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: 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 4: 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 5: Gene associations and regulation of FOXA2 expression.a FOXA2 IHC staining of primary SCLC. Representative images of a FOXA2-negative region and a FOXA2-positive region are shown. Scale bar: 500 μm and 100 μm (inset). b scRNA-seq UMAP including 19 clinical metastatic SCLC tumors indicating FOXA2+ and FOXA2- cells. c Violin plot of FOXA2 expression by scRNA-seq in each of these 19 cases. d Pulmonary neuroendocrine cell signature expression in FOXA2+ cells and FOXA2- cells assessed by scRNA-seq (** < 0.01, P = 0.0000, one-sided Mann–Whitney U test). e Ranking plot of genes highly expressed in FOXA2+ vs. FOXA2- cells at the single-cell level. f Correlation plot of nominated top 100 genes from panel (e) assessed relative to FOXA2 expression in bulk RNA-seq data. g Heatmap of co-expression on ASCL1, FOXA2, and PROX1 in scRNA-seq. h ASCL1 KD effect on protein expression of PROX1, FOXA2, and GAPDH (control) in H1836 and SHP-77 cell lines. The samples derive from the same experiment but different gels for ASCL1, FOXA2, another for PROX1 and GAPDH were processed in parallel. Western blot result is a representative data of one of the 3 individual experiments. i, j ASCL1 ChIP-seq at the FOXA2 and PROX1 gene loci in 2 SCLC cell lines (H1836 and SHP-77). Two independent replicates using anti-ASCL1, and two isotype (IgG) controls are shown. Highlighted regions indicate promoter and enhancer domains. k PROX1 KD and protein expression of FOXA2 and GAPDH (control) in H1836 and SHP-77 cell lines. The samples derive from the same experiment but different gels for FOXA2, another for PROX1 and GAPDH were processed in parallel. Western blot result is a representative data of one of the 3 individual experiments.