Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction.

Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.

NRF2 activates macropinocytosis upon autophagy inhibition.

Su et al. demonstrate that upon inhibiting autophagy, an intracellular nutrient recycling pathway, pancreatic ductal adenocarcinoma cells upregulate NRF2-mediated transcription of macropinocytosis pathway components, thereby triggering an alternate route for tumors to scavenge nutrients from extracellular sources. Accordingly, the combined inhibition of autophagy and macropinocytosis may improve cancer treatment.

The future of gene-targeted therapy for hereditary tyrosinemia type 1 as a lead indication among the inborn errors of metabolism.

INTRODUCTION: Inborn errors of metabolism (IEMs) often result from single-gene mutations and collectively cause liver dysfunction in neonates leading to chronic liver and systemic disease. Current treatments for many IEMs are limited to maintenance therapies that may still require orthotropic liver transplantation. Gene therapies offer a potentially superior approach by correcting or replacing defective genes with functional isoforms; however, they face unique challenges from complexities presented by individual diseases and their diverse etiology, presentation, and pathophysiology. Furthermore, immune responses, off-target gene disruption, and tumorigenesis are major concerns that need to be addressed before clinical application of gene therapy. AREAS COVERED: The current treatments for IEMs are reviewed as well as the advances in, and barriers to, gene therapy for IEMs. Attention is then given to ex vivo and in vivo gene therapy approaches for hereditary tyrosinemia type 1 (HT1). Of all IEMs, HT1 is particularly amenable to gene therapy because of a selective growth advantage conferred to corrected cells, thereby lowering the initial transduction threshold for phenotypic relevance. EXPERT OPINION: It is proposed that not only is HT1 a safe indication for gene therapy, its unique characteristics position it to be an ideal IEM to develop for clinical investigation.

Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition.

Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.

A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers.

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.

A recurrent kinase domain mutation in PRKCA defines chordoid glioma of the third ventricle.

Chordoid glioma is a rare brain tumor thought to arise from specialized glial cells of the lamina terminalis along the anterior wall of the third ventricle. Despite being histologically low-grade, chordoid gliomas are often associated with poor outcome, as their stereotypic location in the third ventricle makes resection challenging and efficacious adjuvant therapies have not been developed. Here we performed genomic profiling on 13 chordoid gliomas and identified a recurrent D463H missense mutation in PRKCA in all tumors, which localizes in the kinase domain of the encoded protein kinase C alpha (PKCα). Expression of mutant PRKCA in immortalized human astrocytes led to increased phospho-ERK and anchorage-independent growth that could be blocked by MEK inhibition. These studies define PRKCA as a recurrently mutated oncogene in human cancer and identify a potential therapeutic vulnerability in this uncommon brain tumor.

SIRT1-Activating Compounds (STAC) Negatively Regulate Pancreatic Cancer Cell Growth and Viability Through a SIRT1 Lysosomal-Dependent Pathway.

PURPOSE: Recent studies suggest that SIRT1-activating compounds (STAC) are a promising class of anticancer drugs, although their mechanism of action remains elusive. The main goal of this study is to determine the role of STACs as a potential therapy for pancreatic cancer. In addition, we also explored the mechanism by which these compounds affect pancreatic cancer. EXPERIMENTAL DESIGN: Using in vitro (cell culture experiments) and in vivo (xenograft experiments) approaches, we studied the role of SIRT1 agonists (STAC) in human pancreatic cancer cell viability and growth. RESULTS: We show that SIRT1 is highly expressed in pancreatic cancer cells and that the STACs SRT1720, SRT1460, and SRT3025 inhibited cell growth and survival of pancreatic cancer cells. STACs enhanced the sensitivity of pancreatic cells to gemcitabine and paclitaxel, indicating that these drugs could be used in combination with other chemotherapy drugs. We also show that STACs were very effective in inhibiting tumor xenograft growth. In mechanistic studies, we observed that STACs activated a SIRT1 lysosomal-dependent cell death. Furthermore, the effect of STACs on cell viability was also dependent on the expression of the endogenous SIRT1 inhibitor DBC1. CONCLUSIONS: Taken together, our results reveal an essential role for SIRT1 and lysosomes in the death pathway regulated by STACs in pancreatic cancer cells. Clin Cancer Res; 22(10); 2496-507. ©2015 AACR.

