SOX11 Overexpression Research Articles

Hypomethylation-associated Sox11 upregulation promotes oncogenesis via the PI3K/AKT pathway in OLP-associated OSCC.

Oral lichen planus (OLP) is a particularly prevalent oral disorder with the potential to progress to oral squamous cell carcinoma (OSCC). SRY-box transcription factor 11 (Sox11) has been reported to serve as a prognostic marker for various cancers. However, the role and mechanism of Sox11 in OLP-related OSCC are unknown. Our results indicated that Sox11 was highly expressed, and that Sox11 promoter methylation was significantly reduced in OLP-associated OSCC tissues. High Sox11 expression and Sox11 promoter hypomethylation indicate a poor patient prognosis. According to invivo and invitro experiments, the knockdown of Sox11 inhibited proliferation, invasion, and migration while driving its apoptotic death in OSSC cells; Sox11 overexpression exerted the opposite effect as Sox11 knockdown. Mechanistically, knockdown of Sox11 inhibited PI3K/AKT and glycolysis pathway, and overexpression of Sox11 enhanced the PI3K/AKT and glycolysis pathways in OSCC cells. In addition, we demonstrated that Sox11 overexpression accelerated the progression of OSCC, at least in part by promoting PI3K/AKT pathway activation. In conclusion, our data indicated that the DNA hypomethylation-associated upregulation of Sox11 could promote oncogenic transformation via the PI3K/AKT pathway in OLP-associated OSCC. Therefore, Sox11 might be a reliable biomarker for predicting the progression of precancerous oral tissues.

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.

Neuropathic pain development following nerve injury is mediated by SOX11-ARID1A-SOCS3 transcriptional regulation in the spinal cord.

Neuropathic pain, a complex condition originating from nervous system damage, remains a significant clinical challenge due to limited understanding of its underlying mechanisms. Recent research highlights the SOX11 transcription factor, known for its role in nervous system development, as a crucial player in neuropathic pain development and maintenance. This study investigates the role of the SOX11-ARID1A-SOCS3 pathway in neuropathic pain modulation within the spinal cord. Using a spinal nerve ligation (SNL) model in mice, we observed a significant upregulation of Sox11 in the spinal cord dorsal horn post-injury. Intrathecal administration of Sox11 shRNA mitigated SNL-induced neuropathic pain behaviors, including mechanical allodynia and heat hyperalgesia. Further, we demonstrated that Sox11 regulates neuropathic pain via transcriptional control of ARID1A, with subsequent modulation of SOCS3 expression. Knockdown of ARID1A and SOCS3 via shRNA resulted in alleviation of Sox11-induced pain sensitization. Additionally, Sox11 overexpression led to an increase in ARID1A binding to the SOCS3 promoter, enhancing chromatin accessibility and indicating a direct regulatory relationship. These findings were further supported by in vitro luciferase reporter assays and chromatin accessibility analysis. The SOX11-ARID1A-SOCS3 pathway plays a pivotal role in the development and maintenance of neuropathic pain. Sox11 acts as a master regulator, modulating ARID1A, which in turn influences SOCS3 expression, thereby contributing to the modulation of neuropathic pain. These findings provide a deeper understanding of the molecular mechanisms underlying neuropathic pain and highlight potential therapeutic targets for its treatment. The differential regulation of this pathway in the spinal cord and dorsal root ganglia (DRG) underscores its complexity and the need for targeted therapeutic strategies.

A large-scale functional analysis of genes expressed differentially in insulin secreting MIN6 sublines with high versus mildly reduced glucose-responsiveness

Molecular mechanisms of glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells are not fully understood. GSIS deteriorations are believed to underlie the pathogenesis of type 2 diabetes mellitus. By comparing transcript levels of 3 insulin secreting MIN6 cell sublines with strong glucose-responsiveness and 3 with mildly reduced responsiveness, we identified 630 differentially expressed genes. Using our recently developed system based on recombinase-mediated cassette exchange, we conducted large-scale generation of stable clones overexpressing such genes in the doxycycline-regulated manner. We found that overexpressions of 18, out of 83, genes altered GSIS. Sox11 ((sex determining region Y)-box 11) was selected to confirm its roles in regulating insulin secretion, and the gene was subjected to shRNA-mediated suppression. While Sox11 overexpression decreased GSIS, its suppression increased GSIS, confirming the role of Sox11 as a negative regulator of insulin secretion. Furthermore, metabolic experiments using radiolabelled glucose showed Sox11 to participate in regulating glucose metabolism. Our data suggested that overexpression screening is a feasible option for systemic functional testing to identify important genes in GSIS.

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.

