Kristin Gurtner

β1 Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy

Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of β₁ integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of β₁ integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of β₁ integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.

Diverse effects of combined radiotherapy and EGFR inhibition with antibodies or TK inhibitors on local tumour control and correlation with EGFR gene expression

PURPOSE To compare functional effects of combined irradiation and EGFR inhibition in different HNSCC tumour models in vivo with the results of molecular evaluations, aiming to set a basis for the development of potential biomarkers for local tumour control. MATERIAL AND METHODS In five HNSCC tumour models, all wild-type for EGFR and KRAS, the effect of radiotherapy alone (30 fractions/6 weeks) and with simultaneous cetuximab or erlotinib treatment on local tumour control were evaluated and compared with molecular data on western blot, immunohistochemistry and fluorescence-in situ-hybridisation (FISH). RESULTS Erlotinib and cetuximab alone significantly prolonged tumour growth time in 4/5 tumour models. Combined irradiation and cetuximab treatment significantly improved local tumour control in 3/5 tumour models, whereas erlotinib did not alter local tumour control in any of the tumour models. The amount of the cetuximab-effect on local tumour control significantly correlated with the EGFR/CEP-7 ratios obtained by FISH. CONCLUSION Both drugs prolonged growth time in most tumour models, but only application of cetuximab during irradiation significantly improved local tumour control in 3/5 tumour models. The significant correlation of this curative effect with the genetic EGFR expression measured by FISH will be further validated in preclinical and clinical studies.

Heterogeneity of tumour response to combined radiotherapy and EGFR inhibitors: Differences between antibodies and TK inhibitors

Purpose: Clinical and preclinical data show a wide variability of tumour response to combined inhibition of the Epidermal Growth Factor Receptor (EGFR) and radiotherapy or chemotherapy. Differences are obvious not only between different tumour entities, but also between different combination schedules and different classes of drugs. The underlying reasons are currently not well understood. Conclusions: In light of the disappointing results of some phase III trials on combined EGFR tyrosine kinase (TK) inhibition and chemotherapy in non-small-cell lung cancer, but also of some early clinical trials on the triple combination of EGFR inhibitors and radio-chemotherapy, negative interactions between the components of the treatment cannot be ruled out. Also, there is increasing evidence for a differential activity of anti-EGFR antibodies and EGFR-TK inhibitors. Potential reasons are an immunogenic component of the cytotoxic effect of chimeric antibodies, alternative signal transduction pathways leading to acquired resistance against the drugs, different effects on tumour micromilieu or nutritional supply, differences in pharmacokinetics and intratumoural distribution or different effects on cancer stem cells. Clarifying these potential mechanisms will require further preclinical and clinical research effort but could in future enable us to individually tailor the use of molecular targeted drugs in order to fully utilise their high potential in cancer therapy.

Epidermal growth factor receptor inhibitors for radiotherapy: biological rationale and preclinical results

Blocking the epidermal growth factor receptor (EGFR) represents a role model for a successful biological targeting approach to improving outcomes after radiotherapy. This review summarizes data from several local tumour control experiments in which EGFR inhibitors were combined with radiation in FaDu human squamous cell carcinomas xenografted into nude mice. BIBX1382BS is an oral bioavailable inhibitor of the intracellular tyrosine kinase domain of EGFR. It was administered in different experimental settings: concurrent with fractionated radiotherapy, following completion of irradiation, and in the period between surgery and adjuvant irradiation. Despite beneficial effects on tumour growth, in none of these experimental settings did BIBX1382BS improve local tumour control. In contrast, cetuximab (Erbitux), an IgG1 monoclonal antibody against the extracellular ligand‐binding domain of EGFR, improved local tumour control when given concurrently with radiation. Results from a series of local tumour control experiments designed to elucidate the underlying mechanisms of cetuximab suggest that multiple radiobiological mechanisms might contribute to the observed effects: decreased number of clonogenic tumour cells, increased cellular radiation sensitivity, decreased repopulation and improved reoxygenation of clonogenic tumour cells during the combined treatment. In summary, the data suggest that different classes of EGFR inhibitors may have a different potential to improve local tumour control after fractionated irradiation.

Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest.

PURPOSE How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. MATERIALS AND METHODS We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. RESULTS When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. CONCLUSION For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation.

Combined treatment of the immunoconjugate bivatuzumab mertansine and fractionated irradiation improves local tumour control in vivo

BACKGROUND AND PURPOSE To test whether BIWI 1 (bivatuzumab mertansine), an immunoconjugate of the humanized anti-CD44v6 monoclonal antibody BIWA 4 and the maytansinoid DM1, given simultaneously to fractionated irradiation improves local tumour control in vivo compared with irradiation alone. MATERIAL AND METHODS For growth delay, FaDu tumours were treated with 5 intravenous injections (daily) of phosphate buffered saline (PBS, control), BIWA 4 (monoclonal antibody against CD44v6) or BIWI 1 (bivatuzumab mertansine) at two different dose levels (50 μg/kg DM1 and 100 μg/kg DM1). For local tumour control, FaDu tumours received fractionated irradiation (5f/5d) with simultaneous PBS, BIWA 4 or BIWI 1 (two dose levels). RESULTS BIWI 1 significantly improved local tumour control after irradiation with 5 fractions already in the lower concentration. The dose modifying factor of 1.9 is substantial compared to the majority of other modifiers of radiation response. CONCLUSION Because of the magnitude of the curative effect, this approach is highly promising and should be further evaluated using similar combinations with improved tumour-specificity.

Radiation Resistance in KRAS-Mutated Lung Cancer Is Enabled by Stem-like Properties Mediated by an Osteopontin–EGFR Pathway

Lung cancers with activating KRAS mutations are characterized by treatment resistance and poor prognosis. In particular, the basis for their resistance to radiation therapy is poorly understood. Here, we describe a radiation resistance phenotype conferred by a stem-like subpopulation characterized by mitosis-like condensed chromatin (MLCC), high CD133 expression, invasive potential, and tumor-initiating properties. Mechanistic investigations defined a pathway involving osteopontin and the EGFR in promoting this phenotype. Osteopontin/EGFR-dependent MLCC protected cells against radiation-induced DNA double-strand breaks and repressed putative negative regulators of stem-like properties, such as CRMP1 and BIM. The MLCC-positive phenotype defined a subset of KRAS-mutated lung cancers that were enriched for co-occurring genomic alterations in TP53 and CDKN2A. Our results illuminate the basis for the radiation resistance of KRAS-mutated lung cancers, with possible implications for prognostic and therapeutic strategies. Cancer Res; 77(8); 2018-28. ©2017 AACR.

EGFR-amplification plus gene expression profiling predicts response to combined radiotherapy with EGFR-inhibition: A preclinical trial in 10 HNSCC-tumour-xenograft models

BACKGROUND AND PURPOSE Improvement of the results of radiotherapy by EGFR inhibitors is modest, suggesting significant intertumoural heterogeneity of response. To identify potential biomarkers, a preclinical trial was performed on ten different human squamous cell carcinoma xenografts of the head and neck (HNSCC) studying in vivo and ex vivo the effect of fractionated irradiation and EGFR inhibition. Local tumour control and tumour growth delay were correlated with potential biomarkers, e.g. EGFR gene amplification and radioresponse-associated gene expression profiles. MATERIAL AND METHODS Local tumour control 120days after end of irradiation was determined for fractionated radiotherapy alone (30f, 6weeks) or after simultaneous EGFR-inhibition with cetuximab. The EGFR gene amplification status was determined using FISH. Gene expression analyses were performed using an in-house gene panel. RESULTS Six out of 10 investigated tumour models showed a significant increase in local tumour control for the combined treatment of cetuximab and fractionated radiotherapy compared to irradiation alone. For 3 of the 6 responding tumour models, an amplification of the EGFR gene could be demonstrated. Gene expression profiling of untreated tumours revealed significant differences between amplified and non-amplified tumours as well as between responder and non-responder tumours to combined radiotherapy and cetuximab. CONCLUSION The EGFR amplification status, in combination with gene expression profiling, may serve as a predictive biomarker for personalized interventional strategies regarding combined treatment of cetuximab and fractionated radiotherapy and should, as a next step, be clinically validated.

