The Efficacy and Safety of Treating Acquired MET Resistance Through Combinations of Parent and MET Tyrosine Kinase Inhibitors in Patients With Metastatic Oncogene-Driven NSCLC.

Introduction: Acquired MET gene amplification, MET exon 14 skip mutations, or MET fusions can emerge as resistance mechanisms to tyrosine kinase inhibitors (TKIs) in patients with lung cancer. The efficacy and safety of combining MET TKIs (such as crizotinib, capmatinib, or tepotinib) with parent TKIs to target acquired MET resistance are not well characterized. Methods: Multi-institutional retrospective chart review identified 83 patients with metastatic oncogene-driven NSCLC that were separated into the following two pairwise matched cohorts: (1) MET cohort (n = 41)-patients with acquired MET resistance continuing their parent TKI with a MET TKI added or (2) Chemotherapy cohort (n = 42)-patients without any actionable resistance continuing their parent TKI with a platinum-pemetrexed added. Clinicopathologic features, radiographic response (by means of Response Evaluation Criteria in Solid Tumors version 1.1), survival outcomes, adverse events (AEs) (by means of Common Terminology Criteria for Adverse Events version 5.0), and genomic data were collected. Survival outcomes were assessed using Kaplan-Meier methods. Multivariate modeling adjusted for lines of therapy, brain metastases, TP53 mutations, and oligometastatic disease. Results: Within the MET cohort, median age was 56 years (range: 36-83 y). Most patients were never smokers (28 of 41, 68.3%). Baseline brain metastases were common (21 of 41, 51%). The most common oncogenes in the MET cohort were EGFR (30 of 41, 73.2%), ALK (seven of 41, 17.1%), and ROS1 (two of 41, 4.9%). Co-occurring TP53 mutations (32 of 41, 78%) were frequent. Acquired MET alterations included MET gene amplification (37 of 41, 90%), MET exon 14 mutations (two of 41, 5%), and MET gene fusions (two of 41, 5%). After multivariate adjustment, the objective response rate (ORR) was higher in the MET cohort versus the chemotherapy cohort (ORR: 69.2% versus 20%, p < 0.001). Within the MET cohort, MET gene copy number (≥10 versus 6-10) did not affect radiographic response (54.5% versus 68.4%, p = 0.698). There was no difference in ORR on the basis of MET TKI used (F [2, 36] = 0.021, p = 0.978). There was no difference in progression-free survival (5 versus 6 mo; hazard ratio = 0.64; 95% confidence interval: 0.34-1.23, p = 0.18) or overall survival (13 versus 11 mo; hazard ratio = 0.75; 95% confidence interval: 0.42-1.35, p = 0.34) between the MET and chemotherapy cohorts. In the MET cohort, dose reductions for MET TKI-related toxicities were common (17 of 41, 41.4%) but less frequent for parent TKIs (two of 41, 5%). Grade 3 AEs were not significant between crizotinib, capmatinib, and tepotinib (p = 0.3). The discontinuation rate of MET TKIs was 17% with no significant differences between MET TKIs (p = 0.315). Among pre- and post-treatment biopsies (n = 17) in the MET cohort, the most common next-generation sequencing findings were loss of MET gene amplification (15 of 17, 88.2%), MET on-target mutations (seven of 17, 41.2%), new Ras-Raf-MAPK alterations (three of 17, 17.6%), and EGFR gene amplification (two of 17, 11.7%). Conclusions: The efficacy and safety of combining MET TKIs (crizotinib, capmatinib, or tepotinib) with parent TKIs for acquired MET resistance are efficacious. Radiographic response and AEs did not differ significantly on the basis of the underlying MET TKI used. Loss of MET gene amplification, development of MET on-target mutations, Ras-Raf-MAPK alterations, and EGFR gene amplification were molecular patterns found on progression with dual parent and MET TKI combinations.

Evolution of acquired resistance in a ROS1+ KRAS G12C+ NSCLC through the MAPK pathway.

