HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. AR KO reduced levels of H3.3 at enhancers, indicating feedback mechanism. In addition, HIRA KO deregulates glucocorticoid-driven transcription, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity. Key points: *H3.3 at enhancers promotes acetylation of H3K27Ac and retention of AR/BRD4 complex for transcription regulation*Knockout of H3.3 chaperone HIRA suppresses PC cells growth and deregulates androgen-induced transcription*H3.3/HIRA pathway regulates both AR and GR, suggesting a common HIRA/H3.3 mechanism of nuclear receptors function.

Inhibition of Mps1 kinase enhances taxanes efficacy in castration resistant prostate cancer.

Androgen ablation therapy is the standard of care for newly diagnosed prostate cancer (PC) patients. PC that relapsed after hormonal therapy, referred to as castration-resistant PC (CRPC), often presents with metastasis (mCRPC) and is the major cause of disease lethality. The few available therapies for mCRPC include the Taxanes Docetaxel (DTX) and Cabazitaxel (CBZ). Alas, clinical success of Taxanes in mCRPC is limited by high intrinsic and acquired resistance. Therefore, it remains essential to develop rationally designed treatments for managing therapy-resistant mCRPC disease. The major effect of Taxanes on microtubule hyper-polymerization is a prolonged mitotic block due to activation of the Spindle Assembly Checkpoint (SAC). Taxane-sensitive cells eventually inactivate SAC and exit mitosis by mitotic catastrophe, resulting in genome instability and blockade of proliferation. Resistant cells remain in mitotic block, and, upon drug decay, resume mitosis and proliferation, underlying one resistance mechanism. In our study we explored the possibility of forced mitotic exit to elevate Taxane efficacy. Inactivation of the SAC component, mitotic checkpoint kinase Mps1/TTK with a small molecule inhibitor (Msp1i), potentiated efficacy of Taxanes treatment in both 2D cell culture and 3D prostasphere settings. Mechanistically, Mps1 inhibition forced mitotic catastrophe in cells blocked in mitosis by Taxanes. Androgen receptor (AR), the main driver of PC, is often mutated or truncated in mCRPC. Remarkably, Mps1i significantly potentiated CBZ cytotoxicity regardless of AR status, in both AR-WT and in AR-truncated CRPC cells. Overall, our data demonstrate that forced mitotic exit by Mps1 inhibition potentiates Taxanes efficacy. Given that several Mps1i's are currently in different stages of clinical trials, our results point to Mps1 as a new therapeutic target to potentiate efficacy of Taxanes in mCRPC patients.

Magnetic nanoparticle hyperthermia potentiates paclitaxel activity in sensitive and resistant breast cancer cells.

INTRODUCTION: Overcoming resistance to antimitotic drugs, such as paclitaxel (PTX), would represent a major advance in breast cancer treatment. PTX induces mitotic block and sensitive cells exit mitosis dying by mitotic catastrophe. Resistant cells remain in block and continue proliferation after drug decay, denoting one of the PTX resistance mechanisms. Mild hyperthermia (HT) triggers mitotic exit of PTX-pretreated cells, overcoming PTX resistance and suggesting HT-forced mitotic exit as a promising strategy to potentiate PTX. METHODS AND RESULTS: Superparamagnetic iron oxide nanoparticles (SPIONs) were used to deliver mild HT at 42°C in PTX-pretreated breast adenocarcinoma MCF-7 cells sensitive and resistant to PTX. To evaluate mechanism of cell death, cells were classified based on nuclear morphology into interphase, mitotic, micronucleated, and apoptotic. The combined PTX→SPION treatment resulted in an increase in the percentage of micronucleated cells, an indication of forced mitotic exit. Importantly, in PTX-resistant cells, the combination therapy using SPION HT helps to overcome resistance by reducing the number of cells relative to the control. CONCLUSION: SPION HT potentiates PTX by significantly reducing cell survival, suggesting potential of combined treatment for future clinical translation.

