Changes in N-Glycosylation Induced By Somatic Hypermutation Modulate the Antigen Reactivity of the Immunoglobulin Receptors in CLL Stereotyped Subset #201

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

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Lysine Propionylation is a Widespread Post-Translational Modification Involved in Regulation of Photosynthesis and Metabolism in Cyanobacteria

International Journal of Molecular Sciences ◽

10.3390/ijms20194792 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4792 ◽

Cited By ~ 1

Author(s):

Mingkun Yang ◽

Hui Huang ◽

Feng Ge

Keyword(s):

Large Fraction ◽

Bioinformatic Analysis ◽

High Light ◽

Site Directed Mutagenesis ◽

Oxygenic Photosynthesis ◽

Post Translational Modification ◽

Functional Studies ◽

Photosynthetic Organisms ◽

Model Combining ◽

And Function

Lysine propionylation is a reversible and widely distributed post-translational modification that is known to play a regulatory role in both eukaryotes and prokaryotes. However, the extent and function of lysine propionylation in photosynthetic organisms remains unclear. Cyanobacteria are the most ancient group of Gram-negative bacteria capable of oxygenic photosynthesis, and are of great importance to global carbon and nitrogen cycles. Here, we carried out a systematic study of lysine propionylaiton in cyanobacteria where we used Synechocystis sp. PCC 6803 (Synechocystis) as a model. Combining high-affinity anti-propionyllysine pan antibodies with high-accuracy mass spectrometry (MS) analysis, we identified 111 unique lysine propionylation sites on 69 proteins in Synechocystis. Further bioinformatic analysis showed that a large fraction of the propionylated proteins were involved in photosynthesis and metabolism. The functional significance of lysine propionylation on the enzymatic activity of fructose-1,6-bisphosphatase (FbpI) was studied by site-directed mutagenesis and biochemical studies. Further functional studies revealed that the propionylation level of subunit II of photosystem I (PsaD) was obviously increased after high light (HL) treatment, suggesting that propionylation may be involved in high light adaption in Synechocystis. Thus, our findings provide novel insights into the range of functions regulated by propionylation and reveal that reversible propionylation is a functional modification with the potential to regulate photosynthesis and carbon metabolism in Synechocystis, as well as in other photosynthetic organisms.

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New Era in Chronic Lymphocytic Leukemia: Consequences of the Arrival of New Drugs

Blood ◽

10.1182/blood-2021-150195 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4696-4696

Author(s):

Jorge Labrador ◽

Covadonga Garcia Diaz ◽

Beatriz Cuevas ◽

Maria Victoria Cuevas ◽

Rodolfo Alvarez ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Statistical Significance ◽

