Fig. 3. Kdm6a Inactivation Alters Chromatin Accessibility and mRNA Expression for ASCL1 to NEUROD1 Subtype Switching.

(a) Immunoblot analysis of an additional cohort of SCLC lung tumors from LSL-Cas9 mice IT injected with sgControl RPP or sgKdm6a RPP adenoviruses. (b) RT-qPCR for Neurod1 from tumors from a where NEUROD1 protein was undetectable. C_t values of each sample are indicated on top. (c) PCA of all chromatin accessibility peaks from ATAC-seq data of 4 Kdm6a^WT SCLC tumors, 7 Kdm6a^MutantASCL1^Only SCLC tumors, and 6 Kdm6a^MutantASCL1^HighNEUROD1^Low SCLC tumors from the 2^nd cohort of mice. (d) PCA of differential chromatin accessibility peaks in Kdm6a^MutantNEUROD1^Only vs. Kdm6a^WT SCLC tumors (see Extended Data Fig. 3d) from both bulk and pseudo-bulk sc-ATAC-seq data of all tumors: 7 Kdm6a^WT SCLC tumors, 7 Kdm6a^MutantASCL1^Only SCLC tumors, 7 Kdm6a^MutantASCL1^HighNEUROD1^Low SCLC tumors, and 2 Kdm6a^MutantNEUROD1^Only SCLC tumors. For tumors in c,d, see Supplementary Table 4. (e, f) Average peak intensity at all peaks upregulated in Kdm6a^MutantNEUROD1^Only vs. Kdm6a^WT (e) or upregulated in Kdm6a^WT vs. Kdm6a^MutantNEUROD1^Only (f) for all tumors in d classified by phenotype. Legend in e also applies to f. p-values for e,f indicates comparisons among the 4 groups. p-values are calculated using Anderson-Darling k-sample test in R package “kSamples”. (g) Average chromatin accessibility tracks for each phenotype at the NEUROD1 target gene Myt1l from ATAC-seq data from d. (h) UMAP of all cells from scRNA-seq of the 10 autochthonous SCLC lung tumors indicated [3 Kdm6a-WT, 7 Kdm6a-Mutant including 5 Kdm6a^MutantASCL1^Only and 2 Kdm6a^MutantASCL1^HighNEUROD1^Low). n=48,651 total cells. Cell types were determined based on representative marker expression (Extended Data Fig. 4a). (I) UMAP of all tumor cells from the scRNA-seq from h. (j,k) Feature plots of Neurod1 (j) or the Neurod47_score (k). (l) Neurod47_score violin plot from k. For l, non-parametric Wilcoxon rank sum test was used to generate a two-tailed p-value. n=35,446 Kdm6a-Mutant and n=6612 Kdm6a-WT tumor cells. Minimum and maximum values define the range of violin plot. Dotted lines represent median and upper and lower quartiles.