ALBERT

Description: Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice. Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36. Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment. Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. Disclosures Zuber: Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Description: Heat shock factor 1 (HSF1) is best known as a key sensor of proteotoxic stress, but accumulating evidence also supports a major role for this transcriptional regulator in cancer biology. In a variety of human solid tumor cells, downregulation of HSF1 inhibits growth, induces cell death and limits metastatic potential. In breast cancers, nuclear accumulation of HSF1 and a tumor-specific gene expression signature reflecting HSF1 activation were found to be strongly associated with poor outcome (Mendillo et al, Cell 2012). In addition, we have recently reported, as a counter-intuitive reversal of the central dogma, that inhibition of protein translation represses the constitutive activation of HSF1 in cancers, and that HSF1 inhibition induced by the potent eIF4a inhibitor rohinitib (RHT) exerts profound, far-ranging anti-tumor effects (Santagata et al, Science 2013). Review of public databases supports targeting of HSF1 and eIF4a in AML: mRNA levels of HSPA8, one of the primary HSF1 targets, are correlated with poor prognosis in AML (Prognoscan, data from Metzeler et al, Blood 2008) and eIF4a mRNA levels were highest in AML among 12 cancer types (Oncomine, data from Ramaswamy et al, PNAS 2001). Here, we demonstrate that inactivation of HSF1 in acute myeloid leukemias (AMLs) by RHT exerts pronounced apoptogeniceffects with preferential activity against FLT3-ITD mutant cells in cell culture and in mice. First, we confirmed our previous finding of inactivation of HSF1 by RHT in AML. In OCI-AML3, MOLM-13 and MV4;11 cells, mRNA levels of HSPA8 were reduced by 70% after RHT treatment compared to untreated controls. OCI-AML3 cells were then infected with lentivirus encoding a reporter GFP-luciferase fusion protein the expression of which is driven by promoter elements from either the HSPA1A or HSPA6 genes; an approximately 50% reduction of reporter induction by heat shock was observed after RHT treatment compared to untreated controls. Next, treatment of 7 human AML cell lines in culture showed that RHT induces marked anti-leukemia effects at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity as determined by flow cytometry at 72hr). The most pronounced cytotoxic effects were observed in FLT3-ITD+ cell lines (LD50s 〈 10 nM in MOLM13 and MV4;11 cells). Using two sets of isogenic cell lines (Ba/F3 and OCI-AML3 cells with FLT3-ITD or wild-type (wt) FLT3), we confirmed that RHT more potently kills FLT3-ITD cells (LD50s; 65.3 vs 20.1 nM in Ba/F3 cells). Furthermore, the combination of FLT3 inhibitor sorafenibwith RHT showed synergistic effects in cell culture (Combination Index: ED50 0.85, ED75 0.86, ED90 0.89). Immunoblot analysis showed higher phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 cells than FLT3-wt cells, suggesting greater dependence of FLT3-ITD cells on HSF1 activation for survival. We also tested primary samples from 17 AML patients and bone marrow (BM) samples from 8 healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (Figure 1A). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem cells (CD45+CD34+CD38-). Moreover, the activity of RHT against the leukemic population was significantly higher in FLT3-ITD than in FLT3-wt cells (Figure 1B). We also evaluated the activity of RHT in a FLT3 mutant AML xenograft model using GFP-luciferase labeled MOLM-13 cells. Significantly decreased luciferase activity was detected by bioluminescence imaging and a dose-dependent reduction in GFP+ leukemic cells was seen in peripheral blood and BM by day 16 (Figure 2). Survival of the treatment groups was significantly prolonged (median; 18 vs 22.5 vs 24 days respectively, p 〈 0.0001). In conclusion, HSF1 function provides an attractive therapeutic target in AML. The eIF4a inhibitor RHT down-regulates HSF1 transcriptional function and exerts robust anti-leukemia activity in cell culture and in mice. Although the relative contributions of HSF1 inactivation and translation inhibition to the net anti-leukemic activity of RHT remain to be defined, promising features of this approach include its activity against AML stem cells, while sparing normal stem cells and its particularly potent cytotoxicity for poor-prognosis FLT3-ITD AMLs. Taken together, these preclinical findings strongly support further development of eIF4a inhibitors in the treatment of AML. Disclosures Ishizawa: Karyopharm: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Description: Development of MDM2 inhibitors enabled successful induction of p53-mediated apoptosis in tumor cells without a risk of DNA damage. Early clinical trials of MDM2 inhibitors demonstrated proof-of-concept (Andreeff et al., Clin Can Res, 2015). However, a clinical challenge is that not all the tumors bearing wild-type TP53 are sensitive to MDM2 inhibition. We here discovered novel gene profiling-based algorithms for predicting tumor sensitivity to MDM2 inhibition, using DS-3032b, a novel potent MDM2 inhibitor, which is currently in early clinical trials. In vitro inhibitory effects of DS-3032b on MDM2-p53 interaction was demonstrated using the homogeneous time resolved fluorescence (HTRF) assay (IC50 5.57 nM). DS-3032b treatment (30-1000 nM) indeed increased p53 protein in a dose-dependent manner, and also the p53 targets MDM2 and p21, in cancer cell lines with wild-type TP53 (SJSA-1, MOLM-13, DOHH-2, and WM-115), showing around 10-fold potent growth inhibition effects compared to Nutlin-3a (Table 1). The xenograft mouse models with SJSA-1 and MOLM-13 cells showed 〉 90% reduction in tumor growth with oral administrations of 25 and 50 mg/kg/day. For discovering predictive gene signatures, we performed two different approaches. In the first approach, 240 cell lines available as OncoPanel were treated with DS-3032b, another prototypic MDM2 inhibitor DS-5272, and Nutlin-3a, and determined 62 sensitive and 164 resistant lines, based on GI50s. Using gene expression profiling (GEP) publicly available for all the cell lines, we selected 175 top-ranked genes with highest expression in the 62 sensitive cell lines. We thus defined the average of Z-scores of the 175 gene expression as "sensitivity score". To validate the 175-gene signature, we evaluated in vivo anti-tumor activities of DS-3032b in 13 patient-derived tumor xenografts (melanoma, NSCLC, colorectal and pancreatic cancers). The prediction accuracy, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) were 85, 88, 88 and 80% respectively. As another validation set, 41 primary AML samples were treated with DS-3032b to define the top and bottom one-third most sensitive or resistant samples (14 each), and GEP was performed in every sample. TP53 mutations were detected in 8 specimens by next generation sequencing and confirmed by Sanger sequencing. The 175-gene signature was applied to the AML dataset, and the accuracy, sensitivity, PPV and NPV to predict the 14 sensitive or resistant samples were 79, 93, 72 and 90% respectively. Importantly, this signature was more predictive than the TP53 mutation status alone applied (68, 93, 62 and 86%). (Table 2A-B) In contrast to the cell line-based approach, the second approach defined an AML-specific gene signature. Specifically, we used the same dataset of 41 primary AML samples described above as training and validation set, by performing random forest methods with cross validation. Using a routine way in bioinformatics analysis of classifying gene signature, we first selected the 1500 top-ranked genes with highest expression variance among all the specimens. In addition, p53-related 32 genes that potentially have predictive values were also selected based on the previous reports. Classification was performed using the random forest method to identify a predictive algorithm with the 1500-gene set, 32-gene set or combined 1525-gene set (7 genes were overlapped), thus we found that the 1525-gene set had highest performance than each gene set alone. However, applying this method to all the 41 samples showed inferior predictive performance than applied only to the 33 wild-type TP53 samples (the prediction accuracy, sensitivity, PPV and NPV were 68, 72, 67 and 69%, vs. 77, 82, 75 and 80%).(Table 2C) Finally, we combined each of the two algorithms (Table 2B-C) with TP53 mutation status. Specifically, the samples with TP53 mutations were predicted as resistant, then either of gene signatures was applied to the rest of the samples with wild-type TP53. Predictive performance (Table 2D-E) was improved in both signatures compared to the others, especially showing the highest PPVs (80 and 82%, respectively). Taken together, gene signatures discovered in the present study, by combining with TP53 mutation status, provided new highly predictive algorithms for therapy of MDM2 inhibition. Our findings will be tested in ongoing clinical trials of DS-3032b. Disclosures Nakamaru: Daiichi Sankyo Co., Ltd: Employment. Seki:2Daiichi Sankyo Co., Ltd.: Employment. Tazaki:2Daiichi Sankyo Co., Ltd.: Employment. DiNardo:Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Abbvie: Research Funding; Agios: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding. Tse:Daiichi Sankyo, Inc.: Employment.

Description: The clinical challenge posed by p53-deficiency in hematological malignancies needs novel therapeutic strategies. ONC201, a first-in-class small molecule, was discovered as a p53-independent activator of apoptosis with a benign preclinical safety profile (Allen et al, Sci Transl Med 2013). Here we report that ONC201 exerts anti-tumor effects in hematological malignancies and leukemia stem cells (LSC) via the induction of ATF4 in the integrated stress response (ISR). ONC201 induced p53-independent apoptosis in cell lines and primary patient samples from mantle cell lymphomas (MCL) and acute myeloid leukemias (AML), independent of genetic alterations that correlate with poor prognosis (e.g., TP53 mutation, FLT3-ITD, or complex karyotype). ONC201 also induced apoptosis in LSC, while sparing normal bone marrow progenitors, as measured in vitro and after transplantation. Specifically, we recovered unfractionated LSC-containing populations of AML cells (t(9;11)(p22; q23), CEBPA and ATM mutant) from secondarily-engrafted mice and cultured them in vitro for 48 hr in two groups, ONC201-treated (5 uM) or Control. For both groups, the same number of Trypan Blue-negative cells was then re-transplanted. The frequency of human CD45+ cells 4 weeks after transplantation was 38.09 ± 2.59 % of tibial BM cells in untreated mice and 0.10 ± 0.05% in treated mice (n = 3 for each, p 〈 0.01). The survival of the treated group was dramatically prolonged (Figure 1). Gene Set Enrichment Analysis (GSEA) of gene expression profiling (GEP) data of Jeko-1 and Z-138 cells treated with ONC201 implicated up-regulated Endoplasmic Reticulum (ER) stress-related genes, such as targets of the ER stress-induced transcription factor CHOP (DDIT3; FDR q = 0.016), and ER component proteins (FDR q = 0.039). We confirmed the ONC201-induced increased mRNA levels of DDIT3, GADD34, DR5 and TRIB3, and increased protein levels of CHOP, ATF4 and IRE1-a in Jeko-1 cells. Knockdown of ATF4 and IRE1-a revealed that ATF4 is an essential protein for ONC201-induced apoptosis in most AML cells and MCL cells, while IRE1-a is only necessary for apoptosis in MCL cells. However, unlike other ER stress inducers, ONC201 did not cause phosphorylation of an eIF2a kinase PERK, a hallmark of classical ER stress. Importantly, ONC201 was also effective in lymphoma cells resistant to bortezomib, an ER stress inducer, supporting the notion that ONC201 uses a unique mechanism to trigger the ISR. In addition, ONC201 inhibited mTORC1 signaling, likely secondary to ATF4 activation via the induction of DDIT4, a negative regulator of mTORC1, which presumably increases the cytotoxicity of ONC201 by global translation inhibition. Investigating the type of apoptosis induced by ONC201, we examined protein levels of anti-apoptotic BCL-2 family members (BCL-2, BCL-XL and MCL-1) after ONC201 treatment. MCL-1 was reduced most notably after 12 hr. We then tested the effect of ONC201 on cells with overexpression or knockdown of BCL-2 family proteins. MCL-1 knockdown in OCI-AML3 cells increased their sensitivity to ONC201 only slightly, but ONC201 efficacy was dramatically reduced in BCL-2-overexpressing HL-60 cells, even more so than in BCL-XL-overexpressing HL-60 cells. Therefore, we investigated whether ONC201 sensitivity could be increased by ABT-199, a small-molecule BH3 mimetic that specifically inhibits BCL-2, and is known to be ineffective in cell lines with MCL-1 overexpression; accordingly, the combination of ONC201 and BCL-2 antagonist ABT-199 was highly synergistic (Figure 2). In conclusion, ONC201 induces p53-independent apoptosis and abrogates LSC function by ATF4 induction, via the ISR. By suppressing MCL-1, ONC201 can also increase the effectiveness of the Bcl-2 inhibitor ABT-199. These findings suggest that ONC201 may provide promising novel therapeutic strategies for TP53 -wild type and TP53 -mutant hematological malignancies. Phase I/II clinical trials have been initiated at the MD Anderson Cancer Center in leukemias and lymphomas to determine safety, efficacy and further characterize mechanism of action. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ishizawa: Karyopharm: Research Funding. Allen:Oncoceutics, Inc: Employment, Equity Ownership. Orlowski:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Forma Therapeutics: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding. Wang:Celgene: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Description: A minor fraction of leukemia cells, leukemia stem cells, have been shown to be highly resistant to current therapies and thought to be responsible for recurrence. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. PTC-209 is a novel selective transcriptional inhibitor of BMI-1, which has been shown to have antitumor activity against cancer-initiating cells in colorectal cancer. We investigated the prognostic significance of BMI-1 in acute myeloid leukemia (AML) using reversed phase protein array and effects of the BMI-1 inhibitor PTC-209 on primary and leukemia cell lines. BMI-1 protein expression was determined in bulk AML blasts from 511 newly diagnosed patients. BMI-1 expression was higher in unfavorable cytogenetics (n=252, median 0.068) compared to intermediate (n=225, median -0.116, P = 0.017) or favorable cytogenetics (n=34, median -0.338, P = 0.0007 versus unfavorable, 0.05 versus intermediate). Higher BMI-1 levels were associated with shorter median overall survival (42.8 versus 55.3 weeks, P = 0.046 Log Rank test). There was no correlation between BMI-1 levels and percentages of CD34 -positive cells (r = 0.07). A total of 6 AML (MOLM-13, OCI-AML3, MV4-11, NB4, HL60 and U-937) and 5 ALL (Reh, NALM6, Jurkat, Raji and MOLT-4) cell lines were exposed to PTC-209 for 48 hours. PTC-209 exhibited dose- and time-dependent anti-proliferative and cytotoxic activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 0.33 ± 0.04 µM (mean ± SEM) for AML and 0.55 ± 0.09 µM for ALL, indicating potent anti-proliferative effects. In contrast, PTC-209 showed differential cytotoxic effects between AML and ALL cells. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V positivity) were no more than 2.5 µM in 5 of 6 AML lines while they were higher than 10 µM in 3 out of 5 ALL cell lines, implicating that BMI-1 is more critical in AML than ALL. Treatment with PTC-209 triggered several molecular events consistent with induction of apoptosis in sensitive lines (e.g. MV4-11 and MOLM-13): conformational change of BAX (i.e., BAX activation), loss of mitochondrial membrane potential (MMP), caspase-3 activation and DNA fragmentation in addition to phosphatidylserine (PS) externalization. Eighteen-hour treatment of MV4-11 cells with 2.5 µM PTC-209 led to compound-specific induction of conformationally active BAX (31%), MMP loss (80%) and caspase-3 cleavage (38%). qRT-PCR showed reduced transcript level of BMI-1 (61% reduction) after 6-hour PTC-209 exposure in MV4-11 cells. PTC-209 induced PS externalization in primary AML cells (82.5 ± 4.3% after 48-hour treatment with 2 µM PTC-209, n = 6) and to a lesser degree, in ALL cells (33.7 ± 13.4%, n = 4, p 〈 0.05). Importantly, CD34+CD38– AML progenitor cells were as sensitive to PTC-209 as C34– more mature AML cells. Normal lymphocytes were resistant to PTC-209 (9.1 ± 4.6% even at 10 µM). Collectively, BMI-1 inhibition by small molecule inhibitors could be developed into a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction TP53 mutations are rare (〈 5%) in de novo acute myeloid leukemia (AML) but if present, they are associated with a very poor prognosis (〈 1% overall survival at 3 years). p53 mutations have been frequently detected in poor-prognosis patients with complex karyotype or therapy-related AML. COTI-2 is a third generation thiosemicarbazone derivative that was identified as a small molecule candidate against a diverse group of human cancer cell lines using a proprietary in silico drug screening method. COTI-2 causes cancer cell death via apoptosis. COTI-2 has been proposed to restore non-functional mutant p53 conformation to functional wild-type conformation in solid cancer cells, and it has recently entered a clinical trial in patients with advanced or recurrent gynecologic malignancies (NCT02433626). The molecular mechanisms of COTI-2 to induce apoptosis, however, remain largely unknown. In this study, we investigated activity and possible modes of action of COTI-2 against AML cells, especially focusing on its p53-independent properties. METHODS AND RESULTS A total of 10 AML cell lines were exposed to COTI-2 for 72 hours and its anti-leukemia effects were determined by viable cell counts and annexin V staining. OCI-AML2, OCI-AML3, MOLM-13, MOLM-14 and MV4;11 express wild-type (WT) p53; THP-1and Kasumi-1 express mutant (MUT) p53; KG-1, U937 and HL-60 does not express p53 protein (p53 NULL). COTI-2 inhibited leukemia cell growth and increased the percentage of annexin V-positive cells, irrespective of cellular p53 status. The IC50 values (concentration at which cell growth is inhibited by 50%) for were not statistically different between p53 WT and MUT/NULL cells (10.3 ± 4.5 nM vs 20.2 ± 11.5 nM, P = 0.44). So did the ED50 values (effective concentration inducing 50% cell killing as measured by Annexin V positivity) (115.0 ± 50.9 nM vs 237.8 ± 109.9 nM, P = 0.34). We next examined the apoptotic effect on primary AML cells from patients with AML. A total of 14 samples [11 p53 WT cases and 3 p53 MUT (C135W, R248Q and C242Y) cases] were examined. COTI-2 induced apoptosis both in p53 WT (40.3 ± 6.4% compound-specific annexin V induction) and MUT samples (48.0 ± 17.7%) (P = 0.62). To elucidate the p53-independent activity of COTI-2, mRNA expression levels of TP53 (p53), CDKN1A (p21) and BBC3 (PUMA) were determined in four AML cell lines (OCI-AML3, MV4;11, HL-60 and Kasumi-1) after exposure to COTI-2. COTI-2 did not induce TP53 or its transcriptional targets CDKN1A or BBC3 in any of the cell lines. To investigate if COTI-2 activates an intrinsic pathway to induce apoptosis in AML, we used p53 NULL HL-60 cells overexpressing BCL-2 (HL-60/BCL-2), BCL-XL (HL-60/BCL-XL) and their controls (HL-60/neo). HL-60/BCL-2 and HL-60/BCL-XL respectively expressed BCL-2 and BCL-XL at greater than 4 times higher levels than HL-60/neo. Interestingly, overexpression of either BCL-2 or BCL-XL almost completely abrogated COTI-2-induced apoptosis. Similar results were obtained in OCI-AML3/BCL-2. MCL-1 overexpression only modestly inhibited COTI-2-induced apoptosis. Involvement of the extrinsic pathway was modest as JurkatI9.2 (a Jurkat clone deficient in caspase-8) and its control showed similar sensitivity to COTI-2. COTI-2 appeared to reduce MCL-1 expression levels through mTORC1 inhibition. Finally, COTI-2 strongly synergized with the BCL-2 inhibitor ABT-199 to induce apoptosis in AML cells. Conclusion COTI-2 induces mitochondrial apoptosis in AML cells, irrespective of their p53 mutational status. COTI-2-induced apoptosis depends on BCL-2 and BCL-XL expression but not on MCL1 expression or p53 activation. COTI-2 has clinical potential in AML that often expresses high levels of MCL-1, especially in combination with BCL-2 inhibition. Disclosures Danter: Critical Outcomes: Employment.

Description: The functional or genetic inactivation of p53 hampers human tumor treatment. Therefore, novel therapeutic strategies are needed. ONC201 is a p53-independent inducer of apoptosis that is the founding member of the imipridone class of novel anti-cancer compounds, which possess a unique pharmacophore. We discovered that ONC201 exerts anti-tumor effects via ATF4 induction through activation of an atypical integrated stress response (ISR) (Ishizawa et al. and Kline et al, Sci Signal, 2016). Several clinical trials of ONC201 are ongoing in advanced cancers, showing a promising safety profiling and signs of clinical activity in both solid tumors and hematopoietic malignancies. In this study, we investigated the effects of ONC212, which has emerged as a highly potent member of the imipridone family, in preclinical models of hematological malignancies. ONC212 exerted potent and prominent apoptogenic effects on acute myeloid leukemia (AML) and mantle cell lymphoma (MCL) cell lines (e.g., ED50s of 141.0 nM in p53 wild-type OCI-AML3 cells, 105.7 nM in MOLM13 cells, and 265.2 nM in p53-null JeKo-1 cell lines). Time course analysis of apoptosis in OCI-AML3 cells showed that ONC212 takes more than 36 hours to start to induce apoptosis, which is similar to observations with ONC201. Next, we further examined similarities between ONC212 and ONC201 by evaluating the in vitro efficacy of ONC212 in ONC201-resistant (ONC201-R) cell lines that we have generated by chronic exposure of MCL and AML cell lines to ONC201, of which ED50s for ONC201 treatment at 72 hrs were all 〉 5 μM. Interestingly, the ONC201-R cell lines were more resistant to ONC212 than the isogenic ONC201-naïve cells (Figure 1), indicating that these cell lines are cross-resistant to ONC212. We previously proved that increased protein translation of the transcription factor ATF4 is one of the major molecular events involved in ONC201-induced apoptosis (Ishizawa et al., Sci Signal, 2016). Similarly, ATF4 protein abundance was increased by 24-hour treatment with ONC212. DDIT3 (CHOP) gene, a target of ATF4, was transcriptionally upregulated in parallel with its target genes GADD34, DR5 and TRIB3 in ONC212-treated JeKo-1 and OCI-AML3 cells by 24 hrs after treatment (Figure 2). Of note, ONC201 was reported to transcriptionally induce TRAIL in a p53-independent manner in solid tumors (Allen et al., Sci Transl Med, 2013), but it was not operational in hematological cell lines (Ishizawa et al., Sci Sig 2016). Consistently, we also confirmed that ONC212 does not increase TRAIL mRNA in MCL (JeKo-1) and AML (OCI-AML3) cells. BCL-2 is a protective factor for cells under endoplasmic reticulum stress, which is one way to activate ISR. Therefore, we investigated whether the BCL-2 inhibitor ABT-199 sensitizes hematopoietic malignant cells to ONC212. Apoptosis was significantly higher in the combination than either drug alone in MCL and AML cell lines even in THP-1 and OCI-AML3 cells that are relatively resistant to ONC201/212 and/or ABT-199 (Figure 3), suggesting that this combination could overcome the resistance to either of agents. Indeed, the combination was also synergistic in the OCI-AML3 ONC201-R cell line (Figure 3). Taken together, our preclinical studies suggest that ONC212 is a promising and potent new member of the impridone class of anti-cancer compounds that warrants further development in hematological malignancies. The combination of ONC212 with ABT-199 is attractive, considering that acquired resistance after a short-term response remains a clinical challenge with ABT-199. Disclosures Konopleva: AbbVie: Research Funding; Genentech: Research Funding. Allen:Oncoceutics Inc.: Employment. Stogniew:Oncoceutics Inc.: Employment, Equity Ownership. Andreeff:Oncoceutics Inc.: Membership on an entity's Board of Directors or advisory committees.

