Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

The protein phosphatase EYA4 promotes homologous recombination (HR) through dephosphorylation of tyrosine 315 on RAD51.

Efficient DNA repair and limitation of genome rearrangements rely on crosstalk between different DNA double-strand break (DSB) repair pathways, and their synchronization with the cell cycle. The selection, timing and efficacy of DSB repair pathways are influenced by post-translational modifications of histones and DNA damage repair (DDR) proteins, such as phosphorylation. While the importance of kinases and serine/threonine phosphatases in DDR have been extensively studied, the role of tyrosine phosphatases in DNA repair remains poorly understood. In this study, we have identified EYA4 as the protein phosphatase that dephosphorylates RAD51 on residue Tyr315. Through its Tyr phosphatase activity, EYA4 regulates RAD51 localization, presynaptic filament formation, foci formation, and activity. Thus, it is essential for homologous recombination (HR) at DSBs. DNA binding stimulates EYA4 phosphatase activity. Depletion of EYA4 decreases single-stranded DNA accumulation following DNA damage and impairs HR, while overexpression of EYA4 in cells promotes dephosphorylation and stabilization of RAD51, and thereby nucleoprotein filament formation. Our data have implications for a pathological version of RAD51 in EYA4-overexpressing cancers.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

UBA80 and UBA52 fine-tune RNF168-dependent histone ubiquitination and DNA repair.

The ubiquitin signaling pathway is crucial for the DNA damage response pathway. More specifically, RNF168 is integral in regulating DNA repair proteins at damaged chromatin. However, the detailed mechanism by which RNF168 is regulated in cells is not fully understood. Here, we identify the ubiquitin-ribosomal fusion proteins UBA80 (also known as RPS27A) and UBA52 (also known as RPL40) as interacting proteins for H2A/H2AX histones and RNF168. Both UBA80 and UBA52 are recruited to laser-induced micro-irradiation DNA damage sites and are required for DNA repair. Ectopic expression of UBA80 and UBA52 inhibits RNF168-mediated H2A/H2AX ubiquitination at K13/15 and impairs 53BP1 recruitment to DNA lesions. Mechanistically, the C-terminal ribosomal fragments of UBA80 and UBA52, S27A and L40, respectively, limit RNF168-nucleosome engagement by masking the regulatory acidic residues at E143/E144 and the nucleosome acidic patch. Together, our results reveal that UBA80 and UBA52 antagonize the ubiquitination signaling pathway and fine-tune the spatiotemporal regulation of DNA repair proteins at DNA damage sites.

EYA4 promotes breast cancer progression and metastasis through its role in replication stress avoidance.

The Eyes Absent (EYA) family of proteins is an atypical group of four dual-functioning protein phosphatases (PP), which have been linked to many vital cellular processes and organogenesis pathways. The four family members of this PP family possess transcriptional activation and phosphatase functions, with serine/threonine and tyrosine phosphatase domains. EYA4 has been associated with several human cancers, with tumor-suppressing and tumor-promoting roles. However, EYA4 is the least well-characterized member of this unique family of PP, with its biological functions and molecular mechanisms in cancer progression, particularly in breast cancer, still largely unknown. In the present study, we found that the over-expression of EYA4 in breast tissue leads to an aggressive and invasive breast cancer phenotype, while the inhibition of EYA4 reduced tumorigenic properties of breast cancer cells in vitro and in vivo. Cellular changes downstream of EYA4, including cell proliferation and migration, may explain the increased metastatic power of breast cancer cells over-expressing EYA4. Mechanistically, EYA4 prevents genome instability by inhibiting the accumulation of replication-associated DNA damage. Its depletion results in polyploidy as a consequence of endoreplication, a phenomenon that can occur in response to stress. The absence of EYA4 leads to spontaneous replication stress characterized by the activation of the ATR pathway, sensitivity to hydroxyurea, and accumulation of endogenous DNA damage as indicated by increased γH2AX levels. In addition, we show that EYA4, specifically its serine/threonine phosphatase domain, plays an important and so far, unexpected role in replication fork progression. This phosphatase activity is essential for breast cancer progression and metastasis. Taken together, our data indicate that EYA4 is a novel potential breast cancer oncogene that supports primary tumor growth and metastasis. Developing therapeutics aimed at the serine/threonine phosphatase activity of EYA4 represents a robust strategy for killing breast cancer cells, to limit metastasis and overcome chemotherapy resistance caused by endoreplication and genomic rearrangements.

