Defining the role of histone H3K4 mono-methyltransferase dysfunction in urothelial carcinoma

Project Summary/Abstract
Urothelial carcinoma (UC) involves the urothelial cells that line the bladder, kidney and ureters and is a major
cause of morbidity and mortality in the US, especially in men. Bladder UC can be clinically separated into
nonmuscle invasive (NMIBC) and muscle invasive (MIBC). MIBC accounts for the vast majority of metastasis
and mortality, having only a ~50% cure rate. Patients with treated NMIBC are at risk of recurrence or
progression to MIBC at prior or de novo sites. Over half of urothelial cancer, regardless of site of origin, harbor
loss of function mutations in the histone demethylase KDM6A (UTX) and in two highly homologous histone
methyltransferases KMT2C (MLL3) and KMT2D (MLL4). These proteins form the MLL3/4-COMPASS
(COMplex of Proteins ASsociated with Set1)-like complex that regulate enhancer function, partly through
methylation of histones at enhancers. Enhancers are regions of DNA that regulate lineage specific
transcriptional programs. Recent studies have shown that patients with two urothelial carcinomas in far away
sites (ureter and bladder) harbor the same COMPASS-like mutation. Further sequencing of histologically
benign urothelium identify frequent mutations in the complex at expand over time. Our hypothesis is that these
mutations under “field-cancerization” of the urothelium. Our lab has generated a genetically engineered mouse
model with deletion of Kmt2c, Kmt2d, or the combination in the urothelium. The urothelium of these mice
exhibit no histologic abnormalities. However, transcriptome analysis shows the urothelium exhibit increased
stemness and functional studies show they exhibit increased organoid forming abilities. When crossed into the
Pten conditional deletion mouse, there was robust cooperativity in tumorigenesis. The overall objective of this
proposal is to utilize our recently generated mouse models of urothelial this COMPASS-like complex loss to
mechanistically understand its role in tumor urothelial suppression. Specifically, in Aim 1, we seek to determine
the stemness, clonal dynamics, oncogene and carcinogen susceptibility of urothelium harboring mutations in
this COMPASS-like complex, using lineage tracing, organoid culture, and single-cell RNA-sequencing. In Aim
2, we seek to determine the functional interplay between MLL3/4-COMPASS dysfunction and oncogene
activation. In Aim 3, we will seek to define how loss of Kmt2c and Kmt2d in urothelial cells affect enhancer and
promoter function. Active enhancers are genomic regions of open chromatin with transcription factor binding,
divergent transcription of enhancer RNA, and looping to promoters. We will study each step by global mapping
of histone marks, chromatin accessibility, mRNA transcription of associated gene and looping to promoters
using state-of the art epigenetics techniques.

Grant Number: 4R01CA265026-04
NIH Institute/Center: NIH
Principal Investigator: Yu Chen