SOX11 expression is restricted to EBV-negative Burkitt lymphoma and is associated with molecular genetic features.

ABSTRACT: SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV-negative (EVB-) BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, whereas in EBV-negative (EBV-)/SOX11-negative (SOX11-) tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11-expressing BL showed higher frequency of SMARCA4 and ID3 mutations than EBV-/SOX11- cases. By RNA sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or B-cell receptor signaling were found between SOX11- and SOX11-positive (SOX11+) BL cells. However, SOX11+ BL showed higher adhesion to vascular cell adhesion molecule 1 (VCAM-1) than SOX11- BL cell lines. Here, we demonstrate that EBV- BL comprises 2 subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma.

ABSTRACT: Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in the bone marrow or blood (BCP acute lymphoblastic leukemia [BCP-ALL]) or are less common in extramedullary tissue (BCP lymphoblastic lymphoma [BCP-LBL]). Although both presentations are similar in morphology and immunophenotype, molecular studies have been virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and restriction on small, mostly formalin-fixed paraffin-embedded (FFPE) tissues. Here, to our knowledge, we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n = 97). Whole-exome sequencing indicated a mutational spectrum of BCP-LBL, strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations, and gene expression by RNA sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even in FFPE tissue, and may be relevant to guide treatment.

Tumor and microenvironment response but no cytotoxic T-cell activation in classic Hodgkin lymphoma treated with anti-PD1.

Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

Molecular features of non-anaplastic peripheral T-cell lymphoma in children and adolescents.

Non-anaplasticperipheral T-cell lymphomas (PTCL) are rare tumors in children, adolescents, and young adults (CAYA) with poor prognosis and scarce genetic data. We analyzed lymphoma tissue from 36 patients up to 18 years old with PTCL, not otherwise specified (PTCL-NOS), hepatosplenic T-cell lymphoma, Epstein-Barr virus (EBV)-positive T-lymphoproliferative diseases, subcutaneous panniculitis-like T-cell lymphoma, and other PTCL types. Twenty-three patients (64%) had at least one genetic variant detectable, including TET2, KMT2C, PIK3D, and DMNT3A. TP53 and RHOA variants, commonly found in adults, were not identified. Eight of 20 (40%) CAYA PTCL-NOS had no detectable mutations. The genetic findings suggest that CAYA PTCL differ from adult cases.

Paracrine recruitment and activation of fibroblasts by c-Myc expressing breast epithelial cells through the IGFs/IGF-1R axis.

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr397 , as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion.

MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

lncRNAs and MYC: An Intricate Relationship.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression networks, acting either at the transcriptional level, by influencing histone modifications, or at the post-transcriptional level, by controlling mRNA stability and translation. Among the gene expression networks known to influence the process of oncogenic transformation, the one controlled by the proto-oncogene MYC is one of the most frequently deregulated in cancer. In B-cell lymphomas, the MYC gene is subject to chromosomal rearrangements that result in MYC overexpression. In many other cancers, the region surrounding MYC is subject to gene amplification. MYC expression is also controlled at the level of protein and mRNA stability. Neoplastic lesions affecting MYC expression are responsible for a drastic change in the number and the type of genes that are transcriptionally controlled by MYC, depending on differential promoter affinities. Transcriptome profiling of tumor samples has shown that several lncRNAs can be found differentially regulated by MYC in different cancer types and many of them can influence cancer cell viability and proliferation. At the same time, lncRNAs have been shown to be able to control the expression of MYC itself, both at transcriptional and post-transcriptional levels. Given that targeting the MYC-dependent transcriptional program has the potential to reach broad anticancer activity, molecular dissection of the complex regulatory mechanisms governing MYC expression will be crucial in the future for the identification of novel therapeutic strategies.

Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide.

Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.

LINC00892 Is an lncRNA Induced by T Cell Activation and Expressed by Follicular Lymphoma-Resident T Helper Cells.

Successful immunotherapy in both solid tumors and in hematological malignancies relies on the ability of T lymphocytes to infiltrate the cancer tissue and mount an immune response against the tumor. Biomarkers able to discern the amount and the types of T lymphocytes infiltrating a given tumor therefore have high diagnostic and prognostic value. Given that lncRNAs are known to have a highly cell-type-specific expression pattern, we searched for lncRNAs specifically expressed by activated T cells and at the same time in a kind of lymphoma, follicular lymphoma, where the microenvironment is known to play a critical role in the regulation of antitumor immunity. We focused on a non-coding transcript, annotated as LINC00892, which reaches extremely high expression levels following cell activation in Jurkat cells. Interestingly LINC00892 has an expression pattern resembling that of genes involved in T cell memory. Accordingly, LINC00892 is mostly expressed by the effector memory and helper CD4+ T cell sub-types but not by naïve T cells. In situ analyses of LINC00892 expression in normal lymph nodes and in follicular lymphoma biopsies show that its expression is limited to CD4+ PD1hi T cells, with a subcellular localization within the germinal center matching that of follicular helper T cells. Our analysis therefore suggests that the previously uncharacterized lncRNA LINC00892 could be a useful biomarker for the detection of CD4+ memory T cells in both normal and tumor tissues.

