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Human Epidermal growth factor Receptor 4 (HER4 or ERBB4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1β. Here, we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1β and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1β and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers.

Understanding the cell cycle at the single-cell level is crucial for cellular biology and cancer research. While current methods using fluorescent markers have improved the study of adherent cells, non-adherent cells remain challenging. In this study, we addressed this gap by combining a specialized surface to enhance cell attachment, the FUCCI(CA)2 sensor, an automated image analysis pipeline, and a custom machine learning algorithm. This approach enabled precise measurement of cell cycle phase durations in non-adherent cells. This method was validated in acute myeloid leukemia cell lines NB4 and Kasumi-1, which have unique cell cycle characteristics, and we tested the impact of cell cycle-modulating drugs on NB4 cells. Our cell cycle analysis system, which is also compatible with adherent cells, is fully automated and freely available, providing detailed insights from hundreds of cells under various conditions. This report presents a valuable tool for advancing cancer research and drug development by enabling comprehensive, automated cell cycle analysis in both adherent and non-adherent cells., Competing Interests: KH, CS, ES, EC, OE, SP, IN, GB, GF, RC, SM, SR No competing interests declared, (© 2024, Hayatigolkhatmi, Soriani, Soda et al.)

Effects from aging in single cells are heterogenous, whereas at the organ- and tissue-levels aging phenotypes tend to appear as stereotypical changes. The mammary epithelium is a bilayer of two major phenotypically and functionally distinct cell lineages: luminal epithelial and myoepithelial cells. Mammary luminal epithelia exhibit substantial stereotypical changes with age that merit attention because these cells are the putative cells-of-origin for breast cancers. We hypothesize that effects from aging that impinge upon maintenance of lineage fidelity increase susceptibility to cancer initiation. We generated and analyzed transcriptomes from primary luminal epithelial and myoepithelial cells from younger <30 (y)ears old and older >55y women. In addition to age-dependent directional changes in gene expression, we observed increased transcriptional variance with age that contributed to genome-wide loss of lineage fidelity. Age-dependent variant responses were common to both lineages, whereas directional changes were almost exclusively detected in luminal epithelia and involved altered regulation of chromatin and genome organizers such as SATB1 . Epithelial expression of gap junction protein GJB6 increased with age, and modulation of GJB6 expression in heterochronous co-cultures revealed that it provided a communication conduit from myoepithelial cells that drove directional change in luminal cells. Age-dependent luminal transcriptomes comprised a prominent signal that could be detected in bulk tissue during aging and transition into cancers. A machine learning classifier based on luminal-specific aging distinguished normal from cancer tissue and was highly predictive of breast cancer subtype. We speculate that luminal epithelia are the ultimate site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype both endows cells with the ability to become cancer-cells-of-origin and represents a biosensor that presages cancer susceptibility., Competing Interests: RS, MM, EC, PS, AZ, SS, MT, VS, SN, MS, DS, ML The authors declare that no competing interests exist.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here, we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single-cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested hepatocyte nuclear factor 4 alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9 , a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers., Competing Interests: JS, JP, JL, JJ, RD, SM, JK, SP, JH, JW, AB No competing interests declared, JC Reviewing editor, eLife, (© 2024, Shin, Park et al.)

A high density of tumor-associated macrophages (TAMs) is associated with poorer prognosis and survival in breast cancer patients. Recent studies have shown that lipid accumulation in TAMs can promote tumor growth and metastasis in various models. However, the specific molecular mechanisms that drive lipid accumulation and tumor progression in TAMs remain largely unknown. Herein, we demonstrated that unsaturated fatty acids (FAs), unlike saturated ones, are more likely to form lipid droplets in murine macrophages. Specifically, unsaturated FAs, including linoleic acids (LA), activate the FABP4/CEBPα pathway, leading to triglyceride synthesis and lipid droplet formation. Furthermore, FABP4 enhances lipolysis and FA utilization by breast cancer cell lines, which promotes cancer cell migration in vitro and metastasis in vivo. Notably, a deficiency of FABP4 in murine macrophages significantly reduces LA-induced lipid metabolism. Therefore, our findings suggest FABP4 as a crucial lipid messenger that facilitates unsaturated FA-mediated lipid accumulation and lipolysis in TAMs, thus contributing to the metastasis of breast cancer., Competing Interests: MY, XJ, SL, JH, JY, AA, ZL, MC, HK, JS, AK, MK, YX, ES, SS, BL No competing interests declared

This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC., Competing Interests: NK, SP, AJ, HE, HK, KL, JC, BK, HJ, JS, SL, JA, JL, JC, DJ, MN, HK, JK, JC, HL, MA No competing interests declared, (© 2024, Kim, Park et al.)

The advent of tyrosine kinase inhibitors (TKIs) as treatment of chronic myeloid leukemia (CML) is a paradigm in molecularly targeted cancer therapy. Nonetheless, TKI-insensitive leukemia stem cells (LSCs) persist in most patients even after years of treatment and are imperative for disease progression as well as recurrence during treatment-free remission (TFR). Here, we have generated high-resolution single-cell multiomics maps from CML patients at diagnosis, retrospectively stratified by BCR::ABL1 IS (%) following 12 months of TKI therapy. Simultaneous measurement of global gene expression profiles together with >40 surface markers from the same cells revealed that each patient harbored a unique composition of stem and progenitor cells at diagnosis. The patients with treatment failure after 12 months of therapy had a markedly higher abundance of molecularly defined primitive cells at diagnosis compared to the optimal responders. The multiomic feature landscape enabled visualization of the primitive fraction as a mixture of molecularly distinct BCR::ABL1 + LSCs and BCR::ABL1 - hematopoietic stem cells (HSCs) in variable ratio across patients, and guided their prospective isolation by a combination of CD26 and CD35 cell surface markers. We for the first time show that BCR::ABL1 + LSCs and BCR::ABL1 - HSCs can be distinctly separated as CD26 + CD35 - and CD26 - CD35 + , respectively. In addition, we found the ratio of LSC/HSC to be higher in patients with prospective treatment failure compared to optimal responders, at diagnosis as well as following 3 months of TKI therapy. Collectively, this data builds a framework for understanding therapy response and adapting treatment by devising strategies to extinguish or suppress TKI-insensitive LSCs., Competing Interests: RW, LG, FS, MS, SS, RT No competing interests declared, PD, GK Is a board member and has equity in Nygen Analytics AB, HH Received honoraria from Pfizer, Novartis, BMS, and Incyte, SM Received honoraria and research funding from BMS, research funding from Novartis, Janpix, and honoraria from Dren Bio, JR Received honoraria and research funding from Novartis and Bristol-Myers Squibb (BMS) and honoraria from Ariad, (© 2023, Warfvinge et al.)

A new approach helps examine the proportion of cancerous and healthy stem cells in patients with chronic myeloid leukemia and how this influences treatment outcomes., Competing Interests: NA, SE No competing interests declared, (© 2024, Arellano and Elf.)

In the realm of cancer immunotherapy, the meticulous selection of neoantigens plays a fundamental role in enhancing personalized treatments. Traditionally, this selection process has heavily relied on predicting the binding of peptides to human leukocyte antigens (pHLA). Nevertheless, this approach often overlooks the dynamic interaction between tumor cells and the immune system. In response to this limitation, we have developed an innovative prediction algorithm rooted in machine learning, integrating T cell receptor β chain (TCRβ) profiling data from colorectal cancer (CRC) patients for a more precise neoantigen prioritization. TCRβ sequencing was conducted to profile the TCR repertoire of tumor-infiltrating lymphocytes (TILs) from 28 CRC patients. The data unveiled both intra-tumor and inter-patient heterogeneity in the TCRβ repertoires of CRC patients, likely resulting from the stochastic utilization of V and J segments in response to neoantigens. Our novel combined model integrates pHLA binding information with pHLA-TCR binding to prioritize neoantigens, resulting in heightened specificity and sensitivity compared to models using individual features alone. The efficacy of our proposed model was corroborated through ELISpot assays on long peptides, performed on four CRC patients. These assays demonstrated that neoantigen candidates prioritized by our combined model outperformed predictions made by the established tool NetMHCpan. This comprehensive assessment underscores the significance of integrating pHLA binding with pHLA-TCR binding analysis for more effective immunotherapeutic strategies., Competing Interests: TP, TN, BT, TD, ND, HP, TC, DL, HN, DT, TT, TP, ML, TV, MP, HG, HN, LT No competing interests declared, (© 2024, Pham et al.)

Air pollution significantly impacts lung cancer progression, but there is a lack of a comprehensive molecular characterization of clinical samples associated with air pollution. Here, we performed a proteogenomic analysis of lung adenocarcinoma (LUAD) in 169 female never-smokers from the Xuanwei area (XWLC cohort), where coal smoke is the primary contributor to the high lung cancer incidence. Genomic mutation analysis revealed XWLC as a distinct subtype of LUAD separate from cases associated with smoking or endogenous factors. Mutational signature analysis suggested that Benzo[a]pyrene (BaP) is the major risk factor in XWLC. The BaP-induced mutation hotspot, EGFR-G719X, was present in 20% of XWLC which endowed XWLC with elevated MAPK pathway activations and worse outcomes compared to common EGFR mutations. Multi-omics clustering of XWLC identified four clinically relevant subtypes. These subgroups exhibited distinct features in biological processes, genetic alterations, metabolism demands, immune landscape, and radiomic features. Finally, MAD1 and TPRN were identified as novel potential therapeutic targets in XWLC. Our study provides a valuable resource for researchers and clinicians to explore prevention and treatment strategies for air-pollution-associated lung cancers., Competing Interests: HZ, CL, SW, QW, XF, HJ, LX, CL, LZ, FH, MZ, ZD, HL, YZ, XS, GL No competing interests declared, (© 2024, Zhang, Liu, Wang et al.)

Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. This study assesses the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative exploration combining functional, morphological, and omics-based evaluation of gastrocnemius muscle from KIC genetically engineered mice developing autochthonous pancreatic tumor and cachexia. Cachectic PDAC KIC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber's size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. Although the number of mitochondria per cell is not altered, mitochondrial mass shows a twofold decrease and the mitochondrial DNA threefold, suggesting a defect in mitochondrial genome homeostasis. In conclusion, this work provides a framework to guide toward the most relevant targets in the clinic to limit PDAC-induced cachexia., Competing Interests: TG, FM, GR, SA, LC, BH, BG, AC No competing interests declared, (© 2024, Gicquel et al.)

Current cancer treatment strategies continue to face significant challenges, primarily due to tumor relapse, drug resistance, and low treatment efficiency. These issues arise because certain tumor cells adapt to the host immune microenvironment and evade the immune system. This study presents a new cancer immunotherapy strategy using serum-based antibodies from mice exposed to mouse cutaneous squamous cell carcinoma (mCSCC). The experiment was conducted in three stages. In the first stage, mCSCC cells were isolated and expanded cultured from DMBA/TPA-induced mCSCC. In the second stage, these expanded tumor cells were injected into healthy mice to stimulate the production of anti-tumor antibodies. In the final stage, therapeutic serum was extracted from these healthy mice and reintroduced into the tumor-bearing mice. An ELISA assay was utilized to analyze the levels of p53, Bcl-xL, NF-κB, and Bax. The results showed that the serum treatment not only reduced tumor volume but also reversed changes in p53, Bcl-xL, NF-κB, and Bax. In conclusion, this study developed a new immunotherapeutic strategy for treating mCSCC. However, further research is needed to fully comprehend the mechanism of this serum treatment., Competing Interests: ZL No competing interests declared, (© 2024, Liu.)

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging., Competing Interests: AU, TH, JN, SS, SS, AU, TB, MM, BM, ZH, JB, PV No competing interests declared, (© 2023, Urbancokova, Hornofova et al.)

Assay for Transposase-Accessible Chromatin sequencing (ATAC-Seq) is a widely used technique to explore gene regulatory mechanisms. For most ATAC-Seq data from healthy and diseased tissues such as tumors, chromatin accessibility measurement represents a mixed signal from multiple cell types. In this work, we derive reliable chromatin accessibility marker peaks and reference profiles for most non-malignant cell types frequently observed in the microenvironment of human tumors. We then integrate these data into the EPIC deconvolution framework (Racle et al., 2017) to quantify cell-type heterogeneity in bulk ATAC-Seq data. Our EPIC-ATAC tool accurately predicts non-malignant and malignant cell fractions in tumor samples. When applied to a human breast cancer cohort, EPIC-ATAC accurately infers the immune contexture of the main breast cancer subtypes., Competing Interests: AG, JR, MF, CJ No competing interests declared, DG has recieved consulting fees from CeCaVa and Gnubiotics, (© 2024, Gabriel et al.)

Despite major successes with inhibitory receptor blockade in cancer, the identification of novel inhibitory receptors as putative drug targets is needed due to lack of durable responses, therapy resistance, and side effects. Most inhibitory receptors signal via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and previous studies estimated that our genome contains over 1600 ITIM-bearing transmembrane proteins. However, testing and development of these candidates requires increased understanding of their expression patterns and likelihood to function as inhibitory receptor. Therefore, we designed a novel bioinformatics pipeline integrating machine learning-guided structural predictions and sequence-based likelihood models to identify putative inhibitory receptors. Using transcriptomics data of immune cells, we determined the expression of these novel inhibitory receptors, and classified them into previously proposed functional categories. Known and putative inhibitory receptors were expressed across different immune cell subsets with cell type-specific expression patterns. Furthermore, putative immune inhibitory receptors were differentially expressed in subsets of tumour infiltrating T cells. In conclusion, we present an inhibitory receptor pipeline that identifies 51 known and 390 novel human inhibitory receptors. This pipeline will support future drug target selection across diseases where therapeutic targeting of immune inhibitory receptors is warranted., Competing Interests: AS AS is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, AM AMB is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, Hv HJR is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, SV, RK, AY No competing interests declared, Mv MV is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, JK JK is cofounder, shareholder and scientific advisor of GADETA. JK is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, CK CK is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, MP IR is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, LM LM has received funding for investigator-initiated studies on inhibitory receptors from Boehringer Ingelheim, NextCure, NGM Biopharmaceuticals and Argenx. LM is named as co-inventor on a patent application (patent application number: PCT/NL2024/050501) based on this work, (© 2024, Singh et al.)

Solitary fibrous tumors have gene expression signatures similar to those of neuroendocrine tumors., Competing Interests: QY, PK, OG No competing interests declared, (© 2024, Yuan et al.)

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1 . However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk., Competing Interests: PH, SM, CB, MH, AB, AR, LM, JT, NL, PL, MC, LB, AD, IR, JB, CD, VA, AM, ML, EP, AV, PG, SM, PL, VR, EC, MC No competing interests declared, (© 2023, Huchede et al.)

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management., Competing Interests: XW, JL, JC, WC, WX, KW, LL, JH, JC, HZ No competing interests declared, (© 2024, Wei, Liu et al.)

The O -GlcNAc transferase (OGT) is an essential enzyme that mediates protein O -GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O -GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT ( Ogt -/- ) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene ( Apc min ). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt -/- cancer cells restored tumor growth, and this correlated with impaired CD8 + T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity., Competing Interests: JC, BZ, HD, TL, XC, WG, JW, JZ, GX, YY, YL, JB, ZL, HW No competing interests declared, (© 2024, Chen, Zhao, Dong et al.)

Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression., Competing Interests: VV, SN, SA, SV, SL No competing interests declared, (© 2023, Vengayil et al.)

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection., Competing Interests: JK, VS, RO, IK, MI, SA, SS, AS, LN, LN, JN No competing interests declared, (© 2023, Karlsson, Sah et al.)

In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR ( p 53 I nactivating T RIM28 A ssociated R NA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR , in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28 -dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR , which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target., Competing Interests: SJ, MM, SM, BG, KP, LK, NJ, AC, VS, CK No competing interests declared, KS Reviewing editor, eLife, (© 2023, Jana et al.)

Cancer patients often experience changes in mental health, prompting an exploration into whether nerves infiltrating tumors contribute to these alterations by impacting brain functions. Using a mouse model for head and neck cancer and neuronal tracing, we show that tumor-infiltrating nerves connect to distinct brain areas. The activation of this neuronal circuitry altered behaviors (decreased nest-building, increased latency to eat a cookie, and reduced wheel running). Tumor-infiltrating nociceptor neurons exhibited heightened calcium activity and brain regions receiving these neural projections showed elevated Fos as well as increased calcium responses compared to non-tumor-bearing counterparts. The genetic elimination of nociceptor neurons decreased brain Fos expression and mitigated the behavioral alterations induced by the presence of the tumor. While analgesic treatment restored nesting and cookie test behaviors, it did not fully restore voluntary wheel running indicating that pain is not the exclusive driver of such behavioral shifts. Unraveling the interaction between the tumor, infiltrating nerves, and the brain is pivotal to developing targeted interventions to alleviate the mental health burdens associated with cancer., Competing Interests: JB, AW, AR, MA, SB, EV, WS, RD, ST, PV No competing interests declared, (© 2024, Barr, Walz et al.)

The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53 +/+ Eμ-Myc males compared to Trp53 ΔAS/ΔAS Eμ-Myc males, but also compared to Trp53 +/+ Eμ-Myc and Trp53 ΔAS/ΔAS Eμ-Myc females. Pre-tumoral splenic cells from Trp53 +/+ Eμ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eμ-Myc -induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis., Competing Interests: AF, IS, JL, MG, AM, VL, AH, JR, WV, EE, MP, AM, JB, BB, FT No competing interests declared, (© 2024, Fajac et al.)

Tumor neoantigen peptide vaccines hold potential for boosting cancer immunotherapy, yet efficiently co-delivering peptides and adjuvants to antigen-presenting cells in vivo remains challenging. Virus-like particle (VLP), which is a kind of multiprotein structure organized as virus, can deliver therapeutic substances into cells and stimulate immune response. However, the weak targeted delivery of VLP in vivo and its susceptibility to neutralization by antibodies hinder their clinical applications. Here, we first designed a novel protein carrier using the mammalian-derived capsid protein PEG10, which can self-assemble into endogenous VLP (eVLP) with high protein loading and transfection efficiency. Then, an engineered tumor vaccine, named ePAC, was developed by packaging genetically encoded neoantigen into eVLP with further modification of CpG-ODN on its surface to serve as an adjuvant and targeting unit to dendritic cells (DCs). Significantly, ePAC can efficiently target and transport neoantigens to DCs, and promote DCs maturation to induce neoantigen-specific T cells. Moreover, in mouse orthotopic liver cancer and humanized mouse tumor models, ePAC combined with anti-TIM-3 exhibited remarkable antitumor efficacy. Overall, these results support that ePAC could be safely utilized as cancer vaccines for antitumor therapy, showing significant potential for clinical translation., Competing Interests: RT, LG, TW, TC, HY, HY, FL, YZ, HY, ZC, XL No competing interests declared, (© 2024, Tang, Guo et al.)

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain., Competing Interests: GE, EL, CM, CE, JM, WC, EC No competing interests declared, NJ E.A.C. is a consultant at IHP Therapeutics, Valar Labs, Tatara Therapeutics and Pear Diagnostics, reports receiving commercial research grants from Pfizer, and has stock ownership in Tatara Therapeutics, HDT Bio, Clara Health, Aqtual, and Guardant Health, JF N.J. is a founder of Rezo Therapeutics and a shareholder of Rezo Therapeutics, Sudo Therapeutics, and Type6 Therapeutics. N.J. is a SAB member of Sudo Therapeutics, Type6 Therapeutic and NIBR Oncology. The Jura laboratory has received sponsored research support from Genentech, Rezo Therapeutics and Type6 Therapeutics, (© 2023, Estevam et al.)

Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model in mice and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer., Competing Interests: YB, YZ, YL, SL, RB, JS, YC, SW, DG No competing interests declared, (© 2023, Ban, Zou et al.)

In T-cell acute lymphoblastic leukemia (T-ALL), more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, cannabidiol (CBD) and cannabidivarin (CBDV), whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca 2+ . This is followed by increased mRNA and protein expression of ATF4, CHOP, and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1 -mutated diseases and cancers such as T-ALL., Competing Interests: EB, AG, SP, PB No competing interests declared, DM A patent pertaining to the results of the paper has been applied for the use of cannabinoids in the treatment of Notch-related diseases. This patent PCT/IL2021/051352 was licensed to Cavnox Ltd. to test the combination of the molecules in human clinical trials, (© 2023, Besser et al.)