CENP-B protects centromere chromatin integrity by facilitating histone deposition via the H3.3-specific chaperone Daxx.

BACKGROUND: The main chromatin unit, the nucleosome, can be modulated by the incorporation of histone variants that, in combination with posttranslational histones modifications, determine epigenetics properties of chromatin. Understanding the mechanism that creates a histone variants landscape at different genomic elements is expected to elevate our comprehension of chromatin assembly and function. The Daxx chaperone deposits transcription-associated histone H3.3 at centromeres, but mechanism of centromere-specific Daxx targeting remains unclear. RESULTS: In this study, we identified an unexpected function of the constitutive centromeric protein CENP-B that serves as a "beacon" for H3.3 incorporation. CENP-B depletion reduces Daxx association and H3.3 incorporation at centromeres. Daxx/CENP-B interaction and Daxx centromeric association are SUMO dependent and requires SIMs of Daxx. Depletion of SUMO-2, but not SUMO-1, decreases Daxx/CENP-B interaction and reduces centromeric accumulation of Daxx and H3.3, demonstrating distinct functions of SUMO paralogs in H3.3 chaperoning. Finally, disruption of CENP-B/Daxx-dependent H3.3 pathway deregulates heterochromatin marks H3K9me3, ATRX and HP1α at centromeres and elevates chromosome instability. CONCLUSION: The demonstrated roles of CENP-B and SUMO-2 in H3.3 loading reveal a novel mechanism controlling chromatin maintenance and genome stability. Given that CENP-B is the only centromere protein that binds centromere-specific DNA elements, our study provides a new link between centromere DNA and unique epigenetic landscape of centromere chromatin.

Inhibitor of H3K27 demethylase JMJD3/UTX GSK-J4 is a potential therapeutic option for castration resistant prostate cancer.

Androgen receptor (AR) mediates initiation and progression of prostate cancer (PCa); AR-driven transcription is activated by binding of androgens to the ligand-binding domain (LBD) of AR. Androgen ablation therapy offers only a temporary relief of locally advanced and metastatic PCa, and the disease eventually recurs as a lethal castration-resistant PCa (CRPC) as there is no effective treatment for CRPC patients. Thus, it is critical to identify novel targeted and combinatorial regimens for clinical management of CRPC. Reduction of the repressive epigenetic modification H3K27me2/3 correlates with PCa aggressiveness, while corresponding demethylases JMJD3/UTX are overexpressed in PCa. We found that JMJD3/UTX inhibitor GSK-J4 reduced more efficiently proliferation of AR-ΔLBD cells (CRPC model) compared with isogenic AR-WT cells. Inhibition of JMJD3/UTX protects demethylation of H3K27Me2/3, thus reducing levels of H3k27Me1. We observed that the reduction dynamics of H3K27Me1 was faster and achieved at lower inhibitor concentrations in AR-ΔLBD cells, suggesting that inhibition of JMJD3/UTX diminished proliferation of these cells by hindering AR-driven transcription. In addition, we observed synergy between GSK-J4 and Cabazitaxel, a taxane derivative that is approved for CRPC treatment. Collectively, our results point at the H3K27 demethylation pathway as a new potential therapeutic target in CRPC patients.

Usp7 protects genomic stability by regulating Bub3.

USP7 (Ubiquitin Specific processing Protease-7) is a deubiquitinase which, over the past decade emerged as a critical regulator of cellular processes. Deregulation of USP7 activity has been linked to cancer, making USP7 inhibition an appealing anti-cancer strategy. The identification of novel USP7 substrates and additional USP7-dependent cellular activities will broaden our knowledge towards potential clinical application of USP7 inhibitors. Results presented in this study uncover a novel and pivotal function of USP7 in the maintenance of genomic stability. Upon USP7 depletion we observed prolonged mitosis and mitotic abnormalities including micronuclei accumulation, lagging chromosomes and karyotype instability. Inhibition of USP7 with small molecule inhibitors stabilizes cyclin B and causes mitotic abnormalities. Our results suggest that these USP7-dependent effects are mediated by decreased levels of spindle assembly checkpoint (SAC) component Bub3, which we characterized as an interacting partner and substrate of USP7. In silico analysis across the NCI-60 panels of cell lines supports our results where lower levels of USP7 strongly correlate with genomic instability. In conclusion, we identified a novel role of USP7 as regulator of the SAC component Bub3 and genomic stability.

Targeting mitotic exit with hyperthermia or APC/C inhibition to increase paclitaxel efficacy.

Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit. To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage. Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells' sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C.

Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions.

Nuclear structures ND10/PML NBs are linked to multiple processes, including the maintenance of intranuclear homeostasis by sequestering proteins into "nuclear depot." This function presumes release of proteins from PML NBs and their redistribution to the alternative, supposedly "active" locations, in response to the external stress application. To further investigate this nuclear depot function, we focused on the intranuclear distribution of protein Daxx that in normal conditions is mainly accumulated at PML NBs, and has a minor association with centromeres and pericentromeres (CEN/periCEN). Here we report that application of physiological Heat Shock (HS) changes this balance forcing very robust and reversible accumulation of Daxx on CEN/periCEN heterochromatin.   Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its maintenance. To understand functional consequences of Daxx deposition at CEN/periCEN, we tested for Daxx-dependency of heterochromatin transcription. Depletion of Daxx reduces accumulation of CEN RNA in normal conditions and periCEN RNA after HS application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. Surprisingly, HS-induced deposition of Daxx does not further elevate incorporation of H3.3 into CEN/periCEN that remained steady during stress and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of active H3K4Me2 modification at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: (1) regulation of H3.3 loading in normal conditions and (2) protection of epigenetic status upon stress-induced accumulation, thus collectively guarding epigenetic identity of CEN/periCEN heterochromatin.

PML NBs (ND10) and Daxx: from nuclear structure to protein function.

Proteins that combine PML NBs (ND10) can be divided into two groups: "transient" (that accumulate at PML NBs upon over-expression, interferon-induced up-regulation, block of proteosomal degradation, environmental stress or viral infection) and "constitutive" that co-localize with PML in the majority of cultured cells. One of the few "constitutive" components of PML NBs is the death domain-associated protein Daxx. While PML NBs are the most obvious depositories of Daxx, there are multiple alternative localization of this protein in the nucleus and cytoplasm, suggesting differential functionality of Daxx at different cellular compartments and stages of the cell cycle. The purpose of this review is to analyze Daxx spatiotemporal behavior within and outside of PML NBs and to discuss functions attributed to these localizations. We suggest that Daxx can participate in numerous cellular functions as a mediator of protein interactions, thus acting as a fine tuning instrument in highly orchestrated cellular processes; we also envision PML NBs accumulation of Daxx as an "out of action" storage depot.

Daxx shortens mitotic arrest caused by paclitaxel.

Resistance to the anti-neoplastic drug paclitaxel is frequent in breast cancer patients. Most studies of paclitaxel resistance have focused on pathways that elicit cellular response, while little is known about players involved in the acquirement of taxane resistance. By screening a cohort of breast cancer cell lines, we observed a correlation between level of protein Daxx and response to paclitaxel. Cells lines expressing increased level of Daxx displayed a robust paclitaxel response with nearly all cells undergoing micronucleation, while cell lines with low amount of Daxx showed a decrease in micronucleation, and accumulation in mitosis. At used paclitaxel concentrations, apoptotic levels were negligible in all cell lines tested. Human cell lines expressing anti-Daxx siRNA as well as Daxx-/- mouse fibroblasts showed similar cellular response to paclitaxel. Importantly, absence or depletion of Daxx resulted in cell survival after paclitaxel treatment, as measured by colony formation assay. We conclude that Daxx may be an important predictive factor in cellular response to paclitaxel, which emphasizes a critical but unknown function of this protein in mitotic progression, which, when disabled, leads to survival advantages upon paclitaxel treatment.