Analysis of secretome changes uncovers an autocrine/paracrine component in the modulation of cell proliferation and motility by c-Myc.

Proteins secreted by cancer cells are a major component of tumor microenvironment. However, little is known on the impact of single oncogenic lesions on the expression of secreted proteins at early stages of tumor development. Because c-Myc overexpression is among the most frequent alterations in cancer, here we investigated the effect of sustained c-Myc expression on the secretome of a nontransformed human epithelial cell line (hT-RPE). By using a quantitative proteomic approach, we have identified 125 proteins in conditioned media of hT-RPE/MycER cells upon c-Myc induction. Analysis of the 49 proteins significantly down-regulated by c-Myc revealed a marked enrichment of factors associated with growth inhibition and cellular senescence. Accordingly, media conditioned by hT-RPE cells expressing c-Myc show an increased ability to sustain hT-RPE cellular proliferation/viability. We also find a marked down-regulation of several structural and regulatory components of the extracellular matrix (ECM), which correlates with an increased chemotactic potency of the conditioned media toward fibroblasts, a major cellular component of tumor stroma. In accordance with these data, the expression of the majority of the genes encoding proteins down-regulated in hT-RPE was significantly reduced also in colorectal adenomatous polyps, early tumors in which c-Myc is invariably overexpressed. These findings help to elucidate the significance of c-Myc overexpression at early stages of tumor development and uncover a remarkable autocrine/paracrine component in the ability of c-Myc to stimulate proliferation, sustain tumor maintenance, and modulate cell migration.

LncRNA as Cancer Biomarkers.

Although the great majority of cancers share a defined group of hallmarks that is responsible for the uncontrolled growth of particular cell types, it is today clear that under the name of cancer we refer to hundreds of different diseases. Furthermore, each of these diseases has an intrinsic variability due to the genetic background in which it develops. The ability to correctly identify these diseases is urgently needed, because each of them may require a specific therapeutic treatment for successful cure. Cancer biomarkers can be extremely valuable tools for efficient diagnosis and prognosis of cancers. In order to succeed in distinguishing between cancer types and progression-associated genetic backgrounds, cancer biomarkers need to have a strong specificity for a particular disease condition. With the development of novel sequencing technologies, it became clear that the set of genes transcribed from human cells is not limited to genes that code for proteins. On the contrary, our cells contain thousands of RNA without any protein-coding potential. The observation that these transcripts have a much higher cell/tissue specificity of expression in comparison to protein-coding genes makes them a potentially very valuable source of novel cancer biomarkers.

Characterization of the mismatch repair defect in the human lymphoblastoid MT1 cells.

Mutations in mismatch repair (MMR) genes predispose to hereditary nonpolyposis colon cancer. Those leading to truncated proteins bring about a MMR defect, but phenotypes of missense mutations are harder to predict especially if they do not affect conserved residues. Several systems capable of predicting the phenotypes of MMR missense mutations were described. We deployed one of these to study the MMR defect in MT1 cells, which carry mutations in both alleles of the hMSH6 gene. In one, an A-->T transversion brings about an Asp(1213)Val amino acid change in the highly conserved ATP binding site, whereas the other carries a G-->A transition, which brings about a Val(1260)Ile change at a nonconserved site. The hMSH2/hMSH6 (hMutS alpha) heterodimers carrying these mutations were expressed in the baculovirus system and tested in in vitro MMR assays. As anticipated, the Asp(1213)Val mutation inactivated MMR by disabling the variant hMutS alpha from translocating along the DNA. In contrast, the recombinant Val(1260)Ile variant displayed wild-type activity. Interestingly, partial proteolytic analysis showed that this heterodimer was absent from MT1 extracts, although both hMSH6 alleles in MT1 cells could be shown to be transcribed with an efficiency similar to each other and to that seen in control cells. The MMR defect in MT1 cells is thus the compound result of one mutation that inactivates the ATPase function of hMutS alpha and a second mutation that apparently destabilizes the Val(1260)Ile hMSH6 protein in human cells in vivo.

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression.

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher's exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; chi(2), P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher's exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.

The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis.

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis). The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.

Understanding metabolic alterations in cancer cells: a promising new/old approach to eradicate cancer.

Alterations in several aspects of cell metabolism have recently been re-discovered as one of the main features of most cancer cells. The three minireviews discuss how alterations in the glycolytic pathway, in lipid metabolism and in amino acid sensing may give a growth advantage to cells with high proliferative rates, experiencing inadequate nutrients availability.

Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Praf2 is a novel Bcl-xL/Bcl-2 interacting protein with the ability to modulate survival of cancer cells.

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.