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Interleukin-1 (IL-1) comprises a family of closely related genes; the two major agonistic proteins, IL-1alpha and IL-1beta, are pleiotropic and affect mainly inflammation, immunity and haemopoiesis. IL-1beta is active solely in its secreted form, whereas IL-1alpha is active mainly as an intracellular precursor. IL-1 is abundant at tumour sites, where it may affect the process of carcinogenesis, tumour growth and invasiveness and the patterns of tumour-host interactions. Here, we review the effects of micro-environment- and tumour cell-derived IL-1 on malignant processes in experimental tumour models. We propose that membrane-associated IL-1alpha expressed on malignant cells stimulates anti-tumour immunity, while secretable IL-1beta derived from the micro-environment or the malignant cells, activates inflammation that promotes invasiveness and induces tumour-mediated suppression. Inhibition of the function of IL-1 by the inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1Ra), reduces tumour invasiveness and alleviates tumour-mediated suppression, pointing to its feasible use in cancer therapy. Differential manipulation of IL-1alpha and IL-1beta in malignant cells or in the tumour's micro-environment may open new possibilities for using IL-1 in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Basket clinical trials targeting the KRAS G12C -mutation in solid tumors have shown initial promise, including in orphan KRAS G12C head and neck cancer (HNC). However, development of resistance to KRAS G12C -mutant-specific inhibitors (KRAS G12C i) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRAS G12C i-MRTX849 and AMG510 in a unique syngenic murine KRAS G12C -mutated HNC cell line., Methods: Western-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRAS G12C i in KRAS G12C -mutated HNC cell line, 4NQO-L. In vitro KRAS G12C -acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8 + T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L- KRAS G12C i-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors., Results: Activation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRAS G12C i in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8 + T-cells after treatment combination, and these CD8 + T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8 + T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8 + T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome., Conclusions: Our study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRAS G12C i-AcR tumors., Competing Interests: Declarations. Ethics approval and consent to participate: Animal experiments were conducted in compliance with protocols established by the Institutional Animal Care and Use Committee (IACUC) of Ben-Gurion University of the Negev for ensuring animal welfare and minimizing discomfort. The animal ethical clearance protocol number used for the study was IL-37–10-2022E. Consent for publication: Not Applicable. Competing interests: All authors have no potential conflicts of interest to declare., (© 2024. The Author(s).)

Background/objectives: Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and the most common form of head and neck cancer, posing significant challenges for disease management. The objective of this review is to assess the utility of single-cell RNA sequencing (scRNAseq) in addressing these challenges by enabling a detailed characterization of the tumor microenvironment (TME) at the cellular level., Methods: This review compiles and analyzes current strategies that utilize scRNAseq and other single-cell technologies in HNSCC research., Results: For HNSCC etiology, scRNAseq allows for the construction of cellular atlases, characterization of different cell types, and investigation of genes and processes involved in cancer initiation, development, and progression within the TME. In terms of HNSCC diagnosis and prognosis, the resolution offered by scRNAseq enables the identification of cell type-specific signatures, enhancing prognostic models and disease stratifiers for patient outcome assessments. Regarding HNSCC treatment, scRNAseq provides insights into cellular responses to various treatments, including radiotherapy, chemotherapy, and immunotherapy, contributing to a better understanding of treatment efficacy and patient outcomes., Conclusions: This review highlights the contributions of scRNAseq to HNSCC research, addressing its cellular and biological complexity, and emphasizes its potential for advancing research and clinical practice in other cancer types.

PARP inhibitors (PARPi) hold substantial promise in treating glioblastoma (GBM). However, the adverse effects have restricted their broad application. Through unbiased transcriptomic and proteomic sequencing, it is discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, beyond the previously reported impact of BET inhibition on homologous recombination repair. Through in vitro experiments using established GBM cell lines and patient-derived primary GBM cells, as well as in vivo orthotopic transplantation tumor experiments in zebrafish and nude mice, it is demonstrated that the concurrent administration of PARPi and BETi can synergistically inhibit GBM. Intriguingly, it is observed that DNA damage lingers after discontinuation of PARPi monotherapy, implying that sequential administration of PARPi followed by BETi can maintain antitumor efficacy while reducing toxicity. In GBM cells with elevated baseline replication stress, the sequential regimen exhibits comparable efficacy to concurrent treatment, protecting normal glial cells with lower baseline replication stress from DNA toxicity and subsequent death. This study provides compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Therapies targeting the PD-1/PD-L1 pathway have transformed head and neck squamous cell carcinoma (HNSCC) treatment. However, predicting the response to anti-PD-1 therapy remains a clinical challenge. This study evaluated the functional binding of PD-1 ligands in 29 HNSCC patients and compared it to the standard PD-L1 Combined Positive Score (CPS). The assessment of PD-1 ligands' functionality advances the current ability to predict the response of HNSCC patients to anti-PD-1 therapy., (© 2024. The Author(s).)

The proto-oncogene MYC encodes a nuclear transcription factor that has an important role in a variety of cellular processes, such as cell cycle progression, proliferation, metabolism, adhesion, apoptosis, and therapeutic resistance. MYC amplification is consistently observed in aggressive forms of several solid malignancies and correlates with poor prognosis and distant metastases. While the tumorigenic effects of MYC in patients with head and neck squamous cell carcinoma (HNSCC) are well known, the molecular mechanisms by which the amplification of this gene may confer treatment resistance, especially to immune checkpoint inhibitors, remains under-investigated. Here we present a unique case of a patient with recurrent/metastatic (R/M) HNSCC who, despite initial response to nivolumab-based treatment, developed rapidly progressive metastatic disease after the acquisition of MYC amplification. We conducted comparative transcriptomic analysis of this patient's tumor at baseline and upon progression to interrogate potential molecular processes through which MYC may confer resistance to immunotherapy and/or chemoradiation and used TCGA-HNSC dataset and an institutional cohort to further explore clinicopathologic features and key molecular networks associated with MYC amplification in HNSCC. This study highlights MYC amplification as a potential mechanism of immune checkpoint inhibitor resistance and suggest its use as a predictive biomarker and potential therapeutic target in R/M HNSCC., (© 2024. The Author(s).)

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells., (© 2024. The Author(s).)

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3 + CD8 + and NKp46 + cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

Background: Phosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models., Methods: KTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC-MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR., Results: KTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8 + T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models., Conclusion: KTC1101's dual mechanism of action-directly inhibiting tumor cell growth and dynamically enhancing the immune response- represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations., (© 2024. The Author(s).)

Given the role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in modulating cellular processes such as proliferation, survival, and migration, we hypothesized its potential as a novel therapeutic agent for wound closure enhancement. In this study, PIP3 was examined in its free form or as a complex with cationic starch (Q-starch) as a carrier. The intracellular bioactivity and localization of free PIP3 and the Q-starch/PIP3 complexes were examined. Our results present the capability of Q-starch to form complexes with PIP3, facilitate its cellular membrane internalization, and activate intracellular paths leading to enhanced wound healing. Both free PIP3 and Q-starch/PIP3 complexes enhanced monolayer gap closure in scratch assays and induced amplified collagen production within HaCAT and BJ fibroblast cells. Western blot presented enhanced AKT activation by free or complexed PIP3 in BJ fibroblasts in which endogenous PIP3 production was pharmacologically inhibited. Furthermore, both free PIP3 and Q-starch/PIP3 complexes expedited wound closure in mice, after single or daily dermal injections into the wound margins. Free PIP3 and the Q-starch/PIP3 complexes inherently activated the AKT signaling pathway, which is responsible for crucial wound healing processes such as migration; this was also observed in wound assays in mice. PIP3 was identified as a promising molecule for enhancing wound healing, and its ability to circumvent PI3K inhibition suggests possible implications for chronic wound healing.

