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Immunotherapy for metastasized non‐small‐cell lung cancer (NSCLC) can show long‐lasting clinical responses. Selection of patients based on programmed death‐ligand 1 (PD‐L1) expression shows limited predictive value for durable clinical benefit (DCB). We investigated whether early treatment effects as measured by a change in circulating tumor DNA (ctDNA) level is a proxy of early tumor response to immunotherapy according to response evaluation criteria in solid tumors v1.1 criteria, progression‐free survival (PFS), DCB, and overall survival (OS). To this aim, blood tubes were collected from advanced‐stage lung adenocarcinoma patients (n = 100) receiving immune checkpoint inhibitors (ICI) at baseline (t0) and prior to first treatment evaluation (4–6 weeks; t1). Nontargetable (driver) mutations detected in the pretreatment tumor biopsy were used to quantify tumor‐specific ctDNA levels using droplet digital PCR. We found that changes in ctDNA levels were strongly associated with tumor response. A > 30% decrease in ctDNA at t1 correlated with a longer PFS and OS. In total, 80% of patients with a DCB of ≥ 26 weeks displayed a > 30% decrease in ctDNA levels. For patients with a PD‐L1 tumor proportion score of ≥ 1%, decreasing ctDNA levels were associated with a higher frequency a DCB (80%) and a prolonged median PFS (85 weeks) and OS (101 weeks) compared with patients with no decrease in ctDNA (34%; 11 and 39 weeks, respectively). This study shows that monitoring of ctDNA dynamics is an easy‐to‐use and promising tool for assessing PFS, DCB, and OS for ICI‐treated NSCLC patients., Predictive markers to reliably monitor response in patients with lung adenocarcinoma treated with immune checkpoint inhibitors are currently lacking. Here, we show that tumor‐informed monitoring of a single bloodborne mutation using droplet digital PCR enabled the identification of early disease progression and durable treatment responses. The association between patient survival and changes in mutant circulating tumor DNA (ctDNA) levels was unrelated to programmed death‐ligand 1 expression. Altogether, our data show that ctDNA dynamics could offer a promising cost‐effective approach to monitor patients with metastasized lung adenocarcinoma.

Background The clinical relevance of epidermal growth factor receptor (EGFR) copy number gain in patients with EGFR mutated advanced non-small cell lung cancer on first-line tyrosine kinase inhibitor treatment has not been fully elucidated. Objective We aimed to estimate EGFR copy number gain using amplicon-based next generation sequencing data and explored its prognostic value. Patients and Methods Next generation sequencing data were obtained for 1566 patients with non-small cell lung cancer. EGFR copy number gain was defined based on an increase in EGFR read counts relative to internal reference amplicons and normal controls in combination with a modified z-score ≥ 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors. Results Specificity and sensitivity of next generation sequencing-based EGFR copy number estimations were above 90%. EGFR copy number gain was observed in 27.9% of EGFR mutant cases and in 7.4% of EGFR wild-type cases. EGFR gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46–6.78, p = 0.003). Besides EGFR copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20–0.91, p = 0.028) and 0.24 (95% confidence interval 0.1–0.59, p = 0.001), respectively. Conclusions Pre-treatment EGFR copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in EGFR-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival. Supplementary Information The online version contains supplementary material available at 10.1007/s11523-021-00798-2.

Circulating cell-free DNA (ccfDNA) may contain DNA originating from the tumor in plasma of cancer patients (ctDNA) and enables noninvasive cancer diagnosis, treatment predictive testing, and response monitoring. A recent multicenter evaluation of workflows by the CANCER-ID consortium using artificial spiked-in plasma showed significant differences and consequently the importance of carefully selecting ccfDNA extraction methods. Here, the quantity and integrity of extracted ccfDNA from the plasma of cancer patients were assessed. Twenty-one cancer patient-derived cell-free plasma samples were selected to compare the Qiagen CNA, Maxwell RSC ccfDNA plasma, and Zymo manual quick ccfDNA kit. High-volume citrate plasma samples collected by diagnostic leukapheresis from six cancer patients were used to compare the Qiagen CNA (2 mL) and QIAamp MinElute ccfDNA kit (8 mL). This study revealed similar integrity and similar levels of amplified short-sized fragments and tumor-specific mutants comparing the CNA and RSC kits. However, the CNA kit consistently showed the highest yield of ccfDNA and short-sized fragments, while the RSC and ME kits showed higher variant allelic frequencies (VAFs). Our study pinpoints the importance of standardizing preanalytical conditions as well as consensus on defining the input of ccfDNA to accurately detect ctDNA and be able to compare results in a clinical routine practice, within and between clinical studies.

Background Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. Conclusions We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Objective The relevance of EGFR gene amplification in patients with EGFR-mutated advanced non-small cell lung cancer (NSCLC) has not been fully elucidated. Moreover, a limited number of studies have been published on methods to estimate gene amplifications based on routinely performed targeted next generation sequencing (NGS). In this study we aimed to determine whether PCR-based targeted NGS data on tumor tissue can be used to estimate presence of gene amplifications and explored the prognostic value of EGFR gene amplification in EGFR mutated NSCLC patients. Materials and methods A total of 3,194 good quality targeted NGS data files were retrieved from 2014 to 2017. Among those, 1,729 NSCLC samples originated from 1,586 NSCLC patients of whom 134 had an EGFR mutation (8.2%). Clinical data were available for 66 of the patients. Raw sequencing data were re-analyzed using a custom designed pipeline. The presence of an amplification was based on the read depth of a given amplicon relative to a set of reference amplicons from the sample or relative to a set of normal control samples. Reference amplicons were selected based on low degree of variation in read depth amongst all tested samples. Amplifications were regarded significant when the ratio was ≥3 and the z score was ≥3.5. Technical validation was done by FISH and MLPA. Cox regression analysis on overall survival was done with each of the amplification parameters using SPSS. Results No amplifications were detected for the ALK, KIT, NRAS, PDGFRA, GNAQ and MAP2K1 gene loci, whereas amplifications for BRAF, ESR1, GNA11, HRAS, KRAS, MET and PIK3CA were observed at a low frequency. Depending on the amplification analysis strategy (within sample or relative to normal controls), 19% and 13% of the EGFR mutated group had an EGFR amplification, respectively. In EGFR wild type patients, amplifications were detected in 5% and 4% of the patients using the two methods, respectively. The sensitivity and specificity of the NGS based estimation of amplifications for EGFR was 94% (14/15) and 97% (34/35) respectively for both data analysis approaches. Patients with concurrent EGFR mutations and amplifications (estimated within sample) treated with EGFR-TKI had a significantly worse overall survival compared with those without concurrent EGFR amplifications (ratio, p=0.047 and z score, p=0.015). Cox regression analysis indicated a borderline significant interaction between ratio and z score (p=0.052). Conclusion Routinely obtained amplicon-based NGS data can be used to identify gene amplifications. The presence of EGFR amplifications in EGFR mutant patients is predictive of a worse overall survival. Keywords: Lung adenocarcinoma, EGFR, survival, tyrosine kinase inhibitor Citation Format: Pei Meng, Jiacong Wei, Miente Martijn Terpstra, Anke van Rijk, Menno Tamminga, Frank Scherpen, Arja ter Elst, Mohamed Z. Alimohamed, Lennart F. Johansson, T. Jeroen N. Hiltermann, Harry J. Groen, Klaas Kok, Anthonie J. van der Wekken, Anke van den Berg. Clinical value of EGFR gene amplifications detected using amplicon based targeted next generation sequencing data in lung adenocarcinoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4335.

Contains fulltext : 155199.pdf (Publisher’s version ) (Open Access) Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric leukemias. In this study, we determined CREB phosphorylation and mRNA levels showing that CREB expression was significantly higher in ALL compared to normal bone marrow (phosphorylation: P < 0.0001, mRNA: P = 0.004). High CREB and phospho-CREB expression was correlated with a lower median overall survival in a cohort of 140 adult ALL patients. ShRNA mediated knockdown of CREB in ALL cell lines blocked leukemic cell growth by inducing cell cycle arrest and apoptosis. Gene expression array analysis showed downregulation of CREB target genes regulating cell proliferation and glucose metabolism and upregulation of apoptosis inducing genes. Similar to CREB knockdown, the CREB inhibitor KG-501 decreased leukemic cell viability and induced apoptosis in ALL cell lines, as well as primary T-ALL samples, with cases showing high phospho-CREB levels being more sensitive than those with lower phospho-CREB levels. Together, these in vitro findings support an important role for CREB in the survival of ALL cells and identify this transcription factor as a potential target for treatment.

