Search

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.

141 Background: Lung cancer is the leading cause of cancer-related lethality globally. Targeted therapies improve the clinical outcome of cancer treatment; however, a subpopulation of cancer cells survive during initial therapy and evolve into drug tolerant persister cells (DTPs) that maintain a residual disease reservoir. Residual disease preludes acquired resistance and tumor progression; therefore, identifying and eliminating DTPs could benefit future treatment paradigms. We have shown that Hippo pathway effector YAP1 (Yes Associated Protein-1) is activated in oncogene-driven lung cancers when cancer cells are exposed to various targeted therapies, such as EGFR, ALK, and RAS inhibitors. YAP1 transcriptional activation during targeted therapy is characterized by its increased nuclear localization and interaction with transcription factors. This activation promotes the expression of genes involved in cell survival, cellular plasticity, and metabolic reprogramming at the residual disease state. We hypothesized that detection of intrinsic or acquired persister cells may aid the development of optimized treatment regimens. In this study, we are building image-based deep learning models to identify YAP1 activation-mediated DTPs from histologically stained slides. Methods: H&E or YAP-stained immunohistochemistry (IHC) images from clinical lung cancer and patient-derived tumor xenograft samples were collected throughout targeted therapy and were annotated with semi-automation using high performance computing clusters. A deep learning model (U-Net algorithm) was used for image segmentation, training, validation, and testing. Results: 1638 images were annotated and over 80,000 patches from these images for YAP positive cells (or regions of interest) comprised the training dataset with semi-supervised automation. Subsequently, we built a customized deep-learning model to detect YAP-mediated DTP cell states from whole histopathological image slides. The deep learning-based model achieved excellent accuracy of 0.8238 and 0.9091 in training, and 0.8040 and 0.8949 in validation datasets for two different annotations, respectively. For a test dataset, the model obtained 0.81 and 0.902 accuracy for the two annotations, respectively. Conclusions: We have constructed a deep learning convolutional neural network model to infer the presence of the YAP1 activation-mediated drug tolerant persister cell state prior to or during targeted treatment of lung cancer. Implementing our AI-based model into routine lung cancer care in the future could identify patient subpopulations with YAP1 activated tumors who would most benefit from receiving YAP1-targeted small molecule inhibitors.

Abstract: Introduction: Identifying parallel survival pathways or compensatory mechanisms that limit the response to targeted therapy is critical to discover effective combinatorial strategies for the treatment of lung cancer and to prevent emergence of drug resistance Results: Using combination drug screening in a panel of cell line models of non-small cell lung cancer (NSCLC), we identified the tumor suppressor STK11 (aka LKB1) as a molecular biomarker of sensitivity to BET and RAS-MAPK pathway inhibitors combination in NSCLC. We found a small molecule inhibitor of BET (bromodomain and extra terminal) family proteins (BRD4, 3, 2), PLX51107, suppressed BRAF and KRAS mutant NSCLC cell proliferation and in combination with RAS (KRAS G12C), RAF or MEK inhibitor led to pronounced loss in cell viability with potent MYC downregulation and synergistic apoptosis induction in vitro and potent tumor regression or stasis in vivo in several KRAS mutant CDX and PDX mouse models of NSCLC. However, BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 were resistant to BET and RAS, RAF or MEK co-inhibition. To unravel the underlying mechanism of resistance to BET and RAS-MAPK inhibitors upon STK11 loss we modeled STK11 deficiency in BRAF and KRAS mutant NSCLC cells and tumors. We found that STK11 loss creates an unprecedented dependence on YAP/TEAD TFs. STK11 silencing in BRAF and KRAS mutant NSCLC cells resulted in nuclear transfer/activation of the HIPPO pathway effector, YAP, upregulation of the YAP target BIRC5 (aka Survivin) and rescued the synthetic lethality due to BET and RAS, RAF or MEK co-inhibition. Phenocopying STK11 loss, constitutively active YAP (YAP5SA), but not transcriptionally inactive YAP (YAPS94A), mitigated the deleterious effects of BET and RAS, RAF or MEK co-suppression in BRAF and KRAS mutant NSCLC cells with potent upregulation of the pro-survival factor BIRC5. Conversely, we found that co-silencing YAP or TEADs with BETs re-sensitized BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 to BET and RAS-MAPK inhibitors combination and caused synthetic lethality. Conclusion: This study uncovered a functional interaction between BET proteins, STK11, HIPPO-YAP and RAS-MAPK signaling pathways in lung cancer. These results will help identify combinatorial strategies for treating aberrant RAS-MAPK pathway driven lung cancer more effectively and forestall drug resistance. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Ben Powell, Trever Bivona. Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3878.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

PurposeAlthough patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.Experimental designTargeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.ResultsProminent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib.ConclusionsOur study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

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.

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.

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 PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

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.

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 BRAFV600E-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, KRASG12C). 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 89Zr-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 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-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 89Zr-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.

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/β-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

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.

Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.

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.

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.

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.

Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE (Apoe(h/h)Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.

ObjectiveTo study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels.Approach and resultsChow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoe(h/h)Ldlr(-/-)Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C(high) monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point.ConclusionsRestoring apoE expression in Apoe(h/h)Ldlr(-/-)Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C(high) monocytes levels and genetic reprogramming of lesional macrophages.