PROJECT SUMMARY During tumor evolution, cancer cells can acquire phenotypic heterogeneity, enabling them to grow more aggressively, invade neighboring tissues, evade the immune system and therapeutic challenges, and metastasize to distant sites. The ability of cancer cells to change their phenotypes can be broadly defined as cancer cell plasticity, which can be influenced by both tumor intrinsic and extrinsic factors. However, the underlying molecular mechanisms that regulate cancer cell state transitions throughout tumor evolution remain poorly understood. A comprehensive understanding of these mechanisms will uncover key gene networks and potentially inform novel therapeutic approaches. Studying cancer cell state transitions in vivo has been challenging due to the lack of tools for tracking cell lineage relationships at high resolution. To overcome this, I have recently contributed to the development of an evolving lineage tracer that leverages CRISPR/Cas9 technology to simultaneously report each cancer cell’s lineage history and transcriptional state in vivo. By applying this tool to the well-characterized Kras;Trp53 (KP) autochthonous mouse model of lung adenocarcinoma, I was able to reconstruct the entire tumor evolution history and measure cancer plasticity by quantifying cell state changes in phylogenetically-related cancer cells. Built on this innovative system, this proposal aims to investigate the mechanisms underlying lung cancer cell plasticity, both cell-autonomous and non-cell-autonomous. Leveraging my expertise in cancer biology and the evolving lineage tracing model, I will quantitatively track cancer cell state transitions within the native microenvironment. Specifically, I will delineate cell-autonomous regulators of cancer cell plasticity by CRISPR knockout of putative regulators of plasticity, identify plasticity-associated tumor microenvironmental factors and dissect non-cell- autonomous regulation of plasticity using spatial transcriptomics, and explore the role of plasticity in resistance development to anti-cancer therapies. Addressing these fundamental questions will provide new insights into the biology of cancer cell state regulation. Moreover, the experimental and computational framework from this proposal can be applicable for studying cancer plasticity in other genetic perturbations and therapeutic contexts, serving as a general template for understanding the molecular basis of cancer cell plasticity and tumor evolution. My diverse expertise in cancer biology, mouse modeling, technology and single cell genomics positions me to execute the proposed projects and help me acquire important preliminary data to apply for future research grants. As part of this grant, I will assemble an advisory team at my institution to support my transition and career development training activities outlined in this proposal. I will also take advantage of the varied resources at my postdoctoral institutions and the institution where I secure an independent position to develop skills related to mentorship, communication, and research ethics. Overall, I believe my focused research plan, detailed training goals, and a strong advisory team will facilitate a successful transition to independence.

PROJECT NARRATIVE The ability to dynamically switch cell states enables cancer cells to adapt to environmental and therapeutic challenges, however, the molecular mechanisms that cancer cells employ to maintain or switch states have not been fully characterized. This proposal aims to dissect the cell-intrinsic and extrinsic mechanisms underlying lung cancer cell plasticity by leveraging a newly developed single-cell lineage tracing technology. A comprehensive understanding of these mechanisms will reveal key gene networks that regulate tumor evolution, and could potentially inform rational therapeutic approaches.