ALBERT

Description: The Activation-Induced Cell Death (AICD) is a stimulation-dependent form of apoptosis used by the organism to shutdown T-cell response once the source of inflammation has been eliminated, while allowing the generation of immune memory. AICD is thought to progress through the activation of the extrinsic Fas/FasL pathway of cell death, leading to cytochrome-C release through caspase-8 and Bid activation. We recently described that, early upon AICD induction, mitochondria undergo structural alterations, which are required to promote cytochrome-C release and execute cell death. Here, we found that such alterations do not depend on the Fas/FasL pathway, which is instead only lately activated to amplify the cell death cascade. Instead, such alterations are primarily dependent on the MAPK proteins JNK1 and ERK1/2, which, in turn, regulate the activity of the pro-fission protein Drp1 and the pro-apoptotic factor Bim. The latter regulates cristae disassembly and cooperate with Drp1 to mediate the Mitochondrial Outer Membrane Permeabilization (MOMP), leading to cytochrome-C release. Interestingly, we found that Bim is also downregulated in T-cell Acute Lymphoblastic Leukemia (T-ALL) cells, this alteration favouring their escape from AICD-mediated control.

Description: Abstract 2492 BACKGROUND: Childhood Acute Myeloid Leukemia (AML) is a heterogeneous disease characterized by recurrent genetic aberrations; impaired apoptosis of leukemia cells can also contribute to disease development. BCL2-associated AthanoGene1 (BAG1) is a multifunctional protein preferentially supporting cell survival. Its aberrant expression was demonstrated in diverse cancer types. We previously reported high expression of BAG1 protein in leukemia cell lines. We showed that leukemic cell survival was impaired after silencing BAG1. Additionally, we confirmed that, in vitro, BAG1 acted in synergy with BCL2 and influenced MCL1 expression, both being important anti-apoptotic players in AML (Aveic et al. PlosOne 2011). AIM: We investigated BAG1 expression and role in pediatric AML samples collected at diagnosis, as well as its partner molecules BCL2, MCL1 and HSP70. We then evaluated whether BAG1 may affect patient survival. METHODS: Expression of BAG1 was studied at mRNA and protein levels, using RQ-PCR and Western Blot (WB). Initially, 10 patients with AML whose samples were collected at diagnosis and during therapy while they were in disease remission, were included. Subsequently, wide protein expression was performed by Reverse-phase protein array (RPPA) on a further cohort of 66 newly diagnosed AML patients enrolled into the AIEOP-LAM 2002 protocol. RESULTS: BAG1 mRNA resulted to be heterogeneously expressed in AML cases. Furthermore, BAG1 protein was highly expressed in leukemia samples when compared with healthy bone marrow (hbm), and with patient bm in remission, where BAG1 was undetectable by WB analysis. By RPPA, we confirmed the overexpression of BAG1 protein in 56/66 AML patients (84.8 %) with respect to hbm (113616 ± 66721 Arbitrary Unit (AU); mean fold increase: 2.36). Then, we studied its anti-apoptotic co-partner BCL2, whose expression was found to correlate with BAG1 over-expression (110033 ± 89117 AU, p

Description: t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients1. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the context of the clonal architecture dynamic evolution. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. The importance of the identification of molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse could be instrumental to improve their cure rate. We performed high throughput DNA methylation profiling (RRBS-seq) and integrated results with gene expression profiling (Affymetrix HTA 2.0) of 16 isolated t(8;21) AML samples collected at diagnosis, and analyzed data by comparing patients who did or did not experience relapse. We applied a logistic regression algorithm to identify differentially methylated regions (DMRs) considering a minimum change in methylation level of 25%. We validated results in a proteome context by reverse phase protein array (RPPA) in an independent cohort of 35 t(8;21) AML patients. DNA methylation profiling analysis identified 337 DMRs able to correctly predict t(8;21) patients who did relapse from those who did not. In particular, 23 DMRs (7%) were located at promoters, while most of them were equally distributed between intronic (48%) and exonic (45%) regions. Globally, we found hypomethylated DMRs being significantly enriched in relapsed patients, in particular in repetitive elements regions of the genome (LINE, SINE, DNA transposon: 38.9% vs 52.4%; p

Description: Key Points Resistance to glucocorticoid treatment in pediatric T-ALL can be reversed by LCK inhibitors in vitro and in vivo. IL-4 overexpression contributes to LCK-induced glucocorticoid resistance.

Description: The presence of the chromosomal rearrangement t(12;21)(ETV6-RUNX1) in childhood B-acute lymphoblastic leukemia (B-ALL) is an independent predictor of favorable prognosis, however relapses still occur many years later after stopping therapy, and patients often display resistance to current treatments. Since spleen tyrosine kinase (SYK), a cytosolic nonreceptor tyrosine kinase interacting with immune receptors, has been previously associated with malignant transformation and cancer cell proliferation, we aimed to assess its role in ETV6-RUNX1 cell survival and prognosis. We evaluated the effects on cell survival of three SYK inhibitors and showed that all of them, in particular entospletinib, are able to induce cell death and enhance the efficacy of conventional chemotherapeutics. By using reverse phase protein arrays we next revealed that activated SYK is upregulated at diagnosis in pediatric ETV6-RUNX1 patients who will experience relapse, and, importantly, hyperactivation is maintained at a high level also at relapse occurrence. We thus treated primary cells from patients both at diagnosis and relapse with the combination entospletinib + chemotherapeutics and observed that SYK inhibition is able to sensitize resistant primary cells to conventional drugs. Entospletinib could thus represent a new therapeutic option supporting conventional chemotherapy for relapsed ETV6-RUNX1 patients, and these evidences encourage further studies on SYK for treatment of other relapsed resistant acute lymphoblastic leukemia (ALL) subgroups.

Description: Although our understanding of pediatric acute lymphoblastic leukemia (ALL) biology has improved through RNA expression profiling, DNA-based somatic mutation analysis and genome-wide DNA methylation assays, these methods are not able to provide insight into altered protein expression and impact on signaling pathways activity. Recent studies clearly indicated the need to target the physiological effects of deregulated pathways rather than their individual gene components, in order to improve therapeutic options. Recently, we identified CD200 deletion as a recurrent genetic alteration in B-cell precursor ALL (BCP-ALL) with prognostic relevance in intermediate risk BCP-ALLs. However, the exact mechanisms by which loss of CD200 triggers precursor B-cell transformation as well as the signaling cascades that are activated downstream of CD200 remain to be elucidated. Given this, we used reverse phase protein array (RPPA) technology in a cohort of 72 ETV6/RUNX1-positive BCP-ALL primary patient samples at diagnosis to study the impact of low CD200 expression on several key cellular signaling pathways. Interestingly, differential protein analysis between CD200-low and CD200-high BCP-ALLs suggested that reduced CD200 levels are associated with enhanced PI3K/AKT/mTOR signaling. More specifically, eIF4G, a scaffold protein that plays a role in initiation of translation, was found to be hyperphosphorylated in CD200-low cases (p = 0.015). Also AKT was found hyperphoshorylated at the activation site (p = 0.033). In addition, hyperphosphorylation of c-RAF (p = 0.026) and B-RAF (p = 0.035) at their activation site were also observed in CD200-low patients. Next to B-RAF, hyperphosphorylation of the p44/42 activation site was noted (p = 0.006), highlighting a global hyperactivation of the RAS/MAPK pathway. Interestingly, the expression of integrin α4/β1 was markedly decreased in CD200-low ETV6-RUNX1-positive patients (p = 0.001). These heterodimeric cell surface receptors play a pivotal role in cell adhesion and migration, as well as in growth and survival and a decreased expression of some subunits of integrin has been observed in several tumor entities. In leukemia, the α4/β1 dimer, also named VLA-4, has been demonstrated several times to contribute to drug resistance and relapse occurrence since its presence allows leukemia cells to adhere to the bone marrow niche and escape from chemotherapeutics action (Hsieh YT, Blood 2013). The relevance of our observation in this context however needs to be further elucidated. Finally, these observations were validated using two different B-ALL cell lines REH and AT-2, both ETV6/RUNX1-positive, but REH is characterized by a CD200 deletion, whereas AT-2 displays no copy number aberrations at this particular genomic locus. Altogether, these results revealed several targetable pathways in ETV6/RUNX1-positive BCP-ALL patients with low CD200 expression, and provide a framework for testing of interesting inhibiting compounds including PI-103, LY294002 and silvestrol. BDM and PVV are shared last authors Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2605 Poster Board II-581 Background. Annexin II (ANXA2) is a member of a peripheral membrane-binding protein family acting in a calcium-dependent manner, is involved in many cellular mechanisms, as cell proliferation and membrane physiology and is related to cancer progression. The aim of this study was to assess the ANXA2 expression in B cell precursor acute lymphoblastic leukemia (Bcp-ALL), in the attempt to finally evaluate it as a new potential therapeutic target. Materials and Methods. The ANXA2 expression was tested in 77 newly diagnosed pediatric Bcp-ALL diagnosed and treated in our centers, according with LLA-2000 protocol of Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP). Diagnostic samples and 3 B-ALL cell lines (REH, SEM, 697) were studied by reverse phase protein array (RPPA), western blot and real-time PCR (RQ-PCR) analyses. Furthermore, immunofluorescence on bone marrow smears and cytofluorimetric studies were performed, in order to visualize the protein subcellular location. The associations between the ANXA2 expression, molecular features and prognosis were evaluated. For statistical purpose, multivariate analyses with Wilcoxon test and t-Test with conservative Bonferroni corrections and Kaplan-Mayer analysis were performed. Pearson correlation was used to compare mRNA and protein levels. Results. Our analyses demonstrated a positive correlation between mRNA and protein ANXA2 expression (Pearson correlation or index 0.6). Comparing ANX2 expression and molecular features, we found a statistically significant difference between patients with unfavourable [t(9;22), t(4;11)] and favourable translocations [t(12;21)], showing a higher level of ANXA2 in the former group (p-value

