The Bruton Tyrosine Kinase Inhibitor, PCI-32765, Inhibits Activation and Proliferation of Human Chronic Lymphocytic Leukemia Cells in the NSG Xenograph Mouse Model of the Tissue Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 596-596 ◽  
Author(s):  
Sarah E. M. Herman ◽  
Xiameng Sun ◽  
Joseph J. Buggy ◽  
Georg Aue ◽  
Patricia Perez-Galan ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Cell Receptor ◽  
Gene Signature ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Tissue Microenvironment

Abstract Abstract 596FN2 PCI-32765, a specific inhibitor of Bruton's tyrosine kinase (Btk), can disrupt several signaling pathways involved in tumor microenvironment interactions. In vitro, PCI-32765 has been demonstrated to induce apoptosis, to varying degrees, in tumor cells and prevent CpG-ODN induced proliferation of cultured chronic lymphocytic leukemia (CLL) cells (Herman et al, Blood 2011). PCI-32765 has been shown to be well tolerated in CLL with preliminary clinical trial data showing that >85% (34/39) of patients remained on therapy at a median follow-up of four months. In addition, a significant shrinkage of lymph nodes has been observed in the majority of patients displaying lymphadenopathy. As with other B-cell receptor (BCR) directed therapies, PCI-32765 results in an initial increase in the absolute lymphocyte count. These observations are not explainable by the available in vitro data, demonstrating the need for in vivo investigation. In order to study the effect of PCI-32765 in vivo we chose to use the recently established NOD scid gamma null (NSG) - human CLL xenograft model with some modifications (Bagnara et al., Blood 2011). NSG mice were conditioned with 25 mg/kg busulfan 24 hours before injection of 1 × 108 CLL peripheral blood mononuclear cells previously labeled with 1μM CFSE. We first demonstrated that xenografted CLL cells isolated from the mouse spleen acquire an activated phenotype and proliferate, mimicking the phenotype of CLL cells isolated from human lymph nodes (Sun et al., abstract submitted). Next we sought to use this model to investigate the effect ot PCI-32765 on CLL cell activation and proliferation. Mice received PCI-32765 or vehicle in their drinking water at 0.16 mg/ml dissolved in 1% HP-beta-CD starting at the time of busulfan treatment. Mice were bled weekly and sacrificed between 3 and 4 weeks post xenografting. We found that PCI-32765 treatment resulted in a significant reduction in proliferation (defined as CFSE low cells) compared to mice that received vehicle water; this was observed in all three biological compartments: peripheral blood (84.5% decrease, p=0.007), spleen (72.4% decrease, p=0.012) and bone marrow (92.5% decrease, p=0.049). In comparison, PCI-32765 treatment did not result in a significant reduction in T-cell proliferation in any of the compartments (p>0.4). Although peripheral blood CLL counts were comparable between treated and untreated mice, we found that there were substantially more CLL cells in the spleens of the vehicle treated mice than in those of the PCI-32765 treated mice. In contrast, no differences in T-cell number or localization were observed between treated and untreated mice. Lastly, we sought to determine whether activation of CLL cells in the microenvironment could be blocked by PCI-32765. As we have previously shown, CLL cells in the human lymph node display a gene signature indicating B-cell receptor (BCR) and NF-kB activation compared to CLL cells in the peripheral blood (Herishanu et al., Blood 2011). We used quantitative RT-PCR (pre-designed Taqman Gene Expression assays) to measure expression of representative BCR and NF-kB target genes. PCI-32765 significantly reduced expression of EGR1 (p=0.049), EGR3 (p=0.023) and GFI1 (p=0.023) (BCR signature) and CCL3 (p=0.013) and CCND2 (p=0.046) (NF-kB signature) compared to vehicle treated mice. In addition, we also observed decreases in the proliferation gene signature (CDT1, PCNA and RRM2) (signature score, p=0.035) in the CLL cells from mice treated with PCI-32765; consistent with the assessed CFSE proliferation measurements. Taken together, our results show that PCI-32765 inhibits CLL activation and proliferation in the tissue microenvironment in vivo without affecting T-cell proliferation. These results demonstrate that targeting Btk is sufficient to block key interactions between tumor cells and the microenvironment and thus warrants the use of PCI-32765 as a targeted agent in CLL. Disclosures: Buggy: Pharmacyclics, Inc.: Employment.

