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Protein List and Enrichment Analysis of KGG-MS

Motif Analysis of Skip Exon Events in T-ALL Compared to CD3 T Cells

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between CD4 T-Cells and T-ALL

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL.Significance:Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241

Supplementary Figures S1-S15 and Legends

Alternative Spliced Genes Between HR and NHR T-ALL Patients and Enrichment Analysis

SRSF Family Proteins Essentiality in Tumor Cell Lines

CRISP Screens of RNA Binding Proteins in T-ALL

KEGG Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

Supplementary Table 1

Supplementary Table 2

Supplementary Figs. 1-17 with legends

Purpose:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.Experimental Design:To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.Results:We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo.Conclusions:These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.

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.

Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Although many reports described the importance of CDK4/CDK6 in different set of human tumors, only few studies have been performed on leukemia. By gene expression analysis performed in a cohort of childhood patients affected by B-acute lymphoblastic leukemia (B-ALL) we found that both CDK4 and CDK6 are highly expressed. Moreover, reverse phase protein array (RPPA) analysis showed that cyclin D1 levels are higher in patients undergoing relapse. Starting from these considerations, we evaluated the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated B-ALL cell lines with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced growth inhibition of B-ALL cell lines, accompanied by strong cell cycle arrest in G1 phase, along with a dose-dependent decrease in phosphorylated retinoblastoma protein. Ribociclib exposure strongly synergizes with dexamethasone in SEM and RCH-ACV, two dexamethasone-resistant cell lines, along with a strong decrease in proliferation and a significant increase in apoptotic cell death. These results were also confirmed on primary cultures derived from bone marrow of pediatric patients affected by B-ALL. Immunoblot analysis showed a significant increase in glucocorticoid receptor (GR) along with some of its target genes, after combined treatment with ribociclib and dexamethasone. Altogether our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

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.

Simple Summary Current treatment protocols for pediatric patients with T-Lymphoblastic lymphoma (T-LBL) allow the achievement of a complete remission in around 85% of T-LBL pediatric patients; however the overall survival rate of second-line treatments for patients with progressive disease or relapse is around 14%. Thus, the major issues to be addressed are the identification of a valuable predictor marker to foresee the disease risk and new therapeutic targets to improve relapsed/resistant patients’ outcome. We identified JAK2 Y1007-1008 as a potential prognosis marker as well as a therapeutic target for patients with progressive disease or relapse and suggest that its inhibition by ruxolitinib, a JAK1/2 FDA approved inhibitor, could represent a novel therapeutic approach to overcome therapy resistance and meliorate the outcome of pediatric T-LBL patients. Abstract Lymphoblastic lymphoma (LBL) is the second most common type of non-Hodgkin lymphoma in childhood, mainly of T cell origin (T-LBL). Although current treatment protocols allow a complete remission in 85% of cases, the second-line treatment overall survival for patients with progressive or relapsed disease is around 14%, making this the major issue to be confronted. Thus, we performed a Reverse Phase Protein Array study in a cohort of 22 T-LBL patients to find reliable disease risk marker(s) and new therapeutic targets to improve pediatric T-LBL patients’ outcome. Interestingly, we pinpointed JAK2 Y1007-1008 as a potential prognosis marker as well as a therapeutic target in poor prognosis patients. Hence, the hyperactivation of the JAK1/2-STAT6 pathway characterizes these latter patients. Moreover, we functionally demonstrated that STAT6 hyperactivation contributes to therapy resistance by binding the glucocorticoid receptor, thus inhibiting its transcriptional activity. This was further confirmed by specific STAT6 gene silencing followed by dexamethasone treatment. Finally, JAK1/2-STAT6 pathway inhibition by ruxolitinib, an FDA approved drug, in cell line models and in one T-LBL primary sample led to cell proliferation reduction and increased apoptosis. Globally, our results identify a new potential prognostic marker and suggest a novel therapeutic approach to overcome therapy resistance in pediatric T-LBL patients.

B cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B lymphocytes from proliferative activity and apoptosis resistance. The increased awareness of the importance of B cell receptor signaling in CLL has raised new opportunities for targeted intervention. Herein, we describe a study performed with the high-throughput RPPA (reverse phase protein array) technique, which allowed us to simultaneously study different molecules in a large series of patients. We analyzed B lymphocytes from 57 patients with CLL and 11 healthy subjects. Different pathways were assessed for activation/expression of key signaling proteins. Data obtained were validated by Western blotting and confocal microscopy. The RPPA investigation and its validation, identified 3 series of proteins: 1) molecules whose expression levels reached statistically significant differences in CLL vs. healthy controls (HSP70, Smac/DIABLO, cleaved PARP, and cleaved caspase-6); 2) proteins with a positive trend of difference in CLL vs. healthy controls (HS1, γ-tubulin, PKC α/β-II Thr-638/641, p38 MAPK Thr-180/Tyr-182, NF-κB Ser-536, Bcl2 Ser-70 and Src Tyr-527); and 3) molecules differentially expressed in patients with IGHV mutations vs. those without mutations (ZAP70, PKC-ζλ, Thr-410/403, and CD45). This study identified some molecules, particularly those involved in apoptosis control, which could be considered for further studies to design new therapeutic strategies in CLL.

ispartof: HAEMATOLOGICA vol:104 issue:1 pages:E17-E20 ispartof: location:Italy status: published

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.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL). The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment. The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death. Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.

PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.

The objective of the study was to quantify the transcriptional profile, as the main T cell lineage-transcription factors on synovial fluid (SF) T cells, in relation to SF cytokines and T cell frequencies (%) of psoriatic arthritis (PsA) patients. Reverse phase protein array was employed to identify interleukin (IL)-23Rp19-, FOXP3- and related orphan receptor gamma T (RORγt)- protein and Janus associated tyrosine kinases 1 (JAK1), signal transducer and activator and transcription 1 (STAT1), STAT3 and STAT5 phosphoproteins in total T cell lysates from SF of PsA patients. IL-1β, IL-2, IL-6, IL-21 and interferon (INF)-γ were measured using a multiplex bead immunoassay in SF from PsA patients and peripheral blood (PB) from healthy controls (HC). Frequencies of CD4(+)CD25(-), CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg, and either mean fluorescence intensity (MFI) of FOXP3(+) on CD4(+) Treg or MFI of classic IL-6 receptor (IL-6R) α expression on CD4(+)CD25(-) helper/effector T cells (Th/eff) and Treg cells, were quantified in SF of PsA patients and in PB from HC by flow cytometry (FC). In PsA SF samples, IL-2, IL-21 and IFN-γ were not detectable, whereas IL-6 and IL-1β levels were higher than in SF of non-inflammatory osteoarthritis patients. Higher levels of IL-23R-, FOXP3- and RORγt proteins and JAK1, STAT1, STAT3 and STAT5 were found in total T cells from SF of PsA patients compared with PB from HC. Direct correlations between JAK1 Y1022/Y1023 and STAT5 Y694, and STAT3 Y705 and IL6, were found in SF of PsA patients. Increased proportion of CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg cells and brighter MFI of IL-6Rα were observed both on CD4(+)CD25(high)- and CD4(+)CD25(-) T cells in PsA SF. The study showed a distinctive JAK1/STAT3/STAT5 transcriptional network on T cells in the joint microenvironment, outlining the interplay of IL-6, IL-23, IL-1β and γC cytokines in the polarization and plasticity of Th17 and Treg cells, which might participate in the perpetuation of joint inflammation in PsA patients.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Treatments1-6 show high response rates but have debilitating effects and carry risk of relapse5,7,8. Previous work implicated NOTCH1 and other oncogenes1,2,9-20. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Here, we demonstrate that ubiquitin-specific protease 7 (USP7)21-32 controls leukemia growth by stabilizing the levels of the NOTCH1 and JMJD3 demethylase. USP7 is overexpressed T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 through deubiquitination. USP7 is bound to oncogenic targets and controls gene expression through H2B ubiquitination and H3K27me3 changes via stabilization of NOTCH1 and JMJD3. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and USP7 inhibition alters associated gene activity. These results provide a new model for deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. USP7 inhibition33 significantly blocked T-ALL cell growth in vitro and in vivo. Our studies also show that USP7 is upregulated in the aggressive high-risk cases of T-ALL and suggest that USP7 expression might be a prognostic marker in ALL and its inhibition could be a therapeutic tool against aggressive leukemia.

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase &alpha, 1 (ChoK&alpha, 1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoK&alpha, 1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.

The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.

