Takahiko Seki

Lead optimization of novel p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold.

With the aim of discovering potent inhibitors of the p53-MDM2 interaction and thus obtaining a potent anticancer drug, we have pursued synthesis and optimization of dihydroimidazothiazole derivatives, which have been discovered via scaffold hopping by mimicing the mode of interaction between MDM2 and Nutlins. Upon the discovery we encountered a problem involving the chemical instability of the scaffold, that is, susceptibility to oxidation which led to imidazothiazole. In order to solve this problem and to obtain further potent compounds, we executed medicinal research and thus furnished the optimal compounds by incorporating the methyl group onto the C-6 position to avoid the oxidation, and by modifying the C-2 moiety of the additional proline motif, which furnished high potency. The incorporation of the pyrrolidine moiety at the C-2 position raised another hydrophobic interaction site with MDM2 protein, which was generated by the induced-fitting observed by co-crystal structure analysis. These optimal molecules showed significant improvement in potency when compared with the early lead (+)-1 or Nutlin-3a.

Discovery of novel dihydroimidazothiazole derivatives as p53–MDM2 protein–protein interaction inhibitors: Synthesis, biological evaluation and structure–activity relationships

Starting with Nutlins as an initial lead, we designed and generated bicyclic scaffolds aiming to place cis-bischlorophenyl moiety at the equivalent location where the hydrophobic interaction with MDM2 could be expected. As a result, we discovered novel MDM2 inhibitors possessing a dihydroimidazothiazole scaffold. Further exploration of the side chains on the dihydroimidazothiazole scaffold aided by molecular modeling resulted in compounds exhibiting almost comparable in vitro potency to Nutlin-3a.

IDH2 and NPM1 Mutations Cooperate to Activate Hoxa9/Meis1 and Hypoxia Pathways in Acute Myeloid Leukemia.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.

Synthesis and evaluation of novel orally active p53–MDM2 interaction inhibitors

We have discovered and reported potent p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold. Our lead showed strong activity in vitro, but did not exhibit antitumor efficacy in vivo for the low metabolic stability. In order to obtain orally active compounds, we executed further optimization of our lead by the improvement of physicochemical properties. Thus we furnished optimal compounds by introducing an alkyl group onto the pyrrolidine at the C-2 substituent to prevent the metabolism; and modifying the terminal substituent of the proline motif improved solubility. These optimal compounds exhibited good PK profiles and significant antitumor efficacy with oral administration on a xenograft model using MV4-11 cells having wild type p53.

Discovery of DS-5272 as a promising candidate: A potent and orally active p53–MDM2 interaction inhibitor

We have published p53-MDM2 interaction inhibitors possessing a novel dihydroimidazothiazole scaffold. Although our lead compound 1 showed strong antitumor activity with single oral administration on a xenograft model using MV4-11 cells harboring wild-type p53, it needed a higher dose (200mg/kg) for distinct efficacy. We executed further optimization with the aim of improvement of potency and physicochemical properties. Thus optimal compounds were furnished by introducing fluorine moieties onto the phenyl ring at the C-6 position and the pyrrolidine part at the C-2 substituent; and modifying the terminal piperazine to 4,7-diazaspiro[2,5]octane variants. Furthermore, replacing 4-chlorophenyl on the C-5 position with pyridyl variant decreased nonspecific cytotoxicity significantly. Our exploration afforded DS-5272 indicating excellent antitumor efficacy from a dose of 25mg/kg on SJSA-1 xenografted models with high safety and good PK profiles, which has appropriate potency as a clinical candidate.

Abstract B5: Preclinical characterization of a novel orally-available MDM2 inhibitor DS-3032b: Anti-tumor profile and predictive biomarkers for sensitivity

The p53 protein exerts its tumor suppressor function by inducing cell cycle arrest, apoptosis, or senescence in cells under stress conditions. Inactivation of p53 by somatic mutations is a frequent tumorigenic event. In human tumors that retain wildtype p53 protein, its activity is often inhibited by interaction with the negative regulator MDM2. DS-3032b, a novel, specific, small-molecule inhibitor of MDM2, disrupts the MDM2-p53 interaction in tumor cells and is being developed as a cancer therapeutic based on a p53 reactivation mechanism. Cell-free study using recombinant MDM2 and p53 proteins demonstrated inhibition of MDM2-p53 binding by DS-3032b with an IC50 value of 5.57nM. Treatment with graded concentrations of DS-3032b resulted in a dose-dependent increase of p21 and PUMA mRNA levels in an osteosarcoma cell line (SJSA-1) harboring MDM2 gene-amplification. DS-3032b inhibited cell growth in p53 wildtype (GI50s: 0.043 - 0.276μM), but not p53 mutant or null cell lines (GI50s: >10μM). Studies in athymic nude mice inoculated with SJSA-1 xenografts showed tumor regression following treatment with DS-3032b given once daily for 10 days at a dose of 50 mg/kg. To identify biomarkers predictive of sensitivity to MDM2 inhibitors, we treated a panel of 240 molecularly-annotated cancer cell lines (OncoPanel, Eurofins Panlabs) with the prototypic MDM2 inhibitor DS-5272 structurally unrelated to DS-3032b. Cells were dichotomized as sensitive or resistant to DS-5272, based on IC50 cut-off value of 4 μM, respectively. As expected, TP53 mutation was identified as a strong predictor for resistance. In addition, a 175-gene signature was identified as an indicator for sensitivity. This gene signature predicts for response to 3 different MDM2 inhibitors (DS-5272, nutlin3a, and DS-3032a (free form of DS-3032b)). A combination of TP53 genotype and gene signature showed further enrichment of sensitive cell lines compared to using either of the biomarker alone. In vivo studies using patients derived tumor xenografts have further validated the predictive value of the gene signature. Bioinformatic reduction of the complexity of the gene signature to a clinically applicable level is on-going. Mapping of this signature to genomic data from 15,000+ clinical samples in the OncoMine database has revealed that only a subset of TP53 wildtype tumors contain high expression of the signature genes. The top-ranked genes in the signature were well-known p53-inducible genes, suggesting that the gene signature might be a surrogate for functional p53. Consistent with this postulate, we showed low signature gene expression and resistance to MDM2 inhibitors in cervical cancers in which p53 function is inactivated by the E6 viral oncoprotein. Cancer subtypes enriched for a low incidence of TP53 mutation and high signature gene expression are melanoma, lymphoma, myeloma, renal cell cancer, and leukemia. In summary, DS-3032b is a specific and potent MDM2 inhibitor showing promising anti-tumor activity in vitro and in vivo. The sensitivity gene signature might serve as a pharmacologically defined surrogate for p53 functionality that is critical for predicting clinical benefits from DS-3032b treatment. Citation Format: Kenji Nakamaru, Takahiko Seki, Koichi Tazaki, Archie Tse. Preclinical characterization of a novel orally-available MDM2 inhibitor DS-3032b: Anti-tumor profile and predictive biomarkers for sensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B5.

