Kelly Slocum

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

Antitumor Activity of the Hsp90 Inhibitor IPI-504 in HER2-Positive Trastuzumab-Resistant Breast Cancer

Hsp90 facilitates the maturation and stability of numerous oncoproteins, including HER2. The aim of this study was to assess the antitumor activity of the Hsp90 inhibitor IPI-504 in trastuzumab-resistant, HER2-overexpressing breast cancer cells. Therapy with trastuzumab, IPI-504, and the combination of trastuzumab and IPI-504 was evaluated in trastuzumab-sensitive and trastuzumab-resistant cells. Inhibition of protein targets, cell proliferation, and tumor growth was assessed in vitro and in xenograft models. IPI-504 inhibited proliferation of both trastuzumab-sensitive and trastuzumab-resistant cells. Administration of IPI-504 markedly reduced total levels of HER2 and Akt, as well as phosphorylated Akt and mitogen-activated protein kinase (MAPK), to an equal extent in trastuzumab-sensitive and trastuzumab-resistant cells. IPI-504, used as single agent or in combination with trastuzumab, also inhibited in vivo the growth of both trastuzumab-sensitive and -resistant tumor xenografts. As a mechanism for the observed antitumor activity, IPI-504 resulted in a marked decrease in the levels of HER2, Akt, p-Akt, and p-MAPK in trastuzumab-resistant xenografts as early as 12 hours after a single dose of IPI-504. IPI-504–mediated Hsp90 inhibition may represent a novel therapeutic approach in trastuzumab refractory HER2-positive breast cancer. Mol Cancer Ther; 10(5); 817–24. ©2011 AACR.

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and Mutant Specific Inhibitors of IDH1

High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure-activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.

Discovery and Evaluation of Clinical Candidate IDH305, a Brain Penetrant Mutant IDH1 Inhibitor

Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Abstract LB-139: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH alters central carbon metabolism, though, remains unclear. To address this question, we performed 13C metabolic flux analysis (MFA) on an isogenic cell panel containing heterozygous IDH1/2 mutations. We observe a dramatic and consistent decrease in the ability of IDH1, but not IDH2, mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate to isocitrate. Additionally we find that cells with IDH1 mutations exhibit increased oxidative tricarboxylic acid (TCA) metabolism. Similar metabolic trends were observed in vivo as well, and also in endogenous, non-engineered IDH1/2 mutant cell lines. Interestingly, IDH1-mutant specific inhibitors were unable to reverse the decrease in reductive metabolism, suggesting that this metabolic phenotype is independent of 2-HG. Furthermore, this metabolic reprogramming increases the sensitivity of IDH1 mutant cells to hypoxia or electron transport chain (ETC) inhibition in vitro . IDH1 mutant cells also grow poorly as subcutaneous xenografts within hypoxic in vivo microenvironments. These results suggest that exploiting metabolic defects specific to IDH1 mutant cells could be an interesting avenue to explore therapeutically. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Ajit Divakaruni, Courtney Green, Xiamei Zhang, Kelly Slocum, Minying Pu, Fallon Lin, Chad Vickers, Carol Joud-Caldwell, Franklin Chung, Hong Yin, Erika Handly, Christopher Straub, Joseph D. Growney, Matt Vander Heiden, Anne Murphy, Raymond Pagliarini, Christian Metallo. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2014-LB-139

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Orally Bioavailable and Brain Penetrant Mutant IDH1 Inhibitors

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

Abstract A144: Inhibition of 2-HG production in IDH mutant xenograft models.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) oxidize isocitrateto α-ketoglutarate (α-KG), a cofactor needed for the function of over 100 enzymes1. Heterozygous mutations of IDH1/2 occur in a variety of tumor types including glioma, acute myeloid leukemia (AML), cholangiosarcoma, chondrosarcoma and melanoma2. Mutant IDH is crippled for wild-type function but gains the ability to convert α-KG to 2-hydroxyglutarate (2-HG), a believed “oncometabolite” that may alter cell biology through, in part, changes in global histone and DNA methylation 1,3,4. However, the dearth of in vivo models dependent upon mutant IDH has made understanding the biological relevance of IDH mutations and 2-HG production in cancer challenging. To better understand the role that IDH mutations play in oncogenesis, and the potential effects of inhibition of these mutations, we introduced a heterozygous point mutation of IDH1 (R132H) into the endogenous locus of the HCT116 colon carcinoma cell line. When compared with the parental IDH1 wild-type HCT116 model, HCT116 IDH1R132H/+ xenograft tumors produce significantly higher levels of 2-HG. In addition, we have identified a patient-derived melanoma model, HMEX2838, which harbors an endogenous IDH1R132C/+ mutation and expresses high levels of 2-HG. To better explore the biology of mutant IDH inhibition, we developed a mutant-selective IDH inhibitor with favorable in vivo properties. Using this compound we demonstrate that 2-HG production is strongly inhibited in both the engineered HCT116 IDH1R132H/+ xenograft model and the endogenously mutant HMEX2838 patient-derived xenograft model. The data we have generated suggests that this inhibition of 2-HG directly correlates with the area under the curve (AUC) of free-drug above the HCT116 IDH1R132H/+ cellular IC50, as determined in vitro. Using these models and our IDH inhibitors we hope to gain a better understanding of how IDH mutations contribute to cancer and how inhibition of the mutant enzyme could benefit patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A144. Citation Format: Kelly L. Slocum, Julia Downall, Ty Gould, Mohammad Zafari, Stephanie Dodd, Brant Firestone, Ray Pagliarini, Julian Levell. Inhibition of 2-HG production in IDH mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A144.

Abstract 1601: Hsp90 inhibition results in a significant delay in tumor progression in a model of emerging EGFR TKI resistance in non-small cell lung cancer

Heat shock protein 90 (Hsp90) plays a role in regulating the stability of key cancer-causing proteins through its role as a protein chaperone. Proteins chaperoned by Hsp90, known as client proteins, include cancer-causing forms of ALK, BCR-ABL, EGFR, FLT3 and HER2. Infinity is developing two drug candidates in its Hsp90 chaperone inhibitor program: IPI-504 (retaspimycin hydrochloride), an intravenously-administered small molecule, and IPI-493, which is administered orally. EGFR tyrosine kinase inhibitors (TKIs) are an effective treatment for lung cancer patients with activating mutations in EGFR. After a dramatic initial response, however, most patients become resistant to drug treatment and progress. In about half of these cases, resistance is due to a second point mutation in EGFR (T790M). It is believed that in at least some of these cases, the TKI resistance mutations are pre-existing and that treatment with TKIs selects for the resistant cells. In an effort to model the emergence of resistance to TKIs from pre-existing mutations, we developed a novel in vivo model, where gefitinib treatment initially leads to tumor regression followed by rebound of tumor growth and outgrowth of drug resistant clones containing the T790M mutation. We show that in this model, treatment with IPI-493 alone and IPI-493 following gefitinib resulted in tumor growth inhibition of 61 and 77%, respectively, when compared with gefitinib treatment alone. Treatment with IPI-493 alone also resulted in a significant delay in time to tumor progression with ∼40% of animals still on study 45 days following tumor implant; all animals treated with either vehicle or gefitinib had been removed due to tumor progression. Interestingly, treatment with IPI-493 following gefitinib resulted in an even more impressive delay in time to progression, with >50% of animals still on study on day 65 post-implant. These results suggest that further studies with Hsp90 inhibitors in EGFR mutant NSCLC patients who have been pre-treated with a TKI may be warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1601. doi:10.1158/1538-7445.AM2011-1601

Abstract A215: Hsp90 inhibitor IPI‐504 demonstrates antitumor activity against a panel of neuroendocrine/carcinoid cell lines in vitro and in vivo

Heat shock protein 90 (Hsp90), an emerging target in cancer, is a chaperone protein that contributes to tumorigenesis by maintaining the stability and activity of multiple oncogenic proteins. Infinity Pharmaceuticals is currently developing two Hsp90 inhibitors: IPI‐504 (retaspimycin hydrochloride), an intravenous agent that has entered several phase II clinical trials, and IPI‐493, an orally administered compound that is currently being tested in a phase I dose‐escalation trial. In pre‐clinical animal models, IPI‐504 and IPI‐493 have demonstrated anti‐tumor activity against a wide variety of tumor types. Neuroendocrine tumors form a heterogeneous group of malignancies, which differ from each other in their biology, prognosis, and genetics. They mainly occur in the gastrointestinal tract and bronchopulmonary system. Conventional cytotoxic therapy has failed to demonstrate effective treatment of neuroendocrine/carcinoid cancers over the past 4 decades, resulting in an unmet need for improved pharmacological treatment of these tumors. To date, no data have been reported on the activity of Hsp90 inhibitors against carcinoid cancer cells. Herein we describe the activity of the Hsp90 inhibitors IPI‐504 and IPI‐493 against a panel of neuroendocrine tumors including: BON‐1, a cell line derived from a metastasis of a primary pancreatic neuroendocrine tumor, NCI‐H720, a lung carcinoid, QGP‐1, a pancreatic carcinoma of islet cells and HC‐45, an ileal carcinoid. In vitro, most of the carcinoid cell lines are sensitive to the Hsp90 inhibitors with EC50 values between 10 and 189 nM for IPI‐493 and 49 and 930 nM for IPI‐504. In vivo, IPI‐504 dosed twice weekly inhibits the tumor growth of BON‐1 cells in mice. To identify the potential mechanism of action of IPI‐504 in BON‐1 cells we have determined that IGF‐1R, a tyrosine kinase receptor and an Hsp90 client protein, is constitutively activated in these cells suggesting that it could participate in the unregulated growth of these tumor cells. Upon treatment of BON‐1 cells with IPI‐504, phospho‐IGF‐1R is degraded in a dose-dependent manner. The EC50 of the protein degradation and the in vitro growth inhibitory activity of IPI‐504 are similar (∼50 nM), suggesting that the anti‐tumor activity of IPI‐504 could be due, in part, to the inactivation of this growth factor receptor. In conclusion, we demonstrated that the Hsp90 inhibitors IPI‐504 and IPI‐493 potently inhibit the growth of neuroendocrine tumor cell lines both in vitro and in vivo and identified IGF‐1R as a potential client protein. These data suggest further exploring IPI‐504 for the possible treatment of neuroendocrine tumors in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A215.