Young Patients ◽

Complete Response ◽

Cell Receptor ◽

Advisory Board ◽

Lymphocytic Leukemia ◽

New Drugs ◽

First Line

Abstract Introduction In the last decade, the incorporation of anti-CD20 monoclonal antibodies (MoAb) (rituximab, obinutuzumab), bendamustine, B-cell receptor inhibitors (ibrutinib) and Bcl-2 antagonists (venetoclax) have changed the paradigm of chronic lymphocytic leukemia (CLL) treatment, improving overall survival (OS). Methods We conducted a retrospective observational study of patients diagnosed with CLL between 2003 and 2016 at our center to evaluate: i) the influence of date of diagnosis, before or after 2010; ii) having received new drugs (MoAb or target therapies) in first line or not. Results A total of 182 patients were evaluated: 104 men (57%) and 78 women (43%); median age 74 years (39 - 97), 132 patients were >65 years (72.5%). At diagnosis, 135 (74%), 19 (10%), 15 (8%), 4 (2%) and 9 (5%) were diagnosed as stages 0, I, II, II, III and IV of the Rai classification. While 154 (84%) 15 (8%) and 14 (8%) were classified as Binet's stages A, B and C. Regarding the presence of comorbidities, the median CIRS scale score was 4 (0 - 15). 71 patients (39%) were diagnosed before 2010 and 111 (61%) from 2010 onwards. With a median follow-up of 76 months (range, 20-212), 77/182 (42%) patients had received ≥1 line(s) of treatment: 1: 53%, 2: 26%, 3: 8%, ≥4: 13%. 20 patients (26%) received the first line of treatment before 2010, and 56 (74%) from 2010 onwards. Half of the patients received new drugs in 1st line (n=39), 92% of them from 2010 onwards. 32 patients (41.5%) achieved complete response after 1st line, and 24 (31%) at least a partial response. The median OS of patients diagnosed before 2010 was 66 months (35.32 - 96.68) vs 143 months (95% CI 90.5 - 195.5) (p=0.032) as of 2010. In those ≤ 65 years median OS has not been reached, being 80.5% at 10 years: 65% for those diagnosed before 2010 and 97% after 2010, p=0.036. The median OS of patients > 65 years was 6 years (95% CI 4.17 - 7.83), and increased from 82 months for those diagnosed before 2010, to 110 months in those diagnosed after 2010, p=0.744. Patients treated with new drugs in 1st line increased the median OS from 76 months and 13% at 10 years to a median not reached and OS of 77% at 10 years, p=0.000. This difference was more evident in those ≤ 65 years: 20% vs 100% at 10 years (p=0.012), while it was not statistically significant for those > 65 years (11% vs 50% at 10 years, p=0.181) (Figure 1). However, in a multivariate model including age, sex, CIRS>6 and year of diagnosis (before or after 2010), treatment with new drugs retained statistical significance (HR 0.414; 95% CI 0.183 - 0.935) along with age ≤ 65 years (HR 0.256; 95% CI 0.085 - 0.771) and CIRS ≤ 6 (HR 0.247; 95% CI 0.111 - 0.551). Conclusions In our experience, the introduction of new drugs in the first line has led to an increase in OS, especially in young patients. Figure 1 Figure 1. Disclosures Gonzalez-Lopez: Grifols: Other: Advisory board honoraria; Sobi: Other: Advisory board honoraria; Amgen: Other: Advisory board and speakers honoraria, Research Funding; Novartis: Other: Advisoryboard and speakers honoraria, Research Funding.

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VH CDR3-Focused Somatic Hypermutation in CLL IGHV-IGHD-IGHJ Gene Rearrangements with 100% IGHV Germline Identity

Blood ◽

10.1182/blood-2019-127979 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4277-4277 ◽

Cited By ~ 1

Author(s):

Katerina Gemenetzi ◽

Andreas Agathangelidis ◽

Fotis Psomopoulos ◽

Kostas Pasentsis ◽

Evdoxia Koravou ◽

...

Keyword(s):

Research Funding ◽

Somatic Hypermutation ◽

Lymphocytic Leukemia ◽

Gene Rearrangements ◽

Rt Pcr ◽

Bioinformatics Pipeline ◽

Sample Mean ◽

Mean Frequency ◽

High Data ◽

Ighv Gene

Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the sequence of the rearranged IGHV gene excluding the VH CDR3. This is mostly due to the difficulty in discriminating actual SHM from random nucleotides added between the recombined IGHV, IGHD and IGHJ genes. Hence, this approach may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Relevant to mention in this respect, studies from our group in CLL with mutated IGHV genes (M-CLL), particularly subset #4, have revealed considerable intra-VH CDR3 diversity attributed to ongoing SHM. Prompted by these findings, here we investigated whether SHM may also be present in cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3), focusing on two well characterized stereotyped subsets i.e. subset #1 (IGHV clan I/IGHD6-19/IGHJ4) and subset #6 (IGHV1-69/IGHD3-16/IGHJ3). These subsets carry germline-encoded amino acid (aa) motifs within the VH CDR3, namely QWL and YDYVWGSY, originating from the IGHD6-19 and IGHD3-16 gene, respectively. However, in both subsets, cases exist with variations in these motifs that could potentially represent SHM. The present study included 12 subset #1 and 5 subset #6 patients with clonotypic IGHV genes lacking any SHM (100% germline identity). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified by subgroup-specific leader primers and a high-fidelity polymerase in order to ensure high data quality. RT-PCR products were subjected to paired-end NGS on the MiSeq platform. Sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis was undertaken using an in-house purpose-built bioinformatics pipeline. Rearrangements with the same IGHV gene and identical VH CDR3 aa sequences were defined as clonotypes. Overall, we obtained 1,570,668 productive reads with V-region identity 99-100%; of these, 1,232,958 (mean: 102,746, range: 20,796-242,519) concerned subset #1 while 337,710 (mean: 67,542, range: 50,403-79,683) concerned subset #6. On average, 64.4% (range: 1.7-77.5%) of subset #1 reads and 49.2% (range: 0.7-70%) of subset #6 reads corresponded to rearrangements with IGHV genes lacking any SHM (100% germline identity). Clonotype computation revealed 1,831 and 1,048 unique clonotypes for subset #1 and #6, respectively. Subset #1 displayed a mean of 157 distinct clonotypes per sample (range: 74-267), with the dominant clonotype having a mean frequency of 96.9% (range: 96-98.2%). Of note, 44 clonotypes were shared between different patients (albeit at varying frequencies), including the dominant clonotype of 11/12 cases, which was present in 2-6 additional subset #1 patients. Subset #6 cases carried a higher number of distinct clonotypes per sample (mean: 219, range: 189-243) while the dominant clonotype had a mean frequency of 95.6% (range: 94.5-96.5%). Shared clonotypes (n=30) were identified also in subset #6 and the dominant clonotype of each subset #6 case was present in 3-5 additional subset #6 patients. Focusing on the VH CDR3, in particular the IGHD-encoded part, the following observations were made: (1) in both subsets, extensive intra-VH CDR3 variation was detected at certain positions within the IGHD gene; (2) in most cases, the observed aa substitutions were conservative i.e. concerned aa sharing similar physicochemical properties. Particularly noteworthy in this respect were the observations in subset #6 that: (i) the valine residue (V) in the D-derived YDYVWGSY motif was very frequently mutated to another aliphatic residue (A, I, L); (ii) in cases were the predominant clonotype carried I (also in the Sanger-derived sequence), several minor clonotypes carried the germline-encoded V, compelling evidence that the observed substitution concerned true SHM. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. While the prognostic/predictive relevance of this observation is beyond the scope of the present work, our findings highlight the possible need to reappraise definitions ('semantics') regarding SHM status in CLL. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

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What Numbers Don't Say: Immunogenetic Evidence Shows That High-Count MBL Resembles Rai 0 CLL While Low-Count MBL Does Not.

Blood ◽

10.1182/blood.v120.21.2883.2883 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2883-2883

Author(s):

Anna Vardi ◽

Antonis Dagklis ◽

Lydia Scarfò ◽

Diane F. Jelinek ◽

Darren J Newton ◽

...

Keyword(s):

