The essential microenvironmental role of oligomannoses specifically inserted into the antigen-binding sites of lymphoma cells.

ABSTRACT: There are 2 mandatory features added sequentially en route to classical follicular lymphoma (FL): first, the t(14;18) translocation, which upregulates BCL2, and second, the introduction of sequence motifs into the antigen-binding sites of the B-cell receptor (BCR), to which oligomannose-type glycan is added. Further processing of the glycan is blocked by complementarity-determining region-specific steric hindrance, leading to exposure of mannosylated immunoglobulin (Ig) to the microenvironment. This allows for interaction with the local lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), expressed by tissue macrophages and follicular dendritic cells. The major function of DC-SIGN is to engage pathogens, but this is subverted by FL cells. DC-SIGN induces tumor-specific low-level BCR signaling in FL cells and promotes membrane changes with increased adhesion to VCAM-1 via proximal kinases and actin regulators but, in contrast to engagement by anti-Ig, avoids endocytosis and apoptosis. These interactions appear mandatory for the early development of FL, before the acquisition of other accelerating mutations. BCR-associated mannosylation can be found in a subset of germinal center B-cell-like diffuse large B-cell lymphoma with t(14;18), tracking these cases back to FL. This category was associated with more aggressive behavior: both FL and transformed cases and, potentially, a significant number of cases of Burkitt lymphoma, which also has sites for N-glycan addition, could benefit from antibody-mediated blockade of the interaction with DC-SIGN.

Exploring the pathways to chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), increasing knowledge of the biology of the tumor cells has led to transformative improvements in our capacity to assess and treat patients. The dependence of tumor cells on surface immunoglobulin receptor signaling, survival pathways, and accessory cells within the microenvironment has led to a successful double-barreled attack with designer drugs. Studies have revealed that CLL should be classified based on the mutational status of the expressed IGHV sequences into 2 diseases, either unmutated (U) or mutated (M) CLL, each with a distinctive cellular origin, biology, epigenetics/genetics, and clinical behavior. The origin of U-CLL lies among the natural antibody repertoire, and dominance of IGHV1-69 reveals a superantigenic driver. In both U-CLL and M-CLL, a calibrated stimulation of tumor cells by self-antigens apparently generates a dynamic reiterative cycle as cells, protected from apoptosis, transit between blood and tissue sites. But there are differences in outcome, with the balance between proliferation and anergy favoring anergy in M-CLL. Responses are modulated by an array of microenvironmental interactions. Availability of T-cell help is a likely determinant of cell fate, the dependency on which varies between U-CLL and M-CLL, reflecting the different cells of origin, and affecting clinical behavior. Despite such advances, cell-escape strategies, Richter transformation, and immunosuppression remain as challenges, which only may be met by continued research into the biology of CLL.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells.

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Critical influences on the pathogenesis of follicular lymphoma.

The development of follicular lymphoma (FL) from a founder B cell with an upregulation of B-cell lymphoma 2 (BCL2), via the t(14;18) translocation, to a proliferating clone, poised to undergo further transformation to an aggressive lymphoma, illustrates the opportunistic Darwinian process of tumorigenesis. Protection against apoptosis allows an innocent cell to persist and divide, with dangerous accumulation of further mutational changes, commonly involving inactivation of chromatin-modifying genes. But this is not all. FL cells reflect normal B cells in relying on expression of surface immunoglobulin. In doing so, they add another supportive mechanism by exploiting the natural process of somatic hypermutation of the IGV genes. Positive selection of motifs for addition of glycan into the antigen-binding sites of virtually all cases, and the placement of unusual mannoses in those sites, reveals a posttranslational strategy to engage the microenvironment. A bridge between mannosylated surface immunoglobulin of FL cells and macrophage-expressed dendritic cell-specific ICAM-3-grabbing nonintegrin produces a persistent low-level signal that appears essential for life in the hostile germinal center. Early-stage FL therefore requires a triad of changes: protection from apoptosis, mutations in chromatin modifiers, and an ability to interact with lectin-expressing macrophages. These changes are common and persistent. Genetic/epigenetic analysis is providing important data but investigation of the posttranslational landscape is the next challenge. We have one glimpse of its operation via the influence of added glycan on the B-cell receptor of FL. The consequential interaction with environmental lectins illustrates how posttranslational modifications can be exploited by tumor cells, and could lead to new approaches to therapy.

Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

Surface IgM expression and function are associated with clinical behavior, genetic abnormalities, and DNA methylation in CLL.

Chronic lymphocytic leukemia (CLL) with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable region (IGHV) displays different states of anergy, indicated by reduced surface immunoglobulin M (sIgM) levels and signaling, consequent to chronic (super)antigen exposure. The subsets also differ in the incidence of high-risk genetic aberrations and in DNA methylation profile, preserved from the maturational status of the original cell. We focused on sIgM expression and function, measured as intracellular Ca(2+) mobilization following stimulation, and probed correlations with clinical outcome. The relationship with genetic features and maturation status defined by DNA methylation of an 18-gene panel signature was then investigated. sIgM levels/signaling were higher and less variable in U-CLL than in M-CLL and correlated with disease progression between and within U-CLL and M-CLL. In U-CLL, increased levels/signaling associated with +12, del(17p) or NOTCH1 mutations. In M-CLL, there were fewer genetic lesions, although the methylation maturation status, generally higher than in U-CLL, varied and was increased in cases with lower sIgM levels/signaling. These features revealed heterogeneity in M-CLL and U-CLL with clear clinical correlations. Multivariate analyses with phenotype, genetic lesions, or DNA methylation maturation status identified high sIgM levels as a new potential independent factor for disease progression. Multiple influences on sIgM include the cell of origin, the clonal history of antigen encounter in vivo, and genetic damage. This simple marker compiles these different factors into an indicator worthy of further investigations for prediction of clinical behavior, particularly within the heterogeneous M-CLL subset.

IL-4 enhances expression and function of surface IgM in CLL cells.

Kinase inhibitors targeting the B-cell receptor (BCR) are now prominent in the treatment of chronic lymphocytic leukemia (CLL). We have focused here on interleukin 4 (IL-4), a cytokine that protects normal and malignant B cells from apoptosis and increases surface immunoglobulin M (sIgM) expression on murine splenic B cells. First, we have demonstrated that IL-4 treatment increased sIgM expression in vitro on peripheral blood B cells obtained from healthy individuals. In CLL, IL-4 target genes are overexpressed in cells purified from the lymph nodes of patients compared with cells derived from matched blood and bone marrow samples. As for normal B cells, IL-4 increased sIgM expression on CLL cells in vitro, especially in samples expressing unmutated V-genes. IL-4-induced sIgM expression was associated with increased receptor signalling activity, measured by anti-IgM-induced calcium mobilization, and with increased expression of CD79B messenger RNA and protein, and the "mature" glycoform of sIgM. Importantly, the ability of the BCR-associated kinase inhibitors idelalisib and ibrutinib, approved for treatment of CLL and other B-cell malignancies, to inhibit anti-IgM-induced signalling was reduced following IL-4 pretreatment in samples from the majority of patients. In contrast to stimulatory effects on sIgM, IL-4 decreased CXCR4 and CXCR5 expression; therefore, CLL cells, particularly within the progressive unmutated V-gene subset, may harness the ability of IL-4 to promote BCR signalling and B-cell retention within lymph nodes. Effects of IL-4 were mediated via JAK3/STAT6 and we propose a potential role for JAK inhibitors in combination with BCR kinase inhibitors for the treatment of CLL.

Engagement of the B-cell receptor of chronic lymphocytic leukemia cells drives global and MYC-specific mRNA translation.

Antigenic stimulation via the B-cell receptor (BCR) is a major driver of the proliferation and survival of chronic lymphocytic leukemia (CLL) cells. However, the precise mechanisms by which BCR stimulation leads to accumulation of malignant cells remain incompletely understood. Here, we investigated the ability of BCR stimulation to increase messenger RNA (mRNA) translation, which can promote carcinogenesis by effects on both global mRNA translation and upregulated expression of specific oncoproteins. Re-analysis of gene expression profiles revealed striking upregulation of pathways linked to mRNA translation both in CLL cells derived from lymph nodes, the major site of antigen stimulation in vivo, and after BCR stimulation in vitro. Anti-IgM significantly increased mRNA translation in primary CLL cells, measured using bulk metabolic labeling and a novel flow cytometry assay to quantify responses at a single-cell level. These translational responses were suppressed by inhibitors of BTK (ibrutinib) and SYK (tamatinib). Anti-IgM-induced mRNA translation was associated with increased expression of translation initiation factors eIF4A and eIF4GI, and reduced expression of the eIF4A inhibitor, PDCD4. Anti-IgM also increased mRNA translation in normal blood B cells, but without clear modulatory effects on these factors. In addition, anti-IgM increased translation of mRNA-encoding MYC, a major driver of disease progression. mRNA translation is likely to be an important mediator of the growth-promoting effects of antigen stimulation acting, at least in part, via translational induction of MYC. Differences in mechanisms of translational regulation in CLL and normal B cells may provide opportunities for selective therapeutic attack.

Lectin binding to surface Ig variable regions provides a universal persistent activating signal for follicular lymphoma cells.

The vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca(2+) flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM(+) and IgG(+) cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site.

Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma.

B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.

The PI3K/mTOR inhibitor PF-04691502 induces apoptosis and inhibits microenvironmental signaling in CLL and the Eµ-TCL1 mouse model.

Current treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti-immunoglobulin M-induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eµ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK.

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

Idiotypic DNA vaccination for the treatment of multiple myeloma: safety and immunogenicity in a phase I clinical study.

We report on the safety and immunogenicity of idiotypic DNA vaccination in a phase I, non-randomised, open-label study in patients with multiple myeloma. The study used DNA fusion gene vaccines encoding patient-specific single chain variable fragment, or idiotype (Id), linked to fragment C (FrC) of tetanus toxin. Patients in complete or partial response following high-dose chemotherapy and autologous stem cell transplant were vaccinated intramuscularly with 1 mg DNA on six occasions, beginning at least 6 months post-transplant; follow-up was to week 52. Fourteen patients were enrolled on study and completed vaccinations. Idiotypic DNA vaccines were well tolerated with vaccine-related adverse events limited to low-grade constitutional symptoms. FrC- and Id-specific T-cell responses were detected by ex vivo ELISPOT in 9/14 and 3/14 patients, respectively. A boost of pre-existing anti-FrC antibody (Ab) was detected by ELISA in 8/14 patients, whilst anti-Id Ab was generated in 1/13 patients. Overall, four patients (29 %) made an immune response to FrC and Id, with six patients (43 %) responding to FrC alone. Over the 52-week study period, serum paraprotein was undetectable, decreased or remained stable for ten patients (71 %), whilst ongoing CR/PR was maintained for 11 patients (79 %). The median time to progression was 38.0 months for 13/14 patients. Overall survival was 64 % after a median follow-up of 85.6 months.