Selective targeting of NOTCH-1 for therapeutic purposes in xenograft models of T-acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an heterogeneous disease, characterized by several genetic alterations and polymorphic clinical features both in children and adults. The Notch pathway, an evolutionary conserved pathway involved in many biological processes including T cell differentiation, has been implicated in the pathogenesis of this disease. Notably, about 50-55% of T-ALL samples show increased Notch1 activity, due to mutations in NOTCH1 or FBW7 genes. Among T-ALL patients, only 70-80% of children and 40% of adults reach long-term remission, therefore new therapeutic approaches are required. Here, we investigated the biologic and therapeutic effects of a human Notch1-specific neutralizing antibody in xenograft models of pediatric T-ALL, obtained from patients with different clinical features and NOTCH1/FBW7 mutational status. We demonstrated that anti-Notch1 treatment greatly delayed engraftment of T-ALL cells bearing NOTCH1/FBW7 mutations, including samples derived from relapsed and clinically difficult-to-treat patients. In these xenografts we observed increased levels of apoptosis, decreased proliferation of leukemic cells and a marked inhibitory effects on Notch transcriptional profile. Moreover, modulation of T-ALL cells metabolism was detected following anti-Notch1 therapy. Serial transplantation experiments suggested that anti-Notch1 therapy could compromise leukemia initiating cell functions and a preliminary experiment showed that resistance may arise in a regimen of continuous administration of anti-Notch1 mAb. Finally, we demonstrated that combination of anti-Notch1 and dexamethasone – a leading drug in T-ALL treatment - could further improve therapeutic effect. Altogether these results indicate that NOTCH1/FBW7 mutations identify suitable candidates for Notch targeted therapy and highlight the potential of Notch target genes and CD7 expression as candidate predictive markers of response to anti-Notch1 therapy

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

AKR1C enzymes sustain therapy resistance in paediatric T-ALL

BACKGROUND: Despite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (TALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors.METHODS: Expression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches.RESULTS: We show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment.CONCLUSIONS: Here, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy

Selective targeting of NOTCH-1 for therapeutic purposes in xenograft models of T-acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an heterogeneous disease, characterized by several genetic alterations and polymorphic clinical features both in children and adults. The Notch pathway, an evolutionary conserved pathway involved in many biological processes including T cell differentiation, has been implicated in the pathogenesis of this disease. Notably, about 50-55% of T-ALL samples show increased Notch1 activity, due to mutations in NOTCH1 or FBW7 genes. Among T-ALL patients, only 70-80% of children and 40% of adults reach long-term remission, therefore new therapeutic approaches are required. Here, we investigated the biologic and therapeutic effects of a human Notch1-specific neutralizing antibody in xenograft models of pediatric T-ALL, obtained from patients with different clinical features and NOTCH1/FBW7 mutational status. We demonstrated that anti-Notch1 treatment greatly delayed engraftment of T-ALL cells bearing NOTCH1/FBW7 mutations, including samples derived from relapsed and clinically difficult-to-treat patients. In these xenografts we observed increased levels of apoptosis, decreased proliferation of leukemic cells and a marked inhibitory effects on Notch transcriptional profile. Moreover, modulation of T-ALL cells metabolism was detected following anti-Notch1 therapy. Serial transplantation experiments suggested that anti-Notch1 therapy could compromise leukemia initiating cell functions and a preliminary experiment showed that resistance may arise in a regimen of continuous administration of anti-Notch1 mAb. Finally, we demonstrated that combination of anti-Notch1 and dexamethasone – a leading drug in T-ALL treatment - could further improve therapeutic effect. Altogether these results indicate that NOTCH1/FBW7 mutations identify suitable candidates for Notch targeted therapy and highlight the potential of Notch target genes and CD7 expression as candidate predictive markers of response to anti-Notch1 therapy.La leucemia linfoblastica acuta a cellule T (T-ALL) è una malattia eterogenea caratterizzata da diverse alterazioni genetiche e caratteristiche cliniche, sia in età pediatrica che adulta. Un ruolo importante in questo tipo di neoplasia è ricoperto dal pathway di Notch, meccanismo evolutivamente conservato coinvolto in numerosi processi biologici tra cui il differenziamento dei linfociti T; difatti in circa il 50-55% dei pazienti affetti da T-ALL si riscontra una mutazione attivante nel gene NOTCH1 o a carico di FBW7. Dal momento che solo il 70-80% dei bambini e il 40% degli adulti affetti da questo tipo di leucemia riesce a raggiungere la remissione a lungo termine, e’ necessario sviluppare ed adottare nuove strategie terapeutiche per poter curare anche i pazienti refrattari alle terapie convenzionali. A questo scopo abbiamo analizzato gli effetti biologici e terapeutici di un anticorpo neutralizzante specifico per il recettore Notch1 umano, avvalendoci di un modello di xenotrapianto di T-ALL. Tale modello è stato generato nel nostro laboratorio utilizzando campioni ottenuti da pazienti pediatrici con caratteristiche cliniche differenti e presentanti diverso stato mutazionale di NOTCH1/FBW7. Il trattamento con anti-Notch1 si è rivelato efficace nel contrastare la crescita della leucemia dei campioni con mutazione di NOTCH1/FBW7, compresi campioni derivati da pazienti in ricaduta o poco responsivi alle terapie convenzionali. In seguito alla somministrazione di anti-Notch1, in questi xenotrapianti abbiamo osservato un aumento dei livelli di apoptosi, una riduzione della proliferazione, un effetto inibitorio molto marcato sui profili trascrizionali dei geni target di Notch e inoltre una modulazione del metabolismo cellulare delle cellule leucemiche. Gli esperimenti di inoculo seriale indicano che la terapia con anti-Notch1 può compromettere la capacità di dare origine a leucemia delle cellule di T-ALL residue dopo il trattamento. Inoltre un esperimento preliminare ha rivelato che la somministrazione continua dell’anticorpo anti-Notch1 può causare l’insorgenza di fenomeni di resistenza alla terapia. Infine abbiamo dimostrato che la combinazione di anti-Notch1 e desametasone, un farmaco comunemente utilizzato nel trattamento delle T-ALL, può ulteriormente migliorare l’efficacia terapeutica. Nel complesso, i nostri risultati indicano che la presenza di mutazioni in NOTCH1/FBW7 identifica dei candidati che potrebbero beneficiare di una terapia mirata contro Notch1 e sottolinea la potenzialità del valutare l’espressione dei geni target di Notch e del CD7 come marcatori predittivi della risposta terapeutica all’anti-Notch1

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

Calcineurin and GSK-3 inhibition sensitizes T-cell acute lymphoblastic leukemia cells to apoptosis through X-linked inhibitor of apoptosis protein degradation

The calcineurin (Cn)-nuclear factor of activated T cells signaling pathway is critically involved in many aspects of normal T-cell physiology; however, its direct implication in leukemogenesis is still ill-defined. Glycogen synthase kinase-3 beta (GSK-3 beta) has recently been reported to interact with Cn in neuronal cells and is implicated in MLL leukemia. Our biochemical studies clearly demonstrated that Cn was able to interact with GSK-3 beta in T-cell acute lymphoblastic leukemia (T-ALL) cells, and that this interaction was direct, leading to an increased catalytic activity of GSK-3 beta, possibly through autophosphorylation of Y216. Sensitivity to GSK-3 inhibitor treatment correlated with altered GSK-3 beta phosphorylation and was more prominent in T-ALL with Pre/Pro immunophenotype. In addition, dual Cn and GSK-3 inhibitor treatment in T-ALL cells promoted sensitization to apoptosis through proteasomal degradation of X-linked inhibitor of apoptosis protein (XIAP). Consistently, resistance to drug treatments in primary samples was strongly associated with higher XIAP protein levels. Finally, we showed that dual Cn and GSK-3 inhibitor treatment in vitro and in vivo is effective against available models of T-ALL, indicating an insofar untapped therapeutic opportunity

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

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