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Ovarian cancer is resistant to immunotherapy, and this is influenced by the immunosuppressed tumor microenvironment (TME) dominated by macrophages. Resistance is also affected by intratumoral heterogeneity, whose development is poorly understood. To identify regulators of ovarian cancer immunity, we employed a spatial functional genomics screen (Perturb-map), focused on receptor/ligands hypothesized to be involved in tumor-macrophage communication. Perturb-map recapitulated tumor heterogeneity and revealed that interleukin-4 (IL-4) promotes resistance to anti-PD-1. We find ovarian cancer cells are the key source of IL-4, which directs the formation of an immunosuppressive TME via macrophage control. IL-4 loss was not compensated by nearby IL-4-expressing clones, revealing short-range regulation of TME composition dictating tumor evolution. Our studies show heterogeneous TMEs can emerge from localized altered expression of cancer-derived cytokines/chemokines that establish immune-rich and immune-excluded neighborhoods, which drive clone selection and immunotherapy resistance. They also demonstrate the potential of targeting IL-4 signaling to enhance ovarian cancer response to immunotherapy., Competing Interests: Declaration of interests B.D.B. has a patent application on the Pro-Codes, which have been licensed to Immunai and Noetik. P.K.S. is co-founder of Glencoe Software and member of the SAB for RareCyte, NanoString, and Montai Health; he holds equity in Glencoe and RareCyte. N.R. is the founder of R&A Data., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Age is a major risk factor for cancer, but how aging impacts tumor control remains unclear. In this study, we establish that aging of the immune system, regardless of the age of the stroma and tumor, drives lung cancer progression. Hematopoietic aging enhances emergency myelopoiesis, resulting in the local accumulation of myeloid progenitor-like cells in lung tumors. These cells are a major source of interleukin (IL)-1⍺, which drives the enhanced myeloid response. The age-associated decline of DNA methyltransferase 3A enhances IL-1⍺ production, and disrupting IL-1 receptor 1 signaling early during tumor development normalized myelopoiesis and slowed the growth of lung, colonic, and pancreatic tumors. In human tumors, we identified an enrichment for IL-1⍺-expressing monocyte-derived macrophages linked to age, poorer survival, and recurrence, unraveling how aging promotes cancer and offering actionable therapeutic strategies.

Cancer cells must develop strategies to adapt to the dynamically changing stresses caused by intrinsic or extrinsic processes, or therapeutic agents. Metabolic adaptability is crucial to mitigate such challenges. Considering metabolism as a central node of adaptability, it is focused on an energy sensor, the AMP-activated protein kinase (AMPK). In a subtype of pancreatic ductal adenocarcinoma (PDAC) elevated AMPK expression and phosphorylation is identified. Using drug repurposing that combined screening experiments and chemoproteomic affinity profiling, it is identified and characterized PF-3758309, initially developed as an inhibitor of PAK4, as an AMPK inhibitor. PF-3758309 shows activity in pre-clinical PDAC models, including primary patient-derived organoids. Genetic loss-of-function experiments showed that AMPK limits the induction of ferroptosis, and consequently, PF-3758309 treatment restores the sensitivity toward ferroptosis inducers. The work established a chemical scaffold for the development of specific AMPK-targeting compounds and deciphered the framework for the development of AMPK inhibitor-based combination therapies tailored for PDAC., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3) + T-cell surface glycoprotein CD4 (CD4) + T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The advent of next-generation sequencing (NGS) and single-cell profiling technologies has revealed the complex and heterogenous ecosystem of human tumors under steady-state and therapeutic perturbation. Breakthroughs in the development of genetically engineered mouse models (GEMM) of human cancers that are based on the combination of two site-specific recombinase systems [dual-recombinase system (DRS)] offer fundamental new possibilities to elucidate and understand critical drivers of the diverse tumor phenotypes and validate potential targets for therapy. Here, we discuss opportunities DRS-based cancer GEMMs offer to model, trace, manipulate, and functionally investigate established cancers, their interactions with the host, and their response to therapy., (©2023 The Authors; Published by the American Association for Cancer Research.)

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer., (© 2023. The Author(s).)

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer entity characterized by a heterogeneous genetic landscape and an immunosuppressive tumor microenvironment. Recent advances in high-resolution single-cell sequencing and spatial transcriptomics technologies have enabled an in-depth characterization of both malignant and host cell types and increased our understanding of the heterogeneity and plasticity of PDAC in the steady state and under therapeutic perturbation. In this Review we outline single-cell analyses in PDAC, discuss their implications on our understanding of the disease and present future perspectives of multimodal approaches to elucidate its biology and response to therapy at the single-cell level., (© 2023. Springer Nature America, Inc.)

SNAIL is a key transcriptional regulator in embryonic development and cancer. Its effects in physiology and disease are believed to be linked to its role as a master regulator of epithelial-to-mesenchymal transition (EMT). Here, we report EMT-independent oncogenic SNAIL functions in cancer. Using genetic models, we systematically interrogated SNAIL effects in various oncogenic backgrounds and tissue types. SNAIL-related phenotypes displayed remarkable tissue- and genetic context-dependencies, ranging from protective effects as observed in KRAS- or WNT-driven intestinal cancers, to dramatic acceleration of tumorigenesis, as shown in KRAS-induced pancreatic cancer. Unexpectedly, SNAIL-driven oncogenesis was not associated with E-cadherin downregulation or induction of an overt EMT program. Instead, we show that SNAIL induces bypass of senescence and cell cycle progression through p16 INK4A -independent inactivation of the Retinoblastoma (RB)-restriction checkpoint. Collectively, our work identifies non-canonical EMT-independent functions of SNAIL and unravel its complex context-dependent role in cancer., (© 2023. The Author(s).)

Immunotherapies have shown benefits across a range of human cancers, but not pancreatic ductal adenocarcinoma (PDAC). Recent evidence suggests that the immunosuppressive tumor microenvironment (TME) constitutes an important roadblock to their efficacy. The landscape of the TME differs substantially across PDAC subtypes, indicating context-specific principles of immunosuppression. In this review, we discuss how PDAC cells, the local TME, and systemic host and environmental factors drive immunosuppression in context. We argue that unraveling the mechanistic drivers of the context-specific modes of immunosuppression will open new possibilities to target PDAC more efficiently by using multimodal (immuno)therapeutic interventions., Significance: Immunosuppression is an almost universal hallmark of pancreatic cancer, although this tumor entity is highly heterogeneous across its different subtypes and phenotypes. Here, we provide evidence that the diverse TME of pancreatic cancer is a central executor of various different context-dependent modes of immunosuppression, and discuss key challenges and novel opportunities to uncover, functionalize, and target the central drivers and functional nodes of immunosuppression for therapeutic exploitation., (©2023 The Authors; Published by the American Association for Cancer Research.)

Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic Kras G12D , to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for Kras G12D -induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF-MEK-ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies., (© 2022. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Two studies by Lodestijn et al. in Cell Stem Cell and Cell Reports reveal a lack of stem cell hierarchies in acinar cell-derived tissue renewal and host instructed clonogenic growth of pancreatic cancer, thereby elucidating determinants of pancreas regeneration and cancer., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype., Significance: We used the first genetically engineered mouse model for extrahepatic bile duct carcinoma to identify cancer genes by genome-wide transposon-based mutagenesis screening. Thereby, we show that PI3K signaling output strength and p27Kip1 function are critical determinants for context-specific ECC formation. This article is highlighted in the In This Issue feature, p. 2945., (©2021 The Authors; Published by the American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP 3 , with a selective decrease of C36:2 PI-3,4,5-P 3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

KRAS G12R mutations occur almost exclusively in pancreatic ductal adenocarcinoma. The results of a study that reveals specific differences in KRAS downstream signaling and metabolic rewiring of pancreatic cancer cells harboring KRAS G12R mutations promise to improve our possibilities to better stratify patients for individualized therapies. See related article by Hobbs et al., p. 104 ., (©2020 American Association for Cancer Research.)

