The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

BACKGROUND: Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. METHODS: Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. RESULTS: Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. CONCLUSIONS: Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

A phase Ib study of the highly selective MET-TKI savolitinib plus gefitinib in patients with EGFR-mutated, MET-amplified advanced non-small-cell lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are recommended first-line treatments in EGFR-mutated (EGFRm) non-small-cell lung cancer (NSCLC). However, acquired resistance (e.g. MET amplification) is frequently observed. Savolitinib (volitinib, HMPL-504, AZD6094) is an oral, potent, and highly selective MET-TKI. In this phase Ib, open-label, multicenter study, we enrolled Chinese patients with EGFRm advanced NSCLC, whose disease progressed following prior EGFR-TKI treatment. In the safety run-in, patients received savolitinib 600 or 800 mg plus gefitinib 250 mg orally once daily, and dose-limiting toxicities were recorded. In the expansion phase, patients with MET amplification received savolitinib plus gefitinib. The primary endpoint was safety/tolerability. Secondary endpoints included antitumor activity. Thirteen patients were enrolled in the safety phase (median age 52 years, 46% female) and 51 enrolled in the expansion phase (median age 61 years, 67% female). No dose-limiting toxicities were reported in either dose group during the safety run-in. Adverse events of grade ≥ 3 in the safety run-in and expansion phases (n = 57) were reported in 21 (37%) patients. The most frequently reported adverse events (all grades) were: vomiting (n = 26, 46%), nausea (n = 23, 40%), increased aspartate aminotransferase (n = 22, 39%). Of four deaths, none were treatment-related. The objective response rates in EGFR T790M-negative, -positive, and -unknown patients were 52% (12/23), 9% (2/23), and 40% (2/5), respectively. Savolitinib 600 mg plus gefitinib 250 mg once daily had an acceptable safety profile and demonstrated promising antitumor activity in EGFRm, MET-amplified advanced NSCLC patients who had disease progression on EGFR-TKIs. NCT02374645, Date of registration: March 2nd 2015.

Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors.

Arp2/3 complex initiates the growth of branched actin-filament networks by inducing actin polymerization from the sides of pre-existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three-dimensional structures at ~2 nm resolution of Arp2/3 complex with three different bound NPFs: N-WASp, Scar-VCA and cortactin. All of these structures adopt a conformation with the two actin-related proteins in an actin-filament-like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin-filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.

Phase II study of the PI3K inhibitor pilaralisib (SAR245408; XL147) in patients with advanced or recurrent endometrial carcinoma.

OBJECTIVE: Patients with endometrial carcinoma who progress after first-line chemotherapy have a poor prognosis. Phosphoinositide 3-kinase (PI3K) inhibitors are investigational treatment options in this setting. This study evaluated the efficacy and safety of the PI3K inhibitor pilaralisib (SAR245408; XL147) in advanced or recurrent endometrial carcinoma. METHODS: This Phase II, multicenter, single-arm, open-label study enrolled patients with histologically confirmed advanced or recurrent endometrial carcinoma, who had received one or two prior chemotherapy regimens. Patients received pilaralisib 600mg capsules or 400mg tablets once daily. Primary endpoints were objective response rate (ORR), proportion of patients with progression-free survival (PFS) >6months and safety. Molecular profiling in archival tumor tissue and circulating tumor DNA were performed to identify molecular markers associated with response or resistance to pilaralisib. RESULTS: 67 patients were enrolled, of which 50 and 17 patients had received one or two prior regimens, respectively. Complete or partial tumor responses occurred in two patients each (ORR 6.0%); three had tumors with normal PTEN expression and PIK3R1 mutations and one had a tumor with PTEN protein deficiency. However, there was no association between molecular alterations and clinical activity. Rate of PFS>6months was 11.9%. The most commonly reported treatment-related adverse events (AEs) were rash (40.3%), diarrhea (37.3%) and fatigue (28.4%). The most commonly reported treatment-related grade ≥3 AEs were rash (9.0%), diarrhea (4.5%) and increased alanine aminotransferase (4.5%). CONCLUSIONS: Pilaralisib was associated with a favorable safety profile and minimal antitumor activity in advanced or recurrent endometrial carcinoma.

The RickA protein of Rickettsia conorii activates the Arp2/3 complex.

Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements. Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement. Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria. We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref. 4). Here we identify a R. conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA. In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails. When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia. Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.

Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions.

Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.

Voxtalisib (XL765) in patients with relapsed or refractory non-Hodgkin lymphoma or chronic lymphocytic leukaemia: an open-label, phase 2 trial.

BACKGROUND: Patients with relapsed or refractory lymphoma or chronic lymphocytic leukaemia have a poor prognosis. Therapies targeting more than one isoform of PI3K, as well as mTOR, might increase antitumour activity. We aimed to investigate the efficacy and safety of voxtalisib (also known as XL765 or SAR245409), a pan-PI3K/mTOR inhibitor, in patients with relapsed or refractory lymphoma, or chronic lymphocytic leukaemia/small lymphocytic lymphoma. METHODS: We did a non-randomised, open-label, phase 2 trial at 30 oncology clinics in the USA, Belgium, Germany, France, the Netherlands, and Australia. Patients aged 18 years or older with Eastern Cooperative Oncology Group (EGOG) performance status score of 2 or lower and relapsed or refractory mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, or chronic lymphocytic leukaemia/small lymphocytic lymphoma were enrolled and treated with voxtalisib 50 mg orally twice daily in 28-day continuous dosing cycles until progression or unacceptable toxicity. The primary endpoint was the proportion of patients in each disease-specific cohort who achieved an overall response, defined as a complete response or partial response. All patients who received more than 4 weeks of treatment and who completed a baseline and at least one post-baseline tumour assessment were analysed for efficacy and all patients were analysed for safety. This study is registered with ClinicalTrials.gov, number NCT01403636, and has been completed. FINDINGS: Between Oct 19, 2011, and July 24, 2013, 167 patients were enrolled (42 with mantle cell lymphoma, 47 with follicular lymphoma, 42 with diffuse large B-cell lymphoma, and 36 with chronic lymphocytic leukaemia/small lymphocytic lymphoma. The median number of previous anticancer regimens was three (IQR 2-4) for patients with lymphoma and four (2-5) for patients with chronic lymphocytic leukaemia/small lymphocytic lymphoma. Of 164 patients evaluable for efficacy, 30 (18·3%) achieved an overall response (partial, n=22; complete, n=8); 19 (41·3%) of 46 with follicular lymphoma, five (11·9%) of 42 with mantle cell lymphoma, two (4·9%) of 41 with diffuse large B-cell lymphoma, and four (11·4%) of 35 with chronic lymphocytic leukaemia/small lymphocytic lymphoma. The safety profile was consistent with that of previous studies of voxtalisib. The most frequently reported adverse events were diarrhoea (in 59 [35%] of 167 patients), fatigue (in 53 [32%]), nausea (in 45 [27%]), pyrexia (in 44 [26%,]), cough (in 40 [24%]), and decreased appetite (in 35 [21%]). The most frequently reported grade 3 or worse adverse events were anaemia (in 20 [12%] of 167 patients), pneumonia (in 14 [8%]), and thrombocytopenia (in 13 [8%]). Serious adverse events occurred in 97 (58·1%) of 167 patients. INTERPRETATION: Voxtalisib 50 mg given orally twice daily had an acceptable safety profile, with promising efficacy in patients with follicular lymphoma but limited efficacy in patients with mantle cell lymphoma, diffuse large B-cell lymphoma, or chronic lymphocytic leukaemia/small lymphocytic lymphoma. FUNDING: Sanofi.

Discovery and Pharmacokinetic and Pharmacological Properties of the Potent and Selective MET Kinase Inhibitor 1-{6-[6-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl]benzothiazol-2-yl}-3-(2-morpholin-4-ylethyl)urea (SAR125844).

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

Efficacy, safety, pharmacokinetics and pharmacodynamics of SAR245409 (voxtalisib, XL765), an orally administered phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor: a phase 1 expansion cohort in patients with relapsed or refractory lymphoma.

The maximum tolerated dose of SAR245409 (voxtalisib), a pan-class I phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor, was determined in a phase 1 dose-escalation study in advanced solid tumors. We report safety, pharmacokinetics (PK), pharmacodynamics and preliminary efficacy of SAR245409 capsules 50 mg twice daily in an expansion cohort of 16 patients with relapsed/refractory lymphoma. The most common treatment-related adverse events (AEs) were nausea (31.3%) and diarrhea (25.0%). The most common grade 3/4 treatment-related AE was increased alanine aminotransferase (12.5%). PK results were consistent with solid tumors, confirming a relatively short steady-state half-life (mean 4.61 h). Among 12 evaluable patients, one complete response and two partial responses were achieved in patients with and without PI3K/mTOR pathway alterations. In a patient with mantle cell lymphoma achieving PR, SAR245409 was associated with significant inhibition of PI3K/mTOR and extracellular signal-related kinase (ERK) pathways. Preliminary efficacy warrants further evaluation of SAR245409 in lymphoma.

Phase I Trial of the Pan-PI3K Inhibitor Pilaralisib (SAR245408/XL147) in Patients with Chronic Lymphocytic Leukemia (CLL) or Relapsed/Refractory Lymphoma.

PURPOSE: This phase I expansion-cohort study evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of the pan-PI3K inhibitor pilaralisib (SAR245408/XL147) in patients with chronic lymphocytic leukemia (CLL) or relapsed or refractory lymphoma. PATIENTS AND METHODS: Patients were treated with the maximum tolerated dose of pilaralisib previously determined in patients with solid tumors (600 mg capsules once daily). Adverse events (AE) and response were evaluated. Plasma pharmacokinetics and pharmacodynamic effects on cytokines and chemokines were also assessed. RESULTS: Twenty-five patients were included in the study: 10 with CLL and 15 with lymphoma. The most frequent AEs of any grade were diarrhea (92.0%), pyrexia (52.0%), and fatigue (44.0%). The most frequent grade ≥3 AEs were neutropenia (32.0%), diarrhea (20.0%), and anemia (16.0%). Pilaralisib exposure on cycle 1 day 28 was similar to exposure in patients with solid tumors. In patients with CLL, pilaralisib significantly reduced plasma levels of several cytokines and chemokines involved in B-cell trafficking. Five patients (50.0%) with CLL and 3 patients (20.0%) with lymphoma had a partial response. Six patients (60.0%) with CLL had nodal shrinkage ≥50%. Overall, 14 patients (56.0%; 7 patients with CLL and 7 patients with lymphoma) had progression-free survival ≥6 months. CONCLUSIONS: Pilaralisib demonstrated an acceptable safety profile in patients with CLL and lymphoma, generally consistent with findings in patients with solid tumors. Single-agent pilaralisib showed preliminary clinical activity in patients with CLL and lymphoma, supporting further development.

Phase I dose-escalation study of the PI3K/mTOR inhibitor voxtalisib (SAR245409, XL765) plus temozolomide with or without radiotherapy in patients with high-grade glioma.

