Cross-talk between GLI transcription factors and FOXC1 promotes T-cell acute lymphoblastic leukemia dissemination.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly malignant pediatric leukemia, where few therapeutic options are available for patients which relapse. We find that therapeutic targeting of GLI transcription factors by GANT-61 is particularly effective against NOTCH1 unmutated T-ALL cells. Investigation of the functional role of GLI1 disclosed that it contributes to T-ALL cell proliferation, survival, and dissemination through the modulation of AKT and CXCR4 signaling pathways. Decreased CXCR4 signaling following GLI1 inactivation was found to be prevalently due to post-transcriptional mechanisms including altered serine 339 CXCR4 phosphorylation and cortactin levels. We also identify a novel cross-talk between GLI transcription factors and FOXC1. Indeed, GLI factors can activate the expression of FOXC1 which is able to stabilize GLI1/2 protein levels through attenuation of their ubiquitination. Further, we find that prolonged GLI1 deficiency has a double-edged role in T-ALL progression favoring disease dissemination through the activation of a putative AKT/FOXC1/GLI2 axis. These findings have clinical significance as T-ALL patients with extensive central nervous system dissemination show low GLI1 transcript levels. Further, T-ALL patients having a GLI2-based Hedgehog activation signature are associated with poor survival. Together, these findings support a rationale for targeting the FOXC1/AKT axis to prevent GLI-dependent oncogenic Hedgehog signaling.

Kidney cancer PDOXs reveal patient-specific pro-malignant effects of antiangiogenics and its molecular traits.

An open debate in antiangiogenic therapies is about their consequence on tumor invasiveness and metastasis, which is undoubtedly relevant for patients currently treated with antiangiogenics, such as renal cell carcinoma patients. To address, this we developed an extensive series of 27 patient biopsy-derived orthotopic xenograft models (Ren-PDOX) that represent inter-patient heterogeneity. In specific tumors, antiangiogenics produced increased invasiveness and metastatic dissemination, while in others aggressiveness remained unchanged. Mechanistically, species-discriminative RNA sequencing identified a tumor cell-specific differential expression profile associated with tumor progression and aggressivity in TCGA RCC patients. Gene filtering using an invasion-annotated patient series pinpointed two candidate genes, of which ALDH1A3 differentiated the pro-invasive subtype of Ren-PDOXs. Validation in an independent series of 15 antiangiogenic-treated patients confirmed that pre-treatment ALDH1A3 can significantly discriminate patients with pro-aggressive response upon treatment. Overall, results confirm that effects of antiangiogenic drugs on tumor invasion and metastasis are heterogeneous and may profoundly affect the natural progression of tumors and promote malignancy. Furthermore, we identify a specific molecular biomarker that could be used to select patients that better benefit from treatment.

Crosstalk between Hedgehog pathway and the glucocorticoid receptor pathway as a basis for combination therapy in T-cell acute lymphoblastic leukemia.

Notwithstanding intensified therapy, a considerable fraction of T-cell acute lymphoblastic leukemia (T-ALL) patients face a dismal prognosis due to primary resistance to treatment and relapse, raising the need for more efficient and targeted therapies. Hedgehog (HH) signaling is a major developmental pathway frequently deregulated in cancer, for which a role in T-ALL is emerging. Mounting evidence suggests that ligand-independent activation of HH pathway occurs in cancer including T-ALL, emphasizing the necessity of dissecting the complex interplay between HH and other signaling pathways regulating activation. In this work, we present a therapeutically relevant crosstalk between HH signaling and the glucocorticoid receptor (NR3C1) pathway acting at the level of GLI1 transcription factor. GLI inhibitor GANT61 and dexamethasone were shown to exert a synergistic anti-leukemic effect in vitro in T-ALL cell lines and patient-derived xenografts. Mechanistically, dexamethasone-activated NR3C1 impaired GLI1 function by dynamically modulating the recruitment of PCAF acetyltransferase and HDAC1 deacetylase. Increased GLI1 acetylation was associated with compromised transcriptional activity and reduced protein stability. In summary, our study identifies a novel crosstalk between GLI1 and NR3C1 signaling pathway which could be exploited in HH-dependent malignancies to increase therapeutic efficacy.

Allelic Imbalance Analysis in Liquid Biopsy to Monitor Locally Advanced Esophageal Cancer Patients During Treatment.

