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Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

Coronavirus disease 2019 (COVID-19) antiviral response in a pan-tumor immune monitoring (CAPTURE) (NCT03226886) is a prospective cohort study of COVID-19 immunity in patients with cancer. Here we evaluated 585 patients following administration of two doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after two doses were 85% and 59% in patients with solid and hematological malignancies, respectively. A lower proportion of patients had detectable titers of neutralizing antibodies (NAbT) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) versus wild-type (WT) SARS-CoV-2. Patients with hematological malignancies were more likely to have undetectable NAbT and had lower median NAbT than those with solid cancers against both SARS-CoV-2 WT and VOC. By comparison with individuals without cancer, patients with hematological, but not solid, malignancies had reduced neutralizing antibody (NAb) responses. Seroconversion showed poor concordance with NAbT against VOC. Previous SARS-CoV-2 infection boosted the NAb response including against VOC, and anti-CD20 treatment was associated with undetectable NAbT. Vaccine-induced T cell responses were detected in 80% of patients and were comparable between vaccines or cancer types. Our results have implications for the management of patients with cancer during the ongoing COVID-19 pandemic.

No abstract available.

OBJECTIVES Increased demand for quality health care has led to lay-press ranking systems, such as the ranking from US News and World Report (US News). Their "Best Hospitals" publication advertises itself as the go-to resource for patients seeking care in a number of specialty areas. We sought to test the relationship between US News rankings and transplant outcomes. MATERIALS AND METHODS Using data from 2014 to 2018, we compared outcomes from the Scientific Registry of Transplant Recipients database for liver and kidney transplants against US News-ranked centers using the categories "Nephrology" and "GI Surgery and Gastroenterology" as substitutes, as US News does not rank transplant centers specifically. P < .05 was set as significant. RESULTS Using hazard ratio data, we found that kidney transplant center rank had only a small impact on postoperative outcomes in terms of patient survival (hazard ratio = 0.996, P = .049) but had no impact on graft survival (hazard ratio = 0.997, P = .077). In addition, liver transplant center rank had no impact on liver graft survival (hazard ratio = 1.003, P = .304). The impact of hospital ranking on survival was minimal compared with other variables. CONCLUSIONS The US News rankings for "Nephrology" and "GI Surgery and Gastroenterology" have minimal values as a measure of liver and kidney transplant outcomes, highlighting that these lay press rankings are not useful to the unique transplant patient population and that providers should help guide patients to transplant-specific resources.

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment. IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

BackgroundRoutine asymptomatic testing using RT-PCR of people who interact with vulnerable populations, such as medical staff in hospitals or care workers in care homes, has been employed to help prevent outbreaks among vulnerable populations. Although the peak sensitivity of RT-PCR can be high, the probability of detecting an infection will vary throughout the course of an infection. The effectiveness of routine asymptomatic testing will therefore depend on testing frequency and how PCR detection varies over time.MethodsWe fitted a Bayesian statistical model to a dataset of twice weekly PCR tests of UK healthcare workers performed by self-administered nasopharyngeal swab, regardless of symptoms. We jointly estimated times of infection and the probability of a positive PCR test over time following infection, we then compared asymptomatic testing strategies by calculating the probability that a symptomatic infection is detected before symptom onset and the probability that an asymptomatic infection is detected within 7 days of infection.FindingsWe estimated that the probability that the PCR test detected infection peaked at 77% (54 - 88%) 4 days after infection, decreasing to 50% (38 - 65%) by 10 days after infection. Our results suggest a substantially higher probability of detecting infections 1–3 days after infection than previously published estimates. We estimated that testing every other day would detect 57% (33-76%) of symptomatic cases prior to onset and 94% (75-99%) of asymptomatic cases within 7 days if test results were returned within a day.InterpretationOur results suggest that routine asymptomatic testing can enable detection of a high proportion of infected individuals early in their infection, provided that the testing is frequent and the time from testing to notification of results is sufficiently fast.FundingWellcome Trust, National Institute for Health Research (NIHR) Health Protection Research Unit, Medical Research Council (UKRI)

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes., Graphical Abstract Graphical abstractHealthy astrocytes (left, blue) have prevalent nuclear intron retention in reactivity transcripts, promoting their nuclear confinement (introns shown as red rectangles). Conversely, ALS astrocytes (right, red) exhibit decreased intron retention in reactivity transcripts, enabling cytoplasmic translocation and translation upon engaging ribosomes, underlying their reactive transformation. A small number of intron retaining transcripts escape to the cytoplasm where they are degraded by nonsense mediated decay (NMD), which is enhanced in ALS astrocytes.

Highlights • We found a high asymptomatic rate in vulnerable people with epilepsy. • Enhanced surveillance allows to quickly contain outbreaks. • We report a low rate of COVID-19 morbidity and mortality in a long-term care facility. • Preventative measures allow reducing resident-to-resident and -to-caregiver transmission. • Children and young adults appear to have lower infection rates., In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance. During 25-week observation period (16 March–6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate. We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth’s (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive. Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes.

Antiretroviral treatment (ART) reduces the risk of developing active tuberculosis (TB) in HIV-1 co-infected persons. In order to understand host immune responses during ART in the context of Mycobacterium tuberculosis (Mtb) sensitization, we performed RNAseq analysis of whole blood-derived RNA from individuals with latent TB infection coinfected with HIV-1, during the first 6 months of ART. A significant fall in RNA sequence abundance of the Hallmark IFN-alpha, IFN-gamma, IL-6/JAK/STAT3 signaling, and inflammatory response pathway genes indicated reduced immune activation and inflammation at 6 months of ART compared to day 0. Further exploratory evaluation of 65 soluble analytes in plasma confirmed the significant decrease of inflammatory markers after 6 months of ART. Next, we evaluated 30 soluble analytes in QuantiFERON Gold in-tube (QFT) samples from the Ag stimulated and Nil tubes, during the first 6 months of ART in 30 patients. There was a significant decrease in IL-1alpha and IL-1beta (Ag-Nil) concentrations as well as MCP-1 (Nil), supporting decreased immune activation and inflammation. At the same time, IP-10 (Ag-nil) concentrations significantly increased, together with chemokine receptor-expressing CD4 T cell numbers. Our data indicate that ART-induced decrease in immune activation combined with improved antigen responsiveness may contribute to reduced susceptibility to tuberculosis in HIV-1/Mtb co-infected persons.

Tissue homeostasis is critically dependent on the function of tissue-resident lymphocytes, including lipid-reactive invariant natural killer T (iNKT) cells. Yet, if and how the tissue environment shapes the antigen specificity of iNKT cells remains unknown. By analysing iNKT cells from lymphoid tissues of mice and humans we demonstrate that their T cell receptor (TCR) repertoire is highly diverse and is distinct for cells from various tissues resulting in differential lipid-antigen recognition. Within peripheral tissues iNKT cell recent thymic emigrants exhibit a different TCR repertoire than mature cells, suggesting that the iNKT population is shaped after arrival to the periphery. Consistent with this, iNKT cells from different organs show distinct basal activation, proliferation and clonal expansion. Moreover, the iNKT cell TCR repertoire changes following immunisation and is shaped by age and environmental changes. Thus, post-thymic modification of the TCR-repertoire underpins the distinct antigen specificity for iNKT cells in peripheral tissues

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1., Targeted CRISPR libraries expand the use of genetic screens across experimental conditions. Here, the authors develop a method for generating and analysing small scale custom CRISPR libraries and use it in the human and livestock pathogen Toxoplasma gondii to identify virulence factors in mice.

Summary The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage., Graphical Abstract, Highlights • UV elicits elongation slowdown and restricts transcription to the 5′ end of genes • UV induces a switch from long to short alternative last exon (ALE) transcript isoforms • ASCC3 short and long ALE isoforms have antagonistic functions in the UV response • The UV-induced ASCC3 short isoform functions as a long non-coding RNA, UV damage generates a functional non-coding RNA through alternative pre-mRNA processing of a damage response factor transcript, identifying a pathway for repurposing protein coding genes under selective conditions.

Cockayne syndrome (CS) is a multisystem disorder with severe neurological symptoms. The majority of CS patients carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled nucleotide excision repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we investigate the connection between the neuropathology of CS and dysregulation of gene expression. Transcriptome analysis of human fibroblasts revealed that even in the absence of DNA damage, CSB affects the expression of thousands of genes, many of which are neuronal genes. CSB is present in a significant subset of these genes, suggesting that regulation is direct, at the level of transcription. Importantly, reprogramming of CS fibroblasts to neuron-like cells is defective unless an exogenous CSB gene is introduced. Moreover, neuroblastoma cells from which CSB is depleted show defects in gene expression programs required for neuronal differentiation, and fail to differentiate and extend neurites. Likewise, neuron-like cells cannot be maintained without CSB. Finally, a number of disease symptoms may be explained by marked gene expression changes in the brain of patients with CS. Together, these data point to dysregulation of gene regulatory networks as a cause of the neurological symptoms in CS.

The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation. Cohesin loading onto chromosomes depends on the Scc2-Scc4 cohesin loader complex, but the chromatin features that form cohesin loading sites remain poorly understood. Here we show that the RSC chromatin remodeling complex recruits budding yeast Scc2-Scc4 to broad nucleosome-free regions, which the cohesin loader helps to maintain. Consequently, inactivation of either the cohesin loader or the RSC complex has similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results show an intimate link between local chromatin structure and higher-order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, which is caused by alterations in the human cohesin loader, and Coffin-Siris syndrome, which results from alterations in human RSC complex components. Both syndromes can arise from gene misregulation due to related changes in the nucleosome landscape.

The pivotal role of LYRIC/AEG‐1 in malignant transformation, tumourigenesis and chemo‐resistance has previously been demonstrated in different cell types and sub‐cellular compartments. The localisation of LYRIC/AEG‐1 appears crucial to its function and is regulated by three lysine‐rich nuclear localisation signal regions, one of which was previously demonstrated to be modified by ubiquitin. Here we show that mutation of LYRIC/AEG‐1 at K486 and K491 results in a loss of ubiquitination. A K486/491R double mutant that is incapable of ubiquitination shows reduced binding to the NFκB subunit p65 or importin‐β resulting in a distinctive peri‐nuclear localisation of LYRIC/AEG‐1. We also provide evidence to suggest that TOPORS, an E3 ligase that also regulates p53 modification may be responsible for LYRIC/AEG‐1 ubiquitin modification. Overall we demonstrate that specific sites of LYRIC/AEG‐1 ubiquitination are essential for regulating LYRIC/AEG‐1 localisation and functionally interacting proteins., Highlights LYRIC/AEG‐1 is an important oncogene.2 specific lysine residues in exNLS‐2 are ubiquitinated.Deletion of both lysine residues changes localisation and interaction with p65.LYRIC/AEG‐1 interacts with TOPORS, a known E3 ligase.

Summary RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes., Graphical Abstract, Highlights • RECQL5 is a general RNAPII elongation factor • RECQL5 reduces the elongation rate while decreasing pausing and arrest events • Loss of RECQL5 results in genome instability in genes and at common fragile sites • Incidents of genome instability colocalize with pause and arrest events, Rapid elongation by RNA polymerase II leads to increased transcriptional stress including a high incidence of pausing and arrests, which correlates with sites of genomic instability. RECQL5 modulates the rate of transcription, mitigating both the stress and instability effects.

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis., Francis Crick Institute (grant no. FCI01), Deutsche Krebshilfe (grant), European Research Council (grant), Association for International Cancer Research (grant)

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy.

Alterations in metabolic activity contribute to the proliferation and survival of cancer cells. We investigated the effect of siRNA-mediated gene silencing of 222 metabolic enzymes, transporters, and regulators on the survival of 3 metastatic prostate cancer cell lines and a nonmalignant prostate epithelial cell line. This approach revealed significant complexity in the metabolic requirements of prostate cancer cells and identified several genes selectively required for their survival. Among these genes was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of phosphofructokinase 2 (PFK2). We show that PFKFB4 is required to balance glycolytic activity and antioxidant production to maintain cellular redox balance in prostate cancer cells. Depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiologic nutrient levels. PFKFB4 mRNA expression was also found to be greater in metastatic prostate cancer compared with primary tumors. Taken together, these results indicate that PFKFB4 is a potential target for the development of antineoplastic agents. Significance: Cancer cells undergo several changes in their metabolism that promote growth and survival. Using an unbiased functional screen, we found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of antioxidants. Targeting PFKFB4 may therefore present new therapeutic opportunities. Cancer Discov; 2(4); 328–43. ©2012 AACR. Read the Commentary on this article by Dang, p. 304 This article is highlighted in the In This Issue feature, 288

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position −3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and −1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr654 in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the −3 position. Finally, the constitutive phosphorylation of EGFR on Thr654 is shown to be modulated by PKN in vivo.

