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In this issue, Lv et al. 1 explore EGFR-driven epitranscriptomic reprogramming in glioblastoma, revealing the pivotal role of the EGFR-ALKBH5-GCLM axis in ferroptosis protection. Their findings offer mechanistic insight and therapeutic strategies involving novel combination targets to enhance tumor responses. In this issue, Lv et al.1 explore EGFR-driven epitranscriptomic reprogramming in glioblastoma, revealing the pivotal role of the EGFR-ALKBH5-GCLM axis in ferroptosis protection. Their findings offer mechanistic insight and therapeutic strategies involving novel combination targets to enhance tumor responses. [ABSTRACT FROM AUTHOR]

Simple Summary: Technological advancements and emerging high throughput molecular data have transformed biology into a more quantitative and multidisciplinary discipline. This has accelerated the translation of laboratory based findings into applied and clinically relevant applications and therapeutics. A shared practice for quantifying and statistical rank-ordering the effects of such translational applications and for understanding their underlying mode-of-action is now critical. In this manuscript, we discuss some of the major types of quantitative translational research and the best practices. We propose that adherence to these guidelines will improve assay design and reduce missteps in translational biomarker and therapeutics clinical application and adoption. Bioscience is an interdisciplinary venture. Driven by a quantum shift in the volume of high throughput data and in ready availability of data-intensive technologies, mathematical and quantitative approaches have become increasingly common in bioscience. For instance, a recent shift towards a quantitative description of cells and phenotypes, which is supplanting conventional qualitative descriptions, has generated immense promise and opportunities in the field of bench-to-bedside cancer OMICS, chemical biology and pharmacology. Nevertheless, like any burgeoning field, there remains a lack of shared and standardized framework for quantitative cancer research. Here, in the context of cancer, we present a basic framework and guidelines for bench-to-bedside quantitative research and therapy. We outline some of the basic concepts and their parallel use cases for chemical–protein interactions. Along with several recommendations for assay setup and conditions, we also catalog applications of these quantitative techniques in some of the most widespread discovery pipeline and analytical methods in the field. We believe adherence to these guidelines will improve experimental design, reduce variabilities and standardize quantitative datasets. [ABSTRACT FROM AUTHOR]

KRAS G12C inhibitors such as sotorasib and adagrasib are often effective in KRAS G12C-driven non-small cell lung cancer (NSCLC) patients. However, acquired resistance limits long-term patient survival. In this issue of the JCI, Tsai et al. present a comprehensive genetic analysis of multiple tumors with acquired sotorasib resistance obtained through an autopsy of a patient with KRAS G12C-mutant NSCLC. This analysis of pre- and posttreatment tumors uncovered cancer cell-intrinsic and -extrinsic features of resistance, including reactivation of KRAS-mediated signaling, reprogramming of metabolism, epithelial-mesenchymal transition, and tumor microenvironment changes. This elegant study demonstrates the multifaceted nature of KRAS G12C inhibitor clinical resistance and potential avenues to overcome resistance. [ABSTRACT FROM AUTHOR]

Relapse of AML patients to FLT3i treatment is the result of a long-term and stepwise process leading to resistance, whereby residual cancer cells initially survive and subsequently expand. Here, Joshi et al. use a multifaceted approach to characterize how microenvironment-driven early resistance to gilteritinib evolves into mutation-driven late resistance. [ABSTRACT FROM AUTHOR]

