Publications by authors named "Emmanuelle Nicolas"

67 Publications

Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer.

Clin Cancer Res 2022 May;28(9):1925-1937

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.

Experimental Design: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.

Results: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.

Conclusions: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-4316DOI Listing
May 2022

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers.

Nat Commun 2022 03 25;13(1):1618. Epub 2022 Mar 25.

Program in Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA.

Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.
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http://dx.doi.org/10.1038/s41467-022-29227-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8956741PMC
March 2022

An autonomous TCR signal-sensing switch influences CD4/CD8 lineage choice in mice.

Commun Biol 2022 01 21;5(1):84. Epub 2022 Jan 21.

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA.

How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases.
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http://dx.doi.org/10.1038/s42003-022-02999-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8783009PMC
January 2022

Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4 T cell identity and function.

Nat Immunol 2021 08 26;22(8):969-982. Epub 2021 Jul 26.

Blood Cell Development and Cancer, Fox Chase Cancer Center, Philadelphia, PA, USA.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4 intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (Sil). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the Sil to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (T) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRPD-1CD25 (Triple) T cells and conversion to CD4 IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.
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http://dx.doi.org/10.1038/s41590-021-00980-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8887595PMC
August 2021

Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1.

Nat Commun 2021 04 14;12(1):2244. Epub 2021 Apr 14.

Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.

Ferroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.
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http://dx.doi.org/10.1038/s41467-021-22471-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046803PMC
April 2021

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Oncogenesis 2021 Mar 15;10(3):29. Epub 2021 Mar 15.

Molecular Therapeutics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA.

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control Kras; Trp53 NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFR). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFR NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFR human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFR NSCLC.
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http://dx.doi.org/10.1038/s41389-021-00317-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961039PMC
March 2021

Cholesterol Pathway Inhibition Induces TGF-β Signaling to Promote Basal Differentiation in Pancreatic Cancer.

Cancer Cell 2020 10 24;38(4):567-583.e11. Epub 2020 Sep 24.

Molecular Therapeutics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA; The Marvin & Concetta Greenberg Pancreatic Cancer Institute, Fox Chase Cancer Center, Philadelphia, PA, USA; Kazan Federal University, Kazan, Russian Federation. Electronic address:

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by Kras expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor β signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.
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http://dx.doi.org/10.1016/j.ccell.2020.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572882PMC
October 2020

X- and Y-Linked Chromatin-Modifying Genes as Regulators of Sex-Specific Cancer Incidence and Prognosis.

Clin Cancer Res 2020 11 30;26(21):5567-5578. Epub 2020 Jul 30.

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Biological sex profoundly conditions organismal development and physiology, imposing wide-ranging effects on cell signaling, metabolism, and immune response. These effects arise from sex-specified differences in hormonal exposure, and from intrinsic genetic and epigenetic differences associated with the presence of an XX versus XY chromosomal complement. In addition, biological sex is now recognized to be a determinant of the incidence, presentation, and therapeutic response of multiple forms of cancer, including cancers not specifically associated with male or female anatomy. Although multiple factors contribute to sex-based differences in cancer, a growing body of research emphasizes a role for differential activity of X- and Y-linked tumor-suppressor genes in males and females. Among these, the X-linked and , and their Y-linked paralogs / and encode lysine demethylases. These epigenetic modulators profoundly influence gene expression, based on enzymatic activity in demethylating H3K27me3 and H3K4me3, and nonenzymatic scaffolding roles for large complexes that open and close chromatin for transcription. In a growing number of cases, mutations affecting these proteins have been recognized to strongly influence cancer risk, prognosis, and response to specific therapies. However, sex-specific patterns of mutation, expression, and activity of these genes, coupled with tissue-specific requirement for their function as tumor suppressors, together exemplify the complex relationship between sex and cancer vulnerabilities. In this review, we summarize and discuss the current state of the literature on the roles of these proteins in contributing to sex bias in cancer, and the status of clinical agents relevant to their function.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642178PMC
November 2020

Suppression of Ca signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

EMBO Rep 2020 05 25;21(5):e48904. Epub 2020 Mar 25.

Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4 T cells leads to enhanced Ca responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4 mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.
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http://dx.doi.org/10.15252/embr.201948904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202224PMC
May 2020

BRCA1 intronic Alu elements drive gene rearrangements and PARP inhibitor resistance.

Nat Commun 2019 12 11;10(1):5661. Epub 2019 Dec 11.

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA.

BRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.
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http://dx.doi.org/10.1038/s41467-019-13530-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906494PMC
December 2019

Modification of the base excision repair enzyme MBD4 by the small ubiquitin-like molecule SUMO1.

DNA Repair (Amst) 2019 10 8;82:102687. Epub 2019 Aug 8.

Cancer Epigenetics and Cancer Biology Programs, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA. Electronic address:

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. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.
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http://dx.doi.org/10.1016/j.dnarep.2019.102687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785017PMC
October 2019

Disease-Associated Genetic Variation in Human Mitochondrial Protein Import.

Am J Hum Genet 2019 05;104(5):784-801

Cancer Prevention and Control Program, Department of Clinical Genetics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address:

Mitochondrial dysfunction has consequences not only for cellular energy output but also for cellular signaling pathways. Mitochondrial dysfunction, often based on inherited gene variants, plays a role in devastating human conditions such as mitochondrial neuropathies, myopathies, cardiovascular disorders, and Parkinson and Alzheimer diseases. Of the proteins essential for mitochondrial function, more than 98% are encoded in the cell nucleus, translated in the cytoplasm, sorted based on the presence of encoded mitochondrial targeting sequences (MTSs), and imported to specific mitochondrial sub-compartments based on the integrated activity of a series of mitochondrial translocases, proteinases, and chaperones. This import process is typically dynamic; as cellular homeostasis is coordinated through communication between the mitochondria and the nucleus, many of the adaptive responses to stress depend on modulation of mitochondrial import. We here describe an emerging class of disease-linked gene variants that are found to impact the mitochondrial import machinery itself or to affect the proteins during their import into mitochondria. As a whole, this class of rare defects highlights the importance of correct trafficking of mitochondrial proteins in the cell and the potential implications of failed targeting on metabolism and energy production. The existence of this variant class could have importance beyond rare neuromuscular disorders, given an increasing body of evidence suggesting that aberrant mitochondrial function may impact cancer risk and therapeutic response.
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http://dx.doi.org/10.1016/j.ajhg.2019.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506819PMC
May 2019

Interaction of germline variants in a family with a history of early-onset clear cell renal cell carcinoma.

Mol Genet Genomic Med 2019 03 24;7(3):e556. Epub 2019 Jan 24.

Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Background: Identification of genetic factors causing predisposition to renal cell carcinoma has helped improve screening, early detection, and patient survival.

Methods: We report the characterization of a proband with renal and thyroid cancers and a family history of renal and other cancers by whole-exome sequencing (WES), coupled with WES analysis of germline DNA from additional affected and unaffected family members.

Results: This work identified multiple predicted protein-damaging variants relevant to the pattern of inherited cancer risk. Among these, the proband and an affected brother each had a heterozygous Ala45Thr variant in SDHA, a component of the succinate dehydrogenase (SDH) complex. SDH defects are associated with mitochondrial disorders and risk for various cancers; immunochemical analysis indicated loss of SDHB protein expression in the patient's tumor, compatible with SDH deficiency. Integrated analysis of public databases and structural predictions indicated that the two affected individuals also had additional variants in genes including TGFB2, TRAP1, PARP1, and EGF, each potentially relevant to cancer risk alone or in conjunction with the SDHA variant. In addition, allelic imbalances of PARP1 and TGFB2 were detected in the tumor of the proband.

Conclusion: Together, these data suggest the possibility of risk associated with interaction of two or more variants.
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http://dx.doi.org/10.1002/mgg3.556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418363PMC
March 2019

Thymine DNA glycosylase as a novel target for melanoma.

Oncogene 2019 05 23;38(19):3710-3728. Epub 2019 Jan 23.

Cancer Epigenetics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA.

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. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
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http://dx.doi.org/10.1038/s41388-018-0640-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563616PMC
May 2019

Cause of Death in Follicular Lymphoma in the First Decade of the Rituximab Era: A Pooled Analysis of French and US Cohorts.

J Clin Oncol 2019 01 27;37(2):144-152. Epub 2018 Nov 27.

1 Hospices Civils de Lyon, Centre Hospitalier Lyon Sud; Université de Lyon, Université Claude Bernard Lyon 1, Centre de Recherche en Cancérologie de Lyon INSERM 1052, Lyon, France.

Purpose: Although the life expectancy of patients with follicular lymphoma (FL) has increased, little is known of their causes of death (CODs) in the rituximab era.

Patients And Methods: We pooled two cohorts of newly diagnosed patients with FL grade 1-3A. Patients were enrolled between 2001 and 2013 in two French referral institutions (N = 734; median follow-up 89 months) and 2002 and 2012 in the University of Iowa and Mayo Clinic Specialized Program of Research Excellence (SPORE; N = 920; median follow-up 84 months). COD was classified as being a result of lymphoma, other malignancy, treatment related, or all other causes.

