Prelinical Evaluation of Lyn Kinase Inhibition for the Treatment of B Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3447-3447
Author(s):  
Vamsidhar Velcheti ◽  
Li Li ◽  
Joseph Philips ◽  
Jyotsna Fuloria ◽  
Francis R Rodwig ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Combined Treatment ◽  
Chemotherapeutic Agents ◽  
Negative Regulator ◽  
Cell Leukemia ◽  
Lyn Kinase ◽  
Control Peptide ◽  
Inhibitor Peptide

Abstract Abstract 3447 Poster Board III-335 B-CLL is characterized by the accumulation of mature B lymphocytes incapable of undergoing apoptosis. Although several mechanisms have been implicated in the apoptotic defects in B-CLL cells, the signal transduction pathways underlying these defects remain unresolved. Lyn kinase is known to be a negative regulator of apoptosis, and is linked to chemo-resistance. Our preliminary study indicated that Lyn activity was 4- to 7-fold higher in primary B cells from six randomly-selected B-CLL patients than in normal B cells from healthy donors. Treatment of the B-CLL cells for 4 h with 10 μM concentration of a Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res. 2004;64:1058) resulted in 40 to 50% inhibition of Lyn kinase activity compared to negligible inhibition with control peptide. Further, treatment of the B-CLL cells for 16 to 20 h with the Lyn inhibitor peptide at 5 μM and 10 μM concentrations, decreased the cell viability by ∼25% and ∼50%, respectively, compared with no peptide or the control peptide. Fludarabine is one of the key chemotherapeutic agents for B-CLL and is known to induce cell death by apoptosis. The combined treatment with Lyn inhibitor peptide (5 μM) and fludarabine (2 μg/ml), decreased cell viability by ∼50% compared to ∼25% decrease with fludarabine or Lyn inhibitor peptide alone. In addition, the combined treatment showed ∼75% increase in caspase-3/7 activity compared to ∼25% increase with the Lyn inhibitor peptide or fludarabine alone. Because, overexpression of antiapoptotic proteins is correlated with apoptotic resistance of B-CLL cells, we examined the effect of Lyn inhibitor peptide on the changes in the expression of antiapoptotic genes, myeloid cell leukemia-1 (Mcl-1), x-linked inhibitor of apoptosis (XIAP), and B-cell leukemia/lymphoma-2 (Bcl-2). Treatment of B-CLL cells with 10 μM Lyn inhibitor peptide for 16 to 20 h resulted in more than 50% decreases in the expression of all the three antiapoptotic genes. Further, the Lyn inhibitor peptide markedly inhibited NF-κB activation (∼60%) and VEGF production (∼80%), both strongly implicated in the apoptotic resistance of B-CLL cells. Collectively, our results suggest that targeting Lyn kinase pathway with a clinically relevant Lyn kinase inhibitor may have a therapeutic potential for B-CLL. Disclosures No relevant conflicts of interest to declare.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

2009 ◽  
Vol 417 (3) ◽  
pp. 673-683 ◽  
Author(s):  
Munetoyo Toda ◽  
Risa Hisano ◽  
Hajime Yurugi ◽  
Kaoru Akita ◽  
Kouji Maruyama ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Signalling ◽  
Negative Regulator ◽  
Mammary Adenocarcinoma ◽  
Marginal Zone B Cells ◽  
Tumour Bearing

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

scholarly journals Polyclonal B-Cell Lymphocytosis With Features Resembling Hairy Cell Leukemia-Japanese Variant

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2008-2014 ◽  
Author(s):  
Takashi Machii ◽  
Mitsuhiro Yamaguchi ◽  
Ryoichi Inoue ◽  
Yukihiro Tokumine ◽  
Hirohiko Kuratsune ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hairy Cell Leukemia ◽  
Leukemia Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Variant Form ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Persistent Lymphocytosis ◽  
Biopsy Specimens

Abstract Polyclonal B lymphocytosis was found in four patients having clinical and hematologic features resembling those of hairy cell leukemia (HCL). All four patients were women between 37 and 67 years of age. Three patients had splenomegaly. Lymphadenopthy was absent or slight. Persistent lymphocytosis was seen in all the patients, and anemia and/or thrombopenia was observed in three of the patients. Abnormal lymphocytes have long microvilli and prominent membranous ruffles on their surfaces. Bone marrow aspirates and biopsy specimens showed increased numbers of abnormal lymphocytes with round nuclei and abundant pale cytoplasm. Although these findings were similar to those of HCL, studies of Ig gene rearrangements and expression showed the polyclonal proliferation of B cells. We called this new disease hairy B-cell lymphoproliferative disorder (HBLD). All four patients exhibited a polyclonal increase in serum IgG. The morphology of the cells in HBLD was more similar to that of leukemia cells of a variant form of HCL (HCL-Japanese variant) than to typical HCL cells. The surface IgG+, CD5−, CD11c+, CD22+, CD24−, CD25− phenotype and the weak tartrate-resistant acid phosphatase activity in the cells were identical to those of HCL cells of the Japanese variant. Our findings suggest that the B cells in HBLD are the nonmalignant counterpart of leukemic B cells in HCL-Japanese variant.

