B Cell Receptor Pathway Activation Research Articles

Antigen-Independent, Autonomous B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) defines the Cell-of-origin (CoO) classification with two major subtypes, germinal center B-cell (GCB) and the more aggressive activated B-cell (ABC) type. Chronic activation of B-cell receptor (BCR) signaling with constitutive activation of the NF-κB pathway is an essential oncogenic mechanism in ABC DLBCL. BCR pathway activation with increased NF-κB activation can be caused by gain-of-function mutations of the BCR signaling cascade. We investigated whether antigen-independent, i.e. autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia, acts as a functional driver mechanism in DLBCL without such gain-of-function mutations. DNA and RNA were extracted from ABC DLBCL cell lines TMD8 and OCI-Ly3 and from fresh-frozen biopsies of 18 histologically confirmed DLBCL cases. Functional BCR transcripts were identified by ARTISAN PCR and transduced into murine triple knock out (TKO) cells. TKO cells lack the recombination activation gene 2 (Rag2) and lambda 5 gene (λ5) and are therefore incapable of expressing an endogenous BCR. In addition, the BCR signal transduction molecule SLP65 is replaced with a tamoxifen-inducible estrogen receptor (ERT2) - SLP65 fusion gene. Upon transduction of both heavy and light BCR chains and staining with Indo-1, background calcium flux can be measured as Indo-1 signal intensity ratio (SIR) by flow cytometry. After restoring functionality of the BCR signaling cascade by tamoxifen, antigen-independent BCR signaling is measured as SIR, followed by maximum BCR stimulation with an isotype-specific crosslinking antibody. Autonomous BCR signaling strength (SSBCR) is quantified by an arithmetic product based on the relative Indo-1 SIR and the relative number of cells with SIR above background in the presence of tamoxifen and calibrated on crosslinking-induced maximum SIR. According to the primary hypothesis, the BCR of CARD11 wild-type, IgM-expressing TMD8 cells demonstrated strong autonomous BCR signaling (SSBCR=0.243), whereas the BCR of CARD11 mutated, IgG-expressing OCI-Ly3 cells completely lacked autonomous signaling (SSBCR=0.022). BCR from 13 primary DLBCL signaled autonomously (SSBCR=0.125-0.619), where 5 samples were negative (SSBCR=0.004-0.023) Autonomous BCR signaling was absolutely dependent on IgM isotype (Fisher's exact test: p=0.0016). CoO classification by digital gene expression (Nanostring) analyses or immunohistochemistry demonstrated autonomous BCR signaling in 8/9 ABC (non-GCB) DLBCL, 2/6 GCB DLBCL, and 2 unclassifiable DLBCL, and a transformed follicular lymphoma DLBCL. Independent of CoO, DLBCL can be assigned to 6-7 clusters based on their genetic aberrations (Chapuy 2018, Wright 2020). Fifteen DLBCL were suited for WES and targeted panel sequencing. Four cases were assigned by a probabilistic calculation to Chapuy cluster C5, 2 cases to C2, and one case to C1, while 8 cases remained unclassifiable. Clustering by the LymphGen tool assigned 3 cases to cluster ST2, one case each into MCD, EZB, and N1, 4 cases into various clusters, and 5 cases unclassified. DLBCL with autonomous BCR signaling fell into clusters C1/unclassified, C2/unclassified, C5/unclassified (n=3), unclassified/EZB, unclassified/ST2, unclassified/N1 and unclassified/unclassified (n=2). Cases lacking autonomous BCR signaling were assigned to unclassified/ST2 (n=2), C5/MCD, and unclassified/unclassified. Eleven of 13 cases with an autonomous signaling BCR lacked activating CARD11 mutations. In conclusion, autonomous BCR signaling is a potential immunological oncogenic driver in DLBCL that cannot be detected by sequencing studies, is independent of external antigen stimulation, and originates from a self-organizing conformation of the surface BCR (Minici 2017). Autonomous BCR signaling is present predominantly in ABC DLBCL and dependent on IgM isotype. Autonomous BCR signaling offers a convincing explanation for oncogenic NF-κB activation in DLBCL. Limited data suggest that the majority of DLBCL with autonomous BCR signaling fall into the ABC-dominated genetic clusters C1/C5, and MCD/N1, respectively. In summary, genetic aberrations, BCR function, and gene expression profile form 3 independent layers of DLBCL pathophysiology that need to be analyzed separately and offer independent targets for therapy.

B-cell receptors of EBV-negative Burkitt lymphoma bind modified isoforms of autoantigens.

Burkitt lymphoma (BL) represents the most aggressive B‐cell‐lymphoma. Beside the hallmark of IG‐MYC‐translocation, surface B‐cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra‐nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom‐made virome array and against self‐antigens, including post‐translationally modified antigens.An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1‐BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1‐BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1‐BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1‐BYSL‐ETA’ immunotoxin, produced by a two‐step intein‐based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1‐BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt‐lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed.These results indicate a pathogenic role of autoreactivity evoked by immunogenic post‐translational modifications in a subgroup of sporadic BL including two EBV‐negative BL cell lines.

Genomics of diffuse large B cell lymphoma.

Next generation sequencing (NGS) technology has revealed the heterogeneity of diffuse large B-cell lymphoma (DLBCL) from a mutation perspective. Accordingly, the conventional cell of origin-based classification of DLBCL has changed to a mutation-based classification. Mutation analysis delineates that B-cell receptor pathway activation, EZH2 mutation, and NOTCH mutations are distinctive drivers of DLBCL. Moreover, the combination of RNA expression data and DNA mutation results suggests similarity between DLBCL subtypes and other non-Hodgkin lymphomas. NGS-based dissection of DLBCL would be the cornerstone for precision treatment in this heterogeneous disease in the near future.

TOSO interacts with SYK and enhances BCR pathway activation in chronic lymphocytic leukemia.

BackgroundTOSO, also named Fas inhibitory molecule 3 (FAIM3), has recently been identified as an immunoglobulin M (IgM) Fc receptor (FcμR). Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia (CLL). However, the functions of TOSO in CLL remain unknown. The B-cell receptor (BCR) signaling pathway has been reported to be constitutively activated in CLL. Here, we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.MethodsWe over-expressed TOSO in B-cell lymphoma cell lines (Granta-519 and Z138) by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells. The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting. Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry (IP/LCMS) was used to identify TOSO interacting proteins. Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2 (BCL-2). Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD. One-way analyses of variance were used for intergroup comparisons, while independent samples t tests were used for two-sample comparisons.ResultsFrom IP/LCMS, we identified spleen tyrosine kinase (SYK) as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation. After stimulation with anti-IgM, TOSO over-expression increased the phosphorylation of SYK, and subsequently activated the BCR signaling pathway, which could be reversed by a SYK inhibitor. TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway. The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were (8.46 ± 2.90)% and (4.20 ± 1.21)%, respectively, significantly lower than the rates of the control groups, which were (25.20 ± 4.60)% and (19.72 ± 1.10)%, respectively (P < 0.05 for both). The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells. In addition, we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis, and vice versa in the cell line.ConclusionsTOSO might be involved in the pathogenesis of CLL by interacting with SYK, enhancing the BCR signaling pathway, and inducing apoptosis resistance.

Mechanisms of B Cell Receptor Activation and Responses to B Cell Receptor Inhibitors in B Cell Malignancies.

