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Basal cell carcinoma (BCC), the most common type of cancer, is characterized by aberrant Hedgehog (Hh) pathway activity. Mutations in pathway components, such as PATCHED1 (PTCH1), are commonly found in BCC. While the tumor suppressor role of PTCH1 in BCC is well established, how Hh pathway activation disrupts normal skin homeostasis to promote BCC formationremains poorly understood. Like Ptc1, Sufu is a major negative regulator of the Hh pathway. Previously, we showed that inactivation of Sufu in the skin does not result in BCC formation. Why loss of Ptc1, but not Sufu, in the epidermis induces BCC formation is unclear. In this report, we utilized gene expression profiling to identify biological pathways and processes that distinguish Sufu from Ptc1 mutants, and discovered a novel role for Sufu in cell cycle regulation. We demonstrated that the Hh pathway activation inSufu and Ptc1 mutant skin is associated with abnormal cell cycle entry, ectopic expression of D-type cyclins and increasedDNA damage. However, despite the presence of DNA damage, p53 stabilization was impaired in the mutant skin. Alternative mechanism to halt genomic instability is the activation of G2/M cell cycle checkpoint, which can occur independent of p53. We found that while Ptc1 mutant cells continue to cycle, which would favor genomic instability, loss of Sufu results in G2/M cell cycle arrest.This finding may explain why inactivation of Sufu is not sufficient to drive BCC formation. Taken together, these studies revealed a unique role for Sufu in G2/M phase progression, and uncovered the molecular and cellular features associated with Hh-driven BCC. [ABSTRACT FROM AUTHOR]

The release of Ca 2+ ions from endoplasmic reticulum calcium stores is a key event in a variety of cellular processes, including gene transcription, migration and proliferation. This release of Ca 2+ often occurs through inositol 1,4,5-triphosphate receptors and the activity of these channels and the levels of stored Ca 2+ in the endoplasmic reticulum are important regulators of cell death in cancer cells. A recently identified Ca 2+ channel of the endoplasmic reticulum is transmembrane and coiled-coil domains 1 (TMCO1). In this study, we link the overexpression of TMCO1 with prognosis in node-positive basal breast cancer patients. We also identify interacting proteins of TMCO1, which include endoplasmic reticulum-resident proteins involved in Ca 2+ regulation and proteins directly involved in nucleocytoplasmic transport. Interacting proteins included nuclear transport proteins and TMCO1 was shown to have both nuclear and endoplasmic reticulum localisation in MDA-MB-231 basal breast cancer cells. These studies also define a role for TMCO1 in the regulation of breast cancer cells in their sensitivity to BCL-2/MCL-1 inhibitors, analogous to the role of inositol 1,4,5-triphosphate receptors in the regulation of cell death pathways activated by these agents., (© 2024. The Author(s).)

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and inter-tumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury-response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K targeted gRNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury-response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury-response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of β1 integrin, FAK, MEK and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity.

Halting breast cancer metastatic relapses following primary tumor removal and the clinical dormant phase, remains challenging, due to a lack of specific vulnerabilities to target during dormancy. To address this, we conducted genome-wide CRISPR screens on two breast cancer cell lines with distinct dormancy properties: 4T1 (short-term dormancy) and 4T07 (prolonged dormancy). We discovered that loss of class-III PI3K, Pik3c3, revealed a unique vulnerability in 4T07 cells. Surprisingly, dormancy-prone 4T07 cells exhibited higher mTORC1 activity than 4T1 cells, due to lysosome-dependent signaling occurring at the cell periphery. Pharmacological inhibition of Pik3c3 counteracted this phenotype in 4T07 cells, and selectively reduced metastasis burden only in the 4T07 dormancy-prone model. This mechanism was also detected in human breast cancer cell lines in addition to a breast cancer patient-derived xenograft supporting that it may be relevant in humans. Our findings suggest dormant cancer cell-initiated metastasis may be prevented in patients carrying tumor cells that display PIK3C3-peripheral lysosomal signaling to mTORC1., Statement of Significance: We reveal that dormancy-prone breast cancer cells depend on the class III PI3K to mediate a constant peripheral lysosomal positioning and mTORC1 hyperactivity. Targeting this pathway might blunt breast cancer metastasis.