Abstract A26: SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice

Abstract Mantle cell lymphoma (MCL) is a highly aggressive B cell lymphoma that accounts for 6% of all non-Hodgkin lymphomas and is characterized by abnormal proliferation of mature antigen- naive B lymphocytes. Despite recent advances in patient risk stratification, MCL remains incurable due to frequent relapses and resistance to therapy. SOX11 is a key transcription factor in the pathogenesis of MCL and is highly expressed in conventional MCL, but it is not expressed in the normal B cells or in the indolent leukemic nonnodal MCL subtype. Although SOX11 was identified as crucial oncogene that supports maintenance of MCL, its role in the initiation of MCL remains largely unknown. Here, we developed a novel mouse model for condition overexpression of SOX11 and a eGFP reporter and found that elevated levels of SOX11 in B-cells are sufficient to drive MCL-like lymphomas in mice. Moreover, SOX11 expression synergized with loss of p53 and overexpression of Ccnd2 to form aggressive MCL-like CD19+CD5+CD23−IgM+IgD+ lymphomas, with a median survival of 314 (p53Mb1 ; n=11), 165 (SOX11/p53Mb1 ; n=19), and 134 (SOX11/Ccnd2/p53Mb1 ; n=15) days. These SOX11-driven lymphomas show transcriptional, immunophenotypic and functional similarities with both murine B1a cells and MCL patients. SOX11 overexpression skews B-cell development towards the B1a and marginal zone B-cell lineage and it block B2-cell development at the Pro-B and Pre-B cell stage, which coincides with onset eGFP that is a measure of expression of Cre and SOX11. In contrast to cyclin D2-driven MCL-like model, SOX11 is inducing a pre-lymphoma stage, in which there is a progressive accumulation of B1a cells in peripheral blood, peritoneal cavity, spleen and bone-marrow. The pre-lymphoma B1a cells have a BCR repertoire which was biased towards binding of self-antigens, such as phosphatidylcholine, and have increased BCR signaling activity. To prove that embryonic-derived B1a cells were the cell-of-origin of SOX11-driven MCL-like lymphoma, we transplanted CD19+ B-cells from SOX11/p53Mb1 fetal livers into secondary recipients and found that these embryonic cells could give rise to MCL-like lymphomas. Next, we wanted to determine how SOX11 is inducing the pre-lymphoma phase, either by expanding existing B1a cells, having a developmental bias towards B1a cells, or by the ability to reprogram B2-cells towards a B1a cell fate. Therefore, we crossed SOX11/p53 mice with a tamoxifen-inducible CreERT2 line and transplanted different sorted B1 and B2 subsets after a short tamoxifen induction ex vivo in immunocompromised mice. We found that SOX11 OE can redirect mature B2 cells to form B1a/MCL lymphomas. Overall, SOX11 is a key B1a lineage factor in mice and its aberrant expression is leading to the formation of MCL-like lymphomas in mice. Citation Format: Tim Pieters, Steven Goossens, Pieter Van Vlierberghe. SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A26.

USP9X Increased Tumor Angiogenesis in Mantle Cell Lymphoma by Upregulation of CCND1-Mediated SOX11.

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy with a poor prognosis. Ubiquitin-specific peptidase 9, X-linked (USP9X), has been associated with multiple physiological pathways and regulates various cellular activities. In this study, we explored the role of USP9X in MCL in vitro and in vivo. USP9X was verified to be increased in peripheral blood mononuclear cells (PBMCs) of MCL patients and MCL cells. Moreover, CCND1 and SOX11 were also upregulated in PBMCs of MCL patients. The positive correlation between USP9X and CCND1, USP9X and SOX11, and CCND1 and SOX11 were identified.Further, USP9X overexpression and knockdown were performed in MCL cells. We proved that USP9X overexpression promoted proliferation and cell cycle and suppressed cell apoptosis in MCL cells. Upregulation of angiogenesis and cell migration were induced by USP9X overexpression in MCL cells. However, the USP9X knockdown showed opposite effects. In addition, USP9X was discovered to decrease Cyclin D1 (CCND1)-mediated SOX11 expression in MCL cells. We demonstrated that SOX11 overexpression reversed USP9X knockdown-mediated angiogenesis in MCL cells. Besides, tumor formation was inhibited by USP9X knockdown in mice in vivo. In conclusion, these results revealed that USP9X promoted tumor angiogenesis in MCL via increasing CCND1-mediated SOX11.

Role of M2 macrophages-derived extracellular vesicles in IL-1β-stimulated chondrocyte proliferation and inflammatory responses.

M2 macrophages-derived extracellular vesicles (M2-EVs) serve as a tool for the delivery of miRNAs and play an anti-inflammatory role in diseases. This study sought to explore the role of (M2-EVs) in the proliferation and inflammatory responses of IL-1β-stimulated chondrocytes. M2 macrophages were induced and characterized, followed by isolation and characterization of M2-EVs. Chondrocytes were treated with 10ng/mL IL-1β and co-cultured with M2 macrophages transfected with Cy3-labeled miR-370-3p. Cell viability, TNF (tumor necrosis factor)-α, IL(Interleukin)-18, IL-10, miR-370-3p, and sex-determining region Y-related high-mobility-group box transcription factor 11 (SOX11) mRNA were determined via cell counting assay kit, colony formation, ELISA, and qRT-PCR. The binding relationship between miR-370-3p and SOX11 was testified via the dual-luciferase assay. The functional rescue experiment was designed to confirm the role of SOX11. M2-EVs improved chondrocyte viability and colony formation, lowered TNF-α and IL-18, and elevated IL-10. M2-EVs delivered miR-370-3p into chondrocytes to upregulate miR-370-3p. Upregulation of miR-370-3p in M2-EVs enhanced the protective role of M2-EVs in chondrocytes. miR-370-3p inhibited SOX11 transcription. SOX11 overexpression attenuated the protective role of M2-EVs in chondrocytes. Overall, our findings suggested that M2-EVs promote proliferation and suppress inflammatory responses in IL-1β-stimulated chondrocytes via the miR-370-3p/SOX11 axis.

SOX11 and CD43 Biomarkers in Mantle Cell Lymphoma

Recently, mantle cell lymphoma (MCL) has been recognized as a heterogeneous clinical and biological entity, as molecular genetics highlighted different pathways of cell survival and progression. Histological diagnosis may be challenging due to atypical MCL variants, immunophenotyping or absence of cyclin D1 (D1), encountered in 10% of cases. SOX11 is a neural transcription factor, involved in blocking mature B cell differentiation and its overexpression is associated with poor prognosis. It is overexpressed in most conventional MCL (cMCL) and in MCL lacking D1 overexpression, but not detected in other mature B cell lymphomas or normal lymphoid cells. Recently, its overexpression has been considered as a specific biomarker for MCL diagnosis.Development of novel targeted therapy approaches renders monitoring of minimal residual disease (MRD) essential to assess treatment effectiveness. Amongst methods developed to monitor MRD, allele-specific oligonucleotide quantitative PCR for BCL1 or IgH, is the most sensitive and commonly used approach. Given the role of SOX11 and BCL1 in the pathophysiology of MCL, we developed an easy set up single step quantitative PCR (qPCR) to assess both markers simultaneously, which was tested on a large panel of MCL patients at diagnosis and MRD monitoring.A reverse transcription qPCR protocol was carried out to quantify SOX11, cyclin D1 and D2 overexpression simultaneously, using specific primers, Taqman probes and B2M house keeping gene (LC480, Roche). RNA was extracted from peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). Sensitivity was determined using 6 fold dilutions starting with 1:10 from 30 subjects with normal lymphocytes. A retrospective study on a total of 170 samples for 145 patients between 2002 and 2017 was conducted. Samples were chosen based on morphology, immunophenotyping and histopathology if available. All samples were CD20+, 162 were CD5+ and 165 were CD10-. All patients had provided an informed written consent in accordance with the declaration of Helsinki. We tested 93 patients with MCL at diagnosis, 79 cMCL and 14 indolent mantle cell lymphoma (iMCL). To assess specificity 52 patients with other lymphoid proliferations CD5/20+, CD10- were studied. 20 samples from 5 patients SOX11+/D1+ were available for MRD monitoring.Sensitivity of the method achieved 1.10-4. At diagnosis, tumor cells ranged between 11-97% of total lymphocytes in PB and 1-91% in BM. SOX11 and D1 overexpression was correlated with the tumor burden. 79/79 cMCL patients (100%) overexpressed SOX11 and D1 (SOX11+/D1+), 2 cMCL were CD5-, SOX11+/ D1+ and 2 cMCL were CD10+, SOX11 +/ D1+. All 14 iMCL patients were (SOX11-/D1+). Sox 11 was not detected in normal lymphocytes or in other lymphoproliferative diseases. 5 patients presented with the blastoid variant, overexpressed SOX11, but only 4 overexpressed D1 and tumor infiltration rate at diagnosis was over 83% in PB. MRD follow up showed negativity of both markers for 2 patients at complete remission. SOX11 overexpression was detected before cyclin D1 in 2 patients at relapse, prior to clinical manifestations. Persistent SOX11 and D1 overexpression was observed for 1 splenctomized patient. Time to treatment was found to be 0.6 vs 18 months in 18 cMCL and 12 iMCL patients respectively (p= 0.0006). CD43 was evaluated for 27 patients, 4 blastoid variants and 23 cMCL. 100% of blastoid variants were positive for CD43, but only 43.4% of cMCL were positive for CD43 (p=0.0368). Overall survival curve for CD43 positive patients showed a median of 2.3 years survival (p=0.0285). This was not reached for the CD43 negative MCL.We developed a feasible single step sensitive method to quantify SOX11, D1 and D2 in MCL which allows differential diagnosis with other lymphoid proliferations. A parallel correlation was found between SOX11, D1 and clinical status of MCL during diagnosis and follow up. As histopathology is unsuitable for MRD evaluation, both markers could be used to assess MRD, though SOX11 is preferred to D1 as it lacks the D1 background overexpression. Considering the higher sensitivity of molecular MRD compared to conventional evaluating procedures, its integration in guided treatment strategies is warranted. To the best of our knowledge, this is the first report stating the association of CD43 with adverse prognosis in MCL which might be interesting as a prognostication marker and a target for a novel immunotherapy development. [Display omitted] DisclosuresTroussard:ROCHE: Honoraria; GILEAD: Honoraria; JANSSEN: Honoraria; ABBVIE: Honoraria.

SOX11 and FAK participate in the stretch‑induced mechanical injury to alveolar type2 epithelial cells.

The aim of the present study was to explore the potential role of SOX11 in the stretch-induced mechanical injury to alveolar type 2 epithelial (AT2) cells. A cell stretch (CS) test was used to induce mechanical injury to primary cultured AT2 cells. Wound healing, adhesion, cell viability assays and flow cytometry were performed to evaluate the migration, adhesion, viability and apoptosis of AT2 cells. Changes in the invasive ability of AT2 cells were detected using a Transwell invasion assay. To further explore the underlying molecular mechanisms, reverse transcription-quantitative PCR and western blot analysis were used to assess the expression levels of SOX11, FAK, Akt, caspase-3/8, p65 and matrix metal-loproteinase (MMP)7. Co-immunoprecipitation (Co-IP) and luciferase reporter assays were used to detect the interaction between SOX11 and FAK. CS reduced the invasion, migration and adhesion, and increased the apoptosis of AT2 cells. It also resulted in the downregulation of SOX11 and FAK expression in AT2 cells. The overexpression of SOX11 reversed these changes, whereas the knockdown of SOX11 aggravated the deterioration of the aforementioned biological behaviors and the apoptosis of the AT2 cells following CS. The overexpression of SOX11 upregulated the FAK and Akt expression levels, and downregulated caspase-3/8 expression, whereas the silencing of SOX11 reversed these changes following CS. Furthermore, the effects of SOX11 overexpression were inhibited by FAK antagonism. The results of Co-IP demonstrated that SOX11 and FAK were bound together, and that the expression of FAK was significantly increased in the SOX11 overexpression group. Luciferase assays revealed that the luciferase activity and the mRNA expression of FAK were significantly increased following transfection with pcDNA SOX11 and pGL3 FAK promoter. Co-IP and luciferase assays revealed that SOX11 directly regulated the expression of FAK. On the whole, the present study demonstrates that the downregulated expression of SOX11 and FAK are involved in the stretch-induced mechanical injury to AT2 cells. The over-expression of SOX11 significantly alleviates AT2 cell injury through the upregulation of FAK and Akt, and the inhibition of apoptosis. These findings suggest that the activation of SOX11 and FAK may be potential preventive and therapeutic options for ventilator-induced lung injury.

LINC00961 inhibits the migration and invasion of colon cancer cells by sponging miR-223-3p and targeting SOX11.

Long noncoding RNAs play essential roles in colon cancer tumorigenesis. This study aimed to explore the potential function and molecular mechanisms of LINC00961 in colon cancer. qPCR results showed that LINC00961 was downregulated in colon cancer cells and tissues. Functional assays demonstrated that LINC00961 suppressed the migration and invasion of colon cancer cells in vitro. LINC00961 functioned as an endogenous sponge for miR‐223‐3p in colon cancer cells. SOX11 was confirmed as a target gene of miR‐223‐3p. The effect of miR‐223‐3p on colon cancer cells was then investigated. MiR‐223‐3p inhibition enhanced their migration and invasion. The effect of SOX11 on colon cancer cells was studied. SOX11 overexpression inhibited the invasion of colon cancer cells. LINC00961 acted as an anti‐oncogene and upregulated SOX11 expression by functioning as a miR‐223‐3p sponge. This research revealed the molecular mechanism of LINC00961 in colon cancer. LINC00961 might act as a potential diagnostic biomarker and therapeutic target for further clinical treatments.