Esophageal cancer (EC) is a highly aggressive tumor, and the current monitoring procedures are partially inadequate to evaluate treatment efficacy. The aim of this study was to investigate whether allelic imbalance analysis in liquid biopsy could be used as an additional tool to monitor tumor burden in EC patients. For this purpose, circulating cell-free DNA (cfDNA) from 52 patients with a locally advanced EC, which underwent neoadjuvant treatment and resection, was analyzed. Data from four representative longitudinally followed patients are also reported. Furthermore, 17 DNAs from formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed and compared to time-matched cfDNAs. To look for allelic imbalance, which is the main genetic alteration in both EC histotypes, we used a panel of five microsatellites (MSs) and three single-nucleotide polymorphisms (SNPs) near genes described as frequently altered. The Fisher exact and Mann-Whitney U tests were used to analyze categorical and continuous data, respectively. The correlation coefficient between cfDNA and FFPE-DNA was calculated with the Pearson's correlation test. We found that the selected tumor-related alterations are present in cfDNA of both adenocarcinoma (EADC) and squamous cell carcinoma (ESCC) with similar frequencies. The only exception were the MSs, one downstream and one upstream, of SMAD4 of which the loss was only observed in EADC (26 vs. 0%, P = 0.018). More interestingly, longitudinal studies disclosed that in patients with disease progression, tumor-related alterations were present in cfDNA before overt clinical or instrumental signs of relapse. In conclusion, our data indicate that the evaluation of tumor-related gene allelic imbalance in cfDNA might be a useful tool to complement the current monitoring procedures for EC patients and to guide their management.

Early assessment of KRAS mutation in cfDNA correlates with risk of progression and death in advanced non-small-cell lung cancer.

BACKGROUND: Liquid biopsy has the potential to monitor biological effects of treatment. KRAS represents the most commonly mutated oncogene in Caucasian non-small-cell lung cancer (NSCLC). The aim of this study was to explore association of dynamic plasma KRAS genotyping with outcome in advanced NSCLC patients. METHODS: Advanced NSCLC patients were prospectively enrolled. Plasma samples were collected at baseline (T1), after 3 or 4 weeks, according to treatment schedule (T2) and at first radiological restaging (T3). Patients carrying KRAS mutation in tissue were analysed in plasma with droplet digital PCR. Semi-quantitative index of fractional abundance of mutated allele (MAFA) was used. RESULTS: KRAS-mutated cohort included 58 patients, and overall 73 treatments (N = 39 chemotherapy and N = 34 immune checkpoint inhibitors) were followed with longitudinal liquid biopsy. Sensitivity of KRAS detection in plasma at baseline was 48.3% (95% confidence interval (CI): 35.0-61.8). KRAS mutation at T2 was associated with increased probability of experiencing progressive disease as best radiological response (adjusted odds ratio: 7.3; 95% CI: 2.1-25.0, p = 0.0016). Increased MAFA (T1-T2) predicted shorter progression-free survival (adjusted hazard ratio (HR): 2.1; 95% CI: 1.2-3.8, p = 0.0142) and overall survival (adjusted HR: 3.2; 95% CI: 1.2-8.4, p = 0.0168). CONCLUSIONS: Longitudinal analysis of plasma KRAS mutations correlated with outcome: its early assessment during treatment has great potentialities for monitoring treatment outcome in NSCLC patients.

Identification of host variants associated with overall survival of esophageal cancer patients receiving platinum-based therapy.

Aim: Clinical features of esophageal cancer (EC) patients have poor prognostic power. Thus, it is paramount to discover biomarkers that can allow a more accurate survival prediction. Methods: To detect genetic variants associated with survival, DNA from 120 patients treated with cisplatin-based neoadjuvant therapy were genotyped using drug metabolism enzymes and transporters array. Results: We identified two variants: the rs2038067 in PPARD (p = 0.0004) and the rs683369 (F160L) in SLC22A1 (p = 0.001). Their prognostic power was greater than that of clinical stage alone (p = 0.017) and comparable to that of response to neoadjuvant therapy (p = 0.71). Interestingly, the prognostic accuracy of response models increased significantly when genetic variables were included (p = 0.003). Conclusion: Our data, though preliminary, strengthen the potential utility of germline variants for a better-tailored management of EC patients.

Comparison of the Genomic Profile of Cancer Stem Cells and Their Non-Stem Counterpart: The Case of Ovarian Cancer.

