Introduction:

Bladder cancer (BLCA), both non-muscle invasive and muscle-invasive, frequently (6-10%) has biallelic inactivation of the tumor suppressor gene TSC1. However, the mechanism by which TSC1 loss promotes BLCA development beyond mTORC1 activation is uncertain. Currently, treatment with rapalogs/other mTOR inhibitors has shown limited effectiveness against TSC1 mutant BLCA.

Methods:

We performed differential gene expression and gene set enrichment analyses (GSEA) using The Cancer Genome Atlas (TCGA) RNA-sequencing data for TSC1-mutated BLCA (TSC1mt-BLCA, n=26) and TSC1 wild-type tumors (TSC1wt-BLCA, n=382) and compared them to an internal cohort of other TSC1/TSC2-mutant tumors (n = 48) seen in patients with Tuberous Sclerosis Complex (TSC). Integrative multi-omics transcriptomic, epigenetic, and functional analyses, including RNA-seq, H3K27ac (a histone to study open chromatin/active transcriptional sites) ChIP-seq, TFE3 CUT&RUN, ATAC-seq and TFE3 knock-out using CRISPR/Cas9 were conducted using various TSC1-mutant/addback BLCA cell lines. Validation studies were performed in a TSC1mt-BLCA patient derived xenograft model.

Results:

: Comparison of TSC1mt-BLCA and TSC1wt-BLCA tumors revealed a conserved TSC-associated gene expression signature in TSC1mt-BLCA that was strikingly similar to that observed in syndromic TSC tumors. GPNMB had the highest increase (median fold change 9.8). GSEA and DESeq2 indicated both mTORC1 hyperactivation and enhanced lysosomal gene expression in TSC1mt-BLCA. Immunohistochemistry confirmed these expression changes in a separate cohort of BLCA, including elevated nuclear-localized TFE3 in TSC1mt-BLCA, a known regulator of lysosomal gene expression. TSC1-mutant BLCA cell lines (HCV29, RT4) recapitulated the human BLCA expression phenotype, and showed TFE3 nuclear localization, which was reversed by rapamycin. TFE3 knockout in HCV29 cells had major effects, reducing growth and lysosomal gene expression, and inducing inflammatory and senescence-associated gene expression. TFE3 CUT&RUN analysis of HCV29 cells confirmed TFE3's direct regulation of lysosomal and other gene pathways. Also, TSC1mt-BLCA patient derived xenografts retain the TFE3-driven expression phenotype.

Conclusion:

Our data reveals TSC1mt-BLCA as a distinct molecular subtype of bladder cancer, and show that human TSC1mt-BLCA tumors and cell lines have a transcriptional signature similar to that of TSC syndromic tumors, characterized by both mTORC1 activation and TFE3-driven gene expression. The essential role of TFE3 expression in TSC1mt-BLCA development suggests that it may represent a therapeutic vulnerability

Funding: N/A