Item does not contain fulltext Still about 20% of patients with acute lymphoblastic leukemia (ALL) struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to give more insights in active signal transduction pathways and point out interesting signaling hubs as well as new potential druggable targets. With this technique the gap between newly designed drugs and ALL may be bridged. The aim of this study was to perform kinome profiling on 20 pediatric ALL samples (14 BCP-ALL and six T-ALL) to identify signaling proteins relevant to ALL. We defined 250 peptides commonly activated in both BCP-ALL and T-ALL representing major signal transduction pathways including MAPK, PI3K/Akt, and regulators of the cell cycle/p53 pathway. For 27 peptides, differentially phosphorylation between BCP-ALL and T-ALL was observed. Among these, ten peptides were more highly phosphorylated in BCP-ALL while 17 peptides showed increased phosphorylation in T-ALL. Furthermore we selected one lead of the list of commonly activated peptides (HGFR_Y1235) in order to test its efficacy as a potential target and provide proof of principle for this approach. In conclusion kinome profiling is an elegant approach to study active signaling and identify interesting potential druggable targets.

Although most children with acute myeloid leukemia (AML) achieve complete remission, the relapse rate is 30% to 40%. Because it is thought that leukemia-initiating cells (LIC) are responsible for AML relapses, targeting these cells might improve outcome. Treatment of pediatric AML blasts with the receptor tyrosine kinase (RTK) inhibitor PTK787/ZK 222584 (PTK/ZK) induces cell death in vitro. However, the role of PTK/ZK inhibition on outgrowth of (pediatric) LICs is unknown. In this study, we cultured CD34+ cells from pediatric patients with AML on MS5 stromal cells in long-term cocultures. In analogy to adult AML, long-term expansion of leukemic cells up to 10 weeks could be generated in 9 of 13 pediatric AMLs. Addition of PTK/ZK to long-term cocultures significantly inhibited leukemic expansion in all samples, ranging from 4% to 80% growth inhibition at week 5 compared with untreated samples. In 75% of the samples, the inhibitory effect was more pronounced at week 10. Proteome profiler array analysis of downstream kinases revealed that PTK/ZK reduced activation of PI3K/Akt kinase signaling. Although main targets of PTK/ZK are VEGF receptors (VEGFR), no effect was seen on outgrowth of LICs when cultured with bevacizumab (monoclonal VEGFA-antibody), specific antibodies against VEGFR2 or VEGFR3, or exposed to stroma-derived VEGFA. These data suggest that the effect of PTK/ZK on LICs is not only dependent on inhibition of VEGFA/VEGFR signaling. Taken together, our data elucidated antileukemic properties of PTK/ZK in long-term expansion cultures, and suggest that targeting multiple RTKs by PTK/ZK might be a potential effective approach in eradicating (pediatric) LICs. Mol Cancer Res; 11(4); 339–48. ©2013 AACR.

The epidermal growth factor receptor (EGFR) inhibitor erlotinib has been shown to induce complete remission of acute myeloid leukemia (AML) in two patients with concurrent lung cancer and raised attention for a role of EGFR in AML whereas a recent phase II clinical study with gefitinib in AML demonstrated a negative result on the outcome. However, from several studies, EGFR expression in AML is poorly defined and the role of EGFR in AML remains unclear. Herein, we report the results of EGFR expression in AML of large cohorts of adult and pediatric AML patients with the data of total protein and phosphorylation levels of EGFR. Our data conclude that there is the expression of EGFR at the protein level in a subset of AML, which was identified to be functionally active in ~15 % of AML patients. This suggests that future studies need to be conducted with a subset of AML patients characterized by high EGFR expression. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0294-x) contains supplementary material, which is available to authorized users.

This review is designed to provide an overview of the current literature concerning vascular endothelial growth factor signaling (VEGF) in acute myeloid leukemia (AML). Aberrant VEGF signaling operates in the bone marrow of AML patients and is related to a poor prognosis. The altered signaling pathway demonstrated to interfere in several autocrine and paracrine signaling pathways. VEGF signaling promotes autocrine AML blast cell proliferation, survival, and chemotherapy resistance. In addition, VEGF signaling can mediate paracrine vascular endothelial cell-controlled angiogenesis in AML. Both effects presumably explain the association of high VEGF levels and poor therapeutic outcome. More recently, researches focusing on bone marrow stem cell niches demonstrate a role for VEGF signaling in the preservation of several cell types within these niches. The bone marrow niches are proposed to be a protective microenvironment for AML cells that could be responsible for relapses in AML patients. This implies the need of sophisticated VEGF-targeted therapeutics in AML therapy strategies. This review highlights our current understanding of aberrant VEGF signaling in AML, appoints the interference of VEGF signaling in the AML-associated microenvironment, and reflects the novelty of current VEGF-targeted therapeutics used in clinical trails for the treatment of AML.

BACKGROUND: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment.METHODS: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n = 7) or VEGFA165 cells (n = 7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays.RESULTS: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P = 0.001), combined with increased angiogenesis (P = 0.002) and enhanced tumour cell proliferation (P = 0.001). Gene expression profiling revealed human genes involved in TGF-beta signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression.CONCLUSION: These results suggest a role for VEGFA-driven tumour growth by TGF-beta signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype. British Journal of Cancer (2011) 105, 1856-1863. doi: 10.1038/bjc.2011.460 www.bjcancer.com Published online 1 November 2011 (C) 2011 Cancer Research UK

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been implicated in tumor progression, making MSCs important targets for anti-cancer strategies. In this study, we show that MSCs promote tumor growth in vivo in a lymphoma xenograft model. We show that MSCs provide direct cell-cell contact interactions and, to a lesser extend, soluble factors that promote tumor cell proliferation and survival in vitro. PTK787/ZK 222584 reduces tumor growth-promoting effects of MSCs both in vitro and in vivo. Our results address the importance of targeting the MSCs for future anti-cancer strategies.

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinase-mediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies. [Cancer Res 2009;69(14):5987–95]

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been shown to favor tumor growth, suggesting the relevance of pharmaceutical inhibition of MSCs for the treatment of malignancies. We tested the effect of PTK787/ZK 222584 (PTK) on the outgrowth of MSCs from human bone marrow-derived mononuclear cells (MNCs) and the migration and tube formation capacity of MSCs in vitro. PTK dose-dependently inhibited the outgrowth of BM-MSCs from BM-MNCs (LC50 1.12 microM PTK), while hematopoietic colony formation (HCF) was only slightly hampered (13 +/- 19% at 1 microM PTK, and stable at approximately 50% at higher concentrations of PTK). Addition of 10 microM PTK inhibited proliferation of MSCs by 74 +/- 6.6% compared to control (p < 0.0001) and increased apoptosis of MSCs by 63 +/- 7.7% (p < 0.01). In addition, upon addition of PTK, BM-MSCs showed impaired tube formation as well as reduced migration (52 +/- 19%, p = 0.006) compared to control. Pepchip array analysis revealed that PTK effectively inhibits activity of kinases involved in cell cycling (WEE1 and several cyclin dependent kinases), and migratory processes (including Rho kinase). In conclusion, we show that PTK impairs outgrowth, proliferation, migration and tube formation of human BM-MSCs. In addition, we show the usability of Pepchip array analysis as a powerful tool for kinase activity profiling in functional studies since the effect of PTK on the kinome profile of MSCs corresponds with the observed functional effects of PTK on proliferation and migration. Inhibition of BM-MSCs and their contribution to tumor growth may be an additional strategy for treatment of cancer in the future.

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express the vascular endothelial growth factors, VEGFA and VEGFC, as well as kinase insert domain-containing receptor (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. This study investigates the role of the VEGFR inhibitor PTK787/ZK 222584 in leukemic cell death, and the possibility of an additional effect on cell death by a chemotherapeutic drug, amsacrine. In three AML cell lines and 33 pediatric AML patient samples, we performed total cell-kill assays to determine the percentages of cell death achieved by PTK787/ZK 222584 and/or amsacrine. Both drugs induced AML cell death. Using a response surface analysis, we could show that, in cell lines as well as in primary AML blasts, an equal magnitude of leukemic cell death could be obtained when lower doses of the more toxic amsacrine were combined with low dosages of the less toxic VEGFR inhibitor. This study shows that PTK787/ ZK 222584 might have more clinical potential in AML when combined with a chemotherapeutic drug such as amsacrine. In future, it will be interesting to study whether the complications and the long-term effects of chemotherapy can be reduced by lowering the dosages of amsacrine, and by replacing it with other drugs with lower toxicity profiles, such as PTK787/ZK 222584.

The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping PI artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity. (c) 2006 Wiley-Liss, Inc.

Introduction Gastrointestinal stromal tumors (GISTs) are rare malignancies of the gastrointestinal tract. In the Netherlands, most patients with GIST are treated in five sarcoma expertise centers. GIST is known to have driver mutations in the tyrosine kinase receptors KIT and PDGFRα in respectively 80% and 10% of the patients. Mutations (single nucleotide variations, insertions and deletions) are mostly found in KIT exon 9 and 11. GIST patients with a primary KIT exon 11 mutation respond generally well to tyrosine kinase inhibitors (TKIs). The aim of this study was to evaluate treatment response by the detection of KIT exon 11 mutations in cell free DNA (cfDNA) of patients with GIST at multiple time points. Materials and methods Since Dec 2014, plasma samples of patients have been collected at every hospital visit and were put in a bio-databank for analysis of mutation detection in cfDNA. Eligibility criteria were histological diagnosis of GIST and treatment with a TKI. Patients with a known KIT exon 11 mutation, of which a baseline (before start of any TKI) sample or a recurrence/progression sample was available, were analyzed. A custom single-tube digital droplet PCR (ddPCR) drop-off assay, designed at the UMCG, was used. This assay detects 80% of the known KIT exon 11 mutations. Results Until Nov 2016, >620 samples of 175 patients have been collected. From 43 patients with a KIT exon 11 mutations in the pretreatment tumor biopsy, a baseline/progression sample was available. Mutations in exon 11 were detected in cfDNA from baseline plasma of 22/43 patients, 20 with metastasized and 2 with localized disease. Of the 21 patients without detectable mutations, 11 had localized and 10 metastatic disease. Three of these patients with metastatic disease had mutations that could not be detected by our probe. Two weeks after start of TKI, an increase in mutant allelic frequency was seen in 5 (of 9 available 2 week samples) patients, while 4 weeks later the mutant allelic frequency had decreased, in parallel with decrease in tumor load on radiological imaging. Eight patients developed radiological disease recurrence (2 patients, after 4 and 20 months) or progression (6 patients, median 10 (6-24) months). This was paralleled by an increase in mutant allelic frequency in cfDNA in 6 of them (baseline median 0% (0-0.9%); at progression median 3.5% (0-30%); p= 0.028; related samples Wilcoxon signed rank test). Summary and conclusion KIT exon 11 mutations in cfDNA could be detected in 74% of the patients with metastatic disease. Disease progression or recurrence, coincides with a rise of mutant alleles detected in the plasma. In conclusion, mutation detection in cfDNA of GIST patients with metastatic disease is feasible. Our study will be extended to include the monitoring of early progression based on cfDNA, which may guide early treatment adaptations. Citation Format: Pieter A. Boonstra, Arja ter Elst, Marco Tibbesma, Ron H. Mathijssen, Florence Atrafi, Frits van Coevorden, Sheima Farag, Neeltje Steeghs, Ingrid M. Desar, Winette T. van der Graaf, Hans Gelderblom, Boudewijn van Etten, Jelle Overbosch, Albert J. Suurmeijer, Jourik A. Gietema, Ed Schuuring, Anna K. Reyners. Dynamics of KIT exon 11 mutations in cell free plasma DNA of patients treated for advanced gastrointestinal stromal tumors: Results from the Dutch GIST bio-databank [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 4951. doi:10.1158/1538-7445.AM2017-4951

Introduction Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size. The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring. Materials and methods The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation (“drop-off”). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment. Results Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected. CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%). Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy. Conclusion The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914). Citation Format: Pieter A. Boonstra, Marco Tibbesma, Lisette J. Bosman, Ron H.J. Mathijssen, Frits van Coevorden, Neeltje Steeghs, Albert J.H. Suurmeijer, Arja ter Elst, Jourik A. Gietema, Anna K.L. Reyners, Ed M.D. Schuuring, Dutch GIST Consortium. A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors. [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 3107.

VEGFA is considered one of the most important regulators of tumor-associated angiogenesis in cancer. In acute myeloid leukemia (AML) VEGFA is an independent prognostic factor for reduced overall and relapse-free survival. Transcriptional activation of the VEGFA promoter, a core mechanism for VEGFA regulation, has not been fully elucidated. We found a significant (P < 0.0001) inverse correlation between expression of VEGFA and AML1/RUNX1 in a large set of gene expression array data. Strikingly, highest VEGFA levels were demonstrated in AML blasts containing a t(8;21) translocation, which involves the AML1/RUNX1 protein (AML1/ETO). Overexpression of AML1/RUNX1 led to downregulation of VEGFA expression, whereas blocking of AML1/RUNX1 with siRNAs resulted in increased VEGFA expression. Cotransfection of AML1/RUNX1 and VEGFA promoter luciferase promoter constructs resulted in a decrease in VEGFA promoter activity. ChIP analysis shows a direct binding of AML1/RUNX1 to the promoter of VEGFA on three AML1/RUNX1 binding sites. Silencing of AML1/ETO caused a decrease in VEGFA mRNA expression and a decrease in secreted VEGFA protein levels in AML1/ETO-positive Kasumi-1 cells. Taken together, these data pinpoint to a model whereby in normal cells AML1/RUNX1 acts as a repressor for VEGFA, while in AML cells VEGFA expression is upregulated due to AML1/RUNX1 aberrations, for example, AML1/ETO. In conclusion, these observations give insight in the regulation of VEGFA at the mRNA level in AML. Cancer Res; 71(7); 2761–71. ©2011 AACR.

Item does not contain fulltext To date, the biology of acute leukemia has been unclear, and defining new therapeutic targets without prior knowledge remains complicated. The use of high-throughput techniques would enable us to learn more about the biology of the disease, and make it possible to directly assess a broader range of therapeutic targets. In this study we have identified comprehensive tyrosine kinase activity profiles in leukemia samples using the PamChip(R) kinase activity profiling system. Strikingly, 31% (44/120) of the detected peptides were active in all three groups of leukemia samples. The recently reported activity of platelet-derived growth factor receptor (PDGFR) and neurotrophic tyrosine kinase receptors (NTRK1 and NTRK2) in leukemia could be appreciated in our array results. In addition, high levels of peptide phosphorylation were demonstrated for peptides related to macrophage stimulating 1 receptor (MST1R). A provisional signal transduction scheme of the common active peptides was constructed and used to specifically select an inhibitor for leukemic blast cell survival assays. As expected, a dose-dependent decrease in leukemic blast cell survival was achieved for all leukemia samples. Our data demonstrate that kinase activity profiling in leukemic samples is feasible and provides novel insights into the pathogenesis of leukemia. This approach can be used for the rapid discovery of potential drug targets.

Increased levels of circulating VEGF-A have been demonstrated in patients with non-Hodgkin lymphoma (NHL) and are associated with progressive disease and poor clinical outcome. We investigated the role of VEGF-A in lymphoma tumour growth on a molecular level in order to identify the mechanism of VEGF-A-promoted tumour growth and to identify the potential targets for therapy. We used a model in which Daudi (human Burkitt lymphoma) tumour cells were transduced with VEGF-A165 or an empty vector (negative control) and subcutaneously injected in NOD/SCID mice. The weight of tumours over-expressing VEGF-A was increased 4-fold compared to that of control tumours (p

It has been hypothesized that the net expression of a gene is determined by the combined effects of various transcriptional system regulators (TSRs). However, characterizing the complexity of regulation of the transcriptome is a major challenge. Principal component analysis on 17,550 heterogeneous human microarray experiments revealed that 50 orthogonal factors (hereafter called TSRs) are able to capture 64% of the variability in expression in a wide range of experimental conditions and tissues. We identified gene clusters controlled in the same direction and show that gene expression can be conceptualized as a process influenced by a fairly limited set of TSRs. Furthermore, TSRs can be linked to biological functions, as we demonstrate a strong relation between TSR-related gene clusters and biological functionality as well as cellular localization, i.e. gene products of similarly regulated genes by a specific TSR are located in identical parts of a cell. Using 3,934 diverse mouse microarray experiments we found striking similarities in transcriptional system regulation between human and mouse. Our results give biological insights into regulation of the cellular transcriptome and provide a tool to characterize expression profiles with highly reliable TSRs instead of thousands of individual genes, leading to a >500-fold reduction of complexity with just 50 TSRs. This might open new avenues for those performing gene expression profiling studies.

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e. g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e. g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data.

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTTI (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTTI, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTTI in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping PI phage clones. Fiber-FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley-Liss, Inc.(dagger).

Acute myeloid leukemia (AML) remains a life threatening malignancy in children. Considerable progress has been made in elucidating the new diagnostic and prognostic markers over the past decades. The precise etiology remains unclear. Therefore, it is essential to evaluate the activation of the components of cellular signaling pathways to understand AML signaling and to design the most successful approach for combinational therapies and new kinase inhibitors. In this study, we used a high-throughput PepChipTMKinomics microarray system containing 976 different kinase substrates and assayed primary leukemic samples of 96 AML patients to produce an exceptionally detailed map of kinome enzymatic activities towards predefined peptide substrates. The generated profiles provide a comprehensive insight in signaling pathways active in AML patients. As expected the activation of proteins belonging to MAPK signaling, PI3K/AKT signaling, cell cycle regulation, apoptosis and insulin signaling pathways along with the signaling receptors and immune system regulators were found. Unsupervised hierarchical cluster analysis separates the AML blast profiles based on 192 peptide activities into two clusters. Cumulative incidence of relapse (CIR) was significantly higher in the patients of cluster-2. Peptide activity patterns were independent of patient characteristics. In addition, with Gaussian network modeling, a total of 540 peptides (55%) showed at least one peptide-peptide association without a prior assumptions whereas 74 peptides (7.5%) had >39 nodes suggesting to be potential interesting signaling hubs. Among these 74 peptides, 10 peptides were identified in cluster-1 and 50 peptides were in cluster-2. Thus, this total analysis defined peptides correlated to low incidence for relapse, for examples AKT1, HGFR, RGS7 and to high incidence for relapse for instance, proteins involve in MAPK pathways (RAF1, RAC1,14-3-3 eta) and cell cycle regulation and cellular growth (c-Myc, FOXO3A, RBL1). In conclusion, our study demonstrates the feasibility of peptide activity profiling to identify two active signaling network clusters in pediatric AML correlated to CIR. Highly correlated peptides belonging to cluster-2 provide stronger leads for selection of novel targets in future therapeutics. Disclosures No relevant conflicts of interest to declare.

In searching for a tumor suppressor gene in the 3p21.3 region, we isolated two genes, RBM5 and RBM6. Sequence analysis indicated that these genes share similarity. RBM5 and-to a lesser extent-RBM6 also have similarity to DXS8237E at Xp11.3-11.23, which maps less than 20 kb upstream of UBE1. A homologue of UBE1, UBE1L, is located at 3p21.3. FISH analysis showed that the distance between UBE1L and RBM5 in 3p21.3 is about 265 kb. DXS8237E and UBE1 on the X chromosome have the same orientation, whereas on chromosome 3 the orientation of RBM5 and that of RBM6 are opposite to the orientation of UBE1L. Presumably, part of the Xp11.3-11.23 region has duplicated to chromosome 3. Part of this region on chromosome 3 may subsequently have duplicated again within the same chromosomal region, Inversion at some stage of the evolution of the human genome would explain the change in orientation of the genes on chromosome 3 compared with that of the genes on the X chromosome. (C) 1999 Academic Press.

In the search for a tumour suppressor gene in the 3P21.3 region we isolated two genes, RBM5 and RBM6. Gene RBM5 maps to the region which is homozygously deleted in the small cell lung cancer cell line GLC20; RBM6 crosses the telomeric breakpoint of this deletion, Sequence comparison revealed that at the amino acid level both genes show 30% identity, They contain two zinc finger motifs, a bipartite nuclear signal and two RNA binding motifs, suggesting that the proteins for which RBM5 and RBM6 are coding have a DNA/RNA binding function and are located in the nucleus. Northern and Southern analysis did not reveal any abnormalities. By SSCP analysis of 16 lung cancer cell lines we found only in RBM5 a single presumably neutral mutation, By RT-PCR we demonstrated the existence of two alternative splice variants of RBM6, one including and one excluding exon 5, in both normal lung tissue and lung cancer cell lines, Exclusion of exon 5 results in a frameshift which would cause a truncated protein of 520 amino acids instead of 1123 amino acids. In normal lung tissue, the relative amount of the shorter transcript was much greater than that in the lung tumour cell lines, which raises the question whether some tumour suppressor function may be attributed to the derived shorter protein.

Two experiments were conducted to investigate the effects of steam processing on the nutritional improvement of full-fat soya beans. In a kinetic study, soya beans were toasted at 102, 120 and 134°C for various durations (19 temperature/time combinations [ T t ]). The raw and processed soya beans were measured for their functional lectins, trypsin inhibitor activity (TIA), urease activity (UA) and protein dispersibility index (PDI). The raw soya beans contained 7638 μg g−1 functional lectins, 23.44 mg g−1 TIA, 2.22 UA (ΔpH) and 85.7% PDI. These criteria all decreased as the heating time increased. Of all criteria measured, the level of functional lectins was found to be most effectively inactivated by high temperature. In the second experiment, 35 piglets of approximately 30 kg liveweight were used. The animals were fitted with a post-valvular T-caecum (PVTC) cannula and divided into seven groups, which were fed a control diet and six experimental diets. The diets consisted of 80% control diet and 20% processed soya beans which were treated under the following conditions: 102°C for 10 min, 102°C for 20 min, 102°C for 40 min, 120°C for 2 min, 120°C for 7.5 min and 134°C for 1.5 min. The apparent ileal digestibilities of the soya beans processed in these conditions [ T t ] were: 37.0, 49.1, 61.3, 48.2, 57.0 and 56.8%, respectively, for dry matter (DM); 51.1, 68.3, 81.2, 70.3, 82.0 and 82.2%, respectively, for nitrogen (N); 82.2, 84.1, 89.2, 89.6, 93.0 and 91.8%, respectively, for ether extract. The apparent digestibilities of DM and N increased as the level of antinutritional factors decreased. It appears that a certain amount of energy is required to achieve the inactivation of antinutritional factors, either by treatment at a high temperature for a short time (e.g. 134°C for 1.5 min), or for a longer time at a lower temperature (e.g. 102°C for 40 min). It was noticed that the residual TIA content of the processed soya beans was correlated to the variation in digestibility among the animals, which indicates that there are considerable differences between individual animals in their responses to the soya bean antinutritional factors.

Abstract 4719 Ephrin signaling has been shown to contribute to the pathogenesis of many solid tumors with respect to tumor growth, tumor cell survival, angiogenesis, and metastasizing capacity (Cytokine Growth Factor Rev. Dec;15(6):419-33, et al. Neuro Oncol. 2012). Recently, an aberrant DNA methylation status of ephrin receptors and ligands was described to be associated with outcome in acute lymphoblastic leukemia (Blood.2010 Mar 25;115(12):2412-9). In acute myeloid leukemia (AML), we found an intriguing heterogeneity in membrane receptor expression levels of EphB1. Therefore, we challenged to evaluate the role of EphB1 receptor forward signaling in AML. We investigated the influence of the EphB1 receptor forward signaling in THP-1 (EphB1high), HL60 (EphB1int), and MOLM13 (EphB1low) AML cell lines through exogenous stimulation with the EphB1 ligand; EfnB1. EfnB1 stimulation of the AML cell lines demonstrated to reduce AML growth solely in the EphB1high and EphB1int cell lines (Fig. 1A,P = 0.001 and P =.056). In addition, EfnB1 stimulation induced apoptosis most pronounced in the EphB1high cells (Fig. 1B). Interestingly, MGG stained cytospins of EfnB1 treated THP-1 showed multinucleation of AML cells (Fig. 1C). We hypothesized that these phenotypic effects could be assigned to cell cycle arrest in THP-1 cells. Additionally, cell cycle regulatory proteins CDC2 and CyclinB1 were evaluated by immunoblotting of EfnB1 stimulated THP-1 cells. Phosphorylation of the inactivating CDC2 Tyr15-site demonstrated to be up-regulated in EfnB1 stimulated THP-1 cells, which might be initiated by the increased total CDC2 protein levels that we found (Fig. 1D). CyclinB1 displays enhanced protein expression in EfnB1 treated THP-1 cells. Moreover, quantitative RT-PCR analysis showed that the expression of cell cycle inhibitor p21 is significantly induced by 3-fold in EfnB1 stimulated THP-1 cells, via increasing levels of p53 (Fig. 1E, both P = To verify whether the EfnB1 induced cell cycle arrest is EphB1 specific, we enforced EphB1 expression in HL60 EphB1int and MOLM13 EphB1low AML cells by introducing a GFP fused EphB1 overexpression construct. EphB1 overexpression increased EphB1 protein expression levels sufficiently in both AML cell lines, as confirmed by flowcytometric analysis and immunoblots. Exogenous EfnB1 stimulation further increased the apoptosis in EphB1 overexpressing cells in both AML cell lines (Fig. 1F). Again, we found increasing levels of phospho-CDC2Tyr15 and CyclinB1 by immunoblots. From this study, we conclude that AML cells with high EphB1 expression can be forced into a cell cycle arrest upon ligand binding in vitro, while AML cells lacking EphB1 expression have a proliferative and anti-apoptotic survival advantage. The clinical significance and exploitation of EphB1 induced cell cycle arrest in AML will be analyzed in the near future. Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis (A) Absolute cell counts represent the growth inhibitory effects of EfnB1 ligand stimulation in AML cell lines THP-1 and HL60. (B) Flowcytometric Annexin V/PI apoptosis assay displayed the induction of apoptosis as a result of EfnB1 ligand stimulation in THP-1 and MOLM-13 AML cells. (C) MGG stained cytospins of EfnB1 treated THP-1 cells promotes the induction of multinucleated cells due to cell cycle arrest. (D) Immunoblots showed an enhanced apoptotic BAX/BCL2 ratio, in synergy with an upregulation of cell cycle inactivating checkpoint kinase CDC2Tyr15 upon EfnB1 stimulation in THP-1. (E) qRT-PCR confirms cell cycle inhibition by a 3-fold upregulation of p21 and a 1.5-fold induced expression of p53 in THP-1 EfnB1 stimulated cells. (F) The flowcytometric Annexin V/PI apoptosis assay showed that EfnB1 ligand induced apoptosis is even further induced in EphB1 overexpressing HL60 and MOLM-13 cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2338 Cancer arises when somatic cells are able to escape the restraints that normally withhold them from unlimited expansion. Cancer progression is thought to be the net result of signaling through various protein-kinase mediated networks driving cell proliferation and survival. The kinome networks can be affected by numerous factors; including acquired or selected mutations as well as environmental cross talk. Additionally, the loss of phosphatases could be a causative factor for activation of multiple tyrosine kinases as well (Cell. 2011 Mar 4;144(5):703–18). Deregulated kinase activity is frequently observed in leukemia, leading to induced proliferation, migration, survival, and chemotherapy resistance of leukemic cells. (Leuk Lymphoma. 2011 Jan;52(1):122–30, Leukemia. 2005 Apr;19(4):586–94, Exp Hematol. 2005 Jun;33(6):660–70) However, single kinase-targeted cancer therapies can default when cancer cells bypass through alternative routes, facilitating therapeutic resistance. In order to circumvent the constraints given by an inihibitor, we need to monitor kinome reprogramming upon mono-treatments to develop the most successful combination therapy approach for disease specific subgroups, as poor prognostic MLL-rearranged AML. Rational designs of kinase inhibitor or RTK antibody combinations require a high-throughput measurement of kinome and proteome activity signatures within this patient subgroup. Figure 1 outlines our approach to explore the intracellular signaling networks and study the dynamic changes resulting in reprogramming of the kinome network, with the goal to define combinational therapeutic strategies. In this study, we succeeded using combined high-throughput approaches for kinomic and proteomic profiling to identify specific aberrant kinase signatures in MLL-rearranged AML as compared to NBM (Fig. 1B/C). The altered activated kinase signatures of a comprehensive set of MLL-rearranged AML patient samples resulted in a detailed map of the overall kinase activity and phosphorylation of signal transduction molecules, which allowed the selection of possible druggable targets i.e. MEK, JNK, and CREB (Fig. 1B/C). Pharmacological MEK inhibition in primary MLL-rearranged AML demonstrated to be most successful in reducing the AML cell survival, without showing cytotoxicity in NBM (mean MEK inhibitor IC50 of 3.5μM +/- 0.7μM in primary MLL-rearranged AML versus a mean IC50 >50μM in NBM), whereas for CREB and JNK inhibitors MLL-rearranged AML cells were equally affected as NBM cells. Dynamic kinome reprogramming of signaling networks in response to MEK therapy did occur, by inducing the activation of RTKs to bypass the initial MEK inhibitory effects in a MLL-rearranged AML cell line. The dynamic escape mechanism allowed us to predict and test the efficacy of novel combination strategies. Combined MEK and VEGFR-2 inhibition demonstrated to induce cell death sufficiently in MLL-rearranged AML (Fig. 1D). This advantageous strategy allows rational design of successful and selective combination therapies for specific target inhibitors. Figure 1. Kinomics, proteomics, and signaling dynamics for novel combination therapies in MLL-rearranged AML (A) Overall study design for novel combination therapies (B) Constructed kinome trees showing the overall kinase derived peptide activity in 15 individual primary MLL-rearranged AML samples versus 5 NBM samples to identify the distribution and the overlap of kinase activity. Combined kinome trees revealed induced JNK annotated peptide activity in MLL-rearranged AML. (C) Representative protein kinase phosphorylation arrays of primary MLL-rearranged AML (n=9 in total) versus NBM (n=4 in total). Phosphoproteomic analysis identified induced phosphorylation levels of MEK and CREB protein kinases in MLL-rearranged AML. (D) Evaluation of AML cell survival of an anticipated successful combined therapeutic strategy in MLL-rearranged AML, showing the synergistic beneficial effect of combined MEK and VEGFR-2 inhibition in MLL-rearranged AML cell line THP-1. Figure 1. Kinomics, proteomics, and signaling dynamics for novel combination therapies in MLL-rearranged AML . / (A) Overall study design for novel combination therapies (B) Constructed kinome trees showing the overall kinase derived peptide activity in 15 individual primary MLL-rearranged AML samples versus 5 NBM samples to identify the distribution and the overlap of kinase activity. Combined kinome trees revealed induced JNK annotated peptide activity in MLL-rearranged AML. (C) Representative protein kinase phosphorylation arrays of primary MLL-rearranged AML (n=9 in total) versus NBM (n=4 in total). Phosphoproteomic analysis identified induced phosphorylation levels of MEK and CREB protein kinases in MLL-rearranged AML. (D) Evaluation of AML cell survival of an anticipated successful combined therapeutic strategy in MLL-rearranged AML, showing the synergistic beneficial effect of combined MEK and VEGFR-2 inhibition in MLL-rearranged AML cell line THP-1. Disclosures: No relevant conflicts of interest to declare.

Abstract 1425 Previously, it was demonstrated that exogenous addition of vascular endothelial growth factor C (VEGFC) increased the leukemic cell viability, reduced apoptosis via activation of Bcl-2, and decreased chemotherapy induced apoptosis via its receptor FLT-4 (Further revert to as VEGFR3) (Dias et al. Blood 2002). Furthermore, it was shown that VEGFC promotes angiogenesis by induction of COX-2 through VEGFR3 activation in THP-1 cells (Chien et al. Carcinogenesis 2005). We have previously found that endogenous VEGFC expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia (AML), both in vitro as well as in vivo (de Jonge et al. Clinical Cancer Research 2008). In addition, high VEGFC mRNA expression is strongly associated with reduced complete remission and overall survival in adult as well as pediatric AML (de Jonge et al. Blood 2010). It was thought that the leukemic blast population is organized as a hierarchy, whereby leukemia initiating cells (LICs) reside at the top of this hierarchy, and it is only these cells that have the capacity to engraft in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The LIC is thought to be enriched in the CD34+ leukemic cell fraction and is shown to expand in vitro using a myeloid cytokine mix of IL-3, TPO, and G-CSF in colony forming cell (CFC) assays and long-term culture-initiating cell (LTC-IC) assays (Guan et al. Exp. Hematol. 2002, van Gosliga et al. Exp. Hematol. 2007). Moreover, LTC-IC assays performed in limiting dilution detect the in vitro outgrowth potential of stem-like cells that reside underneath the stromal cell layer. In this study, we set out to investigate the potential of anti-VEGFC treatment as an inhibitor of the outgrowth of LICs within the CD34+ fraction of primary AML samples. First, we determined the possibility of an autocrine loop for VEGFC in AML. Pediatric AML cell (n=7) derived VEGFC levels were found to be 1.4-fold increased (P =.008) compared to secreted VEGFC levels from normal bone marrow (NBM) cells (n=4). Pediatric AML blast cells showed KDR (further revert to as VEGFR2) membrane expression in 44 out of 50 patient samples (varying 8–99% of the total blast population), whereas on NBM cells VEGFR2 expression was below 5%. VEGFR3 expression was below 5% on both leukemic blasts and NBM cells. We evaluated the effect of anti-VEGFC (VGX-100, kindly provided by Vegenics, used at a concentration of 30 μg/ml) treatment on the CD34+ isolated compartment of pediatric AML bone marrow samples. Anti-VEGFC treatment reduced the outgrowth potential of AML derived CD34+ cells (n=2) with >25% in CFC assays. Interestingly, morphological analysis revealed a 3-fold enhanced formation of macrophages. LTC-IC assays demonstrated a (15% to 50%) decrease in the long-term growth of CD34+ isolated AML cells in 3 out of 4 patient samples. Morphological characterization of the suspension cells suggested a shift in development along the myelomonocytic lineage after two weeks of anti-VEGFC treatment. With FACS analysis, these cells showed a higher number of cells stained positive for CD11b, and CD14, and lower numbers where positive for CD34. Anti-VEGFC treated LTC-IC assays in limiting dilution demonstrated a (44% and 74%) reduction in the outgrowth potential of long-term cultured CD34+ isolated AML cells and blocked the erythroid colony formation in 2 out of 3 patient samples. Anti-VEGFC treatment did not have an effect on the outgrowth of CD34+ sorted NBM cells in the various assays (n=2). In conclusion, anti-VEGFC treatment of the CD34+ isolated fraction from primary pediatric AML samples showed a reduction of AML outgrowth. Differentiating cells are skewed to the myelomonocytic lineage upon anti-VEGFC treatment. We hypothesize that deprivation of VEGFC in primary CD34+ AML cell cultures results in enhanced leukemic cell death and abates an important proliferation signal for AML cells. Yet, further investigations are warranted.Figure 1.Skewing of LTC-IC assay suspension cells towards the myelomonocytic lineage upon anti-VEGFC treatment. MGG stained cytospins of suspension cells of the LTC-IC co-culture obtained during demi-depopulation at week 2.Figure 1. Skewing of LTC-IC assay suspension cells towards the myelomonocytic lineage upon anti-VEGFC treatment. MGG stained cytospins of suspension cells of the LTC-IC co-culture obtained during demi-depopulation at week 2. Disclosures: Baldwin: Circadian Technologies Limited: Employment. Robert:Circadian Technologies Limited: Employment, Membership on an entity's Board of Directors or advisory committees.

Abstract 3626 One of the major pathways involved in the angiogenic process is the VEGF/VEGFR signaling pathway. VEGFA was found to be an independent prognostic factor for therapeutic outcome in AML (de Bont et al, 2002;Aguayo et al, 1999). Moreover, it has been shown that after chemotherapy the expression level of VEGFR2 is restored to normal in the bone marrow of AML patients in complete remission (Padro et al, 2002). Both VEGF and its receptors Flt-1(VEGFR1) and KDR (VEGFR2) are expressed by leukemic blasts which results in both a paracrine and an autocrine VEGF/VEGFR2 pathway in AML (Dias et al, 2001). In response to VEGF stimulation, VEGFR2 has been shown to transmit intracellular signals leading to activation of multiple downstream signaling pathways including the mitogen-activated protein (MAP) kinases, JAK)STAT, and Phosphoinositide 3-kinases (PI3 kinases). In AML it has been described that simultaneous activation of these pathways results in a poor prognosis (Kornblau et al, 2006). Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene at locus 11q23 are associated with a poor outcome. These MLL translocations represent 15–20% of pediatric acute myeloid leukemia's. In this study we show that VEGFR2 expression is significantly higher on blasts of 11q23 translocated acute myeloid leukemia's compared to blasts of AML patients with another karyotype. In addition, using peptide arrays and proteome profiler arrays we have found that the VEGF receptor signaling pathway and part of the MAP kinase signaling pathway are active and or phosphorylated in blasts of 11q23 translocated patients (Fig. 1). Proteins from these active pathway are potential targets in the treatment of pediatric 11q23 translocated AML. To further investigate these potential targets we have performed ex vivo drug target assays using an inhibitor and an antibody against two key proteins from the major signal transduction pathways, VEGFR2 antibody (IMC1121b) and MEK inhibitor (U0126). No effect of VEGFR2 inhibition was measured in two AML cell lines (HL-60 and THP-1). Whereas, MEK inhibition had an effect on both cell lines, LC50 8.7 μM and 7.5 μM for HL-60 and THP-1 respectively. Cell survival of the THP-1 cell line (containing a 11q23 translocation) was substantially inhibited after addition of a combination therapy with an inhibitor against MEK and a antibody against VEGFR2 (LC50 2.9 μM). In contrast, the VEGFR2 antibody in combination with the MEK inhibitor had only a marginal effect on the HL-60 cell line (LC50 7.7 μM). Future experiments to investigate the effect of the combination therapy on primary patient material are underway. Overall these data show for the first time that targeting multiple active pathways in pediatric 11q23 AML could result in promising opportunities concerning future therapy options. Figure 1. Provisional scheme of active kinases and phosphorylated proteins in AML. Green-white, active kinase; Yellow, phosphorylated protein; Green-Yellow, active kinase and phosphorylated protein; thick lining, bold receptors and, bold cell cycle proteins, kinase activity higher in AML cells compared to normal bone marrow. Red arrows, site of inhibition. Figure 1. Provisional scheme of active kinases and phosphorylated proteins in AML. Green-white, active kinase; Yellow, phosphorylated protein; Green-Yellow, active kinase and phosphorylated protein; thick lining, bold receptors and, bold cell cycle proteins, kinase activity higher in AML cells compared to normal bone marrow. Red arrows, site of inhibition. Disclosures: No relevant conflicts of interest to declare.

Abstract 3144 It was previously described that Epidermal Growth Factor Receptor (EGFR) inhibitors can induce a clinical response in AML patients (Boehrer et al., 2008; Chan and Pilichowska, 2007; Pitini et al., 2008). In contrast, it was reported that EGFR is not expressed in AML cells both at protein and mRNA level and that the phenotypic effects of the EGFR inhibitors are off-target effects on the spleen tyrosine kinase (SYK) (Lindhagen et al., 2008; Stegmaier et al., 2005; Hahn et al., 2009). We have, however, detected EGFR mRNA expression in gene expression array data of 534 publicly available adult AML patient samples and 132 publicly available pediatric AML patient samples (obtained from the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo, accession number GSE6891 for adult AML patients and GSE22056 for pediatric AML patients) (Fig. A, and B). EGFR kinase activity was measured in four AML samples using the PamChip® tyrosine kinase microarray system and EGFR phosphorylation measured using Phospho-receptor tyrosine kinase antibody microarray (R&D). EGFR kinase activity could be detected in 2 of 4 of the samples (Fig. C) In addition, protein expression of EGFR and phosphorylated (Tyr-992) EGFR was measured using Reverse Phase Protein Array in 511 cases of newly diagnosed AML. Total EGFR levels were equivalent to normal bone marrow CD34+ cells in 72% and above and below normal in 13.5 and 13.7% of AML patient samples respectively (Fig. D). The phosphorylation of the EGF receptor was equivalent to that observed in normal bone marrow CD34+ cells in 80% and above and below normal in 8 and 11.7% of cases respectively (Fig. E). EGFR protein expression was correlated with the level of NF2, CIAP, Survivin, Stat5p694, CyclinD1, Her3, NRP1, PLAC1, CateninA, IGF1, TAZ, SMAD6, YAP, NF2 and PI3Kp110. The level of phospho-EGFR was correlated with the level of expression of P53, RB, pRB, Cyclin B1 and E, BCLXL, BIM, Cleaved caspase 7 and 9, GATA3, Stat1p701 and ODC. Levels of EGFR protein expression and phosphorylation did not correlate with FAB classification, cytogenetics, or the presence of FLT3-ITD or NPM1 or RAS mutation or with age, gender, or the presence of an antecedent hematological disorder. Neither total or phospho-EGFR levels were prognostic for remission attainment, remission duration, overall or event free survival. These results indicate that EGFR is expressed at both the mRNA and the protein level in AML. In addition, EGFR is commonly phosphorylated and active in primary AML patient samples. Activity of EGFR receptor was correlated with higher expression of proliferation regulating genes (Cyclins, P53, RB) and anti-apoptotic proteins suggesting that signaling through the EGFR axis might be affecting proliferation and apoptosis sensitivity in AML blasts. This suggests that modulation of the EGFR axis in different settings could be used to stimulate proliferation and sensitivity to cell cycle specific agents or inhibited to inhibit proliferation or to reduce anti-apoptotic forces. Future experiments to correlate the responsiveness of AML blasts to therapy with EGFR activators/inhibitors with the endogenous activity of the EGFR receptor and to determine the optimal way to target cells through this pathway are underway. Disclosures: No relevant conflicts of interest to declare.

Tyrosine kinases pose attractive targets for cancer therapy, as their abnormal signaling activity results in enhanced proliferation, migration and angiogenesis. Previously we have been able to generate a comprehensive description of the biological kinase activity present in pediatric brain tumor tissue lysate by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates. To establish which peptide substrates present on the array are most sensitive and discriminative for VEGFR activity we measured the peptide phosphorylation applying recombinant VEGFR1, 2 and 3. Furthermore, a panel of recombinant kinases with highly diverse VEGFR sequence homology was included to study substrate specificity. An aminopeptidase A (APA) derived peptide proved to be highly phosphorylated by VEGFR 1 and 2. No phosphorylation of this peptide could be observed applying any of the other kinases, arguing that this peptide is discriminative for VEGFR1 and 2 activity. In the peptide array data of pediatric brain tumors, phosphorylation of the APA peptide was observed, suggestive for VEGFR activation. Based on these results we hypothesize that APA can be activated through direct phosphorylation by VEGFR2 and exert functional effects on endothelial cell proliferation and migration. To test this hypothesis we stimulated human umbilical vein endothelial cells (HUVECs) with VEGF and observed phosphorylation of Aminopeptidase A. Literature states that inhibition of APA suppresses VEGF-induced migration and proliferation of endothelial cells and inhibits angiogenesis. Based on our data we hypothesize that these APA-mediated effects of VEGF stimulation are induced through direct activation by VEGFR2 kinase activity. Concluding, peptide microarray technology is able to provide a selective view on tyrosine kinase activation, as has been shown here for VEGFR1 and 2. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2377.

Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by a multitude of genetic events. Activating mutations in receptor tyrosine kinases have been identified in 50% of AML primary blasts, and deregulation of one or more signal transduction pathways including the JAK/STAT, RAS/Raf/MEK/ERK, and PI3K/AKT pathways is common (Kornblau et al., 2006). High-throughput procedures which generate comprehensive descriptions of cellular signaling without a priori assumptions in each sample, would enable us to directly assess a broader range of targets for future treatment strategies. In the present study we identified kinase activity profiles in 22 pediatric leukemia samples (6 acute myeloid leukemia, 9 acute lymphoid leukemia, 3 Philadelphia positive acute lymphoid leukemia and 5 chronic myeloid leukemia) and in various normal tissues such as colon and kidney. Peptide phosphorylation profiles were determined using the Pamchip® tyrosine kinase micro array system (Pamgene International B.V., ‘s Hertogenbosch, the Netherlands). This array consists of 144 peptides representing key phosphorylation sites of proteins known to be involved in signal transduction processes. We generated a comprehensive description of the phosphotyrosine proteome of all patient samples. Within the variety of profiles in the various leukemia samples, peptide corresponding with phosphorylation consensus sequences for MAP kinases showed remarkable high levels of phosphorylation, whereas in various normal tissue types substantial lower activity was observed on these substrates. These results imply activated MAPK signaling to be a prominent characteristic of leukemia as already described (Towatari et al., 1997). A differential phosphorylation pattern was seen for the RON peptide (macrophage stimulating 1 receptor (c-met-related tyrosine kinase)), only phosphorylated in one of the AML samples, 4 out of 5 CML samples and one Ph+ ALL sample. According to Phospho-ELM and the literature (Follenzi et al., 2000) this tyrosine kinase residue can be phosphorylated via autophosphorylation by RON kinase or transphosphorylation by MET kinase (met proto-oncogene (hepatocyte growth factor receptor)).To verify the role of MET kinase in AML, a cell survival assay was performed with the selective MET kinase inhibitor PHA 665752. Dose dependent decrease in cell survival was observed in three primary AML samples tested with LC50 concentrations ranging from 2 μM to 5 μM. In conclusion, this study describes a new high throughput technique to generate tyrosine phospho-proteomes or even kinome profiles of various leukemic samples in the future. With this technique we found MET to be a new therapeutic target in AML, and it demonstrates the importance of MAP kinase signaling in various leukemic samples. In the era of a rapidly increasing number of small molecule inhibitors this technique will enable us to rapidly identify new potential targets in different kinds of leukemia.

Vascular Endothelial Growth Factor (VEGFA) at time of diagnosis is an independent prognostic factor for poor treatment outcome in (pediatric) acute myeloid leukemia (AML). Inhibition of VEGFA by Bevacizumab, a recombinant humanized monoclonal antibody, preceded by chemotherapy yields a favorable CR rate and duration in adults with refractory AML (Karp et al., 2004). VEGFA is a powerful stimulator of angiogenesis; VEGFA binds to the tyrosine kinase receptors VEGFR1 and VEGFR2 on endothelial cells, resulting in endothelial cell proliferation. Moreover, binding of VEGFA to VEGF receptors on AML cells in vitro promotes leukemic cell survival via activation of signaling pathways such as RAS/Raf/MEK/ERK and PI3K/AKT pathway. In our study a model was generated to investigate the effect of VEGFA in malignant progression in AML. An HL-60 AML cell line was transduced with VEGFA (the splice variant VEGFA165) or a control vector using a retroviral construct. With RT-PCR we found a threefold induction of VEGFA in VEGFA165 transduced cells. No difference in growth rate and drug sensitivity was found between the HL-60 VEGFA165 cells and HL-60 control cells in vitro. Thus, VEGFA165 overexpression did not result in growth benefit in vitro. To evaluate the in vivo effects of VEGFA165 overexpression, we injected 10 × 106 HL-60 VEGFA165 cells or HL-60 control cells s.c. into NOD/SCID mice (n=14). We observed that overexpression of VEGFA165 increased tumor weight (median weight: HL-60 VEGFA165 tumors 995 mg, range 670–1344; HL-60 control tumors 464 mg, range 413–646; p=0.001). So, the effects of AML overexpressed VEGFA165 are detectable in combination with its environment. Using gene expression profiles (Affymetrix) we found differentially activated signaling pathways, e.g. the PI3K/AKT (p

Cellular VEGFA is an independent adverse prognostic factor related to worse outcome in AML and is significantly upregulated in leukemic blasts (de bont ES. et al., 2002). Transcriptional activation of the VEGFA promoter represents the core mechanism through which expression of VEGFA can be regulated, but is still not identified in AML (Carmeliet P. and Jaine RK., 2000; Kolch W. et al., 1995; Yancoupolus GD. et al., 2000). One of the most common targets of aberrations in acute leukemia is Runt domain transcription factor (AML1/RUNX1), due to point mutations (Roumier C. et al., 2003) or disruption of chromosome 21, which occurs in about 10%-15% of all de novo AML patients. In the current study, functional 5′ deletion analysis of the VEGFA promoter was performed to identify VEGFA promoter regions involved in VEGFA transcriptional regulation in AML. Loss of the region spanning −2274/−507 resulted in a significant increase of promoter activity in two AML cell lines, whereas the promoter activity dropped after deletion of the promoter region −286 to −52. Computer-assisted sequence analysis of the −2247/−507 fragment of the VEGFA promoter, revealed three perfect AML1/RUNX1-binding sites in the first three deletion regions. The loss of each AML1/RUNX1-binding site corresponded to an (further) increase in VEGFA promoter activity. siRNA mediated AML1/RUNX1 depletion caused a 24% to 36% increase in VEGFA mRNA expression in two different AML cell lines (HL-60 and TF-1), whereas VEGFA promoter activity was two-fold increased in HL-60 cells treated with AML1/RUNX1 siRNA compared to the non-silencing control siRNA. In addition, mutation of all three AML1/RUNX1 sites resulted in a 22 fold increase of VEGFA promoter activity in HL-60 cells. VEGFA mRNA expression was found to be higher in AML patients with a t(8;21) translocation compared to patients without this translocation. To investigate whether blocking of the fusion protein AML1-ETO, generated by the t(8;21) translocation, would have an effect on VEGFA mRNA we have used a siRNA against AML-ETO in Kasumi-1. siRNA mediated depletion of AML1-ETO caused a 40% decrease in VEGFA mRNA expression. AML-ETO has the potential to interact with AML1/RUNX1 co-factors such as histone deacetylase (HDAC) which results in a dominant negative effect on AML1/RUNX1 transcriptional regulation. To test whether AML1/RUNX1 transcription repression of VEGFA is histone deacetylase (HDAC) dependent HL-60 cells were treated with 200 nM Trichostatin A (TSA), a potent inhibitor of HDAC, this resulted in an 75% increase of luciferase activity after 24h. In contrast, TSA treatment of Kasumi-1 cells containing the AML-ETO fusion protein had no effect on VEGFA expression. These results indicate that VEGFA is transcriptionally regulated by AML1/RUNX1, one of the most common targets of aberrations in AML, through three AML1/RUNX1 repressor elements located in the promoter of VEGFA. Furthermore, prognostically unfavorable high VEGFA expression in AML is caused by a dominant negative effect of the fusion protein AML-ETO on AML1/RUNX1 mediated repression of VEGFA transcription. These results underscore the importance of AML1/RUNX1 aberrations in AML development and progression and better understanding the molecular basis for aberrant AML/RUNX1 signaling pathway may help design more effective treatment strategies.

VEGF-C, has been characterized as a lymphangiogenic and angiogenic growth factor and mediates Acute Myeloid Leukemic (AML) cell proliferation and survival by FLT-4 signaling. Moreover, exogenously added VEGF-C protected 2 AML cell lines and 5 primary AML samples from in vitro chemotherapy-induced apoptosis for 3 drugs via an increased bcl-2/bax ratio (Dias, S. et al. Blood, Mar 15;99(6):2179–84, 2002). We hypothesized that in vitro as well as in vivo cellular drug resistance of AML cells are increased as a result of downstream effects of endogenous VEGF-C signaling. In primary AML cells from 40 pediatric patients VEGF-C mRNA expression with a quantitative PCR as well as in vitro cellular resistance to 9 drugs with a total cell-kill MTT assay were studied. LC50 values (drug concentration needed to kill 50% of the leukemic cells) were used as a measure of resistance. The best available in vivo equivalent for in vitro drug resistance is the time in days from diagnosis to complete remission (CR). CR is defined as less than 5% leukemic cells in a regenerated bone marrow aspirate. Correlations were calculated using the Spearman rank correlation coefficient. P-values ≤0.05 were considered to be statistically significant. VEGF-C mRNA expression levels varied largely in the samples (median: 0.02, given as arbitrary units, range: 0.004–0.33, n=40). Increased endogenous VEGF-C mRNA expression levels correlated significantly with increased in vitro resistance for 6 of 9 drugs (for cytarabine such a trend was seen), these drugs were all typical AML drugs (Table 1a and 1b). For time to reach CR, cox regression analysis was used to study the effect of VEGF-C, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile and white blood cell counts on time to reach CR. Interestingly, VEGF-C was in vivo an independent prognostic factor for the time to reach CR (p=0.006, Odds ratio:0.224, 95% confidence interval: 0.077–0.655). LC50 values of the various drugs did not correlate to the time to CR (all p-values were above 0.1). So, high VEGF-C mRNA levels are correlated to a slower decrease of the total number of leukemic blasts in vitro as well as in vivo upon drug exposure. In conclusion, these results suggest for the first time that increased endogenous VEGF-C mRNA expression, probably resulting in increased anti/pro-apoptotic ratios, are correlated with in vitro as well as in vivo drug resistance of pediatric AML cells resulting in a longer time to reach CR. Table 1a. Correlations endogenous VEGF-C mRNA expression and LC50 values. Drugs Cytarabine Gemcitabine Fludarabine 6-thioguanine Daunorubicin Etoposide VEGF-C rho 0.28 0.33 0.31 0.40 0.46 0.43 p 0.08 0.04 0.05 0.01 0.005 0.008 Table 1b. Drugs Cladibrine Vincristine L-asparaginase Rho indicates spearman rank correlation coefficient. p ≤ 0.05 was considered statistically significant. VEGF-C rho 0.45 0.28 0.22 p 0.003 0.12 0.21

