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 Apc mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas.

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.

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.

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.

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.

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.

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.

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.

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.

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

Acquired resistance of metastatic melanoma (MM) tumors to 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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

G Protein-coupled receptor kinase 5 is localized to centrosomes and regulates cell cycle progression.

G protein-coupled receptor kinases (GRKs) are important regulators of G protein-coupled receptor function and mediate receptor desensitization, internalization, and signaling. While GRKs also interact with and/or phosphorylate many other proteins and modify their function, relatively little is known about the cellular localization of endogenous GRKs. Here we report that GRK5 co-localizes with γ-tubulin, centrin, and pericentrin in centrosomes.

BubR1 is an effector of multiple mitotic kinases that specifies kinetochore: microtubule attachments and checkpoint.

BubR1 is a critical component of the mitotic checkpoint but has also been shown to play an essential role in establishing kinetochore:microtubule attachments. BubR1 is hyperphosphorylated in mitosis and recent studies in human and Xenopus have identified 9 phosphorylation sites. Plk1-dependent phosphorylations (T792, T1008 and S676) were reported to stimulate BubR1 kinase activity, promote kinetochore microtubule attachments, monitor kinetochore tension, as well as the recruitment of Mad2 checkpoint protein to kinetochores.

Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit.

BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S.

Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells.

We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block.

Targeting mitosis for anti-cancer therapy.

Basic research that has focused on achieving a mechanistic understanding of mitosis has provided unprecedented molecular and biochemical insights into this highly complex phase of the cell cycle. The discovery process has uncovered an ever-expanding list of novel proteins that orchestrate and coordinate spindle formation and chromosome dynamics during mitosis. That many of these proteins appear to function solely in mitosis makes them ideal targets for the development of mitosis-specific cancer drugs.