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Papers by Simon Wagner

Blood, 2014

Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some ... more Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some cases increased cytokine production by the lymphoma is responsible for activation of JAK2 and STAT3 but the regulation of signalling through this pathway is not clear. We constructed a conditional BCL6 deficient cell line through disruption of the endogenous BCL6 loci of a genetically tractable human B-cell lymphoma by homologous recombination, and insertion of a tetracycline regulatable BCL6 transgene. On induction of BCL6 deficiency growth of the cell line slowed by 3 to 4-fold. A synthetic lethal screen employing a library of small molecule inhibitors in genetically BCL6 deficient lymphoma cells showed that lestaurtinib, a JAK2 inhibitor, enhanced loss of viability. We investigated the hypothesis that JAK2 is a direct BCL6 target gene. JAK2 mRNA and protein expression were induced by BCL6 deficiency. Inspection of the JAK2 proximal promoter region demonstrated a potential BCL6 binding...

Translation and Its Regulation in Cancer Biology and Medicine, 2014

Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for norma... more Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD-1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T-cell non-Hodgkin lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and ~20% of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL-NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre-clinical development.

Journal of Cellular and Molecular Medicine, 2020

The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through m... more The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll‐like receptors to regulate NF‐κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B‐cell lymphoma (DLBCL). DLBCL cell lines and patient‐derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non‐germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL‐10 production from Ly10 and repressed NF‐κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF‐κB signalling in lymphoma.

Blood Advances, 2019

Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in norm... more Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in normal human tonsil CD4+ T cells. Primary human PTCL cells alter their functional properties in culture and ITK inhibitors modify these changes.

Journal of Visualized Experiments, 2019

British Journal of Haematology, 2018

Delayed lymphocyte and T-cell immune reconstitution following bendamustine-rituximab (BR) for ind... more Delayed lymphocyte and T-cell immune reconstitution following bendamustine-rituximab (BR) for indolent non-Hodgkin lymphoma (iNHL) has been described, but no information is available for chronic lymphocytic leukaemia (CLL). We present a population-based retrospective analysis of immune reconstitution and risk of infection following BR. Outcomes included timing/correlates of CD4+ recovery and risk of ≥grade 3 infections. Consecutively treated patients (1 April 2014 to 31 January 2017) were included (n = 295),with a median age of 65 years (range 33-92); 57% were 1st line treatments. Median cumulative bendamustine dose was 1080 mg/m 2 (range 140-1440 mg/m 2). CD4/CD8/CD19/NK subsets were available for 148 patients. Median follow-up was 24 months. Median times to lymphocyte count (ALC) recovery (≥1 9 10 9 /l) and CD4+ recovery (≥0Á2 9 10 9 /l) were 26 and 24 months, respectively. Bendamustine total dose >1080 mg/m 2 (hazard ratio [HR] 0Á4; 95% confidence interval [CI]: 0Á2-0Á8), end-of-treatment ALC ≤0Á4 9 10 9 /l (HR 0Á53; 95% CI: 0Á3-0Á9) and CD4+ <0Á1 9 10 9 /l 1-year post-BR (HR 0Á03; 95% CI: 0Á008-0Á15) were covariables for delayed CD4+ recovery. ALC-recovery ≥1 9 10 9 /l was an unreliable predictor of CD4+ recovery (negative predictive vale 74%, positive predictive value 86%, likelihood ratio 3Á3). CD4+ lymphopenia >3 years was a significant risk factor for ≥grade 3 infections (Odds ratio 3Á4; 95% CI: 1Á4-6Á9). CD4+ recovery after BR is unexpectedly delayed and late recovery is associated with risk of serious infections. Monitoring CD4+ following BR could identify patients at high risk of delayed infections.

Diagnostic pathology, Jan 15, 2018

Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone l... more Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4 T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3 expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1 cells showed significantly more clustering than did FOXP3. To investigate the association of infi...

PLOS ONE, 2019

Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large prop... more Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain bears a mutation that increases Tfh cell number and heterozygous animals (Roquin san/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquin san/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or ... more BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in,40 % of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly ...

