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. Author manuscript; available in PMC: 2024 Sep 1.
Published in final edited form as: Nat Cell Biol. 2023 Aug 17;25(9):1346–1358. doi: 10.1038/s41556-023-01210-z

Fig. 1. KDM6A Inactivation in an Autochthonous SCLC Mouse Model Promotes NEUROD1 Expression Leading to SCLC Tumors that Express both ASCL1 and NEUROD1.

Fig. 1.

(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.