Approximately 15% of human cancers utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain the lengths of their telomeres. The Fanconi anaemia protein FANCJ localizes to telomeric foci in human ALT cells, but not in telomerase-positive or primary cells. Telomere-associated FANCJ frequently localizes with FANCD2 and BRCA1, and primarily localizes to ALT-associated PML nuclear bodies. Depletion of FANCJ in human ALT cells causes the loss of BRCA1 at telomeric foci and a decrease in telomeric repeat DNA content primarily as a result of the loss of the brightest telomeric repeat DNA foci. In contrast, depletion of the FANCD2 results in increased telomeric repeat DNA synthesis and this is suppressed upon the codepletion of FANCJ. Together, data from this study suggest that FANCJ is required for telomeric repeat DNA synthesis in human ALT cells, which may or may not be dependent on BRCA1, and FANCD2 restrains this synthesis.

Approximately 15% of human cancers utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain the lengths of their telomeres. The Fanconi anaemia protein FANCJ localizes to telomeric foci in human ALT cells, but not in telomerase-positive or primary cells. Telomere-associated FANCJ frequently localizes with FANCD2 and BRCA1, and primarily localizes to ALT-associated PML nuclear bodies. Depletion of FANCJ in human ALT cells causes the loss of BRCA1 at telomeric foci and a decrease in telomeric repeat DNA content primarily as a result of the loss of the brightest telomeric repeat DNA foci. In contrast, depletion of the FANCD2 results in increased telomeric repeat DNA synthesis and this is suppressed upon the codepletion of FANCJ. Together, data from this study suggest that FANCJ is required for telomeric repeat DNA synthesis in human ALT cells, which may or may not be dependent on BRCA1, and FANCD2 restrains this synthesis.

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Abstract Two major genes are involved in hereditary predisposition to breast and ovarian cancer – BRCA1 and BRCA2. However, germline mutations in these tumor suppressors account for a maximum 20% of the familial breast cancer cases. A significant portion of the genes predisposing to this disease is unknown and therefore needs to be discovered. The aim of this study was to identify novel breast cancer susceptibility genes from the interweaving BRCA/Fanconi anemia (FA) pathway. Five candidate genes – MERIT40, ABRAXAS, BRIP1, CHK1, and FANCA – were screened for mutations by utilizing conformation-sensitive gel electrophoresis and sequencing, or with multiplex ligation-dependent probe amplification in blood DNA samples of Finnish familial breast cancer patients. Investigation of the MERIT40 gene revealed novel nucleotide changes, being the first report on mutation screening of this gene. None of the observed alterations, however, appeared to be disease related, suggesting that germline mutations in MERIT40 are rare or absent in breast cancer patients. A missense alteration (c.1082G>A, leading to Arg361Gln) was identified in ABRAXAS in 3 out of 125 Northern Finnish breast cancer families (2.4%), but not in any of the 867 healthy controls. The prevalence of the mutation between familial and control cases was statistically significantly different (p=0.002). ABRAXAS c.1082G>A appears to have pathological significance based on its exclusive occurrence in cancer cases, evolutionary conservation, disruption of a putative nuclear localization signal, reduced nuclear localization of the protein, and defective accumulation at DNA damage sites. The BRIP1 (FANCJ) and CHK1 genes were screened for large genomic rearrangements, but no abnormalities were detected, ruling out a significant contribution to breast cancer susceptibility in the Northern Finnish population. A novel large heterozygous deletion was identified in the FANCA gene in one out of 100 breast cancer families, removing the promoter and the first 12 exons. The deletion allele was not present in the tested controls, suggesting that it might contribute to breast cancer susceptibility. This is the first report on the association of a large-size germline deletion in a gene acting in the upstream part of the FA signaling pathway with familial breast cancer. / Tiivistelmä BRCA1 ja BRCA2 ovat kaksi tärkeintä perinnöllisen rinta- ja munasarjasyövän alttiusgeeniä. Niissä esiintyvät ituradan muutokset selittävät kuitenkin vain noin 20 % familiaalisista rintasyöpätapauksista. Suurin osa alttiusgeeneistä on edelleen tunnistamatta ja näitä tekijöitä etsitään aktiivisesti. Tämän tutkimuksen tarkoituksena on ollut tunnistaa uusia alttiustekijöitä toisiinsa läheisesti liittyviltä BRCA/Fanconin anemia (FA) signaalinsiirtoreiteiltä. Viisi kandidaattigeeniä - MERIT40, ABRAXAS, BRIP1, CHK1 ja FANCA – kartoitettiin mutaatioiden suhteen suomalaisissa rintasyöpäperheissä käyttämällä konformaatiosensitiivistä geelielektroforeesia ja sekvensointia, tai multiplex ligation-dependent probe amplification- menetelmää. MERIT40-geenissä havaittiin useita aikaisemmin raportoimattomia nukleotidimuutoksia, mutta yhdenkään niistä ei havaittu liittyvän rintasyöpäalttiuteen. MERIT40-geenimuutosten mahdollista yhteyttä rintasyöpäalttiuteen ei ole tutkittu aikaisemmin. ABRAXAS-geenissä havaittiin missense-mutaatio (c.1082G>A, joka johtaa Arg361Gln aminohappokorvautumiseen) kolmessa pohjoissuomalaisessa rintasyöpäperheessä (3/125, 2.4 %). Muutosta ei havaittu terveissä kontrolleissa (N=867), ja ero mutaation esiintyvyydessä familiaalisten rintasyöpätapausten ja terveiden kontrollien välillä oli tilastollisesti merkitsevä (p=0.002). ABRAXAS c.1082G>A-muutos on todennäköisesti patogeeninen, sillä kyseinen aminohappopaikka on evolutiivisesti konservoitunut ja sijaitsee todennäköisellä tumaanohjaussignaalialueella. Funktionaaliset kokeet osoittivat, että mutatoitunut proteiinituote lokalisoitui villityypin proteiinia heikommin tumaan ja sen ohjautuminen DNA-vaurioalueille oli puutteellista. BRIP1- (FANCJ) ja CHK1-geeneistä etsittiin laajoja genomisia uudelleenjärjestelyjä, mutta niitä ei havaittu. Näin ollen kyseisillä muutoksilla ei ole merkittävää roolia perinnöllisessä rintasyöpäalttiudessa suomalaisessa väestössä. FANCA-geenissä havaittiin laaja heterotsygoottinen deleetio yhdessä tutkitusta 100 rintasyöpäperheestä. Deleetio poistaa geenin promoottorialueen lisäksi sen 12 ensimmäistä eksonia. Deleetioalleelia ei havaittu terveissä kontrolleissa, joten se mahdollisesti liittyy perinnölliseen rintasyöpäalttiuteen. Tutkimus on ensimmäinen, jossa raportoidaan laaja genominen deleetio FA-signaalinsiirtoreitin ylävirran geenissä familiaalisessa rintasyövässä.

Mutations in the hereditary breast/ovarian cancer genes BRCA1/2 were shown to be synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). This toxicity is assumed to derive from PARPi-induced DNA double strand breaks (DSBs) that necessitate BRCA function in homologous recombination (HR) and/or fork protection (FP). However, PARPi accelerates replication forks. While high-speed replication could cause DSBs, the finding that PARPi leads to single-stranded DNA (ssDNA) gaps/nicks suggests replication gaps could also or alone be the cause of synthetic lethality. Here, we demonstrate that PARPi toxicity derives from replication gaps. Isogenic cells deficient in BRCA1 or the BRCA1-associated FANCJ, with common DNA repair defects in HR and FP, exhibit opposite responses to PARPi. Deficiency in FANCJ, a helicase also mutated in hereditary breast/ovarian cancer and Fanconi anemia, causes aberrant accumulation of fork remodeling factor HLTF and limits unrestrained DNA synthesis with ssDNA gaps. Thus, we predict replication gaps as a distinguishing factor and further uncouple HR, FP and fork speed from PARPi response. BRCA-deficient cells display excessive gaps that are diminished upon resistance, restored upon re-sensitization and when targeted augment synthetic lethality with PARPi. Furthermore, we define the source of gaps to defects in Okazaki fragment processing (OFP). Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1 but aberrantly low XRCC1 indicating a defective backup OFP pathway. Remarkably, 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. Collectively, our study highlights unprotected lagging strand gaps as a determinant of synthetic lethality, providing a new paradigm and biomarker for PARPi toxicity.