Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification

Background:

MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumour types. We investigated the roles and mechanisms of RTK heterodimerisation in lung cancer with MET amplification.

Methods:

With the use of an RTK array, we identified phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays.

Results:

We identified epidermal growth factor receptor (EGFR), human EGFR (HER)2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signalling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signalling.

Conclusion:

Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET have differential roles in tumour development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerisation partners of MET in lung cancer with MET amplification.     

A randomized,open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification

BackgroundApproximately 5%–10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization.Patients and methodsPatients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m² administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken.ResultsOf 71 patients randomized (AZD4547n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression.ConclusionsAZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.     

HER2, MET and FGFR2 oncogenic driver alterations define distinct molecular segments for targeted therapies in gastric carcinoma

Background:

Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs.

Methods:

Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors.

Results:

Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs'' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074.

Conclusions:

Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.     

Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolour FISH

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained.     

HER2‐positive gastric cancer with concomitant MET and/or EGFR overexpression: A distinct subset of patients for dual inhibition therapy

Growth factor receptors, often carrying tyrosine kinase activities in their cytoplasmic domains, are overexpressed in many cancers. Coactivation of receptor tyrosine kinases (RTKs) plays a critical role in tumor response to targeted therapeutics. We examined concomitant overexpression of EGFR and MET in patients with HER2⁺ and HER2^? gastric cancers (GCs). Tissue microarray samples obtained from 1,589 GC patients who received R0 gastrectomy with extensive node dissection and adjuvant chemoradiationtherapy were analyzed by immunohistochemistry and fluorescence in situ hybridization. HER2⁺ was observed in 169 patients (11%). Out of 169 HER2⁺ patients, 15 (9%) were EGFR⁺ and MET⁺, 29 (17%) were EGFR⁺, 37 (22%) were MET⁺ and the remaining 88 patients (52%) were HER2⁺ only, without concomitant EGFR or MET overexpression. Greater number of overexpressed RTKs correlated with younger age (p < 0.001), larger tumor size (p = 0.027), intestinal histology (p < 0.001) and shorter overall survival (p = 0.002). The mean overall survival was 113 months for HER2^?/EGFR^?/MET^? and 63 months for HER2⁺/EGFR⁺/MET⁺ subgroups. Patients with HER2⁺/EGFR⁺/MET⁺ GCs had a substantial risk of death with a hazard ratio of 3.01 (95% CI: 1.54–5.90), compared with HER2^?/EGFR^?/MET^? GC patients. Using patient‐derived tumor cell models isolated from pericardial effusion of HER2⁺ and MET⁺ GC cases, we demonstrated that the combination of HER2‐inhibitor (lapatinib) and MET‐inhibitor offered a more profound inhibition in the ERK/AKT pathway and cell proliferation than lapatinib alone. Co‐overexpression of RTKs was demonstrated in small subsets of GC associated with aggressive behavior and in these cases, combination therapy may be considered as potential treatment options.     

AZD4547: an orally bioavailable, potent, and selective inhibitor of the fibroblast growth factor receptor tyrosine kinase family

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.     

SWOG S1400D (NCT02965378), a Phase II Study of the Fibroblast Growth Factor Receptor Inhibitor AZD4547 in Previously Treated Patients With Fibroblast Growth Factor Pathway–Activated Stage IV Squamous Cell Lung Cancer (Lung-MAP Substudy)

