(a) Schematic of the adenovirus used for intratracheal injection (IT) into the lungs of lox-stop-lox (LSL)-Cas9 mice to generate autochthonous SCLC tumors that are Kdm6a inactivated or Kdm6a wild-type (WT). RPP=sgRb1, sgTrp53, sgRbl2; sg “T”=sgKdm6a or sgControl (non-targeting sgRNA). (b) Immunoblot analysis of MEFs expressing Cas9 infected with the sgControl RPP, sgKdm6a #3 RPP or sgKdm6a #4 RPP adenoviruses as indicated. (c) Immunoblot analysis of SCLC lung tumors formed in LSL-Cas9 mice injected with sgControl RPP (Kdm6a-WT) or sgKdm6a RPP (Kdm6a-Mutant) adenoviruses. (d) Volcano plot of differential expression analysis from RNA-seq data comparing Kdm6a-Mutant vs. Kdm6a-WT from tumors in c. n=7 Kdm6a-Mutant tumors, n=6 Kdm6a-WT tumors. FDR p-values adjusted for multiple comparisons after log transformation are shown. Neurod1 and one of its target genes Pax6 are highlighted in red. (e) Gene set enrichment analysis (GSEA) of RNA-seq data from ASCL1 and NEUROD1 human SCLC tumors34 of the upregulated genes in Kdm6a-Mutant vs Kdm6a-WT GEMMs (see Supplementary Table 2). FDR q-value adjusted for multiple comparisons is indicated. (f, g) Immunoblot analysis of two mouse SCLC cell lines derived from Kdm6a-WT mice 1014 (f) and 159-1 (g) transduced with 2 independent Kdm6a sgRNAs or a non-targeting control (sgControl) and maintained in culture for 30 days post-transduction. (h, i) Immunoblot analysis of two human ASCL1-positive SCLC cell lines, NCI-H69 (h) and DMS79 (i), nucleofected with Cas9 RNP containing a Kdm6a sgRNA or a non-targeting control (sgControl). Cells were then treated with cisplatin (1 μM) or DMSO for 3 days. (j) Multiplexed immunofluorescence for ASCL1 and NEUROD1 from 3 Kdm6a-WT and 3 Kdm6a-Mutant mouse SCLC lung tumors indicated. Scale Bar= 50 μm.