Proteomic and genomic signatures of repeat instability in cancer and adjacent normal tissues.

Repetitive sequences are hotspots of evolution at multiple levels. However, due to difficulties involved in their assembly and analysis, the role of repeats in tumor evolution is poorly understood. We developed a rigorous motif-based methodology to quantify variations in the repeat content, beyond microsatellites, in proteomes and genomes directly from proteomic and genomic raw data. This method was applied to a wide range of tumors and normal tissues. We identify high similarity between repeat instability patterns in tumors and their patient-matched adjacent normal tissues. Nonetheless, tumor-specific signatures both in protein expression and in the genome strongly correlate with cancer progression and robustly predict the tumorigenic state. In a patient, the hierarchy of genomic repeat instability signatures accurately reconstructs tumor evolution, with primary tumors differentiated from metastases. We observe an inverse relationship between repeat instability and point mutation load within and across patients independent of other somatic aberrations. Thus, repeat instability is a distinct, transient, and compensatory adaptive mechanism in tumor evolution and a potential signal for early detection.

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

Cocaine disrupts histamine H3 receptor modulation of dopamine D1 receptor signaling: σ1-D1-H3 receptor complexes as key targets for reducing cocaine's effects.

The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits ß-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine.

Bladder cancer organoids as a functional system to model different disease stages and therapy response.

Bladder Cancer (BLCa) inter-patient heterogeneity is the primary cause of treatment failure, suggesting that patients could benefit from a more personalized treatment approach. Patient-derived organoids (PDOs) have been successfully used as a functional model for predicting drug response in different cancers. In our study, we establish PDO cultures from different BLCa stages and grades. PDOs preserve the histological and molecular heterogeneity of the parental tumors, including their multiclonal genetic landscapes, and consistently share key genetic alterations, mirroring tumor evolution in longitudinal sampling. Our drug screening pipeline is implemented using PDOs, testing standard-of-care and FDA-approved compounds for other tumors. Integrative analysis of drug response profiles with matched PDO genomic analysis is used to determine enrichment thresholds for candidate markers of therapy response and resistance. Finally, by assessing the clinical history of longitudinally sampled cases, we can determine whether the disease clonal evolution matched with drug response.

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1.

The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis.

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1.

BACKGROUND: Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs. DESIGN AND METHODS: We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells. RESULTS: We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor. CONCLUSIONS: Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.

Allele-specific genomic data elucidate the role of somatic gain and copy-number neutral loss of heterozygosity in cancer.

Pan-cancer studies sketched the genomic landscape of the tumor types spectrum. We delineated the purity- and ploidy-adjusted allele-specific profiles of 4,950 patients across 27 tumor types from the Cancer Genome Atlas (TCGA). Leveraging allele-specific data, we reclassified as loss of heterozygosity (LOH) 9% and 7% of apparent copy-number wild-type and gain calls, respectively, and overall observed more than 18 million allelic imbalance somatic events at the gene level. Reclassification of copy-number events revealed associations between driver mutations and LOH, pointing out the timings between the occurrence of point mutations and copy-number events. Integrating allele-specific genomics and matched transcriptomics, we observed that allele-specific gene status is relevant in the regulation of TP53 and its targets. Further, we disclosed the role of copy-neutral LOH in the impairment of tumor suppressor genes and in disease progression. Our results highlight the role of LOH in cancer and contribute to the understanding of tumor progression.

Antineoplastic activity of lentiviral vectors expressing interferon-alpha in a preclinical model of primary effusion lymphoma.