The classical cancer stem cell (CSC) model places CSCs at the apex of a hierarchical scale, suggesting different genetic alterations in non-CSCs compared to CSCs, since an ill-defined number of cell generations and time intervals separate CSCs from the more differentiated cancer cells that form the bulk of the tumor. Another model, however, poses that CSCs should be considered a functional state of tumor cells, hence sharing the same genetic alterations. Here, we review the existing literature on the genetic landscape of CSCs in various tumor types and as a case study investigate the genomic complexity of DNA obtained from matched CSCs and non-CSCs from five ovarian cancer patients, using a genome-wide single-nucleotide polymorphism (SNP) microarray.

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.

Rewiring of Lipid Metabolism and Storage in Ovarian Cancer Cells after Anti-VEGF Therapy.

Anti-angiogenic therapy triggers metabolic alterations in experimental and human tumors, the best characterized being exacerbated glycolysis and lactate production. By using both Liquid Chromatography-Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) analysis, we found that treatment of ovarian cancer xenografts with the anti-Vascular Endothelial Growth Factor (VEGF) neutralizing antibody bevacizumab caused marked alterations of the tumor lipidomic profile, including increased levels of triacylglycerols and reduced saturation of lipid chains. Moreover, transcriptome analysis uncovered up-regulation of pathways involved in lipid metabolism. These alterations were accompanied by increased accumulation of lipid droplets in tumors. This phenomenon was reproduced under hypoxic conditions in vitro, where it mainly depended from uptake of exogenous lipids and was counteracted by treatment with the Liver X Receptor (LXR)-agonist GW3965, which inhibited cancer cell viability selectively under reduced serum conditions. This multi-level analysis indicates alterations of lipid metabolism following anti-VEGF therapy in ovarian cancer xenografts and suggests that LXR-agonists might empower anti-tumor effects of bevacizumab.

Casein Kinase 1 Delta Regulates Cell Proliferation, Response to Chemotherapy and Migration in Human Ovarian Cancer Cells.

Casein kinase 1 delta (CK1δ) has a tumor-promoting role in different cancers and it is genetically amplified in a portion of human epithelial ovarian cancer (EOC). CK1δ is involved in pleiotropic cellular functions such as cell proliferation, DNA damage, and migration. We specifically knocked down CK1δ by short hairpin RNA (shRNA) in human ovarian cancer cells and we performed proliferation, chemosensitivity, as well as in vitro and in vivo migration assays. CK1δ knocked-down cells displayed reduced proliferation capability both in vitro and in vivo. Nonetheless, these cells were sensitized to the first line chemotherapeutic agent carboplatin (CPT), and this observation could be associated to reduced expression levels of p21(Cip1/Waf1), involved in DNA damage response, and the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP). Moreover, CK1δ knocked-down cells were affected in their migratory and lung homing capability, even if in opposite ways, i.e., IGROV1, SKOV3 and MES-OV lost, while OVCAR3 gained motility potential. The results suggest CK1δ as a potential exploitable target for pharmacological EOC treatment, but they also advise further investigation of its role in cell migration.

Silencing of miR-182 is associated with modulation of tumorigenesis through apoptosis induction in an experimental model of colorectal cancer.

BACKGROUND: miR-182-5p (miR-182) is an oncogenic microRNA (miRNA) found in different tumor types and one of the most up-regulated miRNA in colorectal cancer (CRC). Although this microRNA is expressed in the early steps of tumor development, its role in driving tumorigenesis is unclear. METHODS: The effects of miR-182 silencing on transcriptomic profile were investigated using two CRC cell lines characterized by different in vivo biological behavior, the MICOL-14h-tert cell line (dormant upon transfer into immunodeficient hosts) and its tumorigenic variant, MICOL-14tum. Apoptosis was studied by annexin/PI staining and cleaved Caspase-3/PARP analysis. The effect of miR-182 silencing on the tumorigenic potential was addressed in a xenogeneic model of MICOL-14tum transplant. RESULTS: Endogenous miR-182 expression was higher in MICOL-14tum than in MICOL-14h-tert cells. Interestingly, miR-182 silencing had a strong impact on gene expression profile, and the positive regulation of apoptotic process was one of the most affected pathways. Accordingly, annexin/PI staining and caspase-3/PARP activation demonstrated that miR-182 treatment significantly increased apoptosis, with a prominent effect in MICOL-14tum cells. Moreover, a significant modulation of the cell cycle profile was exerted by anti-miR-182 treatment only in MICOL-14tum cells, where a significant increase in the fraction of cells in G0/G1 phases was observed. Accordingly, a significant growth reduction and a less aggressive histological aspect were observed in tumor masses generated by in vivo transfer of anti-miR-182-treated MICOL-14tum cells into immunodeficient hosts. CONCLUSIONS: Altogether, these data indicate that increased miR-182 expression may promote cell proliferation, suppress the apoptotic pathway and ultimately confer aggressive traits on CRC cells.