Description: The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients. To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCɣ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCɣ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity. We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells. Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

Description: Abstract 744 Background: T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL pediatric patients remain at high risk for early relapse. It is therefore very important to identify new prognostic biomarkers and to develop new therapeutic approaches for these patients. More specific and less toxic therapies might be developed through the identification of molecular targets, such as aberrantly activated phosphoproteins, that offer the possibility to use specific kinase inhibitors. In this research we first explored the phosphoproteomic profile of pediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), and we further investigated the origin and the functional significance of Protein Kinase C alpha (PKCα) downregulation in patients that are liable to relapse. Methods: We analyzed 98 pediatric T-ALL patients at diagnosis using RPPA. This innovative approach allows to profile phosphorylation modifications and to characterize the activation state of cellular protein networks. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with one of 53 different antibodies included in the study. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features through statistical analyses. Gene expression analysis was performed by means of HG-U133 Plus 2.0 array. Permission for this study was obtained following the tenets of the Declaration of Helsinki. A set of commercially available human T-ALL cell lines was studied by Western Blot and cells were treated with Ro-32-0432, a commercial PKC inhibitor. Results: The phosphoproteomic profile of T-ALL patients was compared between patients subgroups at diagnosis. Comparison between relapsed vs non relapsed patients samples revealed the downregulation of PKCα(S657) in relapsed patients (t test with BH multiplicity corrections p=0.02) and Relapse Free Survival analysis through Kaplan-Meier estimates showed that patients with low PKCα(S657) have a high probability to relapse. Notably, 54.5% of patients with low PKCα(S657) relapsed whereas no relapses were observed among patients with high PKCα activity. Multivariate analysis showed no correlation between PKCα(S657) levels and other parameters currently used for clinical characterization of T-ALL patients. Differences in PKCα(S657) between relapsed and non relapsed patients were confirmed by Western Blot in two independent sets of T-ALL patients. Analysis of PKCα total protein form revealed concordance between total protein expression and PKCα(S657) levels. Furthermore, gene expression analysis revealed a significant difference in PKCα gene expression levels, comparable with levels of protein expression among T-ALL patients studied by RPPA. Gene expression data were validated by Sybr Green Real-Time Quantitative PCR. Moreover, gene pathway analysis in a larger cohort of T-ALL pediatric patients, divided by high and low PKCα mRNA levels, revealed the phosphatidylinositol signalling pathway as the most important deregulated pathway. Of note, this pathway is involved in the production of DAG and Ca2+, the PKCα cofactors that play an essential role in mediating its translocation from the cytosol to the plasmatic membrane and subsequent activation. PKCα and PKCα(S657) were then analysed by Western Blot in five T leukemic cell lines (DND41, P12 ICHIKAWA, MOLT3, TALL1 and CEM). These cell lines were treated with a PKCα inhibitor and PKCα inhibition effects were studied by the means of MTT assay and Trypan Blue cell count. An increase in cell proliferation was observed in each treated cell line 9 hours after PKCα inhibition. Conclusions: PKCα is differentially expressed in T-ALL pediatric patients. Low levels of PKCα(S657) are associated with a higher probability to relapse and downregulation or absence of PCKα seems to be related to a more aggressive T-ALL phenotype. Ongoing studies are needed to better define its role as a new prognostic marker. Disclosures: No relevant conflicts of interest to declare.