A main cause of treatment failure for AML patients is resistance to chemotherapy. Survival of AML cells may depend on mechanisms that elude conventional drugs action and/or on the presence of leukemia initiating cells at diagnosis, and their persistence after therapy. MDR1 gene is an ATP-dependent drug efflux pump known to be a risk factor for the emergence of resistance, when combined to unstable cytogenetic profile of AML patients. In the present study, we analyzed the sensitivity to conventional chemotherapeutic drugs of 26 samples of primary blasts collected from AML patients at diagnosis. Detection of cell viability and apoptosis allowed to identify two group of samples, one resistant and one sensitive to in vitro treatment. The cells were then analyzed for the presence and the activity of P-glycoprotein. A comparative analysis showed that resistant samples exhibited a high level of MDR1 mRNA as well as of P-glycoprotein content and activity. Moreover, they also displayed high PI3K signaling. Therefore, we checked whether the association with signaling inhibitors might resensitize resistant samples to chemo-drugs. The combination showed a very potent cytotoxic effect, possibly through down modulation of MDR1, which was maintained also when primary blasts were co-cultured with human stromal cells. Remarkably, dual PI3K/mTOR inactivation was cytotoxic also to leukemia initiating cells. All together, our findings indicate that signaling activation profiling associated to gene expression can be very useful to stratify patients and improve therapy.

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.

According to the new paradigm shift in toxicity testing, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Moreover, pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to be used in these mechanistic studies. In the present study, we compared the neuronal differentiation propensity of hESCs and hiPSCs aiming to develop tolls for mechanistic neurotoxicity testing. The gene expression and signaling pathway analyses demonstrate the activation of a similar neuronal signature in the two cellular models, particularly indicating that the neuronal survival related cAMP responsive element binding protein (CREB) pathway gets activated upon differentiation. Furthermore, analysis of CREB pathway inhibition, using 2-naphthol-AS-E-phosphate, shows a decrease in neuronal cells as well as an inhibition of neurite outgrowth, synaptogenesis and impairment of electrical activity. These data indicate that inhibition of the CREB pathway can be related to relevant endpoints for neurotoxicity testing in our in vitro cell model, and, as such, qualify the use of this cellular model for mechanism-based toxicity testing, JRC.I.5-Systems Toxicology

// Gloria Milani 1 , Paola Rebora 2 , Benedetta Accordi 1 , Luisa Galla 1 , Silvia Bresolin 1 , Gianni Cazzaniga 3 , Barbara Buldini 1 , Rossella Mura 4 , Saverio Ladogana 5 , Eugenia Giraldi 6 , Valentino Conter 7 , Geertruy Te Kronnie 1 , Maria Grazia Valsecchi 2 and Giuseppe Basso 1 1 Laboratory of Oncohematology, Department of Women’s and Children’s Health, University of Padova, Italy 2 Center of Biostatistics for Clinical Epidemiology, Department of Health Sciences, University of Milano-Bicocca, Monza, Italy 3 Centro Ricerca Tettamanti, University of Milano-Bicocca, Ospedale San Gerardo, Monza, Italy 4 Oncoematologia Pediatrica e Patologia della Coagulazione, Ospedale Regionale per le Microcitemie, Cagliari, Italy 5 Oncoematologia Pediatrica, Ospedale “Casa Solievo della Sofferenza”, San Giovanni Rotondo, Italy 6 U.O. Pediatria, Ospedale Papa Giovanni XXIII, Bergamo, Italy 7 Pediatric Department, University of Milano-Bicocca, Ospedale San Gerardo, Monza, Italy Correspondence: Geertruy te Kronnie, email: // Keywords : Low PKCα expression, Childhood T-cell Acute Lymphoblastic Leukemia, T-ALL prognostic marker Received : April 8, 2014 Accepted : June 5, 2014 Published : June 6, 2014 Abstract Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) outcome has improved in the last decades, yet one patient in every four still relapses. Except treatment response and immunophenotype, few markers are reliably prognostic in pediatric T-ALL patients. Aiming to improve T-ALL risk stratification, we investigated a new candidate biomarker with potential prognostic relevance. A phosphoproteomic screening of 98 pediatric T-ALL samples at diagnosis had been performed using the high-throughput Reverse Phase Protein Arrays technique, which led to the identification of PKCαS657 as an activated protein with a broad variation among T-ALL samples. To evaluate PKCα potential as a prognostic biomarker, PKCα expression was analyzed using RQ-PCR in a cohort of 173 patients, representative of ALL2000-ALLR2006 AIEOP study. A threshold of PKCα expression with the highest discrimination for incidence of relapse was identified. Patients with PKCα down-regulation, compared to patients with PKCα levels above the threshold, presented a markedly increased cumulative incidence of relapse (43.8% vs. 10.9%, P

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Resume Objectifs Cette etude realisee en ouvert est basee sur une approche translationnelle dans le but de detecter les changements cliniques, a l’imagerie et des biomarqueurs synoviaux dans le liquide synovial (LS) et le tissu synovial (TS) dans la spondyloarthropathie peripherique (SpA) apres l’inhibition intra-articulaire (IA) du tumor necrosis factor (TNF)-α par des injections repetees d’etanercept (E). Methodes Vingt-sept patients presentant une synovite resistante du genou ont recu quatre injections IA bihebdomadaires d’E (12,5 mg). Le critere d’evaluation principal etait l’indice du genou de Thompson (THOMP) et les criteres secondaires etaient representes par l’indice articulaire du genou (IAG), la proteine C-reactive (CRP), le health assessment questionnaire-disability index (HAQ-DI), l’epaisseur maximale de la synoviale (EMS) evaluee par echographie et imagerie par resonnance magnetique (IRM) de contraste, le taux serique des cellules mononucleaires dans les TS – CD45+ et – CD31+, le taux dans le LS d’interleukine-1β (IL-1β), le recepteur antagoniste de l’IL-1 (IL-1Ra) et d’IL-6 a l’inclusion et a la fin de l’etude. Resultats A la fin de l’etude, les donnees cliniques, de l’imagerie ainsi que les biomarqueurs ont ete tous significativement reduits et ameliores comparativement aux donnees a l’inclusion. Il y avait une correlation significative entre la clinique, l’imagerie et les biomarqueurs. (la CRP avec le THOMP, l’IAG, le HAQ-DI et le taux d’IL-1Ra ; l’EMS a l’echo avec l’IAG, le TS-CD45+ avec le THOMP, IAG, TS-CD31+ et LS-IL-1β ; le LS-IL-6 avec le THOMP, IAG, LS-IL-1β, ou IL-1Ra). Conclusions Cette etude en « preuve de concept » a demontre une amelioration precoce des scores locaux et systemiques, de l’epaisseur synoviale mesuree par IRM et echographie, et de l’expression des biomarqueurs synoviaux. Le CD45+, CD31+ dans le TS et l’IL-6 et IL-1β dans le LS peuvent etre consideres comme des biomarqueurs potentiels dans la reponse de la SpA peripherique a un agent anti-TNF-α IA.

The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.

Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β1-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.

Colonies of the compound ascidian Botryllus schlosseri undergo recurrent generation changes during which massive, natural apoptosis occurs in zooid tissues: for this reason the species is emerging as an interesting model of invertebrate chordate, phylogenetically related to vertebrates, for studies of apoptosis during development. In the present work, we carried out a series of morphological, cytofluorimetrical and biochemical analyses, useful for a better characterization of Botryllus apoptosis. Results are consistent with the following viewpoints: (i) both intrinsic and extrinsic pathways, probably connected by the BH3-only protein Bid, are involved in cell death induction; (ii) phagocytes, once loaded with senescent cells, frequently undergo apoptosis, probably as a consequence of oxidative stress caused by prolonged respiratory burst, and (iii) senescent phagocytes are easily recognized and ingested by other phagocytes, responsible for their clearance. In addition, results suggest the conservation of apoptosis induction mechanisms throughout chordate evolution.

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4(+)IL-17A-F(+) cells, as well as of T CD4(+) cells expressing IL-23Rp19 (T CD4(+) IL-23R(+)), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 Teff cells in SF of clinically active joints of PsA patients.

Expression of c-MET, the HGF (hepatocyte growth factor) tyrosine kinase receptor, was investigated in pediatric B-acute lymphoblastic leukemia (ALL) patients. c-MET was found to be expressed in normal B cells and in B-ALL patients with the t(12;21) TEL-AML1 translocation, but it is not expressed in the most part of B-ALL without the t(12;21). We also found that c-MET, related to proliferation and protection from apoptosis, is associated with the pro-apoptotic protein FAS in TEL-AML1 B-ALL cells and in normal B lymphocytes. The possible role of this protein complex in drug-induced apoptosis was thus investigated in REH TEL-AML1 B-ALL cell line. REH cells prestimulated with HGF and treated with doxorubicin had shown a higher apoptotic rate than non-HGF-prestimulated ones (p = 0.03). REH cells stimulated with IL-3 and treated with doxorubicin did not undergo apoptosis more than nonstimulated cells, demonstrating that increased proliferation in itself is not directly related to the higher apoptotic sensitivity observed with HGF stimulation. These results indicate that c-MET activation enhances specifically FAS-mediated apoptosis in TEL-AML1 ALL cells and, considering that the c-MET/FAS complex is present only in normal B lymphocytes and in TEL-AML1 leukemias, this implies that it may have an important contribution in cellular homeostasis and in high sensitivity of TEL-AML1 ALL to chemotherapeutic regimens.