Results: Ten-year overall survival was comparable in the French (80%) and US (77%) cohorts. We were able to classify COD in 248 (88%) of 283 decedents. In the overall cohort, lymphoma was the most common COD, with a cumulative incidence of 10.3% at 10 years, followed by treatment-related mortality (3.0%), other malignancy (2.9%), other causes (2.2%), and unknown (3.0%). The 10-year cumulative incidence of death as a result of lymphoma or treatment was higher than death as a result of all other causes for each age group (including patients ≥ 70 years of age at diagnosis [25.4% v 16.6%]) Follicular Lymphoma International Prognostic Index score 3 to 5 (27.4% v 5.2%), but not Follicular Lymphoma International Prognostic Index score 0 to 1 (4.0% v 3.7%); for patients who failed to achieve event-free survival within 24 months from diagnosis (36.1% v 7.0%), but not for patients who achieved event-free survival within 24 months of diagnosis (6.7% v 5.7%); and for patients with a history of transformed FL (45.9% v 4.7%), but not among patients without (8.1% v 6.2%). Overall, 77 of 140 deaths as a result of lymphoma occurred in patients whose FL transformed after diagnosis.

Conclusion: Despite the improvement in overall survival in patients with FL in the rituximab era, their leading COD remains lymphoma, especially after disease transformation. Treatment-related mortality also represents a concern, which supports the need for less-toxic therapies.
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http://dx.doi.org/10.1200/JCO.18.00400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366812PMC
January 2019

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

Mol Biol Evol 2019 01;36(1):39-53

Blood Cell Development and Cancer Program, Fox Chase Cancer Center, Philadelphia, PA.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165 Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.
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http://dx.doi.org/10.1093/molbev/msy191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340473PMC
January 2019

BRCA1 Mutation-Specific Responses to 53BP1 Loss-Induced Homologous Recombination and PARP Inhibitor Resistance.

Cell Rep 2018 09;24(13):3513-3527.e7

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address:

BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brca1 mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1;53bp1 mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance.
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http://dx.doi.org/10.1016/j.celrep.2018.08.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219632PMC
September 2018

NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice.

Oncogene 2018 08 18;37(35):4854-4870. Epub 2018 May 18.

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high-grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9 genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin, and AURKA. Cell lines established from MISIIR-TAg;Nedd9 and MISIIR-TAg;Nedd9 mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild-type background Nedd9 allografts exhibited significantly reduced growth, dissemination, and oncogenic signaling compared to Nedd9 allografts. Gene expression analysis revealed that Nedd9 tumors exhibited more mesenchymal "stem-like" transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1, and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression.
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http://dx.doi.org/10.1038/s41388-018-0296-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119087PMC
August 2018

The Optimal PEG for Kidney Preservation: A Preclinical Porcine Study.

Int J Mol Sci 2018 Feb 3;19(2). Epub 2018 Feb 3.

Inserm, U1082, 86000 Poitiers, France.

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).
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http://dx.doi.org/10.3390/ijms19020454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855676PMC
February 2018

Metabolite Profiling Reveals the Glutathione Biosynthetic Pathway as a Therapeutic Target in Triple-Negative Breast Cancer.

Mol Cancer Ther 2018 01 11;17(1):264-275. Epub 2017 Oct 11.

Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry-based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal-like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal-like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth Overall, these findings support the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC and identify the non-basal-like subset as most likely to respond. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892195PMC
January 2018

Functional analysis of rare variants in mismatch repair proteins augments results from computation-based predictive methods.

Cancer Biol Ther 2017 Jul 11;18(7):519-533. Epub 2017 May 11.

a Molecular Therapeutics Program , Fox Chase Cancer Center , Philadelphia , PA , USA.

The cancer-predisposing Lynch Syndrome (LS) arises from germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1, MSH2, MSH6, and PMS2. A major challenge for clinical diagnosis of LS is the frequent identification of variants of uncertain significance (VUS) in these genes, as it is often difficult to determine variant pathogenicity, particularly for missense variants. Generic programs such as SIFT and PolyPhen-2, and MMR gene-specific programs such as PON-MMR and MAPP-MMR, are often used to predict deleterious or neutral effects of VUS in MMR genes. We evaluated the performance of multiple predictive programs in the context of functional biologic data for 15 VUS in MLH1, MSH2, and PMS2. Using cell line models, we characterized VUS predicted to range from neutral to pathogenic on mRNA and protein expression, basal cellular viability, viability following treatment with a panel of DNA-damaging agents, and functionality in DNA damage response (DDR) signaling, benchmarking to wild-type MMR proteins. Our results suggest that the MMR gene-specific classifiers do not always align with the experimental phenotypes related to DDR. Our study highlights the importance of complementary experimental and computational assessment to develop future predictors for the assessment of VUS.
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http://dx.doi.org/10.1080/15384047.2017.1326439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639829PMC
July 2017

Matrix-regulated integrin αβ maintains αβ-dependent desmoplastic traits prognostic of neoplastic recurrence.