The B cell receptor (BCR) pathway has been identified as a potential therapeutic target in a number of common B cell malignancies, including chronic lymphocytic leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, and Waldenstrom’s macroglobulinemia. This finding has resulted in the development of numerous drugs that target this pathway, including various inhibitors of the kinases BTK, PI3K, and SYK. Several of these drugs have been approved in recent years for clinical use, resulting in a profound change in the way these diseases are currently being treated. However, the response rates and durability of responses vary largely across the different disease entities, suggesting a different proportion of patients with an activated BCR pathway and different mechanisms of BCR pathway activation. Indeed, several antigen-dependent and antigen-independent mechanisms have recently been described and shown to result in the activation of distinct downstream signaling pathways. The purpose of this review is to provide an overview of the mechanisms responsible for the activation of the BCR pathway in different B cell malignancies and to correlate these mechanisms with clinical responses to treatment with BCR inhibitors.

Ibrutinib for relapsed/refractory mantle cell lymphoma: 7-year experience

Abstract Background Mantle cell lymphoma (MLC) is nowadays an aggressive and incurable Non-Hodgkin’s lymphoma, characterized by multiple relapses.The persistent activation of B-cell receptor pathway is critical for pathogenesis. Ibrutinib is a potent covalent inhibitor of Bruton’s tyrosine kinase (BTK) that inhibits B-cell antigen receptor signaling downstream of BTK and changed for better how MCL is treated in relapsed/refractory setting. However, resistance is common and response limited. Methods Retrospective analysis and follow-up of all MCL patients treated with Ibrutinib in a Onco-Hematology Department. Results Eleven patients were identified, with a median age of 68 years [58-78], the majority of them were males (83.3%). At diagnosis, 9% were in stage III of Ann Arbor while 81.8% were in stage IV. The median MIPI (MCL International Prognostic Index) was 5.5 [4-8]. These patients were exposed to a median of 3 treatments [3-5]. The majority of them performed as 1oline treatment R-FCM (18.2%) or R-CHOP (54.5%); as 2oline R-CHOP (45.5%), as 3oline Ibrutinib (63.6%), as 4oline PEPC (60%) and as 5oline Ibrutinib (100%). Ibrutinib therapy was prescribed as a 2oline in 9.1% of the patients, as a 3oline in 63.6%, as 4oline in 40% and as a 5oline in 100%. Ibrutinib achieved a complete response rate in 9.1%, a partial response in 45.5% (response rate, RR, of 54.5%), disease progression in 36.4%, stable disease in 9,1%. At the end of the study, 5 patients were alive and still doing Ibrutinib (4 of them in full dose 560 mg/day). The median progression free survival (PFS) was 3 months [1-26]. Overall survival was not reached (3 patients died with disease progression). Conclusions Although a larger sample and a longer follow-up are needed, these data showed worse results when compared to previous phase 2 trials demonstrating a 68% response rate (RR), including for high-risk patients and heavily pretreated. Most recently, a pooled analysis of the three open-label studies of ibrutinib in MCL, PCYC-1104, SPARK, and RAY revealed an ORR of 68%, 63% and 72%. Ibrutinib may be changing the natural history of MCL as many other agents still under development. Enrollment in clinical trials is crucial not only for discovery of new agents and testing combinations but also to understand the natural history of relapsed MCL. Legal entity responsible for the study Onco-Hematology Service of Instituto Portugues de Oncologia do Porto, F.G., E.P.E. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma

Introduction:Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma.Methods:BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities.Results:The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications.Conclusions:A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. DisclosuresStilgenbauer:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.

SHP1 Deficiency Is Responsible for the Constitutive Activation of the BCR Pathway in GCB DLBCL

