Yitzhak Zimmer

The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants

Point mutations constitute a major mode of oncogenic activation of the Met receptor tyrosine kinase. Met is aberrantly activated in many types of human malignancies and its deregulated activity is correlated with aggressive tumor traits such as abnormal proliferation and survival, leading to tumor growth, local invasion and metastasis. Here we report that the Met kinase inhibitor SU11274 differentially affects the kinase activity and subsequent signaling of various mutant forms of Met. Two Met variants tested, M1268T and H1112Y, were potently inhibited by 2 μM SU11274, while two other variants, L1213V and Y1248H, remained resistant under similar experimental conditions. Inhibition of the kinase altered cell proliferation, morphology and motility, while cells containing resistant mutants appeared unaffected by the compound. The basis for the sensitivity or resistance to SU11274 is discussed in terms of the position of the mutations predicted from a homology model.

Prevalence and clinical impact of Met Y1253D-activating point mutation in radiotherapy-treated squamous cell cancer of the oropharynx

Aberrant signalling through the hepatocyte growth factor/scatter factor receptor Met has been implicated in various aspects of the development of human cancer including the promotion of tumour invasion, angiogenesis and metastasis. Moreover, experimental data indicate that activation of the Met receptor may be involved in cellular resistance towards antineoplastic treatments such as chemotherapy and ionizing radiation. We determined the prevalence and clinical impact of the Met-activating mutation Y1253D in patients with squamous cell cancer of the oropharynx treated by radical radiotherapy. To screen archival tissue for the presence of a low-abundance point mutation, we developed a sensitive screening method using real-time polymerase chain reaction along with peptide nucleic acid-based DNA clamping and melting curve analysis. By this approach, Met Y1253D was detected in tumours of 15 out of 138 patients (10.9%). Both univariate and multivariate survival analysis revealed Met Y1253D to be significantly associated with impaired local tumour control. Our results provide evidence that the Met-activating mutation Y1253D is present in a notable subset of patients with oropharyngeal cancer and indicate that it may interfere with radioresponsiveness of these tumours, supporting the notion of aberrant Met signalling as a potential target for radiosensitization.

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3β

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

Quantitative Analysis of Extracapsular Extension of Metastatic Lymph Nodes and its Significance in Radiotherapy Planning in Head and Neck Squamous Cell Carcinoma

PURPOSE We performed a histopathologic analysis to assess the extent of the extracapsular extension (ECE) beyond the capsule of metastatic lymph nodes (LN) in head and neck cancer to determine appropriate clinical target volume (CTV) expansions. METHODS AND MATERIALS All tumor-positive LN of 98 patients who underwent a neck dissection with evidence of ECE in at least one LN were analyzed by a single pathologist. The largest diameters of all LN, and in the case of ECE, the maximal linear distance, from the capsule to the farthest extent of tumor or tumoral reaction were recorded. RESULTS A total of 231 LN with ECE and 200 tumor-positive LN without ECE were analyzed. The incidence of ECE was associated with larger LN size (p < 0.001). Of all tumor-positive LN with a diameter of < 10 mm or < 5 mm, 105/220 (48%) nodes or 17/59 (29%) nodes, respectively, showed evidence of ECE. The mean and median extent values of ECE were 2 and 1 mm (range, 1-10 mm) and the ECE was < or = 5 mm in 97% and < or = 3 mm in 91% of the LN, respectively. Overall, the extent of ECE was significantly correlated with larger LN size (Spearmans correlation coefficient = 0.21; p = 0.001). CONCLUSIONS The incidence of ECE is associated with larger LN size. However, ECE is found in a substantial number of LN with a diameter of < 10 mm. The use of 10-mm CTV margins around the gross tumor volume seems appropriate to account for ECE in radiotherapy planning of head and neck cancer.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal‐Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct‐repeat direct‐repeat green fluorescent protein ((DR)‐GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose‐dependent manner HR. Using cells that express MET‐mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET‐dependent. Furthermore, our data also suggest that decline in HR‐dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51‐BRCA2 complex, which is crucial for error‐free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

MET Inhibition Results in DNA Breaks and Synergistically Sensitizes Tumor Cells to DNA-Damaging Agents Potentially by Breaching a Damage-Induced Checkpoint Arrest.

While recent studies implicate that signaling through the receptor tyrosine kinase MET protects cancer cells from DNA damage, molecular events linking MET to the DNA damage response machinery are largely unknown. Here, we studied the impact of MET inhibition by the small molecule PHA665752 on cytotoxicity induced by DNA-damaging agents. We demonstrate that PHA665752 reduces clonogenic survival of tumor cells with MET overexpression when combined with ionizing radiation and synergistically cooperates with ionizing radiation or adriamycin to induce apoptosis. In search of mechanisms underlying the observed synergism, we show that PHA665752 alone considerably increases γH2AX levels, indicating the accumulation of double-strand DNA breaks. In addition, PHA665752 treatment results in sustained high levels of γH2AX and phosphorylated ATM postirradiation, strengthening the assumption that MET inhibition attenuates postdamage DNA repair. PHA665752, alone or in combination with irradiation, leads also to a massive increase of γH2AX tyrosine phosphorylation and its subsequent interaction with the proapoptotic kinase JNK1. Finally, MET inhibition reduces activation of ATR, CHK1, and CDC25B and abrogates an associated DNA damage-induced S phase arrest. This indicates that MET inhibition compromises a critical damage-dependent checkpoint that may enable DNA-damaged cells to exit cell cycle arrest before repair is completed.

Coupling of Mutated Met Variants to DNA Repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells.

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

Incidence of Small Lymph Node Metastases With Evidence of Extracapsular Extension: Clinical Implications in Patients With Head and Neck Squamous Cell Carcinoma

PURPOSE Small lymph nodes (LN) show evidence of extracapsular extension (ECE) in a significant number of patients. This study was performed to determine the impact of ECE in LN ≤7 mm as compared with ECE in larger LN. METHODS AND MATERIALS All tumor-positive LN of 74 head and neck squamous cell carcinoma (HNSCC) patients with at least one ECE positive LN were analyzed retrospectively for the LN diameter and the extent of ECE. Clinical endpoints were regional relapse-free survival, distant metastasis-free survival, and overall survival. The median follow-up for the surviving patients was 2.1 years (range, 0.3-9.2 years). RESULTS Forty-four of 74 patients (60%) had at least one ECE positive LN ≤10 mm. These small ECE positive LN had a median diameter of 7 mm, which was used as a cutoff. Thirty patients (41%) had at least one ECE positive LN ≤7 mm. In both univariate and multivariate Cox regression analyses, the incidence of at least one ECE positive LN ≤7 mm was a statistically significant prognostic factor for decreased regional relapse-free survival (adjusted hazard ratio [HR]: 2.7, p = 0.03, 95% confidence interval [CI]: 1.1-6.4), distant metastasis-free survival (HR: 2.6, p = 0.04, 95% CI: 1.0-6.6), and overall survival (HR: 2.5, p = 0.03, 95% CI: 1.1-5.8). CONCLUSIONS The incidence of small ECE positive LN metastases is a significant prognostic factor in HNSCC patients. Small ECE positive LN may represent more invasive tumor biology and could be used as prognostic markers.