General Population ◽

B Cell ◽

Research Funding ◽

Somatic Hypermutation ◽

Molecular Genetic ◽

High Sensitivity ◽

Cell Receptor ◽

World Health ◽

High Count ◽

Health Organization

Abstract Abstract 2883 CLL-like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotypic features and cytogenetic abnormalities with CLL and is generally perceived as its premalignant state. The World Health Organization has set a consensus cut-off of 5×109/L circulating B cells to discriminate between what constitutes ‘disease’ and what not. However, the clonal size within MBL is extremely variable. High-count (HC), clinical MBL is associated with absolute lymphocytosis and progresses to CLL requiring treatment at a rate of 1–2% per year, whereas low-count (LC) MBL is found in the general population through high-sensitivity techniques and carries a risk of progression that is limited if any. Given the high frequency of CLL-like MBL in the general population, it is important to understand the underlying mechanisms and also identify biological markers endowing malignant potential that may distinguish between the different forms. To this end, we performed a detailed immunogenetic profiling of 334 CLL-like MBL cases (78 LC and 256 HC) for a total of 355 productive VDJ rearrangements (including double rearrangements), 91 from LC MBL and 264 from HC MBL. We also compared the immunoglobulin (IG) gene repertoires of MBL to 544 CLL Rai Stage 0 (CLL-0) that were part of an IG sequence dataset of 7424 CLL cases previously analyzed by our group. LC and HC MBL had distinct IG gene repertoires, with over-representation of the IGHV1–69 and IGHV4–34 genes in HC and the IGHV4–59/61 genes in LC MBL, respectively (p<0.001). The HC MBL repertoire exhibited clear similarities to CLL-0 in terms of IGHV gene usage (similar frequencies of IGHV1–69 and IGHV4–34). Regarding somatic hypermutation, no differences were identified between LC MBL versus HC MBL versus CLL-0, in that the frequency of mutated rearrangements (<98% identity to the germline) was overall similar (LC MBL: 72.5%, HC MBL: 76.1%, CLL-0: 75%). In this respect, all the aforementioned subgroups differed significantly (p<0.001) from the frequency previously reported by us in CLL where only 55% of rearrangements carried mutated IGHV genes. We finally analyzed the expression of stereotyped B cell receptor (BcR) IGs, identified through a cluster analysis of the MBL sequences together with all CLL sequences from our cohort and 5494 non-CLL IG sequences retrieved from public databases. Overall, only 6/91 (5.5%) LC MBL rearrangements could be clustered with other sequences in subsets with stereotyped BcRs. Two of these six LC MBL cases were clustered together with IG sequences from various entities, including CLL, other lymphomas and autoimmune diseases; thus, they were considered to carry ‘public’ BcR stereotypes. In contrast, HC MBL showed a significantly (p=0.0002) higher frequency of ‘CLL-specific’ BcR stereotypes versus LC MBL, with 60/264 (22.7%) HC MBL cases carrying stereotyped VH CDR3s. This frequency was comparable to the one observed in CLL-0 (20.2%). Notably, a gradation was observed in the frequency of BcR IG stereotypy depending on the absolute count of CLL-like cells, starting with 5.5% in LC MBL, raising to 22.7–20.2% in in HC MBL/CLL-0 and, finally, peaking at 30.4% in the entire CLL cohort as previously reported. Altogether, these findings suggest that rather than a true premalignant condition, LC MBL may merely reflect immune senescence or result from persistent antigen stimulation. On the other hand, HC MBL appears to be a continuum with Rai stage 0 in the evolution to clinically overt CLL, being maybe one step behind where it requires either additional genetic hits or simply extra time to cross the numerical border that discriminates it from CLL. Hence, the identification of molecular genetic markers that may predict progression of HC MBL/CLL-0 into full-fledged CLL is strongly warranted. Disclosures: Shanafelt: Genentech: Research Funding; GlaxoSmith Klein: Research Funding; Teva/Cephalon: Research Funding; Celgene: Research Funding.

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Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment

Blood ◽

10.1182/blood.v126.23.2922.2922 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2922-2922

Author(s):

Elisa ten Hacken ◽

Thomas Oellerich ◽

Maria Gounari ◽

Julia Hoellenriegel ◽

Kuan-Ting Pan ◽

...

Keyword(s):

Mass Spectrometry ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Label Free ◽

Bcr Signaling ◽

Associated Proteins

Abstract Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined. Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells. Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands. Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases. Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment. Disclosures Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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APT1-Mediated Cross-Talk Between Palmitoylation and Phosphorylation Events of the BCR Pathway Sensitizes CLL Cells Towards BCR-Associated Kinase Inhibitors

Blood ◽

10.1182/blood.v128.22.4361.4361 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4361-4361

Author(s):

Valeska Berg ◽

Dunja Baatout ◽

Clemens Wendtner ◽

Michael Hallek ◽

Lukas P. Frenzel

Keyword(s):

