HER-Family Ligands Promote Acquired Resistance to Trastuzumab in Gastric Cancer.

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.

Acquired Resistance to a MET Antibody In Vivo Can Be Overcome by the MET Antibody Mixture Sym015.

Failure of clinical trials due to development of resistance to MET-targeting therapeutic agents is an emerging problem. Mechanisms of acquired resistance to MET tyrosine kinase inhibitors are well described, whereas characterization of mechanisms of resistance toward MET-targeting antibodies is limited. This study investigated mechanisms underlying in vivo resistance to two antibody therapeutics currently in clinical development: an analogue of the MET-targeting antibody emibetuzumab and Sym015, a mixture of two antibodies targeting nonoverlapping epitopes of MET. Upon long-term in vivo treatment of a MET-amplified gastric cancer xenograft model (SNU-5), emibetuzumab-resistant, but not Sym015-resistant, tumors emerged. Resistant tumors were isolated and used to establish resistant cell lines. Characterization of both tumors and cell lines using extensive protein and signaling pathway activation mapping along with next-generation sequencing revealed two distinct resistance profiles, one involving PTEN loss and the other involving activation of the PI3K pathway, likely via MYC and ERBB3 copy number gains. PTEN loss left one model unaffected by PI3K/AKT targeting but sensitive to mTOR targeting, while the PI3K pathway-activated model was partly sensitive to targeting of multiple PI3K pathway proteins. Importantly, both resistant models were sensitive to treatment with Sym015 in vivo due to antibody-dependent cellular cytotoxicity-mediated tumor growth inhibition, MET degradation, and signaling inhibition. Taken together, our data provide key insights into potential mechanisms of resistance to a single MET-targeting antibody, demonstrate superiority of Sym015 in preventing acquired resistance, and confirm Sym015 antitumor activity in tumors resistant to a single MET antibody. Mol Cancer Ther; 17(6); 1259-70. ©2018 AACR.

Sym015: A Highly Efficacious Antibody Mixture against MET-Amplified Tumors.

Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET.Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line-derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans.Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923-35. ©2017 AACR.

Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings.

Different expression of EZH2, BMI1 and Ki67 in low and high grade neuroendocrine tumors of the lung.

BACKGROUND: Enhancer of Zeste Homolog 2 (EZH2) and B lymphoma Mo-MLV Insertion region 1 polycomb ring finger (BMI1) are involved in malignant transformation of many human carcinomas. Still, in neuroendocrine tumors of the lung (NELT) their expression pattern is largely unknown. This study evaluated their expression in 96 NELT and correlated it to clinical features including survival. MATERIALS AND METHODS: Paraffin embedded material from 50 typical carcinoids (TC), 13 atypical carcinoids (AC), 23 large cell neuroendocrine carcinomas (LCNEC) and 10 small cell lung carcinomas (SCLC) was evaluated by immunohistochemisty. RESULTS: Significantly higher expression of EZH2 was found in high grade NELT (LCNEC + SCLC) versus low grade NELT (TC + AC) (p < 0.0001). High expression of BMI1 was observed in low grade NELT (TC + AC) in contrast to the expression in high grade NELT (LCNEC+SCLC) (p=0.004). In multivariate models, diagnosis was the strongest predictor of survival. CONCLUSION: The immunohistochemical expression of EZH2 and BMI1 are significantly different in low versus high grade NELT. This difference might be related to NELT tumorigenesis. These markers have no independent prognostic impact in NELT. Whether EZH2 could become target in new treatments strategies against NELT remains to be elucidated.

A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue.

The systemic delivery of gene therapeutics by non-viral methods has proven difficult. Transfection systems that are performing well in vitro have been reported to have disadvantageous properties such as rapid clearance and short circulation time often resulting in poor transfection efficiency when applied in vivo. Large unilaminary vesicles (LUV) with encapsulated nucleic acids designated stabilized-plasmid-lipo-particle (SPLP) have showed promising results in terms of systemic stability and accumulation in tumor tissue due to the enhanced permeability and retention effect (EPR). We have developed a simple protocol for the research-scale preparation of SPLPs from commercially available reagents with high amounts of encapsulated plasmid DNA. The SPLPs show properties of promising accumulation in tumor tissue in comparison to other organs when intravenously injected into xenograft tumor-bearing nude mice. Although transcriptionally targeted suicide gene therapy was not achieved, the SPLPs were capable of mediating reporter gene transfection in subcutaneous flank tumors originating from human small cell lung cancer.

In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection.

BACKGROUND: DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream. METHOD: A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution. RESULTS: We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing. CONCLUSION: The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.

In vitro cytotoxicity of combinations of dichloroacetate with anticancer platinum compounds.

PURPOSE: Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase (PDK), and thus promotes glucose oxidation over glycolysis and induces apoptotic death of tumor cells. The present study investigated the potential of DCA to increase the antitumor effects of platinum- based compounds against a panel of permanent cell lines, including small cell lung cancer (SCLC), ovarian cancer, and Ewing's sarcoma in vitro. METHODS: DCA at a concentration of 10 mM was combined with cisplatin, carboplatin, satraplatin, the satraplatin metabolite JM118, oxaliplatin, oxoplatin, and picoplatin, and the cytotoxic activity was evaluated in proliferation tests employing a panel of different cell lines. Additionally, cells were pretreated with DCA and then exposed to the platinum drugs and etoposide, or incubated with cisplatin or etoposide followed by application of DCA, respectively. RESULTS: DCA 10 mM significantly increased the cytotoxicity of the platinum-based drugs carboplatin, satraplatin, JM118, and oxoplatin, but not cisplatin, picoplatin, and oxaliplatin in vitro. Preincubation of cell lines with DCA 10 mM for three days reduced the antiproliferative activity of platinum-based agents in sequential application, but exposure of cells pretreated with cisplatin or etoposide to DCA resulted in minor sensitization. The inhibitory effect of DCA showed no correlation with sensitization to the platinum compounds. CONCLUSION: DCA alone in a concentration that shows low antiproliferative activity is capable of increasing the cytotoxicity of selected platinum compounds upon coincubation, and such combinations may be interesting for clinical application in tumors like SCLC, Ewing's sarcoma, and ovarian cancer refractory to cisplatin chemotherapy as standard care. The mechanism of this synergistic effect of DCA in combination with specific platinum species remains to be investigated.

Cre-loxP recombination vectors for promoter studies

For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.