Expressed Barcoding Enables High-Resolution Tracking of the Evolution of Drug Tolerance.

For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. SIGNIFICANCE: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.

Novel Single-Particle Analytical Technique for Inhalable Airborne Microplastic Particles by the Combined Use of Fluorescence Microscopy, Raman Microspectrometry, and SEM/EDX.

This study presents a novel and efficient method for analyzing inhalable airborne microplastics (AMPs) in ambient PM10 aerosols. Although many studies have been conducted on MPs in a variety of environments, the physicochemical characteristics of AMPs of inhalable size (<10 µm) in ambient PM10 are poorly understood because of the lack of suitable analytical methods. The method employed in this study combines fluorescence microscopy, Raman microspectrometry (RMS), and scanning electron microscopy/energy-dispersive X-ray spectrometry (SEM/EDX) for an efficient and reliable investigation of inhalable AMPs, which constitute a small portion of ambient PM10 aerosol particles. Fluorescence microscopy and staining are used to select particles with high MP potential from ambient urban PM10 aerosols. The combination of RMS and SEM/EDX then allows for a detailed characterization of these particles on a single-particle basis. The results of the study show that ∼0.008% of the particles collected using a PM10 sampler had high MP potential, corresponding to ∼800 particles/m3. Among the stained particles of <10 µm, 27% were determined to be plastic, while the remaining 73% were found to be from tire/road wear. The number of inhalable AMPs was estimated to be 192 (±127) particles/m3. This study provides an important insight into the characteristics of inhalable AMPs in ambient PM10 aerosols that are particularly critical in respect of human health and climate change. The authors highlight that the use of a single fluorescence staining method can overestimate the number of inhalable AMPs in ambient air by including tire/road wear particles. To the best of their knowledge, this is the first study to demonstrate the morphological and spectroscopic characteristics of the same individual inhalable AMPs.

Endoplasmin regulates differentiation of tonsil-derived mesenchymal stem cells into chondrocytes through ERK signaling.

It is well-known that some species of lizard have an exceptional ability known as caudal autotomy (voluntary self-amputation of the tail) as an anti-predation mechanism. After amputation occurs, they can regenerate their new tails in a few days. The new tail section is generally shorter than the original one and is composed of cartilage rather than vertebrae bone. In addition, the skin of the regenerated tail distinctly differs from its original appearance. We performed a proteomics analysis for extracts derived from regenerating lizard tail tissues after amputation and found that endoplasmin (ENPL) was the main factor among proteins up-regulated in expression during regeneration. Thus, we performed further experiments to determine whether ENPL could induce chondrogenesis of tonsil-derived mesenchymal stem cells (T-MSCs). In this study, we found that chondrogenic differentiation was associated with an increase of ENPL expression by ER stress. We also found that ENPL was involved in chondrogenic differentiation of T-MSCs by suppressing extracellular signal-regulated kinase (ERK) phosphorylation. [BMB Reports 2022; 55(5): 226-231].

Echinochrome A Treatment Alleviates Fibrosis and Inflammation in Bleomycin-Induced Scleroderma.

Scleroderma is an autoimmune disease caused by the abnormal regulation of extracellular matrix synthesis and is activated by non-regulated inflammatory cells and cytokines. Echinochrome A (EchA), a natural pigment isolated from sea urchins, has been demonstrated to have antioxidant activities and beneficial effects in various disease models. The present study demonstrates for the first time that EchA treatment alleviates bleomycin-induced scleroderma by normalizing dermal thickness and suppressing collagen deposition in vivo. EchA treatment reduces the number of activated myofibroblasts expressing α-SMA, vimentin, and phosphorylated Smad3 in bleomycin-induced scleroderma. In addition, it decreased the number of macrophages, including M1 and M2 types in the affected skin, suggesting the induction of an anti-inflammatory effect. Furthermore, EchA treatment markedly attenuated serum levels of inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, in a murine scleroderma model. Taken together, these results suggest that EchA is highly useful for the treatment of scleroderma, exerting anti-fibrosis and anti-inflammatory effects.

Accurate Prediction of Organic Aerosol Evaporation Using Kinetic Multilayer Modeling and the Stokes-Einstein Equation.

Organic aerosol can adopt a wide range of viscosities, from liquid to glass, depending on the local humidity. In highly viscous droplets, the evaporation rates of organic components are suppressed to varying degrees, yet water evaporation remains fast. Here, we examine the coevaporation of semivolatile organic compounds (SVOCs), along with their solvating water, from aerosol particles levitated in a humidity-controlled environment. To better replicate the composition of secondary aerosol, nonvolatile organics were also present, creating a three-component diffusion problem. Kinetic modeling reproduced the evaporation accurately when the SVOCs were assumed to obey the Stokes-Einstein relation, and water was not. Crucially, our methodology uses previously collected data to constrain the time-dependent viscosity, as well as water diffusion coefficients, allowing it to be predictive rather than postdictive. Throughout the study, evaporation rates were found to decrease as SVOCs deplete from the particle, suggesting path function type behavior.

Transient cavity dynamics and divergence from the Stokes-Einstein equation in organic aerosol.

The diffusion of small molecules through viscous matrices formed by large organic molecules is important across a range of domains, including pharmaceutical science, materials chemistry, and atmospheric science, impacting on, for example, the formation of amorphous and crystalline phases. Here we report significant breakdowns in the Stokes-Einstein (SE) equation from measurements of the diffusion of water (spanning 5 decades) and viscosity (spanning 12 decades) in saccharide aerosol droplets. Molecular dynamics simulations show water diffusion is not continuous, but proceeds by discrete hops between transient cavities that arise and dissipate as a result of dynamical fluctuations within the saccharide lattice. The ratio of transient cavity volume to solvent volume increases with size of molecules making up the lattice, increasing divergence from SE predictions. This improved mechanistic understanding of diffusion in viscous matrices explains, for example, why organic compounds equilibrate according to SE predictions and water equilibrates more rapidly in aerosols.

Comparison of Approaches for Measuring and Predicting the Viscosity of Ternary Component Aerosol Particles.

Measurements of the water activity-dependent viscosity of aerosol particles from two techniques are compared, specifically from the coalescence of two droplets in holographic optical tweezers (HOT) and poke-and-flow experiments on particles deposited onto a glass substrate. These new data are also compared with the fitting of dimer coagulation, isolation, and coalescence (DCIC) measurements. The aerosol system considered in this work are ternary mixtures of sucrose-citric acid-water and sucrose-NaNO3-water, at varying solute mass ratios. Results from HOT and poke-and-flow are in excellent agreement over their overlapping range of applicability (∼103-107 Pa s); fitted curves from DCIC data show variable agreement with the other two techniques because of the sensitivity of the applied modeling framework to the representation of water content in the particles. Further, two modeling approaches for the predictions of the water activity-dependent viscosity of these ternary systems are evaluated. We show that it is possible to represent their viscosity with relatively simple mixing rules applied to the subcooled viscosity values of each component or to the viscosity of the corresponding binary mixtures.

RET fusions observed in lung and colorectal cancers are sensitive to ponatinib.

Genomic studies are revolutionizing clinical oncology, but bridging the lab and the bedside requires the ability to efficiently interrogate rare genetic lesions in unexpected pathological settings using preclinical models. Oncogenes can exhibit intrinsic drug resistance to targeted therapy in different cells of origin, adding complexity to clinical interpretations of genomic findings. Here, we capitalize on the flexibility of engineered cell systems to rapidly profile known multi-kinase inhibitors that harbor rearranged during transfection (RET) kinase activity across multiple RET fusions. Identifying ponatinib as the most potent RET inhibitor tested, we used ponatinib to gauge therapeutic responsiveness in RET fusion-positive patient-derived xenograft (PDX) models. Using a genomics guided outlier approach, we identified 4 RET fusion PDX models with 3 different fusion partners (KIF5B, CCDC6, and NCOA4) in both non-small cell lung cancer and colorectal cancer. By comparing ponatinib activity in RET fusion-positive and RET fusion-negative PDX models alongside a standard of care chemotherapeutic agent, we show that RET fusions in colorectal tumors are therapeutically responsive to RET inhibition. Finally, we suggest that coupling engineered cell systems and genomics guided PDX model selection provides a rapid workflow to triage rare genomics findings.

Characterising the evaporation kinetics of water and semi-volatile organic compounds from viscous multicomponent organic aerosol particles.

The physicochemical changes experienced by organic aerosol particles undergoing dehydration into the surrounding gas phase can be drastic, forcing rapid vitrification of the particle and suppressing internal diffusion. Until recently, experimental studies have concentrated on quantifying diffusional mixing of either water or non-volatile components, while relatively little attention has been paid to the role of semivolatile organic component (SVOC) diffusion and volatilisation in maintaining the equilibrium between the gas and particle phases. Here we present methods to simultaneously investigate diffusivities and volatilities in studies of evolving single ternary aerosol particle size and composition. Analysing particles of ternary composition must account for the multiple chemical species that volatilise in response to a step change in gas phase water activity. In addition, treatments of diffusion in multicomponent mixtures are necessary to represent evolving heterogeneities in particle composition. We find that the contributions to observed size behaviour from volatilisation of water and a SVOC can be decoupled and treated separately. Employing Fickian diffusion modelling, we extract the compositional dependence of the diffusion constant of water and compare the results to recently published parametrisations in binary aerosol particles. The treatment of ideality and activity in each case is discussed, with reference to use in multicomponent core shell models. Meanwhile, the evaporation of an SVOC into an unsaturated gas flow may be treated by Maxwell's equation, with slow diffusional transport manifesting as a suppression in the extracted vapour pressure.

