Journal articles: 'Pathway FA/BRCA2'

1

Wang, XiaoZhe, Paul R. Andreassen, and Alan D. D'Andrea. "Functional Interaction of Monoubiquitinated FANCD2 and BRCA2/FANCD1 in Chromatin." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 5850–62. http://dx.doi.org/10.1128/mcb.24.13.5850-5862.2004.

Full text

Abstract:

ABSTRACT Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2−/− cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2−/− cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.

APA, Harvard, Vancouver, ISO, and other styles

2

Yarde, Danielle N., Lori A. Hazlehurst, Vasco A. Oliveira, Qing Chen, and William S. Dalton. "Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function." Blood 108, no. 11 (November 16, 2006): 840. http://dx.doi.org/10.1182/blood.v108.11.840.840.

Full text

Abstract:

Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.

APA, Harvard, Vancouver, ISO, and other styles

3

Niraj, Joshi, Anniina Färkkilä, and Alan D. D'Andrea. "The Fanconi Anemia Pathway in Cancer." Annual Review of Cancer Biology 3, no. 1 (March 4, 2019): 457–78. http://dx.doi.org/10.1146/annurev-cancerbio-030617-050422.

Full text

Abstract:

Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure (BMF), congenital defects, inability to repair DNA interstrand cross-links (ICLs), and cancer predisposition. FA presents two seemingly opposite characteristics: ( a) massive cell death of the hematopoietic stem and progenitor cell (HSPC) compartment due to extensive genomic instability, leading to BMF, and ( b) uncontrolled cell proliferation leading to FA-associated malignancies. The canonical function of the FA proteins is to collaborate with several other DNA repair proteins to eliminate clastogenic (chromosome-breaking) effects of DNA ICLs. Recent discoveries reveal that the FA pathway functions in a critical tumor-suppressor network to preserve genomic integrity by stabilizing replication forks, mitigating replication stress, and regulating cytokinesis. Homozygous germline mutations (biallelic) in 22 FANC genes cause FA, whereas heterozygous germline mutations in some of the FANC genes (monoallelic), such as BRCA1 and BRCA2, do not cause FA but significantly increase cancer susceptibility sporadically in the general population. In this review, we discuss our current understanding of the functions of the FA pathway in the maintenance of genomic stability, and we present an overview of the prevalence and clinical relevance of somatic mutations in FA genes.

APA, Harvard, Vancouver, ISO, and other styles

4

Giri, Veda N., Laura Gross, Jessica Russo, Ayako Shimada, Christopher McNair, William Kevin Kelly, and Leonard G. Gomella. "Prevalence of Fanconi anemia gene mutations among men undergoing multigene germline testing for prostate cancer: Interim results from the EMPOWeR study." Journal of Clinical Oncology 40, no. 6_suppl (February 20, 2022): 188. http://dx.doi.org/10.1200/jco.2022.40.6_suppl.188.

Full text

Abstract:

188 Background: Germline testing for prostate cancer (PCA) is now central to treatment, screening, and hereditary cancer management. The Fanconi anemia (FA) pathway is a key DNA repair pathway involved in PCA biology and treatment. Prevalence of FA genes BRCA2, PALB2, and BRIP1 is well-described; however, multiple other FA genes are not routinely tested, with limited prevalence data. Here we report mutation prevalence of a spectrum of FA genes among men undergoing PCA multigene testing on the Evaluation and Management for Prostate Oncology, Wellness, and Risk (EMPOWeR) study. Methods: Eligibility includes any male with PCA or at-risk for PCA. Multigene testing includes 51 genes; FA pathway genes include BRCA2, PALB2, BRIP1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, and FANCM. Multiple additional cancer risk genes were tested. Fisher’s exact tests were conducted to compare the prevalence of FA gene mutations between participants in the EMPOWeR study vs population prevalence reported in the literature. Statistical significance level of all tests was set a priori to 0.05. Results: The current cohort includes 235 participants. Characteristics are White (83.3%), Black (13.7%), PCA diagnosis (83.4%), mean age of PCA diagnosis 61.7 + 7.69 years, Gleason score >=7 (66.2%), and T3 or higher (29.4%). Genetic results were available for 179 participants. Overall, 11.1% of participants (n=20) had a pathogenic/likely pathogenic mutation identified. Among mutation carriers, 45% (n=9) had mutations in FA genes, including FANCA (n=3), BRCA2 (n=2), FANCM (n=1), FANCD2 (n=1), PALB2 (n=1), and BRIP1 (n=1). Table shows clinical characteristics of participants with mutations in FANCA, FANCM, and FANCD2. Further mutation spectrum included: CHEK2 (n=3), NBN (n=2), MUTYH (n=2), BRCA (n=1), ATM (n=1), HOXB13 (n=1), APC (n=1). Compared to population prevalence, FA mutation prevalence was significantly higher overall (5.0% vs. 0.6%, p = 0.010) and among mutation carriers (45% vs. 0.6%, p<0.001). Conclusions: While prevalence of FA genes BRCA2, PALB2, and BRIP1 is well-described, our study supports testing a broader range of FA genes given the prevalence rates, potential implications for clinical trials, targeted therapy, inherited syndromes, and reproductive implications.[Table: see text]

APA, Harvard, Vancouver, ISO, and other styles

5

Shain, Kenneth H., Vasco Oliveira, Danielle Yarde, Linda Mathews, and William S. Dalton. "A Novel Upregulation of Fanconi Anemia/BRCA DNA Repair Pathway Is Observed Upon Co-Culture of Bone Marrow Stroma with Multiple Myeloma Cell Lines: A Potential New Participant(s) in Environment Mediated Drug Resistance." Blood 114, no. 22 (November 20, 2009): 2798. http://dx.doi.org/10.1182/blood.v114.22.2798.2798.

Full text

Abstract:

Abstract Abstract 2798 Poster Board II-774 Multiple Myeloma (MM) is an incurable malignancy of mature clonal B cells. The refractory nature of MM has long been attributed to the acquisition of drug resistance. Traditionally, mechanisms of drug resistance have been defined by acquired changes in the expression or function of specific gene products. To this end, we have recently demonstrated that selected resistance to the cytotoxic agent melphalan correlated with increased expression of components of the Fanconi Anemia (FA)/BRCA DNA repair pathway and a concomitant increase in repair of DNA interstrand cross-links (ICLs).(Hazlehurst et al Cancer Res 2003; Chen et al Blood 2005) Further, the exogenous expression of specific FANC components in RPMI 8226 cell lines enhanced ICL repair, favored the release from melphalan-induced S-phase delay, and rendered these cells partially resistant to melphalan treatment. Together, these results suggest a causal relationship between increased expression of FA DNA repair components, increased DNA repair, and acquired resistance to melphalan. Over the past decade a large body of evidence has emerged demonstrating that in addition to drug resistance mechanisms intrinsic to the cancer cell, there exist dynamic, de novo mechanisms coordinated by the tumor microenvironment resulting in an environment-mediated drug resistance (EM-DR). As such, we examined the potential role of the microenvironment in regulating the FA/BRCA DNA repair pathway. FA pathway protein expression was evaluated with anti-sera to FANCD1/BRCA2, FANCC, FANCD2, FANCI, FANCG and BRCA1 in drug sensitive RPMI 8226 cells and melphalan resistant 8226/LR5 cells in co-culture with the HS-5 bone marrow stromal cell line. With these preliminary results we present three novel findings. First, we demonstrate that expression of FA/BRCA pathway components is regulated by intracellular interactions in both MM cells and bone marrow stromal cells (BMSCs). Second, we show that the acquisition of drug resistance alters FANC protein expression profiles upon co-culture. Third, in the HS-5 BMSCs, mono-ubiquitinated FANCD2 is observed in the absence of detectable FANCG. In RPMI 8226 cells, Western blot analysis demonstrated an acute (within 30minutes) and prolonged (up to 48hours) time-dependent increase in expression of FANCD2/BRCA2, FANCC, FANCD2, and BRCA1 upon incubation with BMSCs relative to MM cells incubated alone. However, no appreciated increases in FANCI or FANCG were noted under the same conditions. Incubation of 8226/LR5s with HS-5 BMSCs demonstrated a slightly different up-regulation of FA/BRCA pathway protein expression with addition of increased FANCI expression and no increase in FANCD2 or FANCC expression. We also examined FANC protein expression in the HS-5 cells. Interestingly, in the BMSCs significant differences were noted in the FANC expression profiles. Co-culture of RPMI 8226 cells with HS-5 cells demonstrated only modest elevations in FANCD2; however, co-culture with drug-resistant 8226/LR5s resulted in increased levels of FANCD2, FANCI and BRCA1. These data indicate that different tumor cells may alternately influence FA/BRCA-mediated DNA repair and potentially drug resistance in juxtaposed bone marrow stroma. Curiously, we also observed mono-ubiquitinated FANCD2 in the absence of any detectable levels of FANCG protein under co-culture conditions. As the FA/BRCA DNA repair pathway has been associated with cell cycle progression, we evaluated cell cycle kinetics under the co-culture conditions. The results of BrdU analysis demonstrated that the observed changes in FA/BRCA protein expression in MM and BMSC could not be fully explained by cell-cycle distribution. Therefore, within this report we demonstrate for the first time that microenvironmental interactions can modulate the FA/BRCA DNA repair pathway in MM and BMSCs. These results suggest that the FA/BRCA DNA damage repair pathway may be an important modulatory component of EM-DR. Importantly, the potential de novo drug resistance likely involves both the MM tumor cell and adjacent stromal cells. Current and future studies will attempt to examine a causal relationship between increased FANC expression and melphalan (and other drug) resistance seen in co-culture conditions, as well as to identify specific signaling molecules and mechanisms controlling the enhanced expression in both cell models. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

6

Sullivan, Katherine, Kimberly Cramer-Morales, Daniel L. McElroy, David Ostrov, Kimberly Haas, Margaret Nieborowska-Skorska, Wayne Childers, et al. "Identification of a Small Molecule Inhibitor of RAD52 to Induce Synthetic Lethality in BRCA-Deficient Leukemias." Blood 126, no. 23 (December 3, 2015): 4434. http://dx.doi.org/10.1182/blood.v126.23.4434.4434.

Full text

Abstract:

Abstract Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR. It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations. Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs. RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

7

Shain, Kenneth H., Liang Nong, Danielle Yarde, Vasco Oliveira, and William S. Dalton. "Selected Resistance to Topoisomerase II Inhibitors Correlates with the Over Expression of FANCF In a Fanconi Anemia/BRCA DNA Repair Pathway Independent Manner." Blood 116, no. 21 (November 19, 2010): 3372. http://dx.doi.org/10.1182/blood.v116.21.3372.3372.

