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

Dissecting molecular mechanisms of resistance to NOTCH1-targeted therapy in T-cell acute lymphoblastic leukemia xenografts

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors

HDAC INHIBITORS TARGET TRANSCRIPTION FACTORS DEREGULATED IN T-ACUTE LYMPHOBLASTIC LEUKAEMIA

Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors (HDACi) promote apoptosis, induce cell cycle arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. T-cell acute lymphoblastic leukemia (T-ALL) is a pediatric malignancy characterized by clonal expansion of lymphoid progenitors. Although the majority of pediatric T-ALL patients can be cured by current protocols, about one fourth of patients has chemotherapy-resistant disease or relapse after therapy and novel therapeutic approaches are required. In our study, we analyzed the effects of HDACi on seven transcription factors important in T-ALL pathogenesis (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) using both established T-ALL cell lines and patient-derived T-ALL xenografts previously obtained in our laboratory. In particular, we focused on transcription factors that define specific T-ALL subgroups (TAL/LMO, TLX1, TLX3) and we included members of the Notch family (NOTCH1 and NOTCH3) and c-MYB in view of their transversal role in T-ALL. In vitro analysis highlighted transcriptional down-regulation of C-MYB and TAL1, a post-translation regulation of NOTCH1 and NOTCH3 and the regulation of the transcriptional activity of TLX1 and TLX3 following HDAC inhibition. These biochemical effects were linked to increased apoptosis and impaired proliferation both in T-ALL cell lines and patients-derived cells, partially dependent on NOTCH1 and NOTCH3. We next investigated the in vivo effects of an HDACi in T-ALL xenografts belonging to specific T-ALL subgroups. Interestingly, PD-TALL8 (TLX1) and PD-TALL16 (TLX3) had better response to treatment compared to PD-TALL12 and PD-TALL9 (TAL/LMO). In fact, the HDACi dramatically decreased leukemic cells infiltrating the spleen and the bone marrow in TLX-driven xenografts, whereas this drug had modest or minimal effects on TAL/LMO xenografts. Taken together, these results identify TLX1 and TLX3 T-ALL subgroups as potential candidates for therapeutic treatment with HDACi.Le Istone Deacetilasi (HDACs) sono enzimi coinvolti nel rimodellamento della cromatina. Negli ultimi anni è emerso come l’inibizione delle HDACs potrebbe essere utilizzata come strategia per ripristinare l’alterata regolazione epigenetica che si riscontra nei tumori. Infatti, gli inibitori delle HDAC (HDACi) inducono apoptosi, arresto del ciclo cellulare e differenziamento delle cellule tumorali, ma i meccanismi molecolari alla base di questi fenomeni rimangono poco chiari. La leucemia linfoblastica acuta a cellule T (T-ALL) è un tumore pediatrico caratterizzato dall’espansione clonale di progenitori linfoidi. Nonostante la maggioranza dei pazienti pediatrici affetti da T-ALL siano curati in modo efficace utilizzando gli attuali protocolli terapeutici, circa un quarto dei pazienti manifesta resistenza alla terapia o presenta ricadute e dunque emerge la necessità di nuovi approcci terapeutici. In questo studio abbiamo analizzato gli effetti degli HDACi nei confronti di sette fattori di trascrizione implicati nella patogenesi della T-ALL (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) utilizzando sia linee cellulari stabilizzate, sia modelli murini di T-ALL precedentemente sviluppati nel nostro laboratorio a partire da cellule di pazienti. In particolare, ci siamo concentrati su fattori trascrizionali che identificano specifici sottogruppi di T-ALL (TAL/LMO, TLX1 e TLX3) e abbiamo incluso nell’analisi due membri della famiglia dei recettori Notch (NOTCH1 and NOTCH3) e c-MYB in virtù del loro ruolo oncogenico in questa patologia. Le analisi in vitro hanno evidenziato diversi meccanismi di regolazione dei vari fattori da parte degli HDACi. TAL1 e c-MYB risultano regolati a livello trascrizionale, NOTCH1 e NOTCH3 presentano una regolazione post-traduzionale e, nel caso di TLX 1 e TLX3, è presente una regolazione diretta della loro capacità trascrizionale. Gli effetti a livello di proteina si legano all’induzione di apoptosi e all’inibizione della proliferazione sia nelle linee cellulari, sia nelle cellule derivate da paziente e risultano essere parzialmente dovute alla down-modulazione di NOTCH1 e NOTCH3. In seguito siamo andati ad indagare la risposta in vivo di un HDACi in xenografts di T-ALL appartenenti a specifici sottogruppi genetici. E’ interessante notare che il trattamento ha avuto il maggiore risultato nelle PD-TALL8 (TLX1) e nelle PD-TALL16 (TLX3) rispetto alle PD-TALL12 e le PD-TALL9 (entrambe TAL/LMO). Infatti, il trattamento con HDACi negli xenografts di tipo TLX determina una riduzione dell’infiltrazione da parte delle cellule leucemiche nella milza e nel midollo mentre gli effetti ottenuti negli xenografts TAL/LMO risultano modesti o addirittura nulli. In conclusione, i dati ottenuti identificano i pazienti di T-ALL appartenenti ai sottogruppi TLX1 e TLX3 come potenziali candidati per il trattamento a scopo terapeutico con HDACi

HDAC INHIBITORS TARGET TRANSCRIPTION FACTORS DEREGULATED IN T-ACUTE LYMPHOBLASTIC LEUKAEMIA

Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors (HDACi) promote apoptosis, induce cell cycle arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. T-cell acute lymphoblastic leukemia (T-ALL) is a pediatric malignancy characterized by clonal expansion of lymphoid progenitors. Although the majority of pediatric T-ALL patients can be cured by current protocols, about one fourth of patients has chemotherapy-resistant disease or relapse after therapy and novel therapeutic approaches are required. In our study, we analyzed the effects of HDACi on seven transcription factors important in T-ALL pathogenesis (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) using both established T-ALL cell lines and patient-derived T-ALL xenografts previously obtained in our laboratory. In particular, we focused on transcription factors that define specific T-ALL subgroups (TAL/LMO, TLX1, TLX3) and we included members of the Notch family (NOTCH1 and NOTCH3) and c-MYB in view of their transversal role in T-ALL. In vitro analysis highlighted transcriptional down-regulation of C-MYB and TAL1, a post-translation regulation of NOTCH1 and NOTCH3 and the regulation of the transcriptional activity of TLX1 and TLX3 following HDAC inhibition. These biochemical effects were linked to increased apoptosis and impaired proliferation both in T-ALL cell lines and patients-derived cells, partially dependent on NOTCH1 and NOTCH3. We next investigated the in vivo effects of an HDACi in T-ALL xenografts belonging to specific T-ALL subgroups. Interestingly, PD-TALL8 (TLX1) and PD-TALL16 (TLX3) had better response to treatment compared to PD-TALL12 and PD-TALL9 (TAL/LMO). In fact, the HDACi dramatically decreased leukemic cells infiltrating the spleen and the bone marrow in TLX-driven xenografts, whereas this drug had modest or minimal effects on TAL/LMO xenografts. Taken together, these results identify TLX1 and TLX3 T-ALL subgroups as potential candidates for therapeutic treatment with HDACi.Le Istone Deacetilasi (HDACs) sono enzimi coinvolti nel rimodellamento della cromatina. Negli ultimi anni è emerso come l’inibizione delle HDACs potrebbe essere utilizzata come strategia per ripristinare l’alterata regolazione epigenetica che si riscontra nei tumori. Infatti, gli inibitori delle HDAC (HDACi) inducono apoptosi, arresto del ciclo cellulare e differenziamento delle cellule tumorali, ma i meccanismi molecolari alla base di questi fenomeni rimangono poco chiari. La leucemia linfoblastica acuta a cellule T (T-ALL) è un tumore pediatrico caratterizzato dall’espansione clonale di progenitori linfoidi. Nonostante la maggioranza dei pazienti pediatrici affetti da T-ALL siano curati in modo efficace utilizzando gli attuali protocolli terapeutici, circa un quarto dei pazienti manifesta resistenza alla terapia o presenta ricadute e dunque emerge la necessità di nuovi approcci terapeutici. In questo studio abbiamo analizzato gli effetti degli HDACi nei confronti di sette fattori di trascrizione implicati nella patogenesi della T-ALL (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) utilizzando sia linee cellulari stabilizzate, sia modelli murini di T-ALL precedentemente sviluppati nel nostro laboratorio a partire da cellule di pazienti. In particolare, ci siamo concentrati su fattori trascrizionali che identificano specifici sottogruppi di T-ALL (TAL/LMO, TLX1 e TLX3) e abbiamo incluso nell’analisi due membri della famiglia dei recettori Notch (NOTCH1 and NOTCH3) e c-MYB in virtù del loro ruolo oncogenico in questa patologia. Le analisi in vitro hanno evidenziato diversi meccanismi di regolazione dei vari fattori da parte degli HDACi. TAL1 e c-MYB risultano regolati a livello trascrizionale, NOTCH1 e NOTCH3 presentano una regolazione post-traduzionale e, nel caso di TLX 1 e TLX3, è presente una regolazione diretta della loro capacità trascrizionale. Gli effetti a livello di proteina si legano all’induzione di apoptosi e all’inibizione della proliferazione sia nelle linee cellulari, sia nelle cellule derivate da paziente e risultano essere parzialmente dovute alla down-modulazione di NOTCH1 e NOTCH3. In seguito siamo andati ad indagare la risposta in vivo di un HDACi in xenografts di T-ALL appartenenti a specifici sottogruppi genetici. E’ interessante notare che il trattamento ha avuto il maggiore risultato nelle PD-TALL8 (TLX1) e nelle PD-TALL16 (TLX3) rispetto alle PD-TALL12 e le PD-TALL9 (entrambe TAL/LMO). Infatti, il trattamento con HDACi negli xenografts di tipo TLX determina una riduzione dell’infiltrazione da parte delle cellule leucemiche nella milza e nel midollo mentre gli effetti ottenuti negli xenografts TAL/LMO risultano modesti o addirittura nulli. In conclusione, i dati ottenuti identificano i pazienti di T-ALL appartenenti ai sottogruppi TLX1 e TLX3 come potenziali candidati per il trattamento a scopo terapeutico con HDACi

