Selective Termination of Autophagy-Dependent Cancers.

The goal of cancer research is to identify characteristics of cancer cells that allow them to be selectively eliminated without harming the host. One such characteristic is autophagy dependence. Cancer cells survive, proliferate, and metastasize under conditions where normal cells do not. Thus, the requirement in cancer cells for more energy and macromolecular biosynthesis can evolve into a dependence on autophagy for recycling cellular components. Recent studies have revealed that autophagy, as well as different forms of cellular trafficking, is regulated by five phosphoinositides associated with eukaryotic cellular membranes and that the enzymes that synthesize them are prime targets for cancer therapy. For example, PIKFYVE inhibitors rapidly disrupt lysosome homeostasis and suppress proliferation in all cells. However, these inhibitors selectively terminate PIKFYVE-dependent cancer cells and cancer stem cells with not having adverse effect on normal cells. Here, we describe the biochemical distinctions between PIKFYVE-sensitive and -insensitive cells, categorize PIKFYVE inhibitors into four groups that differ in chemical structure, target specificity and efficacy on cancer cells and normal cells, identify the mechanisms by which they selectively terminate autophagy-dependent cancer cells, note their paradoxical effects in cancer immunotherapy, and describe their therapeutic applications against cancers.

PIKFYVE inhibitors trigger interleukin-24-dependent cell death of autophagy-dependent melanoma.

Inhibitors specifically targeting the 1-phosphatidylinositol 3-phosphate 5-kinase (PIKFYVE) disrupt lysosome homeostasis, thereby selectively terminating autophagy-dependent human cancer cells in vivo as well as in vitro without harming the viability of nonmalignant cells. To elucidate the mechanism by which PIKFYVE inhibition induces cell death, autophagy-dependent melanoma cells were compared with normal foreskin fibroblasts. RNA sequence profiling suggested that PIKFYVE inhibitors upregulated an endoplasmic reticulum (ER) stress response involving interleukin-24 (IL24; also known as MDA7) selectively in melanoma cells. Subsequent biochemical and genetic analyses confirmed these results and extended them to tumor xenografts in which tumor formation and expansion were inhibited. IL24 expression was upregulated by the DDIT3/CHOP/CEBPz transcription factor, a component of the PERK-dependent ER-stress response. Ectopic expression of IL24-induced cell death in melanoma cells, but not in foreskin fibroblasts, whereas ablation of the IL24 gene in melanoma cells prevented death. IL24 upregulation was triggered specifically by PIKFYVE inhibition. Thus, unlike thapsigargin and tunicamycin, which induce ER-stress indiscriminately, PIKFYVE inhibitors selectively terminated PIKFYVE-sensitive melanoma by inducing IL24-dependent ER-stress. Moreover, induction of cell death by a PIKFYVE inhibitor together with ectopic expression of IL24 protein was cumulative, thereby confirming the therapeutic potential of PIKFYVE inhibitors in the treatment of melanoma.

PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells.

