Fig. 5: Prioritisation, network analysis, and experimental validation of non-druggable combination therapy targets for SOX2-dependent LUSC.

A) TRIM28 was identified as a significantly enriched transcription factor (TF) within the WNT pathway community using a TF enrichment analysis of core network communities. TRIM28 directly regulates eight proteins in the core network, highlighting its critical role in the regulatory landscape of LUSC. B Proposed combination therapy strategies involve TRIM28 knockout paired with drugs targeting proteins from the four communities of the iPANDDA prioritised protein network: cell cycle, cytokine signalling, PI3K-AKT pathway, and WNT pathway. C A volcano plot depicts the effects of drug treatment targeting AKT, CDK1, FGFR, mTOR, and PARP in combination with TRIM28 knockout. Experiments were performed in two SOX2-dependent cell lines (LK-2 and KYSE-140). Cells were engineered using guide RNAs (sgRNA) for TRIM28 knockout or a non-targeting control sgRNA, and treatments were applied at 1 µM or 10 µM for 72 h. Cell viability was assessed, and compounds in the top right quadrant of the plot demonstrate significant enhancement of efficacy in combination with TRIM28 knockout. The full list of drug names is provided in Supplementary Fig. 6C for reference D The LK-2 cell line exhibited the strongest response to combination therapy (C). Elevated expression levels of both TRIM28 and SOX2 in LK-2 cells, along with a pronounced SOX2 gene dependency effect, were observed compared to other LUSC cell lines. This highlights the importance of TRIM28 and SOX2 co-dependency in identifying therapeutic vulnerabilities specific to SOX2-dependent LUSC.