Defining splicing factor requirements for androgen receptor variant synthesis in advanced prostate cancer.

Resistance to androgen receptor (AR)-targeted therapies represent a major challenge in prostate cancer (PC). A key mechanism of treatment resistance in patients who progress to castrate-resistant PC (CRPC) is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike full-length AR (FL-AR) isoforms, AR-Vs are constitutively active and refractory to current receptor-targeting agents hence drive tumour progression. Identifying regulators of AR-V synthesis may therefore provide new therapeutic opportunities in combination with conventional AR-targeting agents. Our understanding of AR transcript splicing, and the factors that control the synthesis of AR-Vs, remains limited. While candidate-based approaches have identified a small number of AR-V splicing regulators, an unbiased analysis of splicing factors important for AR-V generation is required to fill an important knowledge gap and furnish the field with novel and tractable targets for PC treatment. To that end, we conducted a bespoke CRISPR screen to profile splicing factor requirements for AR-V synthesis. MFAP1 and CWC22 were shown to be required for the generation of AR-V mRNA transcripts and their depletion resulted in reduced AR-V protein abundance and cell proliferation in several CRPC models. Global transcriptomic analysis of MFAP1-depleted cells revealed both AR-dependent and -independent transcriptional impact, including genes associated with DDR. As such, MFAP1 downregulation sensitised PC cells to ionising radiation suggesting therapeutically targeting AR-V splicing could provide novel cellular vulnerabilities which can be exploited in CRPC. Implications: We have utilised a CRISPR screening approach to identify key regulators of pathogenic AR splicing in prostate cancer.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

The proneural transcription factor ASCL1 regulates cell proliferation and primes for differentiation in neuroblastoma.

Neuroblastoma is believed to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme, but instead become locked in a pro-proliferative developmental state. Achaete-scute homolog 1 (ASCL1) is a proneural master regulator of transcription which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells during development, while its expression has been implicated in the maintenance of an oncogenic programme in MYCN-amplified neuroblastoma. However, the role of ASCL1 expression in neuroblastoma is not clear, especially as its levels vary considerably in different neuroblastoma cell lines. Here, we have investigated the role of ASCL1 in maintaining proliferation and controlling differentiation in both MYCN amplified and Anaplastic Lymphoma Kinase (ALK)-driven neuroblastoma cells. Using CRISPR deletion, we generated neuroblastoma cell lines lacking ASCL1 expression, and these grew more slowly than parental cells, indicating that ASCL1 contributes to rapid proliferation of MYCN amplified and non-amplified neuroblastoma cells. Genome-wide analysis after ASCL1 deletion revealed reduced expression of genes associated with neuronal differentiation, while chromatin accessibility at regulatory regions associated with differentiation genes was also attenuated by ASCL1 knock-out. In neuroblastoma, ASCL1 has been described as part of a core regulatory circuit of developmental regulators whose high expression is maintained by mutual cross-activation of a network of super enhancers and is further augmented by the activity of MYC/MYCN. Surprisingly, ASCL1 deletion had little effect on the transcription of CRC gene transcripts in these neuroblastoma cell lines, but the ability of MYC/MYCN and CRC component proteins, PHOX2B and GATA3, to bind to chromatin was compromised. Taken together, our results demonstrate several roles for endogenous ASCL1 in neuroblastoma cells: maintaining a highly proliferative phenotype, regulating DNA binding of the core regulatory circuit genes to chromatin, while also controlling accessibility and transcription of differentiation targets. Thus, we propose a model where ASCL1, a key developmental regulator of sympathetic neurogenesis, plays a pivotal role in maintaining proliferation while simultaneously priming cells for differentiation in neuroblastoma.

The Pioneering Role of GATA2 in Androgen Receptor Variant Regulation Is Controlled by Bromodomain and Extraterminal Proteins in Castrate-Resistant Prostate Cancer.

The androgen receptor (AR) is a key driver of prostate cancer development. Antiandrogens effectively inactivate the AR, but subsequent AR reactivation progresses the disease to castrate-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-V) that function unchallenged by current AR-targeted therapies are key drivers of CRPC. Currently, very little is known about the regulation of AR-Vs at the chromatin level. Here, we show that the pioneer factor GATA2 is a critical regulator of AR-Vs. Furthermore, we demonstrate that the GATA2 cistrome in CRPC shares considerable overlap with bromodomain and extraterminal (BET) proteins and is codependent for DNA binding. GATA2 activity is compromised by BET inhibitors, which attenuates the pioneering role of GATA2 in CRPC. In all, this study indicates that GATA2 is a critical regulator of AR-V-mediated transactivation and is sensitive to BET inhibitors, signifying these agents may be efficacious in patients with CRPC which overexpress GATA2. IMPLICATIONS: We have defined novel mechanisms of AR-V and GATA2 regulation in advanced prostate cancer that could be therapeutically exploited.

Sporadic and endemic Burkitt lymphoma have frequent FOXO1 mutations but distinct hotspots in the AKT recognition motif.

FOXO1 has an oncogenic role in adult germinal center-derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1, resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples, respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3, or members of the phosphatidylinositol 3-kinase signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

FOXA1 regulates androgen receptor variant activity in models of castrate-resistant prostate cancer.

Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients.