Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

Senescence in Health and Disease.

Many cellular stresses activate senescence, a persistent hyporeplicative state characterized in part by expression of the p16INK4a cell-cycle inhibitor. Senescent cell production occurs throughout life and plays beneficial roles in a variety of physiological and pathological processes including embryogenesis, wound healing, host immunity, and tumor suppression. Meanwhile, the steady accumulation of senescent cells with age also has adverse consequences. These non-proliferating cells occupy key cellular niches and elaborate pro-inflammatory cytokines, contributing to aging-related diseases and morbidity. This model suggests that the abundance of senescent cells in vivo predicts "molecular," as opposed to chronologic, age and that senescent cell clearance may mitigate aging-associated pathology.

Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model.

Monitoring cancer and aging in vivo remains experimentally challenging. Here, we describe a luciferase knockin mouse (p16(LUC)), which faithfully reports expression of p16(INK4a), a tumor suppressor and aging biomarker. Lifelong assessment of luminescence in p16(+/LUC) mice revealed an exponential increase with aging, which was highly variable in a cohort of contemporaneously housed, syngeneic mice. Expression of p16(INK4a) with aging did not predict cancer development, suggesting that the accumulation of senescent cells is not a principal determinant of cancer-related death. In 14 of 14 tested tumor models, expression of p16(LUC) was focally activated by early neoplastic events, enabling visualization of tumors with sensitivity exceeding other imaging modalities. Activation of p16(INK4a) was noted in the emerging neoplasm and surrounding stromal cells. This work suggests that p16(INK4a) activation is a characteristic of all emerging cancers, making the p16(LUC) allele a sensitive, unbiased reporter of neoplastic transformation.

Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis.

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

HMGA2, microRNAs, and stem cell aging.

Mammalian aging results from a replicative decline in the function of somatic stem cells and other self-renewing cells. Recent studies (Monzen et al., 2008; Nishino et al., 2008; Sanna et al., 2008; Weedon et al., 2008) link a chromatin-associated protein, HMGA2, to development, height, and mouse stem cell aging during late fetal development and young adulthood.

Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in All of Us Research Program Participants, 2 January to 18 March 2020.

BACKGROUND: With limited severe acute respiratory syndrome coronavirus (SARS-CoV-2) testing capacity in the United States at the start of the epidemic (January-March 2020), testing was focused on symptomatic patients with a travel history throughout February, obscuring the picture of SARS-CoV-2 seeding and community transmission. We sought to identify individuals with SARS-CoV-2 antibodies in the early weeks of the US epidemic. METHODS: All of Us study participants in all 50 US states provided blood specimens during study visits from 2 January to 18 March 2020. Participants were considered seropositive if they tested positive for SARS-CoV-2 immunoglobulin G (IgG) antibodies with the Abbott Architect SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) and the EUROIMMUN SARS-CoV-2 ELISA in a sequential testing algorithm. The sensitivity and specificity of these ELISAs and the net sensitivity and specificity of the sequential testing algorithm were estimated, along with 95% confidence intervals (CIs). RESULTS: The estimated sensitivities of the Abbott and EUROIMMUN assays were 100% (107 of 107 [95% CI: 96.6%-100%]) and 90.7% (97 of 107 [83.5%-95.4%]), respectively, and the estimated specificities were 99.5% (995 of 1000 [98.8%-99.8%]) and 99.7% (997 of 1000 [99.1%-99.9%]), respectively. The net sensitivity and specificity of our sequential testing algorithm were 90.7% (97 of 107 [95% CI: 83.5%-95.4%]) and 100.0% (1000 of 1000 [99.6%-100%]), respectively. Of the 24 079 study participants with blood specimens from 2 January to 18 March 2020, 9 were seropositive, 7 before the first confirmed case in the states of Illinois, Massachusetts, Wisconsin, Pennsylvania, and Mississippi. CONCLUSIONS: Our findings identified SARS-CoV-2 infections weeks before the first recognized cases in 5 US states.

Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence.

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by "knocking-in" a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16INK4a locus. We used this allele (p16tdTom ) for the enumeration, isolation, and characterization of individual p16INK4a -expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated ß-galactosidase (SA-ß-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.

How stem cells age and why this makes us grow old.

Recent data suggest that we age, in part, because our self-renewing stem cells grow old as a result of heritable intrinsic events, such as DNA damage, as well as extrinsic forces, such as changes in their supporting niches. Mechanisms that suppress the development of cancer, such as senescence and apoptosis, which rely on telomere shortening and the activities of p53 and p16(INK4a), may also induce an unwanted consequence: a decline in the replicative function of certain stem-cell types with advancing age. This decreased regenerative capacity appears to contribute to some aspects of mammalian ageing, with new findings pointing to a 'stem-cell hypothesis' for human age-associated conditions such as frailty, atherosclerosis and type 2 diabetes.

Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Enhancing Next-Generation Sequencing-Guided Cancer Care Through Cognitive Computing.

BACKGROUND: Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. MATERIALS AND METHODS: One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. RESULTS: Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. CONCLUSION: These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. IMPLICATIONS FOR PRACTICE: The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.

A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response.

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Docetaxel-Loaded PLGA Nanoparticles Improve Efficacy in Taxane-Resistant Triple-Negative Breast Cancer.

Novel treatment strategies, including nanomedicine, are needed for improving management of triple-negative breast cancer. Patients with triple-negative breast cancer, when considered as a group, have a worse outcome after chemotherapy than patients with breast cancers of other subtypes, a finding that reflects the intrinsically adverse prognosis associated with the disease. The aim of this study was to improve the efficacy of docetaxel by incorporation into a novel nanoparticle platform for the treatment of taxane-resistant triple-negative breast cancer. Rod-shaped nanoparticles encapsulating docetaxel were fabricated using an imprint lithography based technique referred to as Particle Replication in Nonwetting Templates (PRINT). These rod-shaped PLGA-docetaxel nanoparticles were tested in the C3(1)-T-antigen (C3Tag) genetically engineered mouse model (GEMM) of breast cancer that represents the basal-like subtype of triple-negative breast cancer and is resistant to therapeutics from the taxane family. This GEMM recapitulates the genetics of the human disease and is reflective of patient outcome and, therefore, better represents the clinical impact of new therapeutics. Pharmacokinetic analysis showed that delivery of these PLGA-docetaxel nanoparticles increased docetaxel circulation time and provided similar docetaxel exposure to tumor compared to the clinical formulation of docetaxel, Taxotere. These PLGA-docetaxel nanoparticles improved tumor growth inhibition and significantly increased median survival time. This study demonstrates the potential of nanotechnology to improve the therapeutic index of chemotherapies and rescue therapeutic efficacy to treat nonresponsive cancers.

Total body irradiation causes long-term mouse BM injury via induction of HSC premature senescence in an Ink4a- and Arf-independent manner.

Exposure to total body irradiation (TBI) induces not only acute hematopoietic radiation syndrome but also long-term or residual bone marrow (BM) injury. This residual BM injury is mainly attributed to permanent damage to hematopoietic stem cells (HSCs), including impaired self-renewal, decreased long-term repopulating capacity, and myeloid skewing. These HSC defects were associated with significant increases in production of reactive oxygen species (ROS), expression of p16(Ink4a) (p16) and Arf mRNA, and senescence-associated ß-galacotosidase (SA-ß-gal) activity, but not with telomere shortening or increased apoptosis, suggesting that TBI induces residual BM injury via induction of HSC premature senescence. This suggestion is supported by the finding that SA-ß-gal(+) HSC-enriched LSK cells showed more pronounced defects in clonogenic activity in vitro and long-term engraftment after transplantation than SA-ß-gal(-) LSK cells isolated from irradiated mice. However, genetic deletion of p16 and/or Arf had no effect on TBI-induced residual BM suppression and HSC senescence, because HSCs from irradiated p16 and/or Arf knockout (KO) mice exhibited changes similar to those seen in HSCs from wild-type mice after exposure to TBI. These findings provide important new insights into the mechanism by which TBI causes long-term BM suppression (eg, via induction of premature senescence of HSCs in a p16-Arf-independent manner).

Circular RNAs are abundant, conserved, and associated with ALU repeats.

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a "backsplice") and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.

RAS unplugged: negative feedback and oncogene-induced senescence.

Many normal cells respond to certain stresses, such as oncogene activation, by undergoing a permanent form of growth arrest known as senescence, an intrinsic tumor suppressor program. The predominant view has been that senescence is caused in some settings through a mutant oncogene's ability to induce activation of high levels of sustained MAP kinase and PI3 kinase signaling. A new study in this issue of Cancer Cell has challenged this model with the surprising finding that aberrant activation of the RAS/RAF pathway can induce a negative feedback loop that globally attenuates MAPK and PI3K signaling and that the reduction of signaling in these pathways is required for senescence.

The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies.

To understand the role of human epidermal growth factor receptor (hEGFR) kinase domain mutations in lung tumorigenesis and response to EGFR-targeted therapies, we generated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants seen in human lung cancer. Both bitransgenic lines developed lung adenocarcinoma after sustained hEGFR mutant expression, confirming their oncogenic potential. Maintenance of these lung tumors was dependent on continued expression of the EGFR mutants. Treatment with small molecule inhibitors (erlotinib or HKI-272) as well as prolonged treatment with a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression. These data suggest that persistent EGFR signaling is required for tumor maintenance in human lung adenocarcinomas expressing EGFR mutants.