Functional and transcriptional profiling studies have identified a subset of diffuse large B cell lymphoma (DLBCL) tumors that rely on B cell receptor (BCR) signals for proliferation and survival. These BCR-dependent tumors have been identified among both the ABC and GCB DLBCL subtype. However, differences have been observed between the two subtypes in terms of the mechanism of BCR pathway activation and activity of downstream signaling molecules. BCR-dependent ABC DLBCL tumors are characterized by high basal NF-kB and PI3K/AKT activity and the presence of gain-of-function mutations in the CD79A or CD79B subunit of the BCR that are responsible for the chronic activation of the BCR pathway. In contrast, BCR-dependent GCB DLBCL tumors have low baseline NF-kB activity and lack CD79A/CD79B mutations, suggesting a different mechanism of BCR pathway activation. In this study, we investigated whether activation of the BCR pathway in GCB DLBCL is potentially caused by deficiency of the phosphatase SHP1, which is an important negative regulator of the BCR pathway and has been reported to be downregulated in approximately 40% of primary DLBCL tumors. For this purpose, we first correlated SHP1 expression with the presence of phosphorylated SYK and BLNK in the GCB DLBCL cell lines OCI-Ly1, OCI-Ly7, OCI-Ly18, SU-DHL-4, SU-DHL-6, WSU-NHL, SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage. Immunoblotting analysis revealed that SHP1 is expressed in SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage but not in the remaining 6 cell lines. Expression of SHP1 inversely correlated with expression of phosphorylated SYK (pSYK-Y352) and BLNK (pBLNK-Y84), suggesting that these two BCR-proximal signaling molecules are activated because of SHP1 deficiency. To further evaluate this possibility, we re-expressed SHP1 in OCI-Ly1 and SU-DHL-4 cells by transient transfection with a plasmid or a lentiviral SHP1 expression vector. Both approaches resulted in a substantial reduction of pSYK-Y352 and pBLNK-Y84 levels, confirming that SHP1 deficiency leads to activation of the BCR pathway.To determine whether activation of the BCR pathway contributes to tumor cell survival, we investigated the effects of the SYK inhibitor R406 (active substance of the drug fostamatinib) on the viability of the 6 SHP1-positive and 6 SHP1-negative GCB DLBCL cell lines. At concentrations up to 2 μM, R406 displayed only minimal toxicity against each of the SHP1-positive and SHP1-negative GCB DLBCL cell lines (<20% apoptotic cells), with the exception of WSU-NHL (60% apoptotic cells). However, analysis of the effects of R406 on the expression of BCL-2 family proteins showed downregulation of the antiapoptotic protein MCL-1 and upregulation of the proapoptotic proteins BIM and HRK in all of the SHP1-negative but in none of the SHP1-positive GCB DLBCL cell lines. Such changes were also observed following siRNA-mediated knockdown of SYK or overexpression of SHP1, further suggesting that they are on-target effects of R406. In addition, treatment with the BTK inhibitor ibrutinib or siRNA-mediated knockdown of BTK also resulted in downregulation of MCL-1 and upregulation of BIM in these cell lines. Similar changes were observed in 3 of the 5 investigated primary DLBCL samples following treatment with ibrutinib or fostamatinib. Importantly, downregulation of MCL-1 by RNA interference or upregulation of BIM by mRNA transfection resulted in substantially increased sensitivity of these cell lines to the BCL-2 inhibitor venetoclax, suggesting that SYK or BTK inhibitors could potentiate the cytotoxic effects of this drug. To further evaluate this possibility, we calculated the combination index of the R406 + venetoclax or ibrutinib + venetoclax drug combination in the 12 GCB DLBCL cell lines. Significant synergistic activity was seen in all five BCL-2-positive/SHP1-negative GCB DLBCL cell lines with the R406 + venetoclax combination and in four of them with the ibrutinib + venetoclax combination. In contrast, synergistic activity was detected in only one of the 6 SHP1-positive GCB DLBCL cell lines and was not seen in the BCL-2-negative/SHP1-negative cell line OCI-Ly7. Collectively, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination. DisclosuresNo relevant conflicts of interest to declare.

A Phase 1 Dose Escalation Study of ARQ 531 in Selected Patients with Relapsed or Refractory Hematologic Malignancies

Background: Aberrant activation of B-cell receptor (BCR) signaling is considered to be a major oncogenic mechanism that leads to the development and progression of multiple B-cell malignancies. ARQ 531 is a reversible ATP competitive inhibitor of BTK that inhibits ibrutinib-resistant BTK-C481S mutant CLL cells and has demonstrated antitumor activity in CLL, Richter's transformation, and DLBCL mouse models.Objectives: The primary objectives of the clinical study are to assess the safety and tolerability, and to determine the recommended Phase 2 dose and schedule of ARQ 531. The secondary objectives are to assess the pharmacokinetic (PK) profile, pharmacodynamic (PD) activity, and preliminary evidence of anti-tumor activity of ARQ 531.Methods: This is a first in human, Phase 1 dose escalation study in patients with relapsed or refractory CLL/SLL, Waldenstrom's macroglobulinemia, or B-cell NHL who received at least 2 prior lines of systemic therapy. Prior therapy must have included a BTK inhibitor, if FDA approved for their disease. Dose escalation was performed according to a 3+3 study design. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v.4.03. Tumor responses were evaluated per disease specific guidelines.Results: A total of 16 patients enrolled (median age 65.5 years, male 93.8%, 2 DLBCL, 2 follicular lymphoma, 12 CLL/SLL, 5 median prior systemic regimens) and were treated at dose levels of 5, 10, 15, 20 and 30mg QD. No dose limiting toxicities have been reported with ARQ 531. Drug related TEAEs included diarrhea, nausea, vomiting, fatigue, pneumonia, amylase increased, lipase increased, neutrophil count decreased, platelet count decreased, decreased appetite, hypernatraemia, arthralgia, groin pain, dizziness, facial paralysis, headache, tremor and restlessness in one patient (6.3%) each. Drug related grade 3 or worse TEAEs included lipase increased and platelet count decreased in one patient (6.3%) each. No drug related serious TEAEs were reported. Among the 12 patients who have received at least 1 dose of study drug and have at least 1 post-treatment tumor measurement data, 5 achieved stable disease (SD) (1 follicular lymphoma, 1 DLBCL, 3 CLL) and 7 had progressive disease (6 CLL, 1 follicular lymphoma). Three of 5 SD patients (1 follicular lymphoma, 1 DLBCL, 1 CLL) had 35%, 34% and 29% tumor reduction and 2 of them are ongoing on study treatment at 53 and 18 weeks. The CLL patient with 29% tumor reduction had BTK C481S mutation.Preliminary PK data showed ARQ 531 exposure was close to dose proportional and the estimated plasma half-life generally ranged from 20-24 hours. Consistent with increases in exposure, pBTK knockdown was observed. In Cohort 4 (20mg QD), all three patients showed 100% pBTK knockdown at a mean plasma Cmax exposure of 300nM (~4h post dose). Levels of CCL3 protein, a plasma biomarker for BCR pathway activation, was significantly suppressed in CLL patients.Conclusions: ARQ 531 has demonstrated a manageable safety profile to date. Diminished pBTK signaling and CCL3 protein levels have been observed in CLL patients. Additionally, preliminary anti-tumor activity has been observed at doses of ARQ 531 that were not predicted to completely inhibit BTK. Updated safety, PK, biomarker and anti-tumor activity data will be presented at the meeting. DisclosuresFlinn:Gilead: Research Funding; Infinity: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Kite: Research Funding; Verastem: Consultancy, Research Funding; Pharmacyclics: Research Funding; Novartis: Research Funding; Curis: Research Funding; Seattle Genetics: Research Funding; Merck: Research Funding; Janssen: Research Funding; ArQule: Research Funding; Calithera: Research Funding; Genentech: Research Funding; Forma: Research Funding; Agios: Research Funding; Constellation: Research Funding; Trillium: Research Funding; Verastem: Research Funding; Portola: Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding; Forty Seven: Research Funding; BeiGene: Research Funding. Savage:ArQule, Inc.: Employment. Eathiraj:ArQule, Inc.: Employment. Tith:ArQule, Inc.: Employment.

Targeting B cell receptor signalling in cancer: preclinical and clinical advances.

