Abstract: The patients with relapsed and refractory diffuse large B-cell lymphoma (DLBCL) have poor prognosis, and a novel and effective therapeutic strategy for these patients is urgently needed. Although ubiquitin-specific protease 1 (USP1) plays a key role in cancer, the carcinogenic effect of USP1 in B-cell lymphoma remains elusive. Here we found that USP1 is highly expressed in DLBCL patients, and high expression of USP1 predicts poor prognosis. Knocking down USP1 or a specific inhibitor of USP1, pimozide, induced cell growth inhibition, cell cycle arrest and autophagy in DLBCL cells. Targeting USP1 by shRNA or pimozide significantly reduced tumor burden of a mouse model established with engraftment of rituximab/chemotherapy resistant DLBCL cells. Pimozide significantly retarded the growth of lymphoma in a DLBCL patient-derived xenograft (PDX) model. USP1 directly interacted with MAX, a MYC binding protein, and maintained the stability of MAX through deubiquitination, which promoted the transcription of MYC target genes. Moreover, pimozide showed a synergetic effect with etoposide, a chemotherapy drug, in cell and mouse models of rituximab/chemotherapy resistant DLBCL. Our study highlights the critical role of USP1 in the rituximab/chemotherapy resistance of DLBCL through deubiquitylating MAX, and provides a novel therapeutic strategy for rituximab/chemotherapy resistant DLBCL.

Abstract: Our studies have revealed an unrecognized mechanism for regulating the activity of p53 during the cell cycle. We show, using multiple approaches, that p53 is degraded as a consequence of its phosphorylation by Aurora B. This degradation ultimately prevents p53 from performing its role as a guardian of the genome. Taken together with our earlier study on the effects of the Aurora B kinase inhibitor AZD1152 on breast cancer cells and preclinical evaluations of this drug for treating diverse human tumors, we conclude that Aurora B inhibitors may prove to be effective in treating cancer cells expressing functional p53 molecules. Also, we previously showed that specific inhibition of Aurora B expression is sufficient to inhibit tumor growth and induce the regression of tumors. Significantly, we here show that a specific inhibitor (AZD1152) of Aurora B can cause p53 elevation, thereby increasing p53 target gene expression in a cancer xenograft model. As a result, inhibitor of Aurora B can reduce cell survival through increasing the p53 up-regulated mediator of apoptosis p53 up-regulated modulator of apoptosis (PUMA) and cause cell cycle inhibition through inducing CDK inhibitor p21. These data are consistent with biochemical studies in cell lines. Our present study defines the molecular mechanisms involved in inhibition of tumor growth by an Aurora B inhibitor. General suppression of transcription is observed during mitosis ( 4 ); therefore, Aurora B-mediated p53 transcriptional suppression will not play a role during mitosis. The work by Cross et al. ( 2 ) shows that p53 is involved in facilitating chromosome segregation to ensure the maintenance of diploid cells. Aurora B may coordinate with p53 to mediate the spindle checkpoint ( 2 ) and aid progression through mitosis. Given that Aurora B deregulation also results in polyploidy, the interplay between p53 and Aurora B is conceivably important for spindle checkpoint. The functional significance of Aurora B–p53 interaction during different stages of mitosis remains to be investigated, but p53 is involved in spindle checkpoint ( 2 ); our data serve to confirm its presence in the spindle checkpoint machinery. It is important to point out that the binding between Aurora B and p53 decreases after the end of mitosis because of the down-regulation of Aurora B by E3 ubiquitin ligase anaphase promoting complex. After mitosis, Aurora B is recovering from degradation and again binding p53, and thus, it potentially prevents p53 from arresting the cell cycle at G1 by maintaining a negative impact on p53. The enhanced degradation of phosphorylated p53 leads to the finding that its transcriptional activity on cell cycle control gene was diminished (such as p21 CDK inhibitor) when Aurora B was overexpressed. Moreover, we were able to show that p53-mediated gene transcription was enhanced when Aurora B expression was inhibited. These data provide a rationale for the role of Aurora B in interphase, which is to antagonize the inhibition of cell cycle progression by p53 and allow entry of cells into mitosis. To answer this question, we used bimolecular fluorescence complementation, which allows direct observation of the interaction between two proteins in living cells, and showed that Aurora B and p53 interact during interphase and mitosis ( Fig. P1 ). Immunofluorescence studies showed that, during mitosis, p53 associates with Aurora B located at centromeres at prometaphase or the middle zone of the cleavage furrow at the anaphase–telophase border. We were also able to show that Survivin, a protein component of CPC localized at the centromeres to activate Aurora B ( 3 ), colocalized with Aurora B and p53 to the DNA of prometaphase cells. We also showed that Aurora B and p53 could be coimmunoprecipitated with specific antibodies. Using the technique, we were also able to show the Aurora B–p53 association in every phase of the cell cycle except late M phase, when Aurora B is known to be degraded. The interaction in interphase is quite surprising, because Aurora B has been shown to function exclusively during mitosis. We then showed that Aurora B phosphorylates p53 at three predicted Aurora B sites (S183, T211, and S215). We also showed that these phosphorylations lead to enhanced degradation of p53 through ubiquitination, which is mediated by the murine double minute 2 (MDM2) regulatory protein, an E3 ubiquitin ligase of p53. Moreover, AZD1152-hydroxyquinazoline pyrazole anilide (HQPA), which specifically inhibits the kinase activity of Aurora B, was able to block p53 ubiquitination in a dose-dependent manner. These data provide insights into the contribution of Aurora B-mediated p53 phosphorylation in regulating p53 stability. The regulation of mitosis ensures the equal segregation of chromosomes to daughter cells, and Aurora B plays a critical role in this process as a component of the chromosomal passenger protein complex (CPC). CPC is located on the chromosome arms during prophase and at the centromeres during prometaphase and metaphase ( 1 ). Aurora B subsequently localizes to the midbody during cytokinesis and participates in ensuring the correct attachment of chromosomes to spindle microtubules (spindle checkpoint) and equal distribution of chromosomes. The effects of Aurora B are exerted by its phosphorylation on specific proteins of the mitotic apparatus. The p53 protein is also involved in facilitating chromosome segregation to ensure the maintenance of diploid cells because cells deficient in p53 expression become tetraploid ( 2 ). Thus, it raises the question of whether Aurora B and p53 are functionally related. Aurora B, a protein kinase, plays a key role in mitosis by maintaining correct chromosome segregation and progression of cells through mitosis. Cancer cells frequently express Aurora B at unusually high levels, leading to dysregulated mitosis and therefore, causing unequal chromosome segregation, which may confer a growth advantage. The tumor suppressor protein p53 guards the genome by delaying or arresting the cell cycle at specific checkpoints (G1/S) when DNA is damaged and prevents damaged cells from entering mitosis (G2/M checkpoint). Despite intensive studies on p53 for more than three decades, the exact mechanism by which it regulates mitotic checkpoint is unknown. Whether Aurora B and p53 are coordinately regulated during the cell cycle remains to be determined, and there is no report to our knowledge suggesting that Aurora B functions in processes other than mitosis. By studying cells synchronized to divide at the same time, we show here that Aurora B and p53 interact during all stages of the cell cycle except during late M phase, when Aurora B is degraded. Moreover, we show that Aurora B is a negative regulator of p53 and that, when overexpressed, it affects the ability of p53 to mitigate the detrimental consequences of DNA damage and control the mitotic checkpoint.

Abstract: The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO–containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO–expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO–expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO , is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO–mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.

Abstract: With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N -ethyl- N -nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Abstract: The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE -homologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli , did not differ from that of the parent strain. When 175 μM extrinsic H 2 O 2 was added to the culture medium, bacterial growth of the Δ katE1 strain was delayed and growth of the Δ katE1 Δ katE2 and Δ katE1 Δ ahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the Δ katE1 strain to the inhibition of growth by H 2 O 2 was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H 2 O 2 stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the Δ katE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H 2 O 2 during logarithmic growth and that the function of these genes was influenced by incubation temperature.