Jolien Van den Bossche

APR-246 (PRIMA-1(MET)) strongly synergizes with AZD2281 (olaparib) induced PARP inhibition to induce apoptosis in non-small cell lung cancer cell lines.

APR-246 (PRIMA-1(Met)) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis. We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background.

Deep sequencing of the TP53 gene reveals a potential risk allele for non–small cell lung cancer and supports the negative prognostic value of TP53 variants

The TP53 gene remains the most frequently altered gene in human cancer, of which variants are associated with cancer risk, therapy resistance, and poor prognosis in several tumor types. To determine the true prognostic value of TP53 variants in non–small cell lung cancer, this study conducted further research, particularly focusing on subtype and tumor stage. Therefore, we determined the TP53 status of 97 non–small cell lung cancer adenocarcinoma patients using next generation deep sequencing technology and defined the prognostic value of frequently occurring single nucleotide polymorphisms and mutations in the TP53 gene. Inactivating TP53 mutations acted as a predictor for both worse overall and progression-free survival in stage II–IV patients and patients treated with DNA-damaging (neo)adjuvant therapy. In stage I tumors, the Pro-allele of the TP53 R72P polymorphism acted as a predictor for worse overall survival. In addition, we detected the rare R213R (rs1800372, minor allele frequency: 0.0054) polymorphism in 7.2% of the patients and are the first to show the significant association with TP53 mutations in non–small cell lung cancer adenocarcinoma patients (p = 0.003). In conclusion, Our findings show an important role for TP53 variants as negative predictors for the outcome of non–small cell lung cancer adenocarcinoma patients, especially for TP53 inactivating mutations in advanced stage tumors treated with DNA-damaging agents, and provide the first evidence of the R213R G-allele as possible risk factor for non–small cell lung cancer.

Simultaneous targeting of EGFR, HER2, and HER4 by afatinib overcomes intrinsic and acquired cetuximab resistance in head and neck squamous cell carcinoma cell lines

The epidermal growth factor receptor (EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR‐targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti‐EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first‐generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines, independent of the HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G0/G1 cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.

Towards Prognostic Profiling of Non-Small Cell Lung Cancer: New Perspectives on the Relevance of Polo-Like Kinase 1 Expression, the TP53 Mutation Status and Hypoxia

Background: Currently, prognosis of non-small cell lung cancer (NSCLC) patients is based on clinicopathological factors, including TNM stage. However, there are considerable differences in patient outcome within a similar staging group, even when patients received identical treatments. In order to improve prognostic predictions and to guide treatment options, additional parameters influencing outcome are required. Polo-like kinase 1 (Plk1), a master regulator of mitotic cell division and the DNA damage response, is considered as a new potential biomarker in this research area. While several studies reported Plk1 overexpression in a broad range of human malignancies, inconsistent results were published regarding the clinical significance hereof. A prognostic panel, consisting of Plk1 and additional biomarkers that are related to the Plk1 pathway, might further improve prediction of patient prognosis. Methods: In this study, we evaluated for the first time the prognostic value of Plk1 mRNA and protein expression in combination with the TP53 mutation status (next generation sequencing), induction of apoptotic cell death (immunohistochemistry for cleaved caspase 3) and hypoxia (immunohistochemistry for carbonic anhydrase IX (CA IX)) in 98 NSCLC adenocarcinoma patients. Results: Both Plk1 mRNA and protein expression and CA IX protein levels were upregulated in the majority of tumor samples. Plk1 mRNA and protein expression levels were higher in TP53 mutant samples, suggesting that Plk1 overexpression is, at least partially, the result of loss of functional p53 (<0.05). Interestingly, the outcome of patients with both Plk1 mRNA and CA IX protein overexpression, who also harbored a TP53 mutation, was much worse than that of patients with aberrant expression of only one of the three markers (p=0.001). Conclusion: The combined evaluation of Plk1 mRNA expression, CA IX protein expression and TP53 mutations shows promise as a prognostic panel in NSCLC patients. Moreover, these results pave the way for new combination strategies with Plk1 inhibitors.

Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

The compound APR-246 (PRIMA-1MET) is a known reactivator of (mutant) p53 and inducer of oxidative stress which can sensitize cancer cells to platinum-based chemotherapeutics. However, the effect of a hypoxic tumor environment has been largely overlooked in this interaction. This study focusses on the role of hypoxia-inducible factor-1α (HIF-1α) and the p53 tumor suppressor protein in hypoxia-induced cisplatin resistance in non-small cell lung cancer (NSCLC) cells and the potential of APR-246 to overcome this resistance. We observed that hypoxia-induced cisplatin resistance only occurred in the p53 mutant NCI-H2228Q331* cell line, and not in the wild type A549 and mutant NCI-H1975R273H cell lines. Cisplatin reduced HIF-1α protein levels in NCI-H2228Q331* cells, leading to a shift in expression from HIF-1α-dependent to p53-dependent transcription targets under hypoxia. APR-246 was able to overcome hypoxia-induced cisplatin resistance in NCI-H2228Q331* cells in a synergistic manner without affecting mutant p53Q331* transcriptional activity, but significantly depleting total glutathione levels more efficiently under hypoxic conditions. Synergism was dependent on the presence of mutant p53Q331* and the induction of reactive oxygen species, with depletion of one or the other leading to loss of synergism. Our data further support the rationale of combining APR-246 with cisplatin in NSCLC, since their synergistic interaction is retained or enforced under hypoxic conditions in the presence of mutant p53.

Preclinical data on the combination of cisplatin and anti-CD70 therapy in non-small cell lung cancer as an excellent match in the era of combination therapy

In contrast to the negligible expression of the immunomodulating protein CD70 in normal tissue, we have demonstrated constitutive overexpression of CD70 on tumor cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. This can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Early clinical trials of these antibodies have already shown promising results in CD70-positive malignancies.In this study, we explored the potential of cisplatin to induce CD70 expression in NSCLC. Using real-time measurement tools, we also assessed the efficacy of a combination regimen with cisplatin and anti-CD70 therapy under normoxia and hypoxia. We identified an induction of CD70 expression on lung cancer cells upon low doses of cisplatin, independent of oxygen levels. More importantly, the use of cisplatin resulted in an enhanced ADCC-effect of anti-CD70 therapy. As such, this combination regimen led to a significant decrease in lung cancer cell survival cell survival, broadening the applicability the applicability of CD70-targeting therapy.This is the first study that proves the potential of a combination therapy with cisplatin and CD70-targeting drugs in NSCLC. Based on our data, we postulate that this combination strategy is an interesting approach to increase tumor-specific cytotoxicity and reduce drug-related side effects.In contrast to the negligible expression of the immunomodulating protein CD70 in normal tissue, we have demonstrated constitutive overexpression of CD70 on tumor cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. This can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Early clinical trials of these antibodies have already shown promising results in CD70-positive malignancies. In this study, we explored the potential of cisplatin to induce CD70 expression in NSCLC. Using real-time measurement tools, we also assessed the efficacy of a combination regimen with cisplatin and anti-CD70 therapy under normoxia and hypoxia. We identified an induction of CD70 expression on lung cancer cells upon low doses of cisplatin, independent of oxygen levels. More importantly, the use of cisplatin resulted in an enhanced ADCC-effect of anti-CD70 therapy. As such, this combination regimen led to a significant decrease in lung cancer cell survival cell survival, broadening the applicability the applicability of CD70-targeting therapy. This is the first study that proves the potential of a combination therapy with cisplatin and CD70-targeting drugs in NSCLC. Based on our data, we postulate that this combination strategy is an interesting approach to increase tumor-specific cytotoxicity and reduce drug-related side effects.

MDM2 SNP309 and SNP285 Act as Negative Prognostic Markers for Non-small Cell Lung Cancer Adenocarcinoma Patients

Objectives: Two functional polymorphisms in the MDM2 promoter region, SNP309T>G and SNP285G>C, have been shown to impact MDM2 expression and cancer risk. Currently available data on the prognostic value of MDM2 SNP309 in non-small cell lung cancer (NSCLC) is contradictory and unavailable for SNP285. The goal of this study was to clarify the role of these MDM2 SNPs in the outcome of NSCLC patients. Materials and Methods: In this study we genotyped SNP309 and SNP285 in 98 NSCLC adenocarcinoma patients and determined MDM2 mRNA and protein levels. In addition, we assessed the prognostic value of these common SNPs on overall and progression free survival, taking into account the TP53 status of the tumor. Results and Conclusion: We found that the SNP285C allele, but not the SNP309G allele, was significantly associated with increased MDM2 mRNA expression levels (p = 0.025). However, we did not observe an association with MDM2 protein levels for SNP285. The SNP309G allele was significantly associated with the presence of wild type TP53 (p = 0.047) and showed a strong trend towards increased MDM2 protein levels (p = 0.068). In addition, patients harboring the SNP309G allele showed a worse overall survival, but only in the presence of wild type TP53. The SNP285C allele was significantly associated with an early age of diagnosis and metastasis. Additionally, the SNP285C allele acted as an independent predictor for worse progression free survival (HR = 3.97; 95% CI = 1.51 - 10.42; p = 0.005). Our data showed that both SNP309 (in the presence of wild type TP53) and SNP285 act as negative prognostic markers for NSCLC patients, implicating a prominent role for these variants in the outcome of these patients.

