The developmental stage of the medulloblastoma cell-of-origin restricts Sonic hedgehog pathway usage and drug sensitivity.

Sonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.

CREB signaling activity correlates with differentiation and survival in medulloblastoma.

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy.

Peptide microarray of pediatric acute myeloid leukemia is related to relapse and reveals involvement of DNA damage response and repair.

The majority of acute myeloid leukemia (AML) patients suffer from relapse and the exact etiology of AML remains unclear. The aim of this study was to gain comprehensive insights into the activity of signaling pathways in AML. In this study, using a high-throughput PepChip™ Kinomics microarray system, pediatric AML samples were analyzed to gain insights of active signal transduction pathway. Unsupervised hierarchical cluster analysis separated the AML blast profiles into two clusters. These two clusters were independent of patient characteristics, whereas the cumulative incidence of relapse (CIR) was significantly higher in the patients belonging to cluster-2. In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs. The activated peptides in cluster-1 and cluster-2 reflected the activity of cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT, MAPK, metabolism regulation, transcription factors and GPCRs signaling pathways. The difference between two clusters might be explained by the higher cell cycle arrest response in cluster-1 patients and higher DNA repair mechanism in cluster-2 patients. In conclusion, our study identifies different signaling profiles in pediatric AML in relation with CIR involving DNA damage response and repair.

VEGFC Antibody Therapy Drives Differentiation of AML.

High expression of VEGFC predicts adverse prognosis in acute myeloid leukemia (AML). We therefore explored VEGFC-targeting efficacy as an AML therapy using a VEGFC mAb. VEGFC antibody therapy enforced myelocytic differentiation of clonal CD34+ AML blasts. Treatment of CD34+ AML blasts with the antibody reduced expansion potential by 30% to 50% and enhanced differentiation via FOXO3A suppression and inhibition of MAPK/ERK proliferative signals. VEGFC antibody therapy also accelerated leukemia cell differentiation in a systemic humanized AML mouse model. Collectively, these results define a regulatory function of VEGFC in CD34+ AML cell fate decisions via FOXO3A and serve as a new potential differentiation therapy for patients with AML.Significance: These findings reveal VEGFC targeting as a promising new differentiation therapy in AML. Cancer Res; 78(20); 5940-8. ©2018 AACR.

Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML.

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) which has a need for improved therapeutic strategies. We found PLC-γ1 as one of the highest phosphorylated peptides in t(8;21) AML samples compared to NBM or CN-AML in our previous peptide microarray. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in t(8;21) AML. In this study, PLC-γ1 expression was significantly higher in t(8;21) AML compared to other karyotypes. The PLC-γ1 protein expression was suppressed in AML1-ETO knock down cells indicating that it might induce kasumi-1 cell death. ShRNA-mediated PLC-γ1 knockdown in kasumi-1 cells significantly blocked cell growth, induced apoptosis and cell cycle arrest which was explained by the increased activation of apoptotic related and cell cycle regulatory protein expressions. Gene expression array analysis showed the up-regulation of apoptotic and DNA damage response genes together with the downregulation of cell growth, proliferation and differentiation genes in the PLC-γ1 suppressed kasumi-1 cells, consistent with the observed phenotypic effects. Importantly, PLC-γ1 suppressed kasumi-1 cells showed higher chemosensitivity to the chemotherapeutic drug treatments and lower cell proliferation upon hypoxic stress. Taken together, these in vitro finding strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cells and identify PLC-γ1 as a potential therapeutic target for t(8;21) AML treatment.

Epidermal growth factor receptor is expressed and active in a subset of acute myeloid leukemia.

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.

Effect of IKZF1 deletions on signal transduction pathways in Philadelphia chromosome negative pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

BACKGROUND: IKZF1 deletions are an unfavorable prognostic factor in children with Philadelphia chromosome positive (Ph(+)) as well as negative (Ph(-)) acute lymphoblastic leukemia (ALL). Although IKZF1 deletions occur in 10-15% of Ph(-) ALL cases, effects of IKZF1 deletions on signaling pathways in this group have not been extensively studied. Therefore, in this study we aimed to study the effect of IKZF1 deletions on active signal transduction pathways. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to determine IKZF1 deletions and other copy number alterations in 109 pediatric B-Cell Precursor ALL (BCP-ALL) patients. Kinase activity profiling of 45 primary Ph(-) BCP-ALL patients (31 IKZF1 wild type patients and 14 patients harboring an IKZF1 alteration) and western blot analysis of 14 pediatric BCP-ALL samples was performed to determine active signal transduction pathways. RESULTS: Unsupervised hierarchical cluster analysis of kinome profiles of 45 pediatric Ph(-) ALL cases showed no clustering based on IKZF1 status. Comparing the phosphorylation intensities of peptides associated with signaling pathways known to be involved in BCP-ALL maintenance, we did not observe differences between the two groups. Western blot analysis of 14 pediatric BCP-ALL samples showed large variations in phosphorylation levels between the different ALL samples, independent of IKZF1 status. CONCLUSIONS: Based on these results we conclude that, although IKZF1 deletions appear to be an important clinical prognostic factor, we were unable to identify a unique IKZF1 dependent protein expression signature in pediatric Ph(-) ALL and consequently no specific targets for future therapy of Ph(-) IKZF1 deleted BCP-ALL could be identified.

Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia.

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.

Growth-factor-driven rescue to receptor tyrosine kinase (RTK) inhibitors through Akt and Erk phosphorylation in pediatric low grade astrocytoma and ependymoma.

Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present study will extend our previous observations that vascular endothelial growth factor receptor (VEGFR)-2, platelet derived growth factor receptor (PDGFR)ß, Src, the epidermal growth factor receptor (ErbB) family, and hepatocyte growth factor receptor (HGFR/cMet) are potentially drugable targets in pediatric low grade astrocytoma and ependymoma with investigations concerning growth-factor-driven rescue. This was investigated in pediatric low grade astrocytoma and ependymoma cell lines treated with receptor tyrosine kinase (RTK) inhibitors e.g. sorafenib, dasatinib, canertinib and crizotinib. Flow cytometry analyses showed high percentage of cells expressing VEGFR-1, fibroblast growth factor receptor (FGFR)-1, ErbB1/EGFR, HGFR and recepteur d'origine nantais (RON) (respectively 52-77%, 34-51%, 63-90%, 83-98%, 65-95%). Their respective inhibitors induced decrease of cell viability, measured with WST-1 cell viability assays. At least this was partially due to increased apoptotic levels measured by Annexin V/Propidium Iodide apoptosis assays. EGF, HGF and FGF, which are normally expressed in brain (tumor) tissue, showed to be effective rescue inducing growth factors resulting in increased cell survival especially during treatment with dasatinib (complete rescue) or sorafenib (partial rescue). Growth-factor-driven rescue was less prominent when canertinib or crizotinib were used. Rescue was underscored by significantly activating downstream Akt and/or Erk phosphorylation and increased tumor cell migration. Combination treatment showed to be able to overcome the growth-factor-driven rescue. In conclusion, our study highlights the extensive importance of environmentally present growth factors in developing tumor escape towards RTK inhibitors in pediatric low grade astrocytoma and ependymoma. It is of great interest to anticipate upon these results for the design of new therapeutic trials with RTK inhibitors in these pediatric brain tumors.

Kinase activity profiling reveals active signal transduction pathways in pediatric acute lymphoblastic leukemia: a new approach for target discovery.

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.

EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion.

Eph/ephrin signaling has been implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data published recently, we hypothesized a key role for the Eph/ephrin signaling system in medulloblastoma invasion. In primary medulloblastoma samples, a significantly higher expression of EphB2 and the ligand ephrin-B1 was observed compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an increase in the invasion capacity of cells expressing high levels of EphB2. The cell lines that showed an ephrin-B1-induced phenotype possessed increased levels of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation. Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin ligand-induced effects on adhesion and migration. Analysis of signal transduction identified p38, Erk, and mTOR as downstream signaling mediators potentially inducing the ephrin-B1 phenotype. In conclusion, the observed deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive phenotype, suggesting a potential role in local tumor cell invasion and the formation of metastases.

Repression of vascular endothelial growth factor expression by the runt-related transcription factor 1 in acute myeloid leukemia.

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.

Identification of new possible targets for leukemia treatment by kinase activity profiling.

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® 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.

VEGF-A promotes lymphoma tumour growth by activation of STAT proteins and inhibition of p27(KIP1) via paracrine mechanisms.

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 overexpressing VEGF-A was increased 4-fold compared to that of control tumours (p<0.0001), whereas no in vitro growth advantage was demonstrated upon VEGF-A overexpression. VEGF-A-tumours were associated with increased microvessel densities (p=0.004) and increased tumour cell proliferation (Ki67; p<0.001) compared to control tumours. VEGF-A-tumours were characterised by upregulation of phosphorylated STAT-4 and STAT-6 and downregulation of phospho-p27(KIP1), a crucial cell cycle inhibitor (p<0.05). This was accompanied by increased levels of phosphorylated receptor tyrosine kinases, including EGFR (ErbB-2 and ErbB-4, p<0.05), an upstream regulator of STAT proteins. We demonstrated that various mouse-derived cytokines produced by mouse-derived tumour stromal cells are upregulated in VEGF-A-tumours compared to control tumours (p<0.05). These results indicate an important role for the tumour microenvironment in paracrine promotion of lymphoma tumour growth in response to tumour-derived VEGF-A. In conclusion, lymphoma-derived VEGF-A promoted lymphoma tumour growth in a paracrine loop by activation of tumour stromal cells. Our study reveals VEGF-A and STAT proteins as potential additional targets in the treatment of lymphoma.

Kinome profiling in pediatric brain tumors as a new approach for target discovery.

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.

Endogenous vascular endothelial growth factor-C expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia.

PURPOSE: We hypothesized that downstream effects of endogenous vascular endothelial growth factor (VEGF)/VEGF receptor signaling on acute myelogenous leukemia (AML) cell survival resulted in increased in vitro cellular drug resistance and a longer time to kill most leukemic cells in vivo upon drug exposure. EXPERIMENTAL DESIGN: In primary AML cells from pediatric patients, VEGFA and VEGFC mRNA expression and in vitro cellular resistance to nine cytotoxic drugs were studied. As in vivo equivalents for in vitro drug resistance, in vivo AML blast reduction upon drug exposure, measured as blast cell reduction on day 15 in the bone marrow and as time in days from diagnosis to complete remission (CR) were used. RESULTS: Increased endogenous VEGFC levels significantly correlated with increased in vitro resistance for six typical AML drugs in primary AML cells from pediatric patients. Patients with >5% blasts on day 15 showed a 12.9-fold increase in the median VEGFC level compared with patients with < or =5% blasts (P = 0.002). Time to reach CR was studied using linear regression analysis with VEGFC, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile, and WBC counts as variables. There was a significant positive independent association between VEGFC levels and time to CR (b = 6.02, SE = 1.58, P < or = 0.0001, n = 72). CONCLUSIONS: These results suggest for the first time that higher endogenous VEGFC levels of AML cells are related to decreased in vitro and in vivo drug responsiveness.