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Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.

One of the most compelling approaches to reduce cancer-specific morbidity and mortality is earlier detection. Minimally-invasive screening tests, such as liquid biopsies, hold much promise in this regard. However, prospective clinical studies of these new methods are required to ensure that the anticipated benefits outweigh the potential risks. We describe an interim analysis of a study that enrolled 10,006 women 65 to 75 years of age who had no prior history of cancer, in a population with a high degree of compliance with standard-of-care screening. Enrollment occurred between September 2017 and May 2019. The study evaluated the prospective implementation of a multi-cancer, multi-analyte screening blood test (DETECT-A blood test) incorporating DNA and protein markers. Participants with positive DETECT-A blood test results were followed by an imaging test (generally PET-CT) to localize the putative cancer. The DETECT-A blood test combined with standard-of-care screening doubled the number of cancers detected by screening. The DETECT-A blood test effectively identified subjects with multiple types of cancer, with a positive predictive value higher than existing non-invasive screening tests for individual cancers. The DETECT-A blood test identified ten different cancer types, including seven types for which there is currently no screening test available. No serious adverse events were observed in any participants enrolled in the study. 65% of the cancers detected by the blood test were classified as local or regional disease. Somatic mutations in TP53, BRAF, KRAS, or PIK3CA were the most common alterations identified in plasma; in all cases where tumor tissue was available, the mutations detected in plasma were confirmed to be present in the primary tumor. These results are the first to demonstrate that a minimally-invasive multi-cancer blood test can be safely incorporated into routine clinical care. Moreover, this is the first study to show that it can be used to guide patient management in real-time, leading to surgery with intent to cure in asymptomatic individuals. Citation Format: Nickolas Papadopoulos. A first-of-its-kind prospective study of a multi-cancer blood test to screen and manage 10,000 women with no history of cancer [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 CT022.

Pancreatic cancer is the deadliest of all solid malignancies. Early detection offers the best hope for a cure, but characteristics of this disease, such as the lack of early clinical symptoms, make the early detection difficult. Recent genetic mapping of the molecular evolution of pancreatic cancer suggests that a large window of opportunity exists for the early detection of pancreatic neoplasia, and developments in cancer genetics offer new, potentially highly specific approaches for screening of curable pancreatic neoplasia. We review the challenges of screening for early pancreatic neoplasia, as well as opportunities presented by incorporating molecular genetics into these efforts. Cancer Res; 74(13); 3381–9. ©2014 AACR.

Internal human xenografts provide valuable animal models to study the microenvironments and metastatic processes occurring in human cancers. However, the use of such models is hampered by the logistical difficulties of reproducibly and simply assessing tumor burden. We developed a high-sensitivity assay for quantifying human DNA in small volumes of mouse plasma, enabling in-life monitoring of systemic tumor burden. Growth kinetics analyses of various xenograft models showed the utility of circulating human DNA as a biomarker. We found that human DNA concentration reproducibly increased with disease progression and decreased after successful therapeutic intervention. A marked, transient spike in circulating human tumor DNA occurred immediately after cytotoxic therapy or surgery. This simple assay may find broad utility in target validation studies and preclinical drug development programs. [Cancer Res 2007;67(19):9364–70]