scholarly journals Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

2001 ◽  
Vol 193 (11) ◽  
pp. 1295-1302 ◽  
Author(s):  
Megan K. Levings ◽  
Romina Sangregorio ◽  
Maria-Grazia Roncarolo
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Major Advance ◽  
Loss Of Function

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
Author(s):  
Freda K. Stevenson ◽  
Federico Caligaris-Cappio
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Of Origin ◽  
Regulatory B Cell ◽  
V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

scholarly journals Changes in the peripheral blood T-Cell receptor V beta repertoire in vivo and in vitro during shigellosis.

1996 ◽  
Vol 64 (4) ◽  
pp. 1391-1399 ◽  
Author(s):  
D Islam ◽  
B Wretlind ◽  
A A Lindberg ◽  
B Christensson
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Cell Receptor

scholarly journals In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia

Haematologica ◽  
2017 ◽  
Vol 103 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Eve M. Coulter ◽  
Andrea Pepper ◽  
Silvia Mele ◽  
Najeem’deen Folarin ◽  
William Townsend ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Internalization

The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 832-832
Author(s):  
Matthew D Blunt ◽  
Matthew J Carter ◽  
Marta Larrayoz ◽  
Maria Montserrat Aguilar ◽  
Sarah Murphy ◽  
...  
Keyword(s):  
Mtor Inhibitor ◽  
Annexin V ◽  
Leukemic Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Blood Cell Count ◽  
Mtor Inhibition

Abstract Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL. Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments. To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting. Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients. Disclosures No relevant conflicts of interest to declare.

High-Level Expression of the T Cell Chemokines CCL3 and CCL4 by Chronic Lymphocytic Leukemia B Cells in Nurselike Cell Co-Cultures and in Response to BCR Stimulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 342-342
Author(s):  
Jan A. Burger ◽  
Matthias Niedermeier ◽  
Andrea Bürkle ◽  
Elena Hartmann ◽  
William G. Wierda ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Serum Levels ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
High Level Expression ◽  
High Level

Abstract Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival. Figure Figure

B Cell Receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter Transformation

Blood ◽  
2021 ◽  
Author(s):  
Supriya Chakraborty ◽  
Claudio Martines ◽  
Fabiola Porro ◽  
Ilaria Fortunati ◽  
Alice Bonato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
B Cell ◽  
Critical Role ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Richter Syndrome

B cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A and CDKN2B, which block cell cycle progression. We further show that introduction of genetic lesions that downregulate these cell cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR-dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B and TP53 frequently co-occur in Richter syndrome, and BCR stimulation of human Richter syndrome cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR inhibitor treatment and are synergistically sensitive to the combination of a BCR and CDK4/6 inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

Evaluation of vecabrutinib as a model for non-covalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia

Blood ◽  
2021 ◽  
Author(s):  
Billy Michael Chelliah Jebaraj ◽  
Annika Müller ◽  
Rashmi Priyadharshini Dheenadayalan ◽  
Sascha Endres ◽  
Philipp M. Roessner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Transfer Model ◽  
Treatment Benefit ◽  
Btk Inhibitors

Covalent Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop by a mutation in cysteine 481 of BTK (C481S), which prevents the irreversible binding of the drugs. In the present study we performed pre-clinical characterization of vecabrutinib, a next generation non-covalent BTK inhibitor, with ITK inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wildtype BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, while the naïve populations were increased. Of importance, vecabrutinib treatment significantly reduced frequency of regulatory CD4+ T-cells (Tregs) in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on activation and proliferation of isolated T-cells. Lastly, combination treatment of vecabrutinib with venetoclax was found to augment treatment efficacy, significantly improve survival and lead to favourable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, non-covalent BTK/ITK inhibitors such as vecabrutinib may be efficacious in C481S BTK mutant CLL, while preserving the T-cell immunomodulatory function of ibrutinib.

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