Structural and Functional Biology-based analysis of non-oncogene cancer dependencies

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The Columbia Center for Cancer Systems Therapeutics (CaST) has successfully developed methodologies for the patient-centric prioritization and evaluation of cancer therapeutic strategies. Through this cost extension we seek to complete residual work related to the original CaST aims that was delayed due to the COVID-19 pandemic. Specifically, we will: Assess the tumor replenishing potential of the oncogenic precursor cell population in pancreatic ductal adenocarcinoma (PDA): In PDA we have inferred and experimentally validated the existence of three transcriptionally distinct cell states. The most critical hypothesis emerging from these studies, as supported by RNA velocity studies, is that the oncogenic precursor (OP) population – which unlike the other two, represents a dominant component of all PDAs and was previously undetected – represents the key tumor maintenance reservoir by replenishing both the morphogenic and the lineage sub-populations. To confirm this hypothesis, which would be paradigm shifting in terms of our understanding of this tumor, we are planning barcode-based lineage tracing assays in cell lines that we have already shown to comprise either OP and morphogenic cells (KP4) or OP and lineage cells (CAPAN1). Enhance the structure-informed annotation of the Cancer Homeostasis Proteins Database (CHoPD): One of the key CaST deliverables, the CHoPD is a comprehensive, functionally and structurally annotated inventory of master regulator (MR) proteins, compiled by systematically dissecting tumor checkpoints across samples from publicly available repositories. The CHoPD has been implemented as a graph database in the Neo4j database system and has been partially annotated with the standard cancer hallmarks as derived from PrePPI. We now propose to incorporate the full repertoire of CaST structure-informed resources into this database, so as to create a version of CHoPD that will contain extensive annotation derived from tools that have been developed in the context of CaST Projects 1 and 3. Complete development of a highly scalable platform for joint single-cell DNA and RNA sequencing: One of the major technical objectives of CaST Project 2 is the development of scalable technology for simultaneous single cell DNA- and RNA-seq. As previously reported, we have developed a novel asymmetric microwell array technology for simultaneous co-encapsulation of thousands of individual cells with exactly two beads – one harboring oligonucleotides for direct barcoding of genomic DNA and the other for mRNA capture and barcoding. We have demonstrated that this device facilitates simultaneous solid-phase capture of mRNA and solution-phase capture of gDNA from individual cells. Here we propose to complete the development of our DNA barcoding beads and optimize the performance of our on-chip library construction for a full-scale demonstration of the technology. Infer copy number alterations from scRNA-Seq profiles in a collection of benchmark datasets: We developed PICASSO (Phylogenetic Inference from Copy number Alterations in Scrna-Seq Observations) to infer copy number changes from single-cell RNA sequencing data. PICASSO was demonstrated to outperform state-of- the-art methods, such as HoneyBadger, on simulated data, where ground truth answers are known. We further demonstrated accuracy on a CaST pancreatic cancer dataset. Based on these preliminary data, we propose to systematically apply PICASSO to publicly available scRNA-seq datasets across multiple cancer types, focusing on the Human Tumor Atlas Network.