Somatic mutations are exquisitely specific cancer biomarkers that are not merely associated with cancer, rather they are the root cause of cancer. The realization that this markers are present in bodily fluids of individuals with cancer and the development of digital methods for their detection ushered a new era in the management of cancer patients providing opportunities for the development of noninvasive methods for early detection, detection of minimal residual disease, monitoring of response to therapy, early detection of resistance and diagnosis. There are both technical and biological challenges for the development of noninvasive tests for the detection of somatic mutations. The number of circulating tumor DNA (ctDNA) molecules is very low compared to that of DNA molecules with wild-type sequence and requires very sensitive methods for their detection. To this end, we have developed a method called SafeSeqS that is able to reliably resolve mutations at 0.01% in background of wild-type DNA across multiple regions of interest. To determine the spectrum of cancers in which ctDNA measurements could prove clinically useful, we evaluated the detection of ctDNA in plasma (liquid biopsy) from patients with a large number of tumor types. ctDNA was detectable in the plasma of most patients with advanced tumor, but in less than 50% in patients with certain tumor types, like primary brain cancers. More than half of the patients with localized colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, had detectable ctDNA in their plasma. The sensitivity of detection of advanced colorectal cancer (CRC) was close to 100% and greater than 75% for localized cancers indicating that liquid biopsy could be utilized for a number of clinical applications. One such application is the detection of minimal residual disease after surgery or therapy with curative intent. Currently, there are not efficient ways to determine which patients are cured and which have residual disease that will result in recurrence. In a prospective cohort of 230 patients with resected stage II colon cancer, ctDNA detection after surgery provided direct evidence of residual disease and identified patients at very high risk of recurrence. Another application of liquid biopsy in CRC is monitoring resistance to therapies, like EGFR, providing an early indication that resistance ensues. The holy grail of non-invasive detection of cancer is early detection. Most tumors are incurable because they are detected at a late stage. Early detection of cancer, before symptoms appear, at a stage curable by surgery, holds great promise for decreasing the rate of deaths due to cancer. We will share our efforts on the development of tests for the early detection of cancer. Our overall vision is that non-invasive, somatic mutation based tests become routine in the clinical management of cancer patients. Citation Format: Nickolas Papadopoulos, Yuxuan Wang, Joshua Cohen, Ralph Hruban, Peter Gibbs, Jeanne Tie, Luis Diaz, Kenneth Kinzler, Bert Vogelstein. Non-invasive detection of somatic mutations in the management of CRC. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA17.

Long duration inflammatory bowel disease (IBD) increases the risk for colorectal cancer (CRC). Mutation analysis of limited numbers of genes has suggested that CRCs arising in an IBD background differ from those that do not. We sought to characterize the genetic landscape of these tumors through whole-exome sequencing. Thirty-one IBD patients with CRC were identified, including 15 with ulcerative colitis, 14 with Crohn's disease and 2 with indeterminate colitis. Whole-exome sequencing was performed on micro-dissected tumor and matched non-neoplastic formalin-fixed paraffin-embedded (FFPE) tissues. Somatic alterations were identified by comparing matched specimens. Mutation prevalence in sporadic CRC was obtained from previously published exome-sequencing studies. An average of 11.6 Gb were sequenced per sample. The depth of quality coverage of the targeted region was 29 to 100-fold in the tumors; and 17- to 123-fold in non-tumor samples. Two specimens harbored somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1 and MSH6 and exhibited hypermutable phenotype. The remaining cases had a median mutation rate of 1.33 mutations/Mb and an average of 71 alterations per sample. The pattern of base changes in non-hypermutated tumors showed a preponderance of C to T transitions (62%), a majority of which (48% of all mutations) occurred at 5′-CpG-3′ sites. An excess of A to C transversions at AA dinucleotides, particularly in a context of AAG trinucleotides, was also noted. This pattern has been previously observed in esophageal and gastric cancers, both of which are also tied to chronic inflammation. There were no apparent differences in the spectrum of substitutions between tumors arising in UC and CD patients. We identified 28 genes recurrently mutated in three or more non-hypermutated samples. TP53 was the most commonly mutated gene, with incidence prevalence similar to sporadic CRC (63% of cases), but with different mutation spectrum. APC and KRAS were mutated at significantly lower rates than in sporadic CRCs (13% and 20% of cases, respectively). Genes mutated more commonly or uniquely in IBD-CRC, included SOX9 and EP300, encoding proteins in the WNT pathway, NRG1, encoding an ERBB ligand, and IL16, encoding a cytokine. Three cases featured hotspot PIK3CA mutations. Likely oncogenic driver gene mutations in BRAF, CTNNB1, and CREBBP were found in one case each. Mutated genes were enriched for gene ontologies associated with cell communication, cell-cell signaling, and cell adhesion. Our study also revealed recurrent mutations in components of the Rho/Rac GTPase network, suggesting a role for non-canonical WNT signaling in IBD-CRC. CRCs arising in IBD patients demonstrate a unique molecular profile, which provides clues to their etiology. Our study also sets the stage for improved early detection of IBD-associated CRCs based on their unique genetic compositions as well as for the tailoring of therapies in this patient population. Citation Format: Ana I. Robles, Giovanni Traverso, Ming Zhang, Nicholas Roberts, Mohammed A. Khan, Christine Joseph, Gregory Lauwers, Florin Selaru, Maria Popoli, Xiquan Ke, Ralph H. Hruban, Stephen J. Meltzer, Kenneth W. Kinzler, Bert Vogelstein, Curtis C. Harris, Nickolas Papadopoulos. The genomic landscapes of inflammatory bowel disease-associated colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 127.

