ALBERT

Description: Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of tumor cell-microenvironment interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its specific ligands. In this context, VLA-4 needs to be maintained in an activated state by a continuous stream of inside-out signals from the BCR, which can be triggered by canonical antigens as well as in an autonomous antigen-independent manner, a BCR-mediated signaling recently emphasized to specifically occur in CLL cells. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this still not described feature with the presence of signals from the BCR continuously activating VLA-4. Methods. Expression of the integrin alpha (CD49c, CD49d, CD49e) and beta1 (CD29) chains was analyzed by flow cytometry. The VLA-4 activation state was investigated by flow cytometry using the conformational sensitive anti-CD29 monoclonal antibody HUTS21, employed in conjunction with sources of VLA-4 ligands: either autologous plasma-containing fibronectin (FN) and soluble (s) VCAM-1 or increasing concentrations of exogenously added LDV-containing peptides as a VLA-4 specific ligand. In detail: i) HUTS21 expression was analyzed using whole blood (WB) samples from 727 CLL patients. Negative controls were obtained after plasma depletion through washing of WB samples; ii) the VLA-4 activation state expressed as receptor occupancy (RO) (J Biol Chem, 284,14337, 2009) was analyzed in sequential (t=0, day14, day 30) frozen/thawed samples from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=16) and from a clinical trial (NCT02827617, n=14). BCR engagement was performed using goat F(ab′)2 anti-human IgM. ELISA assays were used to quantify FN and sVCAM-1 in plasma samples. Results. According to the 30% cutoff, CD49d was expressed in 351/727 (48%), while CD29 was expressed in 676/727 (93%), and a strong correlation between CD49d and CD29 was observed (rho=0.7, p20% (arbitrary cut-off) HUTS21 expression. Notably, HUTS21 positive expression was not circumscribed to VLA-4 expressing CLL, being also detected in 64/375 (17%) CD49d-, CD49c+ and/or CD49e+ CLL, thus envisioning a role of other integrins (CD49c and CD49e) in CLL cell interactions in tissues sites. HUTS21 expression did not correlate with a definite cytogenetic group, IGHV mutational status or with a specific IGHV family and stereotype. Depletion of the plasma component from the WB samples before HUTS21 staining significantly reduced the proportion of HUTS21 positive cells compared with those measured in WB samples (p=0.001, n=11), suggesting the need of plasma-borne ligands for HUTS21 epitope exposure. Consistently, both FN (mean 350 μg/ml) and sVCAM-1 (mean 4.8 μg/ml) were detected in the plasma of CLL cases, irrespective to HUTS21 positivity. According to these observations, variable constitutive VLA-4 activation states were observed in CLL cells collected at the pre-treatment stage from patients undergoing IB treatment (VLA-4 RO ranging from 0.22 to 0.40); these values significantly decreased in CLL cells collected at day 14 (p=0.03) and day 30 (p=0.02) on IB, suggesting an IB-dependent impairment of antigen-independent (autonomous) BCR signals. The variability of the constitutive VLA-4 activation levels observed at pre-treatment was paralleled by variable VLA-4 activation levels upon BCR triggering (VLA-4 RO ranging from 0.56 to 0.65). Of note, an inverse correlation between the VLA-4 constitutive level and the extent of anti-IgM induced-VLA-4 activation was found (n=19, rho=-0.5, p=0.0093), implying a competition between antigen-independent and antigen-driven BCR signalling. Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a subset of CLL which might be connected to continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling, which is currently under evaluation. Disclosures Zaja: Takeda: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria.

Description: Introduction. Ibrutinib, a small molecule inhibitor of Bruton's tyrosine kinase (BTK), has proven to be an efficient treatment for chronic lymphocytic leukemia (CLL). A distinct characteristic of ibrutinib therapy is transient lymphocytosis. Recently, we have demonstrated that CLL patients with high levels of CD49d show reduced lymphocytosis and inferior nodal response under ibrutinib due to residual activity of BCR-induced inside-out activation of the CD49d/CD29 integrin VLA-4 (Tissino E et al. J Exp Med. 2018;215(2):681-697). Here, we used Tcl1 transgenic (tg) mice as a model to further validate the observation of VLA-4 activation under ibrutinib and to study involved signaling pathways and the effect of VLA-4 inhibition in vivo. Methods. Surface receptor expression analysis of various receptors was performed by flow cytometry. The phosphorylation of signaling molecules was measured by phosflow and western blotting. VLA-4 affinity state was determined by a real-time kinetic assay described in Chigaev A et al. J Biol Chem. 2001;276(52):48670-8. To analyze the distribution of individual VLA-4 molecules on the cell surface, immunofluorescence approaches and superresolution microscopy (STORM, Abbelight) were employed. Mouse treatment studies were performed upon transplantation of TCL1-tg splenocytes to wild-type C57BL/6J mice using the small molecule VLA-4 inhibitor firategrast in drinking water. Tumor infiltration of different organs was measured by flow cytometry. Results. Analyzing the surface expression of CD49d and other homing receptors, we found that TCL1-tg mice correspond with the CD49d-high CLL cohort. We found that both CLL cells from TCL1-tg mice and human CD49d-high CLL show similar CD49d expression levels as the corresponding healthy B cells (human: N = 116 CD49d-high CLL and 32 healthy donor, P = 0.8717; mouse: N = 12 per group, P = 0.6845). Next, we analyzed the impact of BCR pathway inhibitors on the phosphorylation of signaling molecules involved in the BCR pathway after activation by anti-IgM (aIgM) in TCL1-tg leukemic cells. Ibrutinib and idelalisib showed specific patterns of inhibition of BTK and PI3K, respectively. The combination of ibrutinib and idelalisib proved to be the most efficient in reducing the phosphorylation of BTK, SYK, ERK1/2 and Akt upon IgM activation, compared to the phosphorylation of stimulated cells without inhibition (N = 6, P = 0.0003, 0.0305, 0.0039, 0.0019, respectively). IgM stimulation induced VLA-4 high affinity as well as a reorganization of VLA-4 molecules on the cell surface, forming areas of high VLA-4 density. BCR-induced inside-out activation of VLA-4 remained functional upon treatment with ibrutinib (N = 5, cnt vs aIgM P = 0.0017, cnt vs ibrutinib+aIgM P = 0.0499), while idelalisib reduced VLA-4 activation more effectively (N = 5, cnt vs aIgM P = 0.0014, cnt vs idelalisib+aIgM P = 0.0803), suggesting a pivotal role of PI3K in the transmission of the exogenous antigen signal to the integrin. Finally, to analyze the potential of VLA-4 blockage in a tumor setting similar to VLA-4-high CLL patients, we treated wild-type C57BL/6J mice (N = 6 mice per group), which were transplanted with TCL1-tg splenocytes, with the CD49d inhibitor firategrast. This treatment reduced the tumor load in spleen and bone marrow. Conclusion. We found that the TCL1-tg mouse model is adequate to study the activity of the BCR-VLA-4 axis in CLL. Using this model, we show that a) BCR stimulation induces both, an increase in VLA-4 affinity as well as avidity (clustering), b) that PI3K is an essential transmitter between BCR and VLA-4, and c) that VLA-4 inhibition alters tumor infiltration patterns in vivo. Synergies of VLA-4 blockage with established therapy options as a possible way of reducing microenvironment-induced resistance development are currently been investigated. Disclosures Egle: Celgene: Honoraria, Other: Advisory board and Travel support. Greil:Eisai: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Sandoz: Honoraria; Genentech: Honoraria, Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Honoraria; Janssen-Cilag: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; GSK: Research Funding; Boehringer Ingelheim: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Celgene: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; MSD: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Ratiopharm: Research Funding; Gilead: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding.

Description: Emerging evidence suggests that crosstalk between hematologic tumor cells and the tumor microenvironment contributes to leukemia and lymphoma cell migration, survival, and proliferation. The supportive tumor cell-microenvironment interactions and the resulting cellular processes require adaptations and modulations of the cytoskeleton. The Rac subfamily of the Rho family GTPases includes key regulators of the cytoskeleton, with essential functions in both normal and transformed leukocytes. Rac proteins function downstream of receptor tyrosine kinases, chemokine receptors, and integrins, orchestrating a multitude of signals arising from the microenvironment. As such, it is not surprising that deregulation of Rac expression and activation plays a role in the development and progression of hematological malignancies. In this review, we will give an overview of the specific contribution of the deregulation of Rac GTPases in hematologic malignancies.

Description: Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma–microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

Description: Abstract 4896 Introduction: Chemokines are known to play an important role in the migration and survival of B-CLL cells. The non-signalling chemokine receptors, including DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner, and to variable degrees on B-CLL cells. We have recently shown that it competitively binds CCL19 and that this binding is not followed by classical chemokine responses. CCL19 and its signalling receptor CCR7 are centrally involved in B cell localisation and maturation within the secondary lymphoid tissues. CCR7 is also highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21 which have been shown to be present at higher concentrations in serum of patients with lymphadenopathia compared to patients without. In this study we investigate the influence of CRAM on the CCL19 dependent responses of B-CLL cells and potential correlations to clinical data with a specific focus on lymphadenopathia. Results: We demonstrate that B cells from patients with B-CLL present high, but variable degrees of CCR7 and CRAM expression. Patients with compared to patients without lymphadenopathia show a higher CRAM expression level whereas the CCR7 expression is not significantly different. In single samples showing extremly high CRAM expression the migration towards CCL19 is reduced compared to patients with lower CRAM expression. These observations confirm results in the B-CLL cell line MEC-1 showing increased migration toward CCL19 when CRAM expression is reduced using CRAM-siRNA. On the other hand, CRAM seems to be a chemokine presenter as we can show that it does not degrade its chemokine ligand but presents it on the surface of polarised cell layers. Thus, we assume that CRAM plays a role for cell migration, possibly transmigration and cell localisation within lymph nodes of B-CLL cells. Conclusions: We show that CRAM can act as an integrator of different recruitment and activation factors. It is associated to CCR7 driven recruitment of B cells by regulating CCL19 availability. Expression of CRAM differs in B cell malignancies for which CCL19 and CCL21 have already been shown to be implicated in lymphadenopathia. We therefore suggest that CRAM is an additional player in the localisation and differentiation/maturation processes of malignant B cells of B-CLL patients. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. Especially, a continuous BCR signaling, which is either induced by canonical autoantigen-dependent mechanism, or by an autonomous manner, may augment VLA-4 activation. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this feature with the presence of signals from the BCR. Methods. Constitutive VLA-4 activation was determined in flow cytometry by combining expression of CD49d and activated CD29, the latter using the conformation-sensitive anti-CD29 mAb HUTS21 (Tissino et al, J Exp Med, 2018) in whole blood (WB), as source of VLA-4 ligands, from: i) 1,044 consecutive CLL all with IGHV gene mutations available; ii) sequential samples (0, 14, 30, 90 days) from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=15) and from a clinical trial (NCT02827617, n=15). HUTS21 staining was also performed in the presence of: i) plasma depletion/replacement; ii) soluble (s) VCAM-1; iii) fibronectin (FN); iv) blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). Antigene driven BCR signaling and autonomous BCR signaling were investigated by Ca++ influx assay in a 4-hydroxy tamoxifen (4-OHT) inducible manner in murine TKO cells system as described before (Dühren-von Minden M et al, Nature, 2012). VLA-4 activation was assessed by "real-time" flow cytometry measuring the binding of the VLA-4 ligand LDV-FITC and its replacement by unlabeled LDV. The calculated Koff values indicate the VLA-4 affinity state (Koff 0.06 s−1 low affinity). BCR engagement was performed using goat F(ab′)2 anti-human IgM. Results. 1) A fraction of CD49d+ CLL shows constitutive VLA-4 activation - Out of 1,044 CLL, 534 (51%) expressed CD49d (cutoff 30%), and HUTS21 (cutoff 20%) was scored positive in 112/534 (21%) cases. HUTS21 staining was: i) impaired by depletion of plasma from WB samples (Fig.A), and reconstituted by specific plasma components (sVCAM1, FN; Fig.B); ii) impaired by pre-incubation with anti-CD49d HP2/1 blocking mAbs before addition of plasma, sVCAM-1 and FN (Fig.C). 2) Plasma levels of sVCAM-1 are dependent on the VLA-4 activation state - sVCAM-1 (n=122 plasma samples) was higher in CD49d+ vs CD49d- cases (p50% vs HUTS21

Description: Introduction The development and maturation of B cells is highly dependent on signals provided by the microenvironment of the lymphatic organs. As B cells move from one developmental stage and niche to the next, the integrin family of adhesion molecules provides important cues for their correct positioning and retention. The integrin adaptor protein Kindlin-3 (encoded by the Fermt3 gene) regulates integrin activity and function in a wide range of hematopoietic cell types. In this study, we aimed to define its precise role in the development and function of the different murine B cell subsets. Methods We crossed a Fermt3flox/flox mb1-cre mouse strain (hereforth called K3ΔB mice), harboring a B cell specific Kindlin-3 deletion. B cell subsets in the different lymphoid organs of these K3ΔB mice and control littermates were defined by multicolor flow cytometry. Adoptive transfer, microscopy and real-time flow cytometry were used to analyze the different steps of integrin activation. A co-culture system with OP9 stromal cells and BAFF was used to assess the in vitro differentiation potential of immature progenitors into the different mature B cell subsets. Transcriptional differences between follicular B cells isolated from spleens of K3ΔB- and control mice were assessed by transcriptome array. Results In vitro, we found that integrin activation on B cells was induced upon activation of the chemokine receptors CXCR4 and CXCR5 or the B cell receptor. This stimulation triggered adhesion of wild type B cells to integrin ligands under shear flow. The increase of VLA-4 integrin affinity to its ligand substrates during this process could also be calculated from real-time flow cytometrical analyses. In contrast, K3ΔB-derived B cells could not reach high affinity states of integrins and thus failed to adhere on the substrates upon stimulation, despite slight upregulation of chemokine receptors CXCR4 and CXCR5. B cell migration towards the respective chemokines also required Kindlin-3, even in an integrin ligand-free setting. In vivo, Kindlin-3 was required for homing of mature B cells to the bone marrow and to lymph nodes. When further characterizing K3ΔB mice by flow cytometry and immunohistochemistry we observed increased early B cell numbers in the bone marrow. Of note, marginal zone (MZ) B cells in the spleen were completely absent (Figure 1 A+B). We consequently assessed the potential of immature B cells to develop into B cells with high expression of CD21, a marker for MZ B cells, upon their co-culture with OP9 stromal cells in the presence of the B cell survival factor BAFF. While 18% of B cells differentiating from wild type bone marrow displayed high expression of CD21, the percentage of CD21 high cells recovered from Kindlin-3 deficient progenitors was significantly lower (~12%, Figure 1C). Pathways involved in these developmental differences were analyzed by a transcriptome array, revealing increased activity of the B cell receptor pathway in the knockout situation accompanied by higher, NFkappaB and Notch signaling. Conclusion/Outlook Whereas our results highlight the importance of Kindlin-3 dependent, integrin mediated cell retention and migration during B cell development they also indicate that Kindlin-3 functions in an integrin-independent manner when regulating cell motility and transcription. The complete lack of MZ B cells in the absence of Kindlin-3 is thus most likely a combination of defective retention in the MZ area and transcriptional alterations favoring the development of transitional B cells into follicular- rather than MZ B cells. Figure 1 : B-cell specific Kindlin-3 knockout leads to loss of splenic marginal zone B cells. The percentage of MZ B-cells among total splenic B cells was determined by flow cytometry in K3ΔB mice and wild type (wt) littermates (A). Immunohistochemistry staining of CD19 showed a loss of loosely packed marginal zone B cells (yellow arrows) in the absence of Kindlin-3 (B). B cells were enriched from the bone marrow of K3ΔB mice and wt littermates and cultured on a confluent layer of OP9 cells in the presence of 200 ng/ml BAFF for 72 h. Development of CD21 high/CD23 low B cells was then determined by flow cytometry (C). Figure Disclosures Greil: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Astra zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Daiichi Sankyo, Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS/celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; MSD Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding.