Viacheslav M. Morozov

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.

USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora-A kinase

A large number of patients are resistant to taxane-based chemotherapy. Functional mitotic checkpoints are essential for taxane sensitivity. Thus, mitotic regulators are potential markers for therapy response and could be targeted for anticancer therapy. In this study, we identified a novel function of ubiquitin (Ub)-specific processing protease-7 (USP7) that interacts and cooperates with protein death domain-associated protein (Daxx) in the regulation of mitosis and taxane resistance. Depletion of USP7 impairs mitotic progression, stabilizes cyclin B and reduces stability of the mitotic E3 Ub ligase, checkpoint with forkhead and Ring-finger (CHFR). Consequently, cells with depleted USP7 accumulate Aurora-A kinase, a CHFR substrate, thus elevating multipolar mitoses. We further show that these effects are independent of the USP7 substrate p53. Thus, USP7 and Daxx are necessary to regulate proper execution of mitosis, partially via regulation of CHFR and Aurora-A kinase stability. Results from colony formation assay, in silico analysis across the NCI60 platform and in breast cancer patients suggest that USP7 levels inversely correlate with response to taxanes, pointing at the USP7 protein as a potential predictive factor for taxane response in cancer patients. In addition, we demonstrated that inhibition of Aurora-A attenuates USP7-mediated taxane resistance, suggesting that combinatorial drug regimens of Taxol and Aurora-A inhibitors may improve the outcome of chemotherapy response in cancer patients resistant to taxane treatment. Finally, our study offers novel insights on USP7 inhibition as cancer therapy.

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.

Regulation of mitosis and taxane response by Daxx and Rassf1

Current theories suggest that mitotic checkpoint proteins are essential for proper cellular response to taxanes, a widely used family of chemotherapeutic compounds. We recently showed that absence or depletion of protein Daxx increases cellular taxol (paclitaxel) resistance—a common trait of patients diagnosed with several malignancies, including breast cancer. Further investigation of Daxx-mediated taxol response revealed that Daxx is important for the proper timing of mitosis progression and cyclin B stability. Daxx interacts with mitotic checkpoint protein RAS-association domain family protein 1 (Rassf1) and partially colocalizes with this protein during mitosis. Rassf1/Daxx depletion or expression of Daxx-binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance in cells and mouse xenograft models. In breast cancer patients, we observed the inverse correlation between Daxx and clinical response to taxane-based chemotherapy. These data suggest that Daxx and Rassf1 define a mitotic stress checkpoint that enables cells to exit mitosis as micronucleated cells (and eventually die) when encountered with specific mitotic stress stimuli, including taxol. Surprisingly, depletion of Daxx or Rassf1 does not change the activity of E3 ubiquitin ligase anaphase promotion complex/C in in vitro settings, suggesting the necessity of mitotic cellular environment for proper activation of this checkpoint. Daxx and Rassf1 may become useful predictive markers for the proper selection of patients for taxane chemotherapy.

Daxx shortens mitotic arrest caused by paclitaxel.

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

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.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.

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

BackgroundThe 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.ResultsIn 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.ConclusionThe 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.

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.

Abstract 573: USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora A kinase.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A large number of patients are resistant to taxane-based chemotherapy. Functional mitotic checkpoints are essential for taxane sensitivity. Thus, mitotic regulators are potential markers for therapy response and could be targeted for anticancer therapy. In this study we identified a novel function of Ubiquitin Specific processing Protease-7 (USP7) that interacts and cooperates with protein Daxx in regulation of mitosis and taxane resistance. Depletion of USP7 impairs mitotic progression, stabilizes cyclin B and reduces stability of the mitotic E3 ubiquitin ligase, Checkpoint with Forkhead and RING finger (CHFR). Consequently, cells with depleted of USP7 accumulate Aurora A kinase, a CHFR substrate, thus elevating multipolar mitoses. We further show that these effects are independent from the USP7 substrate p53. Thus, USP7 and Daxx are necessary to regulate proper execution of mitosis, partially via regulation of CHFR and Aurora A kinase stability. Results from colony formation assay, in silico analysis across the NCI60 platform and in breast cancer patients suggest that USP7 levels inversely correlate with response to taxanes, pointing at the USP7 protein as a potential predictive factor for taxane response in cancer patients. In addition, we demonstrated that inhibition of Aurora A attenuates USP7-mediated taxane resistance, suggesting that combinatorial drug regimens of Taxol and Aurora A inhibitors may improve the outcome of chemotherapy response in cancer patients resistant to taxane treatment. Finally our study offers novel insights on USP7 inhibition as cancer therapy. Citation Format: Serena Giovinazzi, Viacheslav M. Morozov, Matthew K. Summers, William C. Reinhold, Alexander M. Ishov. USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora A kinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 573. doi:10.1158/1538-7445.AM2013-573

Abstract 2988: Regulation of mitosis and taxane response by Daxx

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Current theories suggest that mitotic proteins are essential for cellular response to taxanes (paclitaxel and docetaxel), a widely-used family of chemotherapeutic compounds. We found that absence or depletion of protein Daxx increases cellular resistance to paclitaxel _a common trait of patients diagnosed with breast cancer and other malignancies. Particularly, we observed that 1) paclitaxel induced cell death, in both human breast cancer and mouse cell lines, depends on the level of Daxx; 2) cells become paclitaxel-resistant upon experimental depletion of Daxx; 3) breast cancer specimens accumulate differential level of Daxx; 4) docetaxel response in breast cancer patients correlates with level of Daxx mRNA. We also found that Daxx is important for the proper progression of mitosis in human cells: duration of mitotic stages is altered in cells stably depleted of Daxx which also show increased stability of cyclin B1. Thus, protein Daxx, that was previously characterized as a transcription repressor and an apoptotic-related protein, may also have a novel function in mitosis progression as a prometaphase checkpoint release protein in control conditions and upon mitotic stress. In order to understand the mechanism by which Daxx orchestrates mitosis progression and identify new potential targets responsible for taxane resistance, we adopted a functional proteomic approach to identify Daxx-associated mitotic complex. We optimized the protocol for Tag based affinity purification in a way to obtain Daxx expressed downstream of FLAG Tag in tandem with HA Tag (FH) and a Thrombin Cleavage Site (TCS). HEp2 cells transduced with pOZ-FH-TCS-Daxx and the empty pOZ-FH plasmid (control), synchronized by double thymidine block and next arrested in pro-metaphase with paclitaxel, were used to purify Daxx containing mitotic complex. Mitotic specific Daxx interacting proteins were identified by SDS-PAGE followed by mass spectrometry. The functional characterization of newly identified Daxx mitotic partners will shed lights on the complex mechanism/phenomena of taxanes resistance and ultimately offer the molecular basis for successful treatment strategies of breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2011-2988