Full text

Abstract:

Abstract Abstract 3372 Enhanced expression of the Fanconi Anemia (FA)/BRCA DNA repair pathway correlates with melphalan-resistance in multiple myeloma (MM) cell lines. Continued investigation demonstrated a bortezomib sensitive RelB/p50-mediated regulation of the FA/BRCA pathway contributed to the observed melphalan resistance.(Yarde et al 2009) The FA/BRCA pathway represents a co-dependent DNA damage response pathway involving thirteen loss of function complementation groups cloned from FA patients. The key functional event of this pathway is the interdependent mono-ubiquitination (Ub) of FANCD2 and FANCI (ID complex) by the E3 Ub-ligase activity of the FA core complex a multimer consisting of 8 FA (FANCA, B, C, E, F, G, L and M) and three non-FA proteins (FAAP100, FAAP24 and HES1). Formation of the core complex and mono-Ub of the ID complex appears to revolve around the flexible adapter protein FANCF. Nuclear localization of the core complex components requires binding of FANCA/G and FANCC/E subcomplexes to the C- terminal domain (CTD) and NTD domains of FANCF, respectively. This complex associates with FANCM:FAAP24 at sites of interstand crosslinks (ICL) via the FANCM-binding domain of FANCF, culminating in ID complex mono-Ub, recruitment of BRCA1, BRCA2/FANCD1, FANCJ and FANCN, and homologous recombination (HR) repair. Reduced function of this pathway has been associated with increased genomic instability, cancer susceptibility, and increased sensitivity to DNA cross-linking agents in FA. However, as predicted by the role of the FA/BRCA pathway in DNA repair, enhanced expression of the FA/BRCA pathway has been shown to play an important role in resistance to agents requiring HR for ICL repair. We next examined expression of this pathway in models of resistance to DNA damaging agents not predicted to utilize FA/BRCA activity. We screened 8226/Dox40 doxorubicin resistant and 8226/MR20 mitoxantrone resistant MM cell lines for expression of the 12 FA/BRCA pathway members with quantitative PCR (qPCR) using customized micro-fluidic cards. Interestingly, in these models of topoisomerase (topo) II inhibitor resistance qPCR demonstrated a 2.6 (p<0.05) and 1.7 (p<0.05) fold over expression of FANCF mRNA relative to drug sensitive RPMI8226 cells. Importantly, mRNA expression of other the eleven FA/BRCA pathway constituents was not increased relative to sensitive cells. To further characterize the relationship between FANCF and doxorubicin resistance, we examined mRNA and protein expression of FANCF in RMPI8226, 8226/Dox6 and 8226/Dox40 MM cell lines (representing progressive levels of doxorubicin resistance). FANCF qPCR demonstrated a 2 and 4.7 fold increased in mRNA expression in the 8226/Dox6 and 8226/Dox40 cell lines, respectively (p= 0.103 and p= 0.034) suggesting that increasing expression of FANCF correlated with increasing dox resistance. A similar doxorubicin resistance- dependent increase in FANCF protein was demonstrated by Western blot analysis of these cell lines. Consistent with mRNA results, FANCD2 or FANCG protein levels remained unchanged in the doxorubicin resistant versus sensitive cell lines These observations suggest that FANCF may contribute to topoII inhibitor-mediated DNA double strand break repair, a process that primarily thought to involve non-homologous end joining (NHEJ) independent of the FA/BRCA pathway. To determine if FANCF expression alone could facilitate doxorubicin resistance, pQCXIP-control or pQCXIP-FANCF constructs were expressed in RPMI8226 sensitive MM cells. MTT assays demonstrated a greater than 2 fold resistance to doxorubicin in FANCF over expressing cells at 48 and 96 hours (IC50: 1.33 ×10−6 and 5.3×10−9M) as compared to control cells (3.26×10−6 and 1.13×10−8M). Taken together, these results indicate that the flexible adaptor protein FANCF may participate in doxorubicin resistance independently of other FA/BRCA members. However, future studies will be needed to elucidate the nature of FANCF in doxorubicin resistance. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

8

Maia, Nuno, Maria João Nabais Sá, Cláudia Oliveira, Flávia Santos, Célia Azevedo Soares, Catarina Prior, Nataliya Tkachenko, et al. "Can the Synergic Contribution of Multigenic Variants Explain the Clinical and Cellular Phenotypes of a Neurodevelopmental Disorder?" Genes 13, no. 1 (December 28, 2021): 78. http://dx.doi.org/10.3390/genes13010078.

Full text

Abstract:

We describe an infant female with a syndromic neurodevelopmental clinical phenotype and increased chromosome instability as cellular phenotype. Genotype characterization revealed heterozygous variants in genes directly or indirectly linked to DNA repair: a de novo X-linked HDAC8 pathogenic variant, a paternally inherited FANCG pathogenic variant and a maternally inherited BRCA2 variant of uncertain significance. The full spectrum of the phenotype cannot be explained by any of the heterozygous variants on their own; thus, a synergic contribution is proposed. Complementation studies showed that the FANCG gene from the Fanconi Anaemia/BRCA (FA/BRCA) DNA repair pathway was impaired, indicating that the variant in FANCG contributes to the cellular phenotype. The patient’s chromosome instability represents the first report where heterozygous variant(s) in the FA/BRCA pathway are implicated in the cellular phenotype. We propose that a multigenic contribution of heterozygous variants in HDAC8 and the FA/BRCA pathway might have a role in the phenotype of this neurodevelopmental disorder. The importance of these findings may have repercussion in the clinical management of other cases with a similar synergic contribution of heterozygous variants, allowing the establishment of new genotype–phenotype correlations and motivating the biochemical study of the underlying mechanisms.

APA, Harvard, Vancouver, ISO, and other styles

9

Stone, Stacie, Alexandra Sobeck, Igor Landais, Alexis LaChapelle, and Maureen E. Hoatlin. "A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway." Blood 108, no. 11 (November 16, 2006): 4351. http://dx.doi.org/10.1182/blood.v108.11.4351.4351.

Full text

Abstract:

Abstract Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds. We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.

APA, Harvard, Vancouver, ISO, and other styles

10

Kolinjivadi, Arun Mouli, Haresh Sankar, Ramveer Choudhary, Lavina Sierra Tay, Tuan Zea Tan, Naoko Murata-Kamiya, Dominic Chih-Cheng Voon, et al. "The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse." International Journal of Molecular Sciences 23, no. 3 (January 31, 2022): 1661. http://dx.doi.org/10.3390/ijms23031661.

Full text

Abstract:

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

APA, Harvard, Vancouver, ISO, and other styles

11

Palmieri, G., M. Colombino, M. G. Camboni, A. Manca, P. Baldinu, F. Izzo, F. Tatangelo, R. Calemma, A. Cossu, and F. Galimi. "Assessment of the role of Fanconi Anemia (FA) genes in colorectal cancer: A new pathogenetic pathway?" Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 3629. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.3629.

Full text

Abstract:

3629 Background: Fanconi Anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight FA associated genes have been cloned, and their products are thought to function in an integrated pathway which includes BRCA genes and maintains genomic stability. A molecular mechanism involved in the development and progression of some human malignancies, including colorectal cancer, is represented by a defective replication fidelity. Here we tested the role of the main FA genes in colorectal cancer (CRC). Methods: One hundred consecutively-collected patients with histologically-proven diagnosis of CRC and no mutation in the main mismatch repair genes (MLH1 and MSH2) were included into the study. Genomics DNA was screened using DHPLC for FANC-C/G/E/F genes and using MPLA for FANC-A/D2 genes. All PCR products with abnormal DHPLC profiles were sequenced on an automated sequencer. For 28 cases, tumor tissue samples were also analyzed. To date, total RNA was isolated from paired normal and tumor samples of 12 CRC patients. Gene expression was quantified by real-time RT-PCR using the Taqman approach. Results: At germline level, 2 cases presented FANC-C sequence variations (Asp267Ser, Val449Ser) and 5 cases showed a exon 26 deletion in FANC-A as detected by MLPA analysis. Among the 28 tumor samples available from patients negative for mutations in germline DNA, 2 (7%) cases presented a FANC-E mutation (Ala552Thr). Moreover, one of such somatic samples presented a BRCA2–8765delAG mutation (BRCA2 is also recognized as FANC-D1 gene). Assuming as significant difference in gene expression the presence of a variation of more than 50% in mRNA levels between normal and corresponding tumor samples, all tested FA genes (FANC-C/G/E/F) were found overexpressed in 5/12 (42%) tumor samples. Conclusions: Although preliminary, our findings seem to identify a subset of CRC patients with alterations in the FA/BRCA pathway which is required for the activation of DNA repair. Ongoing molecular analyses will better clarify the real frequency of each alteration in FA genes and their relationship with the histological and/or clinical features in colorectal cancer. No significant financial relationships to disclose.

APA, Harvard, Vancouver, ISO, and other styles

12

Alter, Blanche P., Philip S. Rosenberg, and Lawrence C. Brody. "Biallelic Mutations in FANCD1/BRCA2 Are Associated with Extraordinary Risks of Cancer." Blood 108, no. 11 (November 16, 2006): 4326. http://dx.doi.org/10.1182/blood.v108.11.4326.4326.

Full text

Abstract:

Abstract Patients with biallelic mutations in BRCA2 comprise Fanconi Anemia (FA) complementation group FA-D1. We analyzed the severity of the BRCA2 mutations in 26 literature cases and one from our National Cancer Institute Cohort. The functional consequence of each mutant allele was inferred from the Breast Cancer Information Core (BIC) database. Knowledge of gene structure was used to evaluate whether the reported mutations led to inactivation of the BRCA2 gene or diminution of the gene product. We examined genotype/phenotype/cancer correlations between specific BRCA2 mutations and specific malignancies in FA families. We also mapped the location of the FA-related BRCA2 mutations. Five of twenty-seven patients had birth defects consistent with VATER association, a 71,000-fold increase compared with 2.6/106 in the general population, and 5% of unclassified FA patients. Ten patients had acute myeloid leukemia (AML), four acute lymphocytic leukemia (ALL, one had AML as well), twelve brain tumors, seven Wilms tumor, and one neuroblastoma; six patients had ≥2 cancers, and only two patients had no cancer. IVS7+1G>A and IVS7+2T>G were associated with AML (odds ratio 15 compared to those with other mutations in BRCA2, p=0.02), and 886delGT and 6174delT with brain tumors (odds ratios infinite compared to those with other BRCA2 mutations, p=0.006 and 0.03 respectively). Compared with non-FA-D1 patients, the relative risk of any malignancy in FA-D1 was 66 (25/27 patients with 15 solid tumors and 13 leukemias; 3 patient had solid tumors and leukemia); the cumulative probability of any cancer was 97% by age 5. Compared with the age- and sex-adjusted general population, AML was increased 7000-fold, and any malignancy 3300-fold. While half of the families had relatives with BRCA2-type cancers, several of the alleles reported in probands were not associated with cancer in presumed carriers. Two of the reported FA-related mutant BRCA2 alleles were benign polymorphisms (one patient was FA-B with a concurrent mutation and polymorphism in BRCA2), five were missense variants of unknown clinical significance, three involved splice sites, and 20 were frameshift or truncating mutations. Missense mutations formed a distinct cluster between residues 2236 and 2729 of the BRCA2 protein (Pearson statistic p=0.01, range test p<0.001), while the truncation and frameshift mutations did not cluster. This small group of patients with biallelic mutations in BRCA2 is unique in the severity of the physical phenotype, and in the very early onset and inordinately high rates of leukemia and specific solid tumors. In a subset of patients, a single deleterious mutation coupled with a missense mutation seemed sufficient for cancer to occur. Our observations suggest that FA patients with mutations in BRCA2 are distinctly more severely affected than those in other complementation groups. Appropriate counseling strategies need to be developed for family members who are heterozygous for these mutations in BRCA2. BRCA2 regions which are not implicated in cancer in heterozygotes may nonetheless be critical interaction regions for the FA pathway downstream of FANCD2 ubiquitination.

APA, Harvard, Vancouver, ISO, and other styles

13

Hirsch, Betsy, Akiko Shimamura, Lisa Moreau, Shari Baldinger, Maha Hag-alshiekh, Bruce Bostrom, Susan Sencer, and Alan D. D'Andrea. "Association of biallelic BRCA2/FANCD1 mutations with spontaneous chromosomal instability and solid tumors of childhood." Blood 103, no. 7 (April 1, 2004): 2554–59. http://dx.doi.org/10.1182/blood-2003-06-1970.

Full text

Abstract:

Abstract The clinical, cytogenetic, and molecular findings of 2 Fanconi anemia (FA) subtype D1 kindreds, initially identified through a young child with a solid tumor (medullobastoma, Wilms tumor), are described. Each kindred subsequently had a second affected child; one developed Wilms tumor followed by a medulloblastoma, and the other developed T-lineage acute lymphoblastic leukemia. Cytogenetic studies revealed an unusually high spontaneous chromosome aberration rate, contrasting with other FA subtypes. Molecular analysis revealed biallelic BRCA2/FANCD1 mutations. The patients did not exhibit bone marrow failure. Our studies suggest that the D1 subtype represents a severe end of the cytogenetic spectrum within FA, consistent with a critical downstream role of BRCA2 in the FA pathway. Furthermore, this FA subgroup may be preferentially associated with an increased predisposition to solid tumors in early childhood. Recognition of this constellation of findings has significant implications for medical management and genetic counseling of FA families.

APA, Harvard, Vancouver, ISO, and other styles

14

Spardy, Nicole, Anette Duensing, Domonique Charles, Nathan Haines, Tomomi Nakahara, Paul F. Lambert, and Stefan Duensing. "The Human Papillomavirus Type 16 E7 Oncoprotein Activates the Fanconi Anemia (FA) Pathway and Causes Accelerated Chromosomal Instability in FA Cells." Journal of Virology 81, no. 23 (September 26, 2007): 13265–70. http://dx.doi.org/10.1128/jvi.01121-07.

Full text

Abstract:

ABSTRACT Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.

APA, Harvard, Vancouver, ISO, and other styles

15

Levitus, Marieke, Hans Joenje, and Johan P. de Winter. "The Fanconi Anemia Pathway of Genomic Maintenance." Analytical Cellular Pathology 28, no. 1-2 (January 1, 2006): 3–29. http://dx.doi.org/10.1155/2006/974975.

Full text

Abstract:

Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked inheritance, features diverse clinical symptoms, such as progressive bone marrow failure, hypersensitivity to DNA cross-linking agents, chromosomal instability and susceptibility to cancer. At least 12 genetic subtypes have been described (FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked to a distinct gene. Most FA proteins form a complex that activates the FANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream of this step. The FA proteins typically lack functional domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking agents, the FA proteins are thought to function in the repair of DNA interstrand cross-links, which block the progression of DNA replication forks. Here we present a hypothetical model, which not only describes the assembly of the FA pathway, but also positions this pathway in the broader context of DNA cross-link repair. Finally, the possible role for the FA pathway, in particular FANCF and FANCB, in the origin of sporadic cancer is discussed.

APA, Harvard, Vancouver, ISO, and other styles

16

Majidi, Fatemeh, Judith Neukirchen, Ulrich Germing, Ron-Patrick Cadeddu, Stefan Braunstein, Rainer Haas, and Norbert Gattermann. "Concordant Underexpression of Fanconi Anemia/BRCA1 Pathway Proteins in a Major Proportion of Myelodysplastic Syndromes." Blood 128, no. 22 (December 2, 2016): 4311. http://dx.doi.org/10.1182/blood.v128.22.4311.4311.

Full text

Abstract:

Abstract Introduction: BRCA1 plays a key role in maintaining genomic stability and interacts directly with several proteins that regulate hematopoietic stem cell functions and are part of the Fanconi anemia (FA) double-strand break (DSB) repair pathway. Loss of BRCA1 in murine bone marrow causes hematopoietic defects similar to those seen in FA (Vasanthakumar, Blood 2016). BRCA1 is highly expressed in hematopoietic tissues, whereas its expression is lost in CML (Deutsch et al, Blood 2003) and t-AML (Scardocci et al, Br J Cancer 2006). Since FA is associated with an increased risk of developing myelodysplastic syndromes (MDS), we examined the protein expression of BRCA1 and three other important components of the FA DNA repair pathway, as well as PARP-1, whose co-alteration with BRCA causes synthetic lethality. Methods: We recently established a tissue microarray (TMA) with bone marrow biopsies from 119 MDS patients, 40 AML patients, and 11 normal controls. Biopsy material was retrieved from the Institute of Pathology, Heinrich-Heine-University Düsseldorf, Germany. The TMA was produced at SciLifeLab Tissue Profiling Facility in Uppsala, Sweden. Immunohistochemistry (IHC) protocols were established in our lab for the detection of BRCA1, BRCA2, FANCD2, H2AX, and PARP1. Semi-quantitative analysis was done according to Remmele-Stegner immunoreactive score (IRS) with a point system from 0 to 12. Expression was categorized as absent (0-2), weak (3-4), moderate (5-8) or strong (9-12). For correlating protein expression with clinical data from the Düsseldorf MDS Registry, samples were dichotomized into 'low expression' (1-4) and 'higher expression' (5-12). Prognostic analysis was restricted to 80 MDS patients who did not receive disease-modifying therapy. Results: Similar to normal controls, most patients with AML showed strong expression of the proteins under consideration (Fig. 1). In contrast, low expression was found in about 60% of MDS patients. Most MDS patients showed concordant, dichotomous expression of all five proteins (either all up or all down). Correspondingly, the protein expression landscapes look similar (Fig. 1). Almost perfect concordance was noted for PARP1 and H2AX. Low expression of FA/BRCA pathway proteins was not correlated with MDS type (WHO 2008) or IPSS-R risk group. Patients with higher expression of at least 3 of the 5 proteins survived longer (45 vs. 26 months) and had a longer time to AML development (median not reached at 180 months vs. 53 months), but the differences did not reach statistical significance (p=0.108 and p=0.159, respectively). Interestingly, patients with low expression of PARP1 were significantly more likely to show any chromosomal aberration (p=0.02) or an unfavourable karyotype (intermediate, high-risk or very high risk according to IPSS-R) (p=0.016). The same was true for patients with low expression of H2AX (p=0.013 and p=0.01). Conclusions: This is the first TMA-based investigation of FA/BRCA pathway protein expression in MDS. In stark contrast to AML, 60% of MDS patients showed low protein expression in a concordant manner. This could reflect synexpression of genes that share common cis- and trans-acting control elements. Alternatively, it could be the result of aberrant splicing, since at least 50% of MDS patients have spliceosome mutations that render a large spectrum of messenger RNAs susceptible to nonsense-mediated decay. Abnormal splicing factors may also influence the stability (and thus expression) of certain proteins through abnormal protein-protein interactions. Further IHC analyses will show whether the dichotomous protein synexpression pattern we observed in MDS extends to proteins that are unrelated to DNA maintenance. Underexpression of FA/BRCA pathway proteins may cause chromosomal instability, as suggested by our finding of significantly more frequent karyotype anomalies in patients with low PARP1 or H2AX expression. This is in accordance with the known role of H2AX as suppressor of oncogenic chromosome translocations, and with an accelerating effect of PARP1 deficiency on centrosome amplification in BRCA1-deficient cells. FA/BRCA pathway protein underexpression may not only contribute to a better understanding of MDS versus AML pathogenesis, but may also have therapeutic implications, e.g. by affecting the response to treatment with hypomethylating agents, which are known to promote DNA double strand breaks. Figure 1 Figure 1. Disclosures Gattermann: Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

17

Oda, Tsukasa, Hidenobu Miyaso, and Takayuki Yamashita. "Nuclear Localization of Fanconi Anemia Protein FANCA Is Regulated by Hsc70/Hsp90 Chaperone Machinery." Blood 104, no. 11 (November 16, 2004): 2835. http://dx.doi.org/10.1182/blood.v104.11.2835.2835.

Full text

Abstract:

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, cancer susceptibility and cellular hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). Current evidence indicates that formation of a nuclear multiprotein complex (core complex) including six FA proteins FANCA/C/E/F/G/L is essential for FANCL/PHF9 ubiquitin ligase-mediated activation of FANCD2 into a monoubiquinated form, which participates in BRCA1 and FANCD1/BRCA2-mediated DNA repair (the FA/BRCA pathway). Subcellular distribution of FANCA plays a crucial role in the regulation of the FA/BRCA pathway. However, the underlying molecular mechanisms are not fully understood. To address this issue, we tried to identify FANCA-associated proteins. To this end, Flag-FANCA ectopically expressed in HeLa cells was immunopurified from the cytoplasmic fraction, using anti-Flag antibody-conjugated sepharose beads. Analysis of the immune complex on SDS polyacrylamide gel electrophoresis revealed that several proteins of Mr. 60–70 kD specifically associated with Flag-FANCA. These proteins were identified as FANCG and Hsc (heat shock cognate protein) 70 by LC-MS/MS. Immunoblot analysis showed that FANCA associated with Hsp90 as well as Hsc70. Hsc70 is an ATP-dependent molecular chaperone highly homologous to Hsp70 and often cooperates with Hsp90 to form a chaperone machinery involved in the regulation of diverse protein functions. Patient-derived FANCA mutants failed to bind FANCC but associated with larger amounts of Hsc70 than wt-FANCA, indicating that the interaction between FANCA and Hsc70 is not mediated by FANCC, as suggested by previous observations of the interaction of FANCC with Hsp70. To study the role of Hsc70 and Hsp90 in the regulation of FANCA, we examined effects of a dominant-negative (dn) form of Hsc70 with inactivated ATPase activity, and a specific inhibitor of Hsp90, 17-AAG (a geldanamycin analog). Overexpression of dn-Hsc70 inhibited nuclear localization of FANCA and inhibited its core complex formation, whereas wt-Hsc70 did not. 17-AAG induced cytoplasmic distribution and proteosomal degradation of FANCA and suppressed FANCD2 mono-ubiquitination. Taken together, these results suggest that Hsc70/Hsp90 chaperone machinery interacts with FANCA and regulates its subcellular distribution and stability, thereby controlling activation of the FA/BRCA pathway.

APA, Harvard, Vancouver, ISO, and other styles

18

Kupfer, Gary. "Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability." Blood 126, no. 23 (December 3, 2015): 1216. http://dx.doi.org/10.1182/blood.v126.23.1216.1216.

Full text

Abstract:

Background The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored. In this study we explore the incidence of R loops in FA mutant cells. Methods DRIP: duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody. Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide. Bisulfite modification/DNA Sequencing: genomic DNA was extracted and a portion was denatured. After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced. Similarly prepared genomic DNA was prepared, exomic sequencing was performed. Bioinformatic analysis was undertaken on each sample. Immunofluorescence microscopy: cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained. Cell culture: mutant and complemented versions of each Results Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells. Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J. Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH. Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells. BRCA2 mutant cells also demonstrated increases in R loops. Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells. Conclusions RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells. Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability. Translational potential statement The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules. Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

19

Reckamp, Karen L., Thomas Paul Slavin, Stacy W. Gray, Carolyn E. Behrendt, Danielle Castillo, Marianna Koczywas, Mihaela C. Cristea, et al. "Germline mutations and onset of lung adenocarcinoma in smokers and nonsmokers." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 1518. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.1518.

Full text

Abstract:

1518 Background: Eligibility for lung cancer screening is based largely on pack-years of smoking, missing many cases. To propose additional groups for screening, this observational study evaluated whether germline mutations associated with cancer risk accelerate onset of lung adenocarcinoma (LA) in ever- and never-smokers. Methods: Patients with LA and family history of cancer were recruited from our oncology clinic and the Clinical Cancer Genomics Community Research Network. With consent, blood samples were screened by large multi-gene panel for 4 categories of germline mutation [lung cancer-associated genes ( TP53, EGFR); BRCA2; other genes in Fanconi anemia (FA) pathway; other DNA repair genes]. Accelerated failure-time models of age at LA diagnosis, adjusted for sex, ethnicity, and packs per day, were constructed for never-smokers and ever-smokers. Statistical significance, at p<0.05 limited the False Discovery Rate to 5% across 8 hypotheses. Results: In never-smokers with LA (n=104), mutated BRCA2, TP53 or EGFR were associated with younger age at diagnosis, while mutation in other FA or DNA repair gene was not. In ever-smokers with LA (n=65), mutated BRCA2 and other FA gene were associated with younger age at diagnosis, while other mutation categories were not (Table). Conclusions: Regardless of smoking history, BRCA2 mutation carriers experience accelerated onset of LA, as do never-smokers carrying TP53 or EGFR mutation and ever-smokers with mutation in FA gene other than BRCA2. With the exception of TP53 carriers (who merit whole body MRI), lung cancer screening with low-dose computed tomography, starting earlier in adulthood than usual, may be warranted for individuals with germline mutations in these genes. Age at Diagnosis of Lung Adenocarcinoma, by Germline Mutation and Smoking History, Adjusted for Sex, Ethnicity, and Packs per Day. [Table: see text]

APA, Harvard, Vancouver, ISO, and other styles

20

Lachaud, Christophe, and John Rouse. "A route to new cancer therapies: the FA pathway is essential in BRCA1- or BRCA2-deficient cells." Nature Structural & Molecular Biology 23, no. 8 (August 2016): 701–3. http://dx.doi.org/10.1038/nsmb.3276.

Full text

APA, Harvard, Vancouver, ISO, and other styles

21

Sobeck, Alexandra, Stacie Stone, Bendert deGraaf, Vincenzo Costanzo, Johan deWinter, Weidong Wang, Hans Joenje, Jean Gautier, and Maureen E. Hoatlin. "Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition." Blood 104, no. 11 (November 16, 2004): 723. http://dx.doi.org/10.1182/blood.v104.11.723.723.

Full text

Abstract:

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

APA, Harvard, Vancouver, ISO, and other styles

22

Webster, Andrew L., Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, et al. "Abstract 6196: Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6196. http://dx.doi.org/10.1158/1538-7445.am2022-6196.