Patients with metastatic NSCLC bearing a ROS1 gene fusion usually experience prolonged disease control with ROS1-targeting tyrosine kinase inhibitors (TKI), but significant clinical heterogeneity exists in part due to the presence of co-occurring genomic alterations. Here, we report on a patient with metastatic NSCLC with a concurrent ROS1 fusion and KRAS p.G12C mutation at diagnosis who experienced a short duration of disease control on entrectinib, a ROS1 TKI. At progression, the patient continued entrectinib and started sotorasib, a small molecule inhibitor of KRAS p.G12C. A patient-derived cell line generated at progression on entrectinib demonstrated improved TKI responsiveness when treated with entrectinib and sotorasib. Cell-line growth dependence on both ROS1 and KRAS p.G12C was further reflected in the distinct downstream signaling pathways activated by each driver. Clinical benefit was not observed with combined therapy of entrectinib and sotorasib possibly related to an evolving KRAS p.G12C amplification identified on repeated molecular testing. This case supports the need for broad molecular profiling in patients with metastatic NSCLC for potential therapeutic and prognostic information.

Isolation of vasa vasorum endothelial cells from pulmonary artery adventitia: Implementation to vascular biology research.

Isolated endothelial cells are valuable in vitro model for vascular research. At present, investigation of disease-relevant changes in vascular endothelium at the molecular level requires established endothelial cell cultures, preserving vascular bed-specific phenotypic characteristics. Vasa vasorum (VV) form a microvascular network around large blood vessels, in both the pulmonary and systemic circulations, that are critically important for maintaining the integrity and oxygen supply of the vascular wall. However, despite the pathophysiological significance of the VV, methods for the isolation and culture of vasa vasorum endothelial cells (VVEC) have not yet been reported. In our prior studies, we demonstrated the presence of hypoxia-induced angiogenic expansion of the VV in the pulmonary artery (PA) of neonatal calves; an observation which has been followed by a series of in vitro studies on isolated PA VVEC. Here we present a detailed protocol for reproducible isolation, purification, and culture of PA VVEC. We show these cells to express generic endothelial markers, (vWF, eNOS, VEGFR2, Tie1, and CD31), as well as progenitor markers (CD34 and CD133), bind lectin Lycopersicon Esculentum, and incorporate acetylated low-density lipoproteins labeled with acetylated LDL (DiI-Ac-LDL). qPCR analysis additionally revealed the expression of CD105, VCAM-1, ICAM-1, MCAM, and NCAM. Ultrastructural electron microscopy and immunofluorescence staining demonstrated that VVEC are morphologically characterized by a developed actin and microtubular cytoskeleton, mitochondrial network, abundant intracellular vacuolar/secretory system, and cell-surface filopodia. VVEC exhibit exponential growth in culture and can be mitogenically activated by multiple growth factors. Thus, our protocol provides the opportunity for VVEC isolation from the PA, and potentially from other large vessels, enabling advances in VV research.

MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC.

BACKGROUND: ROS1 tyrosine kinase inhibitors (TKIs) have demonstrated significant clinical benefit for ROS1+ NSCLC patients. However, TKI resistance inevitably develops through ROS1 kinase domain (KD) modification or another kinase driving bypass signaling. While multiple TKIs have been designed to target ROS1 KD mutations, less is known about bypass signaling in TKI-resistant ROS1+ lung cancers. METHODS: Utilizing a primary, patient-derived TPM3-ROS1 cell line (CUTO28), we derived an entrectinib-resistant line (CUTO28-ER). We evaluated proliferation and signaling responses to TKIs, and utilized RNA sequencing, whole exome sequencing, and fluorescence in situ hybridization to detect transcriptional, mutational, and copy number alterations, respectively. We substantiated in vitro findings using a CD74-ROS1 NSCLC patient's tumor samples. Last, we analyzed circulating tumor DNA (ctDNA) from ROS1+ NSCLC patients in the STARTRK-2 entrectinib trial to determine the prevalence of MET amplification. RESULTS: CUTO28-ER cells did not exhibit ROS1 KD mutations. MET TKIs inhibited proliferation and downstream signaling and MET transcription was elevated in CUTO28-ER cells. CUTO28-ER cells displayed extrachromosomal (ecDNA) MET amplification without MET activating mutations, exon 14 skipping, or fusions. The CD74-ROS1 patient samples illustrated MET amplification while receiving ROS1 TKI. Finally, two of 105 (1.9%) entrectinib-resistant ROS1+ NSCLC STARTRK-2 patients with ctDNA analysis at enrollment and disease progression displayed MET amplification. CONCLUSIONS: Treatment with ROS1-selective inhibitors may lead to MET-mediated resistance. The discovery of ecDNA MET amplification is noteworthy, as ecDNA is associated with more aggressive cancers. Following progression on ROS1-selective inhibitors, MET gene testing and treatments targeting MET should be explored to overcome MET-driven resistance.

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases.

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples.

INTRODUCTION: ROS1 gene fusions are a well-characterized class of oncogenic driver found in approximately 1% to 2% of NSCLC patients. ROS1-directed therapy in these patients is more efficacious and is associated with fewer side effects compared to chemotherapy and is thus now considered standard-of-care for patients with advanced disease. Consequently, accurate detection of ROS1 rearrangements/fusions in clinical tumor samples is vital. In this study, we compared the performance of three common molecular testing approaches on a cohort of ROS1 rearrangement/fusion-positive patient samples. METHODS: Twenty-three ROS1 rearrangement/fusion-positive clinical samples were assessed by at least two of the following molecular testing methodologies: break-apart fluorescence in situ hybridization, DNA-based hybrid capture library preparation followed by next-generation sequencing (NGS), and RNA-based anchored multiplex polymerase chain reaction library preparation followed by NGS. RESULTS: None of the testing methodologies demonstrated 100% sensitivity in detection of ROS1 rearrangements/fusions. Fluorescence in situ hybridization results were negative in 2 of 20 tested samples, the DNA-based NGS assay was negative in 4 of 18 tested samples, and the RNA-based NGS assay was negative in 3 of 19 tested samples. For all three testing approaches, we identified assay characteristics that likely contributed to false-negative results. Additionally, we report that genomic breakpoints are an unreliable predictor of breakpoints at the transcript level, likely due to alternative splicing. CONCLUSIONS: ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.

Do More With Less: Tips and Techniques for Maximizing Small Biopsy and Cytology Specimens for Molecular and Ancillary Testing: The University of Colorado Experience.

Context .- In an era in which testing of patient tumor material for molecular and other ancillary studies is of increasing clinical importance for selection of therapy, the ability to test on small samplings becomes critical. Often, small samplings are rapidly depleted in the diagnostic workup or are insufficient for multiple ancillary testing approaches. Objective .- To describe technical methodologies that can be implemented to preserve and maximize tissue for molecular and other ancillary testing. Data Sources .- Retrospective analysis of a case cohort from the University of Colorado, description of techniques used at the University of Colorado, and published literature. Conclusions .- Numerous techniques can be deployed to maximize molecular and other ancillary testing, even when specimens are from small samplings. A dedicated process for molecular prioritization has a high success rate, but also increases workload, which must be factored into establishing such a process. Additionally, establishing high-fidelity communication strings is critical for success of dedicated molecular prioritization of samples. Numerous approaches can be deployed for alternative specimen types, and several technical approaches can also aid in maximizing small specimens.

High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

uPAR regulates bronchial epithelial repair in vitro and is elevated in asthmatic epithelium.

BACKGROUND: The asthma-associated gene urokinase plasminogen activator receptor (uPAR) may be involved in epithelial repair and airway remodelling. These processes are not adequately targeted by existing asthma therapies. A fuller understanding of the pathways involved in remodelling may lead to development of new therapeutic opportunities. uPAR expression in the lung epithelium of normal subjects and patients with asthma was investigated and the contribution of uPAR to epithelial wound repair in vitro was studied using primary bronchial epithelial cells (NHBECs). METHODS: Bronchial biopsy sections from normal subjects and patients with asthma were immunostained for uPAR. NHBECs were used in a scratch wound model to investigate the contribution of the plasminogen pathway to repair. The pathway was targeted via blocking of the interaction between urokinase plasminogen activator (uPA) and uPAR and overexpression of uPAR. The rate of wound closure and activation of intracellular signalling pathways and matrix metalloproteinases (MMPs) were measured. RESULTS: uPAR expression was significantly increased in the bronchial epithelium of patients with asthma compared with controls. uPAR expression was increased during wound repair in monolayer and air-liquid interface-differentiated NHBEC models. Blocking the uPA-uPAR interaction led to attenuated wound repair via changes in Erk1/2, Akt and p38MAPK signalling. Cells engineered to have raised levels of uPAR showed attenuated repair via sequestration of uPA by soluble uPAR. CONCLUSIONS: The uPAR pathway is required for efficient epithelial wound repair. Increased uPAR expression, as seen in the bronchial epithelium of patients with asthma, leads to attenuated wound repair which may contribute to the development and progression of airway remodelling in asthma. This pathway may therefore represent a potential novel therapeutic target for the treatment of asthma.

