It is estimated that 40% of cancer patients with EGFR-mutant lung cancer develop brain metastasis during their disease, and the incidence continues to rise in the clinic. Brain metastasis is associated with poor prognosis and a sharp decline in motor and cognitive skills, which compromises day-to-day functioning and accelerates patient death. Fortunately, this dismal scenario has improved with the advent of third- generation EGFR tyrosine kinase inhibitors, such as osimertinib, which show excellent early responses in the clinic, even in patients with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop relapse, often to the brain, and succumb to death. It is therefore important to understand the underlying mechanisms of brain metastasis to improve targeted therapy outcomes, which is an unmet need in the field and can positively impact the lives of lung cancer patients.

The reappearance of cancer after a striking initial response on osimertinib implies that cancer cells continue to reside in organs below the detection limit. This prompts the following questions: How do these cancer cells escape osimertinib? What are the adaptive programs that help them resist osimertinib? Are there cell-intrinsic or cell-extrinsic changes, or a combination of both? Addressing these biological questions is important because residual cancer cells possibly represent the seeds of future relapse. We hypothesize that a combination of cancer-cell-intrinsic and cancer-cell-extrinsic (microenvironment) signaling drives the survival of these early lesions in the brain. Understanding the biology of residual cancer cells could help us design more effective targeting strategies to prevent future brain recurrences and prolong patient survival.

It is challenging to study residual disease in human patients when cancer is below the detection limit of imaging and it is unclear where residual cells are located. In such instances, mouse models that recapitulate the phases of drug response, residual disease, and relapse in the physiological context would enable the profiling and analysis of these cells. However, most studies on osimertinib resistance have relied on in-vitro cultures, short-term subcutaneous tumor implantation models, or sequencing of human tumors to detect genetic mechanisms of osimertinib resistance. To address this deficiency, we recently published a study describing the generation of osimertinib treatment-response-and-relapse mouse models using human lung cancer cells harboring osimertinib-sensitive, EGFR-activating mutations to study the mechanisms of brain relapse (Biswas et al., Cancer Discovery, 2022). Using these two independent mouse models of osimertinib resistance, we defined the distinct phases of osimertinib response, residual disease, and brain relapse. We will further leverage our preclinical models in this proposal to identify and profile osimertinib-refractory residual disease and relapse, which has not been previously studied and represents an unmet need in the field of lung cancer.

We have developed an innovative new platform known as spatially VISBL (Visualize, Interrogate, Small, Brain-Lesions) collaborating with a team of experts in single-cell and spatial transcriptomic profiling. Spatially VISBL enables us to profile cancer cells and their surrounding microenvironment. This new pipeline is modeled after rapid autopsy platforms used in the clinic that allow for the preservation of brain tissues within 3 hours of death for molecular analysis. Spatially VISBL combines rapid whole-brain vibratome slicing from fresh tissue with fluorescence microscopy and spatial and single-cell transcriptomics.

Using this platform, we have designed the following specific aim with associated subaims (a) and (b): Identify cancer-cell-intrinsic and -extrinsic pathways that sustain residual cells in the brain using the spatially VISBL platform; (a) Single-cell and spatial profiling of cancer cells in the brain and their surrounding microenvironment using the spatially VISBL (Visualize, Interrogate, Small, Brain-Lesions) platform; (b) pathway analysis to identify cancer-cell-intrinsic and -extrinsic pathways that sustain residual cells in the brain after osimertinib treatment. These profiles will be analyzed to identify druggable cell signaling pathways that are activated during different stages of brain lesions (from early to late) and can be targeted by pharmacological strategies in the near future. This proposal addresses the following overarching challenges in the FY22 Lung Cancer Research Program (LCRP) mission: (1) identify innovative strategies for the prevention of recurrence of or metastases from lung cancer and (2) understand mechanisms of resistance to treatment (primary and secondary). These studies will be beneficial for preventing brain relapse in patients with lung cancer that affects military personnel and their families, as well as the civilian population.