Head and neck squamous cell carcinoma (HNSCC) is a highly prevalent malignancy worldwide, with a significant proportion of patients developing recurrent and/or metastatic (R/M) disease. Despite recent advances in therapy, the prognosis for patients with advanced HNSCC remains poor. Here, we present the case of a patient with recurrent metastatic HNSCC harboring an HRAS G12S mutation who achieved a durable response to treatment with tipifarnib, a selective inhibitor of farnesyltransferase. The patient was a 48-year-old woman who had previously received multiple lines of therapy with no significant clinical response. However, treatment with tipifarnib resulted in a durable partial response that lasted 8 months. Serial genomic and transcriptomic analyses demonstrated upregulation of YAP1 and AXL in metastatic lesions compared with the primary tumor, the evolution of the tumor microenvironment from an immune-enriched to a fibrotic subtype with increased angiogenesis, and activation of the PI3K/AKT/mTOR pathway in tipifarnib treatment. Lastly, in HRAS-mutated PDXs and in the syngeneic HRAS model, we demonstrated that tipifarnib efficacy is limited by activation of the AKT pathway, and dual treatment with tipifarnib and the PI3K inhibitor, BYL719, resulted in enhanced anti-tumor efficacy. Our case study highlights the potential of targeting HRAS mutations with tipifarnib in R/M HNSCC and identifies potential mechanisms of acquired resistance to tipifarnib, along with immuno-, chemo-, and radiation therapy. Preclinical results provide a firm foundation for further investigation of drug combinations of HRAS-and PI3K -targeting therapeutics in R/M HRAS-driven HNSCC., 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 © 2024 Elsevier Ltd. All rights reserved.)

Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid β-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

RAS genes are known to be dysregulated in cancer for several decades, and substantial effort has been dedicated to develop agents that reduce RAS expression or block RAS activation. The recent introduction of RAS inhibitors for cancer patients highlights the importance of comprehending RAS alterations in head and neck cancer (HNC). In this regard, we examine the published findings on RAS alterations and pathway activations in HNC, and summarize their role in HNC initiation, progression, and metastasis. Specifically, we focus on the intrinsic role of mutated-RAS on tumor cell signaling and its extrinsic role in determining tumor-microenvironment (TME) heterogeneity, including promoting angiogenesis and enhancing immune escape. Lastly, we summarize the intrinsic and extrinsic role of RAS alterations on therapy resistance to outline the potential of targeting RAS using a single agent or in combination with other therapeutic agents for HNC patients with RAS-activated tumors., Competing Interests: Declaration of Competing Interest All authors declare that there is no financial or other conflict of interest in the preparation of this article., (Copyright © 2023. Published by Elsevier B.V.)

Cells sense, manipulate and respond to their mechanical microenvironment in a plethora of physiological processes, yet the understanding of how cells transmit, receive and interpret environmental cues to communicate with distant cells is severely limited due to lack of tools to quantitatively infer the complex tangle of dynamic cell-cell interactions in complicated environments. We present a computational method to systematically infer and quantify long-range cell-cell force transmission through the extracellular matrix (cell-ECM-cell communication) by correlating ECM remodeling fluctuations in between communicating cells and demonstrating that these fluctuations contain sufficient information to define unique signatures that robustly distinguish between different pairs of communicating cells. We demonstrate our method with finite element simulations and live 3D imaging of fibroblasts and cancer cells embedded in fibrin gels. While previous studies relied on the formation of a visible fibrous 'band' extending between cells to inform on mechanical communication, our method detected mechanical propagation even in cases where visible bands never formed. We revealed that while contractility is required, band formation is not necessary, for cell-ECM-cell communication, and that mechanical signals propagate from one cell to another even upon massive reduction in their contractility. Our method sets the stage to measure the fundamental aspects of intercellular long-range mechanical communication in physiological contexts and may provide a new functional readout for high content 3D image-based screening. The ability to infer cell-ECM-cell communication using standard confocal microscopy holds the promise for wide use and democratizing the method., (© 2023. Springer Nature Limited.)

Cancer progression is enhanced by the interaction of programmed death-ligand 1 (PDL1), which is associated with inhibition of the immune response against tumors, and vascular endothelial growth factor (VEGF), which inhibits immune cell activity while inducing angiogenesis and proliferation of cancer cells. Dual inhibition of PDL1 and VEGF may therefore confer a synergistic anti-cancer therapeutic effect. We present a novel strategy for developing a therapeutic that simultaneously binds and inhibits both PDL1 and VEGF. We generated a bi-specific protein, designated DuRan-Bis, comprising a single chain variable fragment (scFv)-based inhibitor of PDL1 fused to an scFv-based inhibitor of VEGF, with the latter being attached to an Fc fragment. We found that DuRan-Bis binds to both PDL1 and VEGF with high affinity. Compared to treatments with mono-specific proteins, alone or in combination, the DuRan-Bis chimera showed superior inhibition of the proliferation of glioblastoma cells. In comparison to treatment with immune cells alone, a combination of immune cells with DuRan-Bis decreased the viability of head and neck cancer cells. To the best of our knowledge, this study is the first to use a single polypeptide chain scFv-scFv-Fc scaffold for engineering a high-affinity bi-specific inhibitor of PDL1 and VEGF., (© 2023. The Author(s).)

Mitochondria-critical metabolic hubs in eukaryotic cells-are involved in a wide range of cellular functions, including differentiation, proliferation, and death. Mitochondria import most of their proteins from the cytosol in a linear form, after which they are folded by mitochondrial chaperones. However, despite extensive research, the extent to which the function of particular chaperones is essential for maintaining specific mitochondrial and cellular functions remains unknown. In particular, it is not known whether mitochondrial chaperones influence the sensitivity to drugs used in the treatment of cancers. By mining gene expression and drug sensitivity data for cancer cell lines from publicly available databases, we identified mitochondrial chaperones whose expression is associated with sensitivity to oncology drugs targeting particular cellular pathways in a cancer-type-dependent manner. Importantly, we found the expression of TRAP1 and HSPD1 to be associated with sensitivity to inhibitors of DNA replication and mitosis. We confirmed experimentally that the expression of HSPD1 is associated with an increased sensitivity of ovarian cancer cells to drugs targeting mitosis and a reduced sensitivity to drugs promoting apoptosis. Taken together, our results support a model in which particular mitochondrial pathways hinge upon specific mitochondrial chaperones and provide the basis for understanding selectivity in mitochondrial chaperone-substrate specificity.

The tailored design of drug delivery systems for specific therapeutic agents is a prevailing approach in the field. In this paper, we present a study that highlights the potential of our modified starch, Q-starch, as a universal and adaptable drug delivery carrier for diverse therapeutic agents. We investigate the ability of Q-starch/cargo complexes to target different organelles within the cellular landscape, based on the specific activation sites of therapeutic agents. Plasmid DNA (pDNA), small interfering RNA (siRNA), and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) were chosen as representative therapeutic molecules, acting in the nucleus, cytoplasm, and membrane, respectively. By carrying out comprehensive characterizations, employing dynamic light scattering (DLS), determining the zeta potential, and using cryo-transmitting electron microscopy (cryo-TEM), we reveal the formation of nano-sized, positively charged, and spherical Q-starch complexes. Our results demonstrate that these complexes exhibit efficient cellular uptake, targeting their intended organelles while preserving their physical integrity and functionality. Notably, the intracellular path of the Q-starch/cargo complex is guided by the cargo itself, aligning with its unique biological activity site. This study elucidates the versatility and potency of Q-starch as a versatile drug delivery carrier, paving the way for novel applications offering targeted delivery strategies for potential therapeutic molecules.

Accurate predictive biomarkers of response to immune checkpoint inhibitors (ICIs) are required for better stratifying patients with cancer to ICI treatments. Here, we present a new concept for a bioassay to predict the response to anti-PD1 therapies, which is based on measuring the binding functionality of PDL1 and PDL2 to their receptor, PD1. In detail, we developed a cell-based reporting system, called the immuno-checkpoint artificial reporter with overexpression of PD1 (IcAR-PD1) and evaluated the functionality of PDL1 and PDL2 binding in tumor cell lines, patient-derived xenografts, and fixed-tissue tumor samples obtained from patients with cancer. In a retrospective clinical study, we found that the functionality of PDL1 and PDL2 predicts response to anti-PD1 and that the functionality of PDL1 binding is a more effective predictor than PDL1 protein expression alone. Our findings suggest that assessing the functionality of ligand binding is superior to staining of protein expression for predicting response to ICIs.

Background: Ovarian cancer (OC) is known for exhibiting low response rates to immune checkpoint inhibitors that activate T cells. However, immunotherapies that activate B cells have not yet been extensively explored and may be a potential target, as B cells that secrete immunoglobulins have been associated with better outcomes in OC. Although the secretion of immunoglobulins is often mediated by the microbiome, it is still unclear what role they play in limiting the progression of OC., Methods: We conducted an in-vivo CRISPR screen of immunodeficient (NSG) and immune-intact wild type (WT) C57/BL6 mice to identify tumor-derived immune-escape mechanisms in a BRAC1- and TP53-deficient murine ID8 OC cell line (designated ITB1). To confirm gene expression and signaling pathway activation in ITB1 cells, we employed western blot, qPCR, immunofluorescent staining, and flow cytometry. Flow cytometry was also used to identify immune cell populations in the peritoneum of ITB1-bearing mice. To determine the presence of IgA-coated bacteria in the peritoneum of ITB1-bearing mice and the ascites of OC patients, we employed 16S sequencing. Testing for differences was done by using Deseq2 test and two-way ANOVA test. Sequence variants (ASVs) were produced in Qiime2 and analyzed by microeco and phyloseq R packages., Results: We identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a tumor-derived immune suppressive mediator in ITB1 cells. Knockout of TRAF3 (TRAF3KO) activated the type-I interferon pathway and increased MHC-I expression. TRAF3KO tumors exhibited a growth delay in WT mice vs. NSG mice, which was correlated with increased B cell infiltration and activation compared to ITB1 tumors. B cells were found to be involved in the progression of TRAF3KO tumors, and B-cell surface-bound and secreted IgA levels were significantly higher in the ascites of TRAF3KO tumors compared to ITB1. The presence of commensal microbiota was necessary for B-cell activation and for delaying the progression of TRAF3KO tumors in WT mice. Lastly, we observed unique profiles of IgA-coated bacteria in the ascites of OC-bearing mice or the ascites of OC patients., Conclusions: TRAF3 is a tumor-derived immune-suppressive modulator that influences B-cell infiltration and activation, making it a potential target for enhancing anti-tumor B-cell responses in OC., (© 2023. The Author(s).)

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression., Significance: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis., (©2023 American Association for Cancer Research.)

Glioblastoma (GBM) is the most aggressive type of cancer. Its current first-line postsurgery regimens are radiotherapy and temozolomide (TMZ) chemotherapy, both of which are DNA damage-inducing therapies but show very limited efficacy and a high risk of resistance. There is an urgent need to develop novel agents to sensitize GBM to DNA-damaging treatments. Here it is found that the triterpene compound stellettin B (STELB) greatly enhances the sensitivity of GBM to ionizing radiation and TMZ in vitro and in vivo. Mechanistically, STELB inhibits the expression of homologous recombination repair (HR) factors BRCA1/2 and RAD51 by promoting the degradation of PI3Kα through the ubiquitin-proteasome pathway; and the induced HR deficiency then leads to augmented DNA damage and cell death. It is further demonstrated that STELB has the potential to rapidly penetrate the blood-brain barrier to exert anti-GBM effects in the brain, based on zebrafish and nude mouse orthotopic xenograft tumor models. The study provides strong evidence that STELB represents a promising drug candidate to improve GBM therapy in combination with DNA-damaging treatments., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Lung cancer cells in the tumor microenvironment facilitate immune evasion that leads to failure of conventional chemotherapies, despite provisionally decided on the genetic diagnosis of patients in a clinical setup. The current study follows three lung cancer patients who underwent "personalized" chemotherapeutic intervention. Patient-derived xenografts (PDXs) were subjected to tumor microarray and treatment screening with chemotherapies, either individually or in combination with the peptide R11-NLS-pep8; this peptide targets both membrane-associated and nuclear PCNA. Ex vivo, employing PDX-derived explants, it was found that combination with R11-NLS-pep8 stimulated antineoplastic effect of chemotherapies that were, although predicted based on the patient's genetic mutation, inactive on their own. Furthermore, treatment in vivo of PDX-bearing mice showed an exactly similar trend in the result, corroborating the finding to be translated into clinical setup.

Harnessing immune effector cells to benefit cancer patients is becoming more and more prevalent in recent years. However, the increasing number of different therapeutic approaches, such as chimeric antigen receptors and armored chimeric antigen receptors, requires constant adjustments of the transgene expression levels. We have previously demonstrated it is possible to achieve spatial and temporal control of transgene expression as well as tailoring the inducing agents using the Chimeric Antigen Receptor Tumor Induced Vector (CARTIV) platform. Here we describe the next level of customization in our promoter platform. We have tested the functionality of three different minimal promoters, representing three different promoters' strengths, leading to varying levels of CAR expression and primary T cell function. This strategy shows yet another level of CARTIV gene regulation that can be easily integrated into existing CAR T systems.