Molecular profiling of cancer patients and modelling of human cancer in mice revealed cell type and tissue-specific differences in tumor development and evolution. However, the context-dependent determinants of cancer remain poorly understood. A systematic characterization of the biological underpinnings of context-specificity will, therefore, be pivotal to design more effective therapies. In this review article, we focus on recent advances on molecular, cellular and microenvironmental aspects of context-dependency. We highlight new strategies to study this phenomenon in tumorigenesis and tumor evolution. Notably, we elucidate tissue and cell type-specific signaling cues as well as tumor microenvironment niches, using novel next-generation dual and triple recombinase-based mouse models of cancer., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC)., Design: Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci ( EYA4 , GRIA4 , ITGA4 , MAP3K14-AS1, MSC ). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy., Results: Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4 , 68.5% for GRIA4 , 69.7% for ITGA4 , 69.1% for MAP3K14-AS1% and 65.1% for MSC . Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival., Conclusion: This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations., Competing Interests: Competing interests: AB reports personal fees (scientific advisory board member) from Horizon Discovery, personal fees (scientific advisory board member) from Biocartis, personal fees (Consultant) from Novartis, personal fees (Consultant) from Roche, personal fees (Consultant) from Illumina. AB and FDN reports grants from Trovagene, outside the submitted work. In addition, FDN and PZ have a patent 102017000072650 pending. All the other authors have nothing to disclose., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Systemic treatment of malignant pleural mesothelioma (MPM) is moderately active for the intrinsic pharmacological resistance of MPM cell and its ability to induce an immune suppressive environment. Here we showed that the expression of bromodomain (BRD) proteins BRD2, BRD4 and BRD9 was significantly higher in human primary MPM cells compared to normal mesothelial cells (HMC). Nanomolar concentrations of bromodomain inhibitors (BBIs) JQ1 or OTX015 impaired patient-derived MPM cell proliferation and induced cell-cycle arrest without affecting apoptosis. Importantly, BBIs primed MPM cells for immunogenic cell death, by increasing extracellular release of ATP and HMGB1, and by promoting membrane exposure of calreticulin and ERp57. Accordingly, BBIs activated dendritic cell (DC)-mediated phagocytosis and expansion of CD8 + T-lymphocyte clones endorsed with antitumor cytotoxic activity. BBIs reduced the expression of the immune checkpoint ligand PD-L1 in MPM cells; while both CD8 + and CD4 + T-lymphocytes co-cultured with JQ1-treated MPM cells decreased PD-1 expression, suggesting a disruption of the immune-suppressive PD-L1/PD-1 axis. Additionally, BBIs reduced the expansion of myeloid-derived suppressor cells (MDSC) induced by MPM cells. Finally, a preclinical model of MPM confirmed that the anti-tumor efficacy of JQ1 was largely due to its ability to restore an immune-active environment, by increasing intra-tumor DC and CD8 + T-lymphocytes, and decreasing MDSC. Thereby, we propose that, among novel drugs, BBIs should be investigated for MPM treatment for their combined activity on both tumor cells and surrounding immune-environment.

Background: O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment., Patients and Methods: Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS)., Results: One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p < 0.001 and 0.004, respectively), but not with OS. By combining both assays, IHC low/MB high patients displayed an 87% reduction in the hazard of progression (p < 0.001) and a 77% in the hazard for death (p = 0.001)., Conclusion: In mCRC selected for MGMT deficiency by MSP, IHC and MB testing improve clinical outcome to alkylating agents. Their combination could enhance patient selection in this setting., (Copyright © 2016 Elsevier Ltd. All rights reserved.)