Rob C. Laister

Cruciform structures are a common DNA feature important for regulating biological processes

DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werners syndrome and others.Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIβ, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a) the junction-resolving enzymes, (b) DNA repair proteins and transcription factors, (c) proteins involved in replication and (d) chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.

Molecular basis of Pirh2-mediated p53 ubiquitylation

Pirh2 (p53-induced RING-H2 domain protein; also known as Rchy1) is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy, we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes, including a RING domain and a left-handed β-spiral in which three zinc ions align three consecutive small β-sheets in an interleaved fashion. We show that Pirh2-p53 interaction is dependent on the C-terminal zinc binding module of Pirh2, which binds to the tetramerization domain of p53. As a result, Pirh2 preferentially ubiquitylates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53.

Small Molecule STAT5-SH2 Domain Inhibitors Exhibit Potent Antileukemia Activity

A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i) < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

CCM3/PDCD10 Heterodimerizes with Germinal Center Kinase III (GCKIII) Proteins Using a Mechanism Analogous to CCM3 Homodimerization

CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization.

The SMX DNA Repair Tri-nuclease

Summary The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81’s conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.Several rearrangements of the MLL gene are associated with acute leukemia, including the fusion of MLL with a RAS effector protein, AF6. Here the authors show that the truncated AF6 can induce AF6-MLL dimerization and drive its oncogenic activity.

Blood-based biomarkers of cancer-related cognitive impairment in non-central nervous system cancer: protocol for a scoping review

Introduction Cancer-related cognitive impairment (CRCI) can have detrimental effects on quality of life, even among patients with non-central nervous system (CNS) cancers. Several studies have been conducted to explore different markers associated with CRCI to understand its pathobiology. It is proposed that the underlying mechanisms of CRCI are related to a cascade of physiological adaptive events in response to cancer and/or treatment. Hence, peripheral blood would be a logical source to observe and identify these physiological events. This paper outlines the protocol for a scoping review being conducted to summarise the extant literature regarding blood-based biomarkers of CRCI among patients with non-CNS cancer. Methods/analysis Methods will be informed by the updated guidelines of Arksey and O’Malley. The systematic search for literature will include electronic databases, handsearching of key journals and reference lists, forward citation tracking and consultation with content experts. Study selection will be confirmed by duplicate review and calculation of inter-rater reliability. Data to be charted will include study design, sample size, cancer and treatment characteristics, demographic characteristics, cognitive variable/s and biomarkers assessed, associations between cognitive functioning and biomarkers (including statistics used), and rigour in biomarker sample collection and processing. Results will be presented through: (1) a descriptive numerical summary of studies, including a flow diagram based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement, (2) a list of blood-based biomarkers associated with CRCI and (3) a narrative overview developed through collaboration among the research team and consultation with content experts. Dissemination The findings of this review will highlight current directions and gaps in the current body of evidence that may lead to improved rigour in future CRCI investigations. The dissemination of this work will be facilitated through the involvement of clinicians and researchers on the research team, an external consultation process and the presentation of the results through scholarly publication and presentation.

The CDK inhibitor AT7519M in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. A Phase II study of the Canadian Cancer Trials Group

Abstract AT7519M is a small molecule inhibitor of cyclin-dependent kinases 1, 2, 4, 5, and 9 with in vitro activity against lymphoid malignancies. In two concurrent Phase II trials, we evaluated AT7519M in relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) using the recommended Phase II dosing of 27 mg/m2 twice weekly for 2 of every 3 weeks. Primary objective was objective response rate (ORR). Nineteen patients were accrued (7 CLL, 12 MCL). Four CLL patients achieved stable disease (SD). Two MCL patients achieved partial response (PR), and 6 had SD. One additional MCL patient with SD subsequently achieved PR 9 months after completion of AT7519M. Tumor lysis syndrome was not reported. In conclusion, AT7519M was safely administered to patients with relapsed/refractory CLL and MCL. In CLL, some patients had tumor reductions, but the ORR was low. In MCL, activity was noted with ORR of 27%.

Inhibition of Glycolysis as a Therapeutic Strategy in Acute Myeloid Leukemias

The metabolic pathways in cancer cells are reprogrammed such that they utilize nutrients quite differently than their normal, nonmalignant counterparts. It has been known for some time that the cancer phenotype results in alterations to glucose metabolism and, more recently, modifications to both glutamine and fatty acid metabolism have been noted. So prevalent is this altered metabolism in malignancy that many now consider it a hallmark of the cancer phenotype. As such, the metabolic discrepancies between cancer cells and normal cells provide a therapeutic window for the potential development of targeted anticancer agents. A number of pharmacological agents that either directly target the enzymes driving tumor glycolysis or the upstream mediators of the glycolytic pathway are currently under investigation with the hope of combining them with existing clinical protocols. Akin to the cytokines and chemokines produced by cancer cells, the intermediates and byproducts of altered tumor glycolysis, upon secretion from cancer cells, are also capable of modulating the phenotypes of normal cells located in the tumor microenvironment. Thus, glycolytic inhibitors may also rescue the effects that tumor derived metabolites have on surrounding cells.