BACKGROUND: This phase I study aimed to evaluate safety, maximum tolerated dose, pharmacokinetics, pharmacodynamics, and preliminary efficacy of voxtalisib (SAR245409, XL765), a pan-class I phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor, in combination with temozolomide (TMZ), with or without radiation therapy (RT), in patients with high-grade glioma. METHODS: Patients received voxtalisib 30-90 mg once daily (q.d.) or 20-50 mg twice daily (b.i.d.), in combination with 200 mg/m(2) TMZ (n = 49), or voxtalisib 20 mg q.d. with 75 mg/m(2) TMZ and RT (n = 5). A standard 3 + 3 dose-escalation design was used to determine the maximum tolerated dose. Patients were evaluated for adverse events (AEs), plasma pharmacokinetics, pharmacodynamic effects in skin biopsies, and tumor response. RESULTS: The maximum tolerated doses were 90 mg q.d. and 40 mg b.i.d. for voxtalisib in combination with TMZ. The most frequently reported treatment-related AEs were nausea (48%), fatigue (43%), thrombocytopenia (26%), and diarrhea (24%). The most frequently reported treatment-related grade ≥3 AEs were lymphopenia (13%), thrombocytopenia, and decreased platelet count (9% each). Pharmacokinetic parameters were similar to previous studies with voxtalisib monotherapy. Moderate inhibition of PI3K signaling was observed in skin biopsies. Best response was partial response in 4% of evaluable patients, with stable disease observed in 68%. CONCLUSIONS: Voxtalisib in combination with TMZ with or without RT in patients with high-grade gliomas demonstrated a favorable safety profile and a moderate level of PI3K/mTOR pathway inhibition.

The selective intravenous inhibitor of the MET tyrosine kinase SAR125844 inhibits tumor growth in MET-amplified cancer.

Activation of the MET/HGF pathway is common in human cancer and is thought to promote tumor initiation, metastasis, angiogenesis, and resistance to diverse therapies. We report here the pharmacologic characterization of the triazolopyridazine derivative SAR125844, a potent and highly selective inhibitor of the MET receptor tyrosine kinase (RTK), for intravenous administration. SAR125844 displayed nanomolar activity against the wild-type kinase (IC50 value of 4.2 nmol/L) and the M1250T and Y1235D mutants. Broad biochemical profiling revealed that SAR125844 was highly selective for MET kinase. SAR125844 inhibits MET autophosphorylation in cell-based assays in the nanomolar range, and promotes low nanomolar proapoptotic and antiproliferative activities selectively in cell lines with MET gene amplification or pathway addiction. In two MET-amplified human gastric tumor xenograft models, SNU-5 and Hs 746T, intravenous treatment with SAR125844 leads to potent, dose- and time-dependent inhibition of the MET kinase and to significant impact on downstream PI3K/AKT and RAS/MAPK pathways. Long duration of MET kinase inhibition up to 7 days was achieved with a nanosuspension formulation of SAR125844. Daily or every-2-days intravenous treatment of SAR125844 promoted a dose-dependent tumor regression in MET-amplified human gastric cancer models at tolerated doses without treatment-related body weight loss. Our data demonstrated that SAR125844 is a potent and selective MET kinase inhibitor with a favorable preclinical toxicity profile, supporting its clinical development in patients with MET-amplified and MET pathway-addicted tumors.

Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245408 (XL147), an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors.

PURPOSE: SAR245408 is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor. This phase I study determined the maximum tolerated dose (MTD) of two dosing schedules [first 21 days of a 28-day period (21/7) and continuous once-daily dosing (CDD)], pharmacokinetic and pharmacodynamic profiles, and preliminary efficacy. EXPERIMENTAL DESIGN: Patients with refractory advanced solid malignancies were treated with SAR245408 using a 3 + 3 design. Pharmacokinetic parameters were determined after single and repeated doses. Pharmacodynamic effects were evaluated in plasma, hair sheath cells, and skin and tumor biopsies. RESULTS: Sixty-nine patients were enrolled. The MTD of both schedules was 600 mg; dose-limiting toxicities were maculopapular rash and hypersensitivity reaction. The most frequent drug-related adverse events included dermatologic toxicities, diarrhea, nausea, and decreased appetite. Plasma pharmacokinetics showed a median time to maximum concentration of 8 to 22 hours, mean terminal elimination half-life of 70 to 88 hours, and 5- to 13-fold accumulation after daily dosing (first cycle). Steady-state concentration was reached between days 15 and 21, and exposure was dose-proportional with doses up to 400 mg. SAR245408 inhibited the PI3K pathway (∼40%-80% reduction in phosphorylation of AKT, PRAS40, 4EBP1, and S6 in tumor and surrogate tissues) and, unexpectedly, also inhibited the MEK/ERK pathway. A partial response was seen in one patient with advanced non-small cell lung cancer. Eight patients were progression-free at 6 months. Pharmacodynamic and clinical activity were observed irrespective of tumor PI3K pathway molecular alterations. CONCLUSIONS: SAR245408 was tolerable at doses associated with PI3K pathway inhibition. The recommended phase II dose of the capsule formulation is 600 mg administered orally with CDD.

Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245409 (XL765), a novel, orally administered PI3K/mTOR inhibitor in patients with advanced solid tumors.

PURPOSE: This phase I, first-in-human study evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and preliminary efficacy of SAR245409, an inhibitor of pan-Class I phosphoinositide 3-kinase (PI3K) and mTOR, administered orally once or twice daily in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Eighty-three patients received SAR245409. Doses ranged from 15 to 120 mg twice daily, and 70 to 100 mg once daily. A 3+3 dose-escalation design was used to determine the MTD. Patients were evaluated for adverse events and response. Assessments included pharmacokinetic, pharmacodynamic impact of SAR245409 on PI3K pathway signaling in hair sheath cells, skin and tumor, and characterization of tumor molecular alterations. RESULTS: The MTDs were 50 mg twice daily and 90 mg once daily. The most frequent treatment-related adverse events were nausea (36.1%), diarrhea (21.7%), vomiting (19.3%), and decreased appetite (16.9%). The most frequent treatment-related grade 3/4 adverse events were increases in alanine aminotransferase (6.0%) and aspartate aminotransferase (4.8%). SAR245409 had a relatively short plasma half-life (2.96-7.52 hours). At MTDs, once- and twice-daily regimens yielded similar mean steady-state plasma exposure. A reduction in PI3K and mTORC1/mTORC2 pathway signaling was observed in serial hair sheath cells, skin, and tumor samples. Best response was stable disease in 48% of evaluable patients; seven patients had minor tumor regression. Twelve patients with stable disease were treated for ≥16 weeks. No trend was observed correlating tumor molecular alteration with antitumor activity. CONCLUSION: SAR245409 had a manageable safety profile, demonstrated reduced PI3K and mTORC1/mTORC2 pathway signaling and was associated with clinically relevant stable disease.

MET genetic abnormalities unreliable for patient selection for therapeutic intervention in oropharyngeal squamous cell carcinoma.

BACKGROUND: Identification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC. METHODS: A retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival. RESULTS: 143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p.Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with ≥10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival. CONCLUSIONS: Our study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.

The structural basis of actin filament branching by the Arp2/3 complex.

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway.

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

Interaction of N-WASP with hnRNPK and its role in filopodia formation and cell spreading.

N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center.

Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection.

Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.

Cortactin regulates cell migration through activation of N-WASP.

Cortactin is an actin-associated scaffolding protein that regulates cell migration. Amplification of the human gene, EMS1, has been detected in breast, head and neck tumors, where it correlates with increased invasiveness. Cortactin can regulate actin dynamics directly via its N-terminal half, which can bind and activate the Arp2/3 complex. The C-terminal portion of cortactin, however, is thought to have limited function in its regulation of the actin polymerization machinery. In this report, we identify a role for the cortactin C-terminus in regulating cell migration and, more specifically, actin dynamics. Overexpression of either full-length cortactin or cortactin C-terminus is sufficient to enhance migration of mammary epithelial cells. In vitro, cortactin binds to and activates, via its SH3 domain, a regulator of the Arp2/3 complex, neural Wiskott Aldrich Syndrome protein (N-WASP). This in vitro activation of N-WASP is likely to be important in vivo, as cortactin-enhanced migration is dependent upon N-WASP. Thus, our results suggest that cortactin has multiple mechanisms by which it can recruit and modulate the actin machinery and ultimately regulate cell migration.