Research Funding ◽

Kinase Inhibitors ◽

Cell Receptor ◽

Fine Tuning ◽

Post Translational Modification ◽

Protein Activity ◽

Post Translational Modifications ◽

Protein Palmitoylation ◽

Bcr Signaling ◽

Support Research

Abstract Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Regulation of phosphorylation events of the BCR is critical for survival and proliferation of CLL cells. Palmitoylation, a common post-translational modification defined as the addition of palmitic acid to internal cysteins, was shown recently to regulate phosphorylation of proteins by controlling their localization and activity. Many proteins in the B cell receptor (BCR) signaling pathway in CLL cells are primarily cytosolic, but upon activation transiently located to the plasma membrane to fulfill their functions. Some of these proteins, like Src kinase family members Lyn, Yes and Fyn, are already reported to be palmitoylated. Previous studies by our group showed that global protein palmitoylation is deregulated in CLL cells and primarily caused by overexpression of the depalmitoylating enzyme APT1. To investigate, if overexpressed APT1 directly targets BCR signaling, we inhibited (genetically and pharmacologically) APT1 in CLL cells and analyzed occurring changes in 45 different phosphorylation-sites of major signaling pathways. Interestingly, we found that APT1 controls the central phosphorylation events within Akt/mTOR/p70S6 signaling. For example, phosphorylation of Akt (T308, S473) and p70S6 (T389, T421, S424) was significantly decreased after interference with protein depalmitoylation. By biochemical dissection of these pathways with acyl-biotin exchange (ABE) assays we identified novel palmitoylation candidates particularly within the PI3K/Akt axis, which are indispensable for phosphorylation of kinases of the Akt/mTOR/p70S6 axis. Functionally, pharmacological inhibition of APTs and genetic knockdown of APT1 sensitizes CLL cells towards BCR-associated KIs like Ibrutinib and Idelalisib. Our data uncovers that central phosphorylation events within the BCR pathway are dependent on palmitoylation controlled by APT1. Future studies should therefore investigate more in detail how addition of APT1 inhibitors can improve clinical outcome of patients treated with Idelalisib or Ibrutinib-based regimens. Disclosures Wendtner: Hoffmann-La Roche, Mundipharma, Janssen, Gilead, Abbvie, Servier, Morphosys: Consultancy, Other: Travle grants, Research Funding. Hallek:Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

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HIGHER ORDER IMMUNOGLOBULIN REPERTOIRE RESTRICTIONS IN CLL: THE ILLUSTRATIVE CASE OF STEREOTYPED SUBSETS #2 AND #169

Blood ◽

10.1182/blood.2020005216 ◽

2020 ◽

Author(s):

Katerina Gemenetzi ◽

Fotis Psomopoulos ◽

Alejandra Carriles ◽

Maria Gounari ◽

Claudia Minici ◽

...

Keyword(s):

Light Chain ◽

Somatic Hypermutation ◽

Cell Receptor ◽

Crystallographic Analysis ◽

Lymphocytic Leukemia ◽

Illustrative Case ◽

Antigen Binding Site ◽

Linker Region ◽

Immunoglobulin Repertoire ◽

Disease Variant

Chronic lymphocytic leukemia (CLL) major stereotyped subset #2 (IGHV3-21/IGLV3-21, ~2.5% of all CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset #169 (IGHV3-48/IGLV3-21, ~0.2% of all CLL) is related to subset #2 as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B cell receptor immunoglobulin (BcR IG). Branching evolution of the predominant clonotype through intraclonal diversification (ID) in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the two subsets were highlighted by the finding of shared SHMs within both the heavy and the light chain genes in all analyzed cases at either clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for IG homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset #169 IG retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset #2, including the SHM at the linker region, and from a molecular standpoint belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets #2 and #169 are very closely related, displaying shared IG features that can be explained only in the context of shared functional selection.

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Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503587112 ◽

2015 ◽

Vol 112 (14) ◽

pp. 4322-4327 ◽

Cited By ~ 23

Author(s):

James S. Blachly ◽

Amy S. Ruppert ◽

Weiqiang Zhao ◽

Susan Long ◽

Joseph Flynn ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Germ Line ◽

Somatic Hypermutation ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Maturation Stage ◽

Rna Seq ◽

Mutation Status ◽

General Test

Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (>1 × 1012) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition. Cancers arising from mature B cells are characterized by clonal production of Ig heavy (IGH@) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was out-of-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.

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Targeting Metabolic Alterations in CLL Microenvironment; Inhibition of Glutamine Import Attenuates Venetoclax Resistance

Blood ◽

10.1182/blood-2021-150236 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3717-3717

Author(s):

Zhenghao Chen ◽

Helga Simon-Molas ◽

Gaspard Cretenet ◽

Beatriz Valle-Argos ◽

Francesco Forconi ◽

...

Keyword(s):

Research Funding ◽

Tca Cycle ◽

Cell Receptor ◽

Steering Committee ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Metabolic Alterations ◽

The Tca Cycle ◽

13C Labelling

Abstract Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model. Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured. Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B). Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance. References 1. Guièze, R. et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, (2019). 2. Chen, Z. et al. Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia. Blood 136, (2020). 3. Herishanu, Y. et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117, 563-574 (2011). Figure 1 Figure 1. Disclosures Forconi: AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Gilead: Research Funding. van der Windt: Genmab: Current Employment. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee.

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Lymphoma-Specific Subversion of B-Cell Receptor Signaling By Macrophage Lectins

Blood ◽

10.1182/blood-2018-99-110954 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2865-2865 ◽

Cited By ~ 1

Author(s):

Beatriz Valle Argos ◽

Giorgia Chiodin ◽

Francesco Forconi ◽

Richard Burack ◽

Philip Rock ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Somatic Hypermutation ◽

Cell Receptor ◽

B Cell Receptor ◽

Antigen Binding ◽

Sequence Motifs ◽

Post Translational Modification ◽

Antigen Binding Site ◽

Variable Regions

Abstract The clue that surface Ig (sIg) is implicated in the pathogenesis of follicular lymphoma (FL) is that in FL the vast majority of the sIg variable regions are structurally modified by insertion of mannose residues into the antigen-binding sites. This is tumor-specific and reflects positive selection of sequence motifs for glycan addition introduced during somatic hypermutation. The termination at high mannoses is very unusual in cell surface molecules and it confers an ability to interact with local lectins expressed by macrophages. In this respect, the mannose cloaking of the sIg receptor resembles that of human immunodeficiency virus (HIV), which acquires a similar mannose coat to facilitate binding to macrophages. The lectin DC-SIGN is upregulated in FL, likely via local IL-4, and is the strong lectin candidate. To address the question of the functional implications of interaction between DC-SIGN and sIgM we used a FL-derived cell line (WSU-FSCCL) which expresses sIg-mannoses. We found that two recombinant derivatives of DC-SIGN (DC-SIGN-Fc or DC-SIGN-HA) bind to the sIgM but not to a cell line which express sIgM without mannose insertion. Although the mannoses are an integral part of the sIgM, and anti-IgM induces a Ca2+ flux, DC-SIGN binds but does not mimic anti-IgM. In the WSU-FSCCL cell line it remains on the surface IgM without inducing a Ca2+ response and without mediating endocytosis of the sIgM. However, DC-SIGN does act on the sIgM as revealed by the effects of pre-exposure of the cells to either DC-SIGN derivative, which, although there is no blocking of access of anti-IgM, alters the ability of the cell to respond to stimulation. Pre-exposure appears to partially paralyze the subsequent sIgM-induced Ca2+ flux indicating a lectin-mediated modification of sIgM function. On engagement by anti-IgM, sIg undergoes conventional endocytosis and this was confirmed in WSU-FSCCL. Inhibitors which block signaling did not affect endocytosis, indicating bifurcation of signaling and endocytic pathways. Knowing that DC-SIGN derivatives, again in contrast to anti-IgM, did not induce endocytosis of sIgM, we then asked if the paralysis of the sIgM-mediated Ca2+ flux by pre-exposure to lectin affected anti-IgM-induced endocytosis, and found that it did not. This confirms the separation of signaling and endocytosis and indicates that whatever change is occurring in sIgM due to lectin exposure does not remove the sIgM from the endocytic machinery. We then investigated primary FL cases, using a splenic FL and 2 lymph node FLs, all carrying N-glycosylation motifs and able to bind DC-SIGN. We focused on the DC-SIGN-HA derivative, which gives no detectable Ca2+ flux signal by itself. Binding of the lectin again paralyzed subsequent anti-IgM-induced Ca2+ flux in all cases (Fig.1A), confirming the results with the cell line. Further investigation revealed that the lectin prevents early events after antigen binding to the sIg receptor reducing SYK phosphorylation and leading to a reduced Ca2+ mobilization (Fig.1B). In conclusion, modulation of the B-cell receptor by mannose addition to the antigen-binding site allows lectin access from innate microenvironmental cells. The effect of this is to provide a low level/null signal without loss by endocytosis. The novel finding is that this interaction then lowers the function of the B-cell receptor and perhaps blocks potential interference by antigen. There may be a parallelism with reports of modification of T-cell receptor function by galectins, with both pointing to the role of post-translational modification adding another layer of control on the operation of the major immune receptors. In the case of FL, this has been exploited to maintain tumor cells in the hostile environment of the germinal center. The apparently lymphoma-specific adaptation offers opportunities for targeted inhibition of the interaction. Figure 1. Figure 1. Disclosures Forconi: Janssen-Cilag: Consultancy; Abbvie: Consultancy. Packham:Aquinox: Research Funding.

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