Lung adenocarcinoma (LUAD) is one of the deadliest cancers, with EGFR mutations—particularly EGFR-TKI sensitizing mutations—being common. Currently, osimertinib is the most used and effective FDA-approved EGFR-TKI; however, resistance is inevitable. Focusing on unknown epigenetic and transcription factors contributing to osimertinib resistance, we recently employed a CRISPR knockout screening that identified the AP1 transcription factor (FOSL1 and JUN) as a key modulator of resistance. Silencing FOSL1 or JUN restored drug sensitivity, revealing a synthetic lethal interaction with the EGFR pathway. Integrative multi-omics analyses (RNA-Seq, ATAC-Seq, CRISPR knock-out screening) highlighted VAV3, a key molecular-switch in Rho-GTPase family, and specifically its major transcript VAV3.1 as a promising resistance-associated gene. Ongoing studies aim to clarify the role of VAV3.1 in the "transcriptional regulation – resistance" axis.

To investigate the role of VAV3.1, multiple bioinformatic analyses and in vitro experiments were conducted. qRT-PCR analysis confirmed the dominance of VAV3.1 over VAV3 in HCC827-OsiR cells, also validated by RNA-Seq. VAV3.1 was then knocked out using CRISPR-Cas9 methodology. Possible synthetic lethal interactions between VAV3.1 knock-out and osimertinib were tested through 2D colony formation, competitive cell proliferation, and BrdU assays. Western blotting was performed to explore the effects of VAV3.1 on survival pathways. Vav3.1 was then overexpressed in parental HCC827 cells to assess the changes in proliferation and survival capabilities.

Our findings validated that VAV3.1, containing a unique 110-nucleotide sequence that suggests a distinct promoter regulation, was the dominant transcript of VAV3 in our cells. Using integrative multi-omics studies, VAV3.1 was identified to have significant changes in expression and chromatin accessibility, suggesting a role in resistance. However, in vitro assays showed a minor effect of VAV3.1 silencing in osimertinib sensitization.

Here we provide the first preliminary findings of a unique VAV3 transcript, VAV3.1, as a downstream target of the AP1 family in osimertinib resistance. Our findings highlight the complexity of VAV3.1's involvement in drug resistance that could stem from the polyclonal nature and heterogeneity of our cells. Ongoing experimental strategies aim to further investigate the precise role of VAV3.1 in resistance mechanisms for a more comprehensive understanding of its function.