The oncogenic STP axis promotes triple-negative breast cancer via degradation of the REST tumor suppressor.

Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 ("STP axis") cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase ßTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo.

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti-angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

BRCA2 localization to the midbody by filamin A regulates cep55 signaling and completion of cytokinesis.

Disruption of the BRCA2 tumor suppressor is associated with structural and numerical chromosomal defects. The numerical abnormalities in BRCA2-deficient cells may partly result from aberrations in cell division caused by disruption of BRCA2 during cytokinesis. Here we show that BRCA2 is a component of the midbody that is recruited through an interaction with Filamin A actin-binding protein. At the midbody, BRCA2 influences the recruitment of endosomal sorting complex required for transport (ESCRT)-associated proteins, Alix and Tsg101, and formation of CEP55-Alix and CEP55-Tsg101 complexes during abscission. Disruption of these BRCA2 interactions by cancer-associated mutations results in increased cytokinetic defects but has no effect on BRCA2-dependent homologous recombination repair of DNA damage. These findings identify a specific role for BRCA2 in the regulation of midbody structure and function, separate from DNA damage repair, that may explain in part the whole-chromosomal instability in BRCA2-deficient tumors.

Tex14, a Plk1-regulated protein, is required for kinetochore-microtubule attachment and regulation of the spindle assembly checkpoint.

Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis-expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects, and whole-chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APC(Cdc20)-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase-to-anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.

Von Hippel-Lindau gene product directs cytokinesis: a new tumor suppressor function.

One of the mechanisms of tumorigenesis is that the failure of cell division results in genetically unstable, multinucleated cells. Here we show that pVHL, a tumor suppressor protein that has been implicated in the pathogenesis of renal cell carcinoma (RCC), plays an important role in regulation of cytokinesis. We found that pVHL-deficient RCC 786-O cells were multinucleated and polyploid. Reintroduction of wild-type pVHL into these cells rescued the diploid cell population, whereas the mutant pVHL-K171G failed to do so. We demonstrate that lysine 171 of pVHL is important for the final step of cytokinesis: the midbody abscission. The pVHL-K171G caused failure to localize the ESCRT-1 interacting protein Alix and the v-SNARE complex component Endobrevin to the midbody in 786-O cells, leading to defective cytokinesis. Moreover, SUMOylation of pVHL at lysine 171 might modulate its function as a cytokinesis regulator. pVHL tumor suppressor function was also disrupted by the K171G mutation, as evidenced by the xenograft tumor formation when 786-O clones expressing pVHL-K171G were injected into mice. Most RCC cell lines show a polyploid chromosome complement and consistent heterogeneity in chromosome number. Thus, this study offers a way to explain the chromosome instability in RCC and reveals a new direction for the tumor suppressor function of pVHL, which is independent of its E3 ubiquitin ligase activity.

Inactivation of Brca2 promotes Trp53-associated but inhibits KrasG12D-dependent pancreatic cancer development in mice.

BACKGROUND & AIMS: Inherited mutations in the BRCA2 tumor suppressor have been associated with an increased risk of pancreatic cancer. To establish the contribution of Brca2 to pancreatic cancer we developed a mouse model of pancreas-specific Brca2 inactivation. Because BRCA2-inactivating mutations cause defects in repair of DNA double-strand breaks that result in chromosomal instability, we evaluated whether Brca2 inactivation induced instability in pancreatic tissue from these mice and whether associated pancreatic tumors were hypersensitive to DNA damaging agents. METHODS: We developed mouse models that combined pancreas-specific Kras activation and Trp53 deletion with Brca2 inactivation. Development of pancreatic cancer was assessed; tumors and nonmalignant tissues were analyzed for chromosomal instability and apoptosis. Cancer cell lines were evaluated for sensitivity to DNA damaging agents. RESULTS: In the presence of disrupted Trp53, Brca2 inactivation promoted the development of premalignant lesions and pancreatic tumors that reflected the histology of human disease. Cancer cell lines derived from these tumors were hypersensitive to specific DNA damaging agents. In contrast, in the presence of KrasG12D, Brca2 inactivation promoted chromosomal instability and apoptosis and unexpectedly inhibited growth of premalignant lesions and tumors. CONCLUSIONS: Trp53 signaling must be modified before inactivation of the Brca2 wild-type allele, irrespective of Kras status, for Brca2-deficient cells to form tumors.

