TISSUE-LOCATION SPECIFIC TRANSCRIPTIONAL PROGRAMS IN COLON DETERMINE MOLECULAR DEPENDENCIES FOR TUMOR INITIATION [ChIP-Seq]

It is not known why cancers arising in anatomically distinct locations but within the same tissue type can exhibit stark differences in molecular, pathological and clinical features. Cancers arising in proximal and distal sites of the colon are emblematic of these above differences as the proximal and distal colon cancers harbor differences in key molecular features, with BRAFV600E oncogene predominantly occurring in proximal colon cancers in the context of the increased promoter DNA methylation phenotype (CIMP-high), while distal colon cancers lack these molecular features. We determined the molecular basis of tissue-location specificity of driver mutations using normal mouse derived organoid models for colon cancer initiation. We show that the homeobox transcription factor, Cdx2, which is downregulated by DNA methylation in proximal colon cancers, plays distinct roles in regulating stem cell and differentiation in proximal compared to distal colon stem cells. Loss of Cdx2 altered the differentiation and stem cell programs and result in transformation by BRAFV600E specifically in the proximal but not distal colon stem cells. Analyses of RNA expression and Cdx2-binding to target genomic regions revealed that Cdx2 was a key effector of the transcriptional response to differentiation cues in proximal colon cells. This proximal colon-specific transcriptional program concurrently is tumor suppressive, and its loss sufficiently creates permissive state for oncogenic-BRAF mutations. The transcriptional program associated with Cdx2 loss in mouse proximal organoids matches that of human proximal colon cancers with downregulated CDX2 expression due to epigenetic silencing. Our analyses reveal that developmental and lineage-restricted transcription factors maintain tissue-location specific transcriptional programs which create critical dependencies for tumor initiation. Proximal and distal colon organoids were generated from 3 mice and cultured in WENR-plus medium for 4 months before collecting sample for ChIP-seq. We applied Chipmentation to identify Cdx2 bound genomic regions in the organoids, with minor adaptions(Schmidl et al., 2015). For WENR-plus group, organoids were cultured in WENR-plus medium, while for WENR-minus group, organoids were cultured in WENR-plus medium for 2 day first after plating in 150 μl of Matrigel, followed with 3 days treatment of WENR-minus medium. Samples were subsequently processed or ChIP-seq (ChIP-mentation) analyses.