The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma.

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

FireLossRate: An R package to estimate the loss rate of residential structures affected by wildfires at the Wildland Urban Interface.

To inform proactive management actions supporting community resilience to wildfires, we developed a new software package called FireLossRate. This package in R helps the user to compute wildfire impacts on residential structures at the Wildland Urban Interface (WUI). The package integrates spatial information about exposed structures, empirical equations that estimate the loss rate of structures affected by wildfires as a function of fireline intensity and distance from fire edge with fire growth modeling outputs from fire simulation software and burn probability models. FireLossRate helps to quantify and produce spatially explicit data on structural exposure and loss for single and multiple fires. The package automates post hoc analyses on simulations that include single or multiple wildfires and enables result mapping when combined with other packages available in R. In this paper, we describe the functionality of the FireLossRate package and introduce users to the interpretation of impact indicators of wildfires at the WUI. FireLossRate is available for download at https://github.com/LFCFireLab/FireLossRate.•FireLossRate allows the computation of wildfire impacts indicators on residential structures at the Wildland Urban Interface in support of community fire risk management.

HER2Δ16 Engages ENPP1 to Promote an Immune-Cold Microenvironment in Breast Cancer.

The tumor-immune microenvironment (TIME) is a critical determinant of therapeutic response. However, the mechanisms regulating its modulation are not fully understood. HER2Δ16, an oncogenic splice variant of the HER2, has been implicated in breast cancer and other tumor types as a driver of tumorigenesis and metastasis. Nevertheless, the underlying mechanisms of HER2Δ16-mediated oncogenicity remain poorly understood. Here, we show that HER2∆16 expression is not exclusive to the clinically HER2+ subtype and associates with a poor clinical outcome in breast cancer. To understand how HER2 variants modulated the tumor microenvironment, we generated transgenic mouse models expressing either proto-oncogenic HER2 or HER2Δ16 in the mammary epithelium. We found that HER2∆16 tumors were immune cold, characterized by low immune infiltrate and an altered cytokine profile. Using an epithelial cell surface proteomic approach, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as a functional regulator of the immune cold microenvironment. We generated a knock-in model of HER2Δ16 under the endogenous promoter to understand the role of Enpp1 in aggressive HER2+ breast cancer. Knockdown of Enpp1 in HER2Δ16-derived tumor cells resulted in decreased tumor growth, which correlated with increased T-cell infiltration. These findings suggest that HER2Δ16-dependent Enpp1 activation associates with aggressive HER2+ breast cancer through its immune modulatory function. Our study provides a better understanding of the mechanisms underlying HER2Δ16-mediated oncogenicity and highlights ENPP1 as a potential therapeutic target in aggressive HER2+ breast cancer.

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer.

How the genetic landscape governs a tumor's response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by KrasG12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies.

Short- and long-term wildfire threat when adapting infrastructure for wildlife conservation in the boreal forest.

Managers designing infrastructure in fire-prone wildland areas require assessments of wildfire threat to quantify uncertainty due to future vegetation and climatic conditions. In this study, we combine wildfire simulation and forest landscape composition modeling to identify areas that would be highly susceptible to wildfire around a proposed conservation corridor in Québec, Canada. In this measure, managers have proposed raising the conductors of a new 735-kV hydroelectric powerline above the forest canopy within a wildlife connectivity corridor to mitigate the impacts to threatened boreal woodland caribou (Rangifer tarandus). Retention of coniferous vegetation, however, can increase the likelihood of an intense wildfire damaging powerline infrastructure. To assess the likelihood of high-intensity wildfires for the next 100 years, we evaluated three time periods (2020, 2070, 2120), three climate scenarios (observed, RCP 4.5, RCP 8.5), and four vegetation projections (static, no harvest, extensive harvesting, harvesting excluded in protected areas). Under present-day conditions, we found a lower probability of high-intensity wildfire within the corridor than in other parts of the study area, due to the protective influence of a nearby, poorly regenerated burned area. Wildfire probability will increase into the future, with strong, weather-induced inflation in the number of annual ignitions and wildfire spread potential. However, a conversion to less-flammable vegetation triggered by interactions between climate change and disturbance may attenuate this trend. By addressing the range of uncertainty of future conditions, we present a robust strategy to assist in decision-making about long-term risk management for both the proposed conservation measure and the powerline.

Skin-resident immune cells actively coordinate their distribution with epidermal cells during homeostasis.