Full text

Abstract:

Abstract Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway comprises at least 22 FANC proteins including BRCA1 and BRCA2 and protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs) with a median onset age of 31 years. The aggressive nature of these tumors and poor patient tolerance of platinum and radiation-based therapy have been associated with short survival in FA. Molecular studies of SCCs from individuals with FA (FA SCCs) have been limited, and it is unclear how they relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of a high number of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions that arise in the context of TP53 loss. The SV breakpoints preferentially localize to early replicating regions, common fragile sites, tandem repeats, and SINE elements. SVs are often connected forming complex rearrangements. Resultant genomic instability underlies elevated copy number alteration (CNA) rates of key HNSCC-associated genes, including PIK3CA, MYC, CSMD1, PTPRD, YAP1, MXD4, and EGFR. In contrast to sporadic HNSCC, we find no evidence of HPV infection in FA HNSCC, although positive cases were identified in gynecologic tumors. A murine allograft model of FA pathway-deficient SCC was enriched in SVs, exhibited dramatic tumor growth advantage, more rapid epithelial-to-mesenchymal transition, and enhanced autonomous inflammatory signaling when compared to an FA pathway-proficient model. In light of the protective role of the FA pathway against SV formation uncovered here, and recent findings of FA pathway insufficiency in the setting of increased formaldehyde load resulting in hematopoietic stem cell failure and carcinogenesis, we propose that high copy-number instability in sporadic HNSCC may result from functional overload of the FA pathway by endogenous and exogenous DNA crosslinking agents. Our work lays the foundation for improved FA patient treatment and demonstrates that FA SCC is a powerful model to study tumorigenesis resulting from DNA crosslinking damage. Citation Format: Andrew L. Webster, Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, Audrey M. Goldfarb, Kevin Hadi, Matthew M. Edwards, Frank X. Donovan, Moonjung Jung, Sunandini Sridhar, Olivier Fedrigo, Huasong Tian, Joel Rosiene, Thomas Heineman, Jennifer Kennedy, Lorenzo Bean, Rasim O. Rosti, Rebecca Tryon, Ashlyn-Maree Gonzalez, Allana Rosenberg, Ji-Dung Luo, Thomas Carrol, Eunike Velleuer, Jeff C. Rastatter, Susanne I. Wells, Jordi Surrallés, Grover Bagby, Margaret L. MacMillan, John E. Wagner, Maria Cancio, Farid Boulad, Theresa Scognamiglio, Roger Vaughan, Amnon Koren, Marcin Imielinski, Settara Chandrasekharappa, Arleen D. Auerbach, Bhuvanesh Singh, David Kutler, Peter J. Campbell, Agata Smogorzewska. Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6196.

APA, Harvard, Vancouver, ISO, and other styles

23

Nong, Liang, Linda Mathews, Mark B. Meads, William Dalton, and Kenneth H. Shain. "Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines." Blood 118, no. 21 (November 18, 2011): 2922. http://dx.doi.org/10.1182/blood.v118.21.2922.2922.

Full text

Abstract:

Abstract Abstract 2922 The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

24

Sater, Zahi Abdul, Richa Sharma, Elizabeth Sierra Potchanant, Ying He, and Grzegorz Nalepa. "FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1K250 Acetylation at the Kinetochores." Blood 128, no. 22 (December 2, 2016): 1040. http://dx.doi.org/10.1182/blood.v128.22.1040.1040.

Full text

Abstract:

Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood. Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2. Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

25

Parmar, Kalindi, Patrizia Vinciguerra, Susana Godinho, Abigail Hamilton, David Pellman, and Alan D. D'Andrea. "Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells." Blood 116, no. 21 (November 19, 2010): 878. http://dx.doi.org/10.1182/blood.v116.21.878.878.

Full text

Abstract:

Abstract Abstract 878 Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

26

Li, Wenliang, Zhu Zhu, Ning Xu, Wei Huang, Junping Shi, Jiaochun Shi, Weifeng Wang, Samuel J. Klempner, Juan Zhao, and Jialiang Gan. "Alterations of DNA damage repair genes in Chinese colorectal cancer patients." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e16121-e16121. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e16121.

Full text

Abstract:

e16121 Background: Colorectal cancer (CRC) is the second most common cancer in women and third in men. DNA damage repair (DDR) deficiency has emerged as a predictive biomarker for chemotherapy, PARP and immune checkpoint inhibitors. However, comprehensive molecular characteristics of DDR variants in Chinese CRC patients is lacking. Methods: Formalin fixed, paraffin embedded (FFPE) tumor tissues and matched blood samples were collected for targeted next-generation sequencing (NGS) assay. The testing was carried out in a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Alterations of six functional gene sets involved in DDR pathways: homologous recombination (HR), mismatch repair (MMR), base excision repair (BER), nonhomologous end-joining (NHEJ), checkpoint factors (CPF) and Fanconi anemia (FA) were analyzed. The association of DDR gene mutations with TMB/MSI was assessed. Results: In total, 319 CRC patients were recruited, 127 female and 192 male with a median age of 59 (range 23-92). Over one third (36%,115/319) patients had at least one mutation in DDR genes. Mutation rates varied in different DDR pathway: HR (20.7%), CPF (15.1%), FA (11.3%), MMR (10.3%), BER (8.8%), and NHEJ (5.3%). The most frequently mutated DDR genes were ARID1A (13.4%), ATM (10.2%), BRCA2 (6.8%), MLH1 (5.6%), MSH6 (5.6%), POLE (5.0%). Germline mutations in MLH1 (2.5%), BRCA2 (1.2%), MSH2 (0.9%), MSH6 (0.9%), FANCA (0.9%), ATM (0.6%), BRCA1 (0.3%), and CHEK2 (0.3%) were detected in 24 patients. DDR variations were enriched in the right-side CRC compared to the left side CRC (50% vs. 32.8%, p= 0.024). Early stage (I-II) harbored more DDR variations. 20.1% of patients had high TMB (≥10 muts/Mb) with a median of 51 muts/Mb (10-326.7 mus/Mb). Patients with DDR mutations had a significantly higher TMB than patients with wild type DDR (8.5 vs. 4.6 muts/Mb, p< 0.001). All CRC tumors with high MSI harbored DDR mutations. Importantly, the mutations in “HM” (HR/MMR), but not BER/CPF/NHEJ/FA mutations, were significantly correlated with high MSI ( p< 0.001). Conclusions: DDR gene alterations occurred in 36% of Chinese CRC patients and were enriched in right sided tumors. DDR pathway alterations are relatively frequent in CRC and consideration for biomarker-enriched clinical trials with PARP, immune checkpoint inhibitors, and novel combinations are warranted.

APA, Harvard, Vancouver, ISO, and other styles

27

Huang, Tingting, Bonnie W. Lau, and Shiyu Wang. "Increased PD-L1 Expression in Acute Myeloid Leukemia with Fanconi Anemia/BRCA Mutations." Blood 136, Supplement 1 (November 5, 2020): 32–33. http://dx.doi.org/10.1182/blood-2020-142949.

Full text

Abstract:

Introduction: Loss of DNA repair fidelity is a common feature of tumors and can drive genomic instability, tumor evolution, and therapy response. 1 Fanconi anemia (FA) is a DNA damage response (DDR) pathway and biallelic germline mutations in the pathway lead to a bone marrow failure syndrome with a predisposition for cancer, especially acute myeloid leukemia (AML). Unfortunately, FA-AML is a secondary AML with very poor prognosis, with an increased risk for relapse even after initial cure with bone marrow transplantation. Therefore FA patients need further options for therapy. DNA damage response (DDR) deficiency has emerged as a predictive biomarker of response to immune checkpoint inhibition. 2 In some solid tumors, defective DDR leads to genomic instability, increased mutational burden, increased neo antigen expression and increased PD-L1 expression; and respond robustly to PD-1 checkpoint inhibitor therapy. The mutational burden and PD-L1 expression in FA-mutated tumors remain to be discovered. Therefore, we hypothesize that FA mutations in AML increase mutational burden and PD-L1 expression. This work can set the stage for future studies involving PD-1 blocking therapies for FA-mutated tumors. Methods/Results: To determine the mutational burden in FA-mutated tumors, we didwhole exome sequencing (WES)of three AML cell lines obtained from pediatric FA patients, all were FANCD1/BRCA2 mutated.3,4 The three AML cell lines also represent a progression of AML disease- an early clone, a late clone and a relapsed AML after bone marrow transplantation. There was an average of 180,000 somatic mutations/case, pared down to 1,000 mutations after limiting calls with >10 reads and allele frequency >0.1%. Mutational burden of pediatric AML in non-FA patients has been reported to be ~10 mutations/case.5Therefore there is about a 100-fold increase in mutations in AML from FA patients compared to AML from non-FA patients.Then we determined PD-L1 mRNA and protein expression of the three FANCD1/BRCA2 mutated AML cells with quantitative PCR and flow cytometry respectively. We found increasing PD-L1 mRNA expression from early to late to relapsed clones (2^-ΔCt values 1.21x10-5, 1.54x10-5, and 7.02x10-5 respectively; p-value<0.05). Interestingly, interferon-gamma (IFN-g), a known inducer and regulator of PD-L1 expression, is detected at high levels in the bone marrow of FA patients.5 We find that PD-L1 expression significantly increases in FANCD1/BRCA2 mutated AML cells after treatment with physiologic doses of IFN-g (fold change in 2^-ΔΔCt before and after treatment with 5ng IFN-g for early clone (x16.55 fold change) late clone (x38.46 fold change) and relapsed clone (x17.57 fold change), p-value<0.05. We also found stable PD-L1 protein expression on the cell surface (% PD-L1-positive cells for early clone (15.2%), late clone (17.7%), and relapsed AML (18.9%). Conclusions: Our data show there is a high mutational load in FA-AML cell lines, which also express PD-L1. Both of these tumor characteristics are predictors of response to PD-1 checkpoint inhibitor immunotherapy. This will lead to future work on PD-1 blockade as a potential therapeutic option for FA-AML. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

28

Nie, Daijing, Fang Wang, Jing Zhang, Xvxin Li, Lili Liu, Wei Zhang, Panxiang Cao, et al. "Fanconi Anemia Gene Associated Germline Predisposition in Aplastic Anemia and Hematologic Malignancies." Blood 136, Supplement 1 (November 5, 2020): 18–19. http://dx.doi.org/10.1182/blood-2020-142452.

Full text

Abstract:

Introduction Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome with a surged cancer incidence, especially in hematologic malignancies, which has been listed in the entity of myeloid neoplasms with germline predisposition in the 2016 revision to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.Whether FA heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is crucial but unsettled. We therefore retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 aplastic anemia (AA) and hematologic malignancies patients to address this issue. Methods Patients diagnosed as AA, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL) from April 2015 to December 2018 in Hebei Yanda Lu Daopei Hospital were enrolled with the ones diagnosed as FA via chromosome breakage test and/or genetic test excluded. Detailed disease histories and workup files were retrieved from the electronic medical record. Amplicon-based high throughput sequencing of aforementioned five genes were performed. The significance of the germline missense variants was assessed by in silico prediction algorithms, including SIFT, Polyphen2, PROVEAN, FATHMM, MutationTaster, and MutationAssessor. Variant predicted as deleterious/possibly deleterious by ≥ 3/6 scoring tools was defined as rare PSV and further included in statistical analysis. For splice site mutations, GeneSplicer, Human Splicing Finder, NetGene2, and FSPLICE were employed and only when ≥2/4 algorithms predict to affect/possibly affect splicing, would the variant be regarded as possibly significant. Same criteria were adopted when stratifying variants recorded in the ExAC. All reported variants in this study were confirmed germline variants by Sanger sequencing with fingernail specimens and/or pedigree analysis. WFisher exact two-tailed test was adopted for variant frequency comparison. Development of disease was analyzed by cumulative incidence method and Kaplan-Meier method. p < 0.05 was considered as statistically significant. Results A total of 788 patients, who were all of the East Asian ancestry, were enrolled, including 341 females and 447 males (Figure 1A). The median age of onset was 10 (1-63), 27 (1-65), 14.5 (1-65), and 6 (1-53) years in AA, MDS, AML, and ALL subgroup, respectively. Sixty-eight variants were identified in 66 patients (8.38%). FANCA is the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20) (Figure 1B). When compared to the ExAC East Asian dataset, there was an overall higher rare PSVs incidence in our cohort (p = 0.016). BRCA2 PSVs showed a higher frequency in ALL (p = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (p = 0.020; p = 0.008). The patients with FANCA heterozygotes also tended to show an increased risk for developing MDS (p = 0.075) (Table 1). No impact of FA-PSV status was found neither on cumulative disease incidence (Figure 1C). FA-PSV + MDS/AML patients have a heavier tumor mutation burden, higher rate of cytogenetic abnormalities, and less epigenetic regulation and spliceosome gene mutations than those of FA-PSV - MDS/AML patients (p = 0.024, p = 0.029, p = 0.024, and p = 0.013) (Figure 1D). Discussion The overall PSVs enrichment in our cohort buttresses that heterozygous mutations of FA genes abate the capacity of DNA homologous recombination repair pathway and contribute to hematopoietic failure. Furthermore, we present the first evidence that BRCA2 heterozygotes have a significantly higher risk of developing into ALL. Instead of all or nothing, the impact of different variant imposed on protein might be seen as a continuous variation. Therefore, contributions of the FA pathway defect could be latent and subtle but be profound as time goes by. We further firstly observed a higher incidence of cytogenetic abnormalities and somatic mutations with statistical significance, and lower frequency of epigenetic regulation and spliceosome gene mutations in FA-PSV + myeloid malignancies. This provides evidence that these FA-PSV carriers are prone to accumulate chromosomal structural abnormalities, and confer the congenital susceptibility of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

29

Taniguchi, Toshiyasu, and Alan D. D'Andrea. "Molecular pathogenesis of Fanconi anemia: recent progress." Blood 107, no. 11 (June 1, 2006): 4223–33. http://dx.doi.org/10.1182/blood-2005-10-4240.

Full text

Abstract:

AbstractA rare genetic disease, Fanconi anemia (FA), now attracts broader attention from cancer biologists and basic researchers in the DNA repair and ubiquitin biology fields as well as from hematologists. FA is a chromosome instability syndrome characterized by childhood-onset aplastic anemia, cancer or leukemia susceptibility, and cellular hypersensitivity to DNAcrosslinking agents. Identification of 11 genes for FA has led to progress in the molecular understanding of this disease. FA proteins, including a ubiquitin ligase (FANCL), a monoubiquitinated protein (FANCD2), a helicase (FANCJ/BACH1/BRIP1), and a breast/ovarian cancer susceptibility protein (FANCD1/BRCA2), appear to cooperate in a pathway leading to the recognition and repair of damaged DNA. Molecular interactions among FA proteins and responsible proteins for other chromosome instability syndromes (BLM, NBS1, MRE11, ATM, and ATR) have also been found. Furthermore, inactivation of FA genes has been observed in a wide variety of human cancers in the general population. These findings have broad implications for predicting the sensitivity and resistance of tumors to widely used anticancer DNA crosslinking agents (cisplatin, mitomycin C, and melphalan). Here, we summarize recent progress in the molecular biology of FA and discuss roles of the FA proteins in DNA repair and cancer biology.

APA, Harvard, Vancouver, ISO, and other styles

30

Houghtaling, Scott, Laura Granville, Yassmine Akkari, Yumi Torimaru, Susan Olson, Milton Finegold, and Markus Grompe. "Heterozygosity for p53 (Trp53 +/−) Accelerates Epithelial Tumor Formation in Fanconi Anemia Complementation Group D2 (Fancd2) Knockout Mice." Cancer Research 65, no. 1 (January 1, 2005): 85–91. http://dx.doi.org/10.1158/0008-5472.85.65.1.

Full text

Abstract:

Abstract Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2−/−/Trp53+/− mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2−/− control mice. Therefore, the Fancd2−/−/Trp53+/− mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2−/− mouse embryonic fibroblasts but not Fancd2−/−/Trp53−/− mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2−/−/Trp53−/− cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

APA, Harvard, Vancouver, ISO, and other styles

31

Stankovic, Tatjana, Philip J. Byrd, Grant S. Stewart, Alexander J. Taylor, Anna Smith, Peggy Fooks, Robert Horsley, et al. "A Novel Role of PALB2 in Lymphoid Tumour Development." Blood 128, no. 22 (December 2, 2016): 5113. http://dx.doi.org/10.1182/blood.v128.22.5113.5113.

Full text

Abstract:

Abstract PALB2 is DNA damage response protein that acts in Homologous recombination Repair (HRR) pathway and promotes recruitment of principal HRR components such as BRCA1, BRCA2 and Rad51. Biallelic PALB2 inactivation is associated with the severe form of Fanconi anemia (FA-N), characterised by severe developmental abnormalities and occurrence of embryonal tumours in infancy. Given the rarity of reported patients with biallelic PALB2 inactivation, the full extent of phenotypes associated with PALB2 mutations is still unknown. Here we describe a family with two inherited PALB2 mutations where the affected individuals presented with few of the clinical features typically exhibited by FA-N patients. The patients, age 19 and 15 were lacking severe developmental abnormalities and displayed impaired growth and mild learning difficulties. Interestingly, however, both individuals developed B cell Non-Hodgkin lymphoma (NHL) at the early age of 12 and 3.5 years respectively, responded well to intensive chemotherapy and stayed in complete remission. Their cellular phenotype included a high level of spontaneous chromosomal damage suggestive of DNA repair defect, but otherwise deviated from the classic FA phenotype revealing an intermediate sensitivity to mitomycin C (MMC) by colony forming assay but an unusually high chromosomal radiosensitivity in G2. We addressed the possibility that this atypical clinical and cellular phenotype could be related to the functional properties of the mutant PALB2 protein(s) expressed from the affected alleles. Indeed, we demonstrated that the PALB2 protein (T839_K862del) produced from one of the mutant alleles carried by these patients retained both its N- and C-terminal domain required to interact with BRCA1 and BRCA2 respectively. We also showed that this mutant protein preserved some function compared with previously identifiedpatient-associated mutant PALB2 proteins and allowed low level recruitment of Rad51 foci at sites of DNA damage. We propose that the presence of this mutant protein accounts for the milder MMC hyper-sensitivity exhibited by cells from these patients and the lack of FA defining clinical characteristics. Finally, the shift in tumour spectrum from embryonal tumours towards B cell NHL observed in this family implies PALB2 as a potential suppressor of lymphoid tumourigenesis and raises the possibility that somatic mutations in this gene may contribute to the development of sporadic lymphoid tumours. Disclosures Janic: Baxter: Other: Paid Instructor, Research Funding; Pfizer: Other: Paid Instructor, Research Funding; Bayer: Other: Paid Instructor, Research Funding; Octopharma: Research Funding; Novo Nordisk: Other: Paid Instructor, Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

32

West, Stephen C. "Links Between Defective DNA Strand Break Repair and Genome Instability in Fanconi Anemia." Blood 120, no. 21 (November 16, 2012): SCI—2—SCI—2. http://dx.doi.org/10.1182/blood.v120.21.sci-2.sci-2.

Full text

Abstract:

Abstract Abstract SCI-2 All living organisms feature DNA repair pathways that safeguard the integrity of the genome, and mutations in proteins that mediate key events in DNA repair have been linked to genome instability and tumorigenesis. Homologous recombination provides an important DNA repair pathway that is needed for the restoration and restart of broken replication forks, for the rejoining of chromosome/chromatid breaks, and for the processing of DNA cross-links. Mutations in genes that encode a variety of recombination proteins are linked to breast cancers and to heritable diseases such as Bloom syndrome (BS) and Fanconi anemia (FA). In recent work, we purified the BLM protein (defective in BS), the BRCA2 (FANCND1) and PALB2 (FANCN) tumor suppressors (mutated in some cases of FA), and the newly discovered FANCP protein, also known as SLX4, and have initiated structure-function analyses to elucidate their molecular functions. How these proteins process DNA, and how they are regulated and controlled to direct the outcome of recombinational repair is now revealing unexpected insights that extend our understanding of efficient DNA repair and tumor avoidance. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

33

Singhal, Deepak, Christopher N. Hahn, Cassandra M. Hirsch, Amilia Wee, Monika M. Kutyna, Milena Babic, Rakchha Chhetri, et al. "Genetic Predisposition to Therapy-Related Myeloid Neoplasm By Rare, Deleterious Germline Variants in DNA Repair Pathway and Myeloid Driver Genes." Blood 132, Supplement 1 (November 29, 2018): 1802. http://dx.doi.org/10.1182/blood-2018-99-117815.

Full text

Abstract:

Abstract Therapy-related myeloid neoplasm (t-MN) is considered to be a direct stochastic complication of chemotherapy and/or radiotherapy for primary cancer or autoimmune diseases. However, genetic predisposition is reported in 8-12% of sporadic adult cancer patients [Lu et al Nature Communication 2015 and Huang et al Cell 2018]. Similarly, genetic predispositions to t-MN have also been reported in limited single institute studies of small numbers of patients [Churpek et al Cancer 2016]. In this study, we performed comprehensive germline and somatic mutation profiling in t-MN using next generation sequencing. Matched germline material was available for 62/194 (32%) patients. Mutation profiling was correlated with clinical features including family history in 194 patients enrolled in the South Australian MDS (SA-MDS) registry and Cleveland Clinic (CC). An in-house well established filtering pipeline was used for identification of somatic mutations. Only variants with Genome Aggregation Database (gnomAD) minor allele frequency (MAF) of ≤0.01% and variant allele frequency (VAF) of ≥35% were selected for further analysis of germline variants. Variants reported in in the Catalogue of Somatic Mutations in Cancer database and MDS/AML were excluded from further analysis. Variants reported pathogenic in Breast Cancer Information Core (BIC) database and Leiden Open Variation Database (LOVD) were retained. Other variants were included if truncating (nonsense, indels, splice alterations), CADD>20, or predicted deleterious by >4/6 scoring algorithms (GERP>4, PhyloP>2, SIFT, PolyPhen2, MutationTaster and FATHMM). Forty-one (21%) t-MN patients harbored 45 rare (MAF<0.001) and deleterious germline mutations in the Fanconi anaemia (FA) pathway and driver myeloid genes including frameshift indels and splice site alterations in BRCA1, BRCA2, FANCA, PALB2, RAD51, DDX41 and TP53 (Figure 1A-B). The highest number of FA germline variants were seen in BRCA1 and FANCA (n=5 each) followed by BRCA2 (n=4), ERCC4, PALB2 and FANCC (n=2 each). We also identified 14 rare, deleterious myeloid germline variants in 13/194 (6.7%) of t-MN patients. These germline myeloid variants were identified in TP53, DDX41, GATA2 and MET; genes with well-known drivers of myeloid malignancies. Of the five acute lymphoblastic leukaemia patients with t-MN, 2/5 (40%) had rare myeloid germline variants in TP53, GATA2 and KMT2A. The frequency of these germline mutations in our t-MN cohort is higher than in the general population (gnomAD; Table 1) and in patients with primary malignancies such as breast cancer and lymphoma [Lu et al Nature Communication 2015 and Churpek et al Cancer 2016]. Intriguingly, the frequency of germline FA gene mutations (FAMT) in our therapy-related myelodysplastic syndrome (t-MDS) patients is also higher than those reported in primary MDS patients (18% vs 9%, p=0.02) [Przychodzen et al 2018]. Additionally, of those with available family history, 62% of t-MN patients have first and/or second degree relatives with non-skin cancers. Significantly more patients with FA mutation (FAMT) had first and second degree relatives with cancers compared to patients without FA (FAWT) mutations (82% vs 58%; p=0.03). Additionally, chromosomes 3 and 7 abnormalities, as well as monosomal karyotype, were more frequent in FAMT cases compared to FAWT. Similarly, somatic mutations in GATA2 (10% vs 2%; p=0.02), BCOR (13% vs 4%; p=0.03) and IDH2 (10% vs 2%; p=0.02) were more frequent in FAMT compared to FAWT cases (Figure 1C). In summary, we show that at least one in five t-MN patients harbor deleterious germline mutations, and 82% of FAMT patients have a first or second degree relative with cancers. These findings have implication in management of not only t-MN patients but genetic testing for their family members. Disclosures Branford: Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cepheid: Honoraria. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy. Hiwase:Celgene: Research Funding; Novartis: Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

34

Alter, Blanche P., Neelam Giri, Lisa James McReynolds, and Philip S. Rosenberg. "Cancer in Heterozygote Carriers of Fanconi Anemia Genes." Blood 132, Supplement 1 (November 29, 2018): 3868. http://dx.doi.org/10.1182/blood-2018-99-116458.

