SOX11 promotes tumor protective microenvironment interactions through CXCR4 and FAK regulation in mantle cell lymphoma.

SOX11 overexpression in mantle cell lymphoma (MCL) has been associated with more aggressive behavior and worse outcome. However, SOX11 oncogenic pathways driving MCL tumor progression are poorly understood. Here, we demonstrate that SOX11 binds to regulatory regions of 2 important genes for microenvironment signals in cancer: (C-X-C motif) chemokine receptor 4 (CXCR4) and PTK2 (encoding for focal adhesion kinase [FAK]). Moreover, SOX11+ xenograft and human primary MCL tumors overexpress cell migration and stromal stimulation gene signatures compared with their SOX11- counterparts. We show that SOX11 directly upregulates CXCR4 and FAK expression, activating PI3K/AKT and ERK1/2 FAK-downstream pathways in MCL. Concordantly, SOX11+ MCL cells have higher cell migration, transmigration through endothelial cells, adhesion to stromal cells, and cell proliferation and display an increased resistance to conventional drug therapies compared with SOX11- MCL cells. Specific FAK inhibition blocks downstream PI3K/AKT- and ERK1/2-mediated phosphorylation. Additionally, specific FAK and PI3K inhibitors reduce SOX11-enhanced MCL cell migration and stromal interactions and revert cell adhesion-mediated drug resistance (CAM-DR) to the same levels as SOX11- MCL cells. In intravenous MCL xenograft models, SOX11+ MCL cells display higher cell migration, invasion, and growth compared with SOX11-knockdown cells, and specific FAK and CXCR4 inhibitors impair SOX11-enhanced MCL engraftment in bone marrow. Overall, our results suggest that SOX11 promotes MCL homing and invasion and increases CAM-DR through the direct regulation of CXCR4 and FAK expression and FAK/PI3K/AKT pathway activation, contributing to a more aggressive phenotype. Inhibition of this pathway may represent an efficient strategy to overcome stromal-mediated chemotherapy refractoriness in aggressive MCL.

SOX11 promotes tumor angiogenesis through transcriptional regulation of PDGFA in mantle cell lymphoma.

SOX11 is overexpressed in several solid tumors and in the vast majority of aggressive mantle cell lymphomas (MCLs). We have recently proven that SOX11 silencing reduces tumor growth in a MCL xenograft model, consistent with the indolent clinical course of the human SOX11-negative mantle cell lymphoma (MCL). However, the direct oncogenic mechanisms and downstream effector pathways implicated in SOX11-driven transformation remain poorly understood. Here, we observed that SOX11-positive xenograft and human primary MCL tumors overexpressed angiogenic gene signatures and had a higher microvascular density compared with their SOX11-negative counterparts. Conditioned media of SOX11-positive MCL cell lines induced in vitro endothelial cell proliferation, migration, tube formation, and activation of downstream angiogenic pathways. We identified PDGFA as a SOX11 direct target gene upregulated in MCL cells whose inhibition impaired SOX11-enhanced in vitro angiogenic effects on endothelial cells. In addition, platelet-derived growth factor A (PDGFA) was overexpressed in SOX11-positive but not in SOX11-negative MCL. In vivo, imatinib impaired tumor angiogenesis and lymphoma growth in SOX11-positive MCL xenograft tumors. Overall, our results demonstrate a prominent role for SOX11 as a driver of proangiogenic signals in MCL, and highlight the SOX11-PDGFA axis as a potential therapeutic target for the treatment of this aggressive disease.

SOX11 regulates PAX5 expression and blocks terminal B-cell differentiation in aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) is one of the most aggressive lymphoid neoplasms whose pathogenesis is not fully understood. The neural transcription factor SOX11 is overexpressed in most MCL but is not detected in other mature B-cell lymphomas or normal lymphoid cells. The specific expression of SOX11 in MCL suggests that it may be an important element in the development of this tumor, but its potential function is not known. Here, we show that SOX11 promotes tumor growth in a MCL-xenotransplant mouse model. Using chromatin immunoprecipitation microarray analysis combined with gene expression profiling upon SOX11 knockdown, we identify target genes and transcriptional programs regulated by SOX11 including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis, and stem cell development. PAX5 emerges as one of the major SOX11 direct targets. SOX11 silencing downregulates PAX5, induces BLIMP1 expression, and promotes the shift from a mature B cell into the initial plasmacytic differentiation phenotype in both primary tumor cells and an in vitro model. Our results suggest that SOX11 contributes to tumor development by altering the terminal B-cell differentiation program of MCL and provide perspectives that may have clinical implications in the diagnosis and design of new therapeutic strategies.

Exploring the Immunogenicity of Noncanonical HLA-I Tumor Ligands Identified through Proteogenomics.

PURPOSE: Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from noncanonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. EXPERIMENTAL DESIGN: Peptides presented on HLA-I were identified in 9 patient-derived tumor cell lines from melanoma, gynecologic, and head and neck cancer through proteogenomics. A total of 507 candidate tumor antigens, including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens, were tested for T-cell recognition of preexisting responses in patients with cancer. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell receptors (TCR) and evaluate their therapeutic potential. RESULTS: We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor-reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor PBL against 170 selected nonC-TL, led to the identification of TCRs specific to three nonC-TL, two of which mapped to the 5' UTR regions of HOXC13 and ZKSCAN1, and one mapping to a noncoding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. CONCLUSIONS: Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies. See related commentary by Fox et al., p. 2173.

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer.

