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.

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)

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 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

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.

Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Jan A. Burger
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Disease Progression ◽  
Drug Testing ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

scholarly journals Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

2018 ◽  
Vol 215 (2) ◽  
pp. 681-697 ◽  
Author(s):  
Erika Tissino ◽  
Dania Benedetti ◽  
Sarah E.M. Herman ◽  
Elisa ten Hacken ◽  
Inhye E. Ahn ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Progression Free Survival ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Independent Manner ◽  
Btk Inhibitor

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 356-356 ◽  
Author(s):  
Yair Herishanu ◽  
Berengere Vire ◽  
Delong Liu ◽  
Federica Gibellini ◽  
Gerald E Marti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Expression Profiling ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Activation Markers ◽  
Bcr Signaling

Abstract The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity &gt;98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p&lt;0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p&lt;0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change &gt;2, FDR &lt;0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2889-2889
Author(s):  
Tom Butler ◽  
Alexander Montoya ◽  
Andrew James Clear ◽  
Rita Coutinho ◽  
David C Taussig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

scholarly journals High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia

Blood ◽  
2009 ◽  
Vol 114 (21) ◽  
pp. 4675-4686 ◽  
Author(s):  
Marco Herling ◽  
Kaushali A. Patel ◽  
Nicole Weit ◽  
Nils Lilienthal ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Stage ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Advanced Clinical Stage ◽  
Therapy Regimen ◽  
Cell Counts

Abstract Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5+ B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70+ tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.

Sign in / Sign up

Export Citation Format

Share Document