A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene.

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.

Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.

The EGFR T790M mutation has been identified in tumors from lung cancer patients that eventually develop resistance to erlotinib. In this study, we generated a mouse model with doxycycline-inducible expression of a mutant EGFR containing both L858R, an erlotinib-sensitizing mutation, and the T790M resistance mutation (EGFR TL). Expression of EGFR TL led to development of peripheral adenocarcinomas with bronchioloalveolar features in alveoli as well as papillary adenocarcinomas in bronchioles. Treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI), HKI-272, shrunk only peripheral tumors but not bronchial tumors. However, the combination of HKI-272 and rapamycin resulted in significant regression of both types of lung tumors. This combination therapy may potentially benefit lung cancer patients with the EGFR T790M mutation.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies.

To understand the role of human epidermal growth factor receptor (hEGFR) kinase domain mutations in lung tumorigenesis and response to EGFR-targeted therapies, we generated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants seen in human lung cancer. Both bitransgenic lines developed lung adenocarcinoma after sustained hEGFR mutant expression, confirming their oncogenic potential. Maintenance of these lung tumors was dependent on continued expression of the EGFR mutants. Treatment with small molecule inhibitors (erlotinib or HKI-272) as well as prolonged treatment with a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression. These data suggest that persistent EGFR signaling is required for tumor maintenance in human lung adenocarcinomas expressing EGFR mutants.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition.

Non-small cell lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Such cancers are "addicted" to EGFR, and treatment with a TKI invariably leads to down-regulation of the PI3K-AKT-mTOR and MEK-ERK signaling pathways, resulting in apoptosis. Using a dual PI3K-mTOR inhibitor, NVP-BEZ235, we evaluated whether PI3K-mTOR inhibition alone induced apoptosis in these cancers. In contrast to HER2-amplified breast cancers, we found that PI3K-mTOR inhibition did not promote substantial apoptosis in the EGFR mutant lung cancers. However, blocking both PI3K-mTOR and MEK simultaneously led to apoptosis to similar levels as the EGFR TKIs, suggesting that down-regulation of these pathways may account for much of the apoptosis promoted by EGFR inhibition. In EGFR mutant lung cancers, down-regulation of both intracellular pathways converged on the BH3 family of proteins regulating apoptosis. PI3K inhibition led to down-regulation of Mcl-1, and MEK inhibition led to up-regulation of BIM. In fact, down-regulation of Mcl-1 by siRNA was sufficient to sensitize these cancers to single-agent MEK inhibitors. Surprisingly, an AKT inhibitor did not decrease Mcl-1 levels, and when combined with MEK inhibitors, failed to induce apoptosis. Importantly, we observed that the combination of PI3K-mTOR and MEK inhibitors effectively shrunk tumors in a transgenic and xenograft model of EGFR T790M-L858R cancers. These data indicate simultaneous inhibition of PI3K-mTOR and MEK signaling is an effective strategy for treating EGFR mutant lung cancers, including those with acquired resistance to EGFR TKIs.

HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy.

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2(YVMA)) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2(YVMA) is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2(YVMA) transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.

Therapeutic anti-EGFR antibody 806 generates responses in murine de novo EGFR mutant-dependent lung carcinomas.

Activating EGFR mutations occur in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%-30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.

Regression of drug-resistant lung cancer by the combination of rosiglitazone and carboplatin.

PURPOSE: Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARgamma ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARgamma/carboplatin combination in these more relevant models of drug resistant non-small cell lung cancer. EXPERIMENTAL DESIGN: Tumorigenesis was induced by activation of either mutant KRAS or EGFR. Mice then received either rosiglitazone or carboplatin monotherapy, or a combination of both drugs. Change in tumor burden, pathology, and evidence of apoptosis and cell growth were assessed. RESULTS: Tumor burden remained unchanged or increased in the mice after monotherapy with either rosiglitazone or carboplatin. In striking contrast, we observed significant tumor shrinkage in mice treated with these drugs in combination. Immunohistochemical analyses showed that this synergy was mediated via both increased apoptosis and decreased proliferation. Importantly, this synergy between carboplatin and rosiglitazone did not increase systemic toxicity. CONCLUSIONS: These data show that the PPARgamma ligand/carboplatin combination is a new therapy worthy of clinical investigation in lung cancers, including those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy.

Mutations in BRAF and KRAS converge on activation of the mitogen-activated protein kinase pathway in lung cancer mouse models.

Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.

PEST domain mutations in Notch receptors comprise an oncogenic driver segment in triple-negative breast cancer sensitive to a γ-secretase inhibitor.

PURPOSE: To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014. EXPERIMENTAL DESIGN: We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014. RESULTS: We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014. CONCLUSIONS: This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs.

[Reconstruction and expression of green fluorescent protein and aquaporin 7 fusion recombinant vector].

OBJECTIVE: To study the membrane mobility of aquaporin 7 (AQP7) by cloning stably transfected CHO cells with expression of pEGFP-C1-AQP7, in which AQP7 cDNA was fused downstream and in frame to pEGFP-C1 gene. METHODS: The full sequence of AQP7 was amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the pEGFP-C1 vector. The recombinant vector pEGFP-C1-AQP7 was stably transfected into CHO cells. With fluorescent microscopy, immunocytochemical stain and Western blot, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. RESULTS: (1) The sequence of AQP7 cDNA of the Wistar rat was logged into the GenBank (access number: AY157737). (2) Identification demonstrated that pEGFP-C1-AQP7 fusion protein stably expressed in CHO cells. (3) With fluorescence microscopy, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. CONCLUSION: The CHO cell line with stable pEGFP-C1-AQP7 expression was set up successfully for advanced research.

ß-catenin contributes to lung tumor development induced by EGFR mutations.

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that ß-catenin is essential for development of EGFR-mutated lung cancers. ß-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated ß-catenin, leading to an increase in ß-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of ß-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the ß-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that ß-catenin plays an essential role in lung tumorigenesis and that targeting the ß-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.

Contrast agents for quantitative microCT of lung tumors in mice.

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (µCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available µCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. µCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative µCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced µCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.

Ganetespib (STA-9090), a nongeldanamycin HSP90 inhibitor, has potent antitumor activity in in vitro and in vivo models of non-small cell lung cancer.

PURPOSE: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. EXPERIMENTAL DESIGN: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. RESULTS: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. CONCLUSIONS: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.

A dual role for the immune response in a mouse model of inflammation-associated lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Both principal factors known to cause lung cancer, cigarette smoke and asbestos, induce pulmonary inflammation, and pulmonary inflammation has recently been implicated in several murine models of lung cancer. To further investigate the role of inflammation in the development of lung cancer, we generated mice with combined loss of IFN-γ and the ß-common cytokines GM-CSF and IL-3. These immunodeficient mice develop chronic pulmonary inflammation and lung tumors at a high frequency. Examination of the relationship between these tumors and their inflammatory microenvironment revealed a dual role for the immune system in tumor development. The inflammatory cytokine IL-6 promoted optimal tumor growth, yet wild-type mice rejected transplanted tumors through the induction of adaptive immunity. These findings suggest a model whereby cytokine deficiency leads to oncogenic inflammation that combines with defective antitumor immunity to promote lung tumor formation, representing a unique system for studying the role of the immune system in lung tumor development.

Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition.

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.

Exploiting cancer cell vulnerabilities to develop a combination therapy for ras-driven tumors.

Ras-driven tumors are often refractory to conventional therapies. Here we identify a promising targeted therapeutic strategy for two Ras-driven cancers: Nf1-deficient malignancies and Kras/p53 mutant lung cancer. We show that agents that enhance proteotoxic stress, including the HSP90 inhibitor IPI-504, induce tumor regression in aggressive mouse models, but only when combined with rapamycin. These agents synergize by promoting irresolvable ER stress, resulting in catastrophic ER and mitochondrial damage. This process is fueled by oxidative stress, which is caused by IPI-504-dependent production of reactive oxygen species, and the rapamycin-dependent suppression of glutathione, an important endogenous antioxidant. Notably, the mechanism by which these agents cooperate reveals a therapeutic paradigm that can be expanded to develop additional combinations.

Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.

Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells.

An ErbB3 antibody, MM-121, is active in cancers with ligand-dependent activation.

ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.