Lou, Zhenkun

Abstract Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm−/− cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm−/− cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm−/− cells Sμ DSBs accumulate as they lack a recombination partner.

Abstract Recently, patients with mutations in DOCK8 have been reported to have a combined immunodeficiency characterized by cutaneous viral infections and allergies. NK cells represent a first-line defense against viral infections, suggesting that DOCK8 might participate in NK cell function. In this study, we demonstrate that DOCK8-suppressed human NK cells showed defects in natural cytotoxicity as well as specific activating receptor-mediated NK cytotoxicity. Additionally, compared with control NK cells, NK cells depleted of DOCK8 showed defective conjugate formation, along with decreased polarization of LFA-1, F-actin, and cytolytic granules toward the cytotoxic synapse. Using a proteomic approach, we found that DOCK8 exists in a macromolecular complex with the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor activated by CDC42, as well as talin, which is required for integrin-mediated adhesion. Taken together, our results demonstrate an important role for DOCK8 in NK cell effector function and provide important new mechanistic insight into how DOCK8 regulates F-actin and integrin-mediated adhesion in immune cells.

Abstract Wolf–Hirschhorn syndrome (WHS) is a genetic disease with characteristic facial features and developmental disorders. Of interest, loss of the MMSET gene (also known as WHSC1) is considered to be responsible for the core phenotypes of this disease. Patients with WHS also display Ab deficiency, although the underlying cause of this deficiency is unclear. Recent studies suggest that the histone methyltransferase activity of MMSET plays an important role in the DNA damage response by facilitating the recruitment of 53BP1 to sites of DNA damage. We hypothesize that MMSET also regulates class switch recombination (CSR) through its effect on 53BP1. In this study, we show that MMSET indeed plays an important role in CSR through its histone methyltransferase activity. Knocking down MMSET expression impaired 53BP1 recruitment as well as the germline transcription of the Igh switch regions, resulting in defective CSR but no effect on cell growth and viability. These results suggest that defective CSR caused by MMSET deficiency could be a cause of Ab deficiency in WHS patients.

Abstract DNA double-strand breaks (DSBs) are highly cytotoxic lesions caused by genotoxin such as ionizing radiation (IR). Following DSB, cells activate the DNA-damage response pathway by activating protein kinases such as ATM, ATR and DNA-PK, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as Mediator of DNA damage checkpoint protein 1 (MDC1), 53BP1, and BRCA1 into IR-induced foci. This process initiates the repair of DSB. Previously we have identified a novel biomarker for radiation response, Bora, by using a genome-wide association approach using data generated with 300 human lymphoblastoid cell liens. Bora is known as a cofactor of Aurora-A, which is involved in mitosis regulation. During G2, Aurora-A, in complex with Bora, phosphorylates Plk1 at Thr-210. Activated Plk1 phosphorylates Cdc25C and Wee1, which induces activation of cyclin B-cdk1 complexes and promotes mitotic entry. In this study, we want to determine the underlying mechanisms by which Bora regulates response to IR. We found that knockdown of Bora in human cancer cell lines including HupT3, MIAPaCa-2 and Hela (siBora) exhibit increased clonogenic survival rate in response to IR, an observation that is opposite from the effect of Plk1 knockdown, suggesting a different mechanism involved. Knockdown of Bora also desensitize cancer cells to chemotherapeutic agents including gemcitabine and oxiplatin. This enhanced resistance to genotoxin is primarily due to increased DNA DSB repair. More than 90% of DSBs were repaired in siBora cells compared with 60% in control cells 8 hours after cell exposure to 2 Gy IR. Upon IR, more cells with Bora knockdown retained in G2-M phase compared with control. Furthermore, Bora directly interacts with BRCT domain of MDC1 via its C terminus, and over expression of Bora can abolish MDC1 foci formation induced by IR. All of these observations suggested a role of Bora in DNA damage response pathway and in turn, in chemo-radio resistance. Indeed, overexpression of the full length or C-terminus of Bora sensitized cancer cells to IR treatment, while the N-terminal Bora did not have significant effect on IR response. In summary, we have identified a novel function of Bora involved in DNA damage and repair pathway, which indicates a broad role of Bora in both tumorgenesis and chemo-radio resistance. 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 2660. doi:10.1158/1538-7445.AM2011-2660

Abstract DNA topoisomerase IIα (Topo IIα) is a critical target for cancer therapy and its protein expression, however, is frequently depleted by its inhibitors and strategies to maintain Topo IIα expression and thereby to sustain the Topo II drug sensitivity are still lacking. Here we show that p38γ MAPK is specifically activated by Topo II inhibitors and activated p38γ in turn phosphorylates Topo IIα, leading to increased Topo IIα protein expression through attenuating its proteasome-dependent degradation. The sensitivity of a panel of human breast cancer cell lines to Topo II inhibitors was found not to correlate with the intrinsic but with inducible Topo IIα protein expression and with endogenous protein expression of p38γ MAPK that is specifically phosphorylated in response to Topo II toxins. p38γ activation and inhibition alone were shown to increase and decrease Topo IIα expression and Topo drug toxicities. Mechanistically, p38γ MAPK phosphorylates Topo IIα at Ser 1524, leading to a decreased proteasome-dependent Topo IIα degradation. These results reveal a novel mechanism by which p38γ MAPK specifically confers Topo II drug toxicity in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 317.

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1's role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc's transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression.

Abstract Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease. [Mol Cancer Ther 2009;8(2):OF407–8]

The product of the Nijmegen breakage syndrome gene (NBS1) plays crucial roles in DNA damage response through its association with many proteins, including MRE11 and RAD50. However, it remains to be determined exactly how NBS1 accumulates at or near DNA double-strand breaks. Here we report that MDC1 directly binds to NBS1 and targets NBS1 to the sites of DNA damage. The MDC1–NBS1 interaction occurs through a specific region (residues 200–420) of MDC1, which contains multiple consensus casein kinase 2 (CK2) phosphorylation sites. In addition, this interaction requires both the forkhead-associated (FHA) and tandem BRCA1 C-terminal (BRCT) domains of NBS1. Disruption of the MDC1–NBS1 interaction results in failure of NBS1 accumulation at DNA double-strand breaks and impairment of intra-S checkpoint activation. These studies provide important mechanistic insights as to how MDC1 regulates NBS1 and the intra-S-phase checkpoint in response to DNA damage.

The importance of the DNA damage response (DDR) pathway in development, genomic stability, and tumor suppression is well recognized. Although 53BP1 and MDC1 have been recently identified as critical upstream mediators in the cellular response to DNA double-strand breaks, their relative hierarchy in the ataxia telangiectasia mutated (ATM) signaling cascade remains controversial. To investigate the divergent and potentially overlapping functions of MDC1 and 53BP1 in the ATM response pathway, we generated mice deficient for both genes. Unexpectedly, the loss of both MDC1 and 53BP1 neither significantly increases the severity of defects in DDR nor increases tumor incidence compared with the loss of MDC1 alone. We additionally show that MDC1 regulates 53BP1 foci formation and phosphorylation in response to DNA damage. These results suggest that MDC1 functions as an upstream regulator of 53BP1 in the DDR pathway and in tumor suppression.

The function of cyclin-dependent kinase 2 (CDK2) is often abolished after DNA damage. The inhibition of CDK2 plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences the survival of cells under genotoxic stress is unknown. Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival. CDK2 specifically phosphorylated FOXO1 at serine-249 (Ser 249 ) in vitro and in vivo. Phosphorylation of Ser 249 resulted in cytoplasmic localization and inhibition of FOXO1. This phosphorylation was abrogated upon DNA damage through the cell cycle checkpoint pathway that is dependent on the protein kinases Chk1 and Chk2. Moreover, silencing of FOXO1 by small interfering RNA diminished DNA damage–induced death in both p53-deficient and p53-proficient cells. This effect was reversed by restored expression of FOXO1 in a manner depending on phosphorylation of Ser 249 . Functional interaction between CDK2 and FOXO1 provides a mechanism that regulates apoptotic cell death after DNA strand breakage.