Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

A novel mouse model of PMS2 founder mutation that causes mismatch repair defect due to aberrant splicing.

Hereditary non-polyposis colorectal cancer, now known as Lynch syndrome (LS) is one of the most common cancer predisposition syndromes and is caused by germline pathogenic variants (GPVs) in DNA mismatch repair (MMR) genes. A common founder GPV in PMS2 in the Canadian Inuit population, NM_000535.5: c.2002A>G, leads to a benign missense (p.I668V) but also acts as a de novo splice site that creates a 5 bp deletion resulting in a truncated protein (p.I668*). Individuals homozygous for this GPV are predisposed to atypical constitutional MMR deficiency with a delayed onset of first primary malignancy. We have generated mice with an equivalent germline mutation (Pms2c.1993A>G) and demonstrate that it results in a splicing defect similar to those observed in humans. Homozygous mutant mice are viable like the Pms2 null mice. However, unlike the Pms2 null mice, these mutant mice are fertile, like humans homozygous for this variant. Furthermore, these mice exhibit a significant increase in microsatellite instability and intestinal adenomas on an Apc mutant background. Rectification of the splicing defect in human and murine fibroblasts using antisense morpholinos suggests that this novel mouse model can be valuable in evaluating the efficacy aimed at targeting the splicing defect in PMS2 that is highly prevalent among the Canadian Inuits.

Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation.

Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle.

Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

A computational model for classification of BRCA2 variants using mouse embryonic stem cell-based functional assays.

Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay.

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

Bypass of premature stop codons and generation of functional BRCA2 by exon skipping.

A pathogenic mutation in BRCA2 significantly increases the risk of breast and ovarian cancers making it imperative to examine the functional consequences of variants of uncertain clinical significance. Variants that are predicted to result in a truncated protein are unambiguously classified as pathogenic. We have previously shown how a pathogenic splice site variant known to generate a premature termination codon (PTC) in exon 9 and a nonsense mutation at exon 7, can generate functional BRCA2 by skipping exons 4-7 and restoring the reading frame. Using a well-established mouse embryonic stem cell-based assay, we functionally characterize here one splice site mutation and 11 pathogenic BRCA2 variants that are either nonsense mutation or generate PTC in different exons ranging from exons 4 to 7. Our study shows that five variants can restore the open reading frame by exon skipping and generate a functional protein. This suggests further need to exercise prudence when classifying clearly pathogenic variants.

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption.

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the 'BRCAness' profile.

Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

Using recombineering to generate point mutations: the oligonucleotide-based "hit and fix" method.

Ability to manipulate the genome or design genes with desired mutation is critical for functional studies. Recombineering has made genetic manipulation of large genomic fragments very feasible and efficient. In the bacteriophage lambda-based recombineering system, three prophage genes, exo, bet, and gam, under the control of a temperature-sensitive lambda cI-repressor, provide the recombination function. The high efficiency of recombineering by oligonucleotides allows generation of subtle alterations in the bacterial chromosomal DNA as well as episomal DNA. We describe here a two-step "Hit and Fix" method, in which a short heterologous sequence is inserted to the target site first (Hit) and this sequence is replaced with the desired mutation in the second step (Fix). Insertion and replacement of the heterologous sequence allows screening of the recombinant clones by PCR or colony hybridization.

Using recombineering to generate point mutations:galK-based positive-negative selection method.

Recombineering is a recombination-based highly efficient method of genetic engineering. It can be used to manipulate the bacterial chromosomal DNA as well as any episomal DNA. Recombineering can be used to insert selectable or nonselectable DNA fragments and subclone DNA fragments without the use of restriction enzymes and also to make precise alterations including single nucleotide changes in the DNA. Here we describe a galactokinase (galK)-based two-step method to generate point mutations in the bacterial artificial chromosome (BAC) insert using the recombineering technology. It takes advantage of the ability to select and also counterselect for the presence of galK.

A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell-based assay.

Biallelic mutations in the human breast cancer susceptibility gene, BRCA2, are associated with Fanconi anemia, implying that some persons who inherit 2 deleterious variants of BRCA2 are able to survive even though it is well established that BRCA2 is indispensable for viability in mice. One such variant, IVS7 + 2T > G, results in premature protein truncation because of skipping of exon 7. Surprisingly, the persons who are either IVS7 + 2T > G homozygous or compound heterozygous are born alive but die of malignancy associated with Fanconi anemia. Using a mouse embryonic stem cell-based functional assay, we found that the IVS7 + 2T > G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2(Δ105)). We demonstrate that BRCA2(Δ105) is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. Evaluation of this transcript in normal and leukemia cells suggests that BRCA2(Δ105) may contribute to the viability of persons inheriting this mutation. In this study, we have also characterized 5 other BRCA2 variants and found 3 of these (p.L2510P, p.R2336H, and p.W2626C) to be deleterious and 2 (p.I2490T and p.K2729N) probably neutral. Such studies are important to understand the functional significance of unclassified BRCA2 variants.

Functional redundancy of exon 12 of BRCA2 revealed by a comprehensive analysis of the c.6853A>G (p.I2285V) variant.

