Cancer progression is driven by both somatic copy number aberrations (CNAs) and chromatin remodeling, yet little is known about the interplay between these two classes of events in shaping the clonal ...diversity of cancers. We present Alleloscope, a method for allele-specific copy number estimation that can be applied to single-cell DNA- and/or transposase-accessible chromatin-sequencing (scDNA-seq, ATAC-seq) data, enabling combined analysis of allele-specific copy number and chromatin accessibility. On scDNA-seq data from gastric, colorectal and breast cancer samples, with validation using matched linked-read sequencing, Alleloscope finds pervasive occurrence of highly complex, multiallelic CNAs, in which cells that carry varying allelic configurations adding to the same total copy number coevolve within a tumor. On scATAC-seq from two basal cell carcinoma samples and a gastric cancer cell line, Alleloscope detected multiallelic copy number events and copy-neutral loss-of-heterozygosity, enabling dissection of the contributions of chromosomal instability and chromatin remodeling to tumor evolution.
Transplantation of exogenous dopaminergic neuron （DA neurons） is a promising approach for treating Parkin- son＇s disease （PD）. However, a major stumbling block has been the lack of a reliable source ...of donor DA neurons. Here we show that a combination of five transcriptional factors Mashl, Ngn2, Sox2, Nurrl, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement thera- py for PD.
The functional properties of circulating CD8
T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph ...nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8
T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8
T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8
T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4
T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8
T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.
Homocysteine and cysteine are associated with oxidative damage and metabolic disorders, which may lead to carcinogenesis. Observational studies assessing the association between circulating ...homocysteine or cysteine and breast cancer are very limited, and findings have been inconsistent. We prospectively evaluated plasma levels of homocysteine and cysteine in relation to breast cancer risk among 812 incident cases of invasive breast cancer and 812 individually matched control subjects from 28,345 women in the Women's Health Study; these women were >or=45 years old, provided blood samples, and had no history of cancer and cardiovascular disease at baseline. Logistic regression controlling for matching factors and risk factors for breast cancer was used to estimate relative risks (RR) and 95% confidence intervals (95% CI). All statistical tests were two sided. Homocysteine levels were not associated with overall risk for breast cancer. However, we observed a positive association between cysteine levels and breast cancer risk; the multivariate RR for the highest quintile group relative to the lowest quintile was 1.65 (95% CI, 1.04-2.61; P for trend = 0.04). In addition, women with higher levels of homocysteine and cysteine were at a greater risk for developing breast cancer when their folate levels were low (P for interaction = 0.04 and 0.002, respectively). Although our study offers little support for an association between circulating homocysteine and overall breast cancer risk, higher homocysteine levels may be associated with an increased risk for breast cancer among women with low folate status. The increased risk of breast cancer associated with high cysteine levels warrants further investigation.
Experimental and epidemiological studies suggest a protective role for vitamin D in colorectal carcinogenesis, but evidence is inconclusive. Circulating 25-hydroxyvitamin D (25(OH)D) concentrations ...that minimize risk are unknown. Current Institute of Medicine (IOM) vitamin D guidance is based solely on bone health.
We pooled participant-level data from 17 cohorts, comprising 5706 colorectal cancer case participants and 7107 control participants with a wide range of circulating 25(OH)D concentrations. For 30.1% of participants, 25(OH)D was newly measured. Previously measured 25(OH)D was calibrated to the same assay to permit estimating risk by absolute concentrations. Study-specific relative risks (RRs) for prediagnostic season-standardized 25(OH)D concentrations were calculated using conditional logistic regression and pooled using random effects models.