Accurate representations of the physicochemical properties of atmospheric aerosols: when are laboratory measurements of value?

Laboratory studies can provide important insights into the processes that occur at the scale of individual particles in ambient aerosol. We examine the accuracies of measurements of core physicochemical properties of aerosols that can be made in single particle studies and explore the impact of these properties on the microscopic processes that occur in ambient aerosol. Presenting new measurements, we examine here the refinements in our understanding of aerosol hygroscopicity, surface tension, viscosity and optical properties that can be gained from detailed laboratory measurements for complex mixtures through to surrogates for secondary organic atmospheric aerosols.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.

Diffusion and reactivity in ultraviscous aerosol and the correlation with particle viscosity.

The slow transport of water, organic species and oxidants in viscous aerosol can lead to aerosol existing in transient states that are not solely governed by thermodynamic principles but by the kinetics of gas-particle partitioning. The relationship between molecular diffusion constants and particle viscosity (for example, as reflected in the Stokes-Einstein equation) is frequently considered to provide an approximate guide to relate the kinetics of aerosol transformation with a material property of the aerosol. We report direct studies of both molecular diffusion and viscosity in the aerosol phase for the ternary system water/maleic acid/sucrose, considering the relationship between the hygroscopic response associated with the change in water partitioning, the volatilisation of maleic acid, the ozonolysis kinetics of maleic acid and the particle viscosity. Although water clearly acts as a plasticiser, the addition of minor fractions of other organic moieties can similarly lead to significant changes in the viscosity from that expected for the dominant component forming the organic matrix (sucrose). Here we highlight that the Stokes-Einstein relationship between the diffusion constant of water and the viscosity of the particle may be more than an order of magnitude in error, even at viscosities as low as 1 Pa s. We show that the thermodynamic relationships of hygroscopic response that underpin such kinetic determinations must be accurately known to retrieve accurate values for diffusion constants; such data are often not available. Further, we show that scaling of the diffusion constants of organic molecules of similar size to those forming the matrix with particle viscosity may be well represented by the Stokes-Einstein equation, suppressing the apparent volatility of semi-volatile components. Finally, the variation in uptake coefficients and diffusion constants for oxidants and small weakly interacting molecules may be much less dependent on viscosity than the diffusion constants of more strongly interacting molecules such as water.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer.

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Variabilities and uncertainties in characterising water transport kinetics in glassy and ultraviscous aerosol.

We present a comprehensive evaluation of the variabilities and uncertainties present in determining the kinetics of water transport in ultraviscous aerosol droplets, alongside new measurements of the water transport timescale in sucrose aerosol. Measurements are performed on individual droplets captured using aerosol optical tweezers and the change in particle size during water evaporation or condensation is inferred from shifts in the wavelength of the whispering gallery mode peaks at which spontaneous Raman scattering is enhanced. The characteristic relaxation timescale (τ) for condensation or evaporation of water from viscous droplets following a change in gas phase relative humidity can be described by the Kohlrausch-Williams-Watts function. To adequately characterise the water transport kinetics and determine τ, sufficient time must be allowed for the particle to progress towards the final state. However, instabilities in the environmental conditions can prevent an accurate characterisation of the kinetics over such long time frames. Comparison with established thermodynamic and diffusional water transport models suggests the determination of τ is insensitive to the choice of thermodynamic treatment. We report excellent agreement between experimental and simulated evaporation timescales, and investigate the scaling of τ with droplet radius. A clear increase in τ is observed for condensation with increase in drying (wait) time. This trend is qualitatively supported by model simulations.

Ponatinib inhibits polyclonal drug-resistant KIT oncoproteins and shows therapeutic potential in heavily pretreated gastrointestinal stromal tumor (GIST) patients.

PURPOSE: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. EXPERIMENTAL DESIGN: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. RESULTS: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. CONCLUSION: Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.

mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1.

The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.

Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2.

PURPOSE: Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. EXPERIMENTAL DESIGN: HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis. RESULTS: A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance. CONCLUSIONS: Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.

Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition.

The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET.

Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models.

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.