Abstract 1368 New treatment options are necessary to improve survival rates for patients with Acute Lymphoblastic Leukemia (ALL), especially for patients with unfavorable prognostic predictors. As a new therapeutic approach specific protein kinase inhibitors are being developed that can down-regulate vital signaling pathways in leukemic blasts (McCubrey et al, 2008). The main goal of the present study is to obtain a better understanding of the kinase signaling pathways active in ALL cells and to identify potential targets for therapeutic intervention, To identify active signaling pathways in ALL we have used kinase activity arrays containing 1024 peptides representing all major signaling pathways and human proteome profiler arrays containing 46 phospho-antibodies on lysates of primary ALL blasts. In 20 patient samples a total of 10.6% 109(1024) peptides were found to be phosphorylated in 90% of the samples. About 46% 50(109). Activities for kinases including PKC, PKA, Akt, CAMK2, CDC2, CDK2, ERK, GSK3beta, JAK and MAPK were detected in these lysates. The human proteome profiler array demonstrated high levels of protein phosphorylation of CREB and RSK. We constructed a provisional signal transduction scheme of active kinases and phosphorylated proteins in ALL cells (Fig. 1A). Consistent with earlier reports, we identified a prominent role for the Raf/MEK/ERK and the PI3K/Akt/mTOR pathways in these ALL cells. Based on this provisional signal transduction scheme we composed a list of possible new druggable targets. Two proteins were selected for further investigation, CREB and RSK. Inhibition of RSK by the p90 RSK inhibitor BI-D1870 had no effect on cell viability as measured with WST-1 cell viability assay in ALL cell lines. Interestingly, inhibition of CREB by the CREB inhibitor KG-501 showed a dose- and time-dependent decrease in cell viability in all cell lines tested (LC50 values after 24h: Jurkat: 18.55 mM, Molt 4: 13.02 mM, RCH-ACV: 38.11 mM, and RS4;11 45.36 mM (Fig. 1B). LC50 values after 48h: Jurkat: 7.36 mM, Molt 4: 6.53 mM, RCH-ACV: 31.73 mM, RS4;11 36.66 mM (Fig. 1C)). In addition, apoptosis measured by AnnexinV/ PI staining showed an increased percentage of apoptotic cells in a dose- and time-dependent manner in all cell lines upon treatment with the CREB inhibitor (apoptosis after 24h: Jurkat 35.83% to 79.7%, Molt 4: 12.19% to 48.5%), RCH-ACV 11.30% to 45.9%, and RS4;11 9.84% to 19.16. Apoptosis after 48h: Jurkat 53.40% to 86.4%, Molt 4: 27.70% to 92.9%, RCH-ACV 14.07% to 63.32%, and RS4;11 7.11% to 20.75%) (Fig. 1D). To investigate the downstream effect of CREB inhibition we measured the mRNA expression of a know CREB target gene: BCL-2. Upon inhibition of CREB (50 mM KG-501) mRNA levels of BCL-2 were found to be significantly decreased compared to vehicle treated cells. In conclusion we have identified the transcription factor CREB in vitro as a potential druggable target for ALL. It is known that CREB plays an important role as a downstream target of hematopoietic growth factor signaling in hematopoiesis (Cheng et al, 2008). Based on these results, we propose CREB as a promising potential druggable target in ALL. Figure 1. (A) Provisional signal transduction scheme of active kinases and phosphorylated proteins in ALL. Green: active kinase; Yellow: phosphorylated protein; Green-Yellow: active kinase and phosphorylated protein. (B) Cell viability percentages plotted against concentration of KG-501 (mM) after 24h. (C) Cell viability percentages plotted against concentration of KG-501 (mM) after 48h. (D) Representative flow cytometric dot-plots of AnnexinV/ PI flow cytometry, inhibition of CREB induced a dose- and time-dependent apoptosis in the Molt 4 cell line. Disclosures: No relevant conflicts of interest to declare.

Overall survival rates for children with Acute Lymphoblastic Leukemia (ALL), the most common type of leukemia in children, are approaching 90% (Mullighan 2013). In the past 5 years, genome wide approaches, studying DNA copy number alterations in ALL, have increased the list of risk stratifications and included IKZF1 deletions to the list of unfavorable prognostic factors. IKZF1 deletions can be identified in approximately 70% of the Philadelphia chromosome-positive (Ph+) and in 15% of the Philadelphia chromosome-negative (Ph-) children with ALL and are associated with an increased risk on relapse and a decreased overall survival (Mullighan 2009, Kuiper 2010, van der Veer 2013). IKZF1 deletions observed in B-cell precursor ALL (BCP-ALL) are typically mono-allelic, resulting in the expression of a dominant-negative isoform (Mullighan 2008). A unique gene expression signature was revealed in IKZF1 deleted BCP-ALL patients, characterized by the downregulation of genes regulating B-cell lineage development and DNA repair upon DNA damage response genes and upregulation of cell cycle/apoptosis genes, JAK/STAT signaling and stem cell self-renewal (Iacobucci 2012). At the level of signal transduction, western blot analysis showed that IKZF1 deletions resulted in B cell receptor (BCR) signaling defects and upregulation of phospho-STAT5 in 2 and 4 Ph+ ALL patients, respectively (Trageser 2009, Iacobucci 2012). However, effects of IKZF1 deletions on signaling pathways in Ph-ALL have not been extensively studied. Pediatric Ph- BCP-ALL patients (N=46) were screened for IKZF1 deletions by multiplex ligation-dependent probe amplification analysis. A total of 15 patients carried an IKZF1 deletion. We performed a kinase activity profile (IKZF1 deleted N=15, IKZF1 wild type N=31) as well as a human phospho-proteome array (IKZF1 deleted N=11, IKZF1 wild type N=17) to elucidate active signal transduction pathways. Kinase activity profiling is a potent high throughput technique using peptides of 11 amino acids in length representing known human phosphorylation sites. In the obtained kinase activity profiles we studied differences in peptide phosphorylation intensities. 37 peptides were differentially expressed between IKZF1 deleted and wild type pediatric Ph- BCP-ALL patients (P ≤ 0.05, Figure 1). From these 37 peptides we first examined peptides derived from proteins involved in the BCR signaling and STAT5. On the kinome array, peptides derived from Src_Y352, CBL_Y371, SYK_Y526, PLCg2_Y753, PLCg2_Y1217, STAT5a_S780, STAT5a_Y694, and STAT5b_Y679 were present but showed no differences in phosphorylation intensities between IKZF1 deleted and IKZF1 wild type Ph- BCP-ALL samples. Neither could we detect differences in phosphorylation intensities of Fyn_Y420, Lyn_Y397, Src_Y419, STAT5a_Y694, STAT5b_Y699, and STAT5a/b_Y694/Y699 using human phospho-proteome arrays, confirming the kinome profiling results. We did, however observe a distinct kinome profile upon hierarchical clustering of 46 BCP-ALL primary samples, based on the 37 peptides identified by t-test (Figure 1). IKZF1 deleted cases showed high phosphorylation of 14 peptides including peptides derived from Akt1_Y326 and Cav1_Y14 (Figure 1). Loss of IKZF1 has been associated with glucocorticoid resistance. Since Akt inhibition reverses glucocorticoid resistance in T cell ALL (Piovan, 2013) and Caveolin 1 is involved in focal adhesion and chemoresistance (Faggi, 2014) we hypothesize that Akt and Caveolin 1 inhibition might convert glucocorticoid resistance in Ph-IKZF1 deleted pediatric BCP-ALL, which requires further investigation. Together, we conclude that kinome profiling revealed a distinct peptide phosphorylation pattern for IKZF1 deleted pediatric Ph- BCP-ALL including novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.