A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer.

Receptors tyrosine kinase (RTK) enable normal and tumor cells to perceive and adapt to stimuli present in the microenvironment. These stimuli, also known as growth factors, are important molecular cues actively supporting cancer stem cell (CSC) self-renewal and viability. Since in epithelial ovarian cancer (EOC) the expression of c-Kit (CD117) has been identified as a CSC hallmark, we investigated the existence of a tumor growth-promoting loop between c-Kit and its ligand Stem Cell Factor (SCF). SCF exists as a soluble or transmembrane protein and through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions. High amounts of SCF were found in the ascitic effusions collected from EOC patients. While tumor cells and CSC only expressed the membrane-associated SCF isoform, both secreted and membrane-bound isoforms were expressed by tumor-associated macrophages (TAM, here shown to be M2-like) and fibroblasts (TAF). Circulating monocytes from EOC-bearing patients and healthy donors did not express both SCF isoforms. However, monocytes isolated from healthy donors produced SCF upon in vitro differentiation into macrophages, irrespectively of M1 or M2 polarization. In vitro, both SCF isoforms were able to activate the Akt pathway in c-Kit+ cells, and this effect was counteracted by the tyrosine kinase inhibitor imatinib. In addition, our results indicated that SCF could help c-Kit+ CSC survival in selective culture conditions and promote their canonical stemness properties, thus indicating the possible existence of a juxtacrine/paracrine circuit in EOC.

Cell membrane protein functionalization of nanoparticles as a new tumor-targeting strategy.

Nanoparticles have seen considerable popularity as effective tools for drug delivery. However, non-specific targeting continues to remain a challenge. Recently, biomimetic nanoparticles have emerged as an innovative solution that exploits biologically-derived components to improve therapeutic potential. Specifically, cell membrane proteins extracted from various cells (i.e., leukocytes, erythrocytes, platelets, mesenchymal stem cells, cancer) have shown considerable promise in bestowing nanoparticles with increased circulation and targeting efficacy. Traditional nanoparticles can be detected and removed by the immune system which significantly hinders their clinical success. Biomimicry has been proposed as a promising approach to overcome these limitations. In this review, we highlight the current trends in biomimetic nanoparticles and describe how they are being used to increase their chemotherapeutic effect in cancer treatment.

Association Between ERCC1 rs3212986 and ERCC2/XPD rs1799793 and OS in Patients With Advanced Esophageal Cancer.

Esophageal cancer (EC) is a very aggressive tumor, and no reliable prognostic markers exist especially for resectable advanced neoplasia. The principal aim of this study was to investigate the association of germline polymorphisms in nucleotide excision repair (NER) pathway genes with the overall survival (OS) of patients with advanced EC. As a second aim, we also studied the association of NER gene variants with response to cisplatin-based chemotherapy. Among the EC patients referred to our Institution between 2004 and 2012, we selected a cohort of 180 patients diagnosed with a clinical tumor stage ranging from IIB and IVA. Patients were genotyped for four NER variants, two in the ERCC1 (rs11615 and rs3212986) and two in the ERCC2/XPD (rs1799793 and rs13181) genes. Kaplan-Meier analyses and Cox proportional hazards model were used to evaluate the associations of the selected variants with OS; association with response to neoadjuvant therapy was investigated using logistic regression. Results showed that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 were significantly associated with shorter OS. On the contrary, response association analysis displayed that, while rs11615 and rs3212986 in ERCC1 were associated with response, both ERCC2/XPD variants were not. By creating survival prediction models, we showed that the rs3212986 and the rs1799793 have a better predictability of the tumor stage alone. Furthermore, they were able to improve the power of the clinical model (AUC = 0.660 vs. AUC = 0.548, p = 0.004). In conclusion, our results indicate that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 could be used as surrogate markers for a better stratification of EC patients with advanced resectable tumor.