Follicular lymphoma has considerable clinical heterogeneity, and there is a need for easily quantifiable prognostic biomarkers. Microvessel density has been shown to be a useful prognostic factor based on numerical assessment of vessel numbers within histologic sections in some studies, but assessment of tumor neovascularization through angiogenic sprouting may be more relevant. We therefore examined the smallest vessels, single-staining structures measuring less than 30 μm2 in area, seen within histologic sections, and confirmed that they were neovascular angiogenic sprouts using extended focal imaging. Tissue microarrays composing diagnostic biopsies from patients at the extremes of survival of follicular lymphoma were analyzed with respect to numbers of these sprouts. This analysis revealed higher angiogenic activity in the poor prognostic group and demonstrated an association between increased sprouting and elevated numbers of infiltrating CD163+ macrophages within the immediate microenvironment surrounding the neovascular sprout.

The expression of hTERT, the catalytic subunit of telomerase, immortalizes normal human urothelial cells (NHUC). Expression of a modified hTERT, without the ability to act in telomere maintenance, did not immortalize NHUC, confirming that effects at telomeres are required for urothelial immortalization. Previous studies indicate that inhibition of telomerase has an immediate effect on urothelial carcinoma (UC) cell line viability, before sufficient divisions to account for telomere attrition, implicating non–telomere effects of telomerase in UC. We analyzed the effects of telomerase on gene expression in isogenic mortal and hTERT-transduced NHUC. hTERT expression led to consistent alterations in the expression of genes predicted to be of phenotypic significance in tumorigenesis. A subset of expression changes were detected soon after transduction with hTERT and persisted with continued culture. These genes (NME5, PSCA, TSPYL5, LY75, IGFBP2, IGF2, CEACAM6, XG, NOX5, KAL1, and HPGD) include eight previously identified as polycomb group targets. TERT-NHUC showed overexpression of the polycomb repressor complex (PRC1 and PRC4) components, BMI1 and SIRT1, and down-regulation of multiple PRC targets and genes associated with differentiation. TERT-NHUC at 100 population doublings, but not soon after transduction, showed increased saturation density and an attenuated differentiation response, indicating that these are not acute effects of telomerase expression. Some of the changes in gene expression identified may contribute to tumorigenesis. Expression of NME5 and NDN was down-regulated in UC cell lines and tumors. Our data supports the concept of both telomere-based and non–telomere effects of telomerase and provides further rationale for the use of telomerase inhibitors in UC. (Mol Cancer Res 2008;6(7):1154–68)

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors. DOI: http://dx.doi.org/10.7554/eLife.08711.001, eLife digest Glioblastomas are the most common and aggressive brain cancers in adults, and currently lack efficient treatment options. Glioblastoma cells contain genetic mutations that enable them to grow and divide more quickly than they would under normal conditions. The occurrence of these mutations often leads to a functional impairment in so-called 'tumor suppressor' proteins that may have a range of roles, including repairing genetic damage or controlling the rate of cell division. Blake et al. have now studied how some of these tumor suppressor proteins interact. Deleting a prominent tumor suppressor called TP53 from the brain of mice caused these animals to develop glioblastomas. If, however, both TP53 and another tumor suppressor called ATMIN were deleted at the same time, the majority of mice did not develop any brain tumors. Further in-depth profiling of these brain tumor cells revealed that TP53-deleted cells had very high levels of the oncogene PDGFRA, which causes cells to divide more rapidly. These high PDGFRA levels were brought back to normal conditions upon deletion of ATMIN. Blake et al. then studied primary human glioblastoma cells that lack TP53 and found that these cells could be efficiently killed by a combination of drugs that block the activity of PDGFRA and the protein ATM, which is known to work in concert with ATMIN. Importantly, this combination of drugs did not adversely affect healthy brain cells, opening up new strategies and potential treatment options for glioblastoma patients. DOI: http://dx.doi.org/10.7554/eLife.08711.002

Background: Chromosomal instability (CIN) has been shown to be associated with drug resistance and poor clinical outcome in several cancer types. However, in oestrogen receptor (ER)-negative breast cancer we have previously demonstrated that extreme CIN is associated with improved clinical outcome, consistent with a negative impact of CIN on tumour fitness and growth. The aim of this current study was to validate this finding using previously defined CIN thresholds in a much larger prospective cohort from a randomised, controlled, clinical trial. Patients and methods: As a surrogate measurement of CIN, dual centromeric fluorescence in situ hybridisation was performed for both chromosomes 2 and 15 on 1173 tumours from the breast cancer TACT trial (CRUK01/ 001). Each tumour was scored manually and the mean percentage of cells deviating from the modal centromere number was used to define four CIN groups (MCD1-4), where tumours in the MCD4 group were defined as having extreme CIN. Results: In a multivariate analysis of disease-free survival, with a median follow-up of 91 months, increasing CIN was associated with improved outcome in patients with ER-negative cancer (P trend = 0.03). A similar pattern was seen in ERnegative/ HER2-negative cancers (Ptrend = 0.007). Conclusions: This prospective validation cohort study further substantiated the association between extreme CIN and improved outcome in ER-negative breast cancers. Identifying such patients with extreme CIN may help distinguish good from poor prognostic groups, and therefore support treatment and risk stratification in this aggressive breast cancer subtype.