Identification of the genomic drivers of cancer has led to the clinical development of targeted therapies that strike at the heart of many malignancies. Nonetheless, many cancers outsmart such precision-medicine efforts, and thus therapeutic resistance contributes significantly to cancer mortality. Attempts to understand the basis for resistance in patient samples and laboratory models has yielded two major benefits: one, more effective chemical inhibitors and rational combination therapies are now employed to prevent or circumvent resistance pathways; and two, our understanding of how oncogenic mutations drive cancer cell survival and oncogene addiction is deeper and broader, highlighting downstream or parallel cellular programs that shape these phenotypes. This review discusses emerging principles of resistance to therapies targeted against key oncogenic drivers. Highlights Genetic and genomic studies of cancer cells have uncovered driving mutations that can in many cases be directly targeted to clinical benefit. The emergence of resistant cancer cells offers both an opportunity to dissect oncogenic and prosurvival cell-biologic pathways and a clear clinical need as resistance is a major cause of cancer mortality. Laboratory models and patient samples highlight resistance mechanisms that include: (i) direct alteration of small-molecule targets; (ii) activation of upstream or downstream signaling nodes; (iii) parallel signaling pathways to activate a common downstream pathway; (iv) epigenetic or transcriptionally effected histologic transformation; and (v) increasingly understood adaptive signaling changes that alter transcriptional cell states to promote survival. Understanding, predicting, and circumventing resistance to targeted cancer therapy is now a major endeavor for translational cancer biologists to undertake. [ABSTRACT FROM AUTHOR]

Simple Summary: Long non-coding RNAs (LncRNAs) are non-protein coding molecules longer than 200 nucleotides. They play essential roles in normal cell function and development, and can contribute to diseases such as cancer when dysregulated. Although lncRNAs have oncogenic or tumor-suppressive properties in lung cancer and can serve as stable biomarkers, this is still an understudied field. Here, we discuss recent evidence for lncRNAs role in lung cancer development, therapy resistance, biomarker potential, and therapeutic strategies. We conclude that understanding the interplay between non-coding and coding molecules in lung cancer should be explored further and may open up new avenues for treatment. Long non-coding RNAs (LncRNAs) are mRNA-like molecules that do not encode for proteins and that are longer than 200 nucleotides. LncRNAs play important biological roles in normal cell physiology and organism development. Therefore, deregulation of their activities is involved in disease processes such as cancer. Lung cancer is the leading cause of cancer-related deaths due to late stage at diagnosis, distant metastasis, and high rates of therapeutic failure. LncRNAs are emerging as important molecules in lung cancer for their oncogenic or tumor-suppressive functions. LncRNAs are highly stable in circulation, presenting an opportunity for use as non-invasive and early-stage cancer diagnostic tools. Here, we summarize the latest works providing in vivo evidence available for lncRNAs role in cancer development, therapy-induced resistance, and their potential as biomarkers for diagnosis and prognosis, with a focus on lung cancer. Additionally, we discuss current therapeutic approaches to target lncRNAs. The evidence discussed here strongly suggests that investigation of lncRNAs in lung cancer in addition to protein-coding genes will provide a holistic view of molecular mechanisms of cancer initiation, development, and progression, and could open up a new avenue for cancer treatment. [ABSTRACT FROM AUTHOR]

The diagnosis and treatment of lung cancer has changed dramatically over the last several years, with a move toward a personalized, precision approach that is more effective and less toxic in patients. This transformation has been enabled by the identification of genetic abnormalities that drive tumor initiation and progression and the development and clinical use of specific targeted therapies that act against these alterations. Despite these recent advances and improved clinical outcomes, lung cancer remains a frequently lethal disease largely because of innate, adaptive and acquired molecular events present in the tumors that promote therapy resistance in patients. Here, we discuss genomically informed management of lung cancer and current and next generation approaches to optimize clinical outcomes in lung cancer patients. [ABSTRACT FROM AUTHOR]