Although PIKFYVE phosphoinositide kinase inhibitors can selectively eliminate PIKFYVE-dependent human cancer cells in vitro and in vivo, the basis for this selectivity has remained elusive. Here we show that the sensitivity of cells to the PIKFYVE inhibitor WX8 is not linked to PIKFYVE expression, macroautophagic/autophagic flux, the BRAFV600E mutation, or ambiguous inhibitor specificity. PIKFYVE dependence results from a deficiency in the PIP5K1C phosphoinositide kinase, an enzyme required for conversion of phosphatidylinositol-4-phosphate (PtdIns4P) into phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2/PIP2), a phosphoinositide associated with lysosome homeostasis, endosome trafficking, and autophagy. PtdIns(4,5)P2 is produced via two independent pathways. One requires PIP5K1C; the other requires PIKFYVE and PIP4K2C to convert PtdIns3P into PtdIns(4,5)P2. In PIKFYVE-dependent cells, low concentrations of WX8 specifically inhibit PIKFYVE in situ, thereby increasing the level of its substrate PtdIns3P while suppressing PtdIns(4,5)P2 synthesis and inhibiting lysosome function and cell proliferation. At higher concentrations, WX8 inhibits both PIKFYVE and PIP4K2C in situ, which amplifies these effects to further disrupt autophagy and induce cell death. WX8 did not alter PtdIns4P levels. Consequently, inhibition of PIP5K1C in WX8-resistant cells transformed them into sensitive cells, and overexpression of PIP5K1C in WX8-sensitive cells increased their resistance to WX8. This discovery suggests that PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors.Abbreviations: DMSO: dimethylsulfoxide; ELISA: enzyme-linked immunosorbent assay; LC3-I: microtubule associated protein light chain 3-I; LC3-II: microtubule associated protein light chain 3-II; MS: mass spectrometry; PtdIns: phosphatidylinositol; PtdIns3P: PtdIns-3-phosphate; PtdIns4P: PtdIns-4-phosphate; PtdIns5P: PtdIns-5-phosphate; PtdIns(3,5)P2: PtdIns-3,5-bisphosphate; PtdIns(4,5)P2/PIP2: PtdIns-4,5-bisphosphate; PtdIns(3,4,5)P3/PIP3: PtdIns-3,4,5-trisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PI4KA: phosphatidylinositol 4-kinase alpha; PI4KB: phosphatidylinositol 4-kinase beta; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PI4K2B: phosphatidylinositol 4-kinase type 2 beta; PIP4K2A: phosphatidylinositol-5-phosphate 4-kinase type 2 alpha; PIP4K2B: phosphatidylinositol-5-phosphate 4-kinase type 2 beta; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PIP5K1A: phosphatidylinositol-4-phosphate 5-kinase type 1 alpha; PIP5K1B: phosphatidylinositol-4-phosphate 5-kinase type 1 beta; PIP5K1C: phosphatidylinositol-4-phosphate 5-kinase type 1 gamma; WX8: 1H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone.

PIKfyve-specific inhibitors restrict replication of multiple coronaviruses in vitro but not in a murine model of COVID-19.

The ongoing COVID-19 pandemic has claimed more than 6 million lives and continues to test the world economy and healthcare systems. To combat this pandemic, the biological research community has shifted efforts to the development of medical countermeasures, including vaccines and therapeutics. However, to date, the only small molecules approved for the treatment of COVID-19 in the United States are the nucleoside analogue Remdesivir and the protease inhibitor Paxlovid, though multiple compounds have received Emergency Use Authorization and many more are currently being tested in human efficacy trials. One such compound, Apilimod, is being considered as a COVID-19 therapeutic in a Phase II efficacy trial. However, at the time of writing, there are no published efficacy data in human trials or animal COVID-19 models. Here we show that, while Apilimod and other PIKfyve inhibitors have potent antiviral activity in various cell lines against multiple human coronaviruses, these compounds worsen disease in a COVID-19 murine model when given prophylactically or therapeutically.

Cell Death and the p53 Enigma During Mammalian Embryonic Development.

Twelve forms of programmed cell death (PCD) have been described in mammalian cells, but which of them occurs during embryonic development and the role played by the p53 transcription factor and tumor suppressor remains enigmatic. Although p53 is not required for mouse embryonic development, some studies conclude that PCD in pluripotent embryonic stem cells from mice (mESCs) or humans (hESCs) is p53-dependent whereas others conclude that it is not. Given the importance of pluripotent stem cells as models of embryonic development and their applications in regenerative medicine, resolving this enigma is essential. This review reconciles contradictory results based on the facts that p53 cannot induce lethality in mice until gastrulation and that experimental conditions could account for differences in results with ESCs. Consequently, activation of the G2-checkpoint in mouse ESCs is p53-independent and generally, if not always, results in noncanonical apoptosis. Once initiated, PCD occurs at equivalent rates and to equivalent extents regardless of the presence or absence of p53. However, depending on experimental conditions, p53 can accelerate initiation of PCD in ESCs and late-stage blastocysts. In contrast, DNA damage following differentiation of ESCs in vitro or formation of embryonic fibroblasts in vivo induces p53-dependent cell cycle arrest and senescence.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

Developmental Acquisition of p53 Functions.

