Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.

Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

BACKGROUND: Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by the inheritance of the serpin family A member 1 "Z" genetic variant driving alpha-1 antitrypsin (AAT) protein misfolding in hepatocytes. There are no approved medicines for this disease. METHODS: We conducted a high-throughput image-based small molecule screen using patient-derived induced pluripotent stem cell-hepatocytes (iPSC-hepatocytes). Identified targets were validated in vitro using 3 independent patient iPSC lines. The effects of the identified target, leucine-rich repeat kinase 2 (LRRK2), were further evaluated in an animal model of A1ATD through histology and immunohistochemistry and in an autophagy-reporter line. Autophagy induction was assessed through immunoblot and immunofluorescence analyses. RESULTS: Small-molecule screen performed in iPSC-hepatocytes identified LRRK2 as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through the induction of autophagy. CONCLUSIONS: Our findings support the use of CZC-25146 and leucine-rich repeat kinase-2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers.

BACKGROUND: The relationship between the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the risk of subsequent reinfection remains unclear. METHODS: We investigated the incidence of SARS-CoV-2 infection confirmed by polymerase chain reaction (PCR) in seropositive and seronegative health care workers attending testing of asymptomatic and symptomatic staff at Oxford University Hospitals in the United Kingdom. Baseline antibody status was determined by anti-spike (primary analysis) and anti-nucleocapsid IgG assays, and staff members were followed for up to 31 weeks. We estimated the relative incidence of PCR-positive test results and new symptomatic infection according to antibody status, adjusting for age, participant-reported gender, and changes in incidence over time. RESULTS: A total of 12,541 health care workers participated and had anti-spike IgG measured; 11,364 were followed up after negative antibody results and 1265 after positive results, including 88 in whom seroconversion occurred during follow-up. A total of 223 anti-spike-seronegative health care workers had a positive PCR test (1.09 per 10,000 days at risk), 100 during screening while they were asymptomatic and 123 while symptomatic, whereas 2 anti-spike-seropositive health care workers had a positive PCR test (0.13 per 10,000 days at risk), and both workers were asymptomatic when tested (adjusted incidence rate ratio, 0.11; 95% confidence interval, 0.03 to 0.44; P = 0.002). There were no symptomatic infections in workers with anti-spike antibodies. Rate ratios were similar when the anti-nucleocapsid IgG assay was used alone or in combination with the anti-spike IgG assay to determine baseline status. CONCLUSIONS: The presence of anti-spike or anti-nucleocapsid IgG antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months. (Funded by the U.K. Government Department of Health and Social Care and others.).

E2F1 proteolysis via SCF-cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition.

Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.

CagA-ASPP2 complex mediates loss of cell polarity and favors H. pylori colonization of human gastric organoids.

The main risk factor for stomach cancer, the third most common cause of cancer death worldwide, is infection with Helicobacter pylori bacterial strains that inject cytotoxin-associated gene A (CagA). As the first described bacterial oncoprotein, CagA causes gastric epithelial cell transformation by promoting an epithelial-to-mesenchymal transition (EMT)-like phenotype that disrupts junctions and enhances motility and invasiveness of the infected cells. However, the mechanism by which CagA disrupts gastric epithelial cell polarity to achieve its oncogenicity is not fully understood. Here we found that the apoptosis-stimulating protein of p53 2 (ASPP2), a host tumor suppressor and an important CagA target, contributes to the survival of cagA-positive H. pylori in the lumen of infected gastric organoids. Mechanistically, the CagA-ASPP2 interaction is a key event that promotes remodeling of the partitioning-defective (PAR) polarity complex and leads to loss of cell polarity of infected cells. Blockade of cagA-positive H. pylori ASPP2 signaling by inhibitors of the EGFR (epidermal growth factor receptor) signaling pathway-identified by a high-content imaging screen-or by a CagA-binding ASPP2 peptide, prevents the loss of cell polarity and decreases the survival of H. pylori in infected organoids. These findings suggest that maintaining the host cell-polarity barrier would reduce the detrimental consequences of infection by pathogenic bacteria, such as H. pylori, that exploit the epithelial mucosal surface to colonize the host environment.