Variants of unknown significance (VUS) in BRCA1 and BRCA2 are common, and present significant challenges for genetic counseling. We observed that BRCA2: c.6853A>G (p.I2285V) (Breast Cancer Information Core [BIC] name: 7081A>G; http://research.nhgri.nih.gov/bic/) co-occurs in trans with the founder mutation c.5946delT (p.S1982RfsX22) (BIC name: 6174delT), supporting the published classification of p.I2285V as a neutral variant. However, we also noted that when compared with wild-type BRCA2, p.I2285V resulted in increased exclusion of exon 12. Functional assay using allelic complementation in Brca2-null mouse embryonic stem cells revealed that p.I2285V, an allele with exon 12 deleted and wild-type BRCA2 were all phenotypically indistinguishable, as measured by sensitivity to DNA-damaging agents, effect on irradiation-induced Rad51 foci formation, homologous recombination, and overall genomic integrity. An allele frequency study showed the p.I2285V variant was identified in 15 out of 722 (2.1%) Ashkenazi Jewish cases and 10 out of 475 (2.1%) ethnically-matched controls (odds ratio, 0.99; 95% confidence interval: 0.44-2.21; P=0.97). Thus the p.I2285V variant is not associated with an increased risk for breast cancer. Taken together, our clinical and functional studies strongly suggest that exon 12 is functionally redundant and therefore missense variants in this exon are likely to be neutral. Such comprehensive functional studies will be important adjuncts to genetic studies of variants.

Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations.

To date, inheritance of a mutant BRCA1 or BRCA2 gene is the best-established indicator of an increased risk of developing breast cancer. Sequence analysis of these genes is being used to identify BRCA1/2 mutation carriers, though these efforts are hampered by the high frequency of variants of unknown clinical significance (VUSs). Functional evaluation of such variants has been restricted due to lack of a physiologically relevant assay. In this study we developed a functional assay using mouse ES cells to study variants of BRCA1. We introduced BAC clones with human wild-type BRCA1 or variants into Brca1-null ES cells and confirmed that only wild-type and a known neutral variant rescued cell lethality. The same neutral variant was also able to rescue embryogenesis in Brca1-null mice. A test of several BRCT domain mutants revealed all to be deleterious, including a VUS. Furthermore, we used this assay to determine the effects of BRCA1 variants on cell cycle regulation, differentiation, and genomic stability. Importantly, we discovered that ES cells rescued by S1497A BRCA1 exhibited significant hypersensitivity after gamma-irradiation. Our results demonstrate that this ES cell-based assay is a powerful and reliable method for analyzing the functional impact of BRCA1 variants, which we believe could be used to determine which patients may require preventative treatments.

Label-free global serum proteomic profiling reveals novel celecoxib-modulated proteins in familial adenomatous polyposis patients.

Celecoxib, a selective inhibitor of cyclooxygenase-2 (Cox-2), was efficacious in clinical prevention trials of patients with familial adenomatous polyposis (FAP) and sporadic colorectal cancer. To identify as yet poorly defined molecular determinants of celecoxib efficacy, a multidimensional serum fractionation approach was used coupled with nanospray tandem mass spectrometry to perform label-free global proteomic profiling of serum samples from the FAP/celecoxib prevention trial. Subsequently, the application of an algorithm for large-scale biomarker discovery on comparative serum proteomic profiles of pre- and post-celecoxib treatment samples identified 83 potentially celecoxib-responsive proteins from various cellular compartments, biological processes and molecular functions. Celecoxib modulation of some of these proteins was confirmed in serum samples of FAP patients and colorectal cancer cell lines by Western blotting. Thus, using a shotgun procedure to rapidly identify important celecoxib-modulated proteins, this pilot study has uncovered novel systemic changes some of which are highly relevant for carcinogenesis and vascular biology. Validation of selected markers, especially those involved in key signaling networks and those considered molecular indicators of cardiovascular pathology, in larger celecoxib clinical trials is expected to provide insights into the molecular mechanisms of celecoxib and the efficacy/toxicity issues related to its use as a chemopreventive agent.

Altered gene expression profiles define pathways in colorectal cancer cell lines affected by celecoxib.

It is well established that celecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2) and a tested chemopreventive agent, has several COX-2-independent activities. In an attempt to better understand COX-2-independent molecular mechanisms underlying the chemopreventive activity of celecoxib, we did global transcription profiling of celecoxib-treated COX-2-positive and COX-2-deficient colorectal cancer cell lines. Celecoxib treatment resulted in significantly altered expression levels of over 1,000 to 3,000 transcripts in these cell lines, respectively. A pathway/functional analysis of celecoxib-affected transcripts, using Gene Ontology and Biocarta Pathways and exploring biological association networks, revealed that celecoxib modulates expression of numerous genes involved in a variety of cellular processes, including metabolism, cell proliferation, apoptotic signaling, cell cycle check points, lymphocyte activation, and signaling pathways. Among these processes, cell proliferation and apoptotic signaling consistently ranked as the highest-scoring Gene Ontology terms and Biocarta Pathways in both COX-2 expresser and nonexpresser cell lines. Altered expression of many of the genes by celecoxib was confirmed by quantitative PCR and at the protein level by Western blotting. Many novel genes emerged from our analysis of global transcription patterns that were not previously reported to be affected by celecoxib. In the future, in-depth work on selected genes will determine if these genes may serve as potential molecular targets for more effective chemopreventive strategies.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.