SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

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.

SOX11, CD70, and Treg cells configure the tumor-immune microenvironment of aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11- primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11- tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory t (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.

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/PRDX2 axis modulates redox homeostasis and chemoresistance in aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell neoplasm characterized by an aggressive behavior, short responses to conventional therapies and SOX11 overexpression, which is associated with aggressive disease features and inferior clinical outcome of patients. Oxidative stress is known to induce tumorigenesis and tumor progression, whereas high expression levels of antioxidant genes have been associated with chemoresistance in different cancers. However, the role of oxidative stress in MCL pathogenesis and the involvement of SOX11 regulating redox homeostasis in MCL cells are largely unknown. Here, by integrating gene set enrichment analysis of two independent series of MCL, we observed that SOX11+ MCL had higher reactive oxygen species (ROS) levels compared to SOX11- MCL primary tumors and increased expression of Peredoxine2 (PRDX2), which upregulation significantly correlated with SOX11 overexpression, higher ROS production and worse overall survival of patients. SOX11 knockout (SOX11KO) significantly reduced PRDX2 expression, and SOX11KO and PRDX2 knockdown (PRDX2KD) had increased ROS levels and ROS-mediated tumor cell death upon treatment with drugs, compared to control MCL cell lines. Our results suggest an aberrant redox homeostasis associated with chemoresistance in aggressive MCL through SOX11-mediated PRDX2 upregulation, highlighting PRDX2 as promising target for new therapeutic strategies to overcome chemoresistance in aggressive MCLs.

Tumorigenic role of Musashi-2 in aggressive mantle cell lymphoma.

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11- MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

A quaternary tetramer assembly inhibits the deubiquitinating activity of USP25.

USP25 deubiquitinating enzyme is a key member of the ubiquitin system, which acts as a positive regulator of the Wnt/ß-catenin signaling by promoting the deubiquitination and stabilization of tankyrases. USP25 is characterized by the presence of a long insertion in the middle of the conserved catalytic domain. The crystal structure of USP25 displays an unexpected homotetrameric quaternary assembly that is directly involved in the inhibition of its enzymatic activity. The tetramer is assembled by the association of two dimers and includes contacts between the coiled-coil insertion domain and the ubiquitin-binding pocket at the catalytic domain, revealing a distinctive autoinhibitory mechanism. Biochemical and kinetic assays with dimer, tetramer and truncation constructs of USP25 support this mechanism, displaying higher catalytic activity in the dimer assembly. Moreover, the high stabilization of tankyrases in cultured cells by ectopic expression of a constitutive dimer of USP25 supports a biological relevance of this tetramerization/inhibition mechanism.