Elife 2017 01 31;6. Epub 2017 Jan 31.

Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, United States.

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active αβ-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αβ-integrin redistribution of pFAK-independent active αβ-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and αβ-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.
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http://dx.doi.org/10.7554/eLife.20600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283834PMC
January 2017

A New Role for ERα: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes.

Mol Cancer Res 2017 02 15;15(2):152-164. Epub 2016 Nov 15.

Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania.

Resistance to hormonal therapies is a major clinical problem in the treatment of estrogen receptor α-positive (ERα) breast cancers. Epigenetic marks, namely DNA methylation of cytosine at specific CpG sites (5mCpG), are frequently associated with ERα status in human breast cancers. Therefore, ERα may regulate gene expression in part via DNA methylation. This hypothesis was evaluated using a panel of breast cancer cell line models of antiestrogen resistance. Microarray gene expression profiling was used to identify genes normally silenced in ERα cells but derepressed upon exposure to the demethylating agent decitabine, derepressed upon long-term loss of ERα expression, and resuppressed by gain of ERα activity/expression. ERα-dependent DNA methylation targets (n = 39) were enriched for ERα-binding sites, basal-up/luminal-down markers, cancer stem cell, epithelial-mesenchymal transition, and inflammatory and tumor suppressor genes. Kaplan-Meier survival curve and Cox proportional hazards regression analyses indicated that these targets predicted poor distant metastasis-free survival among a large cohort of breast cancer patients. The basal breast cancer subtype markers LCN2 and IFI27 showed the greatest inverse relationship with ERα expression/activity and contain ERα-binding sites. Thus, genes that are methylated in an ERα-dependent manner may serve as predictive biomarkers in breast cancer.

Implications: ERα directs DNA methylation-mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; 15(2); 152-64. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308451PMC
February 2017

Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin.

Oncotarget 2017 Mar;8(12):19156-19171

Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Ovarian, head and neck, and other cancers are commonly treated with cisplatin and other DNA damaging cytotoxic agents. Altered DNA damage response (DDR) contributes to resistance of these tumors to chemotherapies, some targeted therapies, and radiation. DDR involves multiple protein complexes and signaling pathways, some of which are evolutionarily ancient and involve protein orthologs conserved from yeast to humans. To identify new regulators of cisplatin-resistance in human tumors, we integrated high throughput and curated datasets describing yeast genes that regulate sensitivity to cisplatin and/or ionizing radiation. Next, we clustered highly validated genes based on chemogenomic profiling, and then mapped orthologs of these genes in expanded genomic networks for multiple metazoans, including humans. This approach identified an enriched candidate set of genes involved in the regulation of resistance to radiation and/or cisplatin in humans. Direct functional assessment of selected candidate genes using RNA interference confirmed their activity in influencing cisplatin resistance, degree of γH2AX focus formation and ATR phosphorylation, in ovarian and head and neck cancer cell lines, suggesting impaired DDR signaling as the driving mechanism. This work enlarges the set of genes that may contribute to chemotherapy resistance and provides a new contextual resource for interpreting next generation sequencing (NGS) genomic profiling of tumors.
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http://dx.doi.org/10.18632/oncotarget.13353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386675PMC
March 2017

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Oncotarget 2017 Mar;8(11):17628-17642

Cancer Biology, Fox Chase Cancer Center, Philadelphia, PA, USA.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.
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http://dx.doi.org/10.18632/oncotarget.12192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392274PMC
March 2017

RING domain-deficient BRCA1 promotes PARP inhibitor and platinum resistance.

J Clin Invest 2016 08 25;126(8):3145-57. Epub 2016 Jul 25.

Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain-deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.
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http://dx.doi.org/10.1172/JCI87033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966309PMC
August 2016

Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer.

Cell Rep 2016 07 7;16(3):657-71. Epub 2016 Jul 7.

Program in Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA; Program in Molecular and Cell Biology and Genetics, Drexel University College of Medicine, Philadelphia, PA 19129, USA. Electronic address:

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.
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http://dx.doi.org/10.1016/j.celrep.2016.06.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956518PMC
July 2016

POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies.

Gene 2016 Sep 16;590(1):128-41. Epub 2016 Jun 16.

Program in Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address:

The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5'-3' DNA polymerase and 3'-5' exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests that POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions.
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http://dx.doi.org/10.1016/j.gene.2016.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969162PMC
September 2016
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