Pharmacological modulation of the Wnt/β-catenin signaling pathway holds promises for both basic research and therapeutic applications. In this issue of Cell Chemical Biology, Kschonsak et al. 1 engineered knotted peptides that promote Wnt signaling by targeting ZNRF3 and serve as pharmacological tools for studying Wnt biology and supporting organoid growth., Competing Interests: Declaration of interests Stephane Angers is a founder, shareholder, and board member of AntlerA Therapeutics. S.A. is also an inventor on patents describing Frizzled antibody agonists (US16/969,909; US17/846,846)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis., (© 2024. The Author(s).)

Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.

The Wnt/β-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of β-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/β-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo., Competing Interests: Competing interests S.A., S.S.S., J.J.A. and L.L.B. hold shares in AntlerA Therapeutics and are inventors on patents for the antibodies described in the manuscript., (© 2024. Published by The Company of Biologists Ltd.)

The identification of genetic regulators of cell secretions is challenging because it requires the sorting of a large number of cells according to their secretion patterns. Here we report the development and applicability of a high-throughput microfluidic method for the analysis of the secretion levels of large populations of immune cells. The method is linked with a kinome-wide loss-of-function CRISPR screen, immunomagnetically sorting the cells according to their secretion levels, and the sequencing of their genomes to identify key genetic modifiers of cell secretion. We used the method, which we validated against flow cytometry for cytokines secreted from primary mouse CD4 + (cluster of differentiation 4-positive) T cells, to discover a subgroup of highly co-expressed kinase-coding genes that regulate interferon-gamma secretion by these cells. We validated the function of the kinases identified using RNA interference, CRISPR knockouts and kinase inhibitors and confirmed the druggability of selected kinases via the administration of a kinase inhibitor in an animal model of colitis. The technique may facilitate the discovery of regulatory mechanisms for immune-cell activation and of therapeutic targets for autoimmune diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

FBXW7 is a commonly mutated tumor suppressor gene that functions to regulate numerous oncogenes involved in cell-cycle regulation. Genome-wide CRISPR fitness screens identified a signature of DNA repair and DNA damage response genes as required for the growth of FBXW7-knockout cells. Guided by these findings, we show that FBXW7-mutant cells have high levels of replication stress, which results in a genotype-specific vulnerability to inhibition of the ATR signaling pathway, as these mutant cells become heavily reliant on a robust S-G2 checkpoint. ATR inhibition induces an accelerated S-phase, leading to mitotic catastrophe and cell death caused by the high replication stress present in FBXW7-/- cells. In addition, we provide evidence in cell and organoid studies, and mining of publicly available high-throughput drug screening efforts, that this genotype-specific vulnerability extends to multiple types of cancer, providing a rational means of identifying responsive patients for targeted therapy., Significance: We have elucidated the synthetic lethal interactions between FBXW7 mutation and DNA damage response genes, and highlighted the potential of ATR inhibitors as targeted therapies for cancers harboring FBXW7 alterations., (© 2023 The Authors; Published by the American Association for Cancer Research.)

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., (© 2023. The Author(s).)

A major impediment to the characterization of mtDNA repair mechanisms in comparison to nuclear DNA repair mechanisms is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of an mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox), we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted doxorubicin (Dox) screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentiality of our top hit, WRNIP1─observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.

Norrin (NDP) and WNT7A/B induce and maintain the blood-brain and blood-retina barrier (BBB, BRB) by stimulating the Frizzled4-LDL receptor related protein 5/6 (FZD4-LRP5/6) complex to induce beta-catenin-dependent signaling in endothelial cells (ECs). Recently developed agonists for the FZD4-LRP5 complex have therapeutic potential in retinal and neurological diseases. Here, we use the tetravalent antibody modality F4L5.13 to identify agonist activities in Tspan12 -/- mice, which display a complex retinal pathology due to impaired NDP-signaling. F4L5.13 administration during development alleviates BRB defects, retinal hypovascularization, and restores neural function. In mature Tspan12 -/- mice F4L5.13 partially induces a BRB de novo without inducing angiogenesis. In a genetic model of impaired BRB maintenance, administration of F4L5.13 rapidly and substantially restores the BRB. scRNA-seq reveals perturbations of key mediators of barrier functions in juvenile Tspan12 -/- mice, which are in large parts restored after F4L5.13 administration. This study identifies transcriptional and functional activities of FZD4-LRP5 agonists., Competing Interests: S.A. is a shareholder of AntlerA Therapeutics and inventor on patent application 20210032352. H.J.J. is scientific advisor for AntlerA Therapeutics and received an honorarium., (Crown Copyright © 2023.)

Nanoneedles are a useful tool for delivering exogenous biomolecules to cells. Although therapeutic applications have been explored, the mechanism regarding how cells interact with nanoneedles remains poorly studied. Here, we present a new approach for the generation of nanoneedles, validated their usefulness in cargo delivery, and studied the underlying genetic modulators during delivery. We fabricated arrays of nanoneedles based on electrodeposition and quantified its efficacy of delivery using fluorescently labeled proteins and siRNAs. Notably, we revealed that our nanoneedles caused the disruption of cell membranes, enhanced the expression of cell-cell junction proteins, and downregulated the expression of transcriptional factors of NFκB pathways. This perturbation trapped most of the cells in G2 phase, in which the cells have the highest endocytosis activities. Taken together, this system provides a new model for the study of interactions between cells and high-aspect-ratio materials.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5 ag ) or Fzd6 ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6 ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury., Competing Interests: Declaration of interests A.N.N., J.D.W., C. Everrett, C. Eidenschenk, A.A., I.F., H.D.B., Z.M., K.N., V.M.D., and J.R.A. are current or former employees of Genentech Inc. J.R.A. is an employee of 23andMe. J.J.A., L.B., S.S., S.A., and S.S.S. are shareholders of AntlerA Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Our objective was to compare [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 and [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 radioimmunotherapy (RIT) agents for decreasing the clonogenic survival fraction (SF) in vitro of EGFR-positive human pancreatic ductal adenocarcinoma (PDAC) cell lines and estimate the relative biological effectiveness (RBE) vs. γ-radiation (XRT)., Methods: EGFR-positive PDAC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1) and EGFR-knockout PANC-1 EGFR KO cells were treated in vitro for 18 h with (0-19.65 MBq; 72 nmols/L) of [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 or [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 or XRT (0-8 Gy) followed by clonogenic assay. The SF was determined after culturing single treated cells for 14 d. Cell fractionation studies were performed for cells incubated with 1 MBq (72 nmols/L) of [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 or [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 for 1, 4, or 24 h to estimate the time-integrated activity (Ã) on the cell surface, cytoplasm, nucleus and medium. Radiation absorbed doses in the nucleus were calculated by multiplying à by S-factors calculated by Monte Carlo N Particle (MCNP) modeling using monolayer cell culture geometry. The SF of PDAC cells was plotted vs. dose and fitted to a linear quadratic model to estimate the dose required to decrease the SF to 0.1 (D 10 ). The D 10 for RIT agents were compared to XRT to estimate the RBE. DNA double-strand breaks (DSBs) caused by [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 or [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 continuous exposure for 5 h or 20 h were probed by immunofluorescence for γ-H2AX. Relative EGFR expression of PDAC cells was assessed by flow cytometry (scored + to +++) and cell doubling times for untreated cells were determined., Results: The D 10 for [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 ranged from 9.1 Gy (PANC-1) to 39.9 Gy (Capan-1). The D 10 for [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 ranged from 11.7 Gy (AsPC-1) to 170.8 Gy (Capan-1). The D 10 for XRT ranged from 2.5 Gy (Capan-1) to 6.7 Gy (PANC-1 EGFR KO). D 10 values were not correlated with EGFR expression over a relatively narrow range (++ to +++) or with cell doubling times. Based on D 10 values, PANC-1 EGFR KO cells were 1.6-fold less sensitive than PANC-1 cells to [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 and 1.9-fold less sensitive to [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 . The RBE for [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 ranged from 0.06 for Capan-1 cells to 0.45 for PANC-1 cells. The RBE for [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 ranged from 0.015 for Capan-1 cells to 0.28 for AsPC-1 cells. DNA DSBs were detected in PDAC cells exposed to [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 or [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 but were not correlated with the SF of the cells., Conclusions: We conclude that at the same dose delivered to the cell nucleus [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 and [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 were less radiobiologically effective than XRT for decreasing the SF of human PDAC cells, but [ 64 Cu]Cu-NOTA-panitumumab F(ab') 2 was more cytotoxic than [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 except for AsPC-1 cells which were more sensitive to [ 177 Lu]Lu-NOTA-panitumumab F(ab') 2 ., Advances in Knowledge and Implications for Patient Care: This study demonstrates that higher radiation doses may be required for RIT than XRT to achieve radiobiologically equivalent effects when used to treat PDAC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Since the recognition that mutations in components of the Wnt-β-catenin pathway underlie some human cancers, considerable attention has been dedicated to developing therapeutic modalities to block its activity. Despite numerous efforts, no drug directly inhibiting Wnt signaling is currently clinically available. Conversely, activating the Wnt pathway in a specific manner has recently been made possible with new molecules mimicking the activity of Wnt proteins, thus offering new possibilities for controlling tissue stem cell activity and for the rational treatment of various degenerative conditions. We describe the landscape of antibody modalities that modulate the Wnt-β-catenin pathway, and detail the advances and challenges in both cancer and regenerative medicine drug development., Competing Interests: Declaration of interests S.A. is a scientific founder and shareholder of AntlerA Therapeutics. The other authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Despite multimodal treatment including surgery, radiotherapy, and chemotherapy, median patient survival has remained at ~15 months for decades. This situation demands an outside-the-box treatment approach. Using magnetic carbon nanotubes (mCNTs) and precision magnetic field control, we report a mechanical approach to treat chemoresistant GBM. We show that GBM cells internalize mCNTs, the mobilization of which by rotating magnetic field results in cell death. Spatiotemporally controlled mobilization of intratumorally delivered mCNTs suppresses GBM growth in vivo. Functionalization of mCNTs with anti-CD44 antibody, which recognizes GBM cell surface-enriched antigen CD44, increases mCNT recognition of cancer cells, prolongs mCNT enrichment within the tumor, and enhances therapeutic efficacy. Using mouse models of GBM with upfront or therapy-induced resistance to temozolomide, we show that mCNT treatment is effective in treating chemoresistant GBM. Together, we establish mCNT-based mechanical nanosurgery as a treatment option for GBM.

During embryo development, cell proliferation, cell fate specification and tissue patterning are coordinated and tightly regulated by a handful of evolutionarily conserved signaling pathways activated by secreted growth factor families including fibroblast growth factor (FGF), Nodal/bone morphogenetic protein (BMP), Hedgehog and Wnt. The spatial and temporal activation of these signaling pathways elicit context-specific cellular responses that ultimately shape the different tissues of the embryo. Extensive efforts have been dedicated to identifying the molecular mechanisms underlying these signaling pathways during embryo development, adult tissue homeostasis and regeneration. In this review, we first describe the role of the Wnt/β-catenin signaling pathway during early embryo development, axis specification and cell differentiation as a prelude to highlight how this knowledge is being leveraged to manipulate Wnt/β-catenin signaling activity with small molecules and biologics for the directed differentiation of pluripotent stem cells into various cell lineages that are physiologically relevant for stem cell therapy and regenerative medicine., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of β-catenin nuclear accumulation, a phenotypic hallmark of APC -mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment., (© 2022 The Authors.)

CDK11 is a cyclin-dependent kinase that controls proliferation by regulating transcription, RNA splicing, and the cell cycle. As its activity is increasingly associated with cancer, CDK11 is an attractive target for the development of small-molecule inhibitors. However, the development of CDK11 inhibitors with limited off-target effects against other CDKs poses a challenge based on the high conservation of sequence across family members. OTS964 is notable as it displays a measure of specificity for CDK11 in cells. To understand the basis for OTS964's specificity for CDK11, we solved a 2.6 Å crystal structure of the CDK11 kinase domain bound to OTS964. Despite the absence of cyclin, CDK11 adopts an active-like conformation when bound to OTS964. We identified amino acids likely to contribute to the specificity of OTS964 for CDK11 and assessed their contribution to OTS964 binding by isothermal titration calorimetry (ITC) in vitro and by resistance to OTS964 in cells., Competing Interests: Declaration of interests F.S. is a founder and consultant of Repare Therapeutics., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Melanoma is the deadliest form of skin cancer and considered intrinsically resistant to chemotherapy. Nearly all melanomas harbor mutations that activate the RAS/mitogen-activated protein kinase (MAPK) pathway, which contributes to drug resistance via poorly described mechanisms. Herein we show that the RAS/MAPK pathway regulates the activity of cyclin-dependent kinase 12 (CDK12), which is a transcriptional CDK required for genomic stability. We find that melanoma cells harbor constitutively high CDK12 activity, and that its inhibition decreases the expression of long genes containing multiple exons, including many genes involved in DNA repair. Conversely, our results show that CDK12 inhibition promotes the expression of short genes with few exons, including many growth-promoting genes regulated by the AP-1 and NF-κB transcription factors. Inhibition of these pathways strongly synergize with CDK12 inhibitors to suppress melanoma growth, suggesting promising drug combinations for more effective melanoma treatment., (© 2022. The Author(s).)

The conclusive identity of Wnts regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of β-catenin. Using single-cell analysis, we identified a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3. EC-elimination of Wnt2 and Wnt9b led to both loss of β-catenin targets in zone 3, and re-appearance of zone 1 genes in zone 3, unraveling dynamicity in the LZ process. Impaired LR observed in the knockouts phenocopied models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts and induced zone 3 gene expression and LR in controls. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF., Competing Interests: Declaration of interests S.P.M. is a consultant for Surrozen. L.L.B., J.J.A., S.S.S., and S.A. are shareholders of AntlerA Therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2 -a gene encoding a secreted protein acting as a cellular migration cue-as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype, resulting in enhanced cancer progression in xenograft models.

Ligands of the Wnt family activate the Frizzled-LRP5/6 receptor complex to initiate intracellular signaling. In this issue of Science Signaling , Mahoney et al. reveal a Wnt-stimulated positive feedback loop that involves local production of the lipid phosphatidylinositol(4,5)bisphosphate [PI(4,5)P 2 ], which promotes Dishevelled recruitment and additional PI(4,5)P 2 production, to facilitate LRP5/6 phosphorylation.

Magnetic cell sorting is an enabling tool for the isolation of specific cellular subpopulations for downstream applications and requires the cells to be labeled by a sufficient number of magnetic nanoparticles to leverage magnetophoresis for efficient separation. This requirement makes it challenging to target weakly expressed biomarkers. Here, we developed a new approach that selectively and efficiently amplifies the magnetic labeling on cells through sequentially connected antibodies and nanoparticles delivered to the surface or interior of the cell. Using this approach, we achieved amplification up to 100-fold for surface and intracellular markers. We also demonstrated the utility of this assay for enabling high-performance magnetic cell sorting when it is applied to the analysis of rare tumor cells for cancer diagnosis and the purification of transfected CAR T cells for immunotherapy. The data presented demonstrate a useful tool for the stratification of rare cell subpopulations.