CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

HELB Is a Feedback Inhibitor of DNA End Resection.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

OBJECTIVE: Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. BACKGROUND: Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. METHODS: We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. RESULTS: Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. CONCLUSIONS: Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Unilateral lupus mastitis.

Lupus mastitis is a rare clinical manifestation associated with systemic lupus erythematosus or discoid lupus erythematosus. It is necessary to make a correct diagnosis to differentiate it from inflammatory breast cancer. The histological study shows involvement of the adipose tissue of the breast with histopathological findings of cutaneous lupus erythematosus. Direct immunofluorescence detects the lupus band at the dermal-epidermal junction. The treatment of choice is hydroxychloroquine. We present a case of unilateral lupus mastitis in a patient with no previous diagnosis of lupus with complete remission after the use of hydroxychloroquine and topical corticosteroids.

Impact of stereotype threat on brain activity during memory tasks in older adults.

We report the first neuroimaging experiment to investigate the impact of explicitly activating aging stereotypes (i.e., stereotype threat) on brain activity during cognitive tasks. Cognitively normal older adults read about aging stereotypes or a control passage prior to taking episodic memory, working memory, and a non-demanding control task during fMRI. At the group level, stereotype activation did not impact cognitive performance or measures sensitive to stress and anxiety (physiological or self-report), but like prior work, highly educated and retired adults exhibited greater stereotype effects on episodic memory. At the neural level, stereotype activation did not impact brain activity in executive control or emotional regulation regions previously linked to stereotype threat effects in younger adults, suggesting that stereotype threat operates differently in older adults. Instead, on each task, the stereotype group showed more brain activity than the control group in parietal midline regions (e.g., precuneus, posterior cingulate). Although activity in these regions can arise from many processes, they have previously been associated with self-referential thinking and error-prevention focus, and in our study, brain activity in these regions was associated with slower responses and lower false alarm errors on the episodic memory task. Collectively, these findings are more consistent with the regulatory fit hypothesis than an executive control interference hypothesis of stereotype threat effects in older adults, whereby older adults adopt an error-prevention mindset in response to explicit stereotype threat.

Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

[Figure: see text].

The effect of feedback and recollection rejection instructions on the development of memory monitoring and accuracy.

Recollection rejection (a form of memory monitoring) involves rejecting false details on the basis of remembering true details (recall to reject), thereby increasing memory accuracy. This study examined how recollection rejection instructions and feedback affect memory accuracy and false recognition in 5-year-olds, 6- and 7-year-olds, 8- and 9-year-olds, and adults. Participants (N = 336) completed three study-test phases. Instructions and item-level feedback were manipulated during the first two phases, with the third phase including a test containing no instructions or feedback to evaluate learning effects. As predicted, in the younger children, as compared with the older children and adults, we found reduced accuracy scores (hits to studied items minus false alarms to related lures), reduced recollection rejection to related lures, and increased false recognition scores. We also found that, in the third phase, prior feedback reduced false recognition scores, potentially by improving monitoring, and typical developmental differences in false recognition were eliminated. However, there were mixed findings of instructions and feedback, and in some conditions these interventions harmed memory. These findings provide initial evidence that combining instructions and feedback with repeated task practice may improve monitoring effectiveness, but additional work is needed on how these factors improve and sometimes harm performance in young children.

Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

Effects of COVID-19 Lockdown on Tumour Burden of Melanoma and Cutaneous Squamous Cell Carcinoma.

The aim of this study was to compare tumour burden in patients who underwent surgery for melanoma and cutaneous squamous cell carcinoma during nationwide lockdown in Spain due to COVID-19 (for the period 14 March to 13 June 2020) and during the same dates in 2019 before the COVID-19 pandemic. In addition, associations between median tumour burden (Breslow thickness for melanoma and maximum clinical diameter for cutaneous squamous cell carcinoma) and demographic, clinical, and medical factors were analysed, building a multivariate linear regression model. During the 3 months of lockdown, there was a significant decrease in skin tumours operated on (41% decrease for melanoma (n = 352 vs n = 207) and 44% decrease for cutaneous squamous cell carcinoma (n = 770 vs n = 429)) compared with the previous year. The proportion of large skin tumours operated on increased. Fear of SARS-CoV-2 infection, with respect to family member/close contact, and detection of the lesion by the patient or doctor, were related to thicker melanomas; and fear of being diagnosed with cancer, and detection of the lesion by the patient or relatives, were related to larger size cutaneous squamous cell carcinoma. In conclusion, lockdown due to COVID-19 has resulted in a reduction in treatment of skin cancer.

Carbon monoxide protects the kidney through the central circadian clock and CD39.

Ischemia reperfusion injury (IRI) is the predominant tissue insult associated with organ transplantation. Treatment with carbon monoxide (CO) modulates the innate immune response associated with IRI and accelerates tissue recovery. The mechanism has been primarily descriptive and ascribed to the ability of CO to influence inflammation, cell death, and repair. In a model of bilateral kidney IRI in mice, we elucidate an intricate relationship between CO and purinergic signaling involving increased CD39 ectonucleotidase expression, decreased expression of Adora1, with concomitant increased expression of Adora2a/2b. This response is linked to a >20-fold increase in expression of the circadian rhythm protein Period 2 (Per2) and a fivefold increase in serum erythropoietin (EPO), both of which contribute to abrogation of kidney IRI. CO is ineffective against IRI in Cd39-/- and Per2-/- mice or in the presence of a neutralizing antibody to EPO. Collectively, these data elucidate a cellular signaling mechanism whereby CO modulates purinergic responses and circadian rhythm to protect against injury. Moreover, these effects involve CD39- and adenosinergic-dependent stabilization of Per2. As CO also increases serum EPO levels in human volunteers, these findings continue to support therapeutic use of CO to treat IRI in association with organ transplantation, stroke, and myocardial infarction.

Miliaria crystallina induced by idarubicin and all-trans-retinoic acid: Two case reports.

Miliaria crystallina is a benign, self-limiting disorder of the eccrine sweat glands characterized by the obstruction of the sweat ducts, which leads to secondary sweat retention into stratum corneum. We present two patients with MC during treatment with idarubicin and all-trans-retinoic acid (ATRA) for acute promyelocytic leukaemia (APL). Anthracyclines can be excreted through sweat and induce MC through exfoliation. The use of idarubicin in combination with ATRA would favour the process of producing a peeling effect. Reports of MC associated with idarubicin and ATRA are scarce. Recognizing this benign entity and its triggers will help to differentiate it from other skin reactions, improving the management of patients by avoiding unnecessary studies and treatments.

Elastosis perforans serpiginosa induced by d-penicillamine treated with cyclosporine and allopurinol.

Elastosis perforans serpiginosa (EPS) is a rare condition within the group of perforating dermatoses. It is characterized by the synthesis of anomalous elastic fibers that are eliminated through perforating channels (transepidermal elimination). It is classified into three subtypes. One of them is drug-induced by prolonged treatment with d-penicillamine. This drug is a heavy metal chelator used to treat diseases such as rheumatoid arthritis, cystinuria, and Wilson's disease. Years of treatment with d-penicillamine at high doses are required for developing EPS, with occasional slow regression after drug withdrawal. There is no established treatment for EPS, with described cases using various treatment options such as corticoids, retinoids, tazarotene, cryotherapy, imiquimod, photodynamic therapy, electrosurgery, and CO2 laser among others with inconsistent results. We present a case of EPS induced by d-penicillamine with favorable response to cyclosporine and allopurinol in a patient with a history of Wilson's disease since childhood. They maybe considered as possible therapeutic options not described so far for an entity with variable response to current treatments. We highlight the extensive involvement of the case with progression, despite the suspension of d-penicillamine and failure to previous treatments with photodynamic therapy and retinoids.

Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner.

Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.

Assembly of Slx4 signaling complexes behind DNA replication forks.

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

Acquired Dermal Melanocytosis Associated With Diffuse Systemic Sclerosis in a White Patient.

We report a case of a 65-year-old man who developed an asymptomatic bluish spot that affected the flank and left lumbar region with the onset 10 years prior. He had a history of diffuse systemic sclerosis with anti-Scl-70-positive antibodies. The appearance of the skin lesion coincided with the onset of his disease. The skin biopsy was consistent with the diagnosis of acquired dermal melanocytosis. The relationship between the appearance of acquired pigmented macules and spots and systemic sclerosis has been known for years, although it is an infrequent finding.