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Supplementary Table 4: List of cell lines evaluated for sensitivity to SHP099 (Tab 1) or an RTK-inhibitor (Tab 2) in 2D or 3D. Included is the lineage and the KRAS mutation status. Where data are blank, no viable data was available, or the cell line did not grow appropriately as a tumor spheroid.

Supplementary Table 3: A total of 246 cell lines were evaluated for both SHP2 knockdown and SHP099 sensitivity. Data displays PTPN11 shRNA ATARIS Quantile Score and SHP099 IC50 and Amax for each cell line tested.

In vivo efficacy of SHP099 (100 mg/kg, daily) in the KYSE-520 esophageal cancer cell line model. Data are plotted as the treatment mean {plus minus} s.e.m (n=7) ( (B-I) In vivo efficacy data for cell line models represented in Fig. 3E. SHP099 and trametinib were orally administered at the doses, schedules and for the duration indicated. Data are plotted as the treatment mean {plus minus} s.e.m.

(A) Immunoblot of p-RSK3 and qPCR for DUSP6 from MIA PaCa-2 xenografts collected 3 hours after the last dose from Fig 5C. (B) Immunoblot for the designated proteins from MIA PaCa-2 cells grown in 2D, 3D and from in vivo xenografts without compound treatment. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor. (C) Dependency of MET by DRIVE pooled shRNA screen (y axis, ATARIS Quantile score of less than -0.5 indicates a significant effect) and expression of HGF (x-axis) by RNAseq in pancreatic cancer cell lines in CCLE (n=21). (D) Immunoblot of p-RSK3 and qPCR for DUSP6 from KP4 xenografts collected 3 hours after the last dose from Fig 5E. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor (E) Immunoblot for the designated proteins from KP4 cells grown in 2D, 3D and from in vivo xenografts without compound treatment (F) Schematic of RTK-SHP2 signaling highlighting that SHP2 acts downstream of one or more activated RTKs to elicit downstream signaling in KRAS mutant and also additional SHP2-specific, non-MAPK signaling. SHP2 inhibition by SHP099 can serve as a surrogate for cancers where KRAS mutant cancers are dependent on upstream RTKs.

Supplementary Table 1: List of all cell lines in the CCLE (Cancer Cell Line Encyclopedia) where sensitivity to SHP2 (PTPN11) knockdown was evaluated in 2D. Lineage, and genetic status of KRAS and BRAF are shown. Blank cells represent cell line data where genetic or sensitivity data do not exist. Values depicted are the ATARIS quantile normalized z-score. Additional details on the methodology of the screen are published (58).

(A) Effects of SHP099 and erlotinib on proliferation of EGFR-dependent and RAS/RAF wildtype Detroit-562 and KYSE520 cells grown in 2D monolayer or 3D spheroids (with 20% matrigel) for 6 days. The colored dotted lines at the bottom of graphs are the percentage of Day 0 reading of DMSO-treated cells normalized to that of Day 6 in 2D and 3D, respectively. The black hyphenated lines visualize IC50 values. Error bars denote standard error of the mean. (B-C) Immunoblot of indicated proteins in T3M-4 cells treated with DMSO, SHP099 (10 µM), or trametinib (10 nM) for 2 hours in 2D and 3D culture, respectively. Tubulin serves as a protein loading control. (D) Heatmap depicts Log2 fold change of genes differentially regulated in 2D and 3D following 19 hours after treatment with SHP099. Duplicate samples were analyzed and are depicted. GO ontology was performed using DAVID (42,43) derived from genes with 2-fold change relative to DMSO control.

Supplementary Material and Methods. File contains the following: Transcriptome sequencing and analysis, Soft agar assay, 2D and 3D Cell proliferation screen and compound characterization information.

(A) In vivo primary human colorectal xenograft model HCOX4087 treated with trametinib (0.3 mg/kg QD), SHP099 (100 mg/kg QD), and a pan-RTK inhibitor, Dovitinib (100 mg/kg QD). (B-C) In vivo efficacy of selective VEGFR2 inhibitor, BFH772 (3 mg/kg QD) in MIA PaCA-2 and T3M-4 cells implanted subcutaneously. Data plotted are tumor volume means {plus minus} s.e.m (T3M-4, n=8. MIA PaCA-2, n=7) (D) Evaluation of SHP099 in firefly- luciferase labeled MIA PaCa-2 pancreatic cells implanted surgically into the mouse pancreas. Data plotted are mean bioluminescent signal (BLI) {plus minus} s.e.m (n=6). (E-F) Immunoblot of SHP2 and soft agar assay with MIA Paca-2 cells with Dox-inducible shRNA targeting SHP2 or control non-targeting shRNA after Dox treatment. (G) In vivo SHP2 expression levels evaluated by Western blot and levels of MAPK pathway suppression by DUSP6, 3 hours after the last dose of SHP099 from MIA PaCa-2 tumors in Fig. 4F (n=3).

Supplementary Table 2: List of all cell lines in the CCLE where sensitivity to SHP099 was evaluated in 2D. Lineage and genetic status of KRAS and BRAF are shown. Blank cells represent cell line data where genetic or screening data do not exist. Data depicted in Figure 1B are IC50 values. This table includes all sensitivity data based on both IC50 and Amax data.

PDF file - 348KB, Figure S1: Isogenic IDH1 mutation compromises metabolic reprogramming under hypoxia. Figure S2: Simulated and measured uncorrected MIDs. Figure S3: Compromised Reductive TCA Metabolism is specific to cells with mutant IDH1. Figure S4: Cells with endogenous IDH1 and IDH2 mutations respond differently to mitochondrial stress. Figure S5: Inhibition of mutant IDH1 does not rescue reprogramming of TCA metabolism. Figure S6: Cells expressing mutant IDH1 are sensitive to pharmacological inhibition of mitochondrial oxidative metabolism.

PDF file - 93KB, Estimates Fluxes for HCT116 Parental and IDH1 R132H/+ 2H1 cells under Normoxia and Hypoxia (2 percent Oxygen).

PDF file - 136KB

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 13C 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. Cancer Res; 74(12); 3317–31. ©2014 AACR.

KRAS, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro, sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo, suggesting a MAPK pathway–dependent mechanism and possible non-MAPK pathway–dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRASG12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.

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 13C 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., National Institutes of Health (U.S.) (Grants R01CA168653 and 5-P30-CA14051-39), David H. Koch Institute for Integrative Cancer Research at MIT. DFHCC Bridge Project, Burroughs Wellcome Fund, Smith Family Foundation, Virginia and D.K. Ludwig Fund for Cancer Research, Damon Runyon Cancer Research Foundation

The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2017-2084

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