Remarkably, the p53 transcription factor, referred to as "the guardian of the genome", is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation.

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage-induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53-/- ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content; annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage-dependent G2-checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53-/- ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53-dependent formation of haploid cells, increased genomic stability, and suppression of the G2-checkpoint. Minimal induction of DNA damage now resulted in p53-facilitated apoptosis, but regulation of pluripotent gene expression remained p53-independent. Primary embryonic fibroblasts underwent p53-dependent total cell cycle arrest (a prelude to cell senescence), and p53-independent apoptosis occurred in the presence of 10-fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.

Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer.

The human positive coactivator 4 (PC4) was originally identified as a multi-functional cofactor capable of mediating transcription activation by diverse gene- and tissue-specific activators. Recent studies suggest that PC4 might also function as a novel cancer biomarker and therapeutic target for different types of cancers. siRNA knockdown studies indicated that down-regulation of PC4 expression could inhibit tumorigeneicity of A549 non-small cell lung cancer tumor model in nude mice. Here we show that AG-1031, a small molecule identified by high throughput screening, can inhibit the double-stranded DNA binding activity of PC4, more effectively than its single-stranded DNA binding activity. AG-1031 also specifically inhibited PC4-dependent transcriptional activation in vitro using purified transcription factors. AG-1031 inhibited proliferation of several cultured cell lines derived from non-small cell lung cancers (NSCLC) and growth of tumors that formed from A549 cell xenografts in immuno-compromised mice. Moreover, pre-injection of AG-1031 in these mice not only reduced tumor size, but also prevented tumor formation in 20% of the animals. AG-1031 treated A549 cells and tumors from AG-1031 treated animals showed a significant decrease in the levels of both PC4 and VEGFC, a key mediator of angiogenesis in cancer. On the other hand, all tested mice remained constant weight during animal trials. These results demonstrated that AG-1031 could be a potential therapy for PC4-positive NSCLC.

The Cdk2-c-Myc-miR-571 Axis Regulates DNA Replication and Genomic Stability by Targeting Geminin.

DNA rereplication leads to genomic instability and has been implicated in the pathology of a variety of human cancers. Eukaryotic DNA replication is tightly controlled to ensure it occurs only once during each cell cycle. Geminin is a critical component of this control, it prevents DNA rereplication from occurring during S, G2, and early M phases by preventing MCM helicases from forming prereplication complexes. Geminin is targeted for degradation by the anaphase-promoting complex (APC/C) from anaphase through G1-phase, however, accumulating evidence indicates that Geminin is downregulated in late S-phase due to an unknown mechanism. Here, we used a high-throughput screen to identify miRNAs that can induce excess DNA replication and found that miR-571 could reduce the protein level of Geminin in late S-phase independent of the APC/C. Furthermore, miR-571 regulated efficient DNA replication and S-phase cell-cycle progression. Strikingly, c-Myc suppressed miR-571 expression by binding directly to the miR-571 promoter. At the beginning of S-phase, Cdk2 phosphorylated c-Myc at Serine 62, promoting its association with the miR-571 promoter region. Collectively, we identify miR-571 as the first miRNA that prevents aberrant DNA replication and the Cdk2-c-Myc-miR-571 axis as a new pathway for regulating DNA replication, cell cycle, and genomic stability in cancer cells. SIGNIFICANCE: These findings identify a novel regulatory mechanism that is critical for maintaining genome integrity by regulating DNA replication and cell-cycle progression.

A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.