Fundamental theories in carcinogenesis and aging evoke the accumulation of rare somatic mutations in normal tissues over time. However, absence of a simple and systematic method to characterize somatic mutations in normal tissues precludes the understanding of their functional consequences. We present Bottleneck Sequencing System (BotSeqS), a next-generation sequencing method that quantitates rare somatic point mutations simultaneously across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. Using BotSeqS, we determine the mutation frequencies and spectra in normal brain, kidney, and colon from a total of 34 individuals ranging from < 1 to 98 years old. We show an age and tissue-dependent accumulation of rare point mutations and demonstrate that the somatic mutational burden in normal tissues can vary by several orders of magnitude depending on biological and environmental factors. For example, individuals with defects in the mismatch repair machinery or exposure to environmental carcinogens (smoking, aristolochic acid) show significant increases in mutational frequencies and altered mutational spectra compared to controls. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, we find that the mutational spectra of normal tissues were different from each other but similar to cancers from the same tissue type, suggesting that the differential mutational spectra observed in cancers are tissue-specific rather than cancer-specific. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues. Citation Format: Margaret L. Hoang, Isaac Kinde, Cristian Tomasetti, Thomas Rosenquist, Arthur P. Grollman, Kenneth W. Kinzler, Bert Vogelstein, Nickolas Papadopoulos. Accumulation of somatic mutations in normal and cancerous tissues with age. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr NG01.

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-γ production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Significance. The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Citation Format: Nicolas Jose Llosa, Michael Cruise, Ada Tam, Elizabeth Wick, Elizabeth Hechenbleikner, Janis Taube, Lee Blosser, Hongni Fan, Hao Wang, Ming Zhang, Brandon Luber, Nickolas Papadopoulos, Kenneth Kinzler, Bert Vogelstein, Cynthia Sears, Robert Anders, Drew Pardoll, Franck Housseau. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. [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 451. doi:10.1158/1538-7445.AM2015-451

INTRO: Pancreatic adenocarcinoma (PDAC) patients are often diagnosed with metastatic disease, yet the extent to which metastasis is determined by genetic evolution is unknown. A better understanding of this lethal process would have great clinical significance. METHODS: We selected four patients from the Johns Hopkins Rapid Medical Donation Program using strict criteria that included diagnosis with Stage IV disease and no treatment prior to death. Tissues from each patient were macrodissected to reduce stromal cell contamination and genomic DNA was extracted and quantified. A set of high quality primary or metastatic samples from these four patients (n = 35, range 4-11 per patient) were whole genome sequenced using the Illumina Hi-Seq 2000 platform, ultimately generating a raw list of 165,815 potential variants. After alignment to the hg19 human reference genome, the variants were filtered for single nucleotide polymorphisms, sequence directionality, and allele frequency in order to limit sequencing artifacts while maximizing detection of clonal changes. Subsequently, we selected 3256 somatic variants for targeted resequencing of the original 35 samples and in an additional 146 samples of the primary carcinoma or metastases (range 5-53 additional samples per patient). RESULTS: Whole genome sequencing resulted in a median high quality distinct coverage of 63X per genome and a median effective coverage of 97.9%. Point mutations in KRAS were identified at allele frequencies ranging from 8-88%. Targeted sequencing resulted in a median distinct coverage depth of 189X across all samples. We developed a robust statistical method to determine the mutation status across the samples of a patient based on an estimated false positive rate of the targeted sequencing of 0.23%. After discarding samples with low purity or low coverage, we found high homogeneity in the mutation profiles across samples from the primary tumor and samples from different metastases in each patient. We show that for many metastases there exists a corresponding sample from the primary tumor differing only in a few positions. The majority of mutations were either present in all or in only one sample of a patient. The low number of remaining parsimony-informative mutations prevents the generation of precise evolutionary trees. However, using hierarchical clustering and our newly developed phylogenetic method, we were able to infer basic evolutionary relationships among the samples. CONCLUSIONS: The natural history of metastatic PDAC is defined by the accumulation of a large number of mutations within the primary tumor, including most of the driver mutations. Indeed, evolutionary analyses indicate that metastases have relatively few unique mutations and are by-products of clonal evolution within the primary tumor over a prolonged period. Citation Format: Alvin P. Makohon-Moore, Ming Zhang, Johannes G. Reiter, Ivana Bozic, Fay Wong, Yuchen Jiao, Krishnendu Chatterjee, Martin Nowak, Nickolas Papadopoulos, Bert Vogelstein, Kenneth W. Kinzler, Christine A. Iacobuzio-Donahue. Clonal evolution defines the natural history of metastatic pancreatic cancer. [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 4137. doi:10.1158/1538-7445.AM2015-4137

The development of minimally-invasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital PCR-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We initially use targeted, exomic, or whole genome sequencing to identify sequence or structural alterations in tumor tissues of 410 patients using targeted, exomic, or whole genome sequencing. We found that at least one tumor-specific mutant molecule could be identified in 75% of patients with advanced ovarian, colorectal, bladder, gastroesophoageal, pancreatic, breast, melanoma, hepatocellular and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73%, 57%, 48% and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. Circulating tumor DNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% while and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor (EGFR) blockade in 24 patients who objectively responded to therapy but who subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase (MAPK) pathway. Remarkably, nearly half (45%) of the observed mutations observed were in codon 61 of either the KRAS or NRAS gene. Taken together, these data suggest that ctDNA is a broadly applicable, sensitive, specific and robust biomarker that can be used for a variety of clinical and research purposes in patients with several multiple different types of cancer. Note: This abstract was not presented at the meeting. Citation Format: Chetan Bettegowda, Mark Sausen, Rebecca Leary, Isaac Kinde, Nishant Agrawal, Bjarne Bartlett, Hao Wang, Brandon Luber, Kenneth Kinzler, Bert Vogelstein, Nickolas Papadopoulos, Luis Diaz. Detection of circulating tumor DNA in early and late stage human malignancies. [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 5606. doi:10.1158/1538-7445.AM2014-5606

Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are still lacking. Here, we report that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of oxidized vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased uptake of DHA in the mutant cell lines causes oxidative stress as intracellular DHA is immediately reduced back to vitamin C by reduced glutathione. Elevated reactive oxygen species inactivate GAPDH and thus divert glycolytic flux into the pentose phosphate pathway. Because KRAS or BRAF mutant cells are highly dependent on glycolysis, this altered glucose metabolism induced by vitamin C ultimately leads to an energy deficit in KRAS or BRAF mutant cells and thereby cell death. There are currently a growing number of clinical trials in phases I/II examining the effect of high dose vitamin C as a treatment for cancers, including colorectal cancers. Our results provide a rationale for the pharmacological use of vitamin C as a therapeutic agent to treat colorectal cancer patients with oncogenic KRAS or BRAF mutations. Citation Format: Jihye Yun, Adam Kavalier, Edouard Mullarky, Kaitlyn Bosch, Jatin Roper, Carlo Rago, Jihye Paik, John Asara, Steven Gross, Bert Vogelstein, Nickolas Papadopoulos, Lewis Cantley. Vitamin C is selectively toxic to cancer cells harboring KRAS or BRAF mutations by targeting GAPDH. [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 2951. doi:10.1158/1538-7445.AM2014-2951

Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are lacking. We previously showed that isogenic human colorectal cancer cell (CRC) lines harboring a KRAS or BRAF mutation up-regulate the expression of glucose transporter 1 (GLUT1), and increase glucose uptake and glycolysis. In this study, we go on to show that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of vitamin C, mainly in its oxidized form, dehydroascorbic acid (DHA), relative to their wild-type counterparts through GLUT1. Notably, CRCs did not uptake vitamin C in its reduced form, possibly due to the fact that its known transporters, sodium vitamin C cotransporters (SVCTs) were generally not present in CRCs. Deletion of GLUT1 in mutant cells, overexpression of GLUT1 in wild-type cells and competition assays between glucose and DHA confirmed that GLUT1 is both necessary and sufficient for the uptake of vitamin C in CRCs. Both mutant and wild-type cells were able to grow in low glucose (1-2 mM) at a similar rate. In the same condition, we found vitamin C to be selectively toxic to KRAS and BRAF mutant cells, causing them to undergo apoptosis. In contrast, in high glucose levels (>10 mM), a similar concentration of vitamin C had no effect on the survival and growth of mutant cells. In addition, vitamin C significantly inhibited the growth of cancer cells with KRAS or BRAF mutations in vivo xenografts models. To find the mechanism by which vitamin C induces cell death in KRAS or BRAF mutant cells, we performed targeted metabolomics using LC/MS. Both unlabeled total metabolite analysis and flux analysis with C13-Glucose isotope strongly suggested that vitamin C re-routes glucose usage from aerobic glycolysis and non-oxidative pentose phosphate pathway (PPP) to the oxidative PPP to compensate for the lower levels of NADPH which was rapidly consumed to reduce GSSH to GSH. Having taken up exclusively DHA, the oxidized state of vitamin C, the mutant cells immediately use GSH to reduce Vitamin C, resulting in the accumulation of GSSH. Shunting glucose to the oxidative PPP reduces aerobic glycolysis, lowering ATP levels, and decreases glucose anabolism, disfavoring the synthesis of nucleic acids, lipid and amino acids, all needed for the survival and growth of mutant cells. Surprisingly the level of reactive oxidative stress (ROS) was largely unchanged following vitamin C treatment possibly because increased levels of vitamin C served as an anti-oxidant in mutant cells. There are currently more than three clinical trials phase I/II examining the effect of high dose vitamin C via intravenous administration in patients against lymphoma or solid cancers including CRCs. Our findings will provide proof of concept that KRAS or BRAF mutations in CRC can be predictive biomarkers for vitamin C therapy. Citation Format: Jihye Yun, Adam Kavalier, Jatin Roper, Steve Gross, Carlo Rago, Nickolas Papadopoulos, Bert Vogelstein, Lewis Cantley. Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C60.

The recent years, genome-wide analyses identified the genetic landscapes of many different tumor types. One of the most frequent class of genes found to be mutated were genes whose products regulate epigenetic mechanisms, like chromatin modification and remodeling, providing a link between genetic alterations and epigenetic changes in cancer. One of these genes is ATRX. This gene has been studied in the context of alpha thalassemia-mental retardation X-linked syndrome. In human sporadic solid tumors, it was first shown to be mutated in pancreatic neuroendocrine tumors (PanNETs). Approximately, 50% of PanNETs had mutations in either ATRX or DAXX. The proteins of these two genes form a complex that deposits H3.3 at heterochromatin and telomeres. ATRX, but not DAXX, has also been found to be mutated in certain types of central nervous system tumors. The prevalence of mutations in ATRX is different for the different types of gliomas. It is mutated more frequently in astrocytomas and oligoastrocytomas, but rarely in primary glioblastomas. Recently, we identified mutations in the promoter of TERT in the same set of glioma samples. TERT mutations were more prevalent in primary glioblastomas and oligodendrogliomas. Overall, mutations in ATRX and TERT were mutually exclusive, suggesting that these two genetic mechanisms result in equivalent growth advantages. Mutations in ATRX have almost perfect correlation with Alternative Lengthening of Telomeres (ALT) phenotype in PanNETs, CNS tumors and other cases with ATRX mutations, thus, TERT activation and ALT phenotype are mutually exclusive. This has implications for understanding the relationship between telomeres and tumorigenesis. These studies, in addition to providing clues about the underlying genetic changes that drive tumorigenesis, they also have clinical implications. Mutations in ATRX or DAXX in PanNETs associate with better prognosis. Mutations of ATRX and TERT aid in the classification and prognostication of brain tumors. Further understanding of the role of ATRX in tumorigenesis should help in uncovering new ways for therapeutic targeting of tumors with mutations in this gene. Citation Format: Nickolas E. Papadopoulos. ATRX mutations in human carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA04.