Mutational profiles of myelodysplastic syndromes (MDS) have established that a relatively small number of genetic aberrations, including SF3B1 and SRSF2 spliceosome mutations, lead to specific phenotypes and prognostic subgrouping. We performed a multi-omics factor analysis (MOFA) on two published MDS cohorts of bone marrow mononuclear cells (BMMNCs) and CD34 + cells with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to identify the latent factors with significant impact on MDS prognosis. SF3B1 was the only mutation among 13 mutations in the BMMNC cohort, indicating a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34 + cohort. Interestingly, the MOFA factor representing the inflammation shows a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases show a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Furthermore, MOFA identified RTE expression as a risk factor for MDS. This work elucidates the efficacy of our integrative approach to assess the MDS risk that goes beyond all the scoring systems described thus far for MDS., Competing Interests: SG, NS, SH, WM, IT, SJ, DK, AI, AP, JB, GN, GM No competing interests declared, MK Reviewing editor, eLife, (© 2024, Gerlevik, Seymen, Hama et al.)

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevent the widespread adoption of these methods. Here, we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hr in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, samples processed with EZ Clear can be subjected to downstream applications, such as tissue embedding and cryosectioning followed by standard histology or immunofluorescent staining without loss of fluorescence signal from endogenous or synthetic reporters. Furthermore, we demonstrate that wholemount adult mouse brains processed with EZ Clear can be successfully immunolabeled for fluorescent imaging while still retaining signal from endogenous fluorescent reporters. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adapt and implement in diverse imaging modalities in biomedical research.

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions. [ABSTRACT FROM AUTHOR]

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent U.S. Food and Drug Administration approval of covalent inhibitors of KRAS G12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRAS G12C -driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients who do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRAS G12C , efforts are underway to develop effective combination therapies. Here, we report that the inhibition of KRAS G12C signaling increases autophagy in KRAS G12C -expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRAS G12C -driven lung cancer cell proliferation in vitro and superior tumor control in vivo. Additionally, in genetically engineered mouse models of KRAS G12C -driven NSCLC, inhibition of either KRAS G12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRAS G12C in lung cancer., Competing Interests: PG, KO, SS, MS, MS, YZ, GL, ES No competing interests declared, MB, BS, DF stockholder of Deciphera Pharmaceuticals, CK, MM Research described here was supported through a Sponsored Research Agreement between the University of Utah and Deciphera Pharmaceuticals award to MM and CK, (© 2024, Ghazi et al.)

Heterogeneity of tumor metabolism is an important, but still poorly understood aspect of tumor biology. Present work is focused on the visualization and quantification of cellular metabolic heterogeneity of colorectal cancer using fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H. FLIM-microscopy of NAD(P)H was performed in vitro in four cancer cell lines (HT29, HCT116, CaCo2 and CT26), in vivo in the four types of colorectal tumors in mice and ex vivo in patients' tumor samples. The dispersion and bimodality of the decay parameters were evaluated to quantify the intercellular metabolic heterogeneity. Our results demonstrate that patients' colorectal tumors have significantly higher heterogeneity of energy metabolism compared with cultured cells and tumor xenografts, which was displayed as a wider and frequently bimodal distribution of a contribution of a free (glycolytic) fraction of NAD(P)H within a sample. Among patients' tumors, the dispersion was larger in the high-grade and early stage ones, without, however, any association with bimodality. These results indicate that cell-level metabolic heterogeneity assessed from NAD(P)H FLIM has a potential to become a clinical prognostic factor., Competing Interests: AK, SS, IS, ID, SS, VT, AM, NI, EN, ES, LS, SG, MS No competing interests declared, VS The author was affiliated with Becker&Hickl GmbH at the time of research. The author has no other competing interests to declare, (© 2024, Komarova, Sinyushkina, Shchechkin et al.)

Cancer is considered a risk factor for COVID-19 mortality, yet several countries have reported that deaths with a primary code of cancer remained within historic levels during the COVID-19 pandemic. Here, we further elucidate the relationship between cancer mortality and COVID-19 on a population level in the US. We compared pandemic-related mortality patterns from underlying and multiple cause (MC) death data for six types of cancer, diabetes, and Alzheimer's. Any pandemic-related changes in coding practices should be eliminated by study of MC data. Nationally in 2020, MC cancer mortality rose by only 3% over a pre-pandemic baseline, corresponding to ~13,600 excess deaths. Mortality elevation was measurably higher for less deadly cancers (breast, colorectal, and hematological, 2-7%) than cancers with a poor survival rate (lung and pancreatic, 0-1%). In comparison, there was substantial elevation in MC deaths from diabetes (37%) and Alzheimer's (19%). To understand these differences, we simulated the expected excess mortality for each condition using COVID-19 attack rates, life expectancy, population size, and mean age of individuals living with each condition. We find that the observed mortality differences are primarily explained by differences in life expectancy, with the risk of death from deadly cancers outcompeting the risk of death from COVID-19., Competing Interests: CH, CV, LS No competing interests declared

MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA., Competing Interests: RV, EL, SH, MM, DF, CS, DP, IR, LU, CA, PG, RB, GB No competing interests declared, ME founder and shareholder of Tucana Biosciences, (© 2024, Vidal et al.)

Hepatocellular carcinoma (HCC), the most common type of liver tumor, is a leading cause of cancer-related deaths, and the incidence of liver cancer is still increasing worldwide. Curative hepatectomy or liver transplantation is only indicated for a small population of patients with early-stage HCC. However, most patients with HCC are not candidates for radical resection due to disease progression, leading to the choice of the conventional tyrosine kinase inhibitor drug sorafenib as first-line treatment. In the past few years, immunotherapy, mainly immune checkpoint inhibitors (ICIs), has revolutionized the clinical strategy for HCC. Combination therapy with ICIs has proven more effective than sorafenib, and clinical trials have been conducted to apply these therapies to patients. Despite significant progress in immunotherapy, the molecular mechanisms behind it remain unclear, and immune resistance is often challenging to overcome. Several studies have pointed out that the complex intercellular communication network in the immune microenvironment of HCC regulates tumor escape and drug resistance to immune response. This underscores the urgent need to analyze the immune microenvironment of HCC. This review describes the immunosuppressive cell populations in the immune microenvironment of HCC, as well as the related clinical trials, aiming to provide insights for the next generation of precision immunotherapy., Competing Interests: YF, XG, LS, TC, CW, JW, YL, LL No competing interests declared, (© 2024, Fu, Guo et al.)

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial-mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p . These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer., Competing Interests: YL, YJ, PQ, TM, YB, JB, YH, MJ, TZ, XG No competing interests declared, (© 2024, Liu, Jiang, Qiu et al.)

It takes more than 20 years for normal colorectal mucosa to develop into metastatic carcinoma. The long time window provides a golden opportunity for early detection to terminate the malignant progression. Here, we aim to enable liquid biopsy of T1a stage colorectal cancer (CRC) and precancerous advanced adenoma (AA) by profiling circulating small extracellular vesicle (sEV)-derived RNAs. We exhibited a full RNA landscape for the circulating sEVs isolated from 60 participants. A total of 58,333 annotated RNAs were detected from plasma sEVs, among which 1,615 and 888 sEV-RNAs were found differentially expressed in plasma from T1a stage CRC and AA compared to normal controls (NC). Then we further categorized these sEV-RNAs into six modules by a weighted gene coexpression network analysis and constructed a 60-gene t-SNE model consisting of the top 10 RNAs of each module that could well distinguish T1a stage CRC/AA from NC samples. Some sEV-RNAs were also identified as indicators of specific endoscopic and morphological features of different colorectal lesions. The top-ranked biomarkers were further verified by RT-qPCR, proving that these candidate sEV-RNAs successfully identified T1a stage CRC/AA from NC in another cohort of 124 participants. Finally, we adopted different algorithms to improve the performance of RT-qPCR-based models and successfully constructed an optimized classifier with 79.3% specificity and 99.0% sensitivity. In conclusion, circulating sEVs of T1a stage CRC and AA patients have distinct RNA profiles, which successfully enable the detection of both T1a stage CRC and AA via liquid biopsy., Competing Interests: LM, FB, JM, QG, XL, SZ, SZ No competing interests declared, XL, LZ, ZL are employed by Echo Biotech Co, Ltd. The authors have no other competing interests to declare, (© 2023, Min et al.)

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in pancreatic ductal adenocardinoma (PDAC) tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single-cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single-cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC., Competing Interests: FB, JS, LD, CB No competing interests declared, (© 2024, Butera et al.)

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors., Competing Interests: DA, RO, BD, LF, AP, HB, AH, GR, NF, ES No competing interests declared, NL N.L. is an inventor on a patent describing Sulfopin (US 2021/0332024 A1), (© 2024, Algranati, Oren et al.)

High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFR vIII and PI3KCA H1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1 :EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations., Competing Interests: AW, CD, BP, MH No competing interests declared, (© 2024, Weiss et al.)

Establishing a zebrafish model of a deadly type of brain tumor highlights the role of the immune system in the early stages of the disease., Competing Interests: IL No competing interests declared, (© 2024, Lorimer.)

The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA , and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes ( DDX6, DCP1A, and LSM14A ), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP 5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP., Competing Interests: XS, XP, YG, ZD, LC, WY, YL, CL No competing interests declared, (© 2023, Shen, Peng, Guo et al.)

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis., Competing Interests: PP, JM, KZ, DP, SW, YR, MH, RR, BG, RC, TS, FD No competing interests declared, PM Founder of Netris Pharma, (© 2023, Perampalam et al.)

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A , two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response., Competing Interests: CD, JV, AB, IM, AR, JD, MP, PB, DP, ND, MV, BB, CB, VM No competing interests declared, (© 2024, Dantzer et al.)

Aberrant alternative splicing is well-known to be closely associated with tumorigenesis of various cancers. However, the intricate mechanisms underlying breast cancer metastasis driven by deregulated splicing events remain largely unexplored. Here, we unveiled that RBM7 is decreased in lymph node and distant organ metastases of breast cancer as compared to primary lesions and low expression of RBM7 is correlated with the reduced disease-free survival of breast cancer patients. Breast cancer cells with RBM7 depletion exhibited an increased potential for lung metastasis compared to scramble control cells. The absence of RBM7 stimulated breast cancer cell migration, invasion, and angiogenesis. Mechanistically, RBM7 controlled the splicing switch of MFGE8, favoring the production of the predominant isoform of MFGE8, MFGE8-L. This resulted in the attenuation of STAT1 phosphorylation and alterations in cell adhesion molecules. MFGE8-L exerted an inhibitory effect on the migratory and invasive capability of breast cancer cells, while the truncated isoform MFGE8-S, which lack the second F5/8 type C domain had the opposite effect. In addition, RBM7 negatively regulates the NF-κB cascade and an NF-κB inhibitor could obstruct the increase in HUVEC tube formation caused by RBM7 silencing. Clinically, we noticed a positive correlation between RBM7 expression and MFGE8 exon7 inclusion in breast cancer tissues, providing new mechanistic insights for molecular-targeted therapy in combating breast cancer., Competing Interests: FH, ZD, JY, KW, CC, DC, JZ, JZ, ML, WZ, XL, YQ, YW No competing interests declared, (© 2024, Huang, Dai, Yu et al.)

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade., Competing Interests: NM, SS, RT, SK, DW, NC, JS, WM, DL, JM, DP, AN, KM, MH, PS No competing interests declared, DS Co-founder and holds equity in Clear Creek Bio, (© 2023, Mullen et al.)

BRAF V600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAF V600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a 'just-right' level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAF V600E -mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAF LSL-V600E/+ ; Ptk2 fl/fl mice, Fak deletion maximized BRAF V600E 's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAF V600E -induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a 'just-right' ERK signaling optimal for BRAF V600E -induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation., Competing Interests: CG, HG, SK, CC, XL, FE, RS, JW, EC, JH No competing interests declared, (© 2024, Gao et al.)