S842 MODELING MANTLE CELL LYMPHOMA IN MICE

Background:Mantle cell lymphoma (MCL) is a highly aggressive subtype of B‐cell lymphoma that is characterized by a poor response to current treatment regimens. Most MCLs carry a prototypical translocation, t(11;14), which juxtaposes the CCND1 gene towards the immunoglobulin heavy chain (IGH) locus, resulting in cyclin D1 overexpression. Notably, a subset of MCL patients are cyclin D1 negative but instead overexpress cyclin D2 (encoded by CCND2)as a consequence of recurrent genomic rearrangements involving the CCND2locus. These CCND2rearrangements were acknowledged in the revised 2016 WHO classification of lymphoid neoplasms and hints towards a putative role for Cyclin D2 in the etiology of MCL. In addition, the transcription factor SOX11 highly expressed in MCL, hinting that it might act as an oncogene in MCL.Aims:We wanted to evaluate if cyclin D2 and SOX11 are true oncogenic drivers of MCL. Furthermore, we aimed to model MCL in mice and use them as a preclinical tool to identify novel therapeutic options for MCL patients.Methods:To unravel the role of Cyclin D2 and SOX11 in the formation of MCL in vivo, we generated R26‐KI mice that allow conditional overexpression of either Ccnd2 or SOX11. For this, we used a targeting vector that contained a floxed stop cassette followed by cDNA (from Ccnd2 or SOX11) and a EGFP/luciferase reporter, which was subsequently targeted in mESCs using recombinase‐mediated cassette exchange (RMCE, Fig. A).Results:We found that hematopoietic‐specific Ccnd2 activation (using Vav‐iCre) is sufficient to drive MCL formation in mice (Fig. B, top). Moreover, we could show that this murine model recapitulates several clinical features of MCL patients, including CD19+, CD5+, CD23‐ immunophenotype, clonal tumor expansion, lack of somatic hypermutation, high expression of Sox11 and Bcl‐2, and infiltration in spleen, liver and GI tract. Furthermore, these murine MLC tumors were transplantable and homed to sites within the lymphatic system, but unfortunately did not show aggressive growth in vivo.Finally, we tested putative synergism between Ccnd2 and SOX11 overexpression and other cooperating genetic lesions that occur in human MCL, such as loss of p53. To this end, we generated mice with B‐cell specific overexpression of Cyclin D2 or SOX11 in combination with deletion of p53 (Fig. B, middle). These mice also formed MCL‐like tumors and developed “full blown” MCL within weeks upon their transplantation into immunocompromised mice (Fig. B, bottom). Importantly, the transplanted tumor cells are luciferase‐positive and are therefore suitable for in vivodrug testing using bioluminescence. As proof‐of‐principle, we tested the efficacy of known MCL drugs, such as the anti‐BCL2 inhibitor Venetoclax in our model system. In conclusion, we generated novel cyclin‐D2 and SOX11‐driven MCL mouse models that can be used for preclinical drug screening and will hopefully lead to a better outcome for MCL patients.Summary/Conclusion:Using novel R26‐KI mice with conditional expression of Ccnd2 or SOX11, we demonstrated that both Ccnd2 and SOX11 are bona fide drivers of MCL.image

Increased Expression of Transcription Factor SRY-box-Containing Gene 11 (Sox11) Enhances Neurite Growth by Regulating Neurotrophic Factor Responsiveness

The peripherally projecting axons of dorsal root ganglion (DRG) neurons readily regenerate after damage while their centrally projecting branches do not regenerate to the same degree after injury. One important reason for this inconsistency is the lack of pro-regeneration gene expression that occurs in DRG neurons after central injury relative to peripheral damage. The transcription factor SRY-box-containing gene 11 (Sox11) may be a crucial player in the regenerative capacity of axons as previous evidence has shown that it is highly upregulated after peripheral axon damage but not after central injury. Studies have also shown that overexpression or inhibition of Sox11 after peripheral nerve damage can promote or block axon regeneration, respectively. To further understand the mechanisms of how Sox11 regulates axon growth, we artificially overexpressed Sox11 in DRG neurons in vitro to determine if increased levels of this transcription factor could enhance neurite growth. We found that Sox11 overexpression significantly enhanced neurite branching in vitro, and specifically induced the expression of glial cell line-derived neurotrophic factor (GDNF) family receptors, GFRα1 and GFRα3. The upregulation of these receptors by Sox11 overproduction altered the neurite growth patterns of DRG neurons alone and in response to growth factors GDNF and artemin; ligands for GFRα1 and GFRα3, respectively. These data support the role of Sox11 to promote neurite growth by altering responsiveness of neurotrophic factors and may provide mechanistic insight as to why peripheral axons of sensory neurons readily regenerate after injury, but the central projections do not have an extensive regenerative capacity.

Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others

At least 30 types of retinal ganglion cells (RGCs) senddistinct messages through the optic nerve to the brain. Available strategies of promoting axon regeneration act on only some of these types. Herewe tested the hypothesis that overexpressing developmentally important transcription factors in adult RGCs could reprogram them to a "youthful" growth-competent state and promote regenerationof other types. From a screen of transcription factors, we identified Sox11 as one that could inducesubstantial axon regeneration. Transcriptomeprofiling indicated that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, α-RGCs, which preferentially regenerate following treatments such as Pten deletion, were killed by Sox11 overexpression. Thus, Sox11 promotes regeneration of non-α-RGCs, which are refractory to Pten deletion-induced regeneration.We conclude that Sox11 can reprogram adult RGCs to a growth-competent state, suggesting that different growth-promoting interventions promote regeneration in distinct neuronal types.

Role of SOX11 and Genetic Events Cooperating with Cyclin D1 in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm, incurable with current therapies. The t(11;14)(q13;q32) involving cyclin D1 is considered the first oncogenic hit found in virtually all MCLs. However, additional secondary genomic alterations are essential for complete transformation. MCLs are genetically very unstable with several genetic alterations associated with its high proliferative behavior involving several oncogenic pathways. Furthermore, SOX11 is overexpressed in the majority of conventional MCLs (cMCL), including cyclin D1-negative cases, but absent in non-nodal leukemic MCL with indolent clinical behavior (nnMCL). Recent data have revealed the potential oncogenic role of SOX11 in MCL biology, highlighting its implication in tumor aggressiveness and progression. This review addresses the implication of SOX11 overexpression and frequent genetic lesions, cooperating with cyclin D1 underlying the pathogenesis of this aggressive disease.

Inhibiting SOX11-DNA Interaction in Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is a fatal subtype of Non-Hodgkins Lymphoma. MCL is characterized by cell cycle dysregulation due to Cyclin D1 (CCND1) overexpression. Murine models overexpressing CCND1 do not develop B-cell proliferation. The SOX11 transcription factor is overexpressed in the majority of nodal human MCL and has been implicated in MCL pathogenesis. We have developed a transgenic Eµ-SOX11-EGFP mouse model that overexpresses SOX11 with aberrant B cell proliferation, lymphadenopathy, spenomegaly and hepatomegaly phenocopying the characteristics of patients with MCL.Using Mass Cytometry (CyTOF), we further characterized this B cell population to be CD23-, CD21/35 dim, CD138-, high surface IgM, and variable IgD expression, an immunophenotype identical to human MCL. SOX11 overexpression drives B cell receptor (BCR) signaling in murine MCL cells, assayed by phosphorylation of p-BTK and p-PLC gamma.To further the study of the role of SOX11 in MCL, compounds inhibiting the binding interaction of SOX11 and DNA are highly desirable and we aim at their discovery by using structure-based virtual screening predictions and experimental validations. No small-molecule inhibitor of SOX11-DNA binding has been developed to date and there is an unmet need in MCL therapeutics for novel and successful treatments.Transcription factors have been considered "undruggable" but since SOX11 binds the minor groove in DNA, we hypothesized that there may be pockets created by SOX11-DNA binding that could be inhibited by small molecules. We therefore built a human SOX11 homology model using the crystal structure of murine SOX4 (98% homology). We first docked molecules from the NIH Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC). From this virtual screening, 70 compounds were purchased for experimental testing. Using a fluorescence anisotropy assay, Flavitan was identified as a potential inhibitor of the SOX11-DNA interaction. Based on inferences from the predicted binding mode of Flavitan on SOX11, we performed another virtual screening of ~12 million molecules from the ZINC database followed by assaying 26 top-scoring compounds, as well as, 16 derivatives of one of them, for SOX11 binding. We thus discovered two compounds, R and T, with binding constants in the low micromolar range. Importantly, compounds R and T were found not to intercalate DNA in a Topoisomerase I based assay.To assess the biological activity of these molecules, we tested whether BCR signaling in MCL cell lines expressing SOX11 would be inhibited by treatment with these compounds. Compounds R and T significantly inhibited p-BTK as compared to vehicle control. Ibrutinib, an FDA approved BTK inhibitor, was used as a positive control in these experiments.To determine whether BCR pathway inhibition through compounds R and T would be cytotoxic in SOX11 positive MCL cell lines, we treated Z138 and JEKO cells with these small-molecules and assayed Annexin-V and PI staining 48 hours later by flow-cytometry. Our results indicate that compound R has greater single agent cytotoxic activity than compound T and Ibrutinib. Compounds R and T also showed synergistic cytotoxicity in combination with Ibrutinib in SOX11 expressing Z138 and JEKO cells, but not in JVM2, a SOX11 negative MCL cell line.In summary, through a combination of in silico predictions and experimental validation, we have identified small-molecule inhibitors of SOX11-DNA binding as candidate leads for optimization for anti-MCL therapy. DisclosuresNo relevant conflicts of interest to declare.