Protonentherapie von Rhabdomyosarkompatienten im Kindesalter

Die hier besprochene Untersuchung ist die erste prospektiv angelegte Studie, welche das Ansprechen sowie die akuten und späten Nebenwirkung einer Protonentherapie im Kindesalter bei Rhabdomyosarkompatienten untersucht hat. Gesamtüberleben sowie lokale Kontrolle sind mit denen vorangegangener Photonentherapiestudien vergleichbar (COG-D9602 für „low risk“ und COG-D9803 für „intermediate risk“; [1–3]). Dies entsprach den Erwartungen, da die Dosisverschreibung für die Protonentherapie gegenüber einer Photonentherapie nicht verändert wurde und die Zielvolumina bei den Patienten durch die Protonentherapie trotz besserer Aussparung der Normalgewebe sehr gut erfasst werden konnten. Die oftmals sehr viel steileren Dosisgradienten, die mit der Protonentherapie im Vergleich zur Photonentherapie resultieren, führen offenbar nicht zu höheren Rezidivraten am Feldrand bzw. außerhalb des bestrahlten Volumens. Ziel der Arbeit Mit einer prospektiven Phase-II-Studie untersuchten Ladra et al. die lokale Kontrolle sowie die akuten und späten Nebenwirkungen einer Protonentherapie von Rhabdomyosarkomen im Kindesund jungen Erwachsenenalter.

Abstract 4756: A SPP1-EGFR pathway links stem-like properties of KRAS-mutated lung cancer to radiation resistance

Lung cancers with activating mutations in the KRAS oncogene present a major clinical challenge due to poor prognosis and frequent treatment resistance, particularly to agents targeting the epidermal growth factor receptor (EGFR). For many years, it has been known that mutations in KRAS enhance cellular resistance to ionizing radiation (IR). Clinical data are emerging suggesting that the radioresistance of KRASmut cancer cells that is observed in the laboratory is also seen in patients. However, the underlying mechanisms of mutant KRAS-associated radioresistance is understudied and poorly understood. Cancer stem cells (CSC), also referred to as tumor-initiating cells, may promote metastases development and recurrence after therapy. Generally, cancer cells with CSC-like phenotypes or markers have been found to be radioresistant. Enhanced DNA damage response and repair pathways have been discussed as one underlying mechanism. Interestingly, mutant KRAS has been recently linked to CSC-like phenotypes such as tumor initiation, anchorage independence, and self-renewal. Here, we characterized a radioresistant phenotype associated with KRAS mutated lung cancer cells, xenografts, and patients. CSC-like cells were defined as tumor-initiating cells. Genomic, biochemical, and cell-based assays were used to identify candidates for targeting KRAS mutation-mediated radiation resistance. Radiation resistance is conferred by a CSC-like subpopulation that is characterized by condensed chromatin, high CD133 expression, invasive potential, and tumor-initiating properties. Osteopontin, the gene product of SPP1, promotes aspects of this phenotype including radiation resistance and acts in the same pathway as the EGFR. SPP1/EGFR-dependent chromatin condensation not only protects cells against radiation-induced DNA damage but also downregulates putative negative regulators of CSC-like properties, including CRMP1 and BIM. This phenotype defines a subset of KRAS mutated lung cancers that is enriched for co-occurring TP53 mutations. In conclusions, markers and mechanisms of resistance to radiation therapy in lung and other cancers are poorly characterized. Our data provide novel insight into the aggressive biology of KRAS mutant cancers and have major implications for strategies to overcome the radiation resistance associated with this genotype. Citation Format: Meng Wang, Jing Han, Lynnette Marcar, Josh Black, Qi Liu, Xiangyong Li, Kshithija Nagulapalli, Lecia V. Sequist, Raymond H. Mak, Cyril H. Benes, Theodore S. Hong, Kristin Gurtner, Mechthild Krause, Michael Baumann, Jing X. Kang, Johnathan R. Whetstine, Henning Willers. A SPP1-EGFR pathway links stem-like properties of KRAS-mutated lung cancer to radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2017-4756