BACKGROUND: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. METHODS: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. RESULTS: We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1- and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1- or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. CONCLUSIONS: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.

Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages.

Toll-like receptor 5 (TLR5) is the receptor of bacterial Flagellin. Reportedly, TLR5 engagement helps to combat infections, especially at mucosal sites, by evoking responses from epithelial cells and immune cells. Here we report that TLR5 is expressed on a previously defined bipotent progenitor of macrophages (MΦs) and osteoclasts (OCs) that resides in the mouse bone marrow (BM) and circulates at low frequency in the blood. In vitro, Flagellin promoted the generation of MΦs, but not OCs from this progenitor. In vivo, MΦ/OC progenitors were recruited from the blood into the lung upon intranasal inoculation of Flagellin, where they rapidly differentiated into MΦs. Recruitment of the MΦ/OC progenitors into the lung was likely promoted by the CCL2/CCR2 axis, since the progenitors expressed CCR2 and type 2 alveolar epithelial cells (AECs) produced CCL2 upon stimulation by Flagellin. Moreover, CCR2 blockade reduced migration of the MΦ/OC progenitors toward lung lavage fluid (LLF) from Flagellin-inoculated mice. Our study points to a novel role of the Flagellin/TLR5 axis in recruiting circulating MΦ/OC progenitors into infected tissue and stimulating these progenitors to locally differentiate into MΦs. The progenitor pathway to produce MΦs may act, next to monocyte recruitment, to fortify host protection against bacterial infection at mucosal sites.

Intratumor Adoptive Transfer of IL-12 mRNA Transiently Engineered Antitumor CD8+ T Cells.

Retroviral gene transfer of interleukin-12 (IL-12) into T cells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ T cells to transiently express IL-12. Engineered T cells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ T cell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating T lymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.

Macrophages and osteoclasts stem from a bipotent progenitor downstream of a macrophage/osteoclast/dendritic cell progenitor.

Monocytes/macrophages (MΦs), osteoclasts (OCs), and dendritic cells (DCs) are closely related cell types of high clinical significance, but the exact steps in their lineage commitment are unclear. In studies on MΦ and DC development, OC development is generally not addressed. Furthermore, findings on DC development are confusing, because monocytes can also differentiate into DC-like cells. To resolve these issues, we have examined the development of monocytes/MΦs, OCs, and DCs from common progenitors, using the homeostatic driver cytokines macrophage colony-stimulating factor, RANK ligand (L), and Flt3L. In mouse bone marrow, B220-CD11blow/-c-Kit+c-Fms+ cells could be dissected into a CD27+Flt3+ population that proved oligopotent for MΦ/OC/DC development (MODP) and a CD27low/-Flt3- population that proved bipotent for MΦ/OC development (MOP). Developmental potential and relationship of MODP and downstream MOP populations are demonstrated by differentiation cultures, functional analysis of MΦ/OC/DC offspring, and genome-wide messenger RNA expression analysis. A common DC progenitor (CDP) has been described as committed to plasmacytoid and conventional DC development. However, the human CDP proved identical to the MODP population, whereas the mouse CDP largely overlapped with the MODP population and was accordingly oligopotent for MΦ, OC, and DC development. The CX3CR1+ MΦ/DC progenitor (MDP) population described in the mouse generated MΦs and OCs but not DCs. Thus, monocytes/MΦs, OCs, and DCs share a common progenitor that gives rise to a bipotent MΦ/OC progenitor, but a dedicated DC progenitor is currently undefined. The definition of these progenitor populations may serve diagnostics and interventions in diseases with pathogenic activity of MΦs, OCs, or DCs.

Assessment of SOX11 expression in routine lymphoma tissue sections: characterization of new monoclonal antibodies for diagnosis of mantle cell lymphoma.

The diagnosis of mantle cell lymphoma (MCL) can be difficult, especially when no t(11;14) translocation and cyclin D1 overexpression can be detected. In such cases, the transcription factor SOX11 represents an important diagnostic marker, as it is expressed in most MCLs and, in particular, in all cyclin D1-negative MCLs reported so far. A reliable anti-SOX11 antibody is therefore a very useful tool for routine diagnosis. Here, we characterize the new monoclonal anti-SOX11 antibodies, suitable for Western blot assay and immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue; we tested them on a large series of primary lymphoid tumors and compared these results with those of other routinely used antibodies. Moreover, we show that IHC results depend on transcription levels of SOX11, which suggests that posttranscriptional and posttranslational modifications do not significantly affect cutoff levels for IHC detection of SOX11.

Molecular subsets of mantle cell lymphoma defined by the IGHV mutational status and SOX11 expression have distinct biologic and clinical features.

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course, whereas others having an indolent behavior. We conducted an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B-cell receptors (BcR) may identify different subsets of tumors. Truly unmutated (100% identity) IGHV genes were found in 24% cases, 40% were minimally/borderline mutated (99.9%-97%), 19% significantly mutated (96.9%-95%), and 17% hypermutated (<95%). Tumors with high or low mutational load used different IGHV genes, and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naive B-cell signatures, respectively. Furthermore, the highly mutated tumors had less genomic complexity, were preferentially SOX11-negative, and showed more frequent nonnodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 97%. Patients with high and low mutational load had significant different outcome with 5-year overall survival (OS) of 59% and 40%, respectively (P = 0.004). Nodal presentation and SOX11 expression also predicted for poor OS. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11-negativity, and nonnodal presentation correspond to a subtype of the disease with more indolent behavior.

Epigenetic activation of SOX11 in lymphoid neoplasms by histone modifications.

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.