Selective ROCK Inhibitor Enhances Blood Flow Recovery after Hindlimb Ischemia.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.

Efficacy of personal protective equipment to prevent environmental infection of COVID-19 among healthcare workers: a systematic review.

BACKGROUND: Healthcare workers (HCWs) employed personal protective equipment (PPE) during the COVID-19 pandemic, crucial to protecting themselves from infection. To highlight the efficacy of PPE in preventing environmental infection among HCWs, a systematic review was conducted in line with PRISMA guidance. METHODS: A search of the PubMed and Web of Science databases was conducted from January 2019 to April 2021 using pre-defined search terms. Articles were screened by three researchers. The approved papers were read in full and included in this review if relevance was mutually agreed upon. Data were extracted by study design and types of PPEs. RESULTS: 47 of 108 identified studies met the inclusion criteria, with seven reviews and meta-analyses, seven cohort, nine case-control, fifteen cross-sectional studies, four before and after, four case series, and one modeling studies. Wearing PPE offered COVID-19 protection in HCWs but required adequate training. Wearing surgical masks provided improved protection over cloth masks, while the benefit of powered air-purifying respirators is less clear, as are individual gowns, gloves, and/or face shields. CONCLUSIONS: Wearing PPE, especially facial masks, is necessary among HCWs, while training in proper use of PPE is also important to prevent COVID-19 infection.

An Observational Cohort Study on the Incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection and B.1.1.7 Variant Infection in Healthcare Workers by Antibody and Vaccination Status.

BACKGROUND: Natural and vaccine-induced immunity will play a key role in controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity. METHODS: In a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, United Kingdom, we investigated the protection from symptomatic and asymptomatic polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after 1 versus 2 vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing. RESULTS: In total, 13 109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses), and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and 2 vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95% confidence interval {CI} < .01-.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [95% CI .02-.38]) and 85% (0.15 [95% CI .08-.26]), respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [95% CI .21-.52]) and any PCR-positive result by 64% (0.36 [95% CI .26-.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7. CONCLUSIONS: Natural infection resulting in detectable anti-spike antibodies and 2 vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.

Carbon Ion Radiotherapy for Locally Recurrent Rectal Cancer of Patients with Prior Pelvic Irradiation.

BACKGROUND: This study aimed to assess the safety and efficacy of carbon-ion radiotherapy (CIRT) for salvage of previously X-ray-irradiated (XRT) locally recurrent rectal cancer (LRRC). METHODS: Between September 2005 and December 2017, 77 patients with LRRC were treated with CIRT re-irradiation. All the patients had received prior XRT with a median dose of 50.0 Gy (range 20-74 Gy), principally for neoadjuvant or adjuvant recurrence prophylaxis in 34 patients and for recurrence in 43 patients. The total CIRT dose of 70.4 Gy (RBE) (gray relative biologic effectiveness) was administered in 16 fixed fractions during 4 weeks (4.4 Gy [RBE] per fraction). RESULTS: All the patients completed the scheduled treatment course. None of the patients received resection after CIRT. Acute grade 3 toxicities occurred for eight patients (10 %), including five grade 3 pelvic infections (2 involving pain and 1 involving neuropathy). Late grade 3 toxicities occurred for 16 patients (21 %): 13 with late grade 3 pelvic infections, 9 with gastrointestinal toxicity, 1 with skin toxicity, 2 with pain, and 4 with neuropathy. No grade 4+ toxicity was noted. The overall local control rates (infield + out-of-field recurrence) were 69 % at 3 years and 62 % at 5 years. In the planning target volume (PTV), the infield recurrence rates were 90 % and 87 % respectively. The control rates for regional recurrence were 85 % at 3 years and 81 % at 5 years. The median overall survival time was 47 months. The survival rates were 61 % at 3 years and 38 % at 5 years. CONCLUSION: Carbon-ion re-irradiation of previously X-ray-irradiated locally recurrent rectal cancer appears to be safe and effective, providing good local control and survival advantage without unacceptable morbidity.

The Duration, Dynamics, and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Responses in Individual Healthcare Workers.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary. METHODS: We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning. RESULTS: Anti-spike IgG levels remained stably detected after a positive result, for example, in 94% (95% credibility interval [CrI] 91-96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% CrI 19-31) days post first polymerase chain reaction (PCR)-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titer, the mean estimated antibody half-life was 85 (95% CrI 81-90) days. Higher maximum observed anti-nucleocapsid titers were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity, and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives. CONCLUSIONS: SARS-CoV-2 anti-nucleocapsid antibodies wane within months and fall faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.

Differential effects of HIF2α antagonist and HIF2α silencing in renal cancer and sensitivity to repurposed drugs.

BACKGROUND: In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed. METHODS: We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385. RESULTS: PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity. CONCLUSIONS: this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable.

Stringent thresholds in SARS-CoV-2 IgG assays lead to under-detection of mild infections.

BACKGROUND: Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear. METHODS: We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds. RESULTS: The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%). CONCLUSION: Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.

The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Adefovir dipivoxil induces DNA replication stress and augments ATR inhibitor-related cytotoxicity.

Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.

Patient-derived malignant pleural mesothelioma cell cultures: a tool to advance biomarker-driven treatments.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.

ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity.

INTRODUCTION: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. METHODS: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. RESULTS: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. DISCUSSION & CONCLUSIONS: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

SARS-CoV-2 antibody prevalence, titres and neutralising activity in an antenatal cohort, United Kingdom, 14 April to 15 June 2020.

SARS-CoV-2 IgG screening of 1,000 antenatal serum samples in the Oxford area, United Kingdom, between 14 April and 15 June 2020, yielded a 5.3% seroprevalence, mirroring contemporaneous regional data. Among the 53 positive samples, 39 showed in vitro neutralisation activity, correlating with IgG titre (Pearson's correlation p<0.0001). While SARS-CoV-2 seroprevalence in pregnancy cohorts could potentially inform population surveillance, clinical correlates of infection and immunity in pregnancy, and antenatal epidemiology evolution over time need further study.

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

Updated long-term outcomes after carbon-ion radiotherapy for primary renal cell carcinoma.

Long-term oncological outcomes for primary renal cell carcinoma (RCC) treated with carbon-ion radiotherapy (CIRT) are poorly understood. Patients with primary RCC were treated with 12 or 16-fraction CIRT at The Hospital of the National Institute of Radiological Sciences outside of clinical trials. Outcome data were pooled and retrospectively analyzed for toxicity, local control, and disease-free, cancer-specific, and overall survival. From 1997 to 2014, 19 RCC patients (11 with T1aN0M0, 4 with T1bN0M0, and 4 with inoperable advanced stage [T4N0M0, T3aN1M0, and T1aN0M1]) were treated with CIRT and followed up for a median of 6.6 (range, 0.7-16.5) years; 9 of these patients were inoperable because of comorbidities or advanced-stage disease. Diagnoses were confirmed by imaging in 11 patients and by biopsy in the remaining 8. In 4 of 5 patients with definitive renal comorbidities, including diabetic nephropathy, sclerotic kidney or solitary kidney pre-CIRT progressed to grade 4 chronic kidney disease (CKD). In contrast, the remaining 14 patients without definitive renal comorbidities did not progress to grade 3 or higher CKD. Furthermore, although 1 case of grade 4 dermatitis was observed, there were no other grade 3 or higher non-renal adverse events. Local control rate, and disease-free, cancer-specific, and overall survival rates at 5 years of all 19 patients were 94.1%, 68.9%, 100%, and 89.2%, respectively. This updated retrospective analysis based on long-term follow-up data suggests that CIRT is a safe treatment for primary RCC patients without definitive renal comorbidities pre-CIRT, and yield favorable treatment outcomes, even in inoperable cases.