Cell cycle checkpoint in cancer: a therapeutically targetable double-edged sword

Major currently used anticancer therapeutics either directly damage DNA or target and upset basic cell division mechanisms like DNA replication and chromosome segregation. These insults elicit activation of cell cycle checkpoints, safeguard mechanisms that cells implement to correctly complete cell cycle phases, repair damage or eventually commit suicide in case damage is unrepairable. Although cancer cells appear to be advantageously defective in some aspects of checkpoint physiology, recent acquisitions on the biochemical mechanisms of the various checkpoints are offering new therapeutic approaches against cancer. Indeed, chemical manipulation of these mechanisms is providing new therapeutic strategies and tools to increase the killing efficacy of major cancer therapeutics as well as to directly promote cancer cell death. In this review we summarize developing concepts on how targeting cell cycle checkpoints may provide substantial improvement to cancer therapy

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle

PP2ARts1 is a master regulator of pathways that control cell size

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth

Analysis of Checkpoint Responses to Histone Deacetylase Inhibitors

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin histones, but they also perturb the cell cycle, causing both Gl and G2 phase arrests, the latter by initiating a checkpoint response. In this chapter we will describe the analysis of the histone deacetylase inhibitor-sensitive G 2 checkpoint using synchronized cell populations

DNA damage stress: Cui prodest?

DNA is an entity shielded by mechanisms that maintain genomic stability and are essential for living cells; however, DNA is constantly subject to assaults from the environment throughout the cellular life span, making the genome susceptible to mutation and irreparable damage. Cells are prepared to mend such events through cell death as an extrema ratio to solve those threats from a multicellular perspective. However, in cells under various stress conditions, checkpoint mechanisms are activated to allow cells to have enough time to repair the damaged DNA. In yeast, entry into the cell cycle when damage is not completely repaired represents an adaptive mechanism to cope with stressful conditions. In multicellular organisms, entry into cell cycle with damaged DNA is strictly forbidden. However, in cancer development, individual cells undergo checkpoint adaptation, in which most cells die, but some survive acquiring advantageous mutations and selfishly evolve a conflictual behavior. In this review, we focus on how, in cancer development, cells rely on checkpoint adaptation to escape DNA stress and ultimately to cell death

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instabilit

Expression of TIM3/VISTA checkpoints and the CD68 macrophage-associated marker correlates with anti-PD1/PDL1 resistance: implications of immunogram heterogeneity.

Although immunotherapies have achieved remarkable salutary effects among subgroups of advanced cancers, most patients do not respond. We comprehensively evaluated biomarkers associated with the "cancer-immunity cycle" in the pan-cancer setting in order to understand the immune landscape of metastatic malignancies as well as anti-PD-1/PD-L1 inhibitor resistance mechanisms. Interrogation of 51 markers of the cancer-immunity cycle was performed in 101 patients with diverse malignancies using a clinical-grade RNA sequencing assay. Overall, the immune phenotypes demonstrated overexpression of multiple checkpoints including VISTA (15.8% of 101 patients), PD-L2 (10.9%), TIM3 (9.9%), LAG3 (8.9%), PD-L1 (6.9%) and CTLA4 (3.0%). Additionally, aberrant expression of macrophage-associated markers (e.g. CD68 and CSF1R; 11-23%), metabolic immune escape markers (e.g. ADORA2A and IDO1; 9-16%) and T-cell priming markers (e.g. CD40, GITR, ICOS and OX40; 4-31%) were observed. Most tumors (87.1%, 88/101) expressed distinct immune portfolios, with a median of six theoretically actionable biomarkers (pharmacologically tractable by Food and Drug Administration approved agents [on- or off-label] or with agents in clinical development). Overexpression of TIM-3, VISTA and CD68 were significantly associated with shorter progression-free survival (PFS) after anti-PD-1/PD-L1-based therapies (among 39 treated patients) (all P < .01). In conclusion, cancer-immunity cycle biomarker evaluation was feasible in diverse solid tumors. High expression of alternative checkpoints TIM-3 and VISTA and of the macrophage-associated markers CD68 were associated with significantly worse PFS after anti-PD-1/PD-L1-based therapies. Most patients had distinct and complex immune expression profiles suggesting the need for customized combinations of immunotherapy