B cell receptor (BCR) signalling is crucial for normal B cell development and adaptive immunity. BCR signalling also supports the survival and growth of malignant B cells in patients with B cell leukaemias or lymphomas. The mechanism of BCR pathway activation in these diseases includes continuous BCR stimulation by microbial antigens or autoantigens present in the tissue microenvironment, activating mutations within the BCR complex or downstream signalling components and ligand-independent tonic BCR signalling. The most established agents targeting BCR signalling are Bruton tyrosine kinase (BTK) inhibitors and PI3K isoform-specific inhibitors, and their introduction into the clinic is rapidly changing how B cell malignancies are treated. B cells and BCR-related kinases, such as BTK, also play a role in the microenvironment of solid tumours, such as squamous cell carcinoma and pancreatic cancer, and therefore targeting B cells or BCR-related kinases may have anticancer activity beyond B cell malignancies.

CCL3 and CCL4 are biomarkers for B cell receptor pathway activation and prognostic serum markers in diffuse large B cell lymphoma.

B cell receptor (BCR) signalling is an important pathway in diffuse large B cell lymphoma (DLBCL). In response to BCR triggering, normal and malignant B cells secrete the chemokines CCL3 and CCL4 to attract accessory cells to the tissue microenvironment. We measured CCL3 and CCL4 serum concentrations in 102 patients with newly diagnosed DLBCL by enzyme-linked immunosorbent assay, investigated their prognostic impact and validated our findings in an independent cohort of 51 patient samples. We also tested CCL3 and CCL4 secretion by DLBCL cells, and the influence of BTK inhibitors on the secretion of these chemokines. High CCL3 (≥40 pg/ml) serum concentrations correlated with higher international prognostic index, lactate dehydrogenase and β2 microglobulin, as did CCL4 (≥180 pg/ml) with advanced Ann Arbor stages. High CCL3 levels correlated with significantly shorter progression-free and overall survival. The in vitro studies demonstrated that activated B cell-like, but not germinal centre B cell-like DLBCL cells, secrete high levels of CCL3 and CCL4 after BCR triggering, which was exquisitely sensitive to BCR pathway inhibition. These findings support CCL3 and CCL4 protein concentrations as biomarkers for BCR pathway activation and prognosis in DLBCL.

Abstract 5317: Preclinical evaluation of IQS019, a novel BCR kinase inhibitor, in in vitro and in vivo models of non-Hodgkin lymphoma

Abstract Constitutive activation of B-cell receptor (BCR) signaling is a hallmark of several B-cell non-Hodgkin lymphoma (B-NHL), including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL). Despite the promising clinical activity of the first BCR kinase inhibitors in some of these entities, the design of new compounds is warranted to improve the survival of B-NHL patients who are poorly responsive, and/or who eventually develop resistance to current BCR-targeting therapies. In this context, we recently described the synthesis of IQS019, a 4-aminopyrido[2,3-d]pyrimidine with improved inhibition pattern against the three BCR-related kinases Lyn, Syk and Btk, when compared to reference BCR kinase inhibitors. Here, by assessing the activity of IQS019 in a panel of 21 human cell lines and 17 primary samples representative of the main B-NHL subtypes, we show that doses of IQS019 in the micromolar range allowed to a rapid and dose-dependent de-phosphorylation of both constitutive and Ig-activated Syk, Lyn and Btk in CLL, MCL, FL and DLBCL cell lines and CLL primary cultures. IQS019 treatment impairs cell proliferation and CXCL12-dependent cell migration, and leads to the induction of caspase-dependent apoptosis. Of interest, in CLL primary cells, these effects were independently of IGVH mutational status. In xenotransplant mouse models of MCL and FL, daily dosing of 2 mg/kg IQS019 for two weeks demonstrated to be safe for the animals and allowed to a 50-60% reduction in tumor outgrowth and glucose uptake, together with a 52% decrease in cell infiltration into the spleen (p &amp;lt; 0.05). These effects were accompanied by a significant (p &amp;lt; 0.05) downregulation of p-Syk, p-Lyn and p-Btk, and consequent mitotic arrest and induction of apoptosis in human malignant B cells. Altogether, these results define the BCR kinase inhibitor IQS019 as a potential candidate in antitumor therapy against a variety of B-NHL subtypes with aberrant activation of BCR pathway. Citation Format: Patricia Balsas, Jocabed Roldan, Laura Jimenez, Vanina Rodriguez, Raimon Puig de la Bellacasa, Jordi Teixido, Alba Matas-Cespedes, Alexandra Moros, Antonio Martinez, Elias Campo, Jose I Borrell, Patricia Perez-Galan, Dolors Colomer, Gael Roue. Preclinical evaluation of IQS019, a novel BCR kinase inhibitor, in in vitro and in vivo models of non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5317. doi:10.1158/1538-7445.AM2015-5317

Racial differences in B cell receptor signaling pathway activation

BackgroundSingle-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity.MethodsThe present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males].ResultsAnalysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies.ConclusionsSCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune cell signaling responses may be one critical component for elucidation of differences in immune-mediated disease prevalence and treatment responses.

Single Cell Network Profiling (SCNP) Reveals Race-Associated Differences in B Cell Receptor Signaling Pathway Activation

Abstract 1125Race-related differences have been documented in the incidence of autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis, in the clinical response to immunotherapies [such as IFNα (in HCV infections) and belimumab (in systemic lupus erythematosus)] and to hematopoietic stem cell transplantation. However, the basis for such race-associated differences remains poorly understood. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based approach that simultaneously measures intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of peripheral blood mononuclear cells (PBMCs) from 60 healthy donors identified a race-associated difference in αIgD induced levels of pS6 and pAkt in B cells. The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors.35 BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured by SCNP in PBMCs from 10 healthy donors [5 African Americans (36–51 yrs), 5 European Americans (36–56 yrs), all males]. Cryopreserved PBMCs were thawed, modulated at 37°C in 96-well plates, fixed and permeabilized. Permeabilized cells were stained with fluorochrome-conjugated antibodies that recognize extracellular surface markers and intracellular signaling molecules. The levels of 7 phospho-proteins [pLck (Y505), pSyk (Y352), pAkt (S473), pS6 (S235/S236), p-p38 (T180/Y182), pErk (T202/Y204), and pNFκB (S529)] were measured in CD20+ B cells at 0, 5, 15, 30, and 60 mins post αIgD exposure. CD20 and IgD surface markers were used to determine the frequency of IgD+ B cells.Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower αIgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and Lck as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NFκB pathway (NFκB) (see example for αIgD induced pS6 levels in Fig. 1A). Overall, 4 (pSyk, pS6, pAkt, and pErk) of the 7 BCR pathway components tested (averaged over all timepoints for each donor) showed statistically significant differences in αIgD induced activation levels between racial groups (p=0.016, Wilcoxon test). Analysis of the frequency of IgD+ B cells showed that PBMCs from African Americans had a lower frequency of IgD+ B cells than PBMCs from European Americans [(p=0.016, Wilcoxon test), Fig. 1B], and that the frequency of IgD+ B cells had a strong positive correlation with BCR pathway activation (i.e. Pearson r>0.6 for most BCR signaling nodes). While race-associated differences in the frequency of IgD+ B cells were detected, the levels of IgD expression (as measured by the median fluorescence intensity) in the IgD+ B cell subpopulation did not differ between the races (p=0.286). Thus, the race-related difference in BCR pathway activation is attributable, at least in part, to a race-associated difference in IgD+ B cell frequencies. [Display omitted] In conclusion, SCNP analysis allowed for the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Further characterization of racial functional differences in additional immune signaling pathways using this assay in samples from both healthy and diseased individuals may be critical for elucidating the basis for race-related differences in immune-mediated disease prevalence and treatment responses. Disclosures:Longo:Nodality: Employment, Equity Ownership. Louie:Nodality: Employment, Equity Ownership. Mathi:Nodality: Employment. Hawtin:Nodality: Employment, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership.