ALBERT

Description: Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system. The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

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: Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such as Ebf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered by conditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions of these genes are downregulated in the AML cells and increased by deletion of IDH2/R140Q. Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN that hydroxylates and marks HIF1α for ubiquitin-proteasomal degradation, it is probable that mutant IDH2-produced 2HG stabilizes HIF1α through inhibition of EGLN. Furthermore, it was reported that Meis1 activates the transcription of HIF1α. In consistent with these information, we showed that IDH2/R140Q increased the protein levels of HIF1α in cultured cells. Disclosures Matsunaga: Daiichi Sankyo Co., Ltd.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Background: MDM2, a negative regulator of the tumor suppressor p53, is overexpressed in several cancers including hematological malignancies. Disrupting the MDM2-p53 interaction represents an attractive approach to treat cancers expressing wild-type functional p53. Anticancer activity of small molecule MDM2 inhibitor milademetan (DS-3032b) has been demonstrated in preclinical studies and in a phase 1 trial in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome. Quizartinib is a highly selective and potent FLT3 inhibitor that has demonstrated single-agent activity and improvement in overall survival in a phase 3 clinical study in relapsed/refractory AML with FLT3-internal tandem duplication (FLT3-ITD) mutations. We present here the preclinical studies exploring the rationale and molecular basis for the combination of quizartinib and milademetan for the treatment of FLT3-ITD mutant/TP53 wild-type AML. Methods: We investigated the effect of quizartinib and milademetan combination on cell viability and apoptosis in established AML cell lines, including MV-4-11, MOLM-13 and MOLM-14, which harbor FLT3-ITD mutations and wild type TP53. Cells were treated with quizartinib and milademetan at specified concentrations; cell viability and caspase activation were determined by chemiluminescent assays, and annexin V positive fractions were determined by flow cytometry. We further investigated the effect of the combination of quizartinib and the murine specific MDM2 inhibitor DS-5272 in murine leukemia cell lines Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD+F691L and Ba/F3-FLT3-ITD+D835Y, which harbor FLT3-ITD, ITD plus F691L and ITD plus D835Y mutations, respectively. F691L or D835Y mutations are associated with resistance to FLT3-targeted AML therapy. In vivo efficacy of combination treatment was investigated in subcutaneous and intravenous xenograft models generated in male NOD/SCID mice inoculated with MOLM-13 and MV-4-11 human AML cells. Results: Combination treatment with milademetan (or DS-5272) and quizartinib demonstrated synergistic anti-leukemic activity compared to the respective single-agent treatments in FLT3 mutated and TP53 wild type cells. Combination indices (CIs) were 0.25 ± 0.06, 0.61 ± 0.03, 0.62 ± 0.06, 0.29 ± 0.004 and 0.50 ± 0.03, respectively, in MV-4-11, MOLM-13, MOLM-14, Ba/F3-FLT3-ITD+F691L and D835Y cell lines, all of which harbor FLT3-ITD or ITD plus TKD point mutations. The combination regimen triggered synergistic pro-apoptotic effect in a p53-dependent manner as shown by annexin-V staining and caspase 3/7 assays. Mechanistically, the combination treatment resulted in significant suppression of phospho-FLT3, phospho-ERK and phospho-AKT and anti-apoptotic Bcl2 family proteins (eg, Mcl-1), as well as up-regulation of p53, p21 and pro-apoptotic protein PUMA, compared to single agent treatments. Of note, the combination regimen also exerted a synergistic pro-apoptotic effect on venetoclax (BCL-2 inhibitor)-resistant MOLM-13 cells (CI: 0.39 ± 0.04) through profound suppression of Mcl-1. In an in vivo study using the MOLM-13 subcutaneous mouse xenograft model, quizartinib at 0.5 and 1 mg/kg and milademetan at 25 and 50 mg/kg demonstrated a significant tumor growth inhibition compared with vehicle treatment or respective single-agent treatments. In MV-4-11 intravenous mouse xenograft model, the combination of quizartinib plus milademetan showed a significantly prolonged survival, with no animal death in the combination group during the study period, compared to respective single agent treatments and untreated control (Figure). Conclusion: Synergistic anti-leukemic activity was observed for quizartinib plus milademetan combination treatment in preclinical AML models. A phase I clinical trial of quizartinib/milademetan combination therapy in patients with FLT3-ITD mutant AML is underway. Figure. Effects of quizartinib, milademetan and their combination on survival of mice intravenously inoculated with human MV-4-11 AML cells Disclosures Andreeff: Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Reata: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership. Kumar:Daiichi Sankyo: Employment, Equity Ownership. Zernovak:Daiichi Sankyo: Employment, Equity Ownership. Daver:Pfizer: Research Funding; ImmunoGen: Consultancy; Otsuka: Consultancy; Karyopharm: Research Funding; Alexion: Consultancy; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Kiromic: Research Funding; Sunesis: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Sunesis: Consultancy. Isoyama:Daiichi SANKYO CO., LTD.: Employment. Iwanaga:Daiichi Sankyo Co., Ltd.: Employment. Togashi:Daiichi SANKYO CO., LTD.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment.

Description: Background The ubiquitin-proteasome pathway is now a validated target for myeloma therapy given the regulatory approvals of proteasome inhibitors such as bortezomib and carfilzomib. Another logical set of targets are the E3 ligases, whose role is to facilitate the protein poly-ubiquitination typically needed for recognition by the constitutive or immunoproteasome prior to proteolysis. One of the more attractive such targets is HDM-2, the E3 ligase best known for its role in turnover of the p53 tumor suppressor, in part because p53 deletion or mutation is less common in myeloma than in solid tumors, and because it has been possible to develop agents targeting the HDM-2 p53 binding pocket. Methods Activity of the specific HDM-2 inhibitor DS-5272 (Daiichi-Sankyo) was evaluated using a panel of p53 wild-type (wt) and mutant (mut) myeloma cell lines and also against primary patient samples. Studies were performed with DS-5272 both alone, and also in combination regimens with novel agents. Tetrazolium dye-based assays were employed to determine cell viability, Annexin V staining was used to examine apoptosis, and quantitative PCR and Western blotting were used to study selected transcripts and gene products, respectively. This study was supported in part by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results DS-5272 induced potent cytotoxicity in wt p53 MM1.S, H929, and MOLP-8 myeloma cell lines, with a median inhibitory concentration (IC50) in the single micromolar range, which was reproduced in studies of primary CD138+ plasma cells but not CD138- cells from myeloma patients. This cytotoxicity was both time- and concentration-dependent, and DS-5272 was more potent than the prototypical HDM-2 inhibitors, Nutlin-3a and MI-219. A dependence of DS-5272 activity on wt p53 was demonstrated by the much lower IC50 in the wt p53 than mut p53 cell lines, and the finding that suppression of p53 with a shRNA, as well as mutation of p53 with a sequence-specific zinc finger nuclease, significantly increased the IC50. Notably, DS-5272 remained active in the presence of conditioned media from stromal cells, or key myeloma cytokines such as IL-6. The reduced viability after exposure to DS-5272 was due at least in part to activation of type I cell death, as determined by increased staining for Annexin V, activation of caspases 9 and 3, and cleavage of PARP. Other downstream effects included induction of transcription of p21, Bax, HDM-2, NOXA, and PUMA. These proteins, as well as p27 and p53 were induced, while the abundance of Survivin, CHK1, Aurora A and B, PLK1, and KIF11 was reduced. Consistent with these latter effects, DS-5272 induced some accumulation of cells at the G2/M phase, and the appearance of cells with a disordered spindle. Suppression of KIF11 (kinesin spindle protein (KSP; Eg5)), which is responsible for centromere separation and bipolar spindle formation, seemed to occur through the binding of p21 to the cell cycle genes homology region (CHR) within the KIF11 promoter. Finally, combinations of DS-5272 with the specific KIF11 inhibitor Ispinesib produced enhanced G2/M arrest, as well as a synergistic reduction in cell viability with increased levels of apoptosis. Conclusion DS-5272 is a potent and novel agent with activity against multiple myeloma, and is active both alone and in rationally designed combination regimens with other drugs. These findings provide a rationale for the clinical translation of HDM-2 inhibitors as monotherapy, and possibly with other agents such as the KSP inhibitor ARRY-520, which is also active against myeloma, for patients with wt p53 relapsed and/or refractory myeloma. Disclosures: Cai: Daiichi-Sankyo Pharma Development: Employment, Equity Ownership. Seki:Daiichi Sankyo Co., Ltd.: Employment, Equity Ownership. Tse:Daiichi-Sankyo Pharma Development: Employment, Equity Ownership.