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 () and (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.

USP25 deubiquitinating enzyme is a key member of the ubiquitin system, which acts as a positive regulator of the Wnt/β-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.

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 (RLN). SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared to 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 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 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.

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Mantle cell lymphoma (MCL) is an incurable B-cell neoplasm characterized by its aggressive behavior, short responses to conventional therapies and SOX11 overexpression. Oxidative stress is known to induce tumorigenesis and tumor progression, whereas high 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, we observed that aggressive SOX11 + MCL presented higher reactive oxygen species (ROS) levels and increased expression of oxidative stress-related genes compared to SOX11- MCL primary cases. Upregulation of the antioxidant gene PRDX2 and the proto-oncogene TCL1A significantly correlated with SOX11 overexpression and associated with worse patients’ overall survival. SOX11 knockout (SOX11KO) significantly reduced TCL1A. Moreover, SOX11KO and TCL1A knockdown (TCL1AKD) reduced PRDX2 expression in MCL cell lines. SOX11KO, TCL1AKD and PRDX2KD increased ROS levels and tumor cell death upon drug treatment in vitro. Interestingly, TCL1AKD reduced tumor growth in vivo, suggesting its involvement in MCL tumorigenesis. Overall, our results suggest an aberrant redox homeostasis associated with chemoresistance in aggressive MCL through TCL1A and PRDX2 upregulation, highlighting them as promising targets for new therapeutic strategies to overcome chemoresistance in aggressive MCLs.

Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cancer stem cells (CSC) able to self-renew and responsible for drug resistance. SOX11 embryonic transcription factor is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-CSC have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL clinical features and aggressive behavior remains unknown. We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. Here, we integrated the differential gene expression profile (GEP) between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, and specific SOX11 ChIP-chip and epigenetic data in MCL cell lines and primary cases, to identify potential mediators of CSC directly regulated by SOX11 in MCL. We observed an enrichment of hematopoietic (HSC) and leukemic stem cells (LSC) gene signatures in SOX11+ compared to SOX11- MCL primary cases. MSI2, an RNA binding protein that maintains self-renewal and prevents differentiation in HSC, emerged as one of the most significant upregulated CSC-related gene in SOX11+ compared to SOX11- MCLs, positively correlating with SOX11 expression in MCL primary cases. Our in vitro experiments showed that SOX11 binds to MSI2 regulatory regions increasing its expression in MCL cells. MSI2 enhancers located in intronic regions were activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/TP53, 11q/ATM and 9p/CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models. Overall, our results suggest that SOX11+ MCL cases acquire stemness features through the upregulation of MSI2 expression, which promotes chemoresistance, self-renewal and tumor survival. MSI2 represents a novel biomarker for MCL-CSC and therapeutic target for relapsed MCL. Citation Format: Marta Sureda-Gómez, Patricia Balsas, Marta Leonor Rodríguez, Ferran Nadeu, Anna De Bolòs, Álvaro Eguileor, Marta Kulis, Giancarlo Castellano, José Ignacio Martin-Subero, Santiago Demajo, Pedro Jares, Eva Giné, Elias Campo, Virginia Amador. Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 777.