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.

Specific properties of shRNA-mediated CCR5 downregulation that enhance the inhibition of HIV-1 infection in combination with shRNA targeting HIV-1 rev.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

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).

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.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene.

Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP-agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.

Influence of rimonabant treatment on peripheral blood mononuclear cells; flow cytometry analysis and gene expression profiling.

The cannabinoid receptor type 1 (CB1) antagonist rimonabant has been used as treatment for obesity. In addition, anti-proliferative effects on mitogen-activated leukocytes have been demonstrated in vitro. We have previously shown that rimonabant (SR141716A) induces cell death in ex vivo isolated malignant lymphomas with high expression of CB1 receptors. Since CB1 targeting may be part of a future lymphoma therapy, it was of interest to investigate possible effects on peripheral blood mononuclear cells (PBMC) in patients treated with rimonabant. We therefore evaluated leukocyte subsets by 6 color flow cytometry in eight patients before and at treatment with rimonabant for 4 weeks. Whole-transcript gene expression profiling in PBMC before and at 4 weeks of rimonabant treatment was done using Affymetrix Human Gene 1.0 ST Arrays. Our data show no significant changes of monocytes, B cells, total T cells or T cell subsets in PBMC during treatment with rimonabant. There was a small but significant increase in CD3-, CD16+ and/or CD56+ cells after rimonabant therapy. Gene expression analysis detected significant changes in expression of genes associated with innate immunity, cell death and metabolism. The present study shows that normal monocytes and leukocyte subsets in blood remain rather constant during rimonabant treatment. This is in contrast to the induction of cell death previously observed in CB1 expressing lymphoma cells in response to treatment with rimonabant in vitro. These differential effects observed on normal and malignant lymphoid cells warrant investigation of CB1 targeting as a potential lymphoma treatment.

Flow cytometric analysis of SOX11: a new diagnostic method for distinguishing B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma from mantle cell lymphoma.

The differential diagnosis between mantle cell lymphoma (MCL) and B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) is essential, since MCL usually has a more aggressive clinical course. By flow cytometry both MCL and B-CLL are CD19, CD20 and usually CD5 positive. However, ambiguities in other immune phenotypic markers of these lymphoma entities sometimes complicate the flow cytometric differential diagnosis. We here demonstrate that the transcription factor SOX11, which is highly up-regulated in most MCL, can be analyzed by flow cytometry. SOX11 protein could be consistently detected in ex vivo isolated MCL but not in B-CLL/SLL. Flow cytometry also enabled protein quantification, and SOX11 protein levels correlated with mRNA expression. We suggest that implementing detection of SOX11 in diagnostic flow cytometry would be beneficial for accurate and reliable diagnosis of MCL, especially for distinguishing cases of MCL and B-CLL/SLL with aberrant immune phenotypes, and for cases of rare cyclin D1 negative MCL.

T-cell levels are prognostic in mantle cell lymphoma.

PURPOSE: The purpose of this study was to investigate the impact of T-cell subsets on pathologic and clinical features including disease outcome in mantle cell lymphoma (MCL). EXPERIMENTAL DESIGN: Cell populations were investigated using flow cytometry in diagnostic MCL (n = 153) and reactive (n = 26) lymph node biopsies. Levels of tumor cells, T cells, T-cell subsets, and the CD4:CD8 ratio were assessed and related to pathologic and clinical parameters. RESULTS: MCL cases with diffuse and nodular histologic subtypes showed lower levels of T cells, especially CD4(+) T cells, than those with mantle zone growth pattern. Both CD3 and CD4 levels were lower in the nodular subtype than in mantle zone (P = 0.007; P = 0.003) and in the diffuse compared with the nodular subtype (P = 0.022; P = 0.015). The CD4:CD8 ratios were inversely correlated to tumor cell proliferation (P = 0.003). Higher levels of CD3(+) and CD4(+) T cells and higher CD4:CD8 ratios were associated with indolent disease (P = 0.043, 0.021, and 0.003 respectively). In univariate analysis, a high CD4:CD8 ratio, but not the histologic subtype, was correlated to longer overall survival (OS). In multivariate analysis, the CD4:CD8 ratio correlated with OS independently of Mantle Cell Lymphoma International Prognostic Index (MIPI) and high p53 expression (P = 0.023). CONCLUSION: CD3(+), CD8(+), and particularly CD4(+) T-cell levels are higher in indolent MCL and decrease with more aggressive histology as reflected by a diffuse growth pattern. High CD4:CD8 ratio correlated independently of other high-risk prognostic factors with longer OS, suggesting a prognostic role for T cells in MCL.

Perturbations of the endocannabinoid system in mantle cell lymphoma: correlations to clinical and pathological features.

The cannabinoid receptors are upregulated in many types of cancers, including mantle cell lymphoma (MCL) and have been suggested to constitute novel therapeutic targets. The expression pattern of the key members of the endocannabinoid system was analyzed in a well-characterized MCL patient cohort and correlated to biological features. 107 tumor tissues were analyzed for the mRNA levels of cannabinoid receptors 1 and 2 (CNR1 and CNR2) and the two main enzymes regulating the endocannabinoid anandamide levels in tissue: NAPEPLD and FAAH (participating in synthesis and degradation, respectively). NAPEPLD, CNR1 and CNR2 were overexpressed while FAAH expression was reduced in MCL compared to non-malignant B-cells. Both low CNR1 and high FAAH levels correlated with lymphocytosis (p=0.016 and p=0.022, respectively) and with leukocytosis (p=0.0018 and p=0.047). Weak to moderate CNR1 levels were a feature of SOX11 negative MCL (p=0.006). Both high CNR2 and high FAAH levels correlated to anemia (p=0.0006 and p=0.038, respectively). In conclusion, the relative expression of the anandamide synthesizing and metabolizing enzymes in MCL is heavily perturbed. This finding, together with high expression of cannabinoid receptors, could favor enhanced anandamide signaling and suggest that targeting the endocannabinoid system might be considered as part of lymphoma therapy.

Higher World Health Organization grades of follicular lymphoma correlate with better outcome in two Nordic Lymphoma Group trials of rituximab without chemotherapy.

Abstract A common treatment for follicular lymphoma is rituximab monotherapy. To identify patients for whom this regimen is adequate as first-line therapy, we applied the World Health Organization (WHO) classification for grading follicular lymphoma in a prospective central pathology review of the biopsies of previously untreated patients in two randomized trials of rituximab without chemotherapy. In the first trial (n1 = 53), higher WHO grades correlated with longer time to next treatment, independently of clinical prognostic factors (p = 0.030); the finding was replicated in the second trial (n2 = 221; p = 0.019). Higher grades were associated with better treatment responses (p = 0.018). Furthermore, also grades externally confirmed by independent local pathologists correlated with time to next treatment (p = 0.048). Flow cytometry in a separate patient series showed that the intensity of CD20 increased with the malignant cell size (p < 0.00005). In conclusion, WHO grade 1 follicular lymphoma correlates with inferior outcome after rituximab monotherapy. WHO grading might provide a clinically useful tool for personalized therapy.

Epstein Barr virus DNA analysis in blood predicts disease progression in a rare case of plasmablastic lymphoma with effusion.

BACKGROUND: In HIV-1-infected patients a long lasting CD4+ cell decline influences the host-EBV balance and thereby increases the risk for EBV related malignancies. In spite of a world-wide access to combination antiretroviral therapy (cART) there are still a considerable number of HIV-1-infected patients who will develop severe immunodeficiency. These undiagnosed HIV-1 infected patients, so called late testers, demonstrate an increased lymphoma risk, compared to patients diagnosed early. Consecutive individual screening for EBV DNA-load in late testers might be a useful predictor of emerging EBV-malignancy. METHODS: Patient biopsies and ascites were analyzed morphologically, by immuncyto-histochemistry and in-situ hybridization. Viral DNA and RNA load were quantified by PCR. Cell lines from primary tumor and from ascites, were established in vitro and further analyzed. RESULT: We here report on a case of EBV-positive lymphoma in an AIDS patient, first presenting with pleural effusion and ascites and was thus initially considered a primary effusion lymphoma (PEL) but was later diagnosed as a plasmablastic lymphoma (PBL). The patient had responded to cART with undetectable HIV-RNA and increased CD4 cell count one year prior to lymphoma presentation. At the time of lymphoma diagnosis the HIV-RNA values were <50 RNA-copies per mL blood (undetectable) and the CD4-positive cell count 170 ×106/L. The lymphoma was CD45-negative and weakly CD22- and CD30-positive. The patient had a history of Kaposi sarcoma and HHV-8 seropositivity. The lymphoma biopsies, and three cell lines derived on different occasions from the tumor cell effusion, were all EBV-positive but HHV-8 negative.A noticeable EBV-DNA load decline was observed during the remission of the lymphoma following CHOP-therapy. The EBV-DNA load increased dramatically at the time of recurrence. CONCLUSION: EBV DNA load might be useful in monitoring the effect of lymphoma treatment as well as in estimating the risk of EBV-associated lymphoma in HIV-1 infected patients with pronounced immunosuppression.

SOXC transcription factors in mantle cell lymphoma: the role of promoter methylation in SOX11 expression.

The related transcription factors SOX11, SOX4 and SOX12 (classified as the SOXC family) compete for the same target genes. SOX11 is expressed in most mantle cell lymphomas (MCL) but a small subset is, like normal lymphocytes, SOX11 negative. Here we report the variable expression of SOX4 and high expression of SOX12 in MCL compared to non-malignant tissue. Our results show that the expression of the SOXC genes is highly correlated in SOX11 positive MCL. SOX11 expression is epigenetically regulated but there are partly conflicting results regarding the underlying mechanisms. Here we report that the SOX11 promoter region is hypomethylated in both MCL and normal B-lymphocytes. Methylation at other sites is important for sustaining high SOX11 in MCL since treatment with 5-azacytidine decreased SOX11 levels in SOX11 positive MCL cell lines: Granta519 and Rec1. Furthermore, 5-azacytidine treatment of the SOX11 negative MCL cell line, JVM2, induced SOX4 but not SOX11.

Prognostic role of SOX11 in a population-based cohort of mantle cell lymphoma.

The prognostic role of the transcription factor SOX11 in mantle cell lymphoma (MCL) is controversial. We investigated prognostic markers in a population-based cohort of 186 MCL cases. Seventeen patients (9%) did not require any therapy within the first 2 years after diagnosis and were retrospectively defined as having an indolent disease. As expected, indolent MCL had less frequent B symptoms and extensive nodal involvement and 88% of these cases expressed SOX11. In our cohort 13 cases (7.5%) lacked nuclear SOX11 at diagnosis. SOX11(-) MCL had a higher frequency of lymphocytosis, elevated level of lactate dehydrogenase (LDH), and p53 positivity. The overall survival in the whole cohort, excluding 37 patients receiving autologous stem cell transplantation, was 3.1 year and in patients with indolent or nonindolent disease, 5.9 and 2.8 years, respectively (P = .004). SOX11(-) cases had a shorter overall survival, compared with SOX11(+) cases, 1.5 and 3.2 years, respectively (P = .014). In multivariate analysis of overall survival, age > 65 (P = .001), Eastern Cooperative Oncology Group score ≥ 2 (P = .022), elevated LDH level (P = .001), and p53 expression (P = .001) remained significant, and SOX11 lost significance. We conclude that most indolent MCLs are SOX11(+) and that SOX11 cannot be used for predicting an indolent disease course.