Microcephalin regulates BRCA2 and Rad51-associated DNA double-strand break repair.

Microcephalin (MCPH1) is a BRCA1 COOH terminal (BRCT) domain containing protein involved in the cellular response to DNA damage that has been implicated in autosomal recessive primary microcephaly. MCPH1 is recruited to sites of DNA double-strand breaks by phosphorylated histone H2AX (gammaH2AX), but the mechanism by which MCPH1 contributes to the repair process remains to be determined. Here, we show that MCPH1 binds to BRCA2 and regulates the localization of BRCA2 and Rad51 at sites of DNA damage. The interaction occurs through the NH(2) terminus of BRCA2 and the COOH terminal BRCT domains of MCPH1. Disruption of the interaction between MCPH1 and BRCA2 has no effect on the ability of BRCA2 to form a complex with Rad51 but is associated with substantially reduced levels of both BRCA2 and Rad51 at sites of DNA double-strand breaks. Uncoupling of MCPH1 from BRCA2 also interferes with Rad51-dependent and BRCA2-dependent homologous recombination repair activity. These results suggest that the role of MCPH1 in the DNA damage response is in part associated with the ability to localize BRCA2 to sites of DNA double-stand breaks.

Overexpression of Cdc20 leads to impairment of the spindle assembly checkpoint and aneuploidization in oral cancer.

Defects in the spindle assembly checkpoint are thought to be responsible for an increased rate of aneuploidization during tumorigenesis. Despite a plethora of information on the correlation between BUB-MAD gene expression levels and defects in the spindle checkpoint, very little is known about alteration of another important spindle checkpoint protein, Cdc20, in human cancer and its role in tumor aneuploidy. We observed overexpression of CDC20 in several oral squamous cell carcinoma (OSCC) cell lines and primary head and neck tumors and provide evidence that such overexpression of CDC20 is associated with premature anaphase promotion, resulting in mitotic abnormalities in OSCC cell lines. We also reconstituted the chromosomal instability phenotype in a chromosomally stable OSCC cell line by overexpressing CDC20. Thus, abnormalities in the cellular level of Cdc20 may deregulate the timing of anaphase promoting complex (APC/C) in promoting premature anaphase, which often results in aneuploidy in the tumor cells.

A new Mad2-interacting domain of Cdc20 is critical for the function of Mad2-Cdc20 complex in the spindle assembly checkpoint.

Interaction between Mad2 and Cdc20 (cell division cycle 20) is a key event during spindle assembly checkpoint activation. In the past, an N-terminal peptide containing amino acid residues 111-150 of Cdc20 was shown to bind Mad2 much better than the full-length Cdc20 protein. Using co-localization, co-immunoprecipitation and peptide inhibition analysis with different deletion mutants of Cdc20, we identified another Mad2-binding domain on Cdc20 from amino acids 342-355 within the WD repeat region. An intervening region between these two domains interferes with its Mad2 binding when present individually with any of these two Mad2-binding sites. We suggest that these three domains together determine the overall strength of Mad2 binding with Cdc20. Functional analysis suggests that an optimum Mad2 binding efficiency of Cdc20 is required during checkpoint arrest and release. Further, we have identified a unique polyhistidine motif with metal binding property adjacent to this second binding domain that may be important for maintaining the overall conformation of Cdc20 for its binding to Mad2.