Non-small-cell lung cancer is often diagnosed at the metastatic stage, with median survival of just 1 year. The identification of driver mutations in the epidermal growth factor receptor (EGFR) as the primary oncogenic event in a subset of lung adenocarcinomas led to a model of targeted treatment and genetic profiling of the disease. EGFR tyrosine kinase inhibitors confer remission in 60% of patients, but responses are short-lived. The pre-existing EGFR Thr790Met mutation could be a subclonal driver responsible for these transient responses. Overexpression of AXL and reduced MED12 function are hallmarks of resistance to tyrosine kinase inhibitors in EGFR-mutant non-small-cell lung cancer. Crosstalk between signalling pathways is another mechanism of resistance; therefore, identification of the molecular components involved could lead to the development of combination therapies cotargeting these molecules instead of EGFR tyrosine kinase inhibitor monotherapy. Additionally, novel biomarkers could be identified through deep sequencing analysis of serial rebiopsies before and during treatment. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) is a well-characterized oncogene that is frequently activated by somatic kinase domain mutations in non-small cell lung cancer (NSCLC). EGFR TKIs are effective therapies for NSCLC patients whose tumors harbor an EGFR activating mutation. However, EGFR TKI treatment is not curative in patients because of both primary and secondary treatment resistance. Studies over the last decade have identified mechanisms that drive primary and secondary resistance to EGFR TKI treatment. The elucidation of mechanisms of resistance to EGFR TKI treatment provides a basis for the development of therapeutic strategies to overcome resistance and enhance outcomes in NSCLC patients. In this paper, we summarize the mechanisms of resistance to EGFR TKIs that have been identified to date and discusses potential therapeutic strategies to overcome EGFR TKI resistance in NSCLC patients. [ABSTRACT FROM AUTHOR]

Human lung adenocarcinomas with activating mutations in EGFR (epidermal growth factor receptor) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs), but the magnitude of tumour regression is variable and transient. This heterogeneity in treatment response could result from genetic modifiers that regulate the degree to which tumour cells are dependent on mutant EGFR. Through a pooled RNA interference screen, we show that knockdown of FAS and several components of the NF-κB pathway specifically enhanced cell death induced by the EGFR TKI erlotinib in EGFR-mutant lung cancer cells. Activation of NF-κB through overexpression of c-FLIP or IKK (also known as CFLAR and IKBKB, respectively), or silencing of IκB (also known as NFKBIA), rescued EGFR-mutant lung cancer cells from EGFR TKI treatment. Genetic or pharmacologic inhibition of NF-κB enhanced erlotinib-induced apoptosis in erlotinib-sensitive and erlotinib-resistant EGFR-mutant lung cancer models. Increased expression of the NF-κB inhibitor IκB predicted for improved response and survival in EGFR-mutant lung cancer patients treated with EGFR TKI. These data identify NF-κB as a potential companion drug target, together with EGFR, in EGFR-mutant lung cancers and provide insight into the mechanisms by which tumour cells escape from oncogene dependence. [ABSTRACT FROM AUTHOR]

Summary: K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-Xl. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-Xl. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181. [Copyright &y& Elsevier]

Abstract: Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1. [Copyright &y& Elsevier]

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Has signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Has isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RED) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Has and K-Has, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRS in lipid microdomains of the Jurkat plasma membrane, N-Has did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RED, a fluorescent reporter of GTP-bound Has, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cϒ- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Has isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells. [ABSTRACT FROM AUTHOR]

Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases. [ABSTRACT FROM AUTHOR]

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane[SUP1,2] but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase C[SUBγ]1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca[SUP2+] positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI[SUP3]. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibro-blasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane. [ABSTRACT FROM AUTHOR]

Until recently, the plasma membrane has been considered to be a unique platform from which emanate the signaling events regulating or regulated by Ras and its close relatives. For the past few years, the role of endosomes derived from the plasma membrane as platforms for Ras/mitogen-activated protein kinase signaling has been appreciated. More recently, the cytoplasmic face of the Golgi apparatus and endoplasmic reticulum have been shown to host Ras signaling. The biological implications of compartmentalized signaling are only beginning to emerge. [Copyright &y& Elsevier]

The article provides information on research into oncogenic RAS has led to the development of new cancer treatment strategies.

