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Human non-small cell lung cancers (NSCLCs) with activating mutations in EGFR frequently respond to treatment with EGFR-targeted tyrosine kinase inhibitors (TKIs), such as erlotinib, but responses are not durable, as tumors acquire resistance. Secondary mut

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response., (© 2024. The Author(s).)

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA -characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09-0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14-0.69) compared with nonregular NSAID users. For PIK3CA -altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE 2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA -altered HNSCC., (© 2019 Hedberg et al.)

Chromosomal rearrangements involving receptor tyrosine kinases (RTK) are a clinically relevant oncogenic mechanism in human cancers. These chimeric oncoproteins often contain the C-terminal kinase domain of the RTK joined in cis to various N-terminal, nonkinase fusion partners. The functional role of the N-terminal fusion partner in RTK fusion oncoproteins is poorly understood. Here, we show that distinct N-terminal fusion partners drive differential subcellular localization, which imparts distinct cell signaling and oncogenic properties of different, clinically relevant ROS1 RTK fusion oncoproteins. SDC4-ROS1 and SLC34A2-ROS1 fusion oncoproteins resided on endosomes and activated the MAPK pathway. CD74-ROS1 variants that localized instead to the endoplasmic reticulum (ER) showed compromised activation of MAPK. Forced relocalization of CD74-ROS1 from the ER to endosomes restored MAPK signaling. ROS1 fusion oncoproteins that better activate MAPK formed more aggressive tumors. Thus, differential subcellular localization controlled by the N-terminal fusion partner regulates the oncogenic mechanisms and output of certain RTK fusion oncoproteins. SIGNIFICANCE: ROS1 fusion oncoproteins exhibit differential activation of MAPK signaling according to subcellular localization, with ROS1 fusions localized to endosomes, the strongest activators of MAPK signaling., (©2018 American Association for Cancer Research.)

Immunohistochemistry (IHC) analysis of YAP in human tissue samples represents an important means to analyze overall expression levels and subcellular localization of YAP in specimen of interest. As transcriptional coactivator, alterations of YAP levels in the cellular nucleus allow important predictions for YAP activity and transcriptional state of target genes. In the following report, IHC procedures optimized for the detection of YAP in tissue slides of FFPE material are provided. Of note, de-paraffinization and heat-induced antigen retrieval are strictly necessary for successful YAP IHC staining. Further, immunostaining using a labelled polymer-HRP system combined with diaminobenzidine (DAB), as signal-amplifying chromogen, allows strong staining results with minimal unspecific background signal.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance.

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAF V600E -driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRAS G12C ). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non-small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89 Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89 Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89 Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89 Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

The success of targeted cancer therapy is limited by drug resistance that can result from tumor genetic heterogeneity. The current approach to address resistance typically involves initiating a new treatment after clinical/radiographic disease progression, ultimately resulting in futility in most patients. Towards a potential alternative solution, we developed a novel computational framework that uses human cancer profiling data to systematically identify dynamic, pre-emptive, and sometimes non-intuitive treatment strategies that can better control tumors in real-time. By studying lung adenocarcinoma clinical specimens and preclinical models, our computational analyses revealed that the best anti-cancer strategies addressed existing resistant subpopulations as they emerged dynamically during treatment. In some cases, the best computed treatment strategy used unconventional therapy switching while the bulk tumor was responding, a prediction we confirmed in vitro. The new framework presented here could guide the principled implementation of dynamic molecular monitoring and treatment strategies to improve cancer control.

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 BRAF V600E -mutant allele, the spectrum of BRAF mutations in LA includes BRAF V600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAF G469A and BRAF G466V The presence of BRAF V600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAF V600E -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 BRAF V600E 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 BRAF V600E 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., Competing Interests: G.B. and A.R. are employees of Plexxikon, Inc., which produced vemurafenib and PLX8394. T.G.B. is a consultant to Driver Group, Novartis, Astellas, Natera, Array Biopharma, Ariad, Teva, Astrazeneca, and a recipient of research grants from Servier and Ignyta.

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 cancer-relevant 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 heat-shock 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.

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS-mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRAS(WT)) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK-positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Although oncogene-targeted therapy often elicits profound initial tumor responses in patients, responses are generally incomplete because some tumor cells survive initial therapy as residual disease that enables eventual acquired resistance. The mechanisms underlying tumor cell adaptation and survival during initial therapy are incompletely understood. Here, through the study of EGFR mutant lung adenocarcinoma, we show that NF-κB signaling is rapidly engaged upon initial EGFR inhibitor treatment to promote tumor cell survival and residual disease. EGFR oncogene inhibition induced an EGFR-TRAF2-RIP1-IKK complex that stimulated an NF-κB-mediated transcriptional survival program. The direct NF-κB inhibitor PBS-1086 suppressed this adaptive survival program and increased the magnitude and duration of initial EGFR inhibitor response in multiple NSCLC models, including a patient-derived xenograft. These findings unveil NF-κB activation as a critical adaptive survival mechanism engaged by EGFR oncogene inhibition and provide rationale for EGFR and NF-κB co-inhibition to eliminate residual disease and enhance patient responses., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Oncogenic mutations in the BRAF kinase occur in 6-8% of nonsmall cell lung cancers (NSCLCs), accounting for more than 90,000 deaths annually worldwide. The biological and clinical relevance of these BRAF mutations in NSCLC is incompletely understood. Here we demonstrate that human NSCLC cells with BRAF(V600E), but not other BRAF mutations, initially are sensitive to BRAF-inhibitor treatment. However, these BRAF(V600E) NSCLC cells rapidly acquire resistance to BRAF inhibition through at least one of two discrete molecular mechanisms: (i) loss of full-length BRAF(V600E) coupled with expression of an aberrant form of BRAF(V600E) that retains RAF pathway dependence or (ii) constitutive autocrine EGF receptor (EGFR) signaling driven by c-Jun-mediated EGFR ligand expression. BRAF(V600E) cells with EGFR-driven resistance are characterized by hyperphosphorylated protein kinase AKT, a biomarker we validated in BRAF inhibitor-resistant NSCLC clinical specimens. These data reveal the multifaceted molecular mechanisms by which NSCLCs establish and regulate BRAF oncogene dependence, provide insights into BRAF-EGFR signaling crosstalk, and uncover mechanism-based strategies to optimize clinical responses to BRAF oncogene inhibition.

Human non-small cell lung cancers (NSCLCs) with activating mutations in EGFR frequently respond to treatment with EGFR-targeted tyrosine kinase inhibitors (TKIs), such as erlotinib, but responses are not durable, as tumors acquire resistance. Secondary mutations in EGFR (such as T790M) or upregulation of the MET kinase are found in over 50% of resistant tumors. Here, we report increased activation of AXL and evidence for epithelial-to-mesenchymal transition (EMT) in multiple in vitro and in vivo EGFR-mutant lung cancer models with acquired resistance to erlotinib in the absence of the EGFR p.Thr790Met alteration or MET activation. Genetic or pharmacological inhibition of AXL restored sensitivity to erlotinib in these tumor models. Increased expression of AXL and, in some cases, of its ligand GAS6 was found in EGFR-mutant lung cancers obtained from individuals with acquired resistance to TKIs. These data identify AXL as a promising therapeutic target whose inhibition could prevent or overcome acquired resistance to EGFR TKIs in individuals with EGFR-mutant lung cancer.