Abstract 258: Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC

Introduction: Polo-like kinase (Plk1), a key regulator of cell division, is considered as an attractive target for mitotic intervention. The observed Plk1 overexpression in several tumor types, including non-small cell lung cancer (NSCLC), has led to the development of small molecule inhibitors. Moreover, previous studies suggested an interplay between Plk1 and the tumor suppressor P53. This led to the hypothesis that the P53 status might be predictive for the response to Plk1 inhibition. Therefore, we investigated the cytotoxic effect of volasertib in NSCLC cell lines with a different P53 background, under normoxia and hypoxia. Material and methods: A549 (TP53 wild type) was transduced with a P53 shRNA lentiviral vector to obtain the P53 deficient sub-cell line A549-920 or with an empty vector as control (A549-NTC). In addition, NCI-H1975 cells were used as a TP53 mutant (R273H) cell line. Cells were treated with 0-85nM volasertib (24-72h). Cell survival was assessed using the sulphorhodamine B assay and IC 50 -values were calculated using WinNonlin software. The effect of Plk1 inhibition (0-20nM) on cell cycle distribution (24h) and apoptosis induction (48h) was determined flow cytometrically. In addition, we investigated the potential of volasertib to inhibit cell migration using a Transwell system. All experiments were performed under both normoxia and hypoxia ( 2 ). Results: Plk1 inhibition established a dose-dependent growth inhibition under both normoxia and hypoxia. Interestingly, a reduced sensitivity to volasertib treatment (24h) was observed in P53 deficient cells (A549-920, IC 50 : 27.59±5.77 nM) compared to P53 wild type cells (A549-NTC, IC 50 : 17.87±0.40 nM) (p Conclusion: Our results show that there is a difference in response to Plk1 inhibition in cancer cells with and without functional P53. While P53 deficient/mutant cells were mostly arrested in the G2/M phase, cell death was induced in P53 wild type cells. These data lead to the hypothesis that P53 might be a predictive marker for response to Plk1 inhibition. Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Deben, Ines De Pauw, Vanessa Deschoolmeester, Evelien Smits, Pol Specenier, Patrick Pauwels, Marc Peeters, An Wouters. Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 258.

Abstract 4328: New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Polo-like kinase 1 (Plk1) overexpression is observed in various tumors, including non-small cell lung cancer (NSCLC), and is correlated with poor patient prognosis and metastasis, suggesting a role in aggressive tumors. Previous studies reported Plk1 downregulation by P53 upon DNA damage, suggesting that TP53 mutations might have an influence on Plk1 expression. In this study, we determined the Plk1 protein level in NSCLC patients and correlated these results with the TP53 status, presence of hypoxia (carbonic anhydrase 9, CA-9) and apoptosis induction (cleaved caspase 3). Material & methods: Tumor tissues of 84 NSCLC patients and 16 control samples were obtained from the Antwerp University Hospital. Immunohistochemistry was performed using antibodies to Plk1 (208G4, 1/50), CA-9 (EPR4151(2), 1/350) and cleaved caspase 3 (9579S, 1/250). Based on the% positive cells and staining intensity, an overall score of negative, weak, moderate or strong expression was assigned for both the Plk1 and CA-9 protein. For the TP53 mutation analysis, DNA was isolated using the QIAamp® DNA FFPE tissue kit. For each sample, DNA concentration and purity were assessed using NanoDrop measurement. The relation between DNA input and FFPE derived DNA quality was determined using the QC-plex assay from Multiplicom. Samples were considered to be usable when the DNA quality coefficient was higher than 0.2. Determination of the TP53 mutation status was performed using Multiplicoms TP53 MASTRTM Plus test with MID for Illumina Miseq®. Results & discussion: Plk1 and CA-9 positivity was detected in 95% and 83% of all tumor samples, respectively. Of the 16 control samples, all samples stained negative, except for 4/16 and 1/16 samples that showed a low expression for Plk1 and CA-9, respectively. In a next phase, cleaved caspase 3 staining will be scored and both CA-9 and cleaved caspase 3 expression patterns will be correlated to Plk1 expression. Furthermore, results will be correlated with clinicopathological parameters, including incidence age, smoking behavior, tumor differentiation and stage, metastasis and survival. For the TP53 mutation analysis, sufficient DNA with acceptable purity was isolated from 82 patients. A QC plex reaction has already been performed for 20 samples, 12 of them showed an acceptable DQC value and 8 sampled were marked as low DNA quality samples. Nonetheless, the latter could be used for further analysis by increasing the DNA input. At present, 15 samples have been sequenced successfully. Besides polymorphisms, 5 exon mutations (c.536A>G, c.916C>T, c.734G>A, c.578A>C, c.559+1G>T) and 4 intron mutations (c.920-2A>T, c.993+352C>T, c.559+1G>T) were observed. As soon as all samples have been processed, it will be evaluated whether a link between Plk1 overexpression and TP53 mutations can be evidenced. Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Hermans, Christophe Deben, Vanessa Deschoolmeester, Julie Jacobs, Karlijn van der Ven, Jurgen Del-Favero, Patrick Pauwels, Marc Peeters, An Wouters. New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2015-4328