Full text

Abstract:

Abstract Background: Fanconi anemia (FA) is an inherited bone marrow failure syndrome, characterized by a defect in DNA repair, increased frequency of birth defects, and high risks of malignancies. Relatives of patients with FA are concerned about the risk of cancer in themselves or other family members. Carrier grandparents with mutations in genes for FA were reported in 2007 to have no increase in overall cancer incidence, except for a higher rate of breast cancer among FANCC carrier grandmothers (Berwick et al, Cancer Research 67:9591, 2007); this study included 944 relatives among 312 families. Tischkowitz et al also did not find increased risks of cancer in 575 relatives among 36 families, except for prostate cancer (Tischkowitz et al, BMC Cancer 8:257, 2008). The relative risk of breast cancer associated with germline mutations in genes in the BRCA/FA DNA repair pathway in the general population not due to BRCA1 or BRCA2 is 5 to 10 fold (Couch et al, JAMA Oncology 3:1190, 2017), suggesting that heterozygotes for FA might be at increased risk. The FA cohort at the National Cancer Institute (NCI) is sufficiently large to again address the question of cancer in FA heterozygotes. Hypothesis: Patients heterozygous for a mutated gene in the BRCA/FA DNA repair pathway have an increased risk of cancer. Identification of FA genes with increased cancer risk in heterozygotes, and specific cancer types in relatives of patients with FA, would guide screening recommendations for these categories of family members, and studies to determine whether FA heterozygotes with cancer should be managed with modifications of drug or radiation doses. Methods: We evaluated cancer risks in relatives of probands in the NCI FA cohort. We collected self-report or proxy report data on the presence or absence of cancer, cancer type, age at cancer diagnosis for individuals with a history of cancer, and age at death from other causes or current age. We studied obligate heterozygotes (parents and offspring of probands with FA), relatives with a 50% probability of heterozygosity (grandparents), and relatives with a 2/3 probability of being carriers (siblings without FA). We determined the ratio of observed/expected cases (O/E) using data from SEER cancer registries, with adjustment for age, sex, and birth cohort. Confirmatory genotyping is being done by targeted panel next-generation sequencing for participants with DNA available. Results and Discussion: Our study includes 94 families. Twenty-one cancers were reported in 188 parents, with 24 expected (O/E 0.86, 95% CI [confidence interval] 0.53-1.31). Three hundred and twenty-seven grandparents had 89 cases with cancer, expected 125 (O/E 0.71, CI 0.57-0.88). There were 122 siblings, with 1 cancer case and 4 expected (O/E 0.26, CI 0.01-1.46). Among 12 offspring there was 1 case of leukemia (O/E 1:0.12, CI 0.2-44.64). Specific cancers had increased O/E in parents (2 cases of salivary gland cancer, O/E 27.3, CI 3.31-98.66), grandparents (5 with liver cancer O/E 4.7, CI 1.53-10.96; 9 with leukemia O/E 2.42, CI 1.11-4.59), and a single case of acute myeloid leukemia in an offspring (O/E of 402.44, CI 10.19-2242.85). Four cases of prostate cancer in parents (O/E of 1.47, CI 0.4-3.76) and 8 in grandparents (O/E 0.47, CI 0.2-0.93) failed to support the suggestion by Tischkowitz et al that prostate cancer was significantly increased in male relatives (5 cases, O/E 3.1, CI 1.1-8.8). Missing data include birth dates, death dates, or cancer dates for 44 grandparents and 2 siblings. Our results suggest that heterozygotes for mutations in FA genes do not appear to have an increased risk of cancer compared with the general population, in agreement with data from other cohorts. Further analyses will be done after completion of genotyping the relatives, with the caveat that DNA may not be available from some of the grandparents. The NCI FA study is limited by the numbers of participants, and the missing data, particularly in the grandparent generation. In addition, data are reported by the individuals or their proxies, and medical record validation may not be possible; the types and ages of cancers cannot be confirmed. Although the types of cancer and risks in FA heterozygotes are similar to those in the general population, the potential concern that management of patients who are carriers of a defect in DNA repair may need to be modified requires future investigation. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

35

Kruper, Laura, Kevin McDonnell, Joseph Bonner, Kevin K. Tsang, Veronica Jones, Joanne Mortimer, Sidney S. Lindsey, et al. "Abstract PD14-03: PD14-03 Reappraising the Fanconi Anemia DNA repair pathway in breast cancer risk and precision intervention: Insights and opportunities from the City of Hope INSPIRE study." Cancer Research 83, no. 5_Supplement (March 1, 2023): PD14–03—PD14–03. http://dx.doi.org/10.1158/1538-7445.sabcs22-pd14-03.

Full text

Abstract:

Abstract Background: Fanconi Anemia (FA) proteins facilitate homologous recombination (HR)-mediated repair of DNA interstrand cross-links. Germline monoallelic, pathogenic/likely pathogenic (P/LP) variants in the highly-penetrant (HP) breast cancer (BC) FA genes, BRCA1 (FANCS), BRCA2 (FANCD1) and PALB2 (FANCN)), compromise HR and predispose to hereditary BC. The effects of monoallelic, pathogenic variants in other non-HP BC FA genes upon HR and BC predisposition remain less understood. In this investigation we report the germline mutational landscape of FA gene P/LP variants and somatic molecular consequences of patients with BC diagnoses from City of Hope’s (COH) INSPIRE (Implementing Next-generation Sequencing for Precision Intervention and Risk Evaluation) study. Methods: COH-INSPIRE is a universal access study open to all patients at COH with a personal and/or family history of cancer. Patients undergo custom panel-based germline genetic testing to detect P/LP single nucleotide variants (SNVs), short insertions/deletions (indels) and exon-level deletions/duplications in 155 cancer-predisposition genes including the HP BC FA genes and 15 non-HP BC FA genes [FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP1), FANCL, FANCM, FANCO (RAD51C), FANCP (SLX4), FANCQ (ERCC4) and FANCU (XRCC2)]. Patients’ tumor specimens undergo somatic tumor (>400X)-normal (>180X) whole exome and transcriptome sequencing (>50 million reads). Somatic sequencing identifies P/LP SNVs, indels, copy number events, and fusions. Secondary analyses assessed somatic homologous recombination deficiency (HRD) by examining tumor mutational signatures, as well as an ensemble HRD score derived by combining individual genomic loss of heterozygosity, telomeric allelic imbalance and large-scale molecular transition scores. Reference comparison of germline and somatic features to current FDA therapeutic guidelines and NIH clinical trials registrations determined eligibility for precision therapeutic intervention and clinical trial enrollment. Results: Of 7,584 patients enrolled in COH-INSPIRE, 1,651 (21.8%) patients had a BC diagnosis. Germline panel testing of BC patients identified 204 (12.4%) with germline P/LP variant in a FA gene. Greater than one third of FA gene-altered BC patients (37.7%) carried a P/LP variant in a non-HP BC FA gene. We observed that BC patients with a non-HP BC FA gene variant may demonstrate HR compromise as evidenced by presence of a Signature 3 mutational profile or an elevated combined HRD score (> 33 and/or > 42). (Table 1) Further, we identified ostensible segregation of triple negative BC in a family harboring a germline pathogenic variant in FANCG. With regard to precision clinical actionability (i.e. qualification for targeted therapeutic intervention [PARP inhibitor (PARPi)] and/or clinical trial) for patients with advanced stage BC: All patients with germline P/LP HP BC FA gene variant and 20.7% (N=16) of patients with a P/LP FA non-HPBC FA gene variant met criteria for treatment with on/off-label PARPi. 100% of patients with advanced BC with germline P/LP HP BC or non-HPBC FA gene variant qualified for a clinical trial. Conclusions: Patients with BC often carry a germline monoallelic, P/LP FA gene variant; in more than one third, the FA gene alteration occurs in a non-HP BC FA gene. BC patients harboring a monoallelic germline non-HP BC P/LP FA gene may exhibit somatic mutational signatures and HRD scoring consistent with compromise of HR. Somatic tumor evaluation of BC patients with germline P/LP non-HP BC FA gene variants expands opportunities for precision therapeutic intervention and clinical trial enrollment. Continued appraisal will clarify emerging questions of germline non-HP P/LP FA gene-associated autosomal dominant BC risk and management as well as facilitate optimization of precision BC care. Table 1 Summary Molecular Features of BC patients with P/LP Variants in FA gene from COH-INSPIRE Citation Format: Laura Kruper, Kevin McDonnell, Joseph Bonner, Kevin K. Tsang, Veronica Jones, Joanne Mortimer, Sidney S. Lindsey, Ilana Solomon, Heather Hampel, Wai Park, Gregory E. Idos, Stacy Gray, Stephen Gruber. PD14-03 Reappraising the Fanconi Anemia DNA repair pathway in breast cancer risk and precision intervention: Insights and opportunities from the City of Hope INSPIRE study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD14-03.

APA, Harvard, Vancouver, ISO, and other styles

36

Ali, Abdullah M., Thiyam R. Singh, and Ruhikanta A. Meetei. "Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method." Blood 108, no. 11 (November 16, 2006): 989. http://dx.doi.org/10.1182/blood.v108.11.989.989.

Full text

Abstract:

Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.

APA, Harvard, Vancouver, ISO, and other styles

37

Wang, Kun, Hongwei Wang, Kemin Jin, Xiaoyu Zhang, Chunhe Yang, and Baocai Xing. "Correlation of existence and number of mutations in DDR signaling pathway genes with poor survival after resection of colorectal liver metastases." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e16004-e16004. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e16004.

Full text

Abstract:

e16004 Background: Patients of colorectal cancer with liver metastatic (CRLM) show various in recurrence and prognosis. Previous researches have revealed that the disfunction of DNA damage repair (DDR) signaling pathways was correlative with oncogenesis and poor prognosis. Here we aimed to determine the impact of DDR gene mutations on survival in patients undergoing CRLM resection. Methods: Tumor tissues from 164 patients with CRLM were collected for next generation sequencing (NGS). Single nucleotide variants, indels, and copy number variations were obtained from a 620-gene panel, including 68 genes in 7 DDR signaling pathways. Correlations of mutations in DDR genes with overall survival (OS) and recurrence-free survival (RFS) were determined by using Kaplan-Meier analysis. Results: 152/164 of patients carried at least one of DDR gene alternations. The most frequently mutated gene was TP53 (132/164), followed by CHEK2 (21/164), BRCA2 (10/164), ATM (9/164), PRKDC (8/164), FANCM (8/164), ATR (7/164), POLE (5/164), BRCA1 (4/164), POLD (3/164). Survival analysis indicated that TP53 mutations were not correlated with OS and RFS (P = 0.38 and 0.21). Further analysis in the TP53mut subgroup showed that the patients with other DDR gene mutations (64/132, 48.48%) exhibited a significantly worse OS than the wild type ones (P = 0.04). Especially, gene alternations involving in the Fanconi anemia (FA) pathway were significantly associated with worse OS and RFS (P = 0.0014 and 0.006). Interestingly, besides the TP53 mutation, patients carrying more than 2 other DDR gene mutations (10/64, 15.63%) also showed worse OS and RFS than patients with single mutation and wild type in DDR genes. (OS: 19 vs 35 months, P = 0.053; RFS: 3 vs 11 months, P = 0.084). Conclusions: Mutations in DDR signaling pathways predicted worse survival in TP53-mutated CRLM patients after surgery. These findings may be useful for clinical decision making in patients with tumor characteristics associated with poor prognosis and for risk stratification of patients in future clinical studies.

APA, Harvard, Vancouver, ISO, and other styles

38

Mori, Minako, Asuka Hira, Kenichi Yoshida, Hideki Muramatsu, Yusuke Okuno, Michiko Anmae, Kazuo Tamura, et al. "Characterization of Pathogenic Variants and Clinical Phenotypes in 117 Japanese Fanconi Anemia Patients." Blood 132, Supplement 1 (November 29, 2018): 3860. http://dx.doi.org/10.1182/blood-2018-99-110362.

Full text

Abstract:

Abstract Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

APA, Harvard, Vancouver, ISO, and other styles

39

Kohli, Manish, Siddhartha Yadav, Winston Tan, Irbaz Bin Riaz, Tiantian Zheng, Amy Wang, Carlos Montesinos, et al. "Plasma cell-free DNA-based prognosis in metastatic hormone sensitive prostate cancer." Journal of Clinical Oncology 37, no. 7_suppl (March 1, 2019): 242. http://dx.doi.org/10.1200/jco.2019.37.7_suppl.242.

