ALBERT

Description: 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.

Description: 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.

Description: 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 =

Description: 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.

Description: Abstract 5235 Aberrant Ephrin signaling has been shown to be an important pathway that contributes to the pathogenesis of many solid tumors (Surawska et al. Cytokine & Growth factor reviews 2004). Deregulated ephrin receptor (Eph) and ligand (Efn) expression is often associated with poor prognosis in solid tumors. Ephrin receptor and ligand overexpression can result in tumorigenesis through induced tumor growth, tumor cell survival, angiogenesis and metastasis (Surawska et al. Cytokine & Growth Factor Reviews 2004; Campbell et al. Curr. Isues Mol. Biol. 2008; Chen et al. Cancer Research 2008). In normal cells Eph receptors and ligands play key roles in vascular patterning, where they function in endothelial cell migration, and proliferation (Adams et al, Genes Dev. 1999; Zhang et al., Blood 2001). Thus far particularly EphB4 receptor and ephrin-B2 ligand have been implicated in the process of normal angiogenesis. In acute myeloid leukemia (AML) patients it was found that bone marrow biopsies at diagnosis exhibited enhanced microvessel density (MVD) (de Bont ES et al., BJH 2001; Byrd JC et al., Blood 2002; Padro et al., Blood 2000). Normal hematopoietic stem cells (HSCs) express the following mRNA transcripts ephrin receptors EphA1, EphA2, EphB2, and EphB4 and ephrin ligands EfnA3, EfnA4, and EfnB2. Moreover, overexpression of EphB4 receptor in HSCs (from cord blood) resulted in enhanced differentiation towards megakaryocytes (Wang et al. Blood 2002). In AML cell lines there is a common co-expression on protein level observed between EphB4 receptor and ephrin-B2 ligand. Recently, an aberrant DNA methylation of ephrin receptors and ligands was described in acute lymphocytic and myelocytic leukemia cell lines (Kuang et al. Blood 2010). In addition, restoration of EphB4 expression in an acute lymphoid leukemia cell line resulted in reduced proliferation and apoptotic cell death. These data suggests that the ephrin signaling pathway might play an important role in leukemia. In a previous study we have found high kinase activity of EphB receptors and high phosphorylation levels of EphB receptors in AML samples, as measured using kinase arrays and proteome profiler arrays. In this study, we have found extensive membrane expression of EphB1 on AML cell lines and primary AML blasts. To identify the role of Ephrin signaling in AML, two AML cell lines THP-1 and HL60 with an EphB1 membrane expressing cell percentage of 70% and 20% respectively were chosen for stimulation with Ephrin-B1 ligand. Treatment of these cell lines with Ephrin-B1 ligand resulted in a decreased proliferation 30% in THP-1 cells versus 22% in HL60 cells and increased apoptosis 23% in THP-1 cells and 4% in HL60 cells. Of note, the most prominent effect of Ephrin-B1 stimulation was found in THP-1 cells, this cell line contained a higher percentage of EphB1 membrane expressing cells. We further investigated the mechanism through which EphB1 reduces leukemic cell growth and induces leukemic cell death in THP-1 cells. Westernblot analysis of cell cycle regulators showed that expression of the anti-apoptotic protein BCL2 is reduced upon Ephrin-B1 ligand stimulation and the expression of the pro-apoptotic protein BAX is induced. In addition, mRNA expression of the cell cycle inhibitor of cell cycle progression p21 was found to be 2,5 fold upregulated in ephrin-B1 ligand treated cells compared to untreated control cells. MGG stainings of Ephrin-B1 treated cells revealed multiple cells with two nuclei in both THP-1 and HL60 cells. These results indicate that a high percentage of AML cells express EphB1 receptor on the membrane and that stimulation of these cells with Ephrin-B1 ligand results in reduced leukemic growth and increased cell death. EphrinB1 activation in AML deserves further investigation considering EphB1 as a putative new treatment option for AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: 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

Description: 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.