Organs consist of multiple cell types that ensure proper architecture and function. How different cell types coexist and interact to maintain their homeostasis in vivo remains elusive. The skin epidermis comprises mostly epithelial cells, but also harbours Langerhans cells (LCs) and dendritic epidermal T cells (DETCs). Whether and how distributions of LCs and DETCs are regulated during homeostasis is unclear. Here, by tracking individual cells in the skin of live adult mice over time, we show that LCs and DETCs actively maintain a non-random spatial distribution despite continuous turnover of neighbouring basal epithelial cells. Moreover, the density of epithelial cells regulates the composition of LCs and DETCs in the epidermis. Finally, LCs require the GTPase Rac1 to maintain their positional stability, density and tiling pattern reminiscent of neuronal self-avoidance. We propose that these cellular mechanisms provide the epidermis with an optimal response to environmental insults.

Computation-guided optimization of split protein systems.

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.

Cx43 Present at the Leading Edge Membrane Governs Promigratory Effects of Osteoblast-Conditioned Medium on Human Prostate Cancer Cells in the Context of Bone Metastasis.

Among the different interacting molecules implicated in bone metastases, connexin43 (Cx43) may increase sensitivity of prostate cancer (PCa) cells to bone microenvironment, as suggested by our in silico and human tissue samples analyses that revealed increased level of Cx43 expression with PCa progression and a Cx43 specific expression in bone secondary sites. The goal of the present study was to understand how Cx43 influences PCa cells sensitivity and aggressiveness to bone microenvironment. By means of Cx43-overexpressing PCa cell lines, we revealed a Cx43-dependent promigratory effect of osteoblastic conditioned media (ObCM). This effect on directional migration relied on the presence of Cx43 at the plasma membrane and not on gap junctional intercellular communication and hemichannel functions. ObCM stimulation induced Rac1 activation and Cx43 interaction with cortactin in protrusions of migrating PCa cells. Finally, by transfecting two different truncated forms of Cx43 in LNCaP cells, we determined that the carboxy terminal (CT) part of Cx43 is crucial for the responsiveness of PCa cells to ObCM. Our study demonstrates that Cx43 level and its membrane localization modulate the phenotypic response of PCa cells to osteoblastic microenvironment and that its CT domain plays a pivotal role.

An ErbB2 splice variant lacking exon 16 drives lung carcinoma.

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2 In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.

Hair follicle regeneration suppresses Ras-driven oncogenic growth.

Mutations associated with tumor development in certain tissues can be nontumorigenic in others, yet the mechanisms underlying these different outcomes remains poorly understood. To address this, we targeted an activating Hras mutation to hair follicle stem cells and discovered that Hras mutant cells outcompete wild-type neighbors yet are integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium leads to benign outgrowths. Follicular Hras mutant cells autonomously and nonautonomously enhance regeneration, which directs mutant cells into continuous tissue cycling to promote integration rather than aberrancy. This follicular tolerance is maintained under additional challenges that promote tumorigenesis in the epidermis, including aging, injury, and a secondary mutation. Thus, the hair follicle possesses a unique, enhanced capacity to integrate and contain Hras mutant cells within both homeostatic and perturbed tissue, demonstrating that in the skin, multiple, distinct mechanisms exist to suppress oncogenic growth.

Positional Stability and Membrane Occupancy Define Skin Fibroblast Homeostasis In Vivo.

Fibroblasts are an essential cellular and structural component of our organs. Despite several advances, the critical behaviors that fibroblasts utilize to maintain their homeostasis in vivo have remained unclear. Here, by tracking the same skin fibroblasts in live mice, we show that fibroblast position is stable over time and that this stability is maintained despite the loss of neighboring fibroblasts. In contrast, fibroblast membranes are dynamic during homeostasis and extend to fill the space of lost neighboring fibroblasts in a Rac1-dependent manner. Positional stability is sustained during aging despite a progressive accumulation of gaps in fibroblast nuclei organization, while membrane occupancy continues to be maintained. This work defines positional stability and cell occupancy as key principles of skin fibroblast homeostasis in vivo, throughout the lifespan of mice, and identifies membrane extension in the absence of migration as the core cellular mechanism to carry out these principles.

Homeostatic Epidermal Stem Cell Self-Renewal Is Driven by Local Differentiation.

Maintenance of adult tissues depends on sustained activity of resident stem cell populations, but the mechanisms that regulate stem cell self-renewal during homeostasis remain largely unknown. Using an imaging and tracking approach that captures all epidermal stem cell activity in large regions of living mice, we show that self-renewal is locally coordinated with epidermal differentiation, with a lag time of 1 to 2 days. In both homeostasis and upon experimental perturbation, we find that differentiation of a single stem cell is followed by division of a direct neighbor, but not vice versa. Finally, we show that exit from the stem cell compartment is sufficient to drive neighboring stem cell self-renewal. Together, these findings establish that epidermal stem cell self-renewal is not the constitutive driver of homeostasis. Instead, it is precisely tuned to tissue demand and responds directly to neighbor cell differentiation.

Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis.

The molecular mechanisms governing the formation of lymphatic vasculature are not yet well understood. Pannexins are transmembrane proteins that form channels which allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. The expression and function of pannexins in blood vessels have been studied in the last few decades. Meanwhile, no studies have been conducted to evaluate the role of pannexins during human lymphatic vessel formation. Here we show, using primary human dermal lymphatic endothelial cells (HDLECs), pharmacological tools (probenecid, Brilliant Blue FCF, mimetic peptides [10Panx]) and siRNA-mediated knockdown that Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. These results newly identify Pannexin-1 as a protein highly expressed in HDLECs and its requirement during in vitro lymphangiogenesis.

Connexins, important players in the dissemination of prostate cancer cells.

Over the past 50years, increasing experimental evidences have established that connexins (Cxs) and gap junctional intercellular communication (GJIC) ensure an important role in both the onset and development of cancerous processes. In the present review, we focus on the impact of Cxs and GJIC during the development of prostate cancer (PCa), from the primary growth mainly localized in acinar glands and ducts to the distant metastasis mainly concentrated in bone. As observed in several other types of solid tumours, Cxs and especially Cx43 exhibit an ambivalent role with a tumour suppressor effect in the early stages and, conversely, a rather pro-tumoural profile for most of invasion and dissemination steps to secondary sites. We report here the current knowledge on the function of Cxs during PCa cells migration, cytoskeletal dynamics, proteinases activities and the cross talk with the surrounding stromal cells in the microenvironment of the tumour and the bones. In addition, we discuss the role of Cxs in the bone tropism even if the prostate model is rarely used to study the complete sequence of cancer dissemination compared to breast cancer or melanoma. Even if not yet fully understood, these recent findings on Cxs provide new insights into their molecular mechanisms associated with progression and bone targeted behaviour of PCa. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

An update on minding the gap in cancer.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

Correction of aberrant growth preserves tissue homeostasis.

Cells in healthy tissues acquire mutations with surprising frequency. Many of these mutations are associated with abnormal cellular behaviours such as differentiation defects and hyperproliferation, yet fail to produce macroscopically detectable phenotypes. It is currently unclear how the tissue remains phenotypically normal, despite the presence of these mutant cells. Here we use intravital imaging to track the fate of mouse skin epithelium burdened with varying numbers of activated Wnt/ß-catenin stem cells. We show that all resulting growths that deform the skin tissue architecture regress, irrespective of their size. Wild-type cells are required for the active elimination of mutant cells from the tissue, while utilizing both endogenous and ectopic cellular behaviours to dismantle the aberrant structures. After regression, the remaining structures are either completely eliminated or converted into functional skin appendages in a niche-dependent manner. Furthermore, tissue aberrancies generated from oncogenic Hras, and even mutation-independent deformations to the tissue, can also be corrected, indicating that this tolerance phenomenon reflects a conserved principle in the skin. This study reveals an unanticipated plasticity of the adult skin epithelium when faced with mutational and non-mutational insult, and elucidates the dynamic cellular behaviours used for its return to a homeostatic state.

Modulation of the inwardly rectifying potassium channel Kir4.1 by the pro-invasive miR-5096 in glioblastoma cells.

Inwardly rectifying potassium channels (Kir), and especially the barium-sensitive Kir4.1 encoded by KCNJ10, are key regulators of glial functions. A lower expression or mislocation of Kir4.1 is detected in human brain tumors. MicroRNAs participate in the regulation of ionic channels and associated neurologic disorders. Here, we analyze effects of miR-5096 on the Kir4.1 expression and function in two glioblastoma cell lines, U87 and U251. Using whole-cell patch-clamp and western-blot analysis, we show that cell loading with miR-5096 decreases the Kir4.1 protein level and associated K+ current. Cell treatment with barium, a Kir4.1 blocker, or cell loading of miR-5096 both increase the outgrowth of filopodia in glioma cells, as observed by time-lapse microscopy. Knocking-down Kir4.1 expression by siRNA transfection similarly increased both filopodia formation and invasiveness of glioma cells as observed in Boyden chamber assay. MiR-5096 also promotes the release of extracellular vesicles by which it increases its own transfer to surrounding cells, in a Kir4.1-dependent manner in U251 but not in U87. Altogether, our results validate Kir4.1 as a miR-5096 target to promote invasion of glioblastoma cells. Our data highlight the complexity of microRNA effects and the role of K+ channels in cancer.

Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice.

Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviours and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviours by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional coexistence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviours and consequent changes in homeostatic behaviours that support tissue-scale organization of wound re-epithelialization.