The nonreceptor protein tyrosine phosphatase SHP2 (encoded by PTPN11) integrates growth and differentiation signals from receptor tyrosine kinases (RTKs) into the RAS/mitogen-activated protein kinase (MAPK) cascade. Considered 'undruggable' over three decades, SHP2 is now a potentially druggable target with the advent of allosteric SHP2 inhibitors. These agents hold promise for improving patient outcomes, showing efficacy in preclinical cancer models, where SHP2 is critical for either oncogenic signaling or resistance to current targeted agents. SHP2 inhibition may also produce immunomodulatory effects in certain tumor microenvironment cells to help cultivate antitumor immune responses. The first generation of allosteric SHP2 inhibitors is under clinical evaluation to determine safety, appropriate tolerability management, and antitumor efficacy, investigations that will dictate future clinical applications. • Allosteric inhibitors stabilize SHP2 in its inactive conformation. • SHP2 inhibition shows efficacy in subsets of RTK-, KRAS-, and BRAF-driven cancers. • Polytherapy approaches address SHP2-mediated signaling in drug resistance. • SHP2 function in T-cells and macrophages suggests immunotherapeutic potential. • Phase I/II clinical trials testing allosteric SHP2 inhibitors are ongoing. [ABSTRACT FROM AUTHOR]

The success of ALK targeted therapy is blunted by resistance. To identify rational polytherapy strategies to improve clinical outcomes, we studied the molecular basis of ALK oncogene dependence in ALK gene rearrangement positive (ALK+) lung adenocarcinoma. We discovered that RAS-RAF-MEK-ERK signaling is the crucial downstream pathway that is required for ALK+ tumor cell survival. Upfront co-inhibition of ALK and MEK improved response and blocked resistance in preclinical ALK+ lung cancer models, providing rationale for a new treatment paradigm for ALK+ patients. [ABSTRACT FROM AUTHOR]

RAF- and MEK-targeted therapies are approved for patients with BRAFV600E melanoma and under investigation in a several other tumor types, but resistance remains a major challenge. We uncovered yes-associated protein 1 (YAP1) as a mechanism of resistance to RAF-MEK inhibition in BRAF- and RAS-mutant cancers, providing a rationale for co-targeting YAP and RAF-MEK to enhance patient outcomes. [ABSTRACT FROM AUTHOR]

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP. Acquired resistance to osimertinib is driven by PDK1 in preclinical models of non-small cell lung cancer and reveals PDK1 as a potential target for restoring osimertinib sensitivity. [ABSTRACT FROM AUTHOR]

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP. Acquired resistance to osimertinib is driven by PDK1 in preclinical models of non-small cell lung cancer and reveals PDK1 as a potential target for restoring osimertinib sensitivity. [ABSTRACT FROM AUTHOR]

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP. Acquired resistance to osimertinib is driven by PDK1 in preclinical models of non-small cell lung cancer and reveals PDK1 as a potential target for restoring osimertinib sensitivity. [ABSTRACT FROM AUTHOR]

Anaplastic lymphoma kinase (ALK) gene rearrangements have been identified in lung cancer at 3-7% frequency, thus representing an important subset of genetic lesions that drive oncogenesis in this disease. Despite the availability of multiple FDA-approved small molecule inhibitors targeting ALK fusion proteins, drug resistance to ALK kinase inhibitors is a common problem in clinic. Thus, there is an unmet need to deepen the current understanding of genomic characteristics of ALK rearrangements and to develop novel therapeutic strategies that can overcome ALK inhibitor resistance. In this review, we present the genomic landscape of ALK fusions in the context of co-occurring mutations with other cancer-related genes, pointing to the central role of genetic epistasis (gene-gene interactions) in ALK-driven advanced-stage lung cancer. We discuss the possibility of targeting druggable domains within ALK fusion partners in addition to available strategies inhibiting the ALK kinase domain directly. Finally, we examine the potential of targeting ALK fusion-specific neoantigens in combination with other treatments, a strategy that could open a new avenue for the improved treatment of ALK positive lung cancer patients. [ABSTRACT FROM AUTHOR]

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors. This study shows that primary and metastatic tumors have discrete requirements for glycolysis- or respiration-derived ATP, suggesting that tumors growing in different anatomic sites develop metabolic heterogeneity, asymmetrically depending upon glycolysis or respiratory metabolism. [ABSTRACT FROM AUTHOR]