Background: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5′, 3′, and exon 9 of the adenomatous polyposis coli ( APC ) gene. These mutations probably encode a limited amount of functional APC protein. Methods and results: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5′ Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes (“third hits”) in some polyps. We found that the “third hit” probably initiated tumorigenesis. Somatic mutation spectra were similar in 5′ and 3′ mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, “third hits” were more common. Most “third hits” left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. Conclusions: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need “three hits” however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell–cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell–cell adhesion, with large intercellular spaces observable at confluence, and diminished cell–substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell–cell and cell–matrix adhesive properties of urothelial cells. Implications: The ability of mutant FGFR3 to drive transcriptional expression profiles involved in tumor cell adhesion suggests a mechanism for expansion of premalignant urothelial lesions. Mol Cancer Res; 13(1); 138–48. ©2014 AACR.

Although microRNAs (miRNA) have been broadly studied in cancer, comparatively less is understood about their role in progression. Here we report that miR-153 has a dual role during progression of colorectal cancer by enhancing cellular invasiveness and platinum-based chemotherapy resistance. miRNA profiling revealed that miR-153 was highly expressed in a cellular model of advanced stage colorectal cancer. Its upregulation was also noted in primary human colorectal cancer compared with normal colonic epithelium and in more advanced colorectal cancer stages compared with early stage disease. In colorectal cancer patients followed for 50 months, 21 of 30 patients with high levels of miR-153 had disease progression compared with others in this group with low levels of miR-153. Functional studies revealed that miR-153 upregulation increased colorectal cancer invasiveness and resistance to oxaliplatin and cisplatin both in vitro and in vivo. Mechanistic investigations indicated that miR-153 promoted invasiveness indirectly by inducing matrix metalloprotease enzyme 9 production, whereas drug resistance was mediated directly by inhibiting the Forkhead transcription factor Forkhead box O3a (FOXO3a). In support of the latter finding, we found that levels of miR-153 and FOXO3a were inversely correlated in matched human colorectal cancer specimens. Our findings establish key roles for miR-153 overexpression in colorectal cancer progression, rationalizing therapeutic strategies to target expression of this miRNA for colorectal cancer treatment. Cancer Res; 73(21); 6435–47. ©2013 AACR.

Genome-wide RNA polymerase II (RNAPII) density profiles provide averaged snapshots of transcription but are difficult to interpret in the context of dynamic gene expression. We performed computational modeling to simulate RNAPII density profiles from individual transcription parameters and constructed simple mathematical models to explore general relationships between transcription parameters and density profiles.

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

The sequential use of non cross-resistant cytotoxic agents is the standard of care for advanced solid tumors in order to enhance survival and optimise quality of life. Nevertheless, drug resistance to non cross-resistant agents is commonly witnessed, with clinical response rates to non cross-resistant regimens declining as the disease advances. Expression of ABC transporters is unlikely to fully explain this phenomenon, and a clear molecular explanation for this process remains uncertain. A statistical analysis of a recently published RNA interference screen targeting 779 kinases in three cell lines deriving from different tumor types reveals a significant correlation between resistance to paclitaxel and a non cross-resistant cytotoxic agent. Furthermore, 20% of kinases that promote resistance to paclitaxel when targeted by RNAi also promote resistance to a non cross-resistant agent within that same cell line, consistent with a tissue-type dependence of multi-drug resistance. Conversely, paclitaxel-specific resistance and sensitising kinases occur less frequently than expected. This indicates that several cell line specific kinases may regulate multi-drug resistance and provide a potential explanation for the phenomenon of drug resistance to non cross-resistant agents witnessed in oncology practice. Furthermore, this work supports efforts to identify common pathways of drug response for future drug discovery programmes.

© 2015 Pender et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogenedriven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCRddPCRMutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Context: Activation of the hypoxia-inducible transcription factors HIF-1 and HIF-2 and a HIF-independent defect in developmental apoptosis have been implicated in the pathogenesis of pheochromocytoma (PCC) associated with VHL, SDHB, and SDHD mutations. Objective: Our objective was to compare protein (HIF-1α, EPAS1, SDHB, JunB, CCND1, CD34, CLU) and gene (VEGF, BNIP3) expression patterns in VHL and SDHB/D associated tumors. Results: Overexpression of HIF-2 was relatively more common in VHL than SDHB/D PCC (12 of 13 vs. 14 of 20, P = 0.02), whereas nuclear HIF-1 staining was relatively more frequent in SDHB/D PCC (19 of 20 vs. 13 of 16, P = 0.04). In addition, CCND1 and VEGF expression (HIF-2 target genes) was significantly higher in VHL than in SDHB/D PCC. These findings suggest that VHL inactivation leads to preferential HIF-2 activation and CCND1 expression as described previously in VHL-defective renal cell carcinoma cell lines but not in other cell types. These similarities between the downstream consequences of VHL inactivation and HIF dysregulation in renal cell carcinoma and PCC may explain how inactivation of the ubiquitously expressed VHL protein results in susceptibility to specific tumor types. Both VHL and SDHB/D PCC demonstrated reduced CLU and SDHB expression. SDHB PCC are associated with a high risk of malignancy, and expression of (proapototic) BNIP3 was significantly lower in SDHB than VHL PCC. Conclusion: Although inactivation of VHL and SDHB/D may disrupt similar HIF-dependent and HIF-independent signaling pathways, their effects on target gene expression are not identical, and this may explain the observed clinical differences in PCC and associated tumors seen with germline VHL and SDHB/D mutations.

Human hematopoietic stem cells (HSCs) are generally regarded as being devoid of the markers expressed by differentiated blood cells, the lineage-specific antigens. However, recent work suggests that genes associated with the myeloid lineage are transcribed in mouse HSCs. Here, we explore whether myeloid genes are actually translated in human HSCs. We show that CD33, CD13, and CD123, well-established myeloid markers, are expressed on human long-term repopulating cells from cord blood and bone marrow. In addition, we demonstrate that nonobese diabetic/severe combined immunodeficiency (NOD/SCID) leukemia-initiating cells (SL-ICs) are restricted to the CD33+ fraction in 11 of 12 acute myeloid leukemia (AML) samples studied, indicating that leukemic stem cells (LSCs) express this antigen. This study changes our view of HSCs and the process of differentiation. Furthermore, based on the phenotypic similarity of HSCs and LSCs, our data provide support for the hypothesis that AML derives from an HSC. Our findings also provide a challenge to contemporary attempts to improve the outcome of AML using myeloid antigen-targeted therapies, given the potential for HSC killing.

Summary In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response., Graphical Abstract, Highlights • A multiomic screening approach examines the UV-induced DNA damage response • Multiple factors are connected to the transcription-related DNA damage response • Melanoma gene STK19 is required for a normal DNA damage response, Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered.

SummaryCytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.

Cutaneous squamous cell carcinomas (cSCCs) are the second most frequent cancers in fair-skinned populations; yet, because of their genetic heterogeneity, the key molecular events in cSCC tumorigenesis remain poorly defined. We have employed single nucleotide polymorphism microarray analysis to examine genome-wide allelic imbalance in 60 cSCCs using paired non-tumour samples. The most frequent recurrent aberrations were loss of heterozygosity (LOH) at 3p and 9p, observed in 39 (65%) and 45 (75%) tumours respectively. Microdeletions at 9p23 within the protein tyrosine phosphatase receptor type D (PTPRD) locus were identified in a total of 9 (15%) samples, supporting a tumour suppressor role for PTPRD in cSCC. In addition, microdeletions at 3p14.2 were detected in 3 (5%) cSCCs, implicating fragile histidine triad (FHIT) gene as a possible target for inactivation. Statistical analysis revealed that well-differentiated cSCCs demonstrated significantly fewer aberrations than moderately and poorly differentiated cSCCs; yet, despite a lower rate of allelic imbalance, some specific aberrations were observed equally frequently in both groups. No correlation was established between the frequency of chromosomal aberrations and immune or human papillomavirus status. Our data suggest that well differentiated tumours are a genetically distinct subpopulation of cSCC.

Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.