EPIGENETIC REGULATION OF NOTCH AND C-MYB IN T-ACUTE LYMPHOBLASTIC LEUKEMIA

Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors promote apoptosis, induce cell cycle arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. In our studies we observed that treatment of T-Acute Lymphoblastic Leukemia (T-ALL) cell lines with the pan-HDAC inhibitor Trichostatin A (TSA), caused marked reduction of Notch1, Notch3 and c-Myb protein levels. The mRNA expression levels of the two Notch receptors did not change, on the contrary c-Myb transcript decreased. This result suggests that Notch1 and Notch3 could be regulated post-transcriptionally and/or post-translationally following TSA treatment. Blockage of HDAC activity also decreased the expression of Notch target transcripts such as pT\u3b1, CR2 and DTX-1, indicating a general down-regulation of Notch signaling. Moreover, inhibition of HDACs exerts strong pro-apoptotic effects in all cell lines tested (n=3). These findings are confirmed in a panel of primary T-ALL cells from xenografts (n=7) and treated in vitro with TSA, albeit heterogeneous responses were observed. To identify HDAC family member(s) responsible for these effects, we are currently exploiting class specific-HDACi as well as shRNA approaches. At the same time, we are investigating whether increased protein degradation may account for Notch reduction. following TSA treatment. To this end, we treated cells with proteasome and lysosome inhibitors, after TSA treatment. Protein levels of the two Notch receptors were rescued using the lysosome inhibitor chloroquine, suggesting involvement of the endocytic pathway, whereas proteasome inhibitors had minimal effects. Prospectively, HDAC inhibitors could represent a novel therapeutic approach for poor prognosis T-ALL patients, alone or in combination with conventional chemotherap

Involvement of NADPH Oxidase 1 in Liver Kinase B1-mediated effects on tumor angiogenesis and growth

The liver kinase B1 (LKB1) gene is a tumor suppressor with an established role in the control of cell metabolism and oxidative stress. However, whether dis-regulated oxidative stress promotes growth of LKB1-deficient tumors remains substantially unknown. Through in vitro studies, we observed that loss of LKB1 perturbed expression of several genes involved in reactive oxygen species (ROS) homeostasis. In particular, this analysis evidenced strongly up-modulated NADPH oxidase 1 (NOX1) transcript levels in tumor cells lacking LKB1. NOX1 accounted in part for enhanced cytotoxic effects of H2O2-induced oxidative stress in A549 LKB1-deficient tumor cells. Notably, genetic and pharmacologic inhibition of NOX1 activity reduced angiogenesis and growth of A549 tumors in mice. These results suggest that NOX1 inhibitors could counteract ROS production and the angiogenic switch in LKB1-deficient tumors

Targeting NOTCH1 in combination with antimetabolite drugs prolongs life span in relapsed pediatric and adult T-acute lymphoblastic leukemia xenografts

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic tumor, characterized by several genetic alterations, that constitutes 15% of pediatric and 25% of adult ALL. While with current therapeutic protocols children and adults’ overall survival (OS) rates reach 85–90% and 40–50%, respectively, the outcome for both pediatric and adult T-ALL patients that relapse or are refractory to induction therapy, remains extremely poor, achieving around 25% OS for both patient groups. About 60% of T-ALL patients show increased NOTCH1 activity, due to activating NOTCH1 mutations or alterations in its ubiquitin ligase FBXW7. NOTCH signaling has been shown to contribute to chemotherapy resistance in some tumor models. Hence, targeting the NOTCH1 signaling pathway may be an effective option to overcome relapsed and refractory T-ALL. Here, we focused on the therapeutic activity of the NOTCH1-specific monoclonal antibody OMP-52M51 in combination either with drugs used during the induction, consolidation, or maintenance phase in mice xenografts established from pediatric and adult relapsed NOTCH1 mutated T-ALL samples. Interestingly, from RNAseq data we observed that anti-NOTCH1 treatment in vivo affects the purine metabolic pathway. In agreement, both in vitro and in vivo, the greatest effect on leukemia growth reduction was achieved by anti-NOTCH1 therapy in combination with antimetabolite drugs. This result was further corroborated by the longer life span of mice treated with the anti-NOTCH1 in combination with antimetabolites, indicating a novel Notch-targeted therapeutic approach that could ameliorate pediatric and adult T-ALL patients outcome with relapse disease for whom so far, no other therapeutic options are available

An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia