Refinement of the assignment to the ACMG/AMP BS3 and PS3 criteria of eight BRCA1 variants of uncertain significance by integrating available functional data with protein interaction assays.

The clinical screening of cancer predisposition genes has led to the identification of a large number of variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is limited by the amount of necessary data, which are difficult to obtain for rare variants. The guidelines for variant interpretation of the American College of Medical Genetics and Genomics along with the Association for Molecular Pathology (ACMG/AMP) state that "well-established" functional studies provide strong support of a pathogenic or benign impact (criteria PS3 and BS3, respectively) and can be used as evidence type to reach a final classification. Moreover, the Clinical Genome Resource Sequence Variant Interpretation Working Group developed rule specifications to refine the PS3/BS3 criteria. Recently, Lira PC et al. developed the "Hi Set" approach that generated PS3/BS3 codes for over two-thousands BRCA1 VUS. While highly successful, this approach did not discriminate a group of variants with conflicting evidences. Here, we aimed to implement the outcomes of the "Hi-set" approach applying Green Fluorescent Protein (GFP)-reassembly assays, assessing the effect of variants in the RING and BRCT domains of BRCA1 on the binding of these domains with the UbcH5a or ABRAXAS proteins, respectively. The analyses of 26 clinically classified variants, including 13 tested in our previous study, showed 100% sensitivity and specificity in identifying pathogenic and benign variants for both the RING/UbcH5a and the BRCTs/ABRAXAS interactions. We derived the strength of evidences generated by the GFP-reassembly assays corresponding to moderate for both PS3 and BS3 criteria assessment. The GFP-reassembly assays were applied to the functional characterization of 8 discordant variants from the study by Lyra et al. The outcomes of these analyses, combined with those reported in the "Hi Set" study, allowed the assignment of ACMG/AMP criteria in favor or against pathogenicity for all 8 examined variants. The above findings were validated with a semi-quantitative Mammalian Two-Hybrid approach, and totally concordant results were observed. Our data contributes in shedding light on the functional significance of BRCA1 VUS and on their clinical interpretation within the ACMG/AMP framework.

LKB1 Down-Modulation by miR-17 Identifies Patients With NSCLC Having Worse Prognosis Eligible for Energy-Stress-Based Treatments.

INTRODUCTION: Preclinical models recently unveiled the vulnerability of LKB1/KRAS comutated NSCLC to metabolic stress-based treatments. Because miR-17 is a potential epigenetic regulator of LKB1, we hypothesized that wild-type LKB1 (LKB1WT) NSCLC with high miR-17 expression may be sensitive to an energetic stress condition, and eligible for metabolic frailties-based therapeutic intervention. METHODS: We took advantage of NSCLC cell lines with different combinations of KRAS mutation and LKB1 deletion and of patient-derived xenografts (PDXs) with high (LKB1WT/miR-17 high) or low (LKB1WT/miR-17 low) miR-17 expression. We evaluated LKB1 pathway impairment and apoptotic response to metformin. We retrospectively evaluated LKB1 and miR-17 expression levels in tissue specimens of patients with NSCLC and PDXs. In addition, a lung cancer series from The Cancer Genome Atlas data set was analyzed for miR-17 expression and potential correlation with clinical features. RESULTS: We identified miR-17 as an epigenetic regulator of LKB1 in NSCLC and confirmed targeting of miR-17 to LKB1 3' untranslated region by luciferase reporter assay. We found that miR-17 overexpression functionally impairs the LKB1/AMPK pathway. Metformin treatment prompted apoptosis on miR-17 overexpression only in LKB1WT cell lines, and in LKB1WT/miR-17 high PDXs. A retrospective analysis in patients with NSCLC revealed an inverse correlation between miR-17 and LKB1 expression and highlighted a prognostic role of miR-17 expression in LKB1WT patients, which was further confirmed by The Cancer Genome Atlas data analysis. CONCLUSIONS: We identified miR-17 as a mediator of LKB1 expression in NSCLC tumors. This study proposes a miR-17 expression score potentially exploitable to discriminate LKB1WT patients with NSCLC with impaired LKB1 expression and poor outcome, eligible for energy-stress-based treatments.

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors.

c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a.

Lung cancer causes approximately one fifth of all cancer deaths. Tumour cells actively communicate with the surrounding microenvironment to support malignant progression. Extracellular vesicles (EVs) play a pivotal role in intercellular communication and modulate recipient cells by delivering their contents, including proteins and nucleic acids such as microRNAs (miRNAs). We isolated EVs from the conditioned medium (CM) of human lung cancer cell lines and plasma of lung cancer patients and cancer-free smokers using an ultracentrifugation method. A significant increase in bronchial HBEC-KRASV12high cell proliferation, confirmed by cell cycle analysis, was observed after treatment with cancer-derived EVs. Lung cancer-derived EVs induced transcription of the pri-miR-92a gene, resulting in the overexpression of mature miR-19b and miR-92a in recipient bronchial cells. Modulation of these two miRNAs using miRNA mimics or inhibitors confirmed their ability to promote proliferation. In silico analysis and experimental validation showed that miR-19b and miR-92a impaired the TGF-beta (TGFB) pathway and identified TGFBRI and TGFBRII as target genes involved in EV-mediated bronchial cell proliferation. Interestingly, the oncoprotein c-Myc, a well-known miR-17-92 cluster activator, was detected only in the EVs derived from lung cancer patients and cell lines and was able to modulate the proliferation of HBEC-KRASV12high recipient cells. These data support the role of c-Myc shuttling in lung cancer-derived EVs in inducing the upregulation of onco-miR-19b and miR-92a expression with concomitant impairment of the TGFB signalling pathway in recipient cells.

GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes.

Genetic testing for BRCA1 and BRCA2 genes has led to the identification of many unique variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is conditioned by the amount of necessary data, which are difficult to obtain if a variant is rare. As an alternative, variants mapping to the coding regions can be examined using in vitro functional assays. BRCA1 and BRCA2 proteins promote genome protection by interacting with different proteins. In this study, we assessed the functional effect of two sets of variants in BRCA genes by exploiting the green fluorescent protein (GFP)-reassembly in vitro assay, which was set-up to test the BRCA1/BARD1, BRCA1/UbcH5a, and BRCA2/DSS1 interactions. Based on the findings observed for the validation panels of previously classified variants, BRCA1/UbcH5a and BRCA2/DSS1 binding assays showed 100% sensitivity and specificity in identifying pathogenic and non-pathogenic variants. While the actual efficiency of these assays in assessing the clinical significance of BRCA VUS has to be verified using larger validation panels, our results suggest that the GFP-reassembly assay is a robust method to identify variants affecting normal protein functioning and contributes to the classification of VUS.

Circulating mir-320a promotes immunosuppressive macrophages M2 phenotype associated with lung cancer risk.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression.

Two Missense Variants Detected in Breast Cancer Probands Preventing BRCA2-PALB2 Protein Interaction.

PALB2 (partner and localizer of BRCA2) was initially identified as a binding partner of BRCA2. It interacts also with BRCA1 forming a complex promoting DNA repair by homologous recombination. Germline pathogenic variants in BRCA1, BRCA2 and PALB2 DNA repair genes are associated with high risk of developing breast cancer. Mutation screening in these breast cancer predisposition genes is routinely performed and allows the identification of individuals who carry pathogenic variants and are at risk of developing the disease. However, variants of uncertain significance (VUSs) are often detected and establishing their pathogenicity and clinical relevance remains a central challenge for the risk assessment of the carriers and the clinical decision-making process. Many of these VUSs are missense variants leading to single amino acid substitutions, whose impact on protein function is uncertain. Typically, VUSs are rare and due to the limited genetic, clinical, and pathological data the multifactorial approaches used for classification cannot be applied. Thus, these variants can only be characterized through functional analyses comparing their effect with that of normal and mutant gene products used as positive and negative controls. The two missense variants BRCA2:c.91T >G (p.Trp31Gly) and PALB2:c.3262C >T (p.Pro1088Ser) were detected in two breast cancer probands originally ascertained at Breast Cancer Units of Institutes located in Milan and Bergamo (Northern Italy), respectively. These variants were located in the BRCA2-PALB2 interacting domains, were predicted to be deleterious by in silico analyses, and were very rare and clinically not classified. Therefore, we initiate to study their functional effect by exploiting a green fluorescent protein (GFP)-reassembly in vitro assay specifically designed to test the BRCA2-PALB2 interaction. This functional assay proved to be easy to develop, robust and reliable. It also allows testing variants located in different genes. Results from these functional analyses showed that the BRCA2:p.Trp31Gly and the PALB2:p.Pro1088Ser prevented the BRCA2-PALB2 binding. While caution is warranted when the interpretation of the clinical significance of rare VUSs is based on functional studies only, our data provide initial evidences in favor of the possibility that these variants are pathogenic.

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10-115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Characterization of an Italian founder mutation in the RING-finger domain of BRCA1.

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area.

Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.

Characterization of the activation domain of the Rad53 checkpoint kinase.

Rad53 protein, the yeast orthologue of the human checkpoint kinase Chk2, presents two highly conserved phosphorylatable threonine residues (T354 and T358) in the activation domain, whose phosphorylation is critical to allow the activation of the kinase. In this study we found that Rad53 protein variants in which alanine and/or aspartate replace the threonine residues 354 and/or 358 do not retain kinase activity and do not undergo auto-phosphorylation, leading to defect in the checkpoint response and iper-sensitivity to DNA damage and DNA replication stress agents. Interestingly, we found that the rad53-T358D mutation severely affects the kinase activity and causes accumulation of the S129-phosphorylated isoform of histone H2A, even during an unperturbed cell cycle, thus indicating the accumulation of spontaneous DNA breaks. We further found that the protein level of Sml1, which is the physiological inhibitor of ribonucleotide reductase, remains high during DNA replication in rad53-T358D cells, suggesting that an inadequate pool of dNTPs in checkpoint defective cells causes the accumulation of spontaneous DNA breaks. In conclusion, our results indicate that phosphorylation of both T354 and T358 residues strongly influences the catalytic activity of Rad53 also in unperturbed cell cycles, and support the notion that Rad53 is essential to preserve genome integrity, by controlling the level of Sml1 and the functionality of ribonucleotide reductase.