Key Points SOX11 mediates regulation of angiogenesis via the PDGFA signaling pathway in MCL. SOX11-dependent increased angiogenesis contributes to a more aggressive MCL phenotype.

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.

MYC-driven B-cell lymphomas are addicted to elevated levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis through decreasing translational capacity and/or through p53-activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eμ-Myc lymphoma cells expressing inducible shRNAs to either ribosomal protein (RP)L7a or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced-p53-mediated apoptosis through the selective proteasomal degradation of anti-apoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the FDA-approved anti-cancer drug Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eμ-Myc, but not Trp53-/-;Eμ-Myc, lymphomas providing a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eμ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild type lymphoma.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC‐292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC‐292 potently inhibited B‐cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC‐292 reduced tumor load and normalized tumor‐associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC‐292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC‐292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

Mantle cell lymphoma (MCL) is an aggressive tumor but recent studies have identified a subtype of this lymphoma with an indolent clinical behavior and long survival of the patients even without chemotherapy that may correspond to a different clinical and biological subtype of the disease. These indolent MCLs differ from conventional MCLs in a particular gene signature that lacks the expression of some transcription factors of the High Mobility Group (HMG). SOX11 is one of the best discriminatory genes between these conventional and indolent MCL tumors. We have recently demonstrated the oncogenic implication of SOX11 expression in the aggressive behavior of MCL as SOX11-knockdown derived tumors display a significant reduction on tumor growth compared to SOX11-control tumors in subcutaneous MCL xenograft experiments (Vegliante MC, Palomero J et al. Blood. 2013; 121:2175-85). To uncover SOX11 regulated genes and transcriptional programs in MCL, we performed an integrative analysis coupling data from human genome-wide promoter analysis by SOX11 ChIP-chip experiments and gene expression profiling (GEP) upon SOX11 silencing in MCL cell lines. In these studies we initially identified the role of SOX11 in blocking the terminal B-cell differentiation by the direct positive regulation of PAX5. However, how SOX11 may enhance tumor growth is still not well known. To further characterize the potential oncogenic mechanisms regulated by SOX11, we have integrated our ChIP-chip data with further analyses including GEP derived from the xenografted SOX11-positive and silenced tumors. The gene ontology term analysis of the genome-wide promoter study revealed “blood vessel development” as one of the most significant biological processes overrepresented among the SOX11-bound genes. Concordantly, a gene set enrichment analysis (GSEA) of the differentially expressed genes in the xenografted tumors showed a significant higher enrichment of gene signatures related to tumor angiogenesis in the SOX11-positive tumors. To validate these results in vivo, we first investigated protein extracts of these tumors using an angiogenesis proteome profiler antibody array and discovered a significant higher expression of 20 pro-angiogenic factors in the SOX11-positive tumors. We have then investigated the angiogenic development in tissue sections from four Z-138 SOX11-positive and nine SOX11-silenced xenografted tumors. The microvascular density area recognized by CD31 staining was significantly larger in SOX11-positive than knockdown tumors (90%±µ2m vs 15%±µ2m, respectively, p<1x10-4). SOX11-silenced tumors had larger necrotic areas than positive tumors (20%±µ2m vs 1%±µ2m, respectively, p<1x10-4), suggesting that vascular paucity could contribute to the significant smaller volume of these tumors. To determine whether angiogenic development was also differentially represented in primary human MCL we have studied the GEP of 16 SOX11-positive and 22 SOX11-negative MCL. A GSEA revealed that SOX11 expressing tumors were enriched in signatures related to tumor vasculature, angiogenesis and vasculature development. Concordantly, the CD34 staining of an independent series of primary SOX11-positive (n=8) and negative (n=9) human MCL showed higher microvascular density in SOX11 expressing tumors (30%±µ2m vs 5%±µ2m, respectively, p<1x10-4). In conclusion, these findings indicate that SOX11 promotes angiogenesis in experimental and primary human MCL and this may be an oncogenic mechanism contributing to the aggressiveness of these tumors. Disclosures: No relevant conflicts of interest to declare.