FER drives drug-tolerant persister cell survival in EGFR-mutant lung cancer

Approximately 20% of patients with metastatic non-small cell lung cancer (NSCLC) harbor an EGFR-mutation (EGFRm). Osimertinib, a third generation EGFR inhibitor, has dramatically improved patient outcomes, but invariably, residual disease or drug-tolerant “persister” (DTP) cells survive treatment, eventually giving way to resistant or progressive disease. Eradicating DTPs remains a key challenge. Although DTPs have been characterized at the transcriptional level, a major gap in our understanding is how the tyrosine kinome is perturbed. We hypothesized that defining the phospho-tyrosine landscape of DTPs would uncover kinases that could be exploited therapeutically. We generated DTPs by treating EGFRm cell lines with osimertinib (IC90) for 14 days and performed tyrosine-enriched, phospho-proteomics. Analysis using Kinase Library revealed that FER, a non-receptor tyrosine kinase that has been implicated in regulating a variety of pathways, is highly active in DTPs. To validate our findings, we confirmed that FER is auto-phosphorylated in DTPs. We then demonstrated that FER and its known substrates are phosphorylated within hours of osimertinib treatment, suggesting FER is activated early and throughout treatment. Next, we tested the functional impact of FER. FER knockdown had no effect on baseline phenotypes such as morphology or proliferation, but FER knockdown markedly decreased DTPs after 14 days of osimertinib treatment. We thus evaluated inhibiting FER as a therapeutic strategy. Alectinib, a well-tolerated FDA approved drug designed to target the ALK kinase, potently inhibits FER. We hypothesized that inhibiting FER using alectinib would eliminate DTPs and prevent osimertinib resistance. We first confirmed that 1) alectinib inhibits FER kinase activity and 2) ALK is not expressed in EGFRm NSCLC, consistent with prior reports. We subsequently treated cells with alectinib at 500nM, the physiologic concentration achievable using low dose alectinib in patients. Combining alectinib with osimertinib dramatically reduced DTPs in four EGFRm cell lines. Phospho-proteomics and RNA-sequencing revealed that alectinib and osimertinib suppressed FER phosphorylation and RHO GTPase signaling, a pathway known to drive DTP survival. Combining alectinib with osimertinib also significantly enhanced osimertinib sensitivity in patient-derived, EGFRm organoids. To test the strategy in vivo , we treated EGFRm xenografts with osimertinib or osimertinib and alectinib for only 21 days. After stopping treatment, 100% of the osimertinib-only treated mice had to be euthanized while 33% of mice treated with combination osimertinib and alectinib were disease-free after 150 days of observation. Collectively, our data show that FER is a key tyrosine kinase that is activated in DTPs and that targeting it with alectinib, an FDA approved drug, could be an effective strategy to enable durable remission in patients with EGFRm NSCLC.