eIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations.

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

Cell-specific models reveal conformation-specific RAF inhibitor combinations that synergistically inhibit ERK signaling in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges for targeted clinical interventions due to prevalent KRAS mutations, rendering PDAC resistant to RAF and MEK inhibitors (RAFi and MEKi). In addition, responses to targeted therapies vary between patients. Here, we explored the differential sensitivities of PDAC cell lines to RAFi and MEKi and developed an isogenic pair comprising the most sensitive and resistant PDAC cells. To simulate patient- or tumor-specific variations, we constructed cell-line-specific mechanistic models based on protein expression profiling and differential properties of KRAS mutants. These models predicted synergy between two RAFi with different conformation specificity (type I½ and type II RAFi) in inhibiting phospho-ERK (ppERK) and reducing PDAC cell viability. This synergy was experimentally validated across all four studied PDAC cell lines. Our findings underscore the need for combination approaches to inhibit the ERK pathway in PDAC.

BTLA and PD-1 signals attenuate TCR-mediated transcriptomic changes.

T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

Cell State Transition Models Stratify Breast Cancer Cell Phenotypes and Reveal New Therapeutic Targets.

Understanding signaling patterns of transformation and controlling cell phenotypes is a challenge of current biology. Here we applied a cell State Transition Assessment and Regulation (cSTAR) approach to a perturbation dataset of single cell phosphoproteomic patterns of multiple breast cancer (BC) and normal breast tissue-derived cell lines. Following a separation of luminal, basal, and normal cell states, we identified signaling nodes within core control networks, delineated causal connections, and determined the primary drivers underlying oncogenic transformation and transitions across distinct BC subtypes. Whereas cell lines within the same BC subtype have different mutational and expression profiles, the architecture of the core network was similar for all luminal BC cells, and mTOR was a main oncogenic driver. In contrast, core networks of basal BC were heterogeneous and segregated into roughly four major subclasses with distinct oncogenic and BC subtype drivers. Likewise, normal breast tissue cells were separated into two different subclasses. Based on the data and quantified network topologies, we derived mechanistic cSTAR models that serve as digital cell twins and allow the deliberate control of cell movements within a Waddington landscape across different cell states. These cSTAR models suggested strategies of normalizing phosphorylation networks of BC cell lines using small molecule inhibitors.

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

Genomic and Transcriptomic Analysis of a Patient with Early-Onset Colorectal Cancer and Therapy-Induced Focal Nodular Hyperplasia: A Case Report.

Early-onset colorectal cancer (EOCRC), defined as colorectal cancer in individuals under 50 years of age, has shown an alarming increase in incidence worldwide. We report a case of a twenty-four-year-old female with a strong family history of colorectal cancer (CRC) but without an identified underlying genetic predisposition syndrome. Two years after primary surgery and adjuvant chemotherapy, the patient developed new liver lesions. Extensive diagnostic imaging was conducted to investigate suspected liver metastases, ultimately leading to a diagnosis of focal nodular hyperplasia. The young age of the patient has prompted comprehensive genomic and transcriptomic profiling in order to identify potential oncogenic drivers and inform further clinical management of the patient. Besides a number of oncogenic mutations identified in the patient's tumour sample, including KRAS G12D, TP53 R248W and TTN L28470V, we have also identified a homozygous deletion of 24.5 MB on chromosome 8. A multivariate Cox regression analysis of this patient's mutation profile conferred a favourable prognosis when compared with the TCGA COADREAD database. Notably, the identified deletion on chromosome 8 includes the WRN gene, which could contribute to the patient's overall positive response to chemotherapy. The complex clinical presentation, including the need for emergency surgery, early age at diagnosis, strong family history, and unexpected findings on surveillance imaging, necessitated a multidisciplinary approach involving medical, radiation, and surgical oncologists, along with psychological support and reproductive medicine specialists. Molecular profiling of the tumour strongly indicates that patients with complex mutational profile and rare genomic rearrangements require a prolonged surveillance and personalised informed interventions.

A Systems Biology Approach to Understand the Racial Disparities in Colorectal Cancer.

