ASCL1 activates neuronal stem cell-like lineage programming through remodeling of the chromatin landscape in prostate cancer

Treatment with androgen receptor pathway inhibitors (ARPIs) in prostate cancer leads to the emergence of resistant tumors characterized by lineage plasticity and differentiation toward neuroendocrine lineage. Here, we find that ARPIs induce a rapid epigenetic alteration mediated by large-scale chromatin remodeling to support activation of stem/neuronal transcriptional programs. We identify the proneuronal transcription factor ASCL1 motif to be enriched in hyper-accessible regions. ASCL1 acts as a driver of the lineage plastic, neuronal transcriptional program to support treatment resistance and neuroendocrine phenotype. Targeting ASCL1 switches the neuroendocrine lineage back to the luminal epithelial state. This effect is modulated by disruption of the polycomb repressive complex-2 through UHRF1/AMPK axis and change the chromatin architecture in favor of luminal phenotype. Our study provides insights into the epigenetic alterations induced by ARPIs, governed by ASCL1, provides a proof of principle of targeting ASCL1 to reverse neuroendocrine phenotype, support luminal conversion and re-addiction to ARPIs.

RNA-seq, ChIP-seq, and ATAC-seq data generated in this study have been deposited and are available in the GEO database under the accession GSE183200 [https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183200]. Publicly available gene expression data from the SU2C/PCF-West Coast Dream Team cohort was downloaded from Aggarwal et al (PMID:29985747)  Sample sizes were determined based on estimates from preliminary experiments and similar studies so that reasonable statistical analysis could be conduced. For in vivo experiments, the number of mice assigned to each arm was selected to provide sufficient statistical power to discern significant differences, based on previous experience with the models used (Davies et al, Nature Cell Biology, 2021;Bishop et al, Cancer Discovery, 2017;Kuruma et al, Molecular Cancer Therapeutics, 2013).
No data was excluded from our study.
All experiments were repeated at least 2 independent times and the replicating experiments produced similar results.
Samples were randomly allocated.
For in vivo studies, mice were inoculated/treated by one researcher and a separate research took tumour measurements to ensure the experiments were run in a blinded manner. Blinding was not performed for in vitro experiments as the individual performing the analysis was also involved in collecting and labeling the samples. However, all analysis within next-generation sequencing experiments were run at the Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. The following antibodies were used for ChIP-seq: ASCL1 (10µg, Abcam, cat #556604), EZH2 (5"g, Active Motif, cat #39933), H3K27Me3 (5µg; Millipore, cat #07-449).
All antibodies used in this study were commercial and validated by the manufactures in human samples. For example the AR (Cell signaling #5153) has been validated by the manufacturer using SimpleChIP® Enzymatic Chromatin IP Kits. EED (Millipore #17-10034) was validated by the manufacturer using chromatin precipitation.
NCI -H660 (cat. #CRL-5813), C4-2 (cat. #CRL-3314), A549 (cat. #CRM-CCL-185) and H2107 (cat. #CRL-5983_FL) cell lines were obtained from ATCC. HEK293T (cat. #R70007) were obtained from ThermoFisher. CRPC (16DCRPC) and ENZ-resistant AR+ NE-like (42DENZR) cell lines were generated from LNCaP cells, previously detailed by our group Bishop et al (PMID: 27784708) and Davies et al (PMID:34489572). We utilized an in vivo model of CRPC and ENZ resistance previously developed by us 54-56 that mirrors clinically reported treatment refractory phenotypes. LNCaP cells were inoculated into mice and upon castration CRPC tumors (16DCRPC) emerged. Further treatment of 16DCRPC tumors with ENZ (10mg/kd/d) lead to the reemergence of tumors with heterogeneous resistance mechanisms, including lineage plasticity. 42DENZR tumors exhibit AR expression, but loss of canonical AR signaling, concomitant with an enrichment in plasticity and neuronal transcriptional Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.

Files in database submission
programs.
All cell lines have been authenticated via STR profiling.
All cell lines tested negative for mycoplasma in routine checks using the Lonza MycoAlert kit.
No commonly misidentified cell lines were used in the study.
This study did not involve wild animals.
This study did not involve samples collected from the field.
All animal studies were performed in accordance with protocols approved by the Animal Care Committee at the University of British Columbia (A16-0246).

GEO:GSE183200
Raw sequencing files (fastq) and coverage files (bigwig) are deposited for ChIP-seq and ATAC-seq experiments. Raw sequencing files (fastq) and read counts (text) are deposited for RNA-seq data.