Synthetic vulnerabilities of mesenchymal subpopulations in pancreatic cancer.

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

SOX9 chromatin folding domains correlate with its real and putative distant cis-regulatory elements.

Evolutionary conserved transcription factor SOX9, encoded by the dosage sensitive SOX9 gene on chromosome 17q24.3, plays an important role in development of multiple organs, including bones and testes. Heterozygous point mutations and genomic copy-number variant (CNV) deletions involving SOX9 have been reported in patients with campomelic dysplasia (CD), a skeletal malformation syndrome often associated with male-to-female sex reversal. Balanced and unbalanced structural genomic variants with breakpoints mapping up to 1.3 Mb up- and downstream to SOX9 have been described in patients with milder phenotypes, including acampomelic campomelic dysplasia, sex reversal, and Pierre Robin sequence. Based on the localization of breakpoints of genomic rearrangements causing different phenotypes, 5 genomic intervals mapping upstream to SOX9 have been defined. We have analyzed the publically available database of high-throughput chromosome conformation capture (Hi-C) in multiple cell lines in the genomic regions flanking SOX9. Consistent with the literature data, chromatin domain boundaries in the SOX9 locus exhibit conservation across species and remain largely constant across multiple cell types. Interestingly, we have found that chromatin folding domains in the SOX9 locus associate with the genomic intervals harboring real and putative regulatory elements of SOX9, implicating that variation in intra-domain interactions may be critical for dynamic regulation of SOX9 expression in a cell type-specific fashion. We propose that tissue-specific enhancers for other transcription factor genes may similarly utilize chromatin folding sub-domains in gene regulation.

In Vivo Functional Platform Targeting Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of Pancreatic Cancer.

Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.

HiCPlotter integrates genomic data with interaction matrices.

Metazoan genomic material is folded into stable non-randomly arranged chromosomal structures that are tightly associated with transcriptional regulation and DNA replication. Various factors including regulators of pluripotency, long non-coding RNAs, or the presence of architectural proteins have been implicated in regulation and assembly of the chromatin architecture. Therefore, comprehensive visualization of this multi-faceted structure is important to unravel the connections between nuclear architecture and transcriptional regulation. Here, we present an easy-to-use open-source visualization tool, HiCPlotter, to facilitate juxtaposition of Hi-C matrices with diverse genomic assay outputs, as well as to compare interaction matrices between various conditions. https://github.com/kcakdemir/HiCPlotter.