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Abstract Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged—the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context—and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

LKB1 tumour suppressor gene is frequently mutated in lung adenocarcinoma. Here the authors show that in genetically engineered mouse models of lung cancer Lkb1 restoration induces growth arrest and drives neoplastic cells toward a more differentiated and less proliferative alveolar type II cell-like state via C/EBP-mediated reprogramming.

Summary: Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

In vivo modeling combined with CRISPR/Cas9-mediated somatic genome editing has contributed to elucidating the functional importance of specific genetic alterations in human tumors. Our recent work uncovered tumor suppressor pathways that affect EGFR-driven lung tumor growth and sensitivity to tyrosine kinase inhibitors and reflect the mutational landscape and treatment outcomes in the human disease.

In a recent issue of Cell, Liu et al. present an innovative mouse model system in which Cre/lox stochastically turns on transgenic expression of one out of up to 100 sgRNAs in somatic cells, creating genetic mosaicism that enables the multiplexed assessment of gene function in vivo.

Abstract Metastasis is a major cause of morbidity and mortality in cancer patients. However, the molecular and cellular mechanisms underlying the ability of cancer cells to metastasize remain relatively poorly understood. Among all solid tumors, small cell lung cancer (SCLC) has remarkable metastatic proclivity, with a majority of patients diagnosed with metastatic disease. Our understanding of SCLC metastasis has been hampered for many years by the paucity of material from primary tumors and metastases, as well as the lack of faithful pre‐clinical models. Here, we review recent advances that are helping circumvent these limitations. These advances include methods that employ circulating tumor cells from the blood of SCLC patients and the development of diverse genetically engineered mouse models of metastatic SCLC. New insights into the cellular mechanisms of SCLC metastasis include observations of cell fate changes associated with increased metastatic ability. Ongoing studies on cell migration and organ tropism promise to expand our understanding of SCLC metastasis. Ultimately, a better molecular understanding of metastatic phenotypes may be translated into new therapeutic options to limit metastatic spread and treat metastatic SCLC.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of the cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

Abstract Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence of disseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.

Genome editing technologies enable the rapid interrogation of genetic alterations. Here, the authors present a CRISPR/Cas9-based platform to simultaneously investigate multiple activating point mutations in de novo cancers in mice; and generate panels of Kras-variants in different tissues to induce cancer.

Jorge Polonia,1,2 André Azevedo,1 Miguel Monte,1 José A Silva,2 Susana Bertoquini1,2 1Department of Medicine, CINTESIS, Faculty of Medicine of Porto, University of Porto, Porto, Portugal; 2Blood Pressure Unit, Hospital Pedro Hispano, Matosinhos, Portugal Background: Chronic kidney disease (CKD) complicates hypertension and diabetes. Knowledge of the deterioration rate of CKD may anticipate adjustment of therapies with renal elimination. This study evaluates the rate of annual deterioration of renal function in hypertensive patients either with type 2 diabetes (DM2) or without it (non-DM) followed for 5 years and relates it with blood pressure (BP) and glycemic control. Materials and methods: Out of a total of 1924 patients, 1023 patients (594 non-DM and 429 DM2, 53% female, aged 62.1±10.2 years) were evaluated over 5 years for the annual evolution of renal function (estimated glomerular filtration rate [eGFR], Modification of Diet in Renal Disease) ambulatory 24-hour blood pressure and metabolic parameters, corresponding to the analysis of 2378 patient-years. Results: DM2 and non-DM did not differ for age, mean 24-hour BP levels, nighttime BP, albuminuria, and body mass index. DM2 versus non-DM showed a higher (p8.0% in DM2 predicted the decline in eGFR. On average, 16.2% of DM2 and 13.1% of non-DM moved each year toward a more severe stage of CKD. For initial eGFR 10% from the previous eGFR value (p=0.042). Conclusion: Progressive deterioration of renal function each year is frequent in hypertensive diabetic and non-diabetic patients. Beyond aging, this is particularly dependent on BP control particularly at nighttime, on drug therapy, and on highly abnormal glucose control. Keywords: renal function, annual deterioration, hypertension, diabetes, chronic kidney disease, diabetes type 2, caucasians, adults

Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC.

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

Abstract As governments around the world seek to develop and implement co-management practices with Indigenous peoples and local communities, there are many questions about how to foster collaborative and just agreements that support self-determination. Despite policy advancements enabling more co-management and shared stewardship within the United States (U.S.), possibilities for co-management are complicated by historical injustices that have hindered Tribal access to ancestral land and cultural practices. We use a multiple case study approach, examining three case studies from the Western U.S. that illustrate challenges facing Tribes and interventions used to improve land management relationships. The first case study focuses on the relationship between the Oglala Sioux Tribe and the U.S. government and highlights that historical injustices continue to affect Tribal land management. The second case study explores the collaborative management of Bears Ears National Monument, showcasing a decolonized approach endorsed by Tribes seeking to reclaim their inherent rights to the land. The third case study examines the Snoqualmie Tribe as an example of a sovereign Tribe embodying the concept of “Land Back.” Tribes have utilized strategies, such as co-management, coalition building, and land reacquisition, to maintain control over lands important to their populations. These strategies promote power-sharing, resource distribution, trust-building, and the integration of Indigenous knowledge with Western science. Nonetheless, failure to recognize and account for the weight of broken treaties, discriminatory laws and policies, and the historical trauma caused by these injustices emphasizes the importance of understanding the dynamics surrounding Tribal land management. As government-led land management paradigms around the world shift to consider self-determination of Indigenous nations and communities as a critical piece of improved stewardship, decision makers have many historically-informed dynamics to consider when shaping future co-management practices. KEYWORDS: co-management, Indigenous, “Land Back”, stewardship, Tribes, and sovereignty.

The cost-effective implementation of nanofibrillated cellulose (CNF) at industrial scale requires optimizing the quality of the nanofibers according to their final application. Therefore, a portfolio of CNFs with different qualities is necessary, as well as further knowledge about how to obtain each of the main qualities. This paper presents the influence of various production techniques on the morphological characteristics and properties of CNFs produced from a mixture of recycled fibers. Five different pretreatments have been investigated: a mechanical pretreatment (PFI refining), two enzymatic hydrolysis strategies, and TEMPO-mediated oxidation under two different NaClO concentrations. For each pretreatment, five high-pressure homogenization (HPH) conditions have been considered. Our results show that the pretreatment determines the yield and the potential of HPH to enhance fibrillation and, therefore, the final CNF properties. These results enable one to select the most effective production method with the highest yield of produced CNFs from recovered paper for the desired CNF quality in diverse applications. [ABSTRACT FROM AUTHOR]

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order. [ABSTRACT FROM AUTHOR]

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 Is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers., Introduction Small-cell lung cancer (SCLC), which originates from neuroendocrine cells of the lung (1, 2), is one of the most lethal subtypes of cancer. Each year, more than 25,000 patients [...]