Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.Leukemia advance online publication, 17 June 2016; doi:10.1038/leu.2016.149.

Chemoprevention in BRCA1 Mutation Carriers—A Proof-of-Concept Study

Background: Women with germline BRCA1 mutations have a high lifetime risk of breast cancer, with the only available risk-reduction strategies being risk-reducing surgery or chemoprevention. These women predominantly develop triple-negative breast cancers; hence, it is unlikely that selective estrogen receptor modulators (serms) will reduce the risk of developing cancer, as these have not been shown to reduce the incidence of estrogen receptor–negative breast cancers. Preclinical data from our laboratory suggest that exposure to estrogen and its metabolites is capable of causing dna double-strand breaks (dsbs) and thus driving genomic instability, an early hallmark of BRCA1-related breast cancer. Therefore, an approach that lowers circulating estrogen levels and reduces estrogen metabolite exposure may prove a successful chemopreventive strategy.

Aims: To provide proof of concept of the hypothesis that the combination of luteinizing-hormone releasing-hormone agonists (lhrha) and aromatase inhibitors (ais) can suppress circulating levels of estrogen and its metabolites in BRCA1 mutation carriers, thus reducing estrogen metabolite levels in breast cells, reducing dna dsbs, and potentially reducing the incidence of breast cancer.

Methods: 12 Premenopausal BRCA1 mutation carriers will undergo baseline ultrasound-guided breast core biopsy and plasma and urine sampling. Half the women will be treated for 3 months with combination goserelin (lhrha) plus anastrazole (ai), and the remainder with tamoxifen (serm) before repeat tissue, plasma, and urine sampling. After a 1-month washout period, groups will cross over for a further 3 months treatment before final biologic sample collection. Tissue, plasma, and urine samples will be examined using a combination of immunohistochemistry, comet assays, and ultrahigh performance liquid chromatography tandem mass spectrometry to assess the impact of lhrha plus ai compared with serm on levels of dna damage, estrogens, and genotoxic estrogen metabolites. Quality of life will also be assessed during the study.

Results: This trial is currently ongoing.

Breast cancer outcomes following predictive BRCA testing in Northern Ireland

Objectives: Since 1995, BRCA testing has identified 445 women in Northern Ireland who carry a pathogenic BRCA1/2 mutation, without breast cancer (bca) at testing. This study examined outcomes with reference to management, bca risk, and incidence following positive predictive testing. Methods: Patients were identified from the regional genetics database. Electronic clinical records were used to obtain management and outcome details. Median follow-up was to bca diagnosis, risk-reducing mastectomy (rrm), death, or last follow-up. Results: 169 women had a BRCA1 mutation, and 276 BRCA2. ■ BRCA1 cohort: Median follow-up post-testing was 3 years. 56 Women (33%) had rrm, and 12 are awaiting rrm (total 68, 40%) at a median age of 36 years. 12 Women (7%) developed bca, at a median of 2 years following testing. 4 Women were diagnosed with bcas incidentally at rrm. 7 Patients had bilateral mastectomies following a cancer diagnosis. 1 Woman developed bca following rrm (1.7%). Three deaths were reported: 1 breast cancer (1.7%), 1 ovarian cancer (1.7%), and 1 with no recorded breast/ovarian cancer diagnosis. ■ BRCA2 cohort: Median follow-up post-testing was 6 years. rrm was carried out in 75 women (27%), with 20 awaiting rrm (total 95, 35%); median age: 39 years. 16 Women developed bca (5.8%), at a median of 5 years from testing. 6 Women were diagnosed with cancer incidentally at rrm; 9 women had bilateral mastectomy following diagnosis, and 1 developed bca following rrm (1.3%). Five deaths were reported: 1 bca, 1 ovarian cancer, and 3 with no recorded breast/ovarian cancer diagnosis. Conclusions: The uptake of rrm following predictive BRCA testing in Northern Ireland is comparable with that reported elsewhere. The incidence of bca following rrm is low (<2%) in our cohort, with low breast and ovarian cancer–specific mortality following positive predictive testing.

Identification of potentially pathogenic variants in candidate breast and ovarian cancer susceptibility genes in BRCAX patients

Mutations within the BRCA1 and BRCA2 genes account for approximately 20% of hereditary breast cancers, with a further 10%–15% being attributable to rare mutations in moderate-risk genes and common variants in low-risk genes. The genes harbouring mutations in the remaining ∼65% of hereditary breast cancers are unknown. The identification of mutation carriers in hereditary breast and ovarian cancer (hboc) families is critical for determining who is most at risk of developing the disease and therefore who should be offered risk-reducing procedures or more intensive screening, or both.

Many of the high- and moderate-risk genes for hereditary breast cancers encode proteins that work in concert to maintain genomic stability and in dna damage signalling and repair. A novel BRCA1 protein complex identified within the research group whose target genes are involved in dna repair provided novel candidates for hboc susceptibility genes. These 12 candidate genes were sequenced in a cohort of 675 affected individuals from the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) with hereditary breast or ovarian cancer, but with no mutations in known susceptibility genes (BRCAx patients). This analysis identified 20 individuals (each from a different BRCAx family) with different potentially pathogenic variants across 6 of the candidate hboc susceptibility genes. The family members of each BRCAx index case were tested for the presence of the specific mutation identified in the proband to examine segregation with disease. To further expand on the potential role of the novel candidate hboc susceptibility genes identified in this study, the genetic variation of a second cohort of 520 Northern Irish BRCAx patients is being characterized using a 61-gene panel.

Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control

Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. 

'That Puissant City': John Milton's Roman Sojourns 1638-1639

John Milton’s sojourns in Rome (1638-9) are attested by his comments in Defensio Secunda, by the minutes of the English College, by Latin encomia which he received from Roman academicians, and, not least, by his Latin letter to Lucas Holstenius (19/29 March 1639), and several Latin poems which he composed in the course of his residency in the capital city: Ad Salsillum, and three Latin epigrams extolling the praises of the virtuosa soprano, Leonora Baroni. Read together, these texts serve to reveal much about Milton’s participation in, and reaction to, the ‘Puissant City’, (History of Britain, Bk 2).

The present monograph presents fresh evidence of Milton's integration into the academic and cultural life of seventeenth-century Rome. It argues that his links with two Roman academies: the Accademia dei Fantastici and Accademia degli Umoristi constitute a sustained participation in an academic community paralleling that of his independently attested performance in Florentine academies (on which I have published extensively). It also investigates his links with Alessandro Cherubini, David Codner, Giovanni Batista Doni, and the Baroni circle hymned in three published anthologies.

Chapter 1: Milton and the Accademia dei Fantastici investigates the cultural climate surrounding Milton's Ad Salsillum by examining two of that academy's publications: the Poesie dei Signori Accademici Fantastici di Roma (Rome, 1637) and the Academia Tenuta da Fantastici a. 12 di Maggio 1655 (Rome, 1655), the latter celebrating the creation of Fabio Chigi as Pope Alexander VII on 5 April 1655. Read in a new light, Milton’s self-fashioning, it is argued, takes its place not only alongside Salzilli’s encomium in Milton's honour, and his Italian sonnets in the 1637 Poesie, but also in relation to other poems in that collection, and the academy's essentially Catholic eulogistic trend. The chapter also provides fresh evidence of Salzilli’s survival of the illness described in Milton’s poem by his epistolary correspondence with Tomaso Stigliani.

Chapter 2: Milton and the Vatican argues for links between Milton’s Latin letter to Holstenius and a range of Holstenius’ published works: his edition of the axioms of the later Pythagoreans gifted by him to Milton, and his published neo-Platonic works. This is achieved by mutual appropriation of Similitudes in a series of Miltonic similes, the anabasis/katabasis motifs in a reworking of the Platonic theory of the transmigration of souls, and allusion to etymological details highlighted in Holstenius’ published editions. The chapter also reveals Milton’s alertness to typographical procedures and, by association, to Holstenius’ recent role (1638) as Director of the press of the Biblioteca Vaticana.

Chapter 3: Milton and the Accademia degli Umoristi argues for Milton’s likely participation in this Roman academy, as suggested by his links with its members. His three Latin epigrams in praise of Leonora Baroni, the only female member of the Umoristi, have hitherto been studied in relation to the 1639 Applausi in her honour. In a new reading, Milton, it is suggested, invokes and interrogates Catholic doctrine before a Catholic audience only to view the whole through the lens of a neo-Platonic Hermeticism (by echoing the phraseology of the sixteenth-century Franciscan Hannibal Rosselli) that refreshingly transcends religious difference. Crucially, the hitherto neglected L’Idea della Veglia (Rome, 1640) includes further encomiastic verse, sonnets to, and by Leonora, and details of the conversazioni hosted by her family at the precise time of Milton’s Roman sojourns. Milton may well have been a participant. The chapter concludes in an assessment of his links with the youthful prodigy Alessandro Cherubini, and of his audience with Francesco Barberini.

Chapter 4: Milton at a Roman Opera analyses the potential impact of ‘Chi Soffre, Speri’, which he attended on 18/28 February 1639, mounted by Francesco Barberini to inaugurate the recently completed theatre of the Palazzo Barberini. A detailed analysis of the opera's libretto, music, and theatricality casts a backward glance to Milton's Comus, and a forward glance to Paradise Lost. It also assesses Milton’s musical interests at this time, as attested by his links with Doni, and his purchase of works by Monteverdi and others.

Chapter 5: Milton’s English Connections in Rome develops the work of Miller and Chaney by investigating Milton’s co-diners at the English College in Rome on 30 October 1638, and by analysing his links between David Codner (alias Matteo Selvaggio), and the family of Jane Savage, Marchioness of Winchester, lamented by Milton in 1631. It also assesses his potential relations with the Englishman Thomas Gawen, who ‘accidentally sometimes fell into the company of John Milton’ (Antony Wood).

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.