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

The 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 haematological 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. Co-immunoprecipitation 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 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.

2-Arachidonoylglycerol Modulates CXCL12-Mediated Chemotaxis in Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia.

To survive chemotherapy, lymphoma cells can relocate to protective niches where they receive support from the non-malignant cells. The biolipid 2-arachidonoylglycerol (2-AG), an agonist for the cannabinoid receptors CB1 and CB2, is released by stromal cells in the bone marrow. To investigate the role of 2-AG in lymphoma, we analyzed the chemotactic response of primary B-cell lymphoma cells enriched from peripheral blood of twenty-two chronic lymphocytic leukemia (CLL) and five mantle cell lymphoma (MCL) patients towards 2-AG alone and/or to the chemokine CXCL12. The expression of cannabinoid receptors was quantified using qPCR and the protein levels visualized by immunofluorescence and Western blot. Surface expression of CXCR4, the main cognate receptor to CXCL12, was analyzed by flow cytometry. Phosphorylation of key downstream signaling pathways activated by 2-AG and CXCL12 were measured by Western blot in three MCL cell lines and two primary CLL samples. We report that 2-AG induces chemotaxis in 80% of the primary samples, as well as 2/3 MCL cell lines. 2-AG induced in a dose-dependent manner, the migration of JeKo-1 cell line via CB1 and CB2. 2-AG affected the CXCL12-mediated chemotaxis without impacting the expression or internalization of CXCR4. We further show that 2-AG modulated p38 and p44/42 MAPK activation. Our results suggest that 2-AG has a previously unrecognized role in the mobilization of lymphoma cells by effecting the CXCL12-induced migration and the CXCR4 signaling pathways, however, with different effects in MCL compared to CLL.

Clinical effects of a single dose of cannabinoids to patients with chronic lymphocytic leukemia.

This phase II clinical trial investigates a one-time oromucosal dose of tetrahydrocannabinol/cannabidiol (THC/CBD) in 23 patients with indolent leukemic B cell lymphomas. Primary endpoint was a significant reduction in leukemic B cells. Grade 1 - 2 adverse events were seen in 91% of the patients; most common were dry mouth (78%), vertigo (70%), and somnolence (43%). After THC/CBD a significant reduction in leukemic B cells (median, 11%) occurred within two hours (p = .014), and remained for 6 h without induction of apoptosis or proliferation. Normal B cells and T cells were also reduced. CXCR4 expression increased on leukemic cells and T cells. All effects were gone by 24 h. Our results show that a single dose of THC/CBD affects a wide variety of leukocytes and only transiently reduce malignant cells in blood. Based on this study, THC/CBD shows no therapeutic potential for indolent B cell lymphomas (EudraCT trial no. 2014-005553-39).

Clinical and biological impact of SAMHD1 expression in mantle cell lymphoma.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that restricts viral replication in infected cells and limits the sensitivity to cytarabine by hydrolysing its active metabolite, as recently shown in acute myeloid leukemia. Cytarabine is an essential component in the Nordic mantle cell lymphoma protocols (MCL2 and MCL3) for induction and high-dose chemotherapy treatment before autologous stem cell transplantation for younger patients with mantle cell lymphoma (MCL). We here investigated the expression of SAMHD1 in a population-based cohort of MCL (N = 150). SAMHD1 was highly variably expressed in MCL (range, 0.4% to 100% of positive tumor cells). Cases with blastoid/pleomorphic morphology had higher SAMHD1 expression (P = 0.028) and SAMHD1 was also correlated to tumor cell proliferation (P = 0.016). SAMHD1 expression showed moderate correlation to the expression of the transcriptional regulator SOX11 (P = 0.036) but genetic silencing of SOX11 and SAMHD1 by siRNA in MCL cell lines did not suggest mutual regulation. We hypothesized that expression of SAMHD1 could predict short time to progression in patients treated with Cytarabine as part of high-dose chemotherapy. Despite the correlation with known biological adverse prognostic factors, neither low or high SAMHD1 expression correlated to PFS or OS in patients treated according to the Nordic MCL2 or MCL3 protocols (N = 158).

Intrinsic 5-lipoxygenase activity regulates migration and adherence of mantle cell lymphoma cells.

Human B-lymphocytes express 5-lipoxygenase (5-LOX) and 5-LOX activating protein (FLAP) and can convert arachidonic acid to leukotriene B4. Mantle cell lymphoma (MCL) cells contain similar amounts of 5-LOX as human neutrophils but the function and mechanism of activation of 5-LOX in MCL cells, and in normal B-lymphocytes, are unclear. Here we show that the intrinsic 5-LOX pathway in the MCL cell line JeKo-1 has an essential role in migration and adherence of the cells, which are important pathophysiological characteristics of B-cell lymphoma. Incubation of JeKo-1 with the FLAP inhibitor GSK2190915 or the 5-LOX inhibitor zileuton, at a concentration below 1 µM, prior to stimulation with the chemotactic agent CXCL12, led to a significant reduction of migration. CRISPR/Cas9 mediated deletion of ALOX5 gene in JeKo-1 cells also led to a significantly decreased migration of the cells. Furthermore, 5-LOX and FLAP inhibitors markedly decreased the adherence of JeKo-1 cells to stromal cells. In comparison, these drugs had a similar effect on adherence of JeKo-1 cells as the Bruton tyrosine kinase inhibitor ibrutinib, which has a proven anti-tumour effect. These results indicate that inhibition of 5-LOX may be a novel treatment for MCL and certain other B-cell lymphomas.

Differential B-Cell Receptor Signaling Requirement for Adhesion of Mantle Cell Lymphoma Cells to Stromal Cells.

Interactions between lymphoma cells and stromal cells play a key role in promoting tumor survival and development of drug resistance. We identified differences in key signaling pathways between the JeKo-1 and REC-1 mantle cell lymphoma (MCL) cell lines, displaying different patterns of stromal cell adhesion and chemotaxis towards stroma-conditioned medium. The identified adhesion-regulated genes reciprocated important aspects of microenvironment-mediated gene modulation in MCL patients. Five-hundred and ninety genes were differently regulated between the cell lines upon adhesion to stromal cells, while 32 genes were similarly regulated in both cell lines. Regulation of B-cell Receptor (BCR) signature genes in adherent cells was specific for JeKo-1. Inhibition of BCR using siRNA or clinically approved inhibitors, Ibrutinib and Acalabrutinib, decreased adhesion of JeKo-1, but not REC-1 cells. Cell surface levels of chemokine receptor CXCR4 were higher in JeKo-1, facilitating migration and adhesion of JeKo-1 but not REC-1 cells. Surface levels of ICAM1 adhesion protein differ for REC-1 and JeKo-1. While ICAM1 played a positive role in adherence of both cell lines to stromal cells, S1PR1 had an inhibitory effect. Our results provide a model framework for further investigation of mechanistic differences in patient-response to new pathway-specific drugs.

Expression of GNAZ, encoding the Gαz protein, predicts survival in mantle cell lymphoma.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gαz protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gαz contribute to the MCL pathobiology.