Responsiveness to Hedgehog Pathway Inhibitors in T-Cell Acute Lymphoblastic Leukemia Cells Is Highly Dependent on 5'AMP-Activated Kinase Inactivation.

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.

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.

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.

miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.

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.

Circulating miRNA-375 as a potential novel biomarker for active Kaposi's sarcoma in AIDS patients.

The aim of this study was to identify circulating microRNAs (miRNAs) that could be used as biomarkers in patients at risk for or affected by AIDS-Kaposi's sarcoma (KS). Screening of 377 miRNAs was performed using low-density arrays in pooled plasma samples of 10 HIV/human herpesvirus 8 (HHV8)-infected asymptomatic and 10 AIDS-KS patients before and after successful combined antiretroviral therapy (cART). MiR-375 was identified as a potential marker of active KS, being the most down-regulated in AIDS-KS patients after cART and the most up-regulated in naïve AIDS-KS patients compared to naïve asymptomatic subjects. Validation on individual plasma samples confirmed that miR-375 levels were higher in AIDS-KS compared to asymptomatic patients, decreased after cART-induced remission in most AIDS-KS patients and increased in patients with active KS. In asymptomatic patients miR-375 was up-regulated after cART in both screening and validation. Statistical analyses revealed an association between miR-375 changes and CD4 cell counts, which could explain the discordant cases and the opposite trend between asymptomatic and AIDS-KS patients. These data suggest that circulating miR-375 might be a good indicator of active AIDS-KS. Moreover, changes in miR-375 levels may have a prognostic value in HIV/HHV8-infected patients undergoing treatment. Further large-scale validation is needed.

Clonal heterogeneity of melanoma in a paradigmatic case study: future prospects for circulating melanoma cells.

The management of metastatic melanoma is a difficult matter. Nevertheless, the advent of target therapy has significantly improved patient outcome, provided that tumor molecular characteristics become available: the detection of drug-resistant clones can contribute to understanding the reasons for resistance onset, influencing the choice of subsequent therapy. This work aimed to provide a possible explanation for the early resistance to vemurafenib developed by a patient with melanoma, and concurrently to assess the extent, and role, of the tumor clonal heterogeneity. We analyzed tissue samples from different sites and time points: first/second primary, three lymph node metastases, and circulating melanoma cells (CMCs). We first investigated these samples by the routine Sanger sequencing for BRAF, NRAS, and KIT, and then, we focused on specific hotspots by droplet digital PCR. We detected a BRAF V600E mutation by Sanger sequencing in the second primary and distant lymph node metastases, but not in the first primary or sentinel lymph node. Interestingly, by droplet digital PCR, the V600E mutation was also detected in the first primary, and the V600K in the second primary and metastases. Moreover, we identified a rare KIT V569G mutation, appearing to be CMC exclusive. This finding confirms the potential of CMCs as a source of tumor material for genetic analysis, reflecting real-time systemic disease evolution and, most likely, the most aggressive, treatment-resistant clones. In summary, this work underlines the importance of CMCs in the early identification of tumor clones putatively responsible for therapy resistance.

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.

A coordinate deregulation of microRNAs expressed in mucosa adjacent to tumor predicts relapse after resection in localized colon cancer.

Up to 20% of colorectal cancer (CRC) node-negative patients develop loco-regional or distant recurrences within 5 years from surgery. No predictive biomarker able to identify the node-negative subjects at high risk of relapse after curative treatment is presently available.Forty-eight localized (i.e. stage I-II) colon cancer patients who underwent radical tumor resection were considered. The expression of five miRNAs, involved in CRC progression, was investigated by qRT-PCR in both tumor tissue and matched normal colon mucosa.Interestingly, we found that the coordinate deregulation of four miRNAs (i.e. miR-18a, miR-21, miR-182 and miR-183), evaluated in the normal mucosa adjacent to tumor, is predictive of relapse within 55 months from curative surgery.Our results, if confirmed in independent studies, may help to identify high-risk patients who could benefit most from adjuvant therapy. Moreover, this work highlights the importance of extending the search for tissue biomarkers also to the tumor-adjacent mucosa.

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.

Identification of a HLA-A*0201-restricted immunogenic epitope from the universal tumor antigen DEPDC1.

The identification of universal tumor-specific antigens shared between multiple patients and/or multiple tumors is of great importance to overcome the practical limitations of personalized cancer immunotherapy. Recent studies support the involvement of DEPDC1 in many aspects of cancer traits, such as cell proliferation, resistance to induction of apoptosis and cell invasion, suggesting that it may play key roles in the oncogenic process. In this study, we report that DEPDC1 expression is upregulated in most types of human tumors, and closely linked to a poorer prognosis; therefore, it might be regarded as a novel universal oncoantigen potentially suitable for targeting many different cancers. In this regard, we report the identification of a HLA-A*0201 allele-restricted immunogenic DEPDC1-derived epitope, which is able to induce cytotoxic T lymphocytes (CTL) exerting a strong and specific functional response in vitro toward not only peptide-loaded cells but also triple negative breast cancer (TNBC) cells endogenously expressing the DEPDC1 protein. Such CTL are also therapeutically active against human TNBC xenografts in vivo upon adoptive transfer in immunodeficient mice. Overall, these data provide evidence that this DEPDC1-derived antigenic epitope can be exploited as a new tool for developing immunotherapeutic strategies for HLA-A*0201 patients with TNBC, and potentially many other cancers.

Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells.

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use.

Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies.

Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies.

T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

Loss of ZFP36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of ZEB1, SOX9 and MACC1.

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/ß-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.

Reverse immunoediting: When immunity is edited by antigen.

Immune selective pressure occurring during cancer immunoediting shapes tumor features revealed at clinical presentation. However, in the "Escape" phase, the tumor itself has the chance to influence the immunological response. Therefore, the capacity of the immune response to sculpt the tumor characteristics is only one side of the coin and even the opposite is likely true, i.e. that an antigen can shape the immune response in a sort of "reverse immunoediting". This reciprocal modeling probably occurs continuously, whenever the immune system encounters a tumor/foreign antigen, and can be operative in the pathogen/immune system interplay, thus possibly permeating the protective immunity as a whole. In line with this view, the characterization of a T cell response as well as the design of both active and passive immunotherapy strategies should also take into account all Ag features (type, load and presentation). Overall, we suggest that the "reverse immunoediting" hypothesis could help to dissect the complex interplay between antigens and the immune repertoire, and to improve the outcome of immunotherapeutic approaches, where T cell responses are manipulated and reprogrammed.

A circulating miRNA assay as a first-line test for prostate cancer screening.

BACKGROUND: Prostate cancer (PCa) screening currently relies on prostate-specific antigen (PSA) testing and digital rectal examination. However, recent large-scale studies have questioned the long-term efficacy of these tests, and biomarkers that accurately identify PCa are needed. METHODS: We analysed the levels of circulating microRNAs (miRNAs) in patients with elevated PSA who were diagnosed with either localised PCa (n=36) or benign prostatic hyperplasia (BPH, n=31) upon biopsy. Real-time RT-PCR with Taqman probes was used to measure plasma levels of miRNAs. To circumvent problems associated with circulating miRNA quantitation, we computed the expression ratios of upregulated and downregulated miRNAs. RESULTS: The miR-106a/miR-130b and miR-106a/miR-223 ratios were significantly different between the biopsy-positive and BPH groups (P<0.0001), and yielded statistical power values that were >0.99. Both miRNA ratios were highly sensitive and more specific than PSA in discriminating localised PCa from BPH. Receiver operating characteristic curve analysis revealed area under curve values of 0.81 (miR-106a/miR-130b) and 0.77 (miR-106a/miR-223). CONCLUSIONS: Testing for circulating miR-106a/miR-130b and miR-106a/miR-223 ratios may reduce the costs and morbidity of unnecessary biopsies and is feasible for large-scale screening, as it requires measuring only three miRNAs.