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The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

PDF File -1 MB, Supplemental data is to further support the statements made it the main text. Captions: Supplemental Table 1: Cell Lines used in the CRISPR pooled screen. Supplemental Table 2: Table of ovarian lines screened for tazemetostat sensitivity along with mutational and protein status for SMARCA2, SMARCA4, and ARID1A. Supplemental Table 3: p-values of COV434 SCCOHT cell line treated with tazemetostat. Supplemental Table 4: p-values of JHOC-5 clear cell ovarian cell line treated with tazemetostat. Supplemental Table 5: p-values of cell cycle analysis after tazemetostat treatment. Supplemental Table 6: p-values of apoptosis analysis after tazemetostat treatment. Supplemental Table 7: LogP scores for SWI/SNF components in ovarian lines +/- tazemetostat treatment. Supplemental Figure 1: Baseline measurement of histone 3 lysine 27 trimethyl mark and EZH2. Supplemental Figure 2a: Growth curves of SCCOHT and ovarian cancer cell lines treated with tazemetostat. Supplemental Figure 2b: Growth curves of SCCOHT and ovarian cancer cell lines treated with EPZ-007210. Supplemental Figure 3: Reduction in H3K27me3 mark after treatment. Supplemental Figure 4a: Sensitivity to PLK1 knockout as pan-essential in CRISPR pooled screen. Supplemental Figure 4b: Control sgRNAs from CRISPR pooled screen. Supplemental Figure 5: TOV112D xenograft tumor growth inhibition, tumor volume, and methyl mark. Supplemental Figure 6: body weights of mice treated with tazemetostat. Supplemental Figure 7: measured blood plasma levels of tazemetostat from xenograft mice.

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.

Gastrointestinal stromal tumor (GIST) is the most common type of sarcoma, with approximately 5,000 patients diagnosed per year in the US. Approximately 80% of patients with GIST present with mutations in the c-KIT oncogene at exon 9 or 11, which leads to constitutive, ligand-independent activation of the KIT receptor tyrosine kinase. For patients with metastatic GIST, frontline therapy with imatinib is effective, with a response rate of approximately 51-54% and median progression-free survival (PFS) of 19-23 months, in a molecularly unselected population. Other agents are approved for advanced GIST, without molecular selection, after progression on imatinib, including sunitinib, regorafenib, and ripretinib; however, response rates are less than 10% with PFS of approximately 5-6 months. Notably, patients who progress on imatinib and other tyrosine kinase inhibitors may develop a variety of on-target resistance mutations in the KIT oncogene, such as those in exon 17 (including at amino acids D816 and D820, activation loop mutation), exon 13 (V654A, ATP-binding region mutation), and less frequently in exon 14 (T670I, gatekeeper mutation). Several KIT inhibitors have been developed to potently target the exon 17 resistance mutations (avapritinib and ripretinib); however, there remains an important medical need in 2nd- and 3rd-line therapy in a molecularly unselected population of imatinib-resistant GIST. This suggests more broad-spectrum KIT inhibition is likely required, a hypothesis supported by the observation of large inter- and intra-patient heterogeneity of KIT secondary mutations across hundreds of samples obtained from patients with GIST treated with avapritinib. Sequencing data from the NAVIGATOR phase 1 trial (NCT02508532) revealed that patients with KIT mutant GIST and with the KIT V654A secondary resistance mutation had a poor response to treatment with avapritinib. To address this, we developed a highly potent and selective inhibitor of KIT V654A. This inhibitor showed dose-dependent modulation of downstream pharmacodynamic markers and induced tumor regression in a mastocytoma xenograft model driven by an exon 11 plus 13 V654A resistance mutation. Importantly, this inhibitor was generally well-tolerated and showed high selectivity over wild-type KIT. These findings suggest this novel KIT inhibitor has the potential to be used as a single agent or combination therapy for patients with imatinib-resistant GIST harboring the KIT V654A mutation. Citation Format: Alexandra R. Grassian, Joseph Kim, Omar Ahmad, Kevin Barvian, Alison Davis, Tom Dineen, Wei Hu, Ebby Job, Ludivine Moine, Kate Newberry, Maria Roche, Doug Shorten, Yeon Sook Choi, Francis Wolenski, Sebastian Bauer, Cesar Serrano, Jonathan Trent, Suzanne George. Efficacy of a highly potent and selective KIT V654A inhibitor for treatment of imatinib resistant GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB565.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

The identification of new therapeutic targets is critical in the fight against cancer. Over the past few decades, the inhibition of kinase cancer drivers has dramatically changed the cancer treatment landscape. Kinases play an important role in modulating the cellular signaling cascades that are often altered in cancer. This role is mainly carried out by the kinase domain(s) which control catalytic activity through binding ATP. Consequently, small molecule inhibition of catalytic activity can disrupt cancer signaling and halt tumor growth. Although examples of kinase drivers in cancer development are well established, there are still numerous understudied kinases with limited information about their biological function. Therefore, we designed a comprehensive kinase-focused CRISPR library specifically aimed at interrogating the contribution of kinase catalytic activity. Our library contains a high density of guides targeting the kinase domain, allowing for quick assessment of the importance of the active site. Our library aims to create opportunities for identifying novel therapeutic targets and for further understanding the cancer signaling mechanisms. We utilized our newly designed library in a high throughput, kinome-wide CRISPR pooled screen applied to multiple cellular models. CRISPR Pooled Screening is a powerful tool for conducting large-scale studies involving hundreds or thousands of genes. However, due to a general preference to increase throughput, most screening efforts include a limited number of guides per gene. This increases chances of detecting false positives and reduces the screen's power to differentiate between the activity of different gene domains. In our library, however, we used a minimum of 24 guides per gene strategically distributed both inside and outside the catalytic domains of each kinase. Additionally, our library allows us to rule out likely false-positive hits due to genomic copy number amplifications through the addition of guides targeting the nearest neighbor gene. As a reference, we used the Broad Institute's Achilles Avana CRISPR Screen. Our library was able to reproduce 100% of Avana's significant kinase hits. Furthermore, we identified novel hits that did not show dependency in Avana. These hits produced a phenotype only when their kinase domain was targeted and did not show any phenotype for the guides outside the kinase domain. Most importantly, our screen highlighted the value of domain-centric CRISPR libraries and how they could be used for identifying kinase targets that have so far eluded previous screens. Citation Format: Ahmad Al Kawam, Alexandra R. Grassian, Fabien Llambi, Erica Evans, Klaus Hoeflich, Chaoyang Ye. Assessing the importance of the kinase domain through kinome-focused CRISPR screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5159.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

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

Purpose of review Exome sequencing studies have recently expanded the genetic characterization of intrahepatic cholangiocarcinomas. Among a number of novel genes, isocitrate dehydrogenase (IDH) is recurrently mutated in intrahepatic cholangiocarcinomas. We review the effects of these mutations on several biochemical pathways, as well as potential changes to downstream signaling pathways. Recent findings Hotspot mutations in IDH isoforms 1 or 2 occur in approximately 15% of intrahepatic cholangiocarcinomas. These mutations result in elevated levels of an oncometabolite, 2-hydroxyglutarate, which is associated with higher DNA CpG methylation and altered histone methylation that accompany a block in cellular differentiation. Exploratory studies have suggested additional phenotypes associated with IDH1/2 mutations. Summary Tumors with IDH1 or IDH2 mutations may represent a distinct subtype of cholangiocarcinomas. Further studies are required to elucidate the exact role that mutant IDH1/2 and 2-hydroxyglutarate play in tumorigenesis, and what are the best strategies to target these tumor types.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival.

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma—a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein—display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECM-attached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation.

Epithelial cells are dependent on extracellular matrix (ECM) attachment for maintenance of metabolic activity and suppression of apoptosis. Here we show that loss of ECM attachment causes down-regulation of epidermal growth factor receptor (EGFR) and β1 integrin protein and mRNA expression and that ErbB2, which is amplified in 25% of breast tumors, reverses these effects of ECM deprivation. ErbB2 rescue of β1 integrin mRNA and protein in suspended cells is dependent on EGFR, however, the rescue of EGFR expression does not require β1 integrin. We show that there is a significant decrease in the stability of EGFR in ECM-detached cells that is reversed by ErbB2 overexpression. Rescue of both EGFR and β1 integrin protein by ErbB2 is dependent on Erk activity and induction of its downstream target Sprouty2, a protein known to regulate EGFR protein stability. Interestingly, expression of EGFR and β1 integrin protein is more dependent on Erk/Sprouty2 in ECM-detached ErbB2-overexpressing cells when compared with ECM-attached cells. These results provide further insight into the ErbB2-driven anchorage independence of tumor cells and provide a new mechanism for regulation of EGFR and β1 integrin expression in ECM-detached cells.

Background Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. Results Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. Conclusion This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection. Electronic supplementary material The online version of this article (doi:10.1186/s12575-015-0028-4) contains supplementary material, which is available to authorized users.

The histone methyltransferase EZH2 is the enzymatic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of H3K27 thereby repressing target gene transcription. EZH2 is amplified, overexpressed, or mutated in multiple cancer types, most notably Follicular Lymphoma (FL) and germinal center Diffuse Large B-cell Lymphoma (GCB-DLBCL). We previously reported that preclinical models of malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SNF5, SMARCB1), are selectively killed by potent and selective inhibitors of EZH2. Here we report another class of SWI/SNF-altered cancers named small cell carcinoma of the ovary hypercalcemic type (SCCOHT) that is dependent on EZH2 activity. SCCOHT is a very aggressive form of cancer that responds poorly to conventional therapy with a one-year overall survival rate of only 50%. Very few novel agents have been approved for this indication; thus there is a need for targeted therapeutics in SCCOHT. SMARCA4 and SMARCA2 are co-inactivated in this tumor type that has many rhabdoid features. We demonstrate that tazemetostat, an EZH2 inhibitor currently in phase 2 clinical trials, induces potent and selective killing in SMARCA2 and SMARCA4-deficient ovarian cell lines. In addition to small molecule inhibitor data, we conducted functional genomics studies with CRISPR pooled screening, and confirmed that SCCOHT is also sensitive to CRISPR-mediated EZH2 gene ablation. Dose-dependent anti-tumor effects were observed upon tazemetostat treatment in SCCOHT xenografts deficient in both SMARCA2 and SMARCA4. We also report on additional non-ovarian tumor types with dual SMARCA2/SMARCA4 loss including NSCLC that exhibit EZH2 dependence representing additional potential therapeutic indications for tazemetostat treatment. Citation Format: Elayne Chan-Penebre, Kelli Armstrong, Allison Drew, Alexandra R. Grassian, Igor Feldman, Maria Roche, Peter Ho, Dorothy Brach, Alejandra Raimondi, Robert A. Copeland, Richard Chesworth, Jesse J. Smith, Scott A. Ribich. Selective killing of SMARCA2- and SMARCA4-deficient tumors by inhibition of EZH2: In vitro and in vivo preclinical models [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 3345. doi:10.1158/1538-7445.AM2017-3345

Target identification is a critical step in drug discovery, but the process has many challenges including non-specific reagents, limited ability to test numerous models, and incomplete target inhibition. Pooled screening with CRISPR/Cas9 permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To determine the specific dependencies of cell lines on epigenetic pathways, we designed a CRISPR/Cas9 library to target 640 epigenetic genes and screened more than 200 cell lines covering a variety of oncology indications, including breast, lung, and renal cell carcinoma (RCC). We find that CRISPR pooled screening is a highly effective approach for target identification and provides robust, highly reproducible data as long as a sufficient number of small guide RNAs are used. We identify known pan-essential genes, including in the transcription (CDK9), translation (EIF4A1 and EIF4A3) and splicing (SRSF2) machinery. We additionally identify many novel pan-essential genes across a variety of epigenetic pathways, including histone acetylases and deacetylases, chromatin remodeling factors, helicases and others. We also investigated epigenetic synthetic lethal interactions that have been previously reported. For example, it has been reported that the SWI/SNF family displays paralog synthetic lethality for SMARCA2 in the context of SMARCA4 mutations, and for ARID1B in the context of ARID1A mutations. While we do see that some of the same trends hold, the synthetic lethal relationship appears to be more complex than previously realized, including the need to examine mRNA levels in addition to mutation type. Most importantly, we identify more than 100 epigenetic genes which show selective sensitivity, i.e. where knockout shows an anti-proliferative effect in only a subset of the cell lines. These are the most promising targets for further drug discovery programs. We have used additionally CRISPR/Cas9-domain based screening to identify the functionally relevant sites for many of these genes. Furthermore, we can overlay gene expression and mutation data to identify novel synthetic lethal relationships. One gene that displays selective sensitivity is EGLN1, the prolyl hydroxylase for the hypoxia-inducible factor, HIF1α. We find that EGLN1 is required for proliferation only in RCC cell lines which retain wild-type VHL, another component of the hypoxia response pathway, which is frequently lost in RCC. As such, EGLN1 loss is synthetically lethal in the presence of wild-type VHL in RCC cells. Thus this approach not only identifies an enzymatic drug target but also a patient stratification method. Other novel synthetic lethal interactions have also been identified. Our data demonstrates that CRISPR pooled screening is a powerful technique for identification of epigenetic synthetic lethal interactions. Citation Format: Alexandra R. Grassian, Darren Harvey, Julian Fowler, Allison E. Drew, Igor Feldman, Richard Chesworth, Robert Copeland, Jesse J. Smith, Scott Ribich. CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships [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 406. doi:10.1158/1538-7445.AM2017-406

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

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 and 2-HG alter signaling pathways to promote cancer, however, remains unclear. Additionally, there exist relatively few cell lines with IDH mutations. To examine the effect of endogenous IDH mutations and 2-HG, we created a panel of isogenic epithelial cell lines with either wild-type IDH1/2 or clinically relevant IDH1/2 mutations. Differences were noted in the ability of IDH mutations to cause robust 2-HG accumulation. IDH1/2 mutants that produce high levels of 2-HG cause an epithelial-mesenchymal transition (EMT)-like phenotype, characterized by changes in EMT-related gene expression and cellular morphology. 2-HG is sufficient to recapitulate aspects of this phenotype in the absence of an IDH mutation. In the cells types examined, mutant IDH-induced EMT is dependent on up-regulation of the transcription factor ZEB1 and down-regulation of the miR-200 family of microRNAs. Furthermore, sustained knockdown of IDH1 in IDH1 R132H mutant cells is sufficient to reverse many characteristics of EMT, demonstrating that continued expression of mutant IDH is required to maintain this phenotype. These results suggest mutant IDH proteins can reversibly deregulate discrete signaling pathways that contribute to tumorigenesis.

Most tumors display enhanced glucose metabolism compared to normal tissues [1-6]. The preferential conversion of glucose to lactate in cancer cells (the Warburg Effect) was one of the first known differences between tumor and normal cells and is believed to contribute to enhanced growth in tumor cells. However, the extent to which specific metabolic fluxes originating from glucose branch from central carbon metabolism and are utilized for anabolic processes is poorly understood. Here, we used an integrated, quantitative metabolomics approach combining NMR experiments with heavy isotope labeling and targeted mass-spectrometry. We carried out direct measurements of metabolic fluxes emanating from glucose metabolism [7]. We found that in some cancer cells, a relatively large amount of glycolytic carbon was diverted into serine and glycine biosynthesis. Serine can be synthesized from a glycolytic intermediate, 3-phosphoglycerate (3PG). 3PG is oxidized by an enzyme phosphoglycerate dehydrogenase (PHGDH) in an NAD-dependent manner to generate phosphohydroxypyruvate which is transaminated and then dephosphorylated to generate serine. Serine hydroxymethyltransferase then donates the side-chain of serine to the Folate pool to generate glycine. We quantified the relative amount of glycolytic flux being diverted into serine and glycine biosynthesis and found that it was comparable to the amount of glucose passing through glycolysis and ultimately being converted to lactate. However, this large flux was observed to be present in only a subset of cultured cells. A non-tumorigenic breast epithelial cell line was found to have no detectable flux into serine and glycine biosynthesis. The observation that some but not all cancer cells divert a large amount of glycolytic flux into serine and glycine metabolism suggested that there might be a context in which this flux might be selected for in the development of cancer. To search for this context, we investigated a pooled analysis of 3131 human cancers [8]. This analysis revealed that PHGDH, the gene that encodes the first enzyme in this biosynthetic pathway branching off of glycolysis is frequently present in a region of recurrent, localized copy number gain at genomic locus 1p12 (q=1.18e-9). The amplification was most commonly found in melanoma but was observed in other cancers such as eosophageal adenocarcinoma and triplenegative breast cancer. We validated this finding in a collection of human melanoma tissue samples by comparing PHGDH expression in human tissues as measured by Immunohistochemistry with matched assessments of PHGDH copy number gain using fluorescence in situ hybridization. It was observed that PHGDH protein expression correlated with genomic copy number gain. To assess whether cells containing the amplification were differentially sensitive to inhibition of PHGDH expression, we considered a panel of tumor-derived human Melanoma cell lines. Each melanoma cell line with PGHDH amplification exhibited significant flux into serine biosynthesis. We then carried out an RNA interference study and found that decreased PHGDH expression by RNA interference impaired growth in cell lines containing amplification of PHGDH and resulted in a distinct metabolic phenotype marked by accumulation of glycolytic intermediates. PHGDH expression was also found to be associated with aggressive breast cancer subtypes. Since we observed no detectable flux in a non-tumorigenic breast epithelial cell line, we questioned whether enhanced PHGDH expression would have any phenotypic consequences. We considered the effects of PHGDH in a model of breast tissue morphogenesis [9]. In this model, ectopic expression of PHGDH in MCF10A cells induced loss of cell polarity, disruptions in nuclear architecture and rescue of anoikis – each being phenotypic alterations that predispose cells to tumorgenicity. Our findings demonstrate that altered metabolic flux stemming from glycolysis can be selected for in the development of cancer and contribute to cell transformation. Furthermore, our studies identify PHGDH as an attractive therapeutic target for subsets of human cancers.

Attachment to extracellular matrix (ECM) is required for the survival and proliferation of normal epithelial cells. Epithelial tumor cells, however, often acquire "anchorage independence," a property that may contribute to their ability to invade and grow in foreign environments. Although apoptosis is the most rapid and effective mechanism that causes the death of matrix-detached cells, it has become apparent that detachment from matrix alters other aspects of cell physiology prior to commitment to cell death and that some of these alterations can lead to cell death under conditions where apoptosis is suppressed. This report provides an overview of death processes that contribute to the death of matrix-detached normal cells and describes mechanisms that confer anchorage independence, with a focus on ECM regulation of cell metabolism. Loss of matrix attachment leads to metabolic stress characterized by reduced nutrient uptake, decreased ATP production, and increased levels of reactive oxygen species (ROS). The decrease in ATP levels is prevented by either constitutive activation of the PI3K/Akt pathway or exogenous antioxidants. Additionally, decreased Erk signaling in matrix-detached cells causes a disproportionate decrease in flux through pyruvate dehydrogenase (PDH), leading to decreased entry of glucose carbons into the citric acid cycle. Interestingly, forced overexpression of a PDH inhibitor suppresses de novo lipogenesis and proliferation, highlighting the importance of mitochondrial metabolism in supplying intermediates for biosynthetic processes required for proliferation. Thus, ECM attachment is a key regulator of cellular metabolism, and alterations in metabolism owing to changes or loss of ECM engagement during tumorigenesis may serve important tumor-suppressive functions.

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood [1, 2]. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer., National Institutes of Health (U.S.), National Cancer Institute (U.S.), Smith Family Foundation, Damon Runyon Cancer Research Foundation, Burroughs Wellcome Fund

Normal epithelial cells require matrix attachment for survival, and the ability of tumour cells to survive outside their natural extracellular matrix (ECM) niches is dependent on acquisition of anchorage independence. Although apoptosis is the most rapid mechanism for eliminating cells lacking appropriate ECM attachment, recent reports suggest that non-apoptotic death processes prevent survival when apoptosis is inhibited in matrix-deprived cells. Here we demonstrate that detachment of mammary epithelial cells from ECM causes an ATP deficiency owing to the loss of glucose transport. Overexpression of ERBB2 rescues the ATP deficiency by restoring glucose uptake through stabilization of EGFR and phosphatidylinositol-3-OH kinase (PI(3)K) activation, and this rescue is dependent on glucose-stimulated flux through the antioxidant-generating pentose phosphate pathway. Notably, we found that the ATP deficiency could be rescued by antioxidant treatment without rescue of glucose uptake. This rescue was found to be dependent on stimulation of fatty acid oxidation, which is inhibited by detachment-induced reactive oxygen species (ROS). The significance of these findings was supported by evidence of an increase in ROS in matrix-deprived cells in the luminal space of mammary acini, and the discovery that antioxidants facilitate the survival of these cells and enhance anchorage-independent colony formation. These results show both the importance of matrix attachment in regulating metabolic activity and an unanticipated mechanism for cell survival in altered matrix environments by antioxidant restoration of ATP generation.

Apoptosis is a cell suicide program that plays a critical role in development and tissue homeostasis. The ability of cancer cells to evade this programmed cell death (PCD) is a major characteristic that enables their uncontrolled growth. The efficiency of chemotherapy in killing such cells depends on the successful induction of apoptosis, since defects in apoptosis signaling are a major cause of drug resistance. Over the past decades, much progress has been made in our understanding of apoptotic signaling pathways and their dysregulation in cancer progression and therapy. These advances have provided new molecular targets for proapoptotic cancer therapies that have recently been used in drug development. While most of those therapies are still at the preclinical stage, some of them have shown much promise in the clinic. Here, we review our current knowledge of apoptosis regulation in cancer progression and therapy, as well as the new molecular targeted molecules that are being developed to reinstate cancer cell death.

Apoptosis is a cell suicide program that plays a critical role in development and tissue homeostasis. The ability of cancer cells to evade this programmed cell death (PCD) is a major characteristic that enables their uncontrolled growth. The efficiency of chemotherapy in killing such cells depends on the successful induction of apoptosis, since defects in apoptosis signaling are a major cause of drug resistance. Over the past decades, much progress has been made in our understanding of apoptotic signaling pathways and their dysregulation in cancer progression and therapy. These advances have provided new molecular targets for proapoptotic cancer therapies that have recently been used in drug development. While most of those therapies are still at the preclinical stage, some of them have shown much promise in the clinic. Here, we review our current knowledge of apoptosis regulation in cancer progression and therapy, as well as the new molecular targeted molecules that are being developed to reinstate cancer cell death.

Target selection for oncology and other indications is a critical step in the successful development of therapeutics, however it remains one of the most challenging areas of drug discovery. In fact, up to two-thirds of oncology relevant targets reported in literature have not been confirmed on follow-up studies, indicating that target validation in oncology is especially challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of specific loci offers an alternative method to RNA interference and complements small molecule inhibitors for determining whether or not a cell line is dependent on a specific gene product for proliferation and/or survival. Importantly, CRISPR-Cas9 may be advantageous for some studies as it offers efficient and specific gene knock-out leading to complete loss of protein function. This may be especially useful for some target classes, including epigenetic targets, which appear to require near complete loss of protein function to observe phenotypes. In our initial studies using CRISPR-Cas9 to verify the essential nature of EZH2 (Enhancer of Zest 2) expression for the proliferation of SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor cell lines, we observed that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity at least one allele of EZH2 remains functional, and this hypothesis suggests that carrying out CRISPR-Cas9 studies for individual target genes without analyzing single cell clones could produce misleading results. To verify this, we developed a medium throughput assay to analyze 10s-100s of single cell clones for target gene disruption using CRISPR-Cas9 gene knockout, followed by a restriction digest and fluorescent undigested fragment length analysis to successfully assess EZH2 allele status. Significantly, these data support our hypothesis that retention of one functional copy of EZH2 is required for the proliferation of EZH2-dependent cell lines and that this can be rapidly assessed by our assay. Thus, the assessment of zygosity of the gene of interest can be evaluated in a medium-throughput manner in single cell clones. The assay is thereby able to unambiguously indicate whether or not a specific gene is essential for survival and/or proliferation in a given cell line, and offers a unique approach for target validation using gene editing. Importantly, this approach should be applicable to any target of interest that is expected to affect cell proliferation or survival. Such data can aid in the development of more robust cancer therapeutics by increasing confidence in target selection. Citation Format: Alexandra R. Grassian, Tim Scales, Sarah K. Knutson, Nicola J. McCarthy, Chris E. Lowe, Jon D. Moore, Robert A. Copeland, Heike Keilhack, Jesse J. Smith, Julie A. Wickendon, Scott Ribich. A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality. [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 C162.

It has become clear in the past decade that dysregulation of epigenetic pathways is fundamental to many if not all tumors. Importantly, a number of epigenetic targeted therapies are now being tested in the clinic and are beginning to show promising efficacy. Identification of new targets for oncology therapeutics is critical, and the ideal target should: 1) be effective in a specific indication or genetically defined patient population; and 2) lead to a minimal amount of deleterious side effects in patients. Thus, target identification must be performed in a large number of cell lines to address both objectives and to ensure specific target dependence. However, it remains challenging to identify specific dependencies on epigenetic genes in preclinical models, which may result in part from the need for near complete loss of protein function for this class of enzymes to observe proliferation phenotypes, and this can be difficult to achieve with RNAi reagents. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology enables the specific and complete knockout of the target protein and allows for observation of proliferation phenotypes that RNAi studies may not uncover. Additionally, the CRISPR-Cas9 system is amenable to pooled cell line screening which permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To examine the specific dependencies of cell lines on epigenetic pathways, we have used pooled CRISPR-Cas9 screening to interrogate over 600 epigenetic-related genes in a panel of more than 60 cells lines. The performance of the screen is highly consistent and able to reproduce findings that have been previously reported. We observe that CRISPR pooled screening is highly effective at identifying targets which are known to be required for cell proliferation either in all cell lines or in a genetically-defined subset of cell lines. We also identify new epigenetic-related genes required for the proliferation of almost all the cell lines tested, and have termed these “pan-essential” epigenetic genes. Intriguingly, these pan-essential genes represent members of almost all classes of epigenetic pathways, including histone methyltransferases, histone acetylases and deacetylases, chromatin remodeling factors, regulators of mRNA splicing, DNA helicases and others. This is an important set of genes to identify, as they represent targets which are likely to induce broad clinical toxicity if inhibited in patients, yet may be identified as potential targets in pre-clinical interrogation which does not fully examine proliferation dependencies in a sufficiently broad panel of cell lines. Importantly, we also identify a variety of epigenetic targets which induce altered proliferation in a subset of cell lines tested. These include epigenetic-related genes from many classes, including histone methyltransferases. Notably, for certain genes, trends are emerging that indicate a specific genetic marker(s) which may predict dependence on these epigenetic targets. These genes represent highly promising targets for epigenetic therapeutics in a variety of oncology indications. Citation Format: Alexandra R. Grassian, Julian Fowler, Igor Feldman, Thomas Riera, Darren Harvey, Allison E. Drew, Richard Chesworth, Robert A. Copeland, Heike Keilhack, Jesse J. Smith, Scott Ribich. CRISPR pooled screening identifies differential dependencies on epigenetic pathways. [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 B78.

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

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA). We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines. The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism). These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [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 B159.

Recent studies from our laboratory revealed the importance of extracellular matrix in regulating cellular metabolism. Briefly, detachment of mammary epithelial cells from extracellular matrix (ECM) causes a significant reduction in ATP levels due to reduced glucose uptake. ErbB2 overexpression restores ATP levels by maintaining glucose uptake in ECM detached cells. The loss of glucose uptake also increases ROS levels, and unexpectedly, antioxidants rescue the metabolic defect, by relieving an ROS-mediated inhibition of ATP generation by fatty acid oxidation. These findings suggest that ECM contact is an important regulator of cellular metabolism, and that tumor cells may be able to circumvent the requirement for ECM-attachment via overexpression of certain oncogenes. We used 13C metabolic flux analysis to more thoroughly investigate the metabolic changes induced by ECM detachment and ErbB2 overexpression in the immortalized, non-transformed human mammary epithelial cell-line, MCF10A. Our results show that matrix-detachment leads to a dramatic decrease in nutrient uptake, including glucose, glutamine and pyruvate. Interestingly, we found that there is a substantial decrease in glucose flux to the tricarboxylic acid (TCA) cycle; this is due both to the decreased glucose uptake, as well as an increased diversion of glucose towards lactate production (aerobic glycolysis). ErbB2 overexpression partially reverses these defects in the ECM-detached cells. Glucose entrance to the TCA cycle is regulated by pyruvate dehydrogenase (PDH), which are in turn negatively regulated by PDH kinases (PDK1-4). Interesting, we find a dramatic increase in PDK4 levels in ECM-detached cells, which is partially prevented by ErbB2 overexpression. While PI3K signaling has previously been shown to negatively regulate PDK4 expression, we find that Mek, but not PI3K, regulates PDK4 levels in our system. Ongoing studies will further examine the regulation of glucose and pyruvate metabolism by ErbB2 and ECM. These analyses will allow us to identify unpredictable nodes of metabolic regulation by ErbB2 and ECM and may provide rational treatment approaches for targeting tumor metabolism. 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 2802. doi:10.1158/1538-7445.AM2011-2802