Compared with the lower range of sufficiency for bone health (50-<62.5 nmol/L), deficient 25(OH)D (<30 nmol/L) was associated with 31% higher colorectal cancer risk (RR = 1.31, 95% confidence interval CI = 1.05 to 1.62); 25(OH)D above sufficiency (75-<87.5 and 87.5-<100 nmol/L) was associated with 19% (RR = 0.81, 95% CI = 0.67 to 0.99) and 27% (RR = 0.73, 95% CI = 0.59 to 0.91) lower risk, respectively. At 25(OH)D of 100 nmol/L or greater, risk did not continue to decline and was not statistically significantly reduced (RR = 0.91, 95% CI = 0.67 to 1.24, 3.5% of control participants). Associations were minimally affected when adjusting for body mass index, physical activity, or other risk factors. For each 25 nmol/L increment in circulating 25(OH)D, colorectal cancer risk was 19% lower in women (RR = 0.81, 95% CI = 0.75 to 0.87) and 7% lower in men (RR = 0.93, 95% CI = 0.86 to 1.00) (two-sided Pheterogeneity by sex = .008). Associations were inverse in all subgroups, including colorectal subsite, geographic region, and season of blood collection.
Higher circulating 25(OH)D was related to a statistically significant, substantially lower colorectal cancer risk in women and non-statistically significant lower risk in men. Optimal 25(OH)D concentrations for colorectal cancer risk reduction, 75-100 nmol/L, appear higher than current IOM recommendations.
Data from laboratory studies, observational research, and/or secondary prevention trials suggest that vitamin D and marine omega-3 fatty acids may reduce risk for cancer or cardiovascular disease ...(CVD), but primary prevention trials with adequate dosing in general populations (i.e., unselected for disease risk) are lacking. The ongoing VITamin D and OmegA-3 TriaL (VITAL) is a large randomized, double-blind, placebo-controlled, 2 x 2 factorial trial of vitamin D (in the form of vitamin D(3) cholecalciferol, 2000 IU/day) and marine omega-3 fatty acid (Omacor fish oil, eicosapentaenoic acid EPA+docosahexaenoic acid DHA, 1g/day) supplements in the primary prevention of cancer and CVD among a multi-ethnic population of 20,000 U.S. men aged ≥ 50 and women aged ≥ 55. The mean treatment period will be 5 years. Baseline blood samples will be collected in at least 16,000 participants, with follow-up blood collection in about 6000 participants. Yearly follow-up questionnaires will assess treatment compliance (plasma biomarker measures will also assess compliance in a random sample of participants), use of non-study drugs or supplements, occurrence of endpoints, and cancer and vascular risk factors. Self-reported endpoints will be confirmed by medical record review by physicians blinded to treatment assignment, and deaths will be ascertained through national registries and other sources. Ancillary studies will investigate whether these agents affect risk for diabetes and glucose intolerance; hypertension; cognitive decline; depression; osteoporosis and fracture; physical disability and falls; asthma and other respiratory diseases; infections; and rheumatoid arthritis, systemic lupus erythematosus, thyroid diseases, and other autoimmune disorders.
Tumor-sequencing studies have revealed the widespread genetic diversity of melanoma. Sequencing of 108 genes previously implicated in melanomagenesis was performed on 462 patient-derived xenografts ...(PDXs), cell lines, and tumors to identify mutational and copy number aberrations. Samples came from 371 unique individuals: 263 were naive to treatment, and 108 were previously treated with targeted therapy (34), immunotherapy (54), or both (20). Models of all previously reported major melanoma subtypes (BRAF, NRAS, NF1, KIT, and WT/WT/WT) were identified. Multiple minor melanoma subtypes were also recapitulated, including melanomas with multiple activating mutations in the MAPK-signaling pathway and chromatin-remodeling gene mutations. These well-characterized melanoma PDXs and cell lines can be used not only as reagents for a large array of biological studies but also as pre-clinical models to facilitate drug development.
