Repurposing Verapamil to Enhance Killing of T-ALL Cells by the mTOR Inhibitor Everolimus.

New therapies are needed for patients with T-cell lymphoblastic leukemia (T-ALL) who do not respond to standard chemotherapy. Our previous studies showed that the mTORC1 inhibitor everolimus increases reactive oxygen species (ROS) levels, decreases the levels of NADPH and glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and induces apoptosis in T-ALL cells. Studies in T-ALL-xenografted NOD/SCID mice demonstrated that everolimus improved their response to the glucocorticoid (GC) dexamethasone. Here we show that verapamil, a calcium antagonist used in the treatment of supraventricular tachyarrhythmias, enhanced the effects of everolimus on ROS and cell death in T-ALL cell lines. The death-enhancing effect was synergistic and was confirmed in assays on a panel of therapy-resistant patient-derived xenografts (PDX) and primary samples from T-ALL patients. The verapamil-everolimus combination produced a dramatic reduction in the levels of G6PD and induction of p38 MAPK phosphorylation. Studies of NOD/SCID mice inoculated with refractory T-ALL PDX cells demonstrated that the addition of verapamil to everolimus plus dexamethasone significantly reduced tumor growth in vivo. Taken together, our results provide a rationale for repurposing verapamil in association with mTORC inhibitors and GC to treat refractory T-ALL.

MiR-150 in HTLV-1 infection and T-cell transformation.

Human T-cell leukemia virus-1 (HTLV-1) is a retrovirus that persistently infects CD4+ T-cells, and is the causative agent of adult T-cell leukemia/lymphoma (ATLL), tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) and several inflammatory diseases. T-cell transformation by HTLV-1 is driven by multiple interactions between viral regulatory proteins and host cell pathways that govern cell proliferation and survival. Studies performed over the last decade have revealed alterations in the expression of many microRNAs in HTLV-1-infected cells and ATLL cells, and have identified several microRNA targets with roles in the viral life cycle and host cell turnover. This review centers on miR-150-5p, a microRNA whose expression is temporally regulated during lymphocyte development and altered in several hematological malignancies. The levels of miR-150-5p are reduced in many HTLV-1-transformed- and ATLL-derived cell lines. Experiments in these cell lines showed that downregulation of miR-150-5p results in activation of the transcription factor STAT1, which is a direct target of the miRNA. However, data on miR-150-5p levels in freshly isolated ATLL samples are suggestive of its upregulation compared to controls. These apparently puzzling findings highlight the need for more in-depth studies of the role of miR-150-5p in HTLV-1 infection and pathogenesis based on knowledge of miR-150-5p-target mRNA interactions and mechanisms regulating its function in normal leukocytes and hematologic neoplasms.

mTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells.

mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing. In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia. These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.

The miR-200 Family of microRNAs: Fine Tuners of Epithelial-Mesenchymal Transition and Circulating Cancer Biomarkers.

The miR-200 family of microRNAs (miRNAs) includes miR-200a, miR-200b, miR-200c, miR-141 and miR-429, five evolutionarily conserved miRNAs that are encoded in two clusters of hairpin precursors located on human chromosome 1 (miR-200b, miR-200a and miR-429) and chromosome 12 (miR-200c and miR-141). The mature -3p products of the precursors are abundantly expressed in epithelial cells, where they contribute to maintaining the epithelial phenotype by repressing expression of factors that favor the process of epithelial-to-mesenchymal transition (EMT), a key hallmark of oncogenic transformation. Extensive studies of the expression and interactions of these miRNAs with cell signaling pathways indicate that they can exert both tumor suppressor- and pro-metastatic functions, and may serve as biomarkers of epithelial cancers. This review provides a summary of the role of miR-200 family members in EMT, factors that regulate their expression, and important targets for miR-200-mediated repression that are involved in EMT. The second part of the review discusses the potential utility of circulating miR-200 family members as diagnostic/prognostic biomarkers for breast, colorectal, lung, ovarian, prostate and bladder cancers.

Prognostic Stratification of Metastatic Prostate Cancer Patients Treated With Abiraterone and Enzalutamide Through an Integrated Analysis of Circulating Free microRNAs and Clinical Parameters.

Androgen Receptor-Targeted Agents (ARTA) have dramatically changed the therapeutic landscape of metastatic Castration-Resistant Prostate Cancer (mCRPC), but 20-40% of these patients progress early after start of ARTA treatment. The present study investigated the potential utility of plasma cell-free microRNAs (cfmiRNAs) as prognostic markers by analyzing a prospective cohort of 31 mCRCP patients treated with abiraterone (N = 10) or enzalutamide (N = 21). Additional potential prognostic factors were extracted from clinical records and outcome was evaluated as overall survival (OS) and progression-free survival (PFS). cfmiRNAs were measured in plasma samples using quantitative real-time RT-PCR. Linear correlation among clinical factors and cfmiRNAs was assessed using the Spearman's rank correlation coefficient. The association with survival was studied using univariate and multivariate Cox proportional hazards models. Continuous variables were dichotomized with the cut points corresponding to the most significant relation with the outcome. Univariate analysis indicated that plasma levels of miR-21-5p, miR-141-3p and miR-223-3p, time to development of castration-resistance (tCRPC), and blood hemoglobin (Hb) levels strongly correlated with both PFS and OS. Multivariate analysis revealed that low plasma levels of miR-21, shorter tCRPC, and lower Hb values were independent factors predicting reduced PFS and OS. These findings suggest that the integrated analysis of cfmiRNAs, tCRPC, and Hb may provide a promising, non-invasive tool for the prognostic stratification of mCRPC patients treated with ARTA.

Near infrared photoimmunotherapy targeting the cutaneous lymphocyte antigen for mycosis fungoides.

Background: Mycosis fungoides (MF) is a low-grade T-cell lymphoma with primary cutaneous involvement accounting for more than half of all primary cutaneous lymphomas. The treatment of MF is very challenging due to the limited therapies available. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, the hydrophilic phthalocyanine IRdye 700DX® (IR700), and near infrared light. In this study, we investigated the effect of NIR-PIT on MF targeting the cell-surface antigen cutaneous lymphocyte antigen (CLA)Matherial and methods: MF derived My-La CD4+ cells were incubated with the anti-CLA antibody conjugated to IR700 and then irradiated with a 690 nm near-infrared light. Cell death was evaluated by propidium iodide staining and flow cytometry 24 hours after irradiation.Results: Treatment with anti-CLA or light irradiation exhibited very modest pro-death effects, whereas treatment with the anti-CLA antibody conjugated to IR700 and then irradiation with a 690 nm near-infrared light induced a substantial increase in death in the MF cell line.Conclusions: NIR-PIT targeting CLA to treat MF showed marked antitumour effects. As such, CLA-targeted NIR-PIT could be a promising treatment for MF and, possibly, other cutaneous diseases characterized by CLA+ skin infiltrating T-cells.

Prognostic Stratification of Bladder Cancer Patients with a MicroRNA-based Approach.

Robust non-invasive tests for prognostic stratification of bladder cancer (BCa) patients are in high demand. Following a comprehensive analysis of studies on BCa, we selected a panel of 29 microRNAs (miRNAs) and analyzed their levels in urine and plasma samples in a prospective cohort of 63 BCa patients (32 at high risk of recurrence and 31 low-risk cases) and 37 healthy controls using RT-qPCR. To design an assay suitable for large-scale testing, we applied a hierarchical pipeline to select the miRNAs that were not affected by confounding factors such as haematuria and urine specific gravity, and exceeded stringent cut-off criteria (fold change >2.5 and p-value < 0.005). Using a two-step decision tree based on the urine levels of miR-34a-5p, miR-200a-3p and miR-193a-5p, normalized against miR-125b-5p, patients could be classified as high- or low-risk with a sensitivity of 0.844, specificity of 0.806 and accuracy of 0.825. Furthermore, univariate Cox proportional hazards regression analyses indicated that increased urine levels of miR-29a-3p, miR-34a-5p, miR-193a-5p, miR-200c-3p, miR-205-5p and miR-532-5p were associated with a shorter event-free survival (hazard ratios > 3.1, p-value < 0.05). Taken together, our findings suggest that measuring the urine levels of these miRNAs could provide a novel cost-effective, noninvasive test for risk assessment of BCa patients.

Functional properties and sequence variation of HTLV-1 p13.

Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K+) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

Nanoparticles as Tools to Target Redox Homeostasis in Cancer Cells.

Reactive oxygen species (ROS) constitute a homeostatic rheostat that modulates signal transduction pathways controlling cell turnover. Most oncogenic pathways activated in cancer cells drive a sustained increase in ROS production, and cancer cells are strongly addicted to the increased activity of scavenging pathways to maintain ROS below levels that produce macromolecular damage and engage cell death pathways. Consistent with this notion, tumor cells are more vulnerable than their normal counterparts to pharmacological treatments that increase ROS production and inhibit ROS scavenging. In the present review, we discuss the recent advances in the development of integrated anticancer therapies based on nanoparticles engineered to kill cancer cells by raising their ROS setpoint. We also examine nanoparticles engineered to exploit the metabolic and redox alterations of cancer cells to promote site-specific drug delivery to cancer cells, thus maximizing anticancer efficacy while minimizing undesired side effects on normal tissues.

Targeting the cutaneous lymphocyte antigen (CLA) in inflammatory and neoplastic skin conditions.

Introduction: The cutaneous lymphocyte antigen interacts with E-selectin on endothelial cells and is expressed on 15% of circulating T-cells. Skin-homing T-cells express the cutaneous lymphocyte antigen and play a role in local cutaneous immunity in inflammatory reactions and neoplastic conditions.Areas covered: Lymphocyte extravasation is the essential para-physiological mechanism enabling immune surveillance of tissues for tumors as well as effector cell recruitment to inflammatory sites.The authors focused on skin inflammatory disorders, on cutaneous lymphoproliferative disease, and on other skin malignancies.Expert opinion: Interfering with leukocyte extravasation has been regarded as an attractive strategy in skin disorders, in the past for inflammatory conditions and more recently for cutaneous T-cell lymphomas. Therapeutic blocking of skin-homing interactions has been attempted in psoriasis and atopic dermatitis and has been achieved in the treatment of cutaneous T-cell lymphomas. Cutaneous lymphocyte antigen is a potential molecular target for both systemic and skin-directed therapy for cutaneous T-cell lymphomas.

Metabolic rewiring and redox alterations in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare malignancy of mesothelial cells with increasing incidence, and in many cases, dismal prognosis due to its aggressiveness and lack of effective therapies. Environmental and occupational exposure to asbestos is considered the main aetiological factor for MPM. Inhaled asbestos fibres accumulate in the lungs and induce the generation of reactive oxygen species (ROS) due to the presence of iron associated with the fibrous silicates and to the activation of macrophages and inflammation. Chronic inflammation and a ROS-enriched microenvironment can foster the malignant transformation of mesothelial cells. In addition, MPM cells have a highly glycolytic metabolic profile and are positive in 18F-FDG PET analysis. Loss-of-function mutations of BRCA-associated protein 1 (BAP1) are a major contributor to the metabolic rewiring of MPM cells. A subset of MPM tumours show loss of the methyladenosine phosphorylase (MTAP) locus, resulting in profound alterations in polyamine metabolism, ATP and methionine salvage pathways, as well as changes in epigenetic control of gene expression. This review provides an overview of the perturbations in metabolism and ROS homoeostasis of MPM cells and the role of these alterations in malignant transformation and tumour progression.

Oncogenic pathways and the electron transport chain: a dangeROS liaison.

Driver mutations in oncogenic pathways, rewiring of cellular metabolism and altered ROS homoeostasis are intimately connected hallmarks of cancer. Electrons derived from different metabolic processes are channelled into the mitochondrial electron transport chain (ETC) to fuel the oxidative phosphorylation process. Electrons leaking from the ETC can prematurely react with oxygen, resulting in the generation of reactive oxygen species (ROS). Several signalling pathways are affected by ROS, which act as second messengers controlling cell proliferation and survival. On the other hand, oncogenic pathways hijack the ETC, enhancing its ROS-producing capacity by increasing electron flow or by impinging on the structure and organisation of the ETC. In this review, we focus on the ETC as a source of ROS and its modulation by oncogenic pathways, which generates a vicious cycle that resets ROS levels to a higher homoeostatic set point, sustaining the cancer cell phenotype.

NF-κB and MicroRNA Deregulation Mediated by HTLV-1 Tax and HBZ.

The risk of developing adult T-cell leukemia/lymphoma (ATLL) in individuals infected with human T-cell lymphotropic virus 1 (HTLV-1) is about 3-5%. The mechanisms by which the virus triggers this aggressive cancer are still an area of intensive investigation. The viral protein Tax-1, together with additional regulatory proteins, in particular HTLV-1 basic leucine zipper factor (HBZ), are recognized as relevant viral factors required for both viral replication and transformation of infected cells. Tax-1 deregulates several cellular pathways affecting the cell cycle, survival, and proliferation. The effects of Tax-1 on the NF-κB pathway have been thoroughly studied. Recent studies also revealed the impact of Tax-1 and HBZ on microRNA expression. In this review, we summarize the recent progress in understanding the contribution of HTLV-1 Tax- and HBZ-mediated deregulation of NF-κB and the microRNA regulatory network to HTLV-1 pathogenesis.

Post-transcriptional Regulation of HTLV Gene Expression: Rex to the Rescue.

Human T-lymphotropic virus type 1 (HTLV-1) and other members of the Deltaretrovirus genus code for a regulatory protein named Rex that binds to the Rex-responsive element present on viral mRNAs. Rex rescues viral mRNAs from complete splicing or degradation and guides them to the cytoplasm for translation. The activity of Rex is essential for expression of viral transcripts coding for the virion components and thus represents a potential target for virus eradication. We present an overview of the functional properties of the HTLV-1 and HTLV-2 Rex proteins (Rex-1 and Rex-2), outline mechanisms controlling Rex function, and discuss similarities and differences in the sequences of Rex coded by HTLV-1, -2, -3, and -4 that may influence their molecular anatomy and functional properties.

Liquid Biopsy in Malignant Pleural Mesothelioma: State of the Art, Pitfalls, and Perspectives.

Malignant pleural mesothelioma (MPM) is an aggressive tumor linked to asbestos exposure. Although the risk factors for MPM are well-known, the majority of MPM patients are diagnosed at an advanced stage and have a very poor prognosis. Circulating biomarkers for early diagnosis remain to be identified, and the current standard for MPM diagnosis relies on pleural biopsies. Robust non-invasive tests for the screening of asbestos-exposed subjects are therefore an important unmet clinical need. This review provides a critical summary of recent liquid biopsy-based studies aimed at discovering novel blood-based circulating biomarkers for the early diagnosis and prognostic stratification of MPM patients.

TRAF3 Is Required for NF-κB Pathway Activation Mediated by HTLV Tax Proteins.

Human T-cell leukemia viruses type 1 (HTLV-1) and type 2 (HTLV-2) share a common genome organization and expression strategy but have distinct pathological properties. HTLV-1 is the etiological agent of Adult T-cell Leukemia (ATL) and of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), whereas HTLV-2 does not cause hematological disorders and is only sporadically associated with cases of subacute myelopathy. Both HTLV genomes encode two regulatory proteins that play a pivotal role in pathogenesis: the transactivating Tax-1 and Tax-2 proteins and the antisense proteins HBZ and APH-2, respectively. We recently reported that Tax-1 and Tax-2 form complexes with the TNF-receptor associated factor 3, TRAF3, a negative regulator of the non-canonical NF-κB pathway. The NF-κB pathway is constitutively activated by the Tax proteins, whereas it is inhibited by HBZ and APH-2. The antagonistic effects of Tax and antisense proteins on NF-κB activation have not yet been fully clarified. Here, we investigated the effect of TRAF3 interaction with HTLV regulatory proteins and in particular its consequence on the subcellular distribution of the effector p65/RelA protein. We demonstrated that Tax-1 and Tax-2 efficiency on NF-κB activation is impaired in TRAF3 deficient cells obtained by CRISPR/Cas9 editing. We also found that APH-2 is more effective than HBZ in preventing Tax-dependent NF-κB activation. We further observed that TRAF3 co-localizes with Tax-2 and APH-2 in cytoplasmic complexes together with NF-κB essential modulator NEMO and TAB2, differently from HBZ and TRAF3. These results contribute to untangle the mechanism of NF-κB inhibition by HBZ and APH-2, highlighting the different role of the HTLV-1 and HTLV-2 regulatory proteins in the NF-κB activation.

Selective killing of human T-ALL cells: an integrated approach targeting redox homeostasis and the OMA1/OPA1 axis.

Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients are currently incurable due to primary or secondary resistance to glucocorticoid-based therapies. Here we employed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (ROS) using NS1619, a benzimidazolone that activates the K+ (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients with refractory T-ALL, but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knockdown of the mitochondrial protease OMA1, indicating the engagement of a ROS-OMA1-OPA1 axis in T-ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-induced apoptosis. In vivo, the combination of dexamethasone and NS1619 significantly reduced the growth of a glucocorticoid-resistant patient-derived T-ALL xenograft. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL.

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.

Expression of miR-34a in T-Cells Infected by Human T-Lymphotropic Virus 1.

Human T-lymphotropic virus 1 (HTLV-1) immortalizes T-cells and is the causative agent of adult T-cell leukemia/lymphoma (ATLL). HTLV-1 replication and transformation are governed by multiple interactions between viral regulatory proteins and host cell factors that remain to be fully elucidated. The present study investigated the impact of HTLV-1 infection on the expression of miR-34a, a microRNA whose expression is downregulated in many types of cancer. Results of RT-PCR assays showed that five out of six HTLV-1-positive cell lines expressed higher levels of miR-34a compared to normal PBMC or purified CD4+ T-cells. ATLL cell line ED, which did not express miR-34a, showed methylation of the miR-34a promoter. Newly infected PBMC and samples from 10 ATLL patients also showed a prominent increase in miR-34a expression compared to PBMC controls. The primary miR-34a transcript expressed in infected cell line C91PL contained binding motifs for NF-κB and p53. Pharmacological inhibition of NF-κB with Bay 11-7082 indicated that this pathway contributes to sustain miR-34a levels in infected cells. Treatment of infected cell lines with the p53 activator nutlin-3a resulted in a further increase in miR-34a levels, thus confirming it as a transcriptional target of p53. Nutlin-3a-treated cells showed downregulation of known miR-34a targets including the deacetylase SIRT1, which was accompanied by increased acetylation of p53, a substrate of SIRT1. Transfection of C91PL cells with a miR-34a mimic also led to downregulation of mRNA targets including SIRT1 as well as the pro-apoptotic factor BAX. Unlike nutlin-3a, the miR-34a mimic did not cause cell cycle arrest or reduce cell viability. On the other hand, sequestration of miR-34a with a sponge construct resulted in an increase in death of C91PL cells. These findings provide evidence for a functional role for miR-34a in fine-tuning the expression of target genes that influence the turnover of HTLV-1-infected cells.

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 pTα, 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 T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL 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 tumors.