SOX11 Cooperates with CCND1 in Mantle Cell Lymphoma Pathogenesis

MCL (Mantle cell lymphoma) is an aggressive and incurable B cell malignancy with a median survival of 5-6 years. Cyclin D1 (CCND1) overexpression is a key diagnostic feature of this disease, observed in more than 90% of MCL tumors. However, murine models over-expressing CCND1 in B cells do not recapitulate the phenotype of MCL. The SOX11 transcription factor is aberrantly expressed in 80-90% of primary MCL. Our published data demonstrated that SOX11 binds and functionally regulates key components in multiple oncogenic pathways in MCL such as WNT and TGFβ pathways. Recent studies have also showed that SOX11 regulates PAX5 and PDGFA to block differentiation and facilitate lymphoma growth. We thus hypothesize that SOX11 expression may contribute directly and functionally cooperate with CCND1 in MCL pathogenesis.To study the role of SOX11 in MCL tumorigenesis in vivo, we have generated a novel SOX11 transgenic mouse model with B cell-specific tissue expression under the E-mu enhancer and an IRES-eGFP tag to monitor the expression of SOX11. The presence of SOX11 can be readily detected in pre-pro-B stage in the bone marrow coincided with the activation of E-mu enhancer and was persistent through all stages of B cells. SOX11 over-expression in our mouse model led to an aberrant oligo-clonal expansion of CD19+/CD5+ B cells. This phenotype was evident in all SOX11 transgenic mice studied (100% penetrance, n= 42 mice) with an average of 7-12 fold increase (p<0.03) of the CD5+ B cell populations as compared to littermate controls starting from 1.5 months. Using Mass Cytometry (CyTOF), we further characterized this B cell population to be CD23-, CD21/35 dim, CD138-, high surface IgM, and variable IgD expression, a naive B cell phenotype consistent with an early precursor stage of human MCL. This MCL phenotype is most prominent in peripheral blood and spleen and, to a much lesser extent, in peritoneal cavity and bone marrow. Transplanting bone marrow from SOX11 transgenic mouse to lethally-irradiated wild type mice successfully transferred the observed phenotypic CD19+/CD5+/CD23- B cell hyperplasia, suggesting that SOX11 overexpression in early B cells drives this MCL phenotype.We next studied the cooperation between CCND1 and SOX11 by crossing SOX11 transgenic mice with a CCND1 transgenic mouse model, which over-expresses CCND1 in a B-cell specific manner under a similar E-mu enhancer. Overexpression of both CCND1 and SOX11 in the double transgenic mice model dramatically enhanced (average 10x, range 6x-30x) the aberrant MCL phenotype (CD19+/CD5+/CD23-) in peripheral blood, spleen, bone marrow, peritoneal cavity and lymph nodes compared to age-matched SOX11 and CCND1 single-transgenic mice.We report here the first direct evidence in vivo that SOX11 expression drives an aberrant expansion of B cells consistent with early human MCL and functionally collaborates with CCND1 in "full blown" MCL pathogenesis, mimicking the commonly observed co-expression of SOX11 and CCND1 in most human MCL tumors. This model captures the underpinning molecular pathogenesis events occurred in the majority of human MCL and overcomes constraints of previous MCL models that develop a phenotype after long latency or with low penetrance, making it a valuable tool for testing anti-MCL therapeutics. We are currently developing small molecule SOX11 inhibitors using SOX11 DNA binding domain models and consensus SOX11 binding nucleotides to screen a large library of compounds to identify new therapeutics for this fatal disease and gain better understanding of the molecular mechanisms of MCL tumorigenesis. DisclosuresNo relevant conflicts of interest to declare.

The role of tumor suppressor gene SOX11 in prostate cancer.

SOX genes play an important role in a number of developmental processes. The transcription factor SOX11 is one of the members of the SOX family emerging as important transcriptional regulators. The aim of this study was to investigate the role of SOX11 in prostate cancer (PCa) and its expression pattern and clinical significance. The gene expression of SOX11 in human PCa tissues compared with benign prostate hyperplasia (BPH) tissues was detected using real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis and immunohositochemistry. SOX11 overexpression cell model was used to examine the role of SOX11 in cell growth and metastasis in vitro. The results showed that the positive rate of SOX11 staining was 16.67 % (10/60) in cases of prostatic carcinoma and 81.67 % (49/60) in cases of BPH, and the difference of SOX11 expression between PCa and BPH was statistically significant (P < 0.001). SOX11 mRNA level was lowly expressed in PCa cell lines compared to RWPE-1. SOX11 overexpression suppresses PCa cell migration and invasion. In conclusion, our findings demonstrate that SOX11 could suppress cell proliferation, migration, and invasion of PCa in vitro.

Overexpression of Sox11 promotes corticospinal tract regeneration after spinal injury while interfering with functional recovery.

Embryonic neurons, peripheral neurons, and CNS neurons in zebrafish respond to axon injury by initiating pro-regenerative transcriptional programs that enable axons to extend, locate appropriate targets, and ultimately contribute to behavioral recovery. In contrast, many long-distance projection neurons in the adult mammalian CNS, notably corticospinal tract (CST) neurons, display a much lower regenerative capacity. To promote CNS repair, a long-standing goal has been to activate pro-regenerative mechanisms that are normally missing from injured CNS neurons. Sox11 is a transcription factor whose expression is common to a many types of regenerating neurons, but it is unknown whether suboptimal Sox11 expression contributes to low regenerative capacity in the adult mammalian CNS. Here we show in adult mice that dorsal root ganglion neurons (DRGs) and CST neurons fail to upregulate Sox11 after spinal axon injury. Furthermore, forced viral expression of Sox11 reduces axonal dieback of DRG axons, and promotes CST sprouting and regenerative axon growth in both acute and chronic injury paradigms. In tests of forelimb dexterity, however, Sox11 overexpression in the cortex caused a modest but consistent behavioral impairment. These data identify Sox11 as a key transcription factor that can confer an elevated innate regenerative capacity to CNS neurons. The results also demonstrate an unexpected dissociation between axon growth and behavioral outcome, highlighting the need for additional strategies to optimize the functional output of stimulated neurons.

Comprehensive Analyses of Mantle Cell Lymphoma with a 17 Gene Panel Reveal an Accumulation of TP53 Mutations in SOX11 Negative Patients

Introduction: Mantle cell lymphoma (MCL) belongs to the mature B-cell neoplasms and is characterized by t(11;14)(q13;q32)/IGH-CCND1 rearrangement resulting in overexpression of CyclinD1 that is encoded by CCND1. CCND1 is a weak oncogene, requiring additional cooperating oncogenic events. MCL show mainly an aggressive course of disease, although a subset of patients have been identified with an indolent clinical course. This has been associated with a mutated IGHV status and the lack of SOX11 expression. However, some additional gene mutations have been identified in recent years, but the underlying biological heterogeneity is still under debate. Also the prognostic impact of SOX11 remains controversially discussed.Aim: To analyze 1) recently identified molecular mutations in CCND1, UBR5, WHSC1 for their specificity for MCL, 2) in more detail SOX11 negative MCL to get more insights in this group of MCL by sequencing a 17 gene panel.Patients and Methods: In total 184 patients with mature B-cell neoplasms were investigated. All cases were diagnosed according to WHO classification by cytomorphology, immunophenotyping and cytogenetics. 81 cases were diagnosed as MCL, 78 as chronic lymphocytic leukemia/prolymphocytic (CLL/PL), and 25 as CLL. The cohort comprised 67 females and 117 males. CyclinD1 and SOX11 expression levels were quantified by real time PCR. The 17 gene panel included ATM, BIRC3, BRAF, CCND1, FBWX7, IGHV, KLHL6, KRAS, MYD88, NOTCH1, NRAS, POT1, SF3B1, TP53, UBR5, WHSC1, and XPO1. Next generation sequencing was performed on MiSeq instruments (Illumina, San Diego, CA), except for CCND1, UBR5, WHSC1, and IGHV mutational status, which were analyzed by Sanger sequencing. The latter 4 genes and gene expression levels were analyzed in the total cohort, while the 17 gene panel was applied to 26 MCL patients only (13 SOX11 negative and 13 SOX11 positive cases matched for CCND1 mutations and IGHV mutation status).Results: 23/184 (13%) patients had CCND1 mutations, while only 3/184 patients (2%) carried a UBR5 mutation, and 4/184 patients (2%) a WHSC1 mutation. Of note, CCND1 and UBR5 mutations occurred exclusively in MCL patients, WHSC1 mutations were found in MCL and CLL/PL. Therefore CCND1 mutation was, as expected, specific for MCL in comparison to the other 2 mature B-cell neoplasms (p<0.001).Of 81 MCL patients 68 (84%) showed SOX11 overexpression, 23 (28%) had CCND1 mutations and 3 (4%) each had UBR5 and WHSC1 mutations, respectively. The IGHV mutational status was evaluable in 73/81 cases, revealing 32/73 (44%) patients with a mutated IGHV status. A negative correlation of CCND1 mutations and SOX11 overexpression was found: 8/13 (62%) SOX11 negative patients were CCND1 mutated as compared to 15/68 (22%) SOX11 positive patients (p=0.007). Furthermore, CCND1 mutations were more frequent in patients with a mutated IGHV status than in those with unmutated status (15/32 (47%) vs. 8/41 (20%), p=0.021). Accordingly, SOX11 overexpression occurred more often in patients with an unmutated than with a mutated IGHV status (88% vs. 75%; n.s.). Regarding clinical data, more males were SOX11 positive (51/54, 94% vs. 17/27, 63%; p=0.001), and correspondingly more females were CCND1 mutated (12/27, 63% vs. 11/54, 20%; p=0.036).To get more insight in the SOX11 negative patients (n=13) we addressed all these cases and a SOX11 positive control group, matched for IGHV mutational status and CCND1 mutations (n=13), by comprehensive mutational analyses. Overall, beside CCND1 the most frequently mutated gene was TP53 (8/26, 31%), followed by ATM (6/26, 23%), BIRC3 (2/21, 10%), and KRAS (2/26, 8%). No mutations were detected in any of the other genes analyzed. Addressing differences in gene mutations between SOX11 negative and SOX11 positive cases revealed that TP53 mutations were found more frequently in SOX11 negative cases (6/13, 46% vs. 2/13, 15%; n.s.), while ATM mutations were more frequent in SOX11 positive cases (5/13, 39% vs. 1/13, 8%; n.s.).Conclusions: 1) CCND1 mutations are specifically found in MCL, correlate with SOX11 negativity and IGHV mutated status, and are more frequent in females. 2) TP53 is frequently mutated in SOX11 negative patients and its prognostic impact has to be further evaluated. 3) Thus, the differences in clinical course between SOX11 positive and negative MCL patients might correlate with a different spectrum of additional molecular alterations. DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.