LSD1 promotes prostate cancer reprogramming by repressing TP53 signaling independently of its demethylase function.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cell survival in cancers such as prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since the widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC versus adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors - but not catalytic inhibitors - reduced NEPC cell survival. RNA-Seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1's catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1's noncatalytic function may be a promising treatment strategy for NEPC.

The Role of Epigenetic Change in Therapy-Induced Neuroendocrine Prostate Cancer Lineage Plasticity.

The androgen receptor (AR) signaling pathway is critical for growth and differentiation of prostate cancer cells. For that reason, androgen deprivation therapy with medical or surgical castration is the principal treatment for metastatic prostate cancer. More recently, new potent AR signaling inhibitors (ARSIs) have been developed. These drugs improve survival for men with metastatic castration-resistant prostate cancer (CRPC), the lethal form of the disease. However, ARSI resistance is nearly universal. One recently appreciated resistance mechanism is lineage plasticity or switch from an AR-driven, luminal differentiation program to an alternate differentiation program. Importantly, lineage plasticity appears to be increasing in incidence in the era of new ARSIs, strongly implicating AR suppression in this process. Lineage plasticity and shift from AR-driven tumors occur on a continuum, ranging from AR-expressing tumors with low AR activity to AR-null tumors that have activation of alternate differentiation programs versus the canonical luminal program found in AR-driven tumors. In many cases, AR loss coincides with the activation of a neuronal program, most commonly exemplified as therapy-induced neuroendocrine prostate cancer (t-NEPC). While genetic events clearly contribute to prostate cancer lineage plasticity, it is also clear that epigenetic events-including chromatin modifications and DNA methylation-play a major role. Many epigenetic factors are now targetable with drugs, establishing the importance of clarifying critical epigenetic factors that promote lineage plasticity. Furthermore, epigenetic marks are readily measurable, demonstrating the importance of clarifying which measurements will help to identify tumors that have undergone or are at risk of undergoing lineage plasticity. In this review, we discuss the role of AR pathway loss and activation of a neuronal differentiation program as key contributors to t-NEPC lineage plasticity. We also discuss new epigenetic therapeutic strategies to reverse lineage plasticity, including those that have recently entered clinical trials.

BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program.

PURPOSE: Lineage plasticity in prostate cancer-most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program-is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear. EXPERIMENTAL DESIGN: Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity. RESULTS: AR inhibition accentuates lineage plasticity in t-NEPC cells-an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial. CONCLUSIONS: E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

LSD1 prevents aberrant heterochromatin formation in Neurospora crassa.

Heterochromatin is a specialized form of chromatin that restricts access to DNA and inhibits genetic processes, including transcription and recombination. In Neurospora crassa, constitutive heterochromatin is characterized by trimethylation of lysine 9 on histone H3, hypoacetylation of histones, and DNA methylation. We explored whether the conserved histone demethylase, lysine-specific demethylase 1 (LSD1), regulates heterochromatin in Neurospora, and if so, how. Though LSD1 is implicated in heterochromatin regulation, its function is inconsistent across different systems; orthologs of LSD1 have been shown to either promote or antagonize heterochromatin expansion by removing H3K4me or H3K9me respectively. We identify three members of the Neurospora LSD complex (LSDC): LSD1, PHF1, and BDP-1. Strains deficient for any of these proteins exhibit variable spreading of heterochromatin and establishment of new heterochromatin domains throughout the genome. Although establishment of H3K9me3 is typically independent of DNA methylation in Neurospora, instances of DNA methylation-dependent H3K9me3 have been found outside regions of canonical heterochromatin. Consistent with this, the hyper-H3K9me3 phenotype of Δlsd1 strains is dependent on the presence of DNA methylation, as well as HCHC-mediated histone deacetylation, suggesting that spreading is dependent on some feedback mechanism. Altogether, our results suggest LSD1 works in opposition to HCHC to maintain proper heterochromatin boundaries.

Selection and Characterization of Mutants Defective in DNA Methylation in Neurospora crassa.

DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The filamentous fungus Neurospora crassa places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render N. crassa an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants d efective i n m ethylation (dim). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of 11 genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene, dim-10 - a homolog of Clr5 in Schizosaccharomyces pombe - that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.

A Light-Inducible Strain for Genome-Wide Histone Turnover Profiling in Neurospora crassa.

In chromatin, nucleosomes are composed of ∼146 bp of DNA wrapped around a histone octamer, and are highly dynamic structures subject to remodeling and exchange. Histone turnover has previously been implicated in various processes including the regulation of chromatin accessibility, segregation of chromatin domains, and dilution of histone marks. Histones in different chromatin environments may turnover at different rates, possibly with functional consequences. Neurospora crassa sports a chromatin environment that is more similar to that of higher eukaryotes than yeasts, which have been utilized in the past to explore histone exchange. We constructed a simple light-inducible system to profile histone exchange in N. crassa on a 3xFLAG-tagged histone H3 under the control of the rapidly inducible vvd promoter. After induction with blue light, incorporation of tagged H3 into chromatin occurred within 20 min. Previous studies of histone turnover involved considerably longer incubation periods and relied on a potentially disruptive change of medium for induction. We used this reporter to explore replication-independent histone turnover at genes and examine changes in histone turnover at heterochromatin domains in different heterochromatin mutant strains. In euchromatin, H3-3xFLAG patterns were almost indistinguishable from that observed in wild-type in all mutant backgrounds tested, suggesting that loss of heterochromatin machinery has little effect on histone turnover in euchromatin. However, turnover at heterochromatin domains increased with loss of trimethylation of lysine 9 of histone H3 or HP1, but did not depend on DNA methylation. Our reporter strain provides a simple yet powerful tool to assess histone exchange across multiple chromatin contexts.