Extended Data Fig. 10. Analyses of KMT2D Loss or Hypoxia with NEUROD1 Expression in SCLC.

(a) Correlation of KDM6A dependency vs. KMT2D depedency across hundreds of cancer cell lines²⁵. KMT2D is the #1 co-dependency with KDM6A (p-value=9.09x10⁻⁶⁷ calculated using dependency map²⁵). (b) GSEA using publicly available datasets of human SCLCs with KMT2D LOF mutations or KMT2D WT⁴ using genes upregulated in Kdm6a-Mutant vs. Kdm6a-WT SCLC GEMMs (see Fig. 1d). FDR q-value generated using GSEA, adjusted for multiple-hypothesis testing, is indicated. (c) Proportion of mixed ASCL1 and NEUROD1 human SCLCs from RNA-seq data from 3 independent data sets^4,41,84 (see methods) in SCLCs with KMT2D LOF mutations vs. KMT2D WT. Fisher’s exact test was used to generate p-value. For b,c, n=14 KMT2D LOF, n=142 KMT2D WT. See also Supplementary Table 9. (d, e) CRISPR amplicon sequencing of 1014 Kdm6a-WT cells transduced with 2 independent Kmt2d sgRNAs. sgRNA sequences are in blue and gene editing is in red. (f) RT-qPCR for Neurod1 in 1014 Kmt2d knockout cells in d,e 3 weeks after transduction. Data are relative to Actb and then normalized to Neurod1 expression in the sgControl (non-targeting) cell line. n=4 biological replicates and error bars represent mean ± SEM. Statistical significance was calculated using unpaired, two-tailed students t-test and p-values are indicated. (g,h) Immunoblot (g) and histone blot (h) analyses in 1014 cells transduced with 4 independent Kmt2c and Kmt2d sgRNAs or a non-targeting control (sgControl) 30 days post-transduction. (i,j) Immunoblot analysis of NEUROD1 and ASCL1 expression of (i) NCI-H69 and (j) DMS79 ASCL1-positive human SCLC cells cultured under 5% or 21% O₂ for 7 days. (k) Immunoblot analysis for HIF1α protein of cells lines in i,j. (l, m) GSEA analysis of scRNA-seq from human ASCL1 or NEUROD1 SCLC tumors³⁴ using (l) HIF1α target gene list⁵¹ or (m) 24 genes induced by hypoxia⁵⁰ (see methods) (see Supplemental Table 10). FDR q-value adjusted for multiple hypothesis testing is indicated. Model-based Analysis of Single Cell Transcriptomics (MAST) was used to generate the GSEA expression profile.