Fig 2: Assessment of in vitro antiproliferative activity of targeted agents in a panel of SCLC cell lines (a); effect of volasertib (b) and onvansertib (c) on proliferation of SCLC cell lines. Cells were treated for 72 h with indicated agents. Cell proliferation was determined using colorimetric or luminescent assays depending on the degree of clustering of SCLC cell lines in culture. Values represent the mean ± S.D. from a minimum of 3 independent experiments. Blue and orange curves define cell lines with non-disruptive and disruptive p53 mutations, respectively. Basal protein expression in SCLC cell lines (d). SCLC subtype based on expression are indicated after each cell line: ASCL1 (A), POU2F3 (P), YAP1 (Y).

Fig 3: ASCL1 binds to the FOXA2 locus in met-associated primary SCLC.a Immunofluorescence (IF) image of never-met primary and met-associated primary tumor clinical samples stained for DAPI, ASCL1, PROX1, and FOXA2 for 2 cases. Insets are indicated in white box. Scale bar: 1000 μm (overlay), 100 μm (DAPI, ASCL1, FOXA2). b Schematic of FFPE-ATAC-seq from never-met primary and met-associated primary tumor clinical samples. Created in BioRender. Rudin C. (2025) https://BioRender.com/mlreujw. c Chromatin accessibility assessed by ATAC-seq at the FOXA2 locus (Peaknorm). Highlighted regions indicate promoter and enhancer domains. d Schematic of ATAC-seq and ChIP-qPCR on PDX tumors. Created in BioRender. Rudin C. (2025) https://BioRender.com/mlreujw. e ATAC-seq comparing ASCL1+FOXA2+ PDX (Lx101, Lx328, Lx891) vs ASCL1+FOXA2- (Lx1231) PDX tumors at the FOXA2 locus. f IgG and ASCL1 ChIP-qPCR of the FOXA2 locus, and negative control region in one ASCL1+FOXA2- (A + F-) and three ASCL1+FOXA2+ (A + F + ) SCLC PDX models from panel. Bars are shown in Mean±S.D of N = 3 experimental replicates. g Schematic of ASCL1 interaction with FOXA2 in never-met versus met-associated SCLC. Created in BioRender. Rudin C. (2025) https://BioRender.com/j34m841.

Fig 4: Metastatic characteristics of SCLC.a Metastatic characteristics of an MSK-SCLC cohort (N = 327). Localized disease (left) including the fraction of never-metastasized cases (dark blue). Frequency (middle) and number (right) of distant metastatic sites are shown among cases with any metastases. LN: Lymph node. b Schematic of the approach to metastatic subline generation in SCLC cell lines. i.c.: intra-cardiac injection. Created in BioRender. Rudin C. (2025) https://BioRender.com/j5rkxx5. c Frequency and distribution of metastases in H1836 (ASCL1 subtype) and H82 (NEUROD1 subtype) SCLC cell lines, and Lx773I SCLC PDX (POU2F3 subtype) parental lines and organ-specific sublines after intracardiac implantation.

Fig 5: Induction of CRBN expression and the enhanced sensitivity to CYRS381 by neuroendocrine cancer drivers. A Description of the combinations of neuroendocrine cancer-driving factors transduced into non-neuroendocrine lung cancer cell lines. RPMA or RPMN represents the combination of Rb1 knockout, TP53 R175H, and N-MYC plus ASCL1 or NeuroD1, respectively. B Upregulation of CRBN protein expression by transduction of RPMA or RPMN in non-neuroendocrine lung cancer cell lines. NCI-H2023 or HCC827 cell lines were transduced with the designated combinations of neuroendocrine cancer drivers (RPMA or RPMN) via lentiviral infection. Rb1 knockdown and overexpression of N-MYC, ASCL1, and NeuroD1 were confirmed by immunoblotting, while TP53 R175H expression was confirmed by qRT-PCR using a specific primer for the mutant. CRBN protein expression was detected by immunoblotting. For the qPCR assay, data represent mean ± SD from three replicates (significantly different compared to wild-type cell lines, **P < 0.01, ***P < 0.01, and **** P < 0.0001). C, D Comparison of CYRS381 effects on GSPT1 degradation in wild-type and RPMA or RPMN-transduced non-neuroendocrine lung cancer cell lines. The concentration-dependent effects of CYRS381 on GSPT1 degradation in transduced NCI-H2023 (C) or HCC827 (D) cell lines were measured by immunoblotting after 24 h of treatment at the indicated concentrations. E, F Comparison of CYRS381 effects on translation rates in wild-type and RPMA or RPMN-transduced non-neuroendocrine lung cancer cell lines. CYRS381 effects on translation rates were measured using puromycin incorporation assays after treatment with CYRS381 (3 μM for 24 h) in multiple cell lines. G, H Effect of transduction of neuroendocrine drivers (RPMA or RPMN) on CYRS381 sensitivity in non-neuroendocrine lung cancer cell lines. The sensitivities of wild-type and RPMA or RPMN-transduced NCI-H2023 (G) or HCC827 (H) cell lines were compared using cell viability assays (WST-8) after 72 h of CYRS381 treatment. Data represent mean ± SD from three replicates