scholarly journals Rare t(X;14)(q28;q32) translocation reveals link between MTCP1 and chronic lymphocytic leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Janek S. Walker ◽  
Zachary A. Hing ◽  
Steven Sher ◽  
James Cronin ◽  
Katie Williams ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Compartment ◽  
Pathogenic Genes ◽  
Immunoglobulin Locus ◽  
Balanced Translocations

AbstractRare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

Abstract The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

scholarly journals Exploring the Role of the Recurrent Exportin 1 (XPO1/CRM1) Mutations E571G and E571K in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 972-972
Author(s):  
Zachary A. Hing ◽  
James S. Blachly ◽  
Virginia M. Goettl ◽  
Guramrit Singh ◽  
John C. Byrd ◽  
...  
Keyword(s):  
Overall Survival ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Transgenic Mice ◽  
B Cell ◽  
Heavy Chain ◽  
Transgenic Animals ◽  
Lymphocytic Leukemia ◽  
Cell Compartment

Abstract Background Exportin 1 (XPO1/CRM1) is a critical component of the nuclear to cytoplasmic export machinery. As such, XPO1 shuttles tumor suppressor proteins (e.g. p53, IkB) out of the nucleus thereby preventing their anti-apoptotic function and contributing to enhanced proliferation. XPO1 also transports a subset of mRNAs (e.g. c-fos, IFN-alpha) by virtue of 3'-UTR AU-rich elements. XPO1 is upregulated in most hematologic malignancies, including CLL, where elevated XPO1 expression correlates with poor prognosis and resistance to therapy. To date, the role of nuclear export in CLL is poorly understood. Interestingly, recurrent mutations at a highly conserved residue (E571G; E571K) in the cargo binding pocket of XPO1 have been described in chronic lymphocytic leukemia (CLL) and are associated with worse overall survival. We hypothesized that the E571G/K mutation confers novel cargo-binding abilities to XPO1 and may contribute to the pathogenesis of CLL. Methods To interrogate this question in an unbiased, transcriptome-wide manner we developed CLL cell lines expressing mutant XPO1 and a novel RNA immunoprecipitation procedure using RNase I digestion followed by RNA-seq to capture direct interaction sites or 'footprints'. Reads were mapped to the transcriptome and peaks (regions where RNA footprints are enriched) were identified. We examined footprints for enriched sequence motifs using the MEME suite. Mass-array screening identified a cohort of CLL patients bearing the E571G/K mutation for validation purposes. The XPO1 transgenic mice were generated via pronuclear injection of linearized plasmid containing human cDNA corresponding to XPO1WT, XPO1E571G, or XPO1E571K under the heavy chain promoter/enhancer. Results We identified a transcriptome-wide pool of >100 XPO1-interacting RNAs in CLL cells and identified RNA footprints of wild-type XPO1 contributing to the first XPO1 RNA profile of a cell line model of CLL. The majority of bound RNA species associated with XPO1 are ribosomal, as expected; however, we identified multiple additional classes of RNAs such as miRs, mRNA, long-non coding RNAs, and many un-annotated RNAs. These data are consistent with additional preliminary data we have generated indicating that XPO1 may regulate mRNAs important in CLL development. We are currently searching for structural motifs. Validation experiments using patient-derived CLL cells with and without XPO1 mutations are ongoing. To further assess the independent transforming role of XPO1 dysfunction in B-cells, we generated transgenic mice overexpressing XPO1WT, XPO1E571G, or XPO1E571K under the heavy chain promoter/enhancer in the B cell compartment of C57BL/6 mice. Overexpression of XPO1 at the protein level in B cells was confirmed in multiple transgenic founder lines. Preliminary studies were performed to characterize the XPO1 transgenic animals for defects or alterations in major stages of B cell maturation. We analyzed the pro-B (IgM-CD43+), pre-B (IgM-CD43-), and immature (IgM+CD43-) B cell stages of development from the bone marrow of XPO1 transgenic animals and littermate controls. We also examined immature (IgM+IgD-) and mature (IgM+IgD+) B cells isolated from the spleen of these animals. In this preliminary analysis no significant differences in the B cell compartment were observed between transgenic and non-transgenic animals. Functional studies of these B-cells are ongoing. A cohort of animals from each founder line is being followed for evidence of B-cell expansion and overall survival analysis. In addition, the XPO1 transgenic mice were crossed with the Eµ-TCL1 mice to establish the XPO1WT/E571K/G/TCL1 double transgenic mouse model whose disease is currently being monitored. Conclusions XPO1 is a recurring, high variant allele mutation that occurs in a small subset of CLL patients. XPO1 transports select mRNA and also novel RNA species whose function in CLL have yet to be characterized, but points to a novel role of this oncoprotein in the pathogenesis of this disease. Disclosures No relevant conflicts of interest to declare.

Serum BAFF (B-Cell Activating Factor of the TNF Family) Compared with Immunoglobulin Heavy-Chain Mutation Status, ZAP-70 and CD38 as a Predictor of Time to First Treatment in Early B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3093-3093
Author(s):  
Stefano Molica ◽  
Gaetano Vitelli ◽  
Giovanna Cutrona ◽  
Giovanna Digiesi ◽  
Rosanna Mirabelli ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
B Cell Activating Factor ◽  
Mutational Status ◽  
Time To First Treatment ◽  
Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently patients with Rai stage 0 (P=0.008) and mutated IgVH (P=0.03). In contrast, peripheral blood lymphocytosis (P=0.06), serum β2-m (P=0.159), LDH (P=0.333) and percentage of ZAP-70-positive (P=0.242) or CD38-positive B-CLL cells (P=0.142) did not reflect circulating levels of BAFF. The relationship among various bio-pathological parameters, analyzed by the multiple correspondence analysis (MCA), showed two different clinico-biological profiles. The first, characterized by higher BAFF serum levels (i.e., > 336 ng/mL), presence of mutation in the IgVH, low percentage of CD38-positive B-CLL cells (< 30%) and low LDH was associated with a stable pattern of disease generally not requiring therapy. The second, defined by lower BAFF levels, absence of mutation in the IgVH, high percentage of CD38- positive B-CLL cells and high LDH was associated with a more progressive pattern of disease and a shorter TFT. After a median follow-up time of 35 months (range, 2–120 months) 26 (37.6%) out of 69 patients experienced a need for chemotherapy. Kaplan-Meier estimates of patientsTFT, plotted after searching the best cut-off for BAFF (i.e., 336 ng/mL), demonstrated that low BAFF concentration was associated with a shorter TFT (median TFT 36 months) while median was not reached by patients with BAFF levels higher than 336 ng/mL (P<0.0001). Along with lower serum levels of BAFF (Hazard Ratio [HR], 0.19; P<0.0001), the univariate Cox proportional hazard model identified absence of mutation in IgVH (HR, 0.17; P<0.0001), CD38-positivity (HR, 3.32; P=0.01) and lower platelet count (HR, 0.19; P=0.03) as predictor of shorter TFT. Finally, in multivariate analysis only mutational status of IgVH (HR, 0.25; P=0.007) and serum concentration of BAFF (HR, 034; P=0.04) affected significantly TFT. Our results indicate that in early B-cell CLL clinico-biological profile including among other parameters BAFF may provide a useful insight into the complex interrelationship of prognostic variables and semplify their interpretation. The possible presence of BAFF isoform in B-CLL could peraphs account for the unexpected correlation between low soluble BAFF levels and poor clinical outcome in patients with early disease.

Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2337-2337
Author(s):  
Lesley-Ann Sutton ◽  
Efterpi Kostareli ◽  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Athanasios Tsaftaris ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
B Cell Receptors ◽  
Valuable Insight ◽  
Cell Receptors ◽  
Mutational Status ◽  
The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia with Stereotyped IGHV4-59/IGKV3-20 B Cell Receptors: Another Manifestation of Hepatitis C Virus-Associated B Cell Lymphoproliferation?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2331-2331
Author(s):  
Efterpi Kostareli ◽  
Agnieszka Janus ◽  
Maria Gounari ◽  
Chrysoula Belessi ◽  
Sarka Pospisilova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
B Cell Lymphoma ◽  
Molecular Techniques ◽  
Lymphocytic Leukemia ◽  
B Cell Receptors ◽  
Cell Receptors

Abstract Abstract 2331 Poster Board II-308 The hepatitis C virus (HCV) has been implicated in the development of B-cell lymphoproliferative disorders, including type II mixed cryoglobulinemia (MC-II) and B-cell lymphoma. MC-II is characterized by the presence of monoclonal IgM autoantibodies with rheumatoid factor (RF) activity. The monoclonal IgMs typically form immune complexes by binding polyclonal IgGs that exhibit anti-HCV reactivity. In a series of 6,196 patients affected by chronic lymphocytic leukemia (CLL), we have identified a subset of 12 cases sharing stereotyped mutated IGHV4-59/IGKV3-20 B cell receptors (BCRs) of the MD isotype (subset #13). Comparison of subset #13 heavy chain sequences to a comprehensive dataset of relevant public-database sequences revealed identical gene usage and remarkable junctional homology with the Ig sequence GenBank/U85234, the heavy chain of a RF detected in a healthy donor, as well as the sequence GenBank/AF303916, the clonotypic heavy chain from a CLL case with a history of HCV-associated MC-II. In addition, the light chain IGKV3-20/IGKJ1 stereotyped rearrangements in subset #13 were closely similar if not identical to the rearrangements expressed by clonally expanded IgM+κ+CD27+ B cells in HCV-associated MC-II. For both heavy and light chains, sequence similarities extended beyond junctional regions to shared, “stereotyped” somatic hypermutations across the entire IGHV and IGKV domain, respectively. We established viable and antibody-secreting heterohybridomas from the leukemic cells of a subset #13 case and confirmed the identity of the produced soluble antibody to the IG expressed by the CLL clone. ELISA tests against various antigens revealed that the soluble stereotyped IGHV4-59/IGKV3-20 antibody exhibited RF activity in vitro, while it was not reactive against HCV antigens. In conclusion, the present study for the first time provides evidence for the potential implication of HCV in the pathogenesis of at least a subset of CLL cases with distinctive stereotyped BCRs. The elucidation of the underlying immune mechanisms may pave the way for tailored anti-viral/anti-leukemic therapy for selected cohorts of patients that can be easily identified by molecular techniques during the diagnostic work-up. Disclosures: No relevant conflicts of interest to declare.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Concomitant, T-Independent TLR9-Mediated and BCR-Mediated Activation Provides Signals For Optimal Telomerase Induction In Chronic Lymphocytic Leukemia Cells Regardless Of IGHV Mutation Status

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4142-4142
Author(s):  
Rajendra N Damle ◽  
Sonal Temburni ◽  
Ryon M. Andersen ◽  
Jacqueline C. Barrientos ◽  
Jonathan E. Kolitz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Telomerase Activity ◽  
Lymphocytic Leukemia ◽  
Tlr9 Activation ◽  
Cells Cultured ◽  
Stimulation Of

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones. B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied. Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures: No relevant conflicts of interest to declare.

Mevalonate pathway inhibitors in the treatment of B-cell chronic lymphocytic leukemia.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 6561-6561
Author(s):  
Ravi Kiran Bobba ◽  
Indira Benakanakere ◽  
Smitha Bearelly ◽  
Monica Arya ◽  
Richard Sleigtholm ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Combination Treatment ◽  
Mevalonate Pathway ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Cell Chronic Lymphocytic Leukemia

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

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