Background: Computer-aided examination of digital tissue images has attracted attention in recent years. Application in the field of parathyroid pathology has not been studied previously. It holds a potential to assist in the examination of parathyroid gland adenoma or hyperplasia., Objectives: To explore parathyroid cell detection of slide images by digital tissue analysis and compare the results to standard human processing., Methods: 47 incisional biopsies of healthy appearing parathyroid glands were evaluated for their cellularity level. First, by the standard examination using microscopy by three independent pathologists. We compared the mean cellularity grading of the pathologists to the output of a computerized cell detection software., Results: A disagreement was found between the standard human cellularity grading and the digital analysis output. However, the digital analysis reaches a 94% specificity and 48% sensitivity to predict high cellularity (>60% parenchymal cells)., Conclusions: Digital analysis of parathyroid tissue can be used as a tool for hypercellularity elimination, therefore assisting in the diagnosis of parathyroid cell hyperplasia. Additional studies using more advanced algorithms are necessary for further precision enhancement., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In light of the development of RAS inhibitors, a reliable assessment of the prevalence of RAS mutations and their correlation with the clinical features of patients with HNC is crucially needed. This meta-analysis compiles the findings of 149 studies with over 8500 HNC patients and assesses the global prevalence of mutations in the HRAS, KRAS and NRAS genes. The available data were stratified according to geographical region, clinical features, and tumor characteristics, including human papillomavirus (HPV) infection status and tumor stage. In addition, the distribution of codon substitutions in each RAS gene was assessed. The estimated mutation rate is highest for HRAS (7%), followed by KRAS (2.89%) and NRAS (2.20%). HRAS prevalence in South Asia (15.28%) is twice as high as the global estimate. HRAS mutations are more prevalent in oral cavity and salivary gland tumors. In contrast, KRAS mutations are found more frequently in sinonasal tumors, and NRAS mutations are found chiefly in tumors of the nasopharynx. OR analyses show a significant association between HRAS mutations and a high tumor stage (OR=3.63). In addition, there is a significant association between HPV-positive status and KRAS mutations (OR=2.09). This study highlights RAS as a potential therapeutic target in certain subsets of HNC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Novoplansky, Jagadeeshan, Regev, Menashe and Elkabets.)

Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.

Background: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance., Methods: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8 + T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment., Results: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8 + T cells. Activation of CD8 + T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R + CD11c + MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME., Conclusion: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination., Competing Interests: Competing interests: JH was paid consultant for Bristol-Myers Squibb and MSD Sharpe & Dohme and has received other commercial research support from CureVac A G and PROGEN Biotechnik, Research funding from AstraZeneca, outside the scope of this work (to LGTM). LGTM is an inventor on a patent held by Memorial Sloan Kettering related to tumor mutational burden and immunotherapy. MS is an employee and stockholder of Astra Zeneca. JF received Honoraria from Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme, Merck Serono, Innate pharma, Roche, serve as an advisor in, Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme, Merck Serono, Innate pharma, Roche, has a research fund by Bristol-Myers Squibb, and had travel grants from Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer., Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining., Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51., Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the "off target" effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer., (© 2021. The Author(s).)

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5' untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS-mt) and KRAS-wild-type (KRAS-wt) patients. We found that KRAS-mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Patients with primary hyperparathyroidism (PHPT) treated surgically occasionally have normalized calcium, but persistently high parathyroid hormone (PTH). We hypothesized that a possible explanation for this phenomenon is an underlying hyperplasia rather than adenoma., Methods: Retrospective cohort of patients who underwent parathyroidectomy for PHPT with biopsy of a normal-appearing parathyroid gland were included. Cellularity level of each biopsy and of the adenoma's rim was determined., Results: Forty-seven patients were included. Of them, 19 (40%) had postoperative normocalcemia but elevated PTH. There was no correlation between cellularity either in the rim or of the normal-appearing parathyroid gland and postoperative PTH. The postoperative high PTH group had higher preoperative PTH ( P  = 0.001) and larger adenomas ( P  = 0.025)., Conclusions: High PTH levels after successful parathyroidectomy in patients with primary hyperparathyroidism do not appear to result from underlying hyperplasia. A possible alternative explanation is that these patients have a higher preoperative burden of disease., Competing Interests: The authors declare no potential conflict of interest., (© 2021 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals LLC on behalf of The Triological Society.)