Valentina Serafin

Modulation of microRNA expression in human T-cell development: targeting of NOTCH3 by miR-150.

Ontogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4(+) and single-positive CD8(+) maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4(+) and CD8(+) T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.

Notch3 signalling promotes tumour growth in colorectal cancer

Increased Notch1 activity has been observed in intestinal tumours, partially accomplished by β‐catenin‐mediated up‐regulation of the Notch ligand Jagged‐1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved is unclear. Microarray data indicated that Notch3 transcript levels are significantly up‐regulated in primary and metastatic CRC samples compared to normal mucosa. Moreover, Notch3 protein was expressed at strong/moderate levels by 19.7% of 158 CRC samples analysed, and at weak levels by 51.2% of the samples. Intrigued by these findings, we sought to investigate whether Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumourigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including Notch3, Delta‐like 4 (DLL4), and Jagged‐1 ligands. Stimulation with immobilized recombinant DLL4 or transduction with DLL4‐expressing vectors dramatically increased Notch3 expression in CRC cells, associated with accelerated tumour growth. Forced expression of an active form of Notch3 mirrored the effects of DLL4 stimulation and increased tumour formation. Conversely, attenuation of Notch3 levels by shRNA resulted in perturbation of the cell cycle followed by reduction in cell proliferation, clonogenic capacity, and inhibition of tumour growth. Altogether, these findings indicate that Notch3 can modulate the tumourigenic properties of CRC cells and contributes to sustained Notch activity in DLL4‐expressing tumours. Copyright

NOTCH3 Signaling Regulates MUSASHI-1 Expression in Metastatic Colorectal Cancer Cells

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.

AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells

The serine/threonine kinase AMP-activated protein kinase (AMPK) and its downstream effectors, including endothelial nitric oxide synthase and BCL-2, are hyperactivated in B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) cells with MLL gene rearrangements. We investigated the role of activated AMPK in supporting leukemic cell survival and evaluated AMPK as a potential drug target. Exposure of leukemic cells to the commercial AMPK inhibitor compound C resulted in massive apoptosis only in cells with MLL gene rearrangements. These results were confirmed by targeting AMPK with specific short hairpin RNAs. Compound C-induced apoptosis was associated with mitochondrial membrane depolarization, reactive oxygen species production, cytochrome c release and caspases cleavage, indicating intrinsic apoptosis pathway activation. Treatment with low concentrations of compound C resulted in a strong antileukemic activity, together with cytochrome c release and cleavage of caspases and poly(ADP-ribose) polymerase, also in MLL-rearranged primary BCP-ALL samples. Moreover, AMPK inhibition in MLL-rearranged cell lines synergistically enhanced the antiproliferative effects of vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase in most of the evaluated conditions. Taken together, these results indicate that the activation of the AMPK pathway directly contributes to the survival of MLL-rearranged BCP-ALL cells and AMPK inhibitors could represent a new therapeutic strategy for this high-risk leukemia.

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

Interleukin‐22 in the diagnosis of active chronic graft‐versus‐host disease in paediatric patients

Burger, J.A., Ghia, P., Rosenwald, A. & CaligarisCappio, F. (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood, 114, 3367–3375. Chen, L., Huynh, L., Apgar, J., Tang, L., Rassenti, L., Weiss, A. & Kipps, T.J. (2008) ZAP-70 enhances IgM signaling independent of its kinase activity in chronic lymphocytic leukemia. Blood, 111, 2685–2692. Crespo, M., Bosch, F., Villamor, N., Bellosillo, B., Colomer, D., Rozman, M., Marce, S., Lopez-Guillermo, A., Campo, E. & Montserrat, E. (2003) ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. The New England Journal of Medicine, 348, 1764–1775. Gobessi, S., Laurenti, L., Longo, P.G., Sica, S., Leone, G. & Efremov, D.G. (2007) ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells. Blood, 109, 2032–2039. ten Hacken, E. & Burger, J.A. (2014) Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia–focus on the B-cell receptor. Clinical Cancer Research, 20, 548–556. Lafarge, S.T., Johnston, J.B., Gibson, S.B. & Marshall, A.J. (2014) Adhesion of ZAP-70 + chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway. Leukemia Research, 38, 109–115. Lagneaux, L., Delforge, A., Bron, D., De Bruyn, C. & Stryckmans, P. (1998) Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood, 91, 2387– 2396. Stamatopoulos, B., Haibe-Kains, B., Equeter, C., Meuleman, N., Soree, A., De Bruyn, C., Hanosset, D., Bron, D., Martiat, P. & Lagneaux, L. (2009) Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA. Haematologica, 94, 790–799. Vroblova, V., Smolej, L. & Krejsek, J. (2012) Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia.Hematology, 17, 268–274. Woyach, J.A., Johnson, A.J. & Byrd, J.C. (2012) The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood, 120, 1175–1184.

EB-3D a novel choline kinase inhibitor induces deregulation of the AMPK-mTOR pathway and apoptosis in leukemia T-cells

Graphical abstract Figure. No caption available. &NA; Choline kinase alpha 1 (ChoK&agr;1) 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 ChoK&agr; 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 &;, 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 ChoK&agr; may be an interesting option for treating T‐ALL and that EB‐3D could represent a valuable therapeutic tool.

Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia

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.

Epigenetic heterogeneity affects the risk of relapse in children with t(8;21)RUNX1-RUNX1T1-rearranged AML.

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.

Preclinical evaluation of second generation PIM inhibitors for the treatment of T-cell acute lymphoblastic leukemia and lymphoma

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are aggressive hematologic malignancies that are currently still treated by highdose chemotherapy possibly followed by hematopoietic stem cell transplantation. Despite important progress in deciphering the genomic landscape of these diseases, the transition of novel targeted therapies towards clinical practice has remained largely unsuccessful. T-ALL and TLBL, which the World Health Organization regards as one single disease entity, can be classified into different molecular genetic subtypes based on the aberrant expression of specific transcription factor oncogenes, such as LYL1, TLX1, TLX3, HOXA, NKX2-1, TAL1 or LMO2. Within these genetic subclasses, a variety of co-operative lesions converge towards activation of specific pathways, such as JAK-STAT or PI3K-AKT signaling. Interestingly, JAK-STAT pathway mutations seem to be more prevalent in LYL1, TLX1, TLX3 and HOXA tumors, whereas mature TAL1 leukemias/lymphomas more often display PI3K/AKT alterations. PIM1 is a highly conserved serine/threonine kinase involved in cell-cycle progression, transcription, apoptosis, drug resistance and cellular metabolism through phosphorylation of a myriad of known downstream targets. Formal proof of its oncogenic activity emerged from the analysis of Pim1 transgenic mice, which spontaneously developed T-cell lymphomas with a latency of several months. At the transcriptional level, PIM1 is a canonical JAK-STAT target gene that can be activated downstream of cytokine signaling. Recently, a number of studies have all shown that PIM1 might act as an attractive molecular target in human T-ALL. Indeed, recent work identified a case of adult T-ALL in which aberrant activation of PIM1 was driven by the T-cell receptor (TCR) translocation t(6;7)(p21;q34) (TCRβ-PIM1). In addition, PIM1 activation was also found to be more broadly implicated in T-ALL disease biology downstream of mutational activation of the JAK-STAT signaling pathway. Although these studies clearly point towards PIM1 as a novel therapeutic target for the treatment of T-ALL, initial drug evaluation experiments have largely been focused on human T-ALL and T-LBL cell lines, which often fail to provide an accurate representation of the primary disease. Therefore, additional in-depth pre-clinical in vivo evaluation of PIM inhibitors using patient-derived xenograft models of human T-ALL and T-LBL will be required to further facilitate the translation of these findings into clinical practice in the near future. Here, we report the identification of a similar TCRβPIM1 translocation, t(6;7)(p21;q34), as previously described, in a case of pediatric T-LBL, suggesting that these PIM1 rearrangements are a rare but recurrent genetic abnormality in both pediatric and adult T-ALL and T-LBL. For this particular T-LBL case (see the Online Supplementary Methods for clinical information), initial FISH analysis revealed the presence of a TCRβ translocation in the major leukemic clone at diagnosis. Using Targeted Locus Amplification (TLA), with the TCRβ