Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Interpreting type 1 diabetes risk with genetics and single-cell epigenomics.

Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.

Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk.

Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.

Intron 1-Mediated Regulation of EGFR Expression in EGFR-Dependent Malignancies Is Mediated by AP-1 and BET Proteins.

The epidermal growth factor receptor (EGFR) is overexpressed in numerous solid tumors and is the subject of extensive therapeutic efforts. Much of the research on EGFR is focused on protein dynamics and downstream signaling; however, few studies have explored its transcriptional regulation. Here, we identified two enhancers (CE1 and CE2) present within the first intron of the EGFR gene in models of glioblastoma (GBM) and head and neck squamous cell carcinoma (HNSCC). CE1 and CE2 contain open chromatin and H3K27Ac histone marks, enhance transcription in reporter assays, and interact with the EGFR promoter. Enhancer genetic deletion by CRISPR/Cas9 significantly reduces EGFR transcript levels, with double deletion exercising an additive effect. Targeted repression of CE1 and CE2 by dCas9-KRAB demonstrates repression of transcription similar to that of genomic deletion. We identify AP-1 transcription factor family members in concert with BET bromodomain proteins as modulators of CE1 and CE2 activity in HNSCC and GBM through de novo motif identification and validate their presence. Genetic inhibition of AP-1 or pharmacologic disruption of BET/AP-1 binding results in downregulated EGFR protein and transcript levels, confirming a role for these factors in CE1 and CE2. Our results identify and characterize these novel enhancers, shedding light on the role that epigenetic mechanisms play in regulating EGFR transcription in EGFR-dependent cancers. IMPLICATIONS: We identify critical constituent enhancers present in the first intron of the EGFR gene, and provide a rationale for therapeutic targeting of EGFR intron 1 enhancers through perturbation of AP-1 and BET in EGFR-positive malignancies.

A Novel Combination Treatment Targeting BCL-XL and MCL1 for KRAS/BRAF-mutated and BCL2L1-amplified Colorectal Cancers.

Colorectal cancer is the third most commonly diagnosed cancer in the world, and exhibits heterogeneous characteristics in terms of genomic alterations, expression signature, and drug responsiveness. Although there have been considerable efforts to classify this disease based on high-throughput sequencing techniques, targeted treatments for specific subgroups have been limited. KRAS and BRAF mutations are prevalent genetic alterations in colorectal cancers, and patients with mutations in either of these genes have a worse prognosis and are resistant to anti-EGFR treatments. In this study, we have found that a subgroup of colorectal cancers, defined by having either KRAS or BRAF (KRAS/BRAF) mutations and BCL2L1 (encoding BCL-XL) amplification, can be effectively targeted by simultaneous inhibition of BCL-XL (with ABT-263) and MCL1 (with YM-155). This combination treatment of ABT-263 and YM-155 was shown to have a synergistic effect in vitro as well as in in vivo patient-derived xenograft models. Our data suggest that combined inhibition of BCL-XL and MCL1 provides a promising treatment strategy for this genomically defined colorectal cancer subgroup. Mol Cancer Ther; 16(10); 2178-90. ©2017 AACR.

High prevalence of TP53 mutations is associated with poor survival and an EMT signature in gliosarcoma patients.

Gliosarcoma (GS) is a rare variant (2%) of glioblastoma (GBM) that poses clinical genomic challenges because of its poor prognosis and limited genomic information. To gain a comprehensive view of the genomic alterations in GS and to understand the molecular etiology of GS, we applied whole-exome sequencing analyses for 28 GS cases (6 blood-matched fresh-frozen tissues for the discovery set, 22 formalin-fixed paraffin-embedded tissues for the validation set) and copy-number variation microarrays for 5 blood-matched fresh-frozen tissues. TP53 mutations were more prevalent in the GS cases (20/28, 70%) compared to the GBM cases (29/90, 32%), and the GS patients with TP53 mutations showed a significantly shorter survival (multivariate Cox analysis, hazard ratio=23.9, 95% confidence interval, 2.87-199.63, P=0.003). A pathway analysis showed recurrent alterations in MAPK signaling (EGFR, RASGRF2 and TP53), phosphatidylinositol/calcium signaling (CACNA1s, PLCs and ITPRs) and focal adhesion/tight junction (PTEN and PAK3) pathways. Genomic profiling of the matched recurrent GS cases detected the occurrence of TP53 mutations in two recurrent GS cases, which suggests that TP53 mutations play a role in treatment resistance. Functionally, we found that TP53 mutations are associated with the epithelial-mesenchymal transition (EMT) process of sarcomatous components of GS. We provide the first comprehensive genome-wide genetic alternation profiling of GS, which suggests novel prognostic subgroups in GS patients based on their TP53 mutation status and provides new insight in the pathogenesis and targeted treatment of GS.

An Integrative Approach to Precision Cancer Medicine Using Patient-Derived Xenografts.

Cancer is a heterogeneous disease caused by diverse genomic alterations in oncogenes and tumor suppressor genes. Despite recent advances in high-throughput sequencing technologies and development of targeted therapies, novel cancer drug development is limited due to the high attrition rate from clinical studies. Patient-derived xenografts (PDX), which are established by the transfer of patient tumors into immunodeficient mice, serve as a platform for co-clinical trials by enabling the integration of clinical data, genomic profiles, and drug responsiveness data to determine precisely targeted therapies. PDX models retain many of the key characteristics of patients' tumors including histology, genomic signature, cellular heterogeneity, and drug responsiveness. These models can also be applied to the development of biomarkers for drug responsiveness and personalized drug selection. This review summarizes our current knowledge of this field, including methodologic aspects, applications in drug development, challenges and limitations, and utilization for precision cancer medicine.

Genomic alterations in BCL2L1 and DLC1 contribute to drug sensitivity in gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases.