Shibin Zhou

Cancer genome landscapes.

Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of “mountains” (genes altered in a high percentage of tumors) and a much larger number of “hills” (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or “drive” tumorigenesis. A typical tumor contains two to eight of these “driver gene” mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality.

Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells

Desperately Seeking Glucose Mutations in oncogenes and tumor suppressor genes allow cancer cells to outgrow their neighboring healthy cells. What microenvironmental conditions provide a selective growth advantage to these cells? Yun et al. (p. 1555, published online 6 August) identify low glucose availability as a microenvironmental factor driving the acquisition of KRAS oncogenic mutations that allow cancer cells to survive and grow. In genetically matched colorectal cancer cells that differed only in the mutational status of the KRAS oncogene, mutant cells selectively overexpressed glucose transporter-1 and exhibited enhanced glucose uptake and glycolysis. When cells with wild-type KRAS were placed in a low-glucose environment, very few cells survived but most of the survivors expressed high levels of glucose transporter-1, and a small percentage of the survivors had acquired new KRAS mutations. Thus, glucose deprivation may help to drive the acquisition of cell growth–promoting oncogenic mutations during tumor development. Glucose deprivation can drive the acquisition of certain oncogenic mutations in human cancer cells. Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently up-regulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low-glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low-glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired KRAS mutations not present in their parents. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade

Predicting responses to immunotherapy Colon cancers with loss-of-function mutations in the mismatch repair (MMR) pathway have favorable responses to PD-1 blockade immunotherapy. In a phase 2 clinical trial, Le et al. showed that treatment success is not just limited to colon cancer (see the Perspective by Goswami and Sharma). They found that a wide range of different cancer types with MMR deficiency also responded to PD-1 blockade. The trial included some patients with pancreatic cancer, which is one of the deadliest forms of cancer. The clinical trial is still ongoing, and around 20% of patients have so far achieved a complete response. MMR deficiency appears to be a biomarker for predicting successful treatment outcomes for several solid tumors and indicates a new therapeutic option for patients harboring MMR-deficient cancers. Science, this issue p. 409; see also p. 358 A pan-cancer biomarker is identified that can predict successful response to cancer immunotherapy in human patients. The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor–1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair–deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair–deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers’ tissue of origin.

Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma

Through exomic sequencing of ten hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCC) and subsequent evaluation of additional affected individuals, we discovered novel inactivating mutations of ARID2 in four major subtypes of HCC (HCV-associated HCC, hepatitis B virus (HBV)-associated HCC, alcohol-associated HCC and HCC with no known etiology). Notably, 18.2% of individuals with HCV-associated HCC in the United States and Europe harbored ARID2 inactivation mutations, suggesting that ARID2 is a tumor suppressor gene that is relatively commonly mutated in this tumor subtype.

Sensitive digital quantification of DNA methylation in clinical samples

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in ∼5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.

Characterization of human FAST-1, a TGFβ and activin signal transducer

We have identified a human homolog of the Xenopus forkhead activin signal transducer-1 (xFAST-1). Although significantly different in sequence from its Xenopus counterpart, hFAST-1 shared with xFAST-1 the ability to bind to human Smad2 and activate an activin response element (ARE). The hFAST-1-dependent activation of ARE was completely dependent on endogenous Smad4 and stimulation by a TGF beta-like ligand. The hFAST-1 protein was shown to bind to a novel DNA motif, TGT (G/T) (T/G)ATT, an exact copy of which was present within the ARE. A single copy of this motif could activate a reporter in a TGF beta-dependent fashion but only when an adjacent Smad-binding element was present in the construct. These data suggest that responses to TGF beta family members may be mediated by a DNA-binding complex formed by hFAST-1, hSmad2, and hSmad4.

Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells

Significance Recent clinical trials have shown highly promising responses in a subset of patients treated with immune checkpoint inhibitory anti–programmed cell death-1, anti–programmed cell death ligand-1, and anti–cytotoxic T-lymphocyte-associated antigen-4 antibodies, but the majority of patients in these trials remained unresponsive. Our results show that elevated myeloid-derived suppressor cells(MDSCs) are responsible for the resistance and that elimination of MDSCs can lead to cures of experimental, metastatic tumors. Impressive responses have been observed in patients treated with checkpoint inhibitory anti–programmed cell death-1 (PD-1) or anti–cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti–PD-1 and anti–CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.

Detection and localization of surgically resectable cancers with a multi-analyte blood test

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancers rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science, this issue p. 926; see also p. 866 A blood test that combines protein and DNA markers may allow earlier detection of eight common cancer types. Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.

A bacterial protein enhances the release and efficacy of liposomal cancer drugs

Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimental tumors. Because C. novyi-NT lyses red blood cells, we hypothesized that its membrane-disrupting properties could be exploited to enhance the release of liposome-encapsulated drugs within tumors. Here, we show that treatment of mice bearing large, established tumors with C. novyi-NT plus a single dose of liposomal doxorubicin often led to eradication of the tumors. The bacterial factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed liposomase. This protein could potentially be incorporated into diverse experimental approaches for the specific delivery of chemotherapeutic agents to tumors.

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses.

Clostridium novyi-NT targets aberrant tumor physiology and can produce a precise, robust, and reproducible antitumor response. Fighting Cancer with Clostridium Tumors are composed of necrotic, hypoxic, and well-oxygenated regions. Hypoxic tumor regions are more resistant to systemic anticancer agents and radiotherapy. However, they provide a fertile ground for the growth of anaerobic bacteria. Roberts et al. use an attenuated strain of the anaerobic, spore-forming bacterium Clostridium novyi (C. novyi-NT) and demonstrate precise, robust, and reproducible antitumor responses when C. novyi-NT spores were injected into tumors of rats, dogs, and one human patient. These results support the further development of intratumoral injections of C. novyi-NT spores as a therapeutic for patients with locally advanced, nonresectable cancers. Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were observed in 6 of 16 dogs (37.5%), with three complete and three partial responses. On the basis of these encouraging results, we treated a human patient who had an advanced leiomyosarcoma with an intratumoral injection of C. novyi-NT spores. This treatment reduced the tumor within and surrounding the bone. Together, these results show that C. novyi-NT can precisely eradicate neoplastic tissues and suggest that further clinical trials of this agent in selected patients are warranted.