma tissue microarrays and archival tumor tissues. Whole exome sequencing was performed in RKO cells with acquired resistance to ONC201. DRD5 wild-type and mutant constructs were generated for overexpression studies. ELISA was used to quantitate serum prolactin and immune effector (perforin) levels. Intratumoral drug concentrations were evaluated by LC-MS assays conducted on glioblastoma tissue resected from patients following the second dose of 625mg ONC201. Results: Evaluation of ONC201 in GDSC cell lines confirmed broad anticancer efficacy with high sensitivity (~1-3 µM) in human brain cancer. The Cancer Genome Atlas (TCGA) revealed that DRD2 is highly expressed in glioblastoma relative to other dopamine receptors and that genetic aberrations are rare. High expression of DRD2 occurred in primary, rather than secondary, glioblastoma and was associated with a poor prognosis. Immunohistochemistry of tissue microarrays revealed DRD2 overexpression in glioblastoma relative to normal brain. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among glioblastoma cell lines in the NCI60 panel. Interestingly, expression of DRD5, a D1-like dopamine receptor that counteracts DRD2 signaling, was significantly inversely correlated with ONC201 potency in the NCI60 and GDSC datasets (P 5 month had no detectable expression of DRD5, unlike those with PFS Citation Format: Varun Vijay Prabhu, Neel Madhukar, C. Leah B. Kline, Rohinton Tarapore, Wafik El-Deiry, Olivier Elemento, Faye Doherty, Alexander VanEngelenburg, Jessica Durrant, Andrew Zloza, Cyril Benes, Isabel Arrillaga, Wolfgang Oster, Joshua E. Allen. Targeting DRD2 dysregulation in recurrent glioblastoma with imipridone ONC201: predictive and pharmacodynamic clinical biomarker analyses [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 A060.

TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G2, showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087–95. ©2012 AACR . This article is featured in Highlights of This Issue, [p. 2075][1] [1]: /lookup/volpage/11/2075?iss=10

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in conjunction with microtubule-targeting agents may be a promising novel anticancer treatment strategy. In vitro studies have suggested that relatively low concentrations of TRAIL enhance the lethality of paclitaxel (Taxol) against human cancer cells. The increased efficacy may be due to the triggering of caspase activation, resulting in mitotic checkpoint abrogation and catastrophe. We show here that wild-type p53 protects cells from caspase-dependent death induced by this therapeutic combination in vitro. We have now also developed an imaging-based model system to test the in vivo efficacy of combined TRAIL and Taxol, in which tumor growth and treatment response can be monitored noninvasively and in real-time. We further utilize bioluminescence, F18-fluorodeoxyglucose-positron emission tomography, and microscale computed tomography imaging to confirm the effects of combined treatment on tumors. These studies together provide the first in vivo confirmation that combined TRAIL plus paclitaxel results in better tumor control compared with either TRAIL or paclitaxel alone, and with no discernable increased normal tissue toxicity in the mouse. Interestingly, the in vivo antitumor response elicited by combined treatment was not affected by the p53 status of the tumor cells. These preclinical observations together suggest the therapeutic potential of combining TRAIL plus paclitaxel in cancer treatment, and support further preclinical and future clinical testing. [Mol Cancer Ther 2009;8(12):3285–95]

Breast cancer is a major cause of cancer related death in United States women, and there is an urgent need for novel targeted therapies with low toxicity and increased efficacy. The TRAIL pathway has been of interest in the field of oncology, but resistance has been identified in cancer cell lines and primary tumors including those of the breast. Only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL, with other breast cancer molecular subtypes being resistant (Rahman et al., Breast Cancer Res. Treat., 2008). Clinical translation of these findings has been limited, as TRAIL receptor agonistic antibodies have shown minimal effects. We identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10 (Allen et al., Sci. Trans. Med., 2013) that functions through dual inhibition of AKT/ERK, induction of the ATF4/CHOP pathway (Kline et al., Submitted, 2015) and cytotoxic effects dependent on TRAIL/DR5 upregulation. Recently, ONC201 completed its first-in-man phase 1 clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-EORTC meeting). The objective of this work was to investigate ONC201 efficacy in TNBC (TRAIL-sensitive) and non-TNBC (TRAIL-resistant) cells. Using cell viability assays, we demonstrate IC50 values for ONC201 in the low micromolar range for both TNBC (n = 6) and non-TNBC cells (n = 5). These doses are achievable based on human PK. The evaluated non-TNBC cell lines, including those with estrogen receptor positivity and HER2 amplifications, have been shown to be highly resistant to TRAIL therapy. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in both TNBC and non-TNBC cells. We also show that a single dose of ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 triple negative human breast cancer xenograft model. The effects of ONC201 on well-known mechanisms of TRAIL-resistance, such as increases in inhibitor of apoptosis (IAP) family proteins, were investigated to explain sensitivity of TRAIL-resistant breast cancer cells to ONC201. We observed that ONC201 mediates a decrease in expression of IAP family proteins XIAP, c-IAP1, and c-IAP2 across TNBC and non-TNBC cell lines. We have shown that XIAP levels correlate with sensitivity to ONC201-induced apoptosis (Kline et al., Submitted, 2015). We also examined levels of death receptors DR4 and DR5 using flow cytometry, as a known mechanism of TRAIL resistance involves constitutive death receptor endocytosis from the cell surface (Zhang et al., Mol. Cancer Res., 2008). Interestingly, increases in cell surface DR4 and DR5 levels are greater in TNBC cell lines when compared with non-TNBC cell lines. Overall, our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC and non-TNBC subtypes. These effects are observed regardless of the TRAIL sensitivity of the cells, and may be mediated through mechanisms involving downregulation of anti-apoptotic proteins and upregulation of cell surface death receptors. Our work will further understanding of these mechanisms and contribute to development of a preclinical rationale for the use of ONC201 as a treatment for breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Christina L.B. Kline, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A16.

We previously reported the anti-tumor and anti-angiogenic activity of a new class of sangivamycin-like molecules (SLMs). In this study we aimed to further the preclinical development of these compounds by identifying tumor types with the highest sensitivity to their cytotoxic effects. Of 35 tumor cell lines from 8 different tissues of origin that we screened, we found that multiple myeloma (MM) cells were approximately 3–10 times more sensitive to SLMs than other tumor types. SLM3 as well as SLM5, SLM6, and SLM7 reduced viability and induced apoptosis of MM cells at sub-micromolar concentrations in vitro. By contrast, SLMs induced negligible amounts of apoptosis in other tumor and normal cell lines. SLM6 exhibited the most potent activity in vivo where it significantly inhibited the growth of xenografted MM tumors. We found that SLM6 inhibited cyclin dependent kinase 9 (CDK9) resulting in hypophosphorylation of RNA polymerase II (Ser2) and transcriptional repression of oncogenes that are known to drive MM progression and resistance to therapy (c-Maf, cyclin D1, c-Myc, L-Myc, and Mcl-1). Furthermore, we found that SLM6 increased the cytotoxic activity of therapies that are currently approved for MM treatment (bortezomib, lenalidomide, dexamethasone, and doxorubicin). These findings suggest that CDK9 inhibition is a particularly effective targeted strategy for MM, and SLM6 is a promising CDK9-targeted agent, both as a single agent and in combination with currently approved therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C77.