Full text

Abstract:

242 Background: We evaluated plasma cell free based genomic aberrations for prognosticating survival of newly diagnosed metastatic hormone sensitive prostate cancer (mHSPC) patients (pts). Methods: Plasma was collected from mHSPC pts enrolled between 2009-2014. Platelet poor plasma (PPP) fractions were processed uniformly and cell free DNA (cfDNA) extracted using Qiagen kits. Pts were followed after initiating hormonal therapy until death. Next Gen Sequencing (NGS) of cfDNA was performed using Illumina HiSeq X for a preselected panel of 128 genes (PredicineDDR-77 cancer driver genes; 29 genes in BRCA-FA homologous recombination deficiency (HRD) pathway; 22 DNA damage repair pathway genes). Statistical analyses of plasma genome based aberrations with overall survival (OS) were performed in R 3.5.1. Cox proportional-hazard models were used for survival analysis. Results: An average of 2.5 ml PPP from 99 pts yielded a median of 10.5 ng (range: 2.8-702) cfDNA per sample. 15/99 pt samples with a yield < 5 ng were excluded from sequencing; 9/99 samples failed NGS. Median follow-up time was 80.2 months (mths) (Range: 74.7, 87]); median OS was 69.1 mths (range: 54,NR). 29 pts with full NGS data had high volume metastatic disease. cfDNA yield correlated with metastatic volume (P = 0.01). Univariate analysis revealed both variables prognostic for OS (Metastatic volume: log-rank P=0.01, HR=2.1, 95% CI: 1.1-3.8; cfDNA yield: P =0.04, HR = 1.3, 95% CI: 1.03-1.7). Multivariate regression showed prognostic value of cfDNA yield remained independent of metastatic volume (P = 0.03, HR = 1.34, 95% CI: 1.02-1.76). 54/67 samples with NGS data had at least one mutation/copy number variation detected. Top mutated genes included TP53 (N=18), ATM (N=9), CHEK2 (N=7), FANCM (N=6), RB1 (N=6), BRCA2 (N=5), PIK3CA (N=4) and 37/67 pts harbored 1≥ variant in HDR pathways. These pts had a shorter survival (median: 58.6 mths) (P=0.04, HR= 2.28, 95% CI: 1.01-5.18) and pts with ATM mutations did significantly worse (median survival: 47.4 mths) (HR=4.03, P=0.0005, 95% CI: 1.73-9.37). Conclusions: Plasma cfDNA yield is prognostic for survival in newly diagnosed mHSPC state and presence of HRD pathway genomic aberrations in plasma cfDNA are associated with poor survival.

APA, Harvard, Vancouver, ISO, and other styles

40

Liang, Zhuobin, Yaqun Teng, Jingchun Liu, Simonne Longerich, Xiaoyong Chen, Allison M. Green, Natalie Collins, Li Lan, Patrick Sung, and Gary M. Kupfer. "FANCI-FANCD2 Binds RNA, Which Stimulates Its Monoubiquitination." Blood 132, Supplement 1 (November 29, 2018): 645. http://dx.doi.org/10.1182/blood-2018-99-118863.

Full text

Abstract:

Abstract Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and a strong cancer predisposition. FA cells are hypersensitive to DNA replicative stress, and accumulate co-transcriptional R-loops. Previous work has demonstrated that BRCA2 binds to R loops, and increased R loops are noted in FA-D2 mutant cells. Additionally, it is understood that at least one FA protein, FANCA, binds RNA. The goal of this study was to understand the relationship between FANCD2 and RNA, especially with regard to manifestation of R loops as a part of the pathophysiology of FA. First, we confirmed the increased presence of R loops in FA mutant cells using the S9.6 monoclonal antibody immunofluorescence microscopy. RNAseH overexpression removes R loop signal and increases cell survival upon mitomycin C treatment. We also showed the presence of increased R loops in an actively transcribed region of the actin gene by bisulfite DNA sequencing. We used the Damage At RNA Transcription (DART) assay, which is designed to combine oxidative DNA damage and the genomic insertion of a hyper transcription site (Fig A). Coactivation of transcription and DNA damage results in colocalization of FANCD2 and S9.6/R loop signal at the transcriptional site (Fig B and C). Consistent with the S9.6 IF, wild type RNAseH overexpression resulted in the abrogation of FANCD2 colocalization. We then asked if FANCD2 binds RNA. FANCD2 in cell lysate bound to biotinylated RNA species, preferring GC rich RNAs. Using recombinant FANCI-FANCD2 (ID2) protein (Fig D), we found that ID2 binds preferably to single stranded RNA in a more robust manner than DNA (Fig E and F). Interestingly, an ID2 complex with a known DNA binding mutation in FANCI also was defective for RNA binding. Furthermore, ID2 bound to R loops but was mediated via the single stranded DNA component of the structure. Importantly, an in vitro monoubiquitination reconstitution system using FANCL as the E3 ligase demonstrated that monoubiquitination of ID2 was stimulated to an equal or greater degree by RNA versus DNA, with greater signal in presence of GC-rich, single-stranded RNA as well as R loops (Fig G and H and data not shown). Collectively, our results support a novel mechanism the ID2 complex suppresses the formation of pathogenic R-loops by binding RNA species, thereby activating the FA pathway (Fig I). Figure. Figure. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

41

Vicens, Alberto, and David Posada. "Selective Pressures on Human Cancer Genes along the Evolution of Mammals." Genes 9, no. 12 (November 28, 2018): 582. http://dx.doi.org/10.3390/genes9120582.

Full text

Abstract:

Cancer is a disease driven by both somatic mutations that increase survival and proliferation of cell lineages and the evolution of genes associated with cancer risk in populations. Several genes associated with cancer in humans, hereafter cancer genes, show evidence of germline positive selection among species. Taking advantage of a large collection of mammalian genomes, we systematically looked for signatures of germline positive selection in 430 cancer genes available in COSMIC. We identified 40 cancer genes with a robust signal of positive selection in mammals. We found evidence for fewer selective constraints—higher number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (dN/dS)—and higher incidence of positive selection—more positively selected sites—in cancer genes bearing germline and recessive mutations that predispose to cancer. This finding suggests a potential association between relaxed selection, positive selection, and risk of hereditary cancer. On the other hand, we did not find significant differences in terms of tissue or gene type. Human cancer genes under germline positive selection in mammals are significantly enriched in the processes of DNA repair, with high presence of Fanconi anaemia/Breast Cancer A (FA/BRCA) pathway components and T cell proliferation genes. We also show that the inferred positively selected sites in the two genes with the strongest signal of positive selection, i.e., BRCA2 and PTPRC, are in regions of functional relevance, which could be relevant to cancer susceptibility.

APA, Harvard, Vancouver, ISO, and other styles

42

Mai, Qicong, Zhiqiang Mo, Jian He, Tingting Chen, Mengli Huang, and Xiaoming Chen. "Analysis of DNA damage repair pathway genes as a predictive biomarker for HAIC in hepatocellular carcinoma." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e16159-e16159. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e16159.

Full text

Abstract:

e16159 Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide.Hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin, fluorouracil, and leucovorin (FOLFOX) is one of the effective treatment for advanced HCC patients. DNA damage repaired (DDR) pathway has been widely researched in many cancers recent years, which was a promising predictive biomarker for the response to platinum-based chemotherapy and Immunotherapy. But the DDR characteristics of different subset pathways in HCC, and its correlation with HAIC efficacy were unknown. Methods: Whole exome sequencing (WES) data of 366 HCC patients was obtained from the Cancer Genome Altas (TCGA). Next generation sequencing (NGS, panel on 381/733-gene) was performed on FFPE tumor samples from 2092 Chinese HCC patients (Chinese cohort). Germ-line or somatic mutations of 32 DDR pathway genes (including Mismatch Repair and Homologous Recombination) were classified as DDR gene mutations. TMB was defined as total number of somatic non-synonymous mutations in coding region. Whole exome sequencing (WES) data and clinical data of 21 HCC advanced patients treated with HAIC were obtained to survival analysis. Results: In total, 59.84% (219/366) of HCC patients in TCGA harboring DDR mutation and 67.38% (1316/2092) in Chinese cohort. In Chinese HCC cohort, the mean TMB level of DDR mutant group was significant higher than wild-type group (mean TMB, mutation vs wild-type = 8.12 vs 6.42 Muts/Mb, P < 0.0001). The top three mutation frequency DDR genes were BRCA2 (17%), ATM (13%) and BARD1 (12%), respectively. The highest mutation frequency DDR subset pathways were Fanconi anemia (FA, 44.14%, 862/2092), homologous recombination repair (HRR, 38.71%, 756/2092) and check point factors (CHP, 17.31%, 338/2092) come in second and third, respectively. Next we compared the TMB level between different DDR subset pathways, the mutant of nucleotide excision repair (NER) pathway harbored the highest medium TMB level (7.26 Muts/Mb). The survival analysis was performed on HCC patients treated with HAIC with FOLFOX. There was no difference in clinical baseline information between DDR mutation group (n = 6) and wild-type group (n = 15). The progression-free survival (PFS) of DDR mutation group were significantly longer than wild-type group (median PFS, mutation vs. wild-type = 8.9 vs. 4.5 months; HR 0.34[95% CI 0.13-0.85]; P = 0.0349), and an extending trend on overall survival (OS) without significant difference (median OS, 15.7 vs 8.9 months; HR 0.37[95% CI 0.13-1.00]; P = 0.0921). Conclusions: The DDR pathways was associated with higher TMB level. Preliminary data from clinical cohorts suggested better treatment outcomes of HAIC with FOLFOX in DDR mutation HCC patients.

APA, Harvard, Vancouver, ISO, and other styles

43

Li, Niu, Jian Wang, Susan S. Wallace, Jing Chen, Jia Zhou, and Alan D. D’Andrea. "Cooperation of the NEIL3 and Fanconi anemia/BRCA pathways in interstrand crosslink repair." Nucleic Acids Research 48, no. 6 (January 25, 2020): 3014–28. http://dx.doi.org/10.1093/nar/gkaa038.

Full text

Abstract:

Abstract The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.

APA, Harvard, Vancouver, ISO, and other styles

44

Roman-Rodriguez, Francisco J., Laura Ugalde, Lara A. Alvarez, Begoña Díez-Cabezas, Jordi Surrallés, Maria Jose Ramirez, Massimo Bogliolo, et al. "NHEJ-Mediated Gene Editing, a Versatile Approach to Correct a Variety of Fanconi Anemia Genes in HSCs." Blood 134, Supplement_1 (November 13, 2019): 4639. http://dx.doi.org/10.1182/blood-2019-129946.

Full text

Abstract:

Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only curative treatment for the bone marrow failure in Fanconi anemia (FA) patients. However, recent advances in lentiviral-mediated gene therapy have shown that corrected FA HSCs develop an in vivo proliferation advantage, facilitating the engraftment of corrected HSCs in non-conditioned FA patients. Based on these observations, we proposed that gene editing might constitute a promising alternative to correct patients' hematopoietic stem and progenitor cells (HSPCs) in this disorder. Since non-homologous end joining (NHEJ) is the most frequent repair pathway in HSCs, particularly in FA-HSCs, we aimed at exploiting this DNA repair mechanism to remove/compensate specific mutations in different FANC genes by the use of CRISPR/Cas9 system, thus mimicking spontaneous genetic reversions observed in FA mosaic patients. Our results in lymphoblastic cell lines from five different complementation groups (FANCA, FANCB, FANCC, FANCD2 and FANCD1/BRCA2) demonstrated the efficiency of this approach to generate potentially corrective events in all the different complementation groups studied. Importantly, corrected cells showed a marked proliferative advantage after in vitro culture and the analysis by next generation sequencing confirmed the expansion of cells harboring therapeutic events. Functional studies showing the reversion of mitomycin C sensitivity, FANCD2 foci formation and chromosomal instability supported the phenotypic correction of different mutations by NHEJ-mediated gene editing. Moving towards the clinical application of NHEJ-mediated repair we focused on improving the gene editing efficiency in HSCs. To this aim, chemically modified small guide RNAs (MS-sgRNAs) enabled us to increase the editing efficacy 8-fold compared to efficacies obtained with in vitro transcribed sgRNAs, reaching up to 89% indels in healthy donor hematopoietic stem/progenitor cells. Moreover, the CRISPR/Cas9 system demonstrated high editing capacity in the primitive HSCs capable of engrafting immunodeficient NSG mice, confirming the efficacy of NHEJ-editing to correct the phenotype of long-term repopulating HSCs. Finally, studies conducted in mobilized peripheral blood and bone marrow CD34+ cells from FA patients demonstrated the feasibility to correct FA HSCs by NHEJ-mediated gene editing and confirmed the proliferative advantage of NHEJ-mediated corrected cells both in vitro and in vivo. Our results suggest that NHEJ-mediated gene editing should constitute a versatile and simple therapeutic approach to efficiently correct specific mutations in FA and other monogenic disorders of the hematopoietic system. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Rio:Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

45

Emelyanova, Marina, Elena Pudova, Darya Khomich, George Krasnov, Anna Popova, Ivan Abramov, Vladimir Mikhailovich, et al. "Platinum-based chemotherapy for pancreatic cancer: impact of mutations in the homologous recombination repair and Fanconi anemia genes." Therapeutic Advances in Medical Oncology 14 (January 2022): 175883592210830. http://dx.doi.org/10.1177/17588359221083050.