Racial disparities between Black/African Americans (AA) and White patients in colorectal cancer are an ever-growing area of concern. Black/AA show the highest incidence and have the highest mortality among major U.S. racial groups. There is no definite cause other than possible sociodemographic, socioeconomic, education, nutrition, delivery of healthcare, screening, and cultural factors. A primary limitation in this field is the lack of and small sample size of Black/AA studies. Thus, this study aimed to investigate whether differences in gene expression contribute to this ongoing unanswered racial disparity issue. In this study, we examined transcriptomic data of Black/AA and White patient cohorts using a bioinformatic and systems biology approach. We performed a Kaplan-Meier overall survival analysis between both patient cohorts across critical colorectal cancer signal transduction networks (STN), to determine the differences in significant genes across each cohort. Other bioinformatic analyses performed included PROGENy (pathway responsive genes for activity inference), RNA sequencing differential expression using DESeq2, multivariable-adjusted regression, and other associated Kaplan-Meier analyses. These analyses identified novel prognostic genes independent from each cohort, 176 differentially expressed genes, and specific patient cohort STN survival associations. Despite the overarching limitation, the results revealed several novel differences in gene expression between the colorectal cancer Black/AA and White patient cohorts, which allows one to dive deeper into and understand the behavior on a systems level of what could be driving this racial difference across colorectal cancer. Concretely, this information can guide precision medicine approaches tailored specifically for colorectal cancer racial disparities. SIGNIFICANCE: The purpose of this work is to investigate the racial disparities in colorectal cancer between Black/AA and White patient cohorts using a systems biology and bioinformatic approach. Our study investigates the underlying biology of each patient cohort. Concretely, the findings of this study include disparity-associated genes and pathways, which provide a tangible starting point to guide precision medicine approaches tailored specifically for colorectal cancer racial disparities.

Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes.

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. Methods: In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days in triplicates.

Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib.

RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.

Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

Reversing pathological cell states: the road less travelled can extend the therapeutic horizon.

Acquisition of omics data advances at a formidable pace. Yet, our ability to utilize these data to control cell phenotypes and design interventions that reverse pathological states lags behind. Here, we posit that cell states are determined by core networks that control cell-wide networks. To steer cell fate decisions, core networks connecting genotype to phenotype must be reconstructed and understood. A recent method, cell state transition assessment and regulation (cSTAR), applies perturbation biology to quantify causal connections and mechanistically models how core networks influence cell phenotypes. cSTAR models are akin to digital cell twins enabling us to purposefully convert pathological states back to physiologically normal states. While this capability has a range of applications, here we discuss reverting oncogenic transformation.

Small Gene Networks Delineate Immune Cell States and Characterize Immunotherapy Response in Melanoma.

Single-cell technologies have elucidated mechanisms responsible for immune checkpoint inhibitor (ICI) response, but are not amenable to a clinical diagnostic setting. In contrast, bulk RNA sequencing (RNA-seq) is now routine for research and clinical applications. Our workflow uses transcription factor (TF)-directed coexpression networks (regulons) inferred from single-cell RNA-seq data to deconvolute immune functional states from bulk RNA-seq data. Regulons preserve the phenotypic variation in CD45+ immune cells from metastatic melanoma samples (n = 19, discovery dataset) treated with ICIs, despite reducing dimensionality by >100-fold. Four cell states, termed exhausted T cells, monocyte lineage cells, memory T cells, and B cells were associated with therapy response, and were characterized by differentially active and cell state-specific regulons. Clustering of bulk RNA-seq melanoma samples from four independent studies (n = 209, validation dataset) according to regulon-inferred scores identified four groups with significantly different response outcomes (P < 0.001). An intercellular link was established between exhausted T cells and monocyte lineage cells, whereby their cell numbers were correlated, and exhausted T cells predicted prognosis as a function of monocyte lineage cell number. The ligand-receptor expression analysis suggested that monocyte lineage cells drive exhausted T cells into terminal exhaustion through programs that regulate antigen presentation, chronic inflammation, and negative costimulation. Together, our results demonstrate how regulon-based characterization of cell states provide robust and functionally informative markers that can deconvolve bulk RNA-seq data to identify ICI responders.

Interactome dynamics of RAF1-BRAF kinase monomers and dimers.

RAF kinases play major roles in cancer. BRAFV600E mutants drive ~6% of human cancers. Potent kinase inhibitors exist but show variable effects in different cancer types, sometimes even inducing paradoxical RAF kinase activation. Both paradoxical activation and drug resistance are frequently due to enhanced dimerization between RAF1 and BRAF, which maintains or restores the activity of the downstream MEK-ERK pathway. Here, using quantitative proteomics we mapped the interactomes of RAF1 monomers, RAF1-BRAF and RAF1-BRAFV600E dimers identifying and quantifying >1,000 proteins. In addition, we examined the effects of vemurafenib and sorafenib, two different types of clinically used RAF inhibitors. Using regression analysis to compare different conditions we found a large overlapping core interactome but also distinct condition specific differences. Given that RAF proteins have kinase independent functions such dynamic interactome changes could contribute to their functional diversification. Analysing this dataset may provide a deeper understanding of RAF signalling and mechanisms of resistance to RAF inhibitors.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

Oncotherapeutic Strategies in Early Onset Colorectal Cancer.

Early onset colorectal cancer (EOCRC), defined as colorectal cancers in patients aged less than 50 years, is becoming an increasingly common issue, globally. Since 1994, the incidence of this condition has been rising by 2% annually. Approximately one in five patients under 50 years of age diagnosed with colorectal cancer have an underlying genetic predisposition syndrome. The detection of cancer among the other 80% of patients poses a considerable task, as there is no family history to advocate for commencing early screening in this group. Patients with EOCRC have distinct social, spiritual, fertility, and financial needs from their older counterparts that need to be addressed. This review discusses the risk factors associated with the development of EOCRC and current best practice for the management of this disease.

Dynamic regulation of RAS and RAS signaling.

RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease.

Targeting KRAS in Pancreatic Cancer.

Pancreatic cancer is mainly driven by mutations in the KRAS oncogene. While this cancer has shown remarkable therapy resistance, new approaches to inhibit mutated KRAS, KRAS activators and effectors show promise in breaking this therapeutic deadlock. Here, we review these innovations in therapies that target RAS signaling in pancreatic cancer from a clinical point of view. A number of promising approaches are currently in clinical trials or in clinical development. We focus on small-molecule drugs but also discuss immunotherapies and tumor vaccines.

CRABP2 - A novel biomarker for high-risk endometrial cancer.

OBJECTIVE: Investigate the clinical and functional implications of elevated CRABP2 expression in endometrial cancer (EC) patients. METHODS: Patients were stratified into high and low CRABP2 expression groups using a decision tree classifier. Univariate and multivariate statistical analyses determined the prognostic and clinicopathological consequences of increased CRABP2 expression. A CRABP2 gene signature was generated using differential expression analysis, and analyzed using network-based approaches. The findings were validated in The Clinical Proteomic Tumor Analysis Consortium (CPTAC), a newly generated cohort of 120 endometrial tissues, and The Cancer Dependency Map (DepMap). RESULTS: 60 (11%) patients in TCGA had high CRABP2 expression, whilst 468 (89%) had low expression. High expression was associated with serous EC, reduced overall survival, advanced stage and grade. Downstream retinoic acid receptors (RARG and RARA) were correlated with CRABP2 expression and were associated with worse prognosis in serous EC. The CRABP2 gene signature was enriched for Polycomb target gene sets, and was regulated by ELP3 and BMP7. BMP7 expression was increased in the CRABP2-high group, was associated with worse prognosis, and CRISPR-Cas9 screens revealed correlations in its cell-fitness score with CRABP2 following gene knockout. The opposite was true for ELP3, suggesting opposing effects from both master regulators. CONCLUSIONS: CRABP2 expression is associated with poor prognosis and advanced EC. The expression of RARA and RARG correlates with CRABP2 and are associated with worse prognosis in advanced histological subtypes. Polycomb target gene sets and two master regulators, ELP3 and BMP7, were identified as functionally relevant mechanisms driving aberrant CRABP2 expression.

Proteasomal down-regulation of the proapoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma.

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase-independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wild-type BRAF protein, binds to MST2 inhibiting its proapoptotic signalling. Down-regulation of MST2 reduces BRAFi-induced apoptosis. In BRAFi-resistant cell lines, MST2 pathway proteins are down-regulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway-induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAFi resistance in melanoma.