TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Active DNA demethylation-The epigenetic gatekeeper of development, immunity, and cancer.

DNA methylation is a critical process in the regulation of gene expression with dramatic effects in development and continually expanding roles in oncogenesis. 5-Methylcytosine was once considered to be an inherited and stably repressive epigenetic mark, which can be only removed by passive dilution during multiple rounds of DNA replication. However, in the past two decades, physiologically controlled DNA demethylation and deamination processes have been identified, thereby revealing the function of cytosine methylation as a highly regulated and complex state-not simply a static, inherited signature or binary on-off switch. Alongside these fundamental discoveries, clinical studies over the past decade have revealed the dramatic consequences of aberrant DNA demethylation. In this review we discuss DNA demethylation and deamination in the context of 5-methylcytosine as critical processes for physiological and physiopathological transitions within three states-development, immune maturation, and oncogenic transformation; and we describe the expanding role of DNA demethylating drugs as therapeutic agents in cancer.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Modification of the base excision repair enzyme MBD4 by the small ubiquitin-like molecule SUMO1.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

Thymine DNA glycosylase as a novel target for melanoma.

Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

Nearly complete deletion of BubR1 causes microcephaly through shortened mitosis and massive cell death.

BUB-related 1 (BubR1) encoded by Budding Uninhibited by Benzimidazole 1B (BUB1B) is a crucial mitotic checkpoint protein ensuring proper segregation of chromosomes during mitosis. Mutations of BUB1B are responsible for mosaic variegated aneuploidy (MVA), a human congenital disorder characterized by extensive abnormalities in chromosome number. Although microcephaly is a prominent feature of MVA carrying the BUB1B mutation, how BubR1 deficiency disturbs neural progenitor proliferation and neuronal output and leads to microcephaly is unknown. Here we show that conditional loss of BubR1 in mouse cerebral cortex recapitulates microcephaly. BubR1-deficient cortex includes a strikingly reduced number of late-born, but not of early-born, neurons, although BubR1 expression is substantially reduced from an early stage. Importantly, absence of BubR1 decreases the proportion of neural progenitors in mitosis, specifically in metaphase, suggesting shortened mitosis owing to premature chromosome segregation. In the BubR1 mutant, massive apoptotic cell death, which is likely due to the compromised genomic integrity that results from aberrant mitosis, depletes progenitors and neurons during neurogenesis. There is no apparent alteration in centrosome number, spindle formation or primary cilia, suggesting that the major effect of BubR1 deficiency on neural progenitors is to impair the mitotic checkpoint. This finding highlights the importance of the mitotic checkpoint in the pathogenesis of microcephaly. Furthermore, the ependymal cell layer does not form in the conditional knockout, revealing an unrecognized role of BubR1 in assuring the integrity of the ventricular system, which may account for the presence of hydrocephalus in some patients.

Chromosome instability in tumor cells due to defects in Aurora B mediated error correction at kinetochores.

We characterized a panel of cancer cells and found that they exhibited chromosome instability (CIN) that was associated with high frequencies of aberrant kinetochore:microtubule attachments. Failure to resolve these defective attachments before anaphase onset can lead to missegregation of chromosomes. Aurora B kinase is concentrated at the inner centromere where it contributes to multiple kinetochore functions, one of which is in error-correction. Analysis of several CIN cell lines showed that many aspects of Aurora B kinase functions were normal. Furthermore, the amount and activity of Aurora B kinase was not reduced at the kinetochores of CIN cells that were examined. However, phosphorylation of a centromeric biosensor for Aurora B in OVCAR10, MCF7 and U2OS cells was consistently reduced relative to non CIN cells. This suggested a localized problem with Aurora B's ability to phosphorylate substrates important for error correction. This possibility was supported by our ability to improve error correction and reduce the frequency of lagging chromosome in CIN cells by directing endogenous Aurora B to the region of centromere that was tested by the biosensor. Our studies suggest that the kinetochores of CIN cells have a defect that limits accessibility of Aurora B to substrates that are important for error-correction.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Methotrexate sensitizes drug-resistant metastatic melanoma cells to BRAF V600E inhibitors dabrafenib and encorafenib.

Acquired resistance of metastatic melanoma (MM) tumors to BRAF V600E inhibitors (BRAFi's) is commonplace in the clinic. Habitual relapse of patients contributes to <20% 5-year survival rates in MM. We previously identified serine synthesis as a critical detrminant of late-stage cancer cell resistance to BRAFi's. Pre-treatment with DNA damaging agent gemcitabine (a nucleoside analog) re-sensitized drug-resistant cancer cells to BRAFi's dabrafenib and vemurafenib. Importantly, the combination treatments were effective against BRAF wild type cancer cells potentially expanding the clinical reach of BRAFi's. In this study, we identify the antifolate methotrexate (MTX) as a sensitizer of acquired- and intrinsically-resistant MM cells to BRAFi's dabrafenib and encorafenib. We identify a novel, positive correlation between dabrafenib treatments and repair delay of MTX induced single-strand DNA (ssDNA) breaks. Cells arrest in G1 phase following simultaneous MTX + dabrafenib treatments and eventually die via apoptosis. Importantly, we identify RAS codon 12 activating mutations as prognostic markers for MTX + BRAFi treatment efficacy. We describe a method of killing drug-resistant MM cells that if translated has the potential to improve MM patient survival.

Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells.

Metastatic melanoma cells commonly acquire resistance to BRAF V600E inhibitors (BRAFi). In this study, we identified serine biosynthesis as a critical mechanism of resistance. Proteomic assays revealed differential protein expression of serine biosynthetic enzymes PHGDH, PSPH, and PSAT1 following vemurafenib (BRAFi) treatment in sensitive versus acquired resistant melanoma cells. Ablation of PHGDH via siRNA sensitized acquired resistant cells to vemurafenib. Inhibiting the folate cycle, directly downstream of serine synthesis, with methotrexate also displayed similar sensitization. Using the DNA-damaging drug gemcitabine, we show that gemcitabine pretreatment sensitized resistant melanoma cells to BRAFis vemurafenib and dabrafenib. We extended our findings to BRAF WT tumor cell lines that are intrinsically resistant to vemurafenib and dabrafenib. Pretreatment of pancreatic cancer and non-small cell lung cancer cell lines with sublethal doses of 50 and 5 nmol/L of gemcitabine, respectively, enhanced killing by both vemurafenib and dabrafenib. The novel aspects of this study are the direct identification of serine biosynthesis as a critical mechanism of BRAF V600E inhibitor resistance and the first successful example of using gemcitabine + BRAFis in combination to kill previously drug-resistant cancer cells, creating the translational potential of pretreatment with gemcitabine prior to BRAFi treatment of tumor cells to reverse resistance within the mutational profile and the WT. Mol Cancer Ther; 16(8); 1596-609. ©2017 AACR.

Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions.

Pixantrone is a novel aza-anthracenedione active against aggressive lymphoma and is being evaluated for use against various hematologic and solid tumors. The drug is an analog of mitoxantrone, but displays less cardiotoxicity than mitoxantrone or the more commonly used doxorubicin. Although pixantrone is purported to inhibit topoisomerase II activity and intercalate with DNA, exact mechanisms of how it induces cell death remain obscure. Here we evaluated the effect of pixantrone on a panel of solid tumor cell lines to understand its mechanism of cell killing. Initial experiments with pixantrone showed an apparent discrepancy between its anti-proliferative effects in MTS assays (short-term) compared with clonogenic assays (long-term). Using live cell videomicroscopy to track the fates of cells, we found that cells treated with pixantrone underwent multiple rounds of aberrant cell division before eventually dying after approximately 5 d post-treatment. Cells underwent abnormal mitosis in which chromosome segregation was impaired, generating chromatin bridges between cells or within cells containing micronuclei. While pixantrone-treated cells did not display γH2AX foci, a marker of DNA damage, in the main nuclei, such foci were often detected in the micronuclei. Using DNA content analysis, we found that pixantrone concentrations that induced cell death in a clonogenic assay did not impede cell cycle progression, further supporting the lack of canonical DNA damage signaling. These findings suggest pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division.

Changing the selectivity of p300 by acetyl-CoA modulation of histone acetylation.

Determining how histone acetylation is regulated is vital for treating the many diseases associated with its misregulation, including heart disease, neurological disorders, and cancer. We have previously reported that acetyl-CoA levels alter p300 histone acetylation in a site-specific manner in vitro. Here, we further investigate how changing acetyl-CoA concentrations alter the histone acetylation pattern by altering p300 specificity. Interestingly, these changes are not a simple global change in acetylation, but rather site specific changes, whereby acetylation at some sites increase while others decrease. We also demonstrate how the p300 inhibitor C646 can pharmacologically alter p300 histone acetylation patterns in vitro and in cells. This study provides insight into the mechanisms regulating p300 residue specificity, a potential means for altering p300 dependent histone acetylation, and an investigation into altering histone acetylation patterns in cells.

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

HuR posttranscriptionally regulates WEE1: implications for the DNA damage response in pancreatic cancer cells.

HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.

Replication stress and mitotic dysfunction in cells expressing simian virus 40 large T antigen.

We previously demonstrated that simian virus 40 (SV40) large T antigen (LT) binds to the Bub1 kinase, a key regulator of the spindle checkpoint and chromosome segregation. Bub1 mutations or altered expression patterns are linked to chromosome missegregation and are considered to be a driving force in some human cancers. Here we report that LT, dependent on Bub1 binding, causes micronuclei, lagging chromatin, and anaphase bridges, which are hallmarks of chromosomal instability (CIN) and Bub1 insufficiency. Using time-lapse microscopy, we demonstrate that LT imposes a Bub1 binding-dependent delay in the metaphase-to-anaphase transition. Kinetochore fibers reveal that LT, via Bub1 binding, causes aberrant kinetochore (KT)-microtubule (MT) attachments and a shortened interkinetochore distance, consistent with a lack of tension. Previously, we showed that LT also induces the DNA damage response (DDR) via Bub1 binding. Using inducible LT cell lines, we show that an activated DDR was observed before the appearance of anaphase bridges and micronuclei. Furthermore, LT induction in serum-starved cells demonstrated γ-H2AX accumulation in cells that had not yet entered mitosis. Thus, DDR activation can occur independently of chromosome segregation defects. Replication stress pathways may be responsible, because signatures of replication stress were observed, which were attenuated by exogenous supplementation with nucleosides. Our observations allow us to propose a model that explains and integrates the diverse manifestations of genomic instability induced by LT.

Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission.

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission.

Dose dependent effects on cell cycle checkpoints and DNA repair by bendamustine.

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.