Complementary effects of extracellular nucleotides and platelet-derived extracts on angiogenesis of vasa vasorum endothelial cells in vitro and subcutaneous Matrigel plugs in vivo.

BACKGROUND: Platelets contribute to vascular homeostasis and angiogenesis through the release of multiple growth factors, cytokines and nucleotides, such as ATP and ADP. Recent reports have demonstrated a marked growth-promoting effect of total platelet extracts and selected platelet growth factors on therapeutic angiogenesis. However, since endogenous adenine nucleotides are rapidly degraded during the platelet isolation and storage, we examined whether supplementing a platelet-derived extract with exogenous adenine nucleotides would augment their pro-angiogenic effects. METHODS: Pulmonary artery vasa vasorum endothelial cells (VVEC) were used to examine the effects of dialyzed platelet-derived soluble extracts and extracellular adenine nucleotides on proliferation, migration and tube formation. In addition, an in vivo Matrigel plug assay was used to examine the effects of platelet extracts and adenine nucleotides on neovascularization of plugs subcutaneously placed in 50 ICR mice. The number of vascular structures in Matrigel plugs were evaluated by histological and statistical methods. RESULTS: Platelet extracts (6.4-64 µg/ml) significantly induced DNA synthesis and at a concentration of 64 µg/ml had a biphasic effect on VVEC proliferation (an increase at 48 hrs followed by a decrease at 60 hrs). Stimulation of VVEC with platelet extracts also significantly (up to several-fold) increased cell migration and tube formation on Matrigel. Stimulation of VVEC with extracellular ATP (100 µM) dramatically (up to ten-fold) increased migration and tube formation on Matrigel; however, no significant effects on cell proliferation were observed. We also found that ATP moderately diminished platelet extract-induced VVEC proliferation (48 hrs) and migration, but potentiated tube formation. Neither ATP, or a mixture of non-hydrolyzable nucleotides (ATPγS, ADPßS, MeSATP, MeSADP) induced vascularization of Matrigel plugs subcutaneously injected in mice, however, the combination of these nucleotides with platelet extracts dramatically increased the number of functional capillaries in the Matrigel plugs. CONCLUSION: Data from this study suggest that platelet-derived growth factors and extracellular nucleotides represent important regulatory signals for angiogenesis. Supplementation of platelet extracts with exogenous adenine nucleotides may reveal new possibilities for therapeutic angiogenesis and tissue regeneration approaches.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development.

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1(-/-) mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-Ras(G12D) markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell-cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.

Glucocorticoids increase repair potential in a novel in vitro human airway epithelial wounding model.

Airway epithelial damage is a cardinal feature of chronic asthma. Agents which enhance epithelial repair without triggering uncontrolled fibrosis of the mesenchyme would be predicted to be useful in the management of asthma. We have developed a repeat wound model using mucociliated human bronchial epithelial cell (HBEC) cultures to define the key pathways involved in airway epithelial repair, and to study the effects of potential therapeutic agents on epithelial repair in a chronic setting. We show that repair occurs primarily by cell migration to close a defect; this process requires activation of the EGF receptor (EGFR) and subsequent tyrosine kinase signalling. Migration is accompanied by up-regulation of CD44 in motile cells at the wound margins with proliferation of non-migrating cells adjacent to the wound area. In long-term studies beta2 adrenoceptor agonists and phosphodiesterase (PDE) inhibitors have no effect on repair potential, in contrast chronic treatment with the glucocorticoid dexamethasone extends the lifespan of repeatedly wounded differentiated cultures. We suggest part of the beneficial effects of glucocorticoids in asthma is related to this ability to prolong repair potential following repeated episodes of epithelial injury.