Abstract Small cell lung cancer (SCLC) is a recalcitrant cancer characterized by loss of TP53 and RB1. POU2F3+ SCLC cells are uniquely dependent on IGF1R signaling although this has not yet led to clinical translation. We therefore aimed to identify efficacious pharmacological targeting approaches for POU2F3+ SCLC and elucidate the underlying mechanisms.Cell viability was measured by CellTiter-Glo and soft agar assays. Drug targets and signaling pathways were identified by quantitative mass spectrometry-based proteomics and validated by Western blot. POU2F3 mutants were cloned to determine the kinase responsible for phosphorylation of POU2F3 the cellular effects of POU2F3 phosphorylation. Loss- and gain-of-function experiments were done using CRISPR, RNAi, chemical probes, or protein expression. Xenograft tumors were obtained by subcutaneous injection of POU2F3+ H526 cells into athymic nude mice. POU2F3 overexpressing, transgenic Rb1fl/flTrp53fl/fl(MycLSL/LSL) (RP/RPM) mice are being generated by genetic engineering and transfer of embryonic stem cells.Combination drug screening identified synergistic combinations between ALK-inhibitors with cross-reactivity for IGF1R and WEE1 inhibitors abrogating cell growth in vitro and in in vivo. Efficacy differences between the IGF1R inhibitors suggested the presence of additional druggable targets. Merging proteome-wide drug targets with changes in protein signaling and expression highlighted mTOR and cell cycle pathways and RSK1/2 as a relevant off-target. Interestingly, changes in both POU2F3 phosphorylation itself and expression of downstream POU2F3 transcriptional targets were identified upon individual or combination drug treatment and confirmed by immunoblotting. Rescue by overexpression of POU2F3 phospho-mutants upon CRISPR knockout of endogenous POU2F3 suggest an inhibitory function for pPOU2F3. Brigatinib and ceritinib, two potent FDA approved ALK/IGF1R inhibitors, were found to synergize with WEE1 inhibitors, in POU2F3+ SCLC. Quantitative, global chemical proteomics analysis found overlapping target profiles indicating favorable polypharmacology beyond IGF1R inhibition. Furthermore, quantitative phospho- and expression proteomics and subsequent genetic validation identified a novel regulatory mechanism of the oncogenic transcription factor POU2F3 by loss-of-function phosphorylation, which is strongly induced by drug combination treatment. This modification may indicate a new actionable vulnerability, which could be targeted to benefit the greatly underserved SCLC patient population. Citation Format: Lily L. Rix, Hayley D. Ackerman, Nicole R. Hackel, Xueli Li, Rachel Leigh O'Rourke, Juhyeon Son, Michelle A. Reiser, Arianna Nenci, Bin Fang, Eric A. Welsh, Brandon J. Klein, John M. Koomen, Eric B. Haura, Florian Karreth, Amanda Garner, Elsa R. Flores, Uwe Rix. Novel therapeutic opportunities in POU2F3 transcription factor-driven small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7653.
Rix et al. (Fri,) studied this question.