Abstract 3563: Unlocking the potential of CD70 as a therapeutic target in non-small cell lung cancer

Introduction: Upon interaction of CD70, a type II transmembrane protein, with its receptor CD27, the NFκB pathway is activated, leading to proliferation and survival. Hence, CD70 expression is tightly regulated and only transient on cells of the lymphoid lineage. In contrast, constitutive overexpression of CD70 has been documented in malignancies, where it appears to mediate immune escape through recruitment of CD27+ regulatory cells. Soluble CD27 (sCD27), the extracellular domain of CD27, can be released after lymphocyte activation and has been detected at increased levels in serum samples of lymphoid malignancies. To our knowledge, expression of CD70 has never been systematically characterized in non-small cell lung cancer (NSCLC). Methods: Fourty-nine primary NSCLC formalin-fixed paraffin embedded and 9 metastatic biopsies were stained by immunohistochemistry (IHC) for expression of CD70 (Clone 301731, 1/40) and CD27 (Clone 137B4, 1/40), using the Dako autostainer Link and Ventana ultra, respectively. Additionally, sCD27 levels were analyzed in 19 serum samples, taken before of biopsy, with the PeliKine Compact™ human sCD27 ELISA kit. Finally, we tested the efficacy of ARGX-110, a CD70-blocking monoclonal IgG1 SIMPLE Antibody™ endowed with enhanced ADCC properties (POTELLIGENT®), using the xCELLigence RTCA technology in CD70+ and CD70- cell lines. Results: In tumor cells, CD70 positivity (>10%) was detected in 16% of cases with a higher percentage in squamous NSCLC (27%) than in adenocarcinoma (9%). Additionally, one neuro-endocrine NSCLC was CD70+. We demonstrated identical CD70 patterns in primary and metastatic tissue in 8/10 biopsies. Furthermore, CD70 was found in the micro-environment of the tumor (23/49). IHC revealed high levels of CD27 expressing tumor infiltrating lymphocytes (TIL) in all NSCLC biopsies, with increasing FOXP3 expression and higher CD4/CD8 ratios in stromal tissue adjacent to CD70+ tumor cells. High sCD27 levels (>500U/ml) were found to be significantly associated with poor overall survival and even though sCD27 levels did not show potential as a blood-based biomarker for CD70 overexpression on tumor cells, dual positivity of CD70 and sCD27 marked even worse prognosis. We also showed that a low concentration of ARGX-110 (0.5μg/ml) induces efficient NK cell based tumor lysis in CD70+ cell lines. Conclusion: Expression of CD70 was demonstrated in tumor cells and TILs of NSCLC. Moreover, all biopsies revealed infiltration of CD27+ TIL in the micro-environment of the tumor and a trend towards increased FOXP3 expression and higher CD4/CD8 ratios in CD70+ biopsies. In addition, serum sCD27 levels have potential as a prognostic marker for NSCLC. Lastly, our in vitro results showed a maximum ADCC of ARGX-110 in CD70+ target cells stained by IHC. Hence, our data suggest that CD70 might be an interesting therapeutic target in NSCLC. Citation Format: Julie Jacobs, Patrick Pauwels, Christian Rolfo, Filip Lardon, Vanessa Deschoolmeester, Christophe Deben, Jolien Van den Bossche, Karen Zwaenepoel, Christophe Hermans, Karen Silence, Alain Thibault. Unlocking the potential of CD70 as a therapeutic target in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2015-3563