Journal of Biological Chemistry

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, t... more B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

Blood Advances

The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencin... more The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencing of lymph node samples, but there has been little work on the mutational load that is present in cell-free DNA (cfDNA) from plasma. We report targeted sequencing of cfDNA from PTCL patients to demonstrate c.50G>T (p.Gly17Val) in RHOA as previously described in angioimmunoblastic T-cell lymphoma (AITL) and a group of PTCL not otherwise specified (NOS) but also detect novel mutations at c.73A>G (p.Phe25Leu) and c.48A>T (p.Cys16*) of exon 2, which were confirmed by Sanger sequencing. In a group of AITL and PTCL-NOS analyzed by droplet digital polymerase chain reaction, 63% (12/19) showed c.50G>T (p.Gly17Val), 53% (10/19) c.73A>G (p.Phe25Leu), and 37% (7/19) c.48A>T (pCys16*). Sequencing of lymph node tissue in 3 out of 9 cases confirmed the presence of c.73A>G (p.Phe25Leu). Inspection of individual sequencing reads from individual patients showed that a single RHOA all...

Journal of virology

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (... more An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 ...

Scientific Reports

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occ... more Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 in...

Blood

We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-xS is induced in chronic lympho... more We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-xS is induced in chronic lymphocytic leukaemia cells during spontaneous and drug induced apoptosis to a greater extent than other pro-apoptotic Bcl-2 family members including Bim, Puma and Noxa. Puma, Noxa and also Bid were induced strongly by stimulation with CD40 ligand, in culture conditions causing CLL cell proliferation and survival, and associated with strong induction of the pro-survival Bcl-x isoform, Bcl-xL. Immunohistochemistry demonstrates Bcl-xL expression in lymph node proliferation centres. We, therefore, conclude that splicing of Bcl-x mRNA is important for regulation of CLL survival such that Bcl-xL is required during proliferation in the lymph node or bone marrow microenvironment, but Bcl-xS is induced during apoptosis. Splicing of Bcl-x mRNA in HeLa cells is known to be controlled by RNA binding proteins including SAM68, heterogeneous ribonucleoprotein-F (hnRNP-F) and hnRNP-H, but expression and regu...

Blood

We analysed outcome for 211 CML patients treated with imatinib in chronic phase (CP) (99 newly di... more We analysed outcome for 211 CML patients treated with imatinib in chronic phase (CP) (99 newly diagnosed and 112 late chronic phase) who were screened for BCR-ABL kinase domain (KD) mutations using direct sequencing regardless of the response status. When a mutation was found all available previous cDNA samples were analysed by pyrosequencing to establish the date of its first occurrence and subsequent kinetics. The median age of patients was 47.4 years. The Sokal risk score was ‘low’ in 57 patients, ‘intermediate’ in 82 and ‘high’ in 72. The median follow up from starting imatinib was 45 months (rage 6 to 89 months). A mutation was detected in 34 of the 211 patients (16%) at a median time of 27 months from starting imatinib. Twenty-two different mutations were identified, the most frequent being M244V (n=6) and F359V (n=3). When studied serially by pyrosequencing the size of the mutant subclone never exceeded 50% of total BCR-ABL transcripts in 8 patients, while in 17 patients it e...

Blood

To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from pati... more To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from patients with lymphoproliferative disorders, we have polymerase chain reaction-amplified and sequenced a total of 26 V kappa genes from a total of 55 cases. Six sequences were obtained both from six cases of prolymphocytic leukemia (PLL) and from nine cases of hairy cell leukemia (HCL). Seven sequences were obtained both from 11 cases of Waldenstrom's macroglobulinemia (WM) and 29 cases of multiple myeloma (MM). Eleven different germline genes have been used in this series, indicating a wide but nonrandom usage of germline Ig gene rearrangements in these disorders. Comparison of the nucleotide sequences of V kappa genes obtained from B-cell malignancies with germline V kappa genes shows that somatic mutation is rare in PLL and HCL and common in WM and MM. Analysis of the pattern of mutations suggests that WM and MM are derived from B cells that have been selected by antigen at a relative...

Blood

The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development... more The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression pla...

Blood

3684 CD4+ T-cells can be distinguished into subsets on the basis of surface marker expression and... more 3684 CD4+ T-cells can be distinguished into subsets on the basis of surface marker expression and growth factor production. Follicular helper T-cells (Tfh cells) are characterized by the co-expression of surface markers (CD4, ICOS, PD1 and CXCR5) and nuclear BCL6. Normal germinal centre formation requires Tfh cells but is repressed by another CD4+ T-cell subset, Tregs, (demonstrating CD4 and CD25 expression with nuclear FoxP3). The numbers and architecture of infiltrating T-cells predict clinical outcome in follicular lymphoma but although T-cells are a component of diffuse large B cell lymphoma (DLBCL), the relative numbers of CD4+ T-cells and their Tfh and Treg subsets or their association with clinical outcome is not known. We used immunohistochemistry to investigate infiltration by total CD4+, Treg and Tfh cells in cases (n=23) from one centre. The male:female was 1.3:1.0, the age range was 30 to 78 years (median 65 years) and the anticipated association between overall survival...

Blood

CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blo... more CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blood rapidly die. We have used mouse fibroblast L-cells transfected with CD40 ligand (CD40L) in combination with IL-4 to reproduce some features of the in vivo microenvironment, which is essential for CLL cell proliferation. As anticipated we found that leukaemic cells survive and proliferate in this system. Also CD23 expression increases, but unexpectedly CD38 expression increased to varying degrees in all patients and CD5 expression decreased. Changes in CD23 and CD38 have been reported in CLL cells in lymph nodes and support the concept that we are able to some extent reproduce the in vivo microenvironment. Next we carried out a systematic analysis of Bcl-2 family proteins by Western Blot analysis. On tissue culture plastic and non-transfected L-cells Bcl-2 is the predominant pro-survival protein. The activator BH3-only protein Bim is expressed together with the sensitiser BH3-only Puma...

Blood, 2014

Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some ... more Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some cases increased cytokine production by the lymphoma is responsible for activation of JAK2 and STAT3 but the regulation of signalling through this pathway is not clear. We constructed a conditional BCL6 deficient cell line through disruption of the endogenous BCL6 loci of a genetically tractable human B-cell lymphoma by homologous recombination, and insertion of a tetracycline regulatable BCL6 transgene. On induction of BCL6 deficiency growth of the cell line slowed by 3 to 4-fold. A synthetic lethal screen employing a library of small molecule inhibitors in genetically BCL6 deficient lymphoma cells showed that lestaurtinib, a JAK2 inhibitor, enhanced loss of viability. We investigated the hypothesis that JAK2 is a direct BCL6 target gene. JAK2 mRNA and protein expression were induced by BCL6 deficiency. Inspection of the JAK2 proximal promoter region demonstrated a potential BCL6 binding...

Translation and Its Regulation in Cancer Biology and Medicine, 2014

Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for norma... more Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD-1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T-cell non-Hodgkin lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and ~20% of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL-NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre-clinical development.

Journal of Cellular and Molecular Medicine, 2020

The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through m... more The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll‐like receptors to regulate NF‐κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B‐cell lymphoma (DLBCL). DLBCL cell lines and patient‐derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non‐germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL‐10 production from Ly10 and repressed NF‐κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF‐κB signalling in lymphoma.

Blood Advances, 2019

Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in norm... more Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in normal human tonsil CD4+ T cells. Primary human PTCL cells alter their functional properties in culture and ITK inhibitors modify these changes.

Journal of Visualized Experiments, 2019

British Journal of Haematology, 2018

Delayed lymphocyte and T-cell immune reconstitution following bendamustine-rituximab (BR) for ind... more Delayed lymphocyte and T-cell immune reconstitution following bendamustine-rituximab (BR) for indolent non-Hodgkin lymphoma (iNHL) has been described, but no information is available for chronic lymphocytic leukaemia (CLL). We present a population-based retrospective analysis of immune reconstitution and risk of infection following BR. Outcomes included timing/correlates of CD4+ recovery and risk of ≥grade 3 infections. Consecutively treated patients (1 April 2014 to 31 January 2017) were included (n = 295),with a median age of 65 years (range 33-92); 57% were 1st line treatments. Median cumulative bendamustine dose was 1080 mg/m 2 (range 140-1440 mg/m 2). CD4/CD8/CD19/NK subsets were available for 148 patients. Median follow-up was 24 months. Median times to lymphocyte count (ALC) recovery (≥1 9 10 9 /l) and CD4+ recovery (≥0Á2 9 10 9 /l) were 26 and 24 months, respectively. Bendamustine total dose >1080 mg/m 2 (hazard ratio [HR] 0Á4; 95% confidence interval [CI]: 0Á2-0Á8), end-of-treatment ALC ≤0Á4 9 10 9 /l (HR 0Á53; 95% CI: 0Á3-0Á9) and CD4+ <0Á1 9 10 9 /l 1-year post-BR (HR 0Á03; 95% CI: 0Á008-0Á15) were covariables for delayed CD4+ recovery. ALC-recovery ≥1 9 10 9 /l was an unreliable predictor of CD4+ recovery (negative predictive vale 74%, positive predictive value 86%, likelihood ratio 3Á3). CD4+ lymphopenia >3 years was a significant risk factor for ≥grade 3 infections (Odds ratio 3Á4; 95% CI: 1Á4-6Á9). CD4+ recovery after BR is unexpectedly delayed and late recovery is associated with risk of serious infections. Monitoring CD4+ following BR could identify patients at high risk of delayed infections.

Diagnostic pathology, Jan 15, 2018

Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone l... more Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4 T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3 expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1 cells showed significantly more clustering than did FOXP3. To investigate the association of infi...

PLOS ONE, 2019

Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large prop... more Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain bears a mutation that increases Tfh cell number and heterozygous animals (Roquin san/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquin san/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or ... more BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in,40 % of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly ...

Journal of Biological Chemistry

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, t... more B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

Blood Advances

The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencin... more The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencing of lymph node samples, but there has been little work on the mutational load that is present in cell-free DNA (cfDNA) from plasma. We report targeted sequencing of cfDNA from PTCL patients to demonstrate c.50G>T (p.Gly17Val) in RHOA as previously described in angioimmunoblastic T-cell lymphoma (AITL) and a group of PTCL not otherwise specified (NOS) but also detect novel mutations at c.73A>G (p.Phe25Leu) and c.48A>T (p.Cys16*) of exon 2, which were confirmed by Sanger sequencing. In a group of AITL and PTCL-NOS analyzed by droplet digital polymerase chain reaction, 63% (12/19) showed c.50G>T (p.Gly17Val), 53% (10/19) c.73A>G (p.Phe25Leu), and 37% (7/19) c.48A>T (pCys16*). Sequencing of lymph node tissue in 3 out of 9 cases confirmed the presence of c.73A>G (p.Phe25Leu). Inspection of individual sequencing reads from individual patients showed that a single RHOA all...

Journal of virology

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (... more An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 ...

Scientific Reports

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occ... more Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 in...

Blood

We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-xS is induced in chronic lympho... more We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-xS is induced in chronic lymphocytic leukaemia cells during spontaneous and drug induced apoptosis to a greater extent than other pro-apoptotic Bcl-2 family members including Bim, Puma and Noxa. Puma, Noxa and also Bid were induced strongly by stimulation with CD40 ligand, in culture conditions causing CLL cell proliferation and survival, and associated with strong induction of the pro-survival Bcl-x isoform, Bcl-xL. Immunohistochemistry demonstrates Bcl-xL expression in lymph node proliferation centres. We, therefore, conclude that splicing of Bcl-x mRNA is important for regulation of CLL survival such that Bcl-xL is required during proliferation in the lymph node or bone marrow microenvironment, but Bcl-xS is induced during apoptosis. Splicing of Bcl-x mRNA in HeLa cells is known to be controlled by RNA binding proteins including SAM68, heterogeneous ribonucleoprotein-F (hnRNP-F) and hnRNP-H, but expression and regu...

Blood

We analysed outcome for 211 CML patients treated with imatinib in chronic phase (CP) (99 newly di... more We analysed outcome for 211 CML patients treated with imatinib in chronic phase (CP) (99 newly diagnosed and 112 late chronic phase) who were screened for BCR-ABL kinase domain (KD) mutations using direct sequencing regardless of the response status. When a mutation was found all available previous cDNA samples were analysed by pyrosequencing to establish the date of its first occurrence and subsequent kinetics. The median age of patients was 47.4 years. The Sokal risk score was ‘low’ in 57 patients, ‘intermediate’ in 82 and ‘high’ in 72. The median follow up from starting imatinib was 45 months (rage 6 to 89 months). A mutation was detected in 34 of the 211 patients (16%) at a median time of 27 months from starting imatinib. Twenty-two different mutations were identified, the most frequent being M244V (n=6) and F359V (n=3). When studied serially by pyrosequencing the size of the mutant subclone never exceeded 50% of total BCR-ABL transcripts in 8 patients, while in 17 patients it e...

Blood

To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from pati... more To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from patients with lymphoproliferative disorders, we have polymerase chain reaction-amplified and sequenced a total of 26 V kappa genes from a total of 55 cases. Six sequences were obtained both from six cases of prolymphocytic leukemia (PLL) and from nine cases of hairy cell leukemia (HCL). Seven sequences were obtained both from 11 cases of Waldenstrom's macroglobulinemia (WM) and 29 cases of multiple myeloma (MM). Eleven different germline genes have been used in this series, indicating a wide but nonrandom usage of germline Ig gene rearrangements in these disorders. Comparison of the nucleotide sequences of V kappa genes obtained from B-cell malignancies with germline V kappa genes shows that somatic mutation is rare in PLL and HCL and common in WM and MM. Analysis of the pattern of mutations suggests that WM and MM are derived from B cells that have been selected by antigen at a relative...

Blood

The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development... more The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression pla...

Blood

3684 CD4+ T-cells can be distinguished into subsets on the basis of surface marker expression and... more 3684 CD4+ T-cells can be distinguished into subsets on the basis of surface marker expression and growth factor production. Follicular helper T-cells (Tfh cells) are characterized by the co-expression of surface markers (CD4, ICOS, PD1 and CXCR5) and nuclear BCL6. Normal germinal centre formation requires Tfh cells but is repressed by another CD4+ T-cell subset, Tregs, (demonstrating CD4 and CD25 expression with nuclear FoxP3). The numbers and architecture of infiltrating T-cells predict clinical outcome in follicular lymphoma but although T-cells are a component of diffuse large B cell lymphoma (DLBCL), the relative numbers of CD4+ T-cells and their Tfh and Treg subsets or their association with clinical outcome is not known. We used immunohistochemistry to investigate infiltration by total CD4+, Treg and Tfh cells in cases (n=23) from one centre. The male:female was 1.3:1.0, the age range was 30 to 78 years (median 65 years) and the anticipated association between overall survival...

Blood

CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blo... more CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blood rapidly die. We have used mouse fibroblast L-cells transfected with CD40 ligand (CD40L) in combination with IL-4 to reproduce some features of the in vivo microenvironment, which is essential for CLL cell proliferation. As anticipated we found that leukaemic cells survive and proliferate in this system. Also CD23 expression increases, but unexpectedly CD38 expression increased to varying degrees in all patients and CD5 expression decreased. Changes in CD23 and CD38 have been reported in CLL cells in lymph nodes and support the concept that we are able to some extent reproduce the in vivo microenvironment. Next we carried out a systematic analysis of Bcl-2 family proteins by Western Blot analysis. On tissue culture plastic and non-transfected L-cells Bcl-2 is the predominant pro-survival protein. The activator BH3-only protein Bim is expressed together with the sensitiser BH3-only Puma...