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•Melanoma cell lines and PDXs more likely to be BRAF/NRAS mutant than patient tumors•Mutations in melanoma PDXs are concordant with tumors from which they are derived•Contrasting MAPK pathway mutation patterns: one high activity, several low activity•Recurrent disease displays intra- and inter-tumor mutational heterogeneity
Garman et al. have characterized melanoma PDXs and cell lines described in Krepler et al. (see the related paper in this issue of Cell Reports), identifying major and minor subtypes, some of which were previously not well defined, targeted and immunotherapy resistance, and tumor heterogeneity, creating a set of reagents for future drug discovery and biological studies.
Persistent inflammation driven by cytokines such as type-one interferon (IFN-I) can cause immunosuppression. We show that administration of the Janus kinase 1 (JAK1) inhibitor itacitinib after ...anti–PD-1 (programmed cell death protein 1) immunotherapy improves immune function and antitumor responses in mice and results in high response rates (67%) in a phase 2 clinical trial for metastatic non–small cell lung cancer. Patients who failed to respond to initial anti–PD-1 immunotherapy but responded after addition of itacitinib had multiple features of poor immune function to anti–PD-1 alone that improved after JAK inhibition. Itacitinib promoted CD8 T cell plasticity and therapeutic responses of exhausted and effector memory–like T cell clonotypes. Patients with persistent inflammation refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve the efficacy of anti–PD-1 immunotherapy by pivoting T cell differentiation dynamics.
Editor’s summary Cancer immunotherapy is a type of treatment that mobilizes a patient’s immune system to kill tumor cells. It has been successful in treating certain tumors, but patients frequently have chronic inflammation and immunosuppression, which can limit treatment response. Two independent clinical trials looked at whether dialing down inflammation using drugs called JAK inhibitors could improve the efficacy of anti–PD-1 immunotherapy in cancer patients (see the Perspective by Gadina and O’Shea). Mathew et al . conducted a phase 2 trial to investigate the drug combination as a first-line therapy for metastatic non-small-cell lung cancer. Delayed administration of itacitinib after treatment with pembrolizumab improved therapeutic response. Zak et al . performed a phase 1/2 trial in patients with relapsed/refractory Hodgkin’s lymphoma. A combination of ruxolitinib and nivolumab resulted in improved clinical efficacy in patients who had previously failed checkpoint blockade immunotherapy. —Priscilla N. Kelly
INTRODUCTION Inflammation is a hallmark of cancer but is also required to generate optimal antitumor immune responses. Although short exposure to cytokines such as interferon (IFN) can enhance tumor immunity, prolonged exposure can result in immunosuppression. The dual nature of inflammation makes it challenging to harness the beneficial effects of cytokine activation during cancer immunotherapy while avoiding detrimental consequences. Thus, a therapeutical strategy to effectively modulate these often-opposing functions of cytokine signaling could improve immunotherapy efficacy and mitigate development of resistance. RATIONALE Preclinical studies in mice have demonstrated that blockade of type-one IFN (IFN-I) signaling can improve immune function during chronic viral infections and enhance the efficacy of immunotherapy for cancer. Moreover, tumors from patients with lung and other cancer types that relapse after—or are resistant to—immune checkpoint blockade (ICB) can have high expression of IFN-stimulated genes. Thus, we conducted a phase 2 clinical trial for first-line metastatic non–small cell lung cancer (NSCLC) with programmed death ligand 1 (PD-L1) expression ≥50%. Patients were administered 6 weeks of anti–PD-1 (PD-1, programmed cell death protein 1) immunotherapy, followed by a combination of anti–PD-1 and itacitinib a selective Janus kinase 1 (JAK1) inhibitor for 6 weeks before continuing with anti–PD-1 alone. To understand the immunological effects of transient JAK inhibition aimed at interfering with persistent inflammation occurring after anti–PD-1, we evaluated the association between clinical response and the evolution of CD8 T cell differentiation, immune signaling, and inflammatory markers. RESULTS In mice, the addition of itacitinib after the start of ICB improved response of interferon-stimulated gene (ISG)–high resistant tumors and increased the proportion of proliferating precursor-like CD8 T cells in the periphery. Use of an IFN-I–receptor blocking antibody similarly improved ICB efficacy, suggesting that blockade of IFN-I signaling is sufficient to phenocopy the effects of delayed JAK inhibition. In humans, the combination of anti–PD-1 with delayed, transient, itactinib treatment in patients with NSCLC led to an overall response rate of 67% and median progression-free survival of 23.8 months. Patients were categorized into three response groups based on the timing of radiographic response: patients with clinical response within the first two cycles of anti–PD-1 monotherapy (αPD1.R), patients who responded only after a 6-week course of concurrent itacitinib was added at the start of the third cycle of anti–PD-1 (JAKi.R), or patients who did not respond regardless of treatment (NR). Each clinical response group had distinctive immunological changes coupled to inflammatory features. The αPD1.R patients had low baseline inflammation and CD8 T cell responses after anti–PD-1 alone. JAKi.R patients had elevated inflammatory markers, poor CD8 T cell responses, and blunted immune signaling after anti–PD-1 alone. However, after addition of itacitinib, subsequent clinical responses in JAKi.R patients were associated with decreased inflammatory signaling accompanied by an increase in a “fate-flexible” CD8 T cell progenitor-like population. This fate-flexible population was linked to features of greater CD8 T cell plasticity. By contrast, NR patients had high baseline inflammation refractory to JAK inhibition. This persistent inflammatory and IFN-I signaling in NR patients was associated with CD8 T cell terminal differentiation and treatment failure. CONCLUSION As a therapeutic strategy to block the immunosuppressive effects of persistent IFN and/or chronic inflammation, JAK inhibition after initial anti–PD-1 is safe, feasible, and associated with durable and high response rates in NSCLC. JAK inhibition may be particularly beneficial in patients with elevated inflammation who have poor CD8 T cell responses to anti–PD-1 alone. In this study, JAK inhibition enhanced CD8 T cell plasticity by decreasing the inflammatory and IFN-I signals that drive terminal differentiation of CD8 T cells. However, some patients with the highest baseline levels of inflammation were largely unaffected by JAK1 inhibition and had progressive terminal CD8 T cell differentiation and disease progression. Our findings suggest that JAK inhibition can target chronic immunoregulatory functions of cytokine signaling that contribute to relapse during cancer immunotherapy and is a strategy warranting further preclinical and clinical investigation. JAK inhibitors to improve clinical and immune response to anti–PD-1 for lung cancer patients. In a clinical trial of patients with NSCLC treated with anti–PD-1, mitigating persistent IFN-I and inflammation with a JAK inhibitor decreased terminal differentiation of CD8 T cells and expanded fate-flexible CD8 T cell progenitors in responding patients. Patients with progressive disease had high baseline inflammation that minimally changed with JAK1 inhibition.
In this study, we explore the distinct reactivity patterns between frontal ring-opening metathesis polymerization (FROMP) and room-temperature solventless ring-opening metathesis polymerization ...(ROMP). Despite their shared mechanism, we find that FROMP is less sensitive to inhibitor concentration than room-temperature ROMP. By increasing the initiator-to-monomer ratio for a fixed inhibitor/initiator quantity, we find reduction in the ROMP background reactivity at room temperature (i.e., increased resin pot life). At elevated temperatures where inhibitor dissociation prevails, accelerated frontal polymerization rates are observed because of the concentrated presence of the initiator. Surprisingly, the strategy of employing higher initiator loading enhances both pot life and front speeds, which leads to FROMP rates exceeding prior reported values by over 5 times. This counterintuitive behavior is attributed to an increase in the proximity of the inhibitor to the initiator within the bulk resin and to whether the temperature favors coordination or dissociation of the inhibitor. A rapid method was developed for assessing resin pot life, and a straightforward model for active initiator behavior was established. Modified resin systems enabled direct ink writing of robust thermoset structures at rates much faster than previously possible.
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