Mapping a Functional Cancer Genome Atlas

Over the past decade, tremendous resources have been devoted to sequencing the genomes of patient cancers. But while the molecular portraits of cancer are now in higher resolution than ever before, it has remained challenging to derive clinically actionable insights from these data. A central issue is that tumor genome sequencing often can only tell us what mutations are present, not which ones are functionally important. Towards this end, I sought to build an improved toolkit for functional cancer genomics, with the overarching goal of creating experimental platforms that are practical, scalable, and flexible. In collaboration with several colleagues, I developed autochthonous AAV-mediated CRISPR/Cas9 screens to quantitatively interrogate the contributions of specific mutations towards tumorigenesis in the murine liver and brain. Importantly, this experimental system is well suited for studies in tumor immunology, as it preserves the native tissue microenvironment in the context of an immunocompetent host. I subsequently utilized the AAV-CRISPR tumor model to further investigate the genetic determinants of response to PD-1 checkpoint blockade immunotherapy. In addition to recovering known regulators of immunotherapy response, I found that deficiency of the histone modifier KMT2D sensitizes diverse tumor types to immune checkpoint blockade by promoting the generation of immunogenic neoantigens. I further sought to comprehensively dissect the genetic regulation of anti-tumor immunity responses from both sides of the aisle, interrogating the immune cells that react against tumors as well as the cancer cells themselves. As a foray into the roles of noncoding RNAs in anti-tumor immunity, I mapped the landscape of sdRNAs expressed in human cancers, finding numerous transcripts that are associated with signatures of anti-tumor immune infiltration and survival across multiple cancer types. My colleagues and I also performed genome-scale CRISPR screens in primary CD8+ T cells to identify genetic regulators of T cell degranulation and tumor infiltration. These studies identified that Dhx37 knockout enhances NF-kB signaling in CD8+ T cells, leading to enhanced anti-tumor function in vitro and in vivo. Using genome-scale CRISPR activation (CRISPRa) screens, I further sought to identify genes that could promote tumorigenesis in immunocompetent hosts when overexpressed. In pursuing this line of study, my colleagues and I unexpectedly found that CRISPRa could be repurposed as a new immunotherapy modality, an approach we termed MAEGI (multiplexed activation of endogenous genes as immunotherapy). By delivering CRISPRa systems through AAVs to forcibly overexpress mutated genes directly in tumors, MAEGI enhanced immune recognition of tumor neoantigens, thereby eliciting robust and long-lasting anti-tumor immunity. The CRISPR screens employed in the aforementioned studies have an important limitation, however. Cancers arise from the sequential acquisition of genetic or epigenetic alterations, and the unique combinations of these alterations can interact in complex ways. To more precisely study these genetic interactions, I established a strategy for in vivo combinatorial knockout screening using massively-parallel CRISPR-Cas12a array profiling. I applied this technique to pinpoint mutation combinations that synergistically promote lung metastasis, demonstrating the utility of this technology to dissect genetic interactions in cancer. Finally, I devised a method for programmable sequential mutagenesis by combining CRISPR-Cas12a arrays with Cre recombination cassettes. I employed this approach to model the stepwise acquisition of resistance mutations against immunotherapy, thus providing a controlled experimental system for exploring strategies to overcome immunotherapy resistance

Activated mammalian target of rapamycin is a potential therapeutic target in gastric cancer

<p>Abstract</p> <p>Background</p> <p>The mammalian target of rapamycin (mTOR) plays a key role in cellular growth and homeostasis. The purpose of our present study is to investigate the expression of activated mTOR (p-mTOR) in gastric cancer patients, their prognostic significance and the inhibition effect of RAD001 on tumor growth and to determine whether targeted inhibition of mTOR could be a potential therapeutic strategy for gastric cancer.</p> <p>Methods</p> <p>The expression of p-mTOR was detected in specimens of 181 gastric cancers who underwent radical resection (R0) by immunohistochemistry. The correlation of p-mTOR expression to clinicopathologic features and survival of gastric cancer was studied. We also determined the inhibition effect of RAD001 on tumor growth using BGC823 and AGS human gastric cancer cell lines.</p> <p>Results</p> <p>Immunostaining for p-mTOR was positive in 93 of 181 (51.4%) gastric cancers, closely correlated with lymph node status and pTNM stage. Patients with p-mTOR positive showed significantly shorter disease-free survival (DFS) and overall survival (OS) rates than those with p-mTOR-negative tumors in univariable analyses, and there was a trend toward a correlation between p-mTOR expression and survival in multivariable analyses. RAD001 markedly inhibited dose-dependently proliferation of human gastric carcinoma cells by down-regulating expression of p70s6k, p-p70s6k, C-myc, CyclinD1 and Bcl-2, up-regulating expression of P53.</p> <p>Conclusions</p> <p>In gastric cancer, p-mTOR is a potential therapeutic target and RAD001 was a promising treatment agent with inducing cell cycle arrest and apoptosis by down-regulating expression of C-myc, CyclinD1 and Bcl-2, up-regulating expression of P53.</p

Relationship between H.Pylori infection and clinicopathological features and prognosis of gastric cancer

<p>Abstract</p> <p>Background</p> <p>Aimed to assess the relationship between H.Pylori and the clinicopathological features and prognosis of gastric cancer by quantitative detection of H.Pylori.</p> <p>Methods</p> <p>157 patients were enrolled, all patients had a record of clinicopathological parameters. Specimens including the tumor and non-neoplastic were detected for H.Pylori by Real-Time PCR and analyzed clinical data retrospectively. Variables independently affecting prognosis were investigated by means of multivariate analysis using the Cox proportional hazards model.</p> <p>Results</p> <p>H.Pylori infection was greater in non-neoplastic tissue than the tumor tissue (p < 0.05), H.Pylori infection and its copies were related to the tumor site and N staging (p < 0.05). Overall survival (OS) in all 157 patients has no correlation with the H.Pylori infection status (p = 0.715). As to the patients who underwent a curative surgery, relapse-free survival (RFS) has no correlation with the H.Pylori infection status (p = 0.639). Among the H.Pylori positive patients, OS and RFS of those with higher copies were longer than in patients with low copies, but there was no significant statistical difference.</p> <p>Conclusions</p> <p>H.Pylori infection status and its copies were related to N staging. The OS and RFS in patients with positive H.Pylori status has no significant difference from the patients with negative H.Pylori status.</p

Space-time variation of malaria incidence in Yunnan province, China

Abstract Background Understanding spatio-temporal variation in malaria incidence provides a basis for effective disease control planning and monitoring. Methods Monthly surveillance data between 1991 and 2006 for Plasmodium vivax and Plasmodium falciparum malaria across 128 counties were assembled for Yunnan, a province of China with one of the highest burdens of malaria. County-level Bayesian Poisson regression models of incidence were constructed, with effects for rainfall, maximum temperature and temporal trend. The model also allowed for spatial variation in county-level incidence and temporal trend, and dependence between incidence in June–September and the preceding January–February. Results Models revealed strong associations between malaria incidence and both rainfall and maximum temperature. There was a significant association between incidence in June–September and the preceding January–February. Raw standardised morbidity ratios showed a high incidence in some counties bordering Myanmar, Laos and Vietnam, and counties in the Red River valley. Clusters of counties in south-western and northern Yunnan were identified that had high incidence not explained by climate. The overall trend in incidence decreased, but there was significant variation between counties. Conclusion Dependence between incidence in summer and the preceding January–February suggests a role of intrinsic host-pathogen dynamics. Incidence during the summer peak might be predictable based on incidence in January–February, facilitating malaria control planning, scaled months in advance to the magnitude of the summer malaria burden. Heterogeneities in county-level temporal trends suggest that reductions in the burden of malaria have been unevenly distributed throughout the province

5-FU-hydrogel inhibits colorectal peritoneal carcinomatosis and tumor growth in mice

<p>Abstract</p> <p>Background</p> <p>Colorectal peritoneal carcinomatosis (CRPC) is a common form of systemic metastasis of intra-abdominal cancers. Intraperitoneal chemotherapy is a preferable option for colorectal cancer. Here we reported that a new system, 5-FU-loaded hydrogel system, can improve the therapeutic effects of intraperitoneal chemotherapy.</p> <p>Methods</p> <p>A biodegradable PEG-PCL-PEG (PECE) triblock copolymer was successfully synthesized. The biodegradable and temperature sensitive hydrogel was developed to load 5-FU. Methylene blue-loaded hydrogel were also developed for visible observation of the drug release. The effects and toxicity of the 5-FU-hydrogel system were evaluated in a murine CRPC model.</p> <p>Results</p> <p>The hydrogel system is an injectable flowing solution at ambient temperature and forms a non-flowing gel depot at physiological temperature. 5-FU-hydrogel was subsequently injected into abdominal cavity in mice with CT26 cancer cells peritoneal dissemination. The results showed that the hydrogel delivery system prolonged the release of methylene blue; the 5-FU-hydrogel significantly inhibited the peritoneal dissemination and growth of CT26 cells. Furthermore, intraperitoneal administration of the 5-FU-hydrogel was well tolerated and showed less hematologic toxicity.</p> <p>Conclusions</p> <p>Our data indicate that the 5-FU-hydrogel system can be considered as a new strategy for peritoneal carcinomatosis, and the hydrogel may provide a potential delivery system to load different chemotherapeutic drugs for peritoneal carcinomatosis of cancers.</p

Support for a synaptic chain model of neuronal sequence generation

In songbirds, the remarkable temporal precision of song is generated by a sparse sequence of bursts in the premotor nucleus HVC. To distinguish between two possible classes of models of neural sequence generation, we carried out intracellular recordings of HVC neurons in singing zebra finches (Taeniopygia guttata). We found that the subthreshold membrane potential is characterized by a large, rapid depolarization 5–10 ms before burst onset, consistent with a synaptically connected chain of neurons in HVC. We found no evidence for the slow membrane potential modulation predicted by models in which burst timing is controlled by subthreshold dynamics. Furthermore, bursts ride on an underlying depolarization of ~10-ms duration, probably the result of a regenerative calcium spike within HVC neurons that could facilitate the propagation of activity through a chain network with high temporal precision. Our results provide insight into the fundamental mechanisms by which neural circuits can generate complex sequential behaviours.National Institutes of Health (U.S.) (Grant MH067105)National Institutes of Health (U.S.) (Grant DC009280)National Science Foundation (U.S.) (IOS-0827731)Alfred P. Sloan Foundation (Research Fellowship

Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

Gene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.The genome-wide status of H3K27me3 modifications on blood lymphocytes in lactating Holsteins was performed via ChIP-Seq approach. Combined with digital gene expression (DGE) technique, the regulation effects of H3K27me3 on genes expressions were analyzed.The ChIP-seq results showed that the peaks of H3K27me3 in cows lymphocytes were mainly enriched in the regions of up20K (~50%), down20K (~30%) and intron (~28%) of the genes. Only ~3% peaks were enriched in exon regions. Moreover, the highest H3K27me3 modification levels were mainly around the 2 Kb upstream of transcriptional start sites (TSS) of the genes. Using conjoint analysis with DGE data, we found that H3K27me3 marks tended to repress target genes expressions throughout whole gene regions especially acting on the promoter region. A total of 53 differential expressed genes were detected in third parity cows compared to first parity, and the 25 down-regulated genes (PSEN2 etc.) were negatively correlated with H3K27me3 levels on up2Kb to up1Kb of the genes, while the up-regulated genes were not showed in this relationship.The first blueprint of bovine H3K27me3 marks that mediates gene silencing was generated. H3K27me3 plays its repressed role mainly in the regulatory region in bovine lymphocytes. The up2Kb to up1Kb region of the down-regulated genes in third parity cows could be potential target of H3K27me3 regulation. Further studies are warranted to understand the regulation mechanisms of H3K27me3 on somatic cell count increases and milk losses in latter parities of cows

DNA Immunization with Fusion of CTLA-4 to Hepatitis B Virus (HBV) Core Protein Enhanced Th2 Type Responses and Cleared HBV with an Accelerated Kinetic

BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses

The Formation and Stabilization of a Novel G-Quadruplex in the 5′-Flanking Region of the Relaxin Gene

It has been reported that binding of STAT3 protein to the 5′-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes

Hybridization thermodynamics of NimbleGen Microarrays

Background While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets. Results We demonstrate the benefits of an improved model for microarray hybridization and assess the relative contributions of the probe-target binding strength and the different competing structures. Remarkably, specific and unspecific hybridization were apparently driven by different energetic contributions: For unspecific hybridization, the melting temperature Tm was the best predictor of signal variation. For specific hybridization, however, the effective interaction energy that fully considered competing structures was twice as powerful a predictor of probe signal variation. We show that this was largely due to the effects of secondary structures in the probe and target molecules. The predictive power of the strength of these intramolecular structures was already comparable to that of the melting temperature or the free energy of the probe-target duplex. Conclusions This analysis illustrates the importance of considering both the effects of probe-target binding strength and the different competing structures. For specific hybridization, the secondary structures of probe and target molecules turn out to be at least as important as the probe-target binding strength for an understanding of the observed microarray signal intensities. Besides their relevance for the design of new arrays, our results demonstrate the value of improving thermodynamic models for the read-out and interpretation of microarray signals