BackgroundS1400D is a biomarker-driven therapeutic substudy of Lung-MAP evaluating the fibroblast growth factor (FGF) receptor (FGFR) inhibitor AZD4547 in patients with FGF pathway-activated squamous cell. This is the first phase II trial to evaluate AZD4547 as a targeted approach in patients with previously treated FGFR-altered squamous cell NSCLC and is the first demonstration of successful implementation and conduct of a national umbrella protocol in this disease setting.MethodsEligible patients had tumoral FGFR alteration or mutation and had progressive disease after at least one line of platinum-based systemic therapy. Patients received AZD4547 80 mg twice daily orally. Primary endpoint was response by Response Evaluation Criteria in Solid Tumors version 1.1; secondary endpoints included progression-free survival, overall survival, and duration of response (DoR).ResultsNinety-two patients were assigned to S1400D, 43 were enrolled, and 27 AZD4547-treated patients were evaluable. Evaluable patients were predominantly white (n = 24, 89%), median age 66 years (range, 49–88 years old), and female (n = 7, 26%). FGFR alterations included FGFR1 amplification (n = 23; 85%), FGFR3 amplification (n = 2; 7%), FGFR3 S249C (n = 2; 7%), and FGFR3 fusion (n = 1; 4%). Treatment with ADZ4547 was well tolerated; grade 3 adverse events occurred in six patients, and one patient had grade 4 sepsis. Of 27 response-evaluable patients, 1 patient with FGFR3 S249C had unconfirmed partial response with a DoR of 1.5 months and 1 patient with FGFR1 amplification had a confirmed partial response with a DoR of 2.9 months (7%, 95% confidence interval [CI]: 0%–17%). Median progression-free survival and overall survival for the AZD4547-treated cohort were 2.7 months (95% CI: 1.4– 4.5 months) and 7.5 months (95% CI: 3.7–9.3 months).ConclusionsAZD4547 had an acceptable safety profile but minimal activity in this predominantly FGFR1/FGFR3–amplified cohort. Evaluation of other targeted agents in Lung-MAP is ongoing.     

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models

Introduction

The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2⁺ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.

Methods

Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs.

Results

The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2⁺ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2⁺ breast cancers, providing potential clinical relevance to our findings.

Conclusions

Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.     

Prognostic relevance of receptor tyrosine kinase expression in breast cancer: A meta-analysis

Background

Receptor tyrosine kinases (RTKs) may facilitate tumor progression if activated aberrantly. The prognostic impact of human epidermal growth factor receptor 2 (HER2) overexpression and effectiveness of its therapeutic targeting is well established, but the effects on prognosis of overexpression of other RTKs is unknown. Here we evaluate the association of RTK expression and survival in breast cancer.

Methods

PubMed was searched to identify studies evaluating the association between expression of RTKs other than HER2 and survival of women with breast cancer. Published data were extracted and computed into odds ratios (OR) for death at 5 years with 95% confidence intervals (CI). Data were pooled in a meta-analysis using the Mantel–Haenszel random-effect model. For studies reporting data for more than one RTK the lowest and highest OR were used for separate analyses.

Results

Sixteen studies comprising 11,056 patients were included in the analysis. There was an association between overexpression of RTKs and decreased 5-year OS and this was highly significant when using highest ORs from studies reporting more than one RTK (OR = 2.42; 95% CI = 1.92–3.06, P < 0.001). Similar results were observed for 5-year BCSS. Worse OS was seen with overexpression of fibroblast growth factor receptor 2/3 (FGFR) (OR = 3.81; 95% CI = 1.79–8.11) and epidermal growth factor receptor (EGFR)/HER1 (OR = 2.45; 95% CI = 1.90–3.15).

Conclusion

Overexpression of various RTKs is associated with poor outcomes. This data suggests the clinical evaluation of combination of agents against RTKs or relevant oncogenic nodes.     

ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma

Vestibular schwannomas (VS) arising sporadically or in patients with neurofibromatosis type 2 (NF2) consistently lack expression of Merlin, a tumor suppressor. Conventional treatment options include surgery and radiotherapy but there is no validated medical option. Recent evidence suggests that Merlin deficiency may result in abnormal activation of receptor tyrosine kinases (RTKs) and downstream signaling, promoting tumor growth. Although small-molecule RTK inhibitors are widely available for clinical use, no such therapy has been validated in patients with VS. To screen for RTK activation, surgical VS specimens from patients with and without NF2 were analyzed by phospho-RTK profiling arrays. Downstream signaling pathway activation was analyzed by phospho-MAPK arrays. Activated RTKs and downstream kinases were validated immunohistochemically in corresponding formalin-fixed, paraffin-embedded tissues. Phospho-RTK arrays and immunohistochemistry showed consistent overexpression and activation of EGFR family receptors and evidence of ERK1/2 downstream signaling was observed in all samples analyzed (n = 11). Based on the findings, the small-molecule EGFR/ErbB2 kinase inhibitor lapatinib was selected for evaluation of target inhibition and treatment efficacy in our in vitro human schwannoma model. EGFR/ErbB2 targeted therapy with lapatinib inhibited ErbB2 phosphorylation and survivin upregulation, as well as downstream ERK1/2 and AKT activation, resulting in decreased proliferation. We conclude that EGFR family receptor activation is a consistent feature of both sporadic and NF2-related VS. Molecular targeted therapy with lapatinib downregulates survivin and has antiproliferative activity in a preclinical VS model. Based on these findings, a clinical trial with lapatinib for the treatment of VS is currently underway.     

Comprehensive analyses using next-generation sequencing and immunohistochemistry enable precise treatment in advanced gastric cancer

BackgroundIn advanced gastric cancer (AGC), most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Recently, next-generation sequencing (NGS) allowed us to comprehensively profile the tumor gene status. This study aimed to elucidate the profiling between gene alterations and protein expression in AGC to aid in future clinical trials on AGC.Patients and methodsFormalin-fixed, paraffin-embedded tumor samples from 121 stage III/IV gastric cancer patients were examined for protein expression of tyrosine kinase receptors (RTKs; ERBB2, EGFR, c-MET, and FGFR2) using immunohistochemistry (IHC). Furthermore, 409 cancer-related genes were sequenced to detect mutations and copy number variations using NGS.ResultsMost ERBB2 overexpression (IHC 3+) cases (80.0%) had ERBB2 amplification and did not have other RTK amplification or oncogene mutations. However, one-fourth of MET overexpression cases (25.0%) had ERBB2 alterations. EGFR and FGFR2 overexpression cases had ERBB2 alterations or other gene alterations such as KRAS or PIK3CA. On the other hand, most of the four RTK amplification cases (88.2%) were mutually exclusive with each amplification. However, RTK amplification did not simply correlate with protein overexpression, whereas cases with RTK high-level amplification had protein overexpression and rarely showed other co-existing gene alterations.ConclusionAGC involves a complicated arrangement of protein expression and gene alterations. Comprehensive analyses of NGS and IHC will be necessary to design the optimal therapy for treating the appropriate population of patients in future clinical trials.     

Protein expression and gene copy number changes of receptor tyrosine kinase in thymomas and thymic carcinomas

BackgroundInsulin-like growth factor-1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-type 2 (HER2), and c-Met are members of the receptor tyrosine kinases (RTKs). The associations between the RTK status [protein expression and gene copy number (GCN)] and patient characteristics and between the RTK status and prognosis remain undetermined.Materials and methodsThe study included 140 patients who underwent surgery for thymic tumors. Protein expression was evaluated by immunohistochemistry (IHC) and GCN was evaluated by bright-field in situ hybridization (BISH). The correlations between the RTK status and clinicopathological findings were examined.ResultsIGF-1R protein was frequently detected in thymic carcinoma (83.8%) and EGFR in thymic tumors (91.4%). Thirty-six and 39 tumors were BISH high for IGF-1R and EGFR, respectively: 28 and 25 exhibited high polysomy; 8 and 14 exhibited gene amplification. No tumor was positive for HER2 or c-Met by IHC and BISH. Multivariate analysis revealed that IGF-1R gene amplification (P = 0.027), thymic carcinoma histology, and higher tumor stage were significantly correlated with an adverse prognosis.ConclusionsThymic epithelial tumors frequently express IGF-1R and/or EGFR proteins. IGF-1R gene amplification is suggested to define an unfavorable subset for thymic epithelial tumors.     

AZD8931, an equipotent,reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 non-amplified inflammatory breast cancer models

Introduction

Epidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER2, and HER3 signaling. In this study, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel using preclinical models of EGFR-overexpressed and HER2 non-amplified IBC cells.

Methods

Two IBC cell lines SUM149 and FC-IBC-02 derived from pleural effusion of an IBC patient were used in this study. Cell growth and apoptotic cell death were examined in vitro. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. AZD8931 was given by daily oral gavage at doses of 25 mg/kg, 5 days/week for 4 weeks. Paclitaxel was subcutaneously injected twice weekly.

Results

AZD8931 significantly suppressed cell growth of IBC cells and induced apoptosis of human IBC cells in vitro. Significantly, we showed that AZD8931 monotherapy inhibited xenograft growth and the combination of paclitaxel + AZD8931 was demonstrably more effective than paclitaxel or AZD8931 alone treatment at delaying tumor growth in vivo in orthotopic IBC models.

Conclusion

AZD8931 single agent and in combination with paclitaxel demonstrated signal inhibition and antitumor activity in EGFR-overexpressed and HER2 non-amplified IBC models. These results suggest that AZD8931 may provide a novel therapeutic strategy for the treatment of IBC patients with HER2 non-amplified tumors.     

Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17-demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of downstream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G₂ phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G₁ apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.     

Pancreatic endocrine tumours: mutational and immunohistochemical survey of protein kinases reveals alterations in targetable kinases in cancer cell lines and rare primaries

BackgroundKinases represent potential therapeutic targets in pancreatic endocrine tumours (PETs).Patients and methodsThirty-five kinase genes were sequenced in 36 primary PETs and three PET cell lines: (i) 4 receptor tyrosine kinases (RTK), epithelial growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tyrosine-protein kinase KIT (KIT), platelet-derived growth factor receptor alpha (PDGFRalpha); (ii) 6 belonging to the Akt/mTOR pathway; and (iii) 25 frequently mutated in cancers. The immunohistochemical expression of the four RTKs and the copy number of EGFR and HER2 were assessed in 140 PETs.ResultsSomatic mutations were found in KIT in one and ATM in two primary neoplasms. Among 140 PETs, EGFR was immunopositive in 18 (13%), HER2 in 3 (2%), KIT in 16 (11%), and PDGFRalpha in 135 (96%). HER2 amplification was found in 2/130 (1.5%) PETs. KIT membrane immunostaining was significantly associated with tumour aggressiveness and shorter patient survival. PET cell lines QGP1, CM and BON harboured mutations in FGFR3, FLT1/VEGFR1 and PIK3CA, respectively.ConclusionsOnly rare PET cases, harbouring either HER2 amplification or KIT mutation, might benefit from targeted drugs. KIT membrane expression deserves further attention as a prognostic marker. ATM mutation is involved in a proportion of PET. The finding of specific mutations in PET cell lines renders these models useful for preclinical studies involving pathway-specific therapies.     

Receptor Tyrosine Kinase Fusions and BRAF Kinase Fusions are Rare but Actionable Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors

Introduction

We analyzed a large set of EGFR-mutated (EGFR+) NSCLC to identify and characterize cases with co-occurring kinase fusions as potential resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs).

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte‐derived invasive and metastatic tumor of the skin. It is the second‐most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65‐ to 100‐fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B‐induced epidermal hyperplasia and hyperproliferation in SKH‐1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell‐specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan‐FGFR inhibitor; AZD4547 significantly decreased cSCC cell‐cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR‐mediated effects. To further bolster the in vitro studies, NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle‐only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.     

AMP-activated kinase (AMPK) regulates activity of HER2 and EGFR in breast cancer

AMP-activated Protein Kinase (AMPK) activity retards growth of many types of cancers. Investigating effects of AMPK activation on breast cancer cell signaling and survival, we found that breast cancer cell lines with amplification and over-expression of HER2 or EGFR are 2- to 5-fold more sensitive to cytotoxic effects of AICAR, a canonical pharmacological activator of AMPK, than breast cancer cell lines lacking HER2 or EGFR overexpression. Paralleling effects on cell survival, AICAR leads to dose- and time-dependent inhibition of HER2 and EGFR in HER2-amplified breast cancer cells, with activation of AMPK and suppression of HER2/EGFR activity preceding commitment to cell death. Transfection of constitutively active AMPKα also leads to decreased HER2 and EGFR phosphorylation, reduced downstream signaling associated with these receptor tyrosine kinases (RTKs), and reduced breast cancer cell growth, confirming effects of AMPK activity on HER2/EGFR. Ensuing co-immunoprecipitation experiments demonstrated an interaction of HER2 with AMPK and an in vitro phosphorylation assay found that HER2 and EGFR contain sequences that are potential substrates for AMPK. Our results lead us to postulate that AMPK regulates HER2 and EGFR activity in HER2-amplified breast cancer cells and thus activation of AMPK might provide therapeutic benefit in such cancers.