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

The SWI/SNF subunit SMARCD3 regulates cell cycle progression and predicts survival outcome in ER+ breast cancer.

PURPOSE: Chromatin remodeling plays an essential role in regulating transcriptional networks and timing of gene expression. Chromatin remodelers such as SWItch/Sucrose Non-Fermentable (SWI/SNF) harbor many protein components, with the catalytic subunit providing ATPase activity to displace histones along or from the DNA molecules, and associated subunits ensuring tissue specificity and transcriptional or co-transcriptional activities. Mutations in several of the SWI/SNF subunits have been linked to cancer. Here, we investigate between SMARCD3/Baf60c expression and hormone-positive (ER+) breast cancer. METHODS:  The level of SMARCD3 was detected by immunohistochemistry in breast cancer patient samples, and expression levels of SMARCD1, SMARCD2, and SMARCD3 were investigated using publicly available datasets from large cohorts of breast cancer patients. Using molecular biology and microscopy, we interrogated the cellular consequences of lower SMARCD3 expression. RESULTS:  Lower proliferation rates were observed in SMARCD3-depleted cells, which reflects a failure of the cell cycle progression and an increase in endoreplication. In the absence of SMARCD3, p21 accumulates in cells, but does not halt the cell cycle, and DNA damage accumulates and remains unrepaired. CONCLUSION:  Taken together, our data begin to explain why ER+ breast cancer patients with low-SMARCD3 expressing tumors exhibit reduced survival rates compared to patients expressing normal or higher levels of SMARCD3. SMARCD3 might act as a tumor suppressor through regulation of cell cycle checkpoints and could be a reliable and specific breast cancer prognostic biomarker.

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination.

Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

Tunable "nano-shearing": a physical mechanism to displace nonspecific cell adhesion during rare cell detection.

We report a tunable alternating current electro-hydrodynamic (ac-EHD) force which drives lateral fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells.

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as "nanoshearing" which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target­sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over nonspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific adsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1).

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

Polyploidy in Cancer: Causal Mechanisms, Cancer-Specific Consequences, and Emerging Treatments.

Drug resistance is the major determinant for metastatic disease and fatalities, across all cancers. Depending on the tissue of origin and the therapeutic course, a variety of biological mechanisms can support and sustain drug resistance. Although genetic mutations and gene silencing through epigenetic mechanisms are major culprits in targeted therapy, drug efflux and polyploidization are more global mechanisms that prevail in a broad range of pathologies, in response to a variety of treatments. There is an unmet need to identify patients at risk for polyploidy, understand the mechanisms underlying polyploidization, and to develop strategies to predict, limit, and reverse polyploidy thus enhancing efficacy of standard-of-care therapy that improve better outcomes. This literature review provides an overview of polyploidy in cancer and offers perspective on patient monitoring and actionable therapy.

Aurkin-A, a TPX2-Aurora A small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma.

Chemotherapy induced polyploidy is a mechanism of inherited drug resistance resulting in an aggressive disease course in cancer patients. Alisertib, an Aurora Kinase A (AK-A) ATP site inhibitor, induces cell cycle disruption resulting in polyaneuploidy in Diffuse Large B Cell Lymphoma (DLBCL). Propidium iodide flow cytometry was utilized to quantify alisertib induced polyploidy in U2932 and VAL cell lines. In U2932 cells, 1µM alisertib generated 8n+ polyploidy in 48% of the total cell population after 5 days of treatment. Combination of Aurkin A an AK-A/TPX2 site inhibitor, plus alisertib disrupted alisertib induced polyploidy in a dose-dependent manner with associated increased apoptosis. We generated a stable FUCCI U2932 cell line expressing Geminin-clover (S/G2/M) and cdt1-mKO (G1), to monitor cell cycle progression. Using this system, we identified alisertib induces polyploidy through endomitosis, which was eliminated with Aurkin A treatment. In a VAL mouse xenograft model, we show polyploidy generation in alisertib treated mice versus vehicle control or Aurkin A. Aurkin A plus alisertib significantly reduced polyploidy to vehicle control levels. Our in vitro and in vivo studies show that Aurkin A synergizes with alisertib and significantly decreases the alisertib dose needed to disrupt polyploidy while increasing apoptosis in DLBCL cells.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Evolved histone tail regulates 53BP1 recruitment at damaged chromatin.

The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair.

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization.

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.