Description: ONC201 is a novel agent with profound anti-tumor effects, reported to induce p53-independent apoptosis in diverse types of cancers while sparing normal cells (Allen JE et al., Sci Transl Med, 2013). We further investigated its activity and mechanism(s) of action (MOA) in hematological malignancies. We studied mantle cell lymphoma (MCL) cell lines Z-138 and JVM-2 with wild-type (WT) p53, MINO and Jeko-1 with mutant (mut) p53, and Z-138 and JVM-2 cells with knockdown of p53 by lentiviral shRNA, as well as acute myeloid leukemia (AML) cell lines OCI-AML and MOLM-13 with WT p53, and HL-60 with null p53. We also studied primary cells of MCL (n = 8, 2 with mut p53) and AML (n = 11, 2 with mut p53). In vitro treatment with ONC201, ranging from 2.5 to 10 μM, confirmed that ONC201 induces apoptosis independent of p53 status, and has consistent efficacy against primary AML stem/progenitor cells (CD45 dim+/ CD34+/ CD38-) (Figure 1A). In addition to cytotoxic effects, cell cycle analysis using PI/EdU demonstrated induction of p53-independent cell cycle arrest with S phase delay (diminished incorporation of EdU into S phase cells). The IC50 in most samples was 〈 5 μM, well within the therapeutic window of ONC201 according to previous pharmacokinetic studies with mice, rats and dogs. Consistent with this, ONC201 showed little toxicity in vitro in normal bone marrow and stem/progenitor cells, suggesting a very favorable therapeutic window (Figure 1B). In contrast to previous reports of its MOA, ONC201 did not induce TRAIL in AML and MCL cell lines, although DR5 mRNA was induced 2-fold. As further evidence of a TRAIL-independent MOA of ONC201, its toxicity was not diminished by FOXO3a knockdown in OCI-AML3 and Z-138 cells, or by mutation of caspase 8 (i.e., the Jurkat clone I9.2 was as sensitive as Jurkat cells with WT caspase 8). Gene Set Enrichment Analysis (GSEA) of gene expression profiling (GEP) data of Jeko-1 and Z-138 cells treated with ONC201 implicated upregulated Endoplasmic Reticulum (ER) stress-related genes, such as targets of the ER stress-induced transcription factor CHOP (DDIT3; FDR q = 0.016), and ER component proteins (FDR q = 0.039). Direct investigation of ER stress-induced changes found that XBP-1 splicing begins 6 hr after ONC201 exposure (Figure 2A), followed by transcriptional induction of DDIT3 and its targets (GADD34, TRIB3 and DR5) at 12-24 hr (Figure 2B). In addition, GEP of Jeko-1 cells treated with the ER stress inducer tunicamycin showed high overlap with the changes (both up and down) caused by ONC201. GSEA also implicated mTOR inhibition, such as modulation of the Molecular Signatures Database gene set "mTOR UP.N4.V1 UP" (FDR q = 0.000), and we found dephosphorylation of p70S6K and S6 12 hr after ONC201 exposure (Figure 3). Therefore, we hypothesized that post-translational inhibition of p70S6K/S6 could be another activity of ONC201. In support of this, the combination of tunicamycin and mTORC1 inhibitor rapamycin revealed synergistic effects in Jeko-1 cells. Although the correlation between ER stress and mTOR pathway inhibition has been reported previously, the mechanisms underlying this correlation are not known. However, we found that the oxidative stress gene DDIT4, known as a negative regulator of the TSC1/2 complex (thus inhibiting the mTORC1 pathway) and HIF-1-alpha, is upregulated much more in Jeko-1 cells by ONC201 than by tunicamycin, possibly explaining mTOR pathway inhibition by ONC201. In conclusion, we confirmed that ONC201 induces p53-independent cell death, with little toxicity to normal cells. We identified a novel MOA in leukemias and lymphomas for ONC201 that involves induction of ER stress and inhibition of mTOR pathway signaling. ONC201 is the first clinical candidate known to both induce ER stress and inhibit mTOR signaling, and will soon enter Phase I trials in hematological malignancies. This finding could provide important biomarkers to predict and monitor the efficacy of ONC201. Disclosures Allen: Oncoceutics, Inc.: Employment. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Description: Key Points FZR1 loss causes increased sensitivity of B-ALL cells to oncogene- or chemotherapy-induced DNA damage. Prolonged loss of FZR1 contributes to the development of treatment-resistant clones in mouse and human B-ALL.