Full text

Abstract:

Background: Mutations in homologous recombination (HR) and Fanconi anemia (FA) genes may predispose to pancreatic cancer (PC) and enable the prediction of sensitivity to platinum-based chemotherapy. FOLFIRINOX is a standard treatment option for non-selected PC patients and could be effective due to undiagnosed DNA repair deficiency. Here, we aimed to determine the frequency of mutations in genes involved in the HR and FA pathways, evaluate their clinical implications, and determine the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) of PC patients treated with platinum. Methods: We performed targeted DNA sequencing of 30 genes ( ABRAXAS1, ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDKN2A, CHEK1, CHEK2, FANCC, FANCF, FANCG, FANCI, FANCL, FANCM, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, RAD52, RAD54B, RBBP8, RINT1, SLX4, and XRCC2) for 543 PC patients. Results: In BRCA/PALB2-mutated patients with advanced PC (33 patients, 6.1%), the PFS and OS were higher for first-line platinum therapy than for non-platinum therapy [PFS: HR = 0.28, 95% confidence interval (CI) = 0.10–0.81, p = 0.02; OS: HR = 0.31, 95% CI = 0.08–1.16, p = 0.08]. Among 93 patients (17.1%) with mutations in other HR/FA genes, no statistically significant difference in PFS and OS was observed between first-line platinum therapy and non-platinum therapy (PFS: HR = 0.83, 95% CI = 0.43–1.62, p = 0.59; OS: HR = 0.58, 95% CI = 0.28–1.22, p = 0.15). For patients with early PC, no prognostic value was observed for BRCA1/2, PALB2, or other HR/FA genes mutations. Moreover, a personal history of breast, ovarian, pancreatic, or prostate cancer was identified as the only independent predictor of the risk of BRCA/PALB2 mutations (HR = 5.83, 95% CI = 2.16–15.73, p < 0.01). Conclusion: Mutations in the BRCA1/2 and PALB2 genes increase the sensitivity of PC to platinum agents. Thus, alterations in these genes in PC patients must be determined prior to anticancer therapy.

APA, Harvard, Vancouver, ISO, and other styles

46

Mulligan, Jude M., Laura A. Hill, Steve Deharo, Katherine E. Keating, Olaide Raji, Fionnuala A. McDyer, Jacqueline James, et al. "Identification and validation of an assay predictive of response and prognosis following anthracycline-based chemotherapy for early breast cancer." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): TPS11120. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.tps11120.

Full text

Abstract:

TPS11120 Background: Currently there is no biomarker to predict specific benefit from DNA-damaging anthracycline and cyclophosphamide-based chemotherapy in the clinic. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA-damage response pathway occurs in approximately 25% of breast cancer and results in sensitivity to DNA-damaging agents. We therefore developed an assay to detect loss of the FA/BRCA pathway, for the purpose of predicting benefit from chemotherapy. Methods: 21 FA patient samples were analyzed to identify genetic processes associated with loss of the FA/BRCA pathway. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples and a molecular subgroup was identified that was defined by the molecular processes representing loss of the FA/BRCA pathway. A 44-gene DNA Damage response deficient (DDRD) assay was developed that could identify this subgroup from formalin fixed, paraffin embedded (FFPE) samples in the clinic. Results: In a publicly available independent cohort of 204 patients, the assay predicted response to neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline and cyclophosphamide) with an odds ratio of 4.01, (95% Cl:1.69-9.54). We also analysed samples from an independent cohort of 114 node-negative breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin and cyclophosphamide treatment at the Northern Ireland Cancer Centre. The DDRD assay significantly predicted 5-year relapse free survival with a hazard ratio of 0.27 (95% Cl:0.10-0.83). The assay was not predictive of survival in patients who did not receive chemotherapy. Conclusions: An FFPE tissue-based assay that detects loss of the FA/BRCA pathway has been developed and independently validated as a predictor of response and prognosis following DNA damaging anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings.

APA, Harvard, Vancouver, ISO, and other styles

47

Freie, Brian, Xiaxin Li, Samantha L. M. Ciccone, Kathy Nawa, Scott Cooper, Catherine Vogelweid, Laurel Schantz, et al. "Fanconi anemia type C and p53 cooperate in apoptosis and tumorigenesis." Blood 102, no. 12 (December 1, 2003): 4146–52. http://dx.doi.org/10.1182/blood-2003-03-0971.

Full text

Abstract:

Abstract Fanconi anemia (FA) is a recessive genomic instability syndrome characterized by developmental defects, progressive bone marrow failure, and cancer. FA is genetically heterogeneous, however; the proteins encoded by different FA loci interact functionally with each other and with the BRCA1, BRCA2, and ATM gene products. Although patients with FA are highly predisposed to the development of myeloid leukemia and solid tumors, the alterations in biochemical pathways responsible for the progression of tumorigenesis in these patients remain unknown. FA cells are hypersensitive to a range of genotoxic and cellular stresses that activate signaling pathways mediating apoptosis. Here we show that ionizing radiation (IR) induces modestly elevated levels of p53 in cells from FA type C (Fancc) mutant mice and that inactivation of Trp53 rescues tumor necrosis factor α-induced apoptosis in myeloid cells from Fancc-/- mice. Further, whereas Fancc-/- mice failed to form hematopoietic or solid malignancies, mice mutant at both Fancc and Trp53 developed tumors more rapidly than mice mutant at Trp53 alone. This shortened latency was associated with the appearance of tumor types that are found in patients with FA but not in mice mutant at Trp53 only. Collectively, these data demonstrate that p53 and Fancc interact functionally to regulate apoptosis and tumorigenesis in Fancc-deficient cells. (Blood. 2003;102:4146-4152)

APA, Harvard, Vancouver, ISO, and other styles

48

Soulier, Jean, Thierry Leblanc, Jérôme Larghero, Hélène Dastot, Akiko Shimamura, Philippe Guardiola, Hélène Esperou, et al. "Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway." Blood 105, no. 3 (February 1, 2005): 1329–36. http://dx.doi.org/10.1182/blood-2004-05-1852.

Full text

Abstract:

AbstractFanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. Eight FA-associated genes have been identified so far, the products of which function in the FA/BRCA pathway. A key event in the pathway is the monoubiquitination of the FANCD2 protein, which depends on a multiprotein FA core complex. In a number of patients, spontaneous genetic reversion can correct FA mutations, leading to somatic mosaicism. We analyzed the FA/BRCA pathway in 53 FA patients by FANCD2 immunoblots and chromosome breakage tests. Strikingly, FANCD2 monoubiquitination was detected in peripheral blood lymphocytes (PBLs) in 8 (15%) patients. FA reversion was further shown in these patients by comparison of primary fibro-blasts and PBLs. Reversion was associated with higher blood counts and clinical stability or improvement. Once constitutional FANCD2 patterns were determined, patients could be classified based on the level of FA/BRCA pathway disruption, as “FA core” (upstream inactivation; n = 47, 89%), FA-D2 (n = 4, 8%), and an unidentified downstream group (n = 2, 4%). FA-D2 and unidentified group patients were therefore relatively common, and they had more severe congenital phenotypes. These results show that specific analysis of the FA/BRCA pathway, combined with clinical and chromosome breakage data, allows a comprehensive characterization of FA patients.

APA, Harvard, Vancouver, ISO, and other styles

49

Lyakhovich, Alex, and Jordi Surralles. "New Roads to FA/BRCA Pathway: H2AX." Cell Cycle 6, no. 9 (May 2, 2007): 1019–23. http://dx.doi.org/10.4161/cc.6.9.4223.

Full text

APA, Harvard, Vancouver, ISO, and other styles

50

Hong, Lih En, Deepak Singhal, Amilia Wee, Rakchha Chhetri, Mihir D. Wechalekar, Susanna Proudman, and Devendra K. Hiwase. "The Mutation Profile of Myelodysplastic Syndrome Associated with Auto-Immune Rheumatological Disorders." Blood 132, Supplement 1 (November 29, 2018): 3081. http://dx.doi.org/10.1182/blood-2018-99-119965.

Full text

Abstract:

Abstract Introduction: A subset of patients with MDS and related myeloid disorders present with concomitant autoimmune rheumatological diseases (AIRD); however the prevalence ranges from 10-48% based on limited literature. Further, use of immunosuppressive agents in AIRD patients could confound the secondary diagnosis of MDS and in some cases cause it (therapy-related myeloid neoplasm; t-MN). The prevalence of cytopenia in AIRD patients is unknown and the genetic characteristics of MDS patients with concomitant AIRD have not been described. Hence, we interrogated two large multi-institutional databases -Royal Adelaide Hospital Rheumatology Database (RAH-RD) and South-Australian MDS (SA-MDS) registry in this study. Methods: Demographic, clinical, laboratory and treatment data of 2663 AIRD and 1157 MDS patients were analysed. In AIRD patients (autoimmune inflammatory arthritis, spondyloarthritis, vasculitis and connective tissue diseases), cytopenia (persisting >6 months) were defined as follows: hemoglobin <100g/dL, absolute neutrophil <1800/mm3 and platelet <100,000/mm3. Targeted massively parallel sequencing of a custom panel of 43 myeloid neoplasms associated genes and 20 Fanconi (FA) DNA repair pathway genes (all coding regions) was performed on diagnosis bone marrow samples (n=237). An in-house well established filtering pipeline was used for identification of somatic mutations. Matched germline material was available for 62/194 (32%) patients. Only variants with Genome Aggregation Database minor allele frequency of ≤0.01% and variant allele frequency of ≥35% were selected for further analysis of germline variants. Results: During follow up of 2663 AIRD patients, 36 (1.3%) patients satisfied the criteria for at least one cytopenia. Anemia (19/36, 53%) was most common followed by neutropenia (8/36, 22%), thrombocytopenia (4/36, 11%) and bi-cytopenia (5/36, 14%). Twenty-two patients had bone marrow examination which was non-diagnostic in 16 patients, while 7/2663 (0.3%) patients were diagnosed with MDS. Importantly, 5 patients with MDS and 11 patients with cytopenia did not receive any cytotoxic agents. In the MDS database, 69(5.4%) were diagnosed with AIRD, with rheumatoid arthritis (n=20, 29%) being the most common AIRD. Among these 69 patients, 24 (34.8%) had low risk MDS and 15 (21.7%) had higher risk MDS. The remaining 30 patients had t-MN (n=19, 27.5%), MDS/MPN (n=8, 11.6%) and AML (n=3, 4.3%). Overall, in a combined population of 2663 RAH-RD and 1157 SA-MDS, 76(2%) had concomitant MDS and AIRD. Genetic profile of patients with MDS and AIRD: The cytogenetic and mutational profile of MDS patients with (n=20) or without (n=217) AIRD were compared. No significant difference was seen in the cytogenetic profile (normal, complex or monosomal karyotype, chr. 5 or 7 abnormalities) between the two groups but in mutational analysis, 56 mutations were seen in 20 MDS patients with AIRD (Fig1). In these patients, mutations in epigenetic pathways were most common (23/56, 41%) followed by transcription pathway (10/56, 18%). Splicing mutations were seen in 5 patients, with SRSF2 mutations being more common than SF3B1. Mutations in TP53 were present in 4 (24%) patients; 3/4 patients developed MDS following therapy for AIRD (t-MDS). IDH1 mutations were found in significantly higher frequency in MDS patients with AIRD compared to MDS without AIRD (30% vs 3%, p=0.04). There was no significant difference in the frequency of other mutations or overall mutation frequency between the two groups. Interestingly, 2 (10%) patients with MDS and AIRD also had rare, deleterious germline mutations in FA pathway genes (BRCA2 V2601M and L2512F) which could suggest either genetic predisposition to both these conditions or compromised DNA repair capability increasing susceptibility to t-MN. Conclusions: In a large multi-institutional cohort of autoimmune rheumatological disorders, 1.3% patients developed persistent cytopenia with 0.3% diagnosed with MDS. This is significantly higher than incidence of MDS in the general population (30-50/100,000). Similarly, 5% MDS patients had AIRD. The mutation profile of MDS patients with AIRD shows higher frequency of IDH1 and SRSF2 mutations. A small proportion of cases also had deleterious rare germline mutations in the DNA repair pathway. Our findings warrant further study and have potential implications for selection of immunosupressive agents for AIRD. Disclosures Hiwase: Novartis: Research Funding; Celgene: Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Pathway FA/BRCA2'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles