Fast-Track proposals will be accepted. Direct-to-Phase II proposals will NOT be accepted. Number of anticipated awards: 2-3 Budget (total costs, per award): Phase I: up to $400,000 for up to 12 months Phase II: up to $2,000,000 for up to 2 years PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED. Summary Numerous studies over the last several decades have reported on extrachromosomal circular DNAs (eccDNAs) that appear alongside coiled linear chromosomes in the cells of normal tissues. These DNAs are found in many eukaryotic species and have been observed in various forms including telomeric circles, small polydispersed DNA elements, and microDNAs. New research has revealed that cancer cells contain large numbers of a specific type of extrachromosomal DNA known Page 77 simply as ‘ecDNA.’ As compared to the other forms of DNA listed above, ecDNAs are relatively large (1-3 MB) and contain multiple full genes and regulatory regions. Recently, ecDNA has been increasingly recognized as a potent source of driver oncogene copy number amplification events in human tumors. ecDNAs are subject to non-Mendelian inheritance and can multiply rapidly while maintaining intratumoral genetic heterogeneity, which likely plays an important role in helping cancers to adapt, evolve and become resistant to treatment. Driver “undruggable” oncogenic targets, such as NMyc, are subject to ecDNA driven amplifications, and other enzymes and oncogenic proteins responsible for drug resistance may also be transiently driven by such events. Recent interest in this emerging and important area of research is reflected in the latest round of Cancer Grand Challenges, a major collaborative funding initiative between the NCI and Cancer Research United Kingdom (CRUK), which identified as one of its new 2020 challenges, “Understand the biology of ecDNA generation and action, and develop approaches to target these mechanisms in cancer.” Unfortunately, little is known about the genomic organization of ecDNA or the mechanisms that drive their formation, due in part to the challenges involved in their detection. ecDNAs can reintegrate into the genome and are distributed unequally to daughter cells during cell division, both of which contribute to the difficulty in their detection. Although recent advances in commercially available long-read sequencing platforms may play a role in addressing some of these challenges, many technology gaps still exist for the reliable analysis of ecDNAs, especially if limited samples are available. To keep pace with our rapidly evolving understanding of ecDNAs and their role in cancer, this contract topic aims to develop new tools that are critically needed to analyze ecDNA sequence, structure and regulation. This topic is agnostic as to specific technological approaches, which could involve optimizing ecDNA enrichment and purification, improving existing sequencing technologies, and/or developing new informatics tools. In the near term, technologies developed under this topic are expected to enable important basic research on ecDNA and cancer. Ultimately, such tools may also play a key role in revealing new therapeutic vulnerabilities in cancers that are currently intractable. Project Goals The goal of this contract topic is to spur the development of new and/or advanced analytical approaches that can support research into the mechanisms giving rise to ecDNA formation and organization, and its role in cancer. This solicitation seeks both completely new approaches, as well as “better, faster, cheaper” versions of existing technologies, to advance this field. Responsive proposals may include novel methods and/or reagents to selectively enrich, isolate, detect, and/or visualize ecDNA targets. Possible approaches that would be considered responsive to this solicitation include (but are not necessarily limited to): • Biochemical approaches to selectively enrich or purify ecDNA • Sequencing approaches that distinguish ecDNA from other forms of DNA • Affinity reagents or other biochemical detection strategies specific for ecDNA • Imaging probes that are specific for ecDNA targets • IT approaches that allow novel data analysis to interpret/detect ecDNA Phase I projects must demonstrate that the proposed technology/approach is capable of selectively detecting, analyzing and/or characterizing ecDNA in cancer-relevant biological systems (e.g., cancer cell lines). Offerors should conduct feasibility studies in cancer models for which there exists a sufficient understanding of the ecDNA biology to reliably interpret the results of the novel assay or technique. Phase I activities should focus on characterizing the relevant analytical parameters of the technique and should include target performance measures for key analytical parameters. Phase II activities should demonstrate the assay throughput, as well as the ability to analyze ecDNA in systems of increasing biological complexity (e.g., patient-derived xenografts, tumor tissue sections, human plasma). Phase II activities should demonstrate the ability of the proposed approach to detect temporal changes in ecDNA that are biologically relevant in human cancers (e.g., ecDNA biogenesis, replication, genomic organization, distribution to daughter cells). Offerors are encouraged, but not required, to conduct experiments in which changes in ecDNA can be monitored in a cancer-relevant model(s) following drug treatment or some other biological perturbation. Phase II activities should include other necessary validation activities to advance the technology as a commercially available research tool. Activities not responsive to announcement: Activities involving the detection of ecDNAs in non-cancer biological systems will be considered non-responsive to this announcement. Phase I Activities and Deliverables: • Demonstrate the ability to selectively analyze (e.g., enrich, purify, isolate, detect, image) ecDNAs found in cancer-relevant biological systems • Provide a clear justification for the biological systems (e.g., cell lines) used for analytical validation; include a summary of what is currently known about the role of ecDNA in these systems and how this may impact the interpretation of the proposed validation experiments • Demonstrate the specificity of the assay/technique to distinguish ecDNA from all other forms of cellular DNA in Page 78 the chosen cancer cells • Fully characterize the relevant analytical parameters of the assay/technique including sensitivity, specificity, limit of detection, dynamic range, etc. (as appropriate) • Describe target performance measures (i.e., quantitative milestones) for key analytical parameters, including the methods by which they will be assessed Phase II Activities and Deliverables: • Demonstrate the maximum throughput of the approach, and define appropriate measures of performance reliability for large-scale screening of cancer samples • Demonstrate the ability to analyze ecDNA in systems of increasing biological complexity (e.g., patient-derived xenografts, tumor tissue sections, human plasma) • Demonstrate the ability to use the proposed approach to detect temporal changes in ecDNA that are biologically relevant in human cancers (e.g., ecDNA biogenesis, replication, genomic organization, distribution to daughter cells) • Offerors are encouraged, but not required, to demonstrate that the technique can be used to monitor biologically relevant ecDNA changes in a cancer-relevant model(s) following drug treatment or some other biological perturbation • Conduct additional validation studies to advance the technology as a commercially available research tool, including manufacturing scale up, commercial partnerships, beta testing, etc. (as appropriate).