A Novel t(8;14)(q24;q11) Rearranged Human Cell Line as a Model for Mechanistic and Drug Discovery Studies of NOTCH1-Independent Human T-Cell Leukemia.

MYC-translocated T-lineage acute lymphoblastic leukemia (T-ALL) is a rare subgroup of T-ALL associated with CDKN2A/B deletions, PTEN inactivation, and absence of NOTCH1 or FBXW7 mutations. This subtype of T-ALL has been associated with induction failure and aggressive disease. Identification of drug targets and mechanistic insights for this disease are still limited. Here, we established a human NOTCH1-independent MYC-translocated T-ALL cell line that maintains the genetic and phenotypic characteristics of the parental leukemic clone at diagnosis. The University of Padua T-cell acute lymphoblastic leukemia 13 (UP-ALL13) cell line has all the main features of the above described MYC-translocated T-ALL. Interestingly, UP-ALL13 was found to harbor a heterozygous R882H DNMT3A mutation typically found in myeloid leukemia. Chromatin immunoprecipitation coupled with high-throughput sequencing for histone H3 lysine 27 (H3K27) acetylation revealed numerous putative super-enhancers near key transcription factors, including MYC, MYB, and LEF1. Marked cytotoxicity was found following bromodomain-containing protein 4 (BRD4) inhibition with AZD5153, suggesting a strict dependency of this particular subtype of T-ALL on the activity of super-enhancers. Altogether, this cell line may be a useful model system for dissecting the signaling pathways implicated in NOTCH1-independent T-ALL and for the screening of targeted anti-leukemia agents specific for this T-ALL subgroup.

A Preclinical Model for the ATLL Lymphoma Subtype With Insights Into the Role of Microenvironment in HTLV-1-Mediated Lymphomagenesis.

Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-γc-/- mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2Rγc KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype.

Chemotactic Cues for NOTCH1-Dependent Leukemia.

The NOTCH signaling pathway is a conserved signaling cascade that regulates many aspects of development and homeostasis in multiple organ systems. Aberrant activity of this signaling pathway is linked to the initiation and progression of several hematological malignancies, exemplified by T-cell acute lymphoblastic leukemia (T-ALL). Interestingly, frequent non-mutational activation of NOTCH1 signaling has recently been demonstrated in B-cell chronic lymphocytic leukemia (B-CLL), significantly extending the pathogenic significance of this pathway in B-CLL. Leukemia patients often present with high-blood cell counts, diffuse disease with infiltration of the bone marrow, secondary lymphoid organs, and diffusion to the central nervous system (CNS). Chemokines are chemotactic cytokines that regulate migration of cells between tissues and the positioning and interactions of cells within tissue. Homeostatic chemokines and their receptors have been implicated in regulating organ-specific infiltration, but may also directly and indirectly modulate tumor growth. Recently, oncogenic NOTCH1 has been shown to regulate infiltration of leukemic cells into the CNS hijacking the CC-chemokine ligand 19/CC-chemokine receptor 7 chemokine axis. In addition, a crucial role for the homing receptor axis CXC-chemokine ligand 12/CXC-chemokine receptor 4 has been demonstrated in leukemia maintenance and progression. Moreover, the CCL25/CCR9 axis has been implicated in the homing of leukemic cells into the gut, particularly in the presence of phosphatase and tensin homolog tumor suppressor loss. In this review, we summarize the latest developments regarding the role of NOTCH signaling in regulating the chemotactic microenvironmental cues involved in the generation and progression of T-ALL and compare these findings to B-CLL.

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.

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia.

Loss-of-function mutations and deletions in Wilms tumor 1 (WT1) gene are present in approximately 10% of T-cell acute lymphoblastic leukemia. Clinically, WT1 mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here we demonstrate that WT1 plays a critical role in the response to DNA damage in T-cell leukemia. WT1 loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, WT1 loss positively affected the expression of the X-linked inhibitor of apoptosis protein, XIAP, and genetic or chemical inhibition with embelin (a XIAP inhibitor) significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient-derived xenografts. These results reveal an important role for the WT1 tumor suppressor gene in the response to DNA damage, and support the view that anti-XIAP targeted therapies could have a role in the treatment of WT1-mutant T-cell leukemia.