Search

ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

ONC201, the founding member of the imipridone class of anti-cancer compounds, is a highly selective small molecule GPCR antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated the anti-cancer effects of ONC212, an ONC201 analogue that possess the same unique core chemical structure shared by imipridones. The in vitro efficacy of ONC212 was assessed in the Genomics of Drug Sensitivity in Cancer collection of cell lines (>1,000 cell lines). Cell viability assays were performed to generate dose responses curves at concentrations from 78nM upto 20uM and at 72 hours post-treatment. ONC212 was broadly efficacious across most solid tumors and hematological malignancies in the low nanomolar range. Ranking the ONC212 sensitivity dataset revealed that leukemia is the most responsive tumor type based on completeness of response (area under the dose-response curve, AUC). ONC212 was tested in 65 leukemia cell lines in this study that is comprised of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and hairy cell leukemia. ONC212 demonstrated broad spectrum anti-leukemic activity and was equally efficacious across all leukemia subtypes tested, in terms of AUC. Most cell lines (63/65) were responsive to ONC212 with GI50 ranging from Thus, ONC212 possesses robust anti-leukemic activity irrespective of subtype and provides further validation of the anti-cancer efficacy of the novel imipridone class of small molecules. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership. Stogniew:Oncoceutics Inc.: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership.

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.

A newly cloned gene named wild-type p53-activated fragment 1 (WAF1; also known as p21, Pic-1, Cip-1, or SDI1) is directly regulated by p53 and can itself suppress tumor cell growth in culture. Induction of expression of WAF1 may be an important means by which cells with DNA injury arrest their growth to repair DNA or undergo apoptosis. Based on the hypothesis that mutations of this gene may play a role in carcinogenesis, we have studied 351 DNAs from 14 kinds of malignancies, as well as 36 human transformed cell lines, for alterations of WAF1 gene by single-strand conformation polymorphism analysis of polymerase chain reaction amplification of the DNA coding region of the WAF1 gene. No abnormal band shifts of WAF1 were noted in any of the samples or cell lines, but three major variants in exons 2 and 3 of the gene were found that are consistent with the existence of two different DNA polymorphisms. Sequence analysis of the amplified products producing these three variants in each exon from normal DNAs confirmed the presence of the polymorphisms in the WAF1 gene. Of 290 selected tumor samples previously evaluated for p53 mutations by single-strand conformation polymorphism, 90% had no detectable p53 alterations. In summary, mutations within the coding portion of the WAF1 gene were undetectable in a large series of human tumors, many of which had a normal p53 gene. This suggests that WAF1 alterations are generally caused indirectly, through p53 mutations rather than through intragenic mutation of the WAF1 itself.

Background: Diffuse Large B cell lymphoma, the most commonly diagnosed type of non-Hodgkin lymphoma is curable in many cases, despite this, up to 30% of patients will relapse after initial therapy, necessitating salvage chemotherapy and transplantation if feasible. There is a pressing need for novel treatment strategies in highly chemo refractory cases. ONC201 is a small molecule that induces p53-independent cell death in tumor cells while sparing normal cells through a number of putative mechanisms, including inactivation of the pro-survival kinases Akt and ERK. ONC201 is currently entering Phase I/II clinical trials as a monoagent in adult advanced cancers. ONC201 promotes up-regulation of TRAIL gene transcription by inactivating AKT and ERK kinases which leads to translocation of transcription factor Foxo3a into the nucleus. It appears to act on a p53 independent pathway. ABT-199 is a selective, potent and orally bioavailable small molecule that selectively inhibits BCL-2 and triggers apoptosis. The Bcl-2 family of proteins is key regulator of the apoptotic process, comprising proapoptotic and prosurvival proteins. BH3-only proteins (such as Bim) bind to pro-survival proteins and cause increased permeability of mitochondrial membrane, release of cytochrome c and activation of caspases through release of Bax and others. Methods: Cell lines Pfeiffer and Toledo were purchased from ATCC and one patient DLBCL cell specimen from the ascitic fluid was cultured for use in experiments. ABT-199 was purchased from MedKoo Biosciences and ONC201 was provided from Oncoceutics. Cytotoxicity was evaluated by using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturerÕs instructions. Cell viability was measured over time in response to treatment with ONC201(1-64μM) and ABT-199(128 nM-4μM). Western Blotting was performed on treated cells with well-established methodologies, with antibodies to c-Myc, Bcl-2, pAKT, pERK. Results: Immunohistochemical Staining Pattern of Cell Lines Table 1. Patient Toledo Pfeiffer Bcl-2 +++ ++ + Bcl-6 + ++ ++ c-Myc +++ ++ + p-ERK + + ++ p-AKT + ++ + Bax ++ NA NA Bim ++ NA NA Mean IC50 Calculated for Cell Lines Table 2. Cell Line Therapeutic Agent ABT199 ONC201 Patient Sample 8 μM 5 nM Toledo 9 μM 28 nM Pfeiffer 6 μM 2 μM SHAPE Conclusion/Discussion: The patient cell line, an ascitic fluid sample of DLBCL was sensitive to both ONC201 and ABT-199 and manifested bright Bcl-2 expression, the target of ABT-199. In this series there was a higher sensitivity to ABT199 in DLBCL cells with higher Bcl-2 expression. ONC201 down regulated pAKT expression, as seen in Western Blots in treated cells, consistent with prior investigation with the agent. We further found that ONC201 synergizes to potentiate cytotoxicity with ABT199, as demonstrated in the cell viability assay for Toledo cell lines (at the 24 hour time point), which were the least sensitive to ONC201 (highest IC50) when given as a single agent. Yet, when combined with increasing doses of ABT199, there was synergistic lymphoma cell kill with a fixed dose of ONC201. Together these results suggest that ONC201 has potential as an antitumor agent in NHL as monoagent and in combination with ABT-199, which may be further explored in phase Ib/II trials. Further analysis in larger patient sample series may elucidate the biomarkers that predict for greater therapeutic sensitivity to these highly potent lymphoma agents. Figure 1. Figure 1. Disclosures Allen: Oncoceutics, Inc: Employment, Equity Ownership. Eldeiry:Oncoceutics, Inc: Equity Ownership.

ONC201 is a small molecule that is being developed as a novel anticancer drug based on its compelling antitumor activity (Allen et al, Sci Transl Med, 2013). ONC201 possesses a mutation-agnostic efficacy profile that is not impaired by oncogenic mutations that commonly drive disease progression and therapeutic resistance. Given the strong apoptotic potential of ONC201 in a variety of human malignancies, there was a potential concern for normal cell toxicity based on historical experience with potent cytotoxic agents. To establish the safety profile of ONC201, a series of studies was conducted with ONC201 in normal human cells at efficacious and exaggerated doses. With respect to cell viability, dose response relationships between normal human fibroblasts and tumor cell lines revealed a similar inflection point at ~2-5 µM that subsequently saturated in all tested cell lines. Interestingly, saturation of the effects occurred at a much higher level of viability reduction in tumor cells as compared to normal cells. Further investigation revealed that ONC201 does not cause any appreciable levels of cell death in normal cells, which are drastically increased in tumor cells, and that the small effect on normal cell viability is transient and reversible. Studying downstream signaling effects in tumor vs normal cells revealed that ONC201 did not induce DR5 in normal cells under conditions that induced DR5 in tumor cells. This differential DR5 induction is explained by recent mechanistic findings with ONC201 that implicate the ER stress response in its antitumor mechanism, which activates CHOP that positively regulates the human DR5 gene as part of the maladaptive response (Ishizawa J et al, ASH 2014). Thus ONC201 induces a strong apoptotic response in tumor cells at doses that do not affect normal cells, which is reflective of an attractive therapeutic window. The lack of cytotoxicity in normal cells was also confirmed in a panel of normal human bone marrow specimens, including examination of normal progenitor cells. The absence of bone marrow toxicity despite extremely high apoptotic activity in refractory primary human lymphoma samples is a unique and compelling feature of ONC201 compared to available clinical agents in this setting. The genotoxic potential of ONC201 was also assessed in normal and tumor human cell lines. Unlike chemotherapy positive controls, ONC201 did not induce gamma-H2AX, the genotoxic stress marker, in tumor or normal cell lines. The lack of genotoxicity in addition to absence of normal cell toxicity exhibited by ONC201 in preclinical studies enables a range of potential therapeutic settings for application, such as pediatric indications as well as adjuvant and neoadjuvant settings. To enable translation and confirm the safety profile of ONC201, GLP toxicology studies were conducted in Sprague Dawley rats and beagle dogs with single oral doses of ONC201. Dogs received a single dose of 0, 4.2, 42, or 120 mg/kg by oral gavage, which represents a human equivalent of 0, 125 mg, 1.25 g, and 3.57 g, respectively. Rats received 0, 12.5, 125, or 225 mg/kg ONC201 by oral gavage, which represents a human equivalent of 0, 125 mg, 1.25 g, and 2.25 g, respectively. There were no deaths or dose-limiting toxicities. The only findings that were observed in both rats and dogs occurred at the highest doses tested and were mild and reversible: decreased activity (no indications of anemia), decreased food consumption (weight loss only seen in rats), and salivation. The NOAEL was at least 42 mg/kg in dogs and at least 125 mg/kg in rats. Based on regulatory guidance documents, the starting dose in human clinical trials will be a 125 mg oral dose given once every three weeks, which represents a starting efficacious dose based on preclinical tumor xenograft studies in mice. The strong cytotoxic potential of ONC201 in human cancer cells without compromising safety bodes well for its scalability into clinical settings where traditional therapies are too toxic to be implemented. Disclosures Allen: Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics: Equity Ownership, Patents & Royalties. Andreeff:Oncoceutics: Equity Ownership.

ONC201/TIC10 is a potent small molecule anti-tumor agent in several types of solid tumors and lymphomas. ONC201/TIC10 is on track to enter clinical trials for patients with advanced cancer in 2014, with IND issued by the FDA in March, 2014. Early trials will evaluate the safety and efficacy of ONC201/TIC10 as a monoagent in hematological malignancies. In the current study, we evaluated the anti-cancer effects of the small molecule in Acute Lymphoblastic Leukemia (ALL). Analysis of cell viability by the CellTiter-Glo method revealed that ONC201/TIC10 treatment reduces the viability of three ALL cell lines (Reh, Jurkat, MOLT-4) in a dose- (2.5/5/10 μM) and time-dependent manner (24/48/72 h). We have previously reported that ONC201/TIC10-mediated reduction in cell viability and apoptosis in various types of solid tumors occurs at 60/72 h. Interestingly, ONC201/TIC10 reduces the viability of ALL cell lines within 24/48 h at the indicated doses. An inactive TIC10 isomer compound synthesized by Medkoo Biosciences with a structure related to the active ONC201/TIC10 compound does not reduce the viability of ALL cells. Sub-G1 analysis indicated that ONC201/TIC10 induces apoptosis in ALL cells and a pan-caspase inhibitor reduces ONC201/TIC10-mediated apoptosis. Western blot analysis was used to further investigate the mechanism of ONC201/TIC10-mediated apoptosis. ONC201/TIC10-mediated apoptosis involves PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl are downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201/TIC10 treatment. ONC201/TIC10 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2. We have previously shown that the anti-tumor effect of ONC201/TIC10 involves inhibition Akt and ERK phosphorylation resulting in Foxo3a activation and TRAIL-gene transcription. We observed inhibition of Akt phosphorylation upon ONC201/TIC10 treatment of ALL cells. Thus, ONC201/TIC10 holds promise as a novel agent for the treatment of ALL based on its robust activity in preclinical models of the disease. Our ongoing studies are evaluating the impact of this novel therapy on ALL cells with different translocations, and are introducing combination therapy with ONC201/TIC10 for ALL. Figure 1 Figure 1. Disclosures Allen: Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

The antitumor protein recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer therapy although it has notable shortcomings that have limited its clinical development. Our lab previously identified a novel use for TRAIL-inducing compound 10 (ONC201/TIC10) as an efficacious antitumor therapeutic that induces TRAIL pathway activation through dual blockade of Akt and ERK that releases Foxo3a to transcriptionally activate the TRAIL and its death receptor DR5 genes. ONC201/TIC10 is being developed by Oncoceutics for the clinical therapy of cancer. Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with an increasing rate of refractory/relapsed population of patients, making it a challenging disease to treat. Therefore, new therapeutic options as single dose or combinational treatments are required. Therapeutic agonist TRAIL antibodies have been investigated as a therapy for solid tumors; however, the biologic agents have not been explored extensively as a potential single or combinational therapy for the treatment of ALL. The aim of this study was to begin to characterize ONC201/TIC10 as a single agent for ALL therapy and in combination with bortezomib to increase induction of tumor cell death. Single agent ONC201/TIC10 decreased ALL cell viability at IC50 values ranging from 2.5-5 µM after a 48 hour treatment. ONC201/TIC10 was combined with various accepted clinical treatments of ALL to determine if a synergistic or additive effect exists. Treatments of compounds at doses below their IC50, including the proteasome inhibitor bortezomib and monoclonal antibody-based rituximab therapy, showed synergistic or additive anti-leukemic effects when treated mutually with ONC201/TIC10 in ALL and T-cell ALL. TRAIL and Foxo3a expression was monitored by western blot analysis and apoptosis was indicated by both subG1 and caspase activity to investigate the effect of these combination treatments. Bortezomib-ONC201/TIC10 combinational treatments decreased cell viability by approximately 30% in MALT-4 cells and 20% in Jurkat compared to single dose bortezomib treatments. Rituximab and the steroid compound dexamethasone were administered both below their IC50 in combination with ONC201/TIC10 and exhibited 20-40% cell viability decrease in ALL in vitro beyond the monoagent effects. Pharmacological enhancement by the addition of ONC201/TIC10 increased apoptotic efficacy and significantly decreased cell viability during combination treatments as compared to single agent therapy. The formation of the TRAIL death-inducing signaling complex could provide a rationale for further investigation into ONC201/TIC10 combinational treatment in ALL. These preclinical results suggest that there may be a clinical therapeutic advantage to modifying current ALL patient therapies to include the addition of ONC201/TIC10. Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

Quinacrine is a bioactive acridine derivative which has been used for treatment of malaria, giardiasis, systemic lupus erythematosus, and rheumatoid arthritis. In searching for p53 pathway activating agents for cancer therapy, we found that quinacrine stabilizes p53 and induces p53-dependent and p53-independent tumor cell death. Quinacrine also induces expression of TRAIL Death Receptor 5 (DR5) and reduces expression of anti-apoptotic Mcl-1 in tumor cells. These activities predict synergies with TRAIL (tumor necrosis factor-related apoptosis inducing ligand) and chemotherapeutic agents in inducing extrinsic and intrinsic pathway mediated apoptosis. In addition, quinacrine suppresses NFkB activity in tumor cells. Clinical trials have been ongoing for treatment of solid tumors including colon cancer, renal cancer, prostate cancer, and non-small cell lung cancer with quinacrine in combination with chemotherapy or tyrosine-kinase inhibitors, however, the therapeutic potential of quinacrine in blood cancer cells has not been established. We tested quinacrine on hematopoietic malignant cells, which included cell lines of myeloid leukemia, lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and multiple myeloma. We found that quinacrine induces massive cell death in the cell lines tested, at concentrations from as less as 1 microM to 5 microM, 2-10 times lower than required to induce solid tumor cell death. Quinacrine synergizes with TRAIL in inducing cell death of TRAIL-sensitive cells and reverses resistance in TRAIL-resistant cells. Quinacrine also synergizes with chemotherapeutic agents, such as antimetabolites, alkylating agents, and tyrosine kinase inhibitors, in inducing apoptosis of hematopoietic cancer cell lines. Our work supports translational efforts to advance the use of quinacrine from bench to clinic and provides rationale for combination chemotherapeutic regimes for treatment of hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

PI3K/Akt and Ras/MAPK pathways are attractive therapeutic targets in almost all tumor types, including AML and MM. Apo2L/TRAIL has been deemed a promising therapeutic given its selectivity towards cancer cells although its clinical development has been hampered by various limitations including short half-life and general shortcoming of protein-based therapeutics. ONC201/TIC10 (Oncoceutics, Inc.) is a first-in-class small molecule inducer of TRAIL expression. ONC201/TIC10 has previously been shown to up-regulate TRAIL and its death inducing receptor DR5 in HCT116 colon cancer cells, in part through the inhibition of Foxo3a phosphorylation mediated by dual inhibition of Akt and ERK (Allen JE et al, Sci Transl Med., 2013). Currently, ONC201/TIC10 is set to enter clinical trials for patients with advanced malignancies after the IND was approved by the FDA in March, 2014. We thus investigated the therapeutic potential of ONC201/TIC10 in AML and MM given a major unmet need when conventional therapy fails. We explored the possibility that ONC201/TIC10 induces apoptosis in MM and AML in part through dual inhibition of the PI3K/Akt and Ras/MAPK pathways. We tested a panel of four human MM cell lines (KMS18, MM.1S, MM.1S 33X and RPMI-8226) and three human AML cell lines (Kasumi-1, HL60, HL60/VCR). The Cell-titer Glo assay demonstrated a time and dose-dependent decrease in viability in the entire panel of MM and AML cells. EC50 values ranged from 1-2.5 µM for the MM and 2-5µM for the AML cell lines, respectively. Bortezomib-resistant cells MM.1S 33X and vincristine- resistant cells HL60/VCR were also significantly sensitive to ONC201/ TIC10 as a single agent with EC50s on par with the corresponding parental cell lines. Given the previously reported pro-apoptotic effects of ONC201/TIC10 against solid tumor cells, we assessed apoptosis by performing Sub-G1 analyses and assessing caspase-3 cleavage as two widely used methods to analyze apoptotic cell death. We observed an average of 10-fold induction of ONC201/TIC10–mediated apoptosis in MM cells at 5 mM at 48 hrs post-treatment. Rescue of ONC201/TIC10-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Dose and time dependent induction of apoptosis was noted in western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201/TIC10 for 48 hrs prior to analysis. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously proposed mechanism of ONC201/TIC10 against solid tumor cells. To further investigate the therapeutic potential of ONC201/TIC10 in the context of AML, fresh AML cells were treated with ONC201/TIC10. The primary cells were also found to be sensitive to ONC201/TIC10 (60% decrease in cell viability 72 hrs post 5mM ONC201/TIC10 treatment). Similarly, caspase 3/7 activity was significantly increased as assessed by the Caspase Glo 3/7 assay (~5 fold induction in activity 72 hrs post 5mM ONC201/TIC10 treatment). To explore further the therapeutic potential of ONC201/TIC10, we performed combinatorial experiments with bortezomib and vincristine using the MM.1S 33X MM cells and the HL60/VCR AML cell lines. ONC201/ TIC10 showed an additive effect with both these compounds against the MM and AML lines. Our work demonstrates activity of ONC201/TIC10 against AML and MM cell lines including fresh AML tumor cells. The efficacy data with resistant cells is in par with the applicability of TIC10 in patients with refractory/relapsed hematological malignancies. The long-term goal of this project is to provide a rationale for a phase 1b trial of ONC201/TIC10 for refractory/relapsed MM and AML in combination with existing therapies. Figure 1: Efficacy of ONC201/TIC10 in AML and MM cells Figure 1:. Efficacy of ONC201/TIC10 in AML and MM cells Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

Outcome for Non-Hodgkin lymphoma (NHL) patients using conventional treatment standards remains unsatisfactory, particularly in advanced stage/ relapsed disease creating an imminent need for investigating novel treatment strategies. ONC201/TIC10 is a small molecule (Allen et al, 2014) that induces p53-independent cell death in tumor cells (sparing normal cells) through inactivation of prosurvival kinases Akt and ERK. ONC201 is scheduled to enter Phase I/II clinical trials as a monoagent for advanced cancers in adults in 2014. We have previously shown that ONC201 induces significant cytotoxicity in preclinical models of human lymphomas (Talekar et al, ASPHO 2014). Here, we show that ONC201 is not only effective as a monoagent across several NHL cell lines, but that it also synergizes with several chemotherapeutic agents to cooperatively induce cell death in vitro. We found that ONC201 induces significant apoptosis in a diverse panel of seven human NHL cell lines at low micromolar concentrations (1.3 to 5.06 uM). Increased surface TRAIL and surface DR5 expression was noted in a dose-dependent manner across representative cell lines. The increase in surface TRAIL correlated with increase in subG1 DNA content, which suggests TRAIL as a potential biomarker of ONC201 response. ONC201-induced apoptosis was inhibited using a pan-caspase inhibitor and was blocked by an anti-TRAIL antibody RIK-2. Western blot analysis of ONC201-treated representative cell lines suggests ERK inhibition and Foxo3a activation as a potential mechanism of cytotoxicity via TRAIL induction. In agreement with this notion, we observed upregulation of PARP & DR5 and caspase-3 activation in response to ONC201 treatment. We further found that ONC201 synergizes to potentiate cytotoxicity with several chemotherapeutic agents approved for NHL treatment, particularly anthracyclines (doxorubicin), nitrogen mustard (bendamustine), antimetabolite (cytarabine) and proteasome inhibitor (bortezomib) via CellTiter-Glo cell viability experiments that were corroborated by apoptosis assays. Together these results suggest that ONC201 is a potent antitumor agent in NHL as monoagent and in combination with approved therapies. The ultimate goal of this project is to provide a preclinical rationale for a phase Ib/II trial of ONC201 as a combination therapy in pediatric lymphoma. Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Ownership interests (including stock options) in a start-up company, the stock of which is not publicly traded Other, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Founder and Shareholder in Oncoceutics, Inc. The potential conflict of interest has been disclosed to and is being managed by his academic employer. Other.

ONC201 is a small molecule that is being developed as a novel anticancer drug based on its strong anti-tumor efficacy in preclinical models of advanced cancer (Allen et al, Sci Transl Med, 2013). ONC201 has demonstrated strong antitumor effects in vitro and in vivo in a variety of solid and liquid tumors. Cancer stem cells (CSCs) are known to be responsible for tumor propagation and maintenance as well as driving resistance to many cancer therapies. Based on the potent efficacy of ONC201 in preclinical models, we investigated the therapeutic impact of ONC201 on CSCs to rationalize its strong antitumor effects and realize the extent of its therapeutic potential. In primary refractory AML patient samples, ONC201 induced very high levels of apoptosis in leukemia stem/progenitor cells (CD45 dim+/ CD34+/ CD38-) to a similar extent observed in the bulk tumor population. In addition to this effect in hematological malignancies, ONC201 has also exhibited strong anti-CSC efficacy in solid tumors such as colorectal cancer (CRC) and glioblastoma multiforme (GBM). ONC201 eradicated CSC-enriched 3-dimensional neurosphere culture models of primary GBM samples, including newly diagnosed and recurrent samples. ONC201 potently inhibited in vitro cell proliferation of all 5 neurosphere lines tested, with IC50 values of 433 nM (MGG18), 1.46 µM (MGG4), 1.09 µM (MGG8), 3.97 µM (MGG67R) and 688 nM (MGG152). In CRC, ONC201 significantly depleted CD133, CD44 and Aldefluor positive CSCs in HCT116, DLD1 and SW480 cells. ONC201 significantly inhibited colonosphere formation of unsorted and sorted Aldefluor positive 5-Fluorouracil-resistant DLD1 and SW480 cells. ONC201 significantly reduced tumor growth of CSC-initiated tumors and prevented the passage of these tumors. CD44 and CD133 expression was reduced in ONC201-treated tumors. ONC201-treatment decreased tumor initiation relative to 5-Fluorouracil treatment in a limiting dilution tumor initiation assay. The ONC201-mediated anti-CSC effect was significantly blocked by the TRAIL sequestering antibody RIK-2. ONC201-mediated TRAIL-induction was also observed in CSC-initiated tumors. ONC201 inhibited Akt and ERK leading to Foxo3a activation and surface TRAIL induction in sorted CSCs and non-CSCs. Overexpression of myristoylated Akt significantly reduced the induction of surface TRAIL by ONC201 in CSCs and prevented ONC201-mediated depletion of CSCs and colonosphere inhibition. In conclusion, ONC201 cytotoxicity is extended to CSCs in hematological and solid malignancies as well as the bulk tumor population. The ability of ONC201 to deplete CSCs suggests the clinical potential for ONC201 to reduce the incidence of disease relapse, which is frequently a fatal event in hematological malignancies, in addition to its immediately apparent antitumor effects. Disclosures Allen: Oncoceutics: Employment, Equity Ownership, Patents & Royalties. Andreeff:Oncoceutics: Membership on an entity's Board of Directors or advisory committees. El-Deiry:Oncoceutics: Equity Ownership, Patents & Royalties.

Background The outcome for pediatric Non-Hodgkin Lymphomas (NHL) using conventional treatment standards is still inferior compared to other childhood malignancies, particularly in advanced stage and relapsed disease. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous protein that induces apoptosis selectively in cancer cells by binding to death receptors. Previous TRAIL-targeting strategies have had limited potential due to unfavorable biodistribution and pharmacokinetics. Our laboratory identified a small molecule, ONC201(TIC10), which induces p53 independent TRAIL gene transcription and TRAIL-dependent cell death and overcomes the most critical efficacy-limiting drug properties of available TRAIL-based therapies. Selective advantages of ONC201 include longer half-life, stimulation of TRAIL and death receptor expression, stability, lower production cost, and ability to cross the intact blood-brain barrier. Our lab has reported that ONC201 is orally active, has potent anti-tumor effects in several preclinical animal cancer models including prolonging survival in lymphoma bearing Eu-myc transgenic mice, exhibits synergy with several antitumor agents (Allen JE et al, Sci Transl Med.,2013) and is being developed into a first-in-man study in advanced cancer patients in early 2014. Objective Assess monoagent activity and mechanism of action of ONC201 in pediatric lymphoma. Derive preclinical rationale to evaluate ONC201 as a novel targeted therapy for pediatric lymphoma as a monoagent or in combination in clinical trials. Methods A diverse sub-type panel of human lymphoma cell lines [Ramos, Raji and Daudi (Burkitt's lymphoma); Karpas299 (T-cell NHL); UPN2 and Granta (Mantle Cell lymphoma)] was selected to assess lymphoma sensitivity to ONC201. Luminescent cell viability assay was used to generate inhibition curves at 72 hours post-treatment with ONC201 yielding IC50's. Caspase-based Apoptosis assay was performed on cells treated with low micromolar dose ranges of ONC201 and data generated via flow cytometry at 72 hours post-treatment. Surface TRAIL expression was analyzed via FACS (Fluorescence-activated cell-sorting) at 60 hours post-treatment. Inhibition of ONC201-induced Apoptosis was demonstrated using Pan-caspase inhibitor (Z-VAD-FMK) at 72 hour post-treatment. Western blot analysis of representative cell lines from the three sub-types (Ramos, Karpas299 and UPN2) at 48 hour post-treatment with ONC201 for AKT and ERK was used to validate the mechanism of induction of apoptosis of lymphoma cells by ONC201. Results Cell viability assays demonstrated sharp dose-response curves at 72 hours post-treatment with ONC201. Quantitative analysis of these relationships yielded ONC201 IC50 values in the range of 1.3 to 5.06 uM, which is comparable to reported activity of ONC201 in other tumor types. Sub-G1 analysis by flow cytometry revealed significant levels of apoptosis at 72 hours post-treatment in a dose-dependent manner (Fig.1) with substantial increase in sub-G1 content (range 1.8-fold to 4.15-fold) in comparison with that of untreated cells. Treatment with ONC201 revealed increase in surface TRAIL expression in a dose dependent manner across the representative cell lines. ONC201 induced apoptosis was demonstrably inhibited using Pan-caspase inhibitor. Western blot analysis of representative cell lines from the three sub-types (Ramos, Karpas299 and UPN2) at 48 hour post-treatment with ONC201 confirmed dual inhibition of Akt and ERK as potential mechanism of induction of apoptosis of lymphoma cells by ONC201. Conclusion ONC201 is a first-in-class therapy that has apoptotic activity in human lymphoma cells at low micromolar concentrations and promotes up-regulation of TRAIL. ONC201 is promising as a monoagent and may be enhanced by combination with approved therapies to improve the standard of care for pediatric NHL. We are currently assessing in vitro synergistic tumor cell-killing by standard chemotherapeutic agents used to treat lymphoma in combination with ONC201. We plan to translate our work in vivo using xenograft mice models. The ultimate goal of this project is to provide the preclinical rationale for a potential phase Ib trial of ONC201 as a combination therapy in pediatric lymphoma. Disclosures: Allen: ONCOCEUTICS: Employment; ONCOCEUTICS: Equity Ownership. El-Deiry:ONCOCEUTICS: Equity Ownership.

ONC201 (TIC10) is a novel small molecule that induces TRAIL-dependent apoptosis in various cancer cell types and is under development to enter a first-in-man study in advanced cancer patients .It was identified in a screen for small molecules capable of up-regulating endogenous TRAIL gene transcription in a p53-independent manner (Allen JE et al, Sci Transl Med., 2013). ONC201 triggers FOXO3a activation through dual inhibition of ERK and AKT, which transcriptionally upregulates TRAIL and TNFRSF10B (TRAIL-R2/DR5) in solid tumors. Because PI3K/AKT and MEK/ERK activation have been shown to be major contributors to drug resistance, ONC201 is potentially promising since it not only promotes TRAIL activation, but also upregulates its pro-apoptotic receptor DR5. Here we report the anti-lymphoma effects of ONC201 in MCL, a presently incurable disease. We treated three human MCL cell lines with wild-type p53 (Z-138, JVM-2, and Granta-519) and two similar lines with mutant p53 (MINO and Jeko-1) with ONC201. A 72-hour ONC201 treatment induced apoptosis in all MCL cell lines. Surprisingly, the p53 mutant MINO and Jeko-1 cells were more susceptible in apoptosis assays to ONC201 than cells with wild-type p53 (Fig.1) The effective concentrations inducing cell killing (as measured by annexin V positivity) in 50%/75% of the cells in the Z-138, JVM-2, MINO, Jeko-1, and Granta-519 cells were 9.9/>10, >10/>10, 2.6/5.2, 2.7/4.6 and >10/ >10 micromolar, respectively. We also treated five primary human MCL samples (three with wild-type p53 and two with mutant p53), and found that one of the two mutant p53 samples was highly sensitive to ONC201 as were the three samples with wild-type p53. One mutant p53 sample that was less sensitive to ONC201, was also resistant to Nutlin-3a and Ibrutinib suggesting an extremely drug-resistant phenotype. Real-time PCR analysis revealed that both DR5 and TRAIL mRNAs were transcriptionally upregulated in the primary MCL samples (a relative ratio of 7.25 compared to 3.13 in controls) after 72-hour treatment with ONC201. To determine the significance of p53 functional status in ONC201-induced apoptosis, p53 wild-type Z-138 and JVM-2 cells were stably transduced with lentivirus encoding either negative control shRNA or p53-specific shRNA and were exposed to ONC201 and results demonstrated complete p53-independence. Normal human bone marrow cells and mesenchymal stem cells were completely resistant to the cytotoxic effects of ONC201, which illustrated this agent's low toxicity against normal tissues. In order to examine the role of p53 activation in ONC201-induced apoptosis in MCL cells, we combined ONC201 with the MDM2 inhibitor Nutlin-3a. The combination cytotoxic effects of this combination were synergistic in p53 wild-type Z-138 and JVM-2 cells (combination index 0.87 and 0.63, respectively). Similar synergistic effects of ONC201 combined with the BTK inhibitor Ibrutinib were observed in Z-138 and MINO cells (combination index 0.63 and 0.61, respectively). This combination also triggered synergistic apoptotic effects in two primary MCL samples with combination indexes of 0.0011 and 0.073, respectively. Conclusion ONC201 induces p53-independent apoptosis in MCL cells, and may have significant clinical impact by targeting both p53 wild type and p53 mutant drug-resistant MCL cells. ONC201 exerts synergistic effects with MDM2 and BTK inhibitors that may be explored clinically. Disclosures: Allen: Drug Company: Employment. Andreeff:Oncoceutics: SAB Other.