Aberrant Expression of ERG Promotes Resistance to Combined PI3K and AR Pathway Inhibition through Maintenance of AR Target Genes

Abstract

On the basis of our previous work defining the molecular rationale for combined targeting of the PI3K and AR pathways in PTEN loss prostate cancer, the first clinical trial was recently reported demonstrating a significant benefit for combination therapy in patients with metastatic prostate cancer. In this phase II trial, loss of PTEN was a biomarker predictive of response to combined AKT and AR inhibition. Given that PTEN loss prostate cancers are significantly enriched for ERG genomic rearrangements, we evaluated how the aberrant expression of ERG may impact response to PI3K/AR-targeted therapy. Here, we show that overexpression of ERG in the setting of Pten loss promotes resistance to combined PI3K and AR pathway inhibition with associated maintenance of AR target gene expression. Importantly, following AR knockout in the setting of ERG overexpression, there is maintenance of a subset of AR lineage-specific target genes, making AR dispensable in this context. This has important clinical implications as even in the setting of the androgen-regulated TMPRSS2:ERG genomic rearrangement, ERG expression is never abolished following AR inhibition and may allow for cell survival following AR (lineage)-targeted therapies.

Figure 1.. ERG aberrant expression promotes resistance to PI3K and AR targeted therapy.

A) Waterfall plots for proportional change in tumor volume in Pten^lox/lox (n=4) and ERG Pten^lox/lox mice (n=4) treated with BEZ235 (30mg/kg/day) and MDV3100 (30mg/kg/day). B) Representative MRI images of the ERG Pten^lox/lox mice pre- and post-treatment. C) Tumor volume measurements for ERG Pten^lox/lox tumor grafts (n=10 per group) treated with vehicle, castration+MDV3100, BEZ235, and castration+MDV3100+Bez235. D) Western blot confirming PI3K pathway target inhibition following acute treatment with BEZ235. E) AR immunohistochemistry showing cytoplasmic sequestering following combined PI3K/AR inhibition. F) qRT-PCR of canonical AR target genes across the treatment groups (3 tumors per group) demonstrates maintenance of AR target gene expression despite complete androgen blockade.

Figure 2.. Establishment and characterization of Pten−/− and ERG Pten−/− prostate cancer organoids.

A) Tumors derived from our Pten^lox/lox and ERG Pten^lox/lox GEM models were used to establish prostate cancer organoids (3 clones for each genotype) and characterized in 3-D culture conditions for histology, and immunohistochemistry was performed for ERG and AR. B) Western blotting confirming loss of Pten, activation of PI3K pathway, and ERG over-expression. C) Pten−/− and ERG Pten−/− organoids underwent AR Crispr (3 individual clones for each genotype) and were characterized in 3-D culture conditions for histology, and immunohistochemistry was performed for ERG and AR. D) Western blotting confirming loss of Pten, activation of PI3K pathway, ERG over-expression, and loss of AR following AR Crispr.

Figure 3.. AR target and luminal gene expression is maintained in the presence of ERG following AR knock-out.

A) RNAseq expression profiling (3 clones for each genotype) for genes differentially expressed greater than 1.5 fold following AR Crispr in Pten−/− and ERG Pten−/− organoids shows substantial overlap in AR targets and a subset of genes that are not differentially regulated in the context of ERG. B) AR gene signature was developed in the Pten−/− organoids following AR knock-out and then applied across our organoid models. C) Gene set enrichment analysis was performed revealing loss of enrichment for the AR regulated luminal gene set in Pten−/− organoids following AR knock-out. D) Gene set enrichment analysis was performed comparing Pten−/− and ERG Pten−/− following AR knock-out which demonstrated enrichment for the luminal signature in the ERG Pten−/− AR Crispr organoids. E) Gene set enrichment analysis revealed loss of enrichment for luminal gene sets in our Pten−/− organoids following AR knock-out. F) Gene set enrichment analysis demonstrating enrichment of basal signatures (loss of luminal signatures) in the Pten−/− versus ERG Pten−/− following AR Crispr.

Figure 4.. Interaction of ERG and AR on transcription start site and enhancer regions.

A) ChIP seq analyses for ERG and AR shows that in ERG Pten−/− organoids there is significant overlap of AR and ERG binding peaks at transcription start site and enhancer regions. B) AR knock-out revealed an increase in ERG binding at enhancer regions in a subset of genes. C) Integrated genomics view of AR and ERG chromatin binding at the enhancer regions of Tmprss2 and Fkbp5.

Figure 5.. Over-expression of ERG promotes resistance to combined PI3K and AR inhibition in a TMPRSS2:ERG pre-clinical model.

A) VCaP cells harboring the TMPRSS2: ERG genomic rearrangement display resistance to PI3K (BEZ235 30mg/kg/day) and AR (castration+enzalutamide 30mg/kg/day) pathway inhibition following modest exogenous over expression of ERG (n=10 tumors per group). B) Western blot following acute in vivo treatment with PI3K and AR pathway inhibition. C) Hisotlogic and immunohistochemical analyses of VCaP tumors. D) Model of ERG and AR interaction in established prostate cancers.