Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pT alpha, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in TALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of TALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumor

Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis

Studio del ruolo dei microRNA nella maturazione e nella leucemogenesi T

Maturation of T cells occurs through a series of steps and is driven by factors and mechanisms which remain poorly defined. Recent work shows that some microRNAs (miRs) are dynamically regulated in their expression during hematopoietic development, inflammation and leukemogenesis. However, little is known regarding the possible role of miRs during the physiologic development of human T cells or their significance in T cell-derived malignancies. In order to identify miRs which could be involved in the differentiation of T lymphocytes, we analyzed the expression profile of miRs in human T cell progenitors at different stages of maturation: Double Positive (DP; CD4+CD8+), Single Positive CD4+ (SP CD4; CD4+CD8-) and Single Positive CD8+ (SP CD8, CD4-CD8+). In parallel, in order to deepen our knowledge about microRNA expression in T lymphoid cells, smallRNA libraries were generated from total RNA of unsorted thymocytes, DP thymocytes, CD4+ and CD8+ mature peripheral blood lymphocytes. The array-based analysis showed that each thymic population displays a distinct miR expression profile, which reflected the developmental relationships between maturation stages in thymocytes. A general up-regulation of miR expression was observed in T precursors during the maturation from DP to SP stage. The generation of small-RNA libraries enabled us to study the expression of both known and new candidate miRs in different T lymphoid populations. In order to identify known and new candidate miRNAs among the short-RNA sequences, a computational pipeline was developed. Computational analysis of the 29.744 small-RNA sequences obtained from our libraries led to the identification of 139 sequences corresponding to known miRs and 98 sequences of new candidate miRs. A bootstrap analysis estimated that the sequence data covered more than 80% of the total content of miRs in the 4 libraries. The analysis of the libraries confirmed the general up-regulation of miR expression during T cell maturation. By comparing the array and sequence data, we identified a group of known miRs which were consistently regulated during normal T cell maturation. The modulation of the expression during T cell differentiation of some of these miR, such as miR-150, was further validated by qRT-PCR. Subsequently, for this subset of miR, we searched for potential target genes by crossing the results of 3 different target prediction softwares (Miranda, TargetScan and PicTar). Among the predicted targets for this group of miRs we found many genes involved in biologically relevant processes, including cell cycle regulation, apoptosis, differentiation and tumorigenesis. Moreover, we compared the gene expression profiles from thymic subpopulations with the list of computationally predicted targets for the most significantly regulated miRs between mature SP and DP thymocytes. By combining this approach to the pure bioinformatic approach, we identified a gene of the family of Notch receptors (Notch3), referred to as Candidate Target 1, which is known to be important in T cell differentiation and in leukemic transformation and which is concordantly predicted by 3 different target prediction softwares as a target of miR-150, one of the top up-regulated miR during T cell maturation from DP to SP stage. Moreover, Candidate Target 1 is regulated in thymocytes maturation in the opposite direction compared to miR-150. In particular, we identified a high complementarity between miR-150 and the 3’UTR of Candidate Target 1. We are now working in order to validate experimentally the association between miR-150 and Candidate Target 1. In parallel, we decided to study the functional effects of miR-150 over-expression in T-ALL cell lines. MiR-150 is expressed at very low levels in all the T-ALL cell lines tested. Interestingly, when we forced the expression of miR-150 in Jurkat T-ALL cells, we observed a significant reduction of the proliferation rate associated with the accumulation of cells in the G2 phase of cell cycle. Eventually, in order to identify patterns of miR expression that can be associated with malignant transformation, we compared the miR expression profiles of human thymocyte subpopulations to the profile of a group of T-cell lymphoblastic lymphomas (T-LBL) of childhood and to that of a group of reactive non-neoplastic lymph nodes (LN) (provided by Dr. Rosolen laboratory, Department of Pediatrics, University of Padova). The hierarchical clustering of the samples indicated that TLBLs have a group of miRs which are differentially expressed both in respect to reactive lymph nodes and to thymic healthy subsets. Interestingly, we observed that all the top 25 differentially regulated miRs during T differentiation from DP to SP stage (excluding miR-128) show a significant differential expression in T-LBL compared to at least one of the thymus populations. In the future, we plan to investigate the biological significance of some of the miRs regulated in the maturation and neoplastic transformation of T cells, playing particular attention to miR-150 role in these processes.La maturazione delle cellule T avviene attraverso una serie di complesse modificazioni fenotipiche e genotipiche ed è guidata da fattori e meccanismi in parte ancora poco compresi. Recenti lavori hanno mostrato che l'espressione di alcuni microRNA (miR) è dinamicamente regolata nel corso dello sviluppo ematopoietico, della risposta immunitaria e della leucemogenesi. Tuttavia, attualmente, è ancora in gran parte sconosciuto il ruolo dei miR nello sviluppo fisiologico delle cellule T, nonché il significato della loro alterata espressione nella leucemogenesi T. Allo scopo di identificare miR potenzialmente coinvolti nella differenziazione dei linfociti T, abbiamo analizzato il profilo d’espressione dei miR in timociti umani a diversi stadi di maturazione: Doppi Positivi (DP; CD4+CD8+), Singoli Positivi CD4+ (SP CD4; CD4+CD8-) e Singoli Positivi CD8+ (SP CD8, CD4-CD8+). Parallelamente, al fine di approfondire la nostra conoscenza sull’espressione dei miR nelle popolazioni linfoidi T, abbiamo generato delle librerie di small-RNA a partire dall’RNA totale di timociti non frazionati, timociti DP, linfociti maturi CD4+ e CD8+ del sangue periferico. L’analisi dei dati degli array ha mostrato che ogni popolazione timica presenta un profilo d’espressione dei miR caratteristico e distinto, che riflette le relazioni fra gli stadi di sviluppo dei precursori T. Nel corso della maturazione dei precursori T dallo stadio DP a quello SP si osserva una generale up-regolazione dell’espressione dei miR. La generazione delle librerie di small-RNA ci ha permesso di studiare l’espressione sia dei miR noti, che di nuovi candidati miR nelle diverse popolazioni linfoidi T. Al fine di identificare i miR noti e nuovi potenziali fra le sequenze delle librerie di small-RNA, è stata sviluppata una pipeline bioinformatica. L’analisi computazionale delle 29.744 sequenze di small-RNA ricavate dalle nostre librerie ha portato all’identificazione di 139 sequenze corrispondenti a miR noti e 98 sequenze di candidati nuovi miR. Mediante un'analisi bootstrap, è stato calcolato che, per tutte e 4 le librerie, il set di miR maturi sequenziati rappresenta più dell'80% della totalità dei miR che si stima siano espressi nei campioni. L’analisi delle librerie ha confermato la generale up-regolazione dell’espressione dei miR nel corso della maturazione delle cellule T. Comparando i dati degli array e del sequenziamento delle librerie, è stato individuato un gruppo di miR noti che sono consistentemente regolati durante la differenziazione T. Il pattern di espressione nei diversi stadi di sviluppo T di alcuni di questi miR, tra cui miR-150, è stato validato mediante qRT-PCR. In seguito, abbiamo fatto una ricerca dei target potenziali di questi miR integrando i risultati di 3 diversi software di predizione di target (Miranda, TargetScan and PicTar). Fra i candidati target del gruppo di miR d’interesse sono stati identificati molti geni coinvolti in processi biologici rilevanti, come la regolazione del ciclo cellulare, l’apoptosi, il differenziamento e la tumorigenesi. Inoltre, abbiamo confrontato i profili di espressione genica delle popolazioni timocitarie con la lista di target predetti computazionalmente per i miR regolati con maggiore fold-change nel corso della differenziazione dei timociti da DP a SP. Combinando quest’ultimo approccio alla ricerca bioinformatica integrata di target, abbiamo identificato un gene della famiglia dei recettori Notch (Notch3), definito Candidate Target 1, che è noto giocare un ruolo importante nella differenziazione T e nella trasformazione leucemica e che viene predetto, in modo concorde da tre diversi software di predizione di target, come bersaglio di miR-150, uno dei miR maggiormente up-regolati nelle popolazioni timocitarie mature rispetto ai DP. Inoltre, il trascritto di Candidate Target 1 risulta regolato in modo opposto al miR-150 nel passaggio dei timociti dallo stadio DP a quello di SP CD4. In particolare, abbiamo identificato un'elevata complementarietà fra il miR-150 e l'UTR-3' del gene Candidate Target 1. Attualmente stiamo lavorando per validare sperimentalemente l’associazione fra miR-150 e Candidate Target 1. In parallelo, abbiamo deciso di studiare gli effetti funzionali dell’over-espressione di miR-150 in linee cellulari di T-ALL. MiR-150 è espresso a livelli molto bassi in tutte le linee cellulari di T-ALL analizzate. Inducendo l’espressione forzata di miR-150 nella linea di T-ALL Jurkat, abbiamo osservato un significativo rallentamento del tasso di proliferazione cellulare associato ad un accumulo delle cellule in fase G2. Infine, allo scopo di identificare pattern di espressione dei miR associati alla trasformazione neoplastica T, abbiamo confrontato i profili d’espressione dei miR delle sottopopolazioni timocitarie umane con il proflo di un gruppo di linfomi linfoblastici T (T-LBL) pediatrici ed un gruppo di linfonodi reattivi non neoplastici (LN) (forniti dal laboratorio del Dott. Rosolen, Dipartimento di Pediatria, Università di Padova). Il clustering gerarchico dei campioni ha mostrato che i T-LBL hanno un profilo d’espressione dei miR distinto sia da quello delle sottopopolazioni timocitarie, sia da quello dei linfonodi reattivi. Abbiamo inoltre osservato che tutti i 25 miR maggiormente regolati nel passaggio dei timociti dallo stadio DP a SP (a parte miR-128) risultano espressi in modo differenziale nei T-LBL rispetto ad almeno una delle popolazioni timiche. Nel futuro, ci proponiamo di investigare il significato biologico di alcuni dei miR regolati nella maturazione e la trasformazione neoplastica delle cellule T, ponendo particolare attenzione al ruolo di miR-150 in questi processi

Id2 represses E2A-mediated activation of IL-10 expression in T cells

Interleukin-10 (IL-10) is a key immunoregulatory cytokine that functions to prevent inflammatory and autoimmune diseases. Despite the critical role for IL-10 produced by effector CD8 T cells during pathogen infection and autoimmunity, the mechanisms regulating its production are poorly understood.Weshowthat loss of the inhibitor ofDNA binding 2 (Id2) in T cells resulted in aberrant IL-10 expression in vitro and in vivo during influenza virus infection and in a model of acute graft-versus-host disease (GVHD). Furthermore, IL-10 overproduction substantially reduced the immunopathology associated with GVHD. We demonstrate that Id2 acts to repress the E2A-mediated transactivation of the Il10 locus. Collectively, our findings uncover a key regulatory role of Id2 during effector T cell differentiation necessary to limit IL-10 production by activated T cells and minimize their suppressive activity during the effector phase of disease control

The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Abstract Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy. Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation. Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. Disclosures No relevant conflicts of interest to declare

Id2 and E Proteins Orchestrate the Initiation and Maintenance of MLL-Rearranged Acute Myeloid Leukemia

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