The peculiar site of development of primary effusion lymphoma (PEL) highlights a specific role of body cavities in the pathogenesis of this neoplasia. We used a xenograft murine model of PEL to characterize the contribution of the host microenvironment to PEL growth. The activity of a murine (ie, host-specific) interferon-alpha(1) (IFN-alpha(1))-expressing lentiviral vector (mIFN-alpha(1)-LV) was compared with that of a human (h) IFN-alpha(2)b-LV. LVs efficiently delivered the transgene to PEL cells and conferred long-term transgene expression in vitro and in vivo. Treatment of PEL-injected severe combined immunodeficiency mice with hIFN-alpha(2)b-LV significantly prolonged mice survival and reduced ascites development. Interestingly, mIFN-alpha(1)-LV showed an antineoplastic activity comparable with that observed with hIFN-alpha(2)b-LV. As mIFN-alpha(1) retained species-restricted activity in vitro, it probably acted in vivo on the intracavitary murine milieu. mIFN-alpha(1)-treated murine mesothelial cells were found to express tumor necrosis factor-related apoptosis-inducing ligand and to significantly trigger apoptosis of cocultured PEL cells in a tumor necrosis factor-related apoptosis-inducing ligand-dependent manner. These data suggest that the interaction between lymphomatous and mesothelial cells lining the body cavities may play a key role in PEL growth control and also indicate that the specific targeting of microenvironment may impair PEL development.

Fast mutual exclusivity algorithm nominates potential synthetic lethal gene pairs through brute force matrix product computations.

Mutual Exclusivity analysis of genomic aberrations contributes to the exploration of potential synthetic lethal (SL) relationships thus guiding the nomination of specific cancer cells vulnerabilities. When multiple classes of genomic aberrations and large cohorts of patients are interrogated, exhaustive genome-wide analyses are not computationally feasible with commonly used approaches. Here we present Fast Mutual Exclusivity (FaME), an algorithm based on matrix multiplication that employs a logarithm-based implementation of the Fisher's exact test to achieve fast computation of genome-wide mutual exclusivity tests; we show that brute force testing for mutual exclusivity of hundreds of millions of aberrations combinations can be performed in few minutes. We applied FaME to allele-specific data from whole exome experiments of 27 TCGA studies cohorts, detecting both mutual exclusivity of point mutations, as well as allele-specific copy number signals that span sets of contiguous cytobands. We next focused on a case study involving the loss of tumor suppressors and druggable genes while exploiting an integrated analysis of both public cell lines loss of function screens data and patients' transcriptomic profiles. FaME algorithm implementation as well as allele-specific analysis output are publicly available at https://github.com/demichelislab/FaME.

Modulation of dopamine D1 receptors via histamine H3 receptors is a novel therapeutic target for Huntington's disease.

Early Huntington's disease (HD) include over-activation of dopamine D1 receptors (D1R), producing an imbalance in dopaminergic neurotransmission and cell death. To reduce D1R over-activation, we present a strategy based on targeting complexes of D1R and histamine H3 receptors (H3R). Using an HD mouse striatal cell model and HD mouse organotypic brain slices we found that D1R-induced cell death signaling and neuronal degeneration, are mitigated by an H3R antagonist. We demonstrate that the D1R-H3R heteromer is expressed in HD mice at early but not late stages of HD, correlating with HD progression. In accordance, we found this target expressed in human control subjects and low-grade HD patients. Finally, treatment of HD mice with an H3R antagonist prevented cognitive and motor learning deficits and the loss of heteromer expression. Taken together, our results indicate that D1R - H3R heteromers play a pivotal role in dopamine signaling and represent novel targets for treating HD.

Contribution of viral and cellular cytokines to Kaposi's sarcoma-associated herpesvirus pathogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus is associated with the proliferative/malignant disorders KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD) in patients with AIDS. In spite of recent advances in the treatment of KS, PEL and MCD represent therapeutic challenges. Recent advances in dissecting the pathogenesis of these diseases have indicated that the viral cytokine IL-6 and the cellular cytokines/growth factors IL-10, IL-6, stromal cell-derived factor 1, and vascular endothelial growth factor are important contributors to the growth, survival, and spread of PEL and MCD and are therefore potential targets for drug development.

RB1 Heterogeneity in Advanced Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal but clinically heterogeneous disease, with patients having variable benefit from endocrine and cytotoxic treatments. Intrapatient genomic heterogeneity could be a contributing factor to this clinical heterogeneity. Here, we used whole-genome sequencing (WGS) to investigate genomic heterogeneity in 21 previously treated CRPC metastases from 10 patients to investigate intrapatient molecular heterogeneity (IPMH).Experimental Design: WGS was performed on topographically separate metastases from patients with advanced metastatic prostate cancer. IPMH of the RB1 gene was identified and further evaluated by FISH and IHC assays. RESULTS: WGS identified limited IPMH for putative driver events. However, heterogeneous genomic aberrations of RB1 were detected. We confirmed the presence of these RB1 somatic copy-number aberrations, initially identified by WGS, with FISH, and identified novel structural variants involving RB1 in 6 samples from 3 of these 10 patients (30%; 3/10). WGS uncovered a novel deleterious RB1 structural lesion constituted of an intragenic tandem duplication involving multiple exons and associating with protein loss. Using RB1 IHC in a large series of mCRPC biopsies, we identified heterogeneous expression in approximately 28% of mCRPCs. CONCLUSIONS: mCRPCs have a high prevalence of RB1 genomic aberrations, with structural variants, including rearrangements, being common. Intrapatient genomic and expression heterogeneity favors RB1 aberrations as late, subclonal events that increase in prevalence due to treatment-selective pressures.

Inherited determinants of early recurrent somatic mutations in prostate cancer.

Prostate cancer is a highly heritable molecularly and clinically heterogeneous disease. To discover germline events involved in prostate cancer predisposition, we develop a computational approach to nominate heritable facilitators of somatic genomic events in the context of the androgen receptor signaling. Here, we use a ranking score and benign prostate transcriptomes to identify a non-coding polymorphic regulatory element at 7p14.3 that associates with DNA repair and hormone-regulated transcript levels and with an early recurrent prostate cancer-specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene. The locus shows allele-specific activity that is concomitantly modulated by androgen receptor and by CCAAT/enhancer-binding protein (C/EBP) beta (CEBPB). Deletion of this locus via CRISPR-Cas9 leads to deregulation of the genes predicted to interact with the 7p14.3 locus by Hi-C chromosome conformation capture data. This study suggests that a polymorphism at 7p14.3 may predispose to SPOP mutant prostate cancer subclass through a hormone-dependent DNA damage response.Prostate cancer is a heterogeneous disease, and many cases show somatic mutations of SPOP. Here, the authors show that a non-coding polymorphic regulatory element at 7p14.3 may predispose to SPOP mutant prostate cancer subclass through a hormone dependent DNA damage response.

Long-term clinical outcome of AIDS-related Kaposi's sarcoma during highly active antiretroviral therapy.

The long-term impact of highly active antiretroviral therapy (HAART) in AIDS patients with Kaposi's sarcoma (KS) was evaluated in 22 consecutive, HAART-naïve KS patients attending a single Italian referral centre for HIV/AIDS. Clinical, virologic and immunologic responses to HAART were assessed at baseline and every three months during the follow-up. Peripheral blood mononuclear cell (PBMC)-associated human herpesvirus 8 (HHV-8) load was also evaluated by real-time PCR in 13 patients with durable clinical KS complete response (CR). In a median follow-up of 40 months (range 17-78), the KS overall clinical response rate was 91%: 18 complete and 2 partial responses were achieved, and two patients experienced disease progression. CR persisted in all 18 patients, including the 5 poor-risk KS patients in whom CR lasted for > 60 months, and was significantly linked to an increase in CD4+ cell counts and a drop in HIV-1-RNA copies. Compared to baseline levels, a decrease in PBMC HHV-8 load was observed at CR, and a significant further reduction was found at the end of follow-up. In this monocentric study, AIDS-KS patients treated with HAART showed high clinical response rate. Patients with CR showed a prolonged remission, lasting more than 5 years in a group of poor-risk patients, and a persistent reduction in circulating HHV-8-infected cells. These findings highlight that HAART deeply modifies the natural history of this tumour in AIDS patients, and that this long-lasting approach may be considered a first-line treatment for the majority of HIV-1-infected patients developing KS.

Kaposi sarcoma herpesvirus promotes endothelial-to-mesenchymal transition through Notch-dependent signaling.

Endothelial-to-mesenchymal transition (EndMT) is now widely considered a pivotal contributor to cancer progression. In this study, we show that the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a sufficient cause of EndMT, potentially helping to explain the aggressiveness of KS that occurs commonly in AIDS patients. Upon KSHV infection, primary dermal microvascular endothelial cells lost expression of endothelial markers and acquired expression of mesenchymal markers, displaying new invasive and migratory properties along with increased survival. KSHV activated Notch-induced transcription factors Slug and ZEB1, and canonical Notch signaling was required for KSHV-induced EndMT. In contrast, KSHV did not utilize the TGFß signaling pathway, which has also been linked to EndMT. Within KS lesions, KSHV-infected spindle cells displayed features compatible with KSHV-induced EndMT including a complex phenotype of endothelial and mesenchymal properties, Notch activity, and nuclear ZEB1 expression. Our results show that KSHV engages the EndMT program to increase the invasiveness and survival of infected endothelial cells, traits that likely contribute to viral persistence and malignant progression. One important implication of our findings is that therapeutic approaches to disrupt the Notch pathway may offer novel approaches for KS treatment.

Impaired recruitment of Grk6 and beta-Arrestin 2 causes delayed internalization and desensitization of a WHIM syndrome-associated CXCR4 mutant receptor.

WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of beta-Arrestin 2, but not beta-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since beta-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome.

Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells.

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)-induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF-induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

Pregnancy and human herpesvirus 8 reactivation in human immunodeficiency virus type 1-infected women.

To investigate the impact of pregnancy on human herpesvirus 8 (HHV-8) reactivation in human immunodeficiency virus type 1 (HIV-1)-infected women, the HHV-8 DNA presence and load were analyzed in peripheral blood mononuclear cells (PBMCs) and cervicovaginal secretions (CVSs) from 15 pregnant women coinfected with HIV-1 and HHV-8. HHV-8 detection was analyzed in relation to anti-HHV-8 antibodies and HIV-1-related parameters. Nucleotide sequence analysis of an ORFK1 hypervariable region of the HHV-8 strains was performed. HHV-8 was detected in maternal PBMCs (5/15 women) from the second trimester and in CVSs (5/15 women) mainly from the third trimester. The HHV-8 load significantly increased late in pregnancy in both maternal compartments and was associated with a significant increase in HIV-1 shedding in the genital tract. Antilytic antibodies were significantly more common in HHV-8 DNA-positive women. An elevated HHV-8 load was found in the PBMCs of an infant born to a mother with large amounts of HHV-8 in both compartments at delivery. Different ORFK1 subtypes were found in maternal samples, whereas the same subtype was identified in the mother-child pair. These data suggest that pregnancy may induce HHV-8 replication in HIV-1-infected women. An augmented HHV-8 load may, in turn, influence mother-to-child transmission, since one of the HIV-1-infected mothers with HHV-8 reactivation transmitted her ORFK1 subtype to the infant, who showed a high level of HHV-8 viremia indicative of a primary infection. This finding documents for the first time the perinatal transmission of a specific HHV-8 subtype. Vertical transmission may thus play a role in HHV-8 spread also in areas of subendemicity among HIV-1-infected women.

Use of a BJAB-derived cell line for isolation of human herpesvirus 8.

Establishment of latently infected cell lines from primary effusion lymphomas (PEL) presently is the most efficient system for the propagation of clinical strains of human herpesvirus 8 (HHV-8) in culture. Here we describe a new approach to culture productively replicating HHV-8 from patient samples. A BJAB-derived B-cell line, BBF, was found to retain HHV-8 longer, to support the latent and lytic replication programs, and to produce transmissible virus. Supernatants from n-butyrate-treated peripheral blood mononuclear cells of 24 HHV-8-seropositive renal transplant recipients were used to infect BBF cells, and replicating virus was detected in cultures from 11 patients. Moreover, BBF cells infected with saliva strains showed a highly productive profile regardless of the initial viral load, which confirms that infectious HHV-8 can be present in saliva and also suggests that saliva strains may exhibit a high tropism for B lymphocytes. In conclusion, we established an in vitro system that efficiently detects HHV-8 in samples with low viral loads and that produces infectious progeny. BBF cells can be used to propagate HHV-8 from different biological samples as well as to clarify important issues related to virus-cell interactions in a context distinct from endothelial and PEL-derived cell lines.