High-throughput screening identified 5 chemical analogs (termed the WX8-family) that disrupted 3 events in lysosome homeostasis: (1) lysosome fission via tubulation without preventing homotypic lysosome fusion; (2) trafficking of molecules into lysosomes without altering lysosomal acidity, and (3) heterotypic fusion between lysosomes and autophagosomes. Remarkably, these compounds did not prevent homotypic fusion between lysosomes, despite the fact that homotypic fusion required some of the same machinery essential for heterotypic fusion. These effects varied 400-fold among WX8-family members, were time and concentration dependent, reversible, and resulted primarily from their ability to bind specifically to the PIKFYVE phosphoinositide kinase. The ability of the WX8-family to prevent lysosomes from participating in macroautophagy/autophagy suggested they have therapeutic potential in treating autophagy-dependent diseases. In fact, the most potent family member (WX8) was 100-times more lethal to 'autophagy-addicted' melanoma A375 cells than the lysosomal inhibitors hydroxychloroquine and chloroquine. In contrast, cells that were insensitive to hydroxychloroquine and chloroquine were also insensitive to WX8. Therefore, the WX8-family of PIKFYVE inhibitors provides a basis for developing drugs that could selectively kill autophagy-dependent cancer cells, as well as increasing the effectiveness of established anti-cancer therapies through combinatorial treatments. Abbreviations: ACTB: actin beta; Baf: bafilomycin A1; BECN1: beclin 1; BODIPY: boron-dipyrromethene; BORC: BLOC-1 related complex; BRAF: B-Raf proto-oncogene, serine/threonine kinase; BSA: bovine serum albumin; CTSD: cathepsin D; CQ: chloroquine; DNA: deoxyribonucleic acid; EC50: half maximal effective concentration; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HCQ: hydroxychloroquine; HOPS complex: homotypic fusion and protein sorting complex; Kd: equilibrium binding constant; IC50: half maximal inhibitory concentration; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3A: microtubule associated protein 1 light chain 3 alpha; MES: 2-(N-morpholino)ethanesulphonic acid; MTOR: mechanistic target of rapamycin kinase; µM: micromolar; NDF: 3-methylbenzaldehyde (2,6-dimorpholin-4-ylpyrimidin-4-yl)hydrazine;NEM: N-ethylmaleimide; NSF: N-ethylmaleimide sensitive factor; PBS: phosphate-buffered saline; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PtdIns3P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P2: phosphatidylinositol 3,5-biphosphate; RFP: red fluorescent protein; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TWEEN 20: polysorbate 20; V-ATPase: vacuolar-type H+-translocating ATPase; VPS39: VPS39 subunit of HOPS complex; VPS41: VPS41 subunit of HOPS complex; WWL: benzaldehyde [2,6-di(4-morpholinyl)-4-pyrimidinyl]hydrazone; WX8: 1H-indole-3-carbaldehyde [4-anilino-6-(4-morpholinyl)-1,3,5-triazin-2-yl]hydrazine; XBA: N-(3-chloro-4-fluorophenyl)-4,6-dimorpholino-1,3,5-triazin-2-amine hydrochloride; XB6: N-(4-ethylphenyl)-4,6-dimorpholino-1,3,5-triazin-2-amine hydrochloride.

DHS (trans-4,4'-dihydroxystilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2).

DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Since inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs) that are essential for DNA replication and DNA damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to this drug during treatment. Thus, new drugs that inhibit RNR are needed to be developed. In this study, we identified a synthetic analog of resveratrol (3,5,4'-trihydroxy-trans-stilbene), termed DHS (trans-4,4'-dihydroxystilbene), that acts as a potent inhibitor of DNA replication. Molecular docking analysis identified the RRM2 (ribonucleotide reductase regulatory subunit M2) of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F-mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently decreased synthesis of dNTPs with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anti-cancer agent that targets RRM2 with therapeutic potential either alone or in combination with other agents to arrest cancer development.

CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human Chorionic Gonadotropin.

AIMS: The human placental syncytiotrophoblast (STB) cells play essential roles in embryo implantation and nutrient exchange between the mother and the fetus. STBs are polyploid which are formed by fusion of diploid cytotrophoblast (CTB) cells. Abnormality in STBs formation can result in pregnancy-related disorders. While a number of genes have been associated with CTB fusion the initial events that trigger cell fusion are not well understood. Primary objective of this study was to enhance our understanding about the molecular mechanism of placental cell fusion. METHODS: FACS and microscopic analysis was used to optimize Forskolin-induced fusion of BeWo cells (surrogate of CTBs) and subsequently, changes in the expression of different cell cycle regulator genes were analyzed through Western blotting and qPCR. Immunohistochemistry was performed on the first trimester placental tissue sections to validate the results in the context of placental tissue. Effect of Cyclin Dependent Kinase 1 (CDK1) inhibitor, RO3306, on BeWo cell fusion was studied by microscopy and FACS, and by monitoring the expression of human Chorionic Gonadotropin (hCG) by Western blotting and qPCR. RESULTS: The data showed that the placental cell fusion was associated with down regulation of CDK1 and its associated cyclin B, and significant decrease in DNA replication. Moreover, inhibition of CDK1 by an exogenous inhibitor induced placental cell fusion and expression of hCG. CONCLUSION: Here, we report that the placental cell fusion can be induced by inhibiting CDK1. This study has a high therapeutic significance to manage pregnancy related abnormalities.

Links between DNA Replication, Stem Cells and Cancer.

Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories; cancer driver mutations that initiate carcinogenesis and genome destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid missegregation. Increased genome instability results in accelerated clonal evolution leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, results from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all of the cell lineages in the embryo and adult, but when they mislocate to ectopic sites, they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the Geminin protein to prevent excess DNA replication from triggering DNA damage-dependent apoptosis. This link between the control of DNA replication during early development and germ cell neoplasia reveals Geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells.

Geminin Is Essential for Pluripotent Cell Viability During Teratoma Formation, but Not for Differentiated Cell Viability During Teratoma Expansion.

Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA rereplication-dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that geminin is a haplosufficient gene that is essential for ESC viability before they differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, geminin protein, and pluripotent stem cells. Thus, differentiated cells did not require geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. These results provide proof-of-principle that preventing geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

Identification of genes that are essential to restrict genome duplication to once per cell division.

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.

Genome Duplication: The Heartbeat of Developing Organisms.

The mechanism that duplicates the nuclear genome during the trillions of cell divisions required to develop from zygote to adult is the same throughout the eukarya, but the mechanisms that determine where, when and how much nuclear genome duplication occur regulate development and differ among the eukarya. They allow organisms to change the rate of cell proliferation during development, to activate zygotic gene expression independently of DNA replication, and to restrict nuclear DNA replication to once per cell division. They allow specialized cells to exit their mitotic cell cycle and differentiate into polyploid cells, and in some cases, to amplify the number of copies of specific genes. It is genome duplication that drives evolution, by virtue of the errors that inevitably occur when the same process is repeated trillions of times. It is, unfortunately, the same errors that produce age-related genetic disorders such as cancer.

Geminin is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells.

Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability.

And-1 coordinates with Claspin for efficient Chk1 activation in response to replication stress.

The replisome is important for DNA replication checkpoint activation, but how specific components of the replisome coordinate with ATR to activate Chk1 in human cells remains largely unknown. Here, we demonstrate that And-1, a replisome component, acts together with ATR to activate Chk1. And-1 is phosphorylated at T826 by ATR following replication stress, and this phosphorylation is required for And-1 to accumulate at the damage sites, where And-1 promotes the interaction between Claspin and Chk1, thereby stimulating efficient Chk1 activation by ATR. Significantly, And-1 binds directly to ssDNA and facilitates the association of Claspin with ssDNA. Furthermore, And-1 associates with replication forks and is required for the recovery of stalled forks. These studies establish a novel ATR-And-1 axis as an important regulator for efficient Chk1 activation and reveal a novel mechanism of how the replisome regulates the replication checkpoint and genomic stability.