Abstract B1: Gene expression and TP53 mutation analysis predict sensitivity of leukemia cells to MDM2 inhibition by DS-3032b

Background: MDM2 overexpression, by preventing p53 activation, contributes to the growth and development of a variety of solid tumors and hematologic malignancies; hence, MDM2 inhibition could be a promising novel therapeutic strategy. Several MDM2 inhibitors have shown promise in early clinical trials. While preclinical studies generally reveal a requirement of wild-type (wt) TP53 for activity, tumor response to MDM2 inhibitors varies widely in the clinic and may not be strictly linked to TP53 mutational status. Identification of predictive biomarkers is therefore needed to enrich for patients with high likelihood of response. We here propose two gene signature-based models to predict the sensitivity of AML cells to MDM2 inhibition using two different methods. Methods: Leukemia samples isolated from peripheral blood or bone marrow of patients with newly diagnosed or relapsed/refractory AML were treated using DS-3032b (Daiichi-Sankyo), a dispiropyrrolidine-based, highly potent MDM2 inhibitor currently undergoing clinical trials in solid and hematological malignancies. Forty-one primary AML samples were treated ex vivo for 48 hours with DS-3032b (0, 25, 50, 100, 250, 500, and 1000 nM), and live cell numbers were determined. To define drug sensitivity/resistance, area under the curve (AUC) values, based on%live cell number measured at each concentration, were calculated. Baseline whole-genome RNA expression profile (Affymetrix Human Genome U133 Plus 2.0 Array) and TP53 mutation status (next generation sequencing) were determined. In the first model, we validated a predictive 175-gene signature that was established in a wide range of cancer cells by Daiichi Sankyo. In the second model, we used the random forest method with cross validation to establish a new predictive gene signature. Results: Eight samples (20%) had TP53 mutations. 6/8 (75%) p53 mutant and 8/33 (24%) of p53 wt samples were resistant (p = 0.01). In the first model, 11 each p53 wt samples were selected as sensitive or resistant to DS-3032 based on AUC values, and the 175-gene signature was applied. The prediction accuracy was 72%. In the genotype mixed samples, 14 each sensitive and resistant samples were selected, and the prediction accuracy was 79%. In the second model, we focused on 33 p53 wt samples and trichotomize the samples in the same way as in the first model, and investigated the accuracy of gene expression-derived prediction model with (A) 1500 gene set with the highest variance in mRNA expression (unbiased approach), (B) 32 gene set derived from previous studies (referenced approach), (C) combined (A+B) gene set. The sensitivities to predict cases with high drug sensitivity were 72%, 73% and 82% in scenarios (A), (B) and (C), respectively. The analysis was then extended to all 41 samples and the sensitivity to predict cases with high drug sensitivity remained high (64%, 64% and 72%). The results indicate that an unbiased approach can create a prediction model as accurate as the referenced approach, and moreover, that the combining approach can provide the highest prediction of sensitivity to the MDM2 inhibitor. Conclusion: The two models reported here could provide a novel strategy to identify the optimal gene signatures for predicting the cases most sensitive to MDM2 inhibitors prior to therapy. These models will be tested in an ongoing AML phase 1 clinical study of DS-3032b. Citation Format: Jo Ishizawa, Kenji Nakamaru, Takahiko Seki, Koichi Tazaki, Kensuke Kojima, Dhruv Chachad, Archie Tse, Arvind Rao, Michael Andreeff. Gene expression and TP53 mutation analysis predict sensitivity of leukemia cells to MDM2 inhibition by DS-3032b. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B1.

Predictive Gene Signatures Determine Tumor Sensitivity to MDM2 Inhibition

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

Abstract LB-252: IDH mutations are promising targets for acute myeloid leukemia

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. 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 IDH2/R140Q decreased the level of 5hmC and the expression of differentiation-inducing genes, including Ebf1, Spib and Pax5. Gene expression analysis revealed that IDH2/R140Q activated the hypoxia pathway and the expression of Meis1. 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. Citation Format: Yoko Ogawara, Hironori Matsunaga, Takahiko Seki, Yukino Machida, Kazushi Araki, Issay Kitabayashi. IDH mutations are promising targets for acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-252. doi:10.1158/1538-7445.AM2015-LB-252