Activation of the PI3K and epidermal growth factor receptor (EGFR) pathway is able to drive oncogenesis in multiple human cancers, including head and neck squamous cell carcinoma. Targeted agents such as cetuximab and erlotinib are currently used in patients with head and neck squamous cell carcinoma, but, in this disease, the genomic alterations that cause pathway activation and determine response to pharmacologic inhibition remain ill-defined. Here, we present a detailed dissection of the EGFR/PI3K pathway, composed of sequencing of the core pathway components, and high-resolution genomic copy number assessment. Mutations were found in PIK3CA (6%), but no point mutations were observed in other pathway genes such as PTEN and EGFR. In contrast, we observed frequent copy number alterations of genes in the pathway, including PIK3CA, EGFR, protein tyrosine phosphatase receptor S (PTPRS), and RICTOR. In total, activating genetic pathway alterations were identified in 74% of head and neck tumors. Importantly, intragenic microdeletions of the EGFR phosphatase PTPRS were frequent (26%), identifying this gene as a target of 19p13 loss. PTPRS loss promoted EGFR/PI3K pathway activation, modulated resistance to EGFR inhibition, and strongly determined survival in lung cancer patients with activating EGFR mutations. These findings have important implications for our understanding of head and neck cancer tumorigenesis and for the use of targeted agents for this malignancy. [ABSTRACT FROM AUTHOR]

Talin and RapL are components of molecular pathways that regulate the avidity of the integrin lymphocyte function-associated antigen 1 (LFA-1) for its ligand, intercellular adhesion molecule 1. In this review, we discuss recent advances in our understanding of LFA-1 affinity regulation and signaling and discuss a scenario for how Talin and Rap1 might act in synergy to achieve regulation of LFA-1 that is tailored to the specific functional requirements of different situations. Speedy delivery of signals may be crucial, and membrane trafficking from endosomes and the Golgi apparatus seem to be essential in delivering the messages from spatially segregated surface receptors. [ABSTRACT FROM AUTHOR]

Background: Early recurrence is a major obstacle to prolonged postoperative survival in squamous cell lung carcinoma (SqCLC). The molecular mechanisms underlying early SqCLC recurrence remain unclear, and effective prognostic biomarkers for predicting early recurrence are needed. Methods: We analyzed primary tumor samples of 20 SqCLC patients using quantitative proteomics to identify differentially-expressed proteins in patients who experienced early versus late disease recurrence. The expression and prognostic significance of DDX56 was evaluated using a SqCLC tumor tissue microarray and further verified using different online databases. We performed in vitro and in vivo experiments to obtain detailed molecular insight into the functional role of DDX56 in SqCLC. Results: We found that DDX56 exhibited increased expression in tumors of patients who experienced early versus late disease recurrence. Increased DDX56 expression in SqCLC tumors was subsequently confirmed as an independent prognostic factor of poor recurrence-free survival in independent SqCLC cohorts. Functionally, DDX56 promotes SqCLC cell growth and migration in vitro, and xenograft tumor progression in vivo. Mechanistically, DDX56 post-transcriptionally promotes expression of multiple Wnt signaling pathway-related genes, including CTNNB1, WNT2B, and represses a subset of miRNAs, including miR-378a-3p, a known suppressor of Wnt signaling. Detailed analysis revealed that DDX56 facilitated degradation of primary miR-378a, leading to down-regulation of mature miR-378a-3p and thus derepression of the target gene WNT2B. Conclusion: We identified DDX56 as a novel independent prognostic biomarker that exerts its oncogenic effects through miRNA-mediated post-transcriptional regulation of Wnt signaling genes to promote early SqCLC recurrence. DDX56 may assist in identifying SqCLC patients at increased risk of early recurrence and who could benefit from Wnt signaling-targeted therapies. [ABSTRACT FROM AUTHOR]

Intersectin (ITSN) is a molecular scaffold involved in regulating endocytosis and mitogenic signaling. We previously demonstrated that ITSN transformed rodent fibroblasts, accelerated hormone-induced maturation of Xenopus oocytes, and activated the Elk-1 transcription factor through an MEK- and Erk-independent mechanism. We now demonstrate that ITSN complexes with the Ras guanine nucleotide exchange factor Sos1 leading to increased RasGTP levels. Using fluorescence resonant energy transfer analysis, we demonstrate that ITSN complexes with Ras in living cells leading to Ras activation on intracellular vesicles. These vesicles contain epidermal growth factor receptor but are distinct from transferrin-positive vesicles. However, Ras is not required for ITSN stimulation of transcription. Rather, we demonstrate that ITSN signals through JNK to activate Elk-1. Although ITSN activation of Elk-1 was Ras-independent, ITSN cooperates with Ras to synergistically activate JNK. These findings indicate that ITSN activates multiple intracellular signaling pathways and suggest that this adaptor protein may coordinately regulate the activity of these pathways in vivo. [ABSTRACT FROM AUTHOR]

The expression of lncRNA ESCCAL-1 is upregulated in esophageal squamous cell carcinoma (ESCC). However, the molecular pathways regulated by ESCCAL-1 in esophageal cancer remain obscure. We found that high expression of the lncRNA ESCCAL-1 in human ESCC tumors correlated with worse clinicopathologic features. Furthermore, depletion of ESCCAL-1 in ESCC models inhibited the cellular processes associated with malignancy, including proliferation, migration and invasion, resistance to apoptosis, and impaired tumor growth in mice. Using a combinatorial approach, we discovered that ESCCAL-1 regulates malignant phenotypes in ESCC by acting as a molecular sponge for miR-590-3p. This interaction prevents miR-590-3p from suppressing APOBEC3G expression. Increased APOBEC3G was also a biomarker of worse clinicopathologic features in human ESCC tumors. Depletion of ESSCAL-1 or APOBEC3G, or overexpression of miR-590-3p resulted in increased apoptosis due to downregulation of the PI3K/Akt signaling. This study demonstrates that the lncRNA ESCCAL-1 promotes malignant features of ESCC by relieving the inhibitory effect of miR-590-3p on APOBEC3G expression and identifies potential biomarkers or therapeutic targets to improve ESCC treatment outcomes. [ABSTRACT FROM AUTHOR]

Identifying recurrent somatic genetic alterations of, and dependency on, the kinase BRAF has enabled a "precision medicine" paradigm to diagnose and treat BRAF-driven tumors. Although targeted kinase inhibitors against BRAF are effective in a subset of mutant BRAF tumors, resistance to the therapy inevitably emerges. In this review, we discuss BRAF biology, both in wild-type and mutant settings. We discuss the predominant BRAF mutations and we outline therapeutic strategies to block mutant BRAF and cancer growth. We highlight common mechanistic themes that underpin different classes of resistance mechanisms against BRAF-targeted therapies and discuss tumor heterogeneity and co-occurring molecular alterations as a potential source of therapy resistance. We outline promising therapy approaches to overcome these barriers to the long-term control of BRAF-driven tumors and emphasize how an extensive understanding of these themes can offer more pre-emptive, improved therapeutic strategies. [ABSTRACT FROM AUTHOR]

Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua-double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4-expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4-expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications. [ABSTRACT FROM AUTHOR]

Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy, and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 in rhabdomyosarcoma triggers UPR induction and apoptosis. To define how these cancer cells respond to compromised proteostasis, we compared rhabdomyosarcoma cells that were sensitive (RMS13) or resistant (RMS13-R) to an Hsp70 inhibitor, MAL3-101. We discovered that endoplasmic reticulum associated degradation (ERAD) and autophagy were activated in RMS13-R cells, suggesting that resistant cells overcome Hsp70 ablation by increasing misfolded protein degradation. Indeed, RMS13-R cells degraded ERAD substrates more rapidly than RMS cells and induced the autophagy pathway. Surprisingly, inhibition of the proteasome or ERAD had no effect on RMS13-R cell survival, but silencing of select autophagy components or treatment with autophagy inhibitors restored MAL3-101 sensitivity and led to apoptosis. These data indicate a route through which cancer cells overcome a chaperone-based therapy, define how cells can adapt to Hsp70 inhibition, and demonstrate the value of combined chaperone and autophagy-based therapies. [ABSTRACT FROM AUTHOR]

Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response.Methods: We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided.Results: We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort.Conclusions: Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling that limits targeted therapy response in lung cancer and identifies an unforeseen rational upfront polytherapy strategy to minimize residual disease and enhance clinical outcomes. [ABSTRACT FROM AUTHOR]

Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ~2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (~60% of cases) and non-V600E mutant alleles (~40% of cases) such as BRAFG469A and BRAFG466V. The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients. [ABSTRACT FROM AUTHOR]

Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR(+) mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types. [ABSTRACT FROM AUTHOR]

Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancerrelevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heatshock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor α (eIF2α)-CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum-cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70-UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities. [ABSTRACT FROM AUTHOR]

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53 (KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression. [Display omitted] • KP-tracer mice enable continuous, high-resolution in vivo cancer lineage tracing • Rare subclones with distinct expression programs expand during tumor evolution • Lineage tracing reveals cellular plasticity and evolutionary paths • Metastases are derived from spatially localized, expanding subclones of the tumor Yang et al. developed a genetically engineered mouse model of lung cancer capable of continuous lineage tracing with single-cell RNA-seq readout. They identified the subclonal dynamics of tumors, gene modules underlying expansion, transient increases in cellular plasticity, stereotypical evolutionary paths to aggressiveness across tumor genotypes, and the spatial and phylogenetic origins of metastases. [ABSTRACT FROM AUTHOR]

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival. [ABSTRACT FROM AUTHOR]

The activation of bypass signals, such as MET and AXL, has been identified as a possible mechanism of EGFR-TKI resistance. Because various oncoproteins depend on HSP90 for maturation and stability, we investigated the effects of AUY922, a newly developed non-geldanamycin class HSP90 inhibitor, in lung cancer cells with MET- and AXL-mediated resistance. We established resistant cell lines with HCC827 cells harboring an exon 19-deletion mutation in of the EGFR gene via long-term exposure to increasing concentrations of gefitinib and erlotinib (HCC827/GR and HCC827/ER, respectively). HCC827/GR resistance was mediated by MET activation, whereas AXL activation caused resistance in HCC827/ER cells. AUY922 treatment effectively suppressed proliferation and induced cell death in both resistant cell lines. Accordingly, the downregulation of EGFR, MET, and AXL led to decreased Akt activation. The inhibitory effects of AUY922 on each receptor were confirmed in gene-transfected LK2 cells. AUY922 also effectively controlled tumor growth in xenograft mouse models containing HCC827/GR and HCC827/ER cells. In addition, AUY922 reduced invasion and migration by both types of resistant cells. Our study findings thus show that AUY922 is a promising therapeutic option for MET- and AXL-mediated resistance to EGFR-TKI in lung cancer. [ABSTRACT FROM AUTHOR]

The Gαo/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through Gαo/i to induce neurite outgrowth were studied. The expression of Gαo/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. Gαo/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. Gαo/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the Gαo/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous Gαo/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the Gαo/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth. [ABSTRACT FROM AUTHOR]

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras. [ABSTRACT FROM AUTHOR]

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcγ1, is decreased. Decreased Plcγ1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility. [Copyright &y& Elsevier]

Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling. [Display omitted] • RTK oncoproteins can form de novo membraneless cytoplasmic protein granules • RTK protein granules activate RAS in a lipid membrane-independent manner • Higher-order protein assembly is critical for oncogenic RAS/MAPK signaling • Protein granules serve as a subcellular platform for organizing RTK signaling Receptor tyrosine kinase fusion oncoproteins undergo higher-order assembly to form membraneless cytoplasmic protein granules that coordinate local RAS activation and organize RAS/MAPK signaling in cancer [ABSTRACT FROM AUTHOR]