The Suppressor of Cytokine Signaling-3 Gene Is Overexpressed in Human De Novo Follicular Lymphomas and Promotes IL-3-Independent Proliferation of the BaF3 Pro-B Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1107-1107
Author(s):  
Jacqueline C. Barrientos ◽  
Sofya Rodov ◽  
Arthur W. Zieske ◽  
K. Gary J. Vanasse
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Myeloid Cells ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Socs3 Protein

Abstract The recent generation of mice lacking functional SOCS3 in hepatocytes, macrophages, and neutrophils reveals SOCS3 to be an essential regulator of IL-6 signaling via mediation of gp130-related cellular complexes, as well as a negative regulator of G-CSF signaling in myeloid cells. Although SOCS3 would appear to be a critical physiologic regulator of inflammatory responses, its possible role in hematologic malignancies and the underlying mechanisms which regulate its expression in B cells remain to be clearly defined. We previously showed that CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice express high levels of SOCS3 in addition to overexpression of Bcl-2. Moreover, hematopoietic cell lines transduced to stably overexpress Bcl-2 exhibited marked induction of SOCS3 compared to controls, suggesting Bcl-2-associated pathways may play a role in the induction of SOCS3. In the current study, we describe SOCS3 overexpression limited to neoplastic follicular lymphoma (FL) cells in Bcl-2-associated human de novo FL and show that overexpression of SOCS3 is capable of stimulating cytokine-independent cellular proliferation of the BaF3 pro-B cell line. We measured SOCS3 protein levels by immunohistochemistry in paraffin-embedded biopsies from twelve patients diagnosed with de novo, untreated histologic grade I or II FL which harbored t(14;18) and Bcl-2 overexpression. In 9/12 de novo FL cases examined, immunostaining with two distinct antibodies to SOCS3 revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic FL cells and co-localized with Bcl-2 primarily in the nucleus of positive cells. In contrast, SOCS3 protein was not detected by immunostaining in germinal center follicular B cells from benign hyperplastic tonsil tissue. To further evaluate the role of SOCS3 in B cell biology, the IL-3-dependent BaF3 pro-B cell line was stably transduced with either a retroviral expression construct containing a 675bp human SOCS3 cDNA (BaF3SOCS3) or with vector only control (BaF3Δ). Whereas no SOCS3 protein was detected in control cells, high level expression of SOCS3 in transduced BaF3SOCS3 cells was confirmed by Western analysis using SOCS3 anti-sera. Furthermore, Bcl-2 protein was not detected in either BaF3SOCS3 or control cell lines. 2 x 105 BaF3SOCS3, BaF3Δ, and non-transduced BaF3 cell lines were initially grown in the presence 10% fetal bovine serum (FBS) and 5% WEHI 3B cell-conditioned medium as a source of IL-3. IL-3 was then removed by washing with DMEM/10% FBS. Cell viability was then measured by recording absorbance at 490nm using incorporation of the MTS tetrazolium compound. Interestingly, BaF3SOCS3 cells overexpressing SOCS3 did not undergo apoptosis but were able to proliferate in the absence of IL-3, with percent viable cells approaching 400% at > 96 hours, which represented the final time-point measured. In contrast, BaF3Δ and non-transduced BaF3 cells underwent apoptotic cell death between 8 and 36 hours in response to IL-3 withdrawal. Thus, SOCS3 overexpression confers IL-3-independent cell proliferation to the BaF3 cell line. These data indicate that unlike its negative regulatory effect on G-CSF signaling in myeloid cells, overexpression of SOCS3 in B cells may promote B cell proliferation rather than growth suppression and may play an important role in the pathogenesis of de novo FL in humans.

CDDO and CDDO-Im Reduce Tumor Burden in a Transgenic Mouse Model of CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3477-3477
Author(s):  
Juan M. Zapata ◽  
Christina L. Kress ◽  
Marina Konopleva ◽  
Maryla Krajewska ◽  
Mark Hyer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Chemotherapeutic Agents ◽  
Tumor Burden ◽  
Leukemic Cells ◽  
Medium Size ◽  
Control Group

Abstract Transgenic mice over-expressing in B lymphocytes both Bcl-2 and a TRAF2 mutant lacking the N-terminal RING and zinc finger domains (TRAF2DN), which mimics TRAF1, develop small B cell lymphoma and leukemia that have remarkably similar characteristics to human chronic lymphocytic leukemia (CLL). TRAF2DN/Bcl-2 mice develop over time leukemia, severe splenomegaly, and lymphadenopathy, which are associated with monoclonal and oligoclonal B cell neoplasms. The lifespan of TRAF2DN/Bcl-2 mice is markedly reduced compared to Bcl-2 and TRAF2DN single transgenics or wild-type littermates. The expanded B cell population in the blood of leukemic TRAF2DN/Bcl-2 double transgenic mice is primarily comprised of small-medium size, non-cycling B220M/IgMH/IgDL/CD21L/CD23−/CD11b+/CD5+ cells that were Bcl-6 negative, consistent with a B-1 phenotype, closely resembling their human CLL counterparts. Indeed, these B cells showed comparable proliferation rates to normal B-cells, but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. We studied the effects of synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and its imidazolide derivative (CDDO-Im) on cultured B-cells from the TRAF2DN/Bcl-2 transgenic mice. Both CDDO and CDDO-Im efficiently induced apoptosis of these cells in vitro, although CDDO-Im was approximately 10-times more potent than CDDO (LD50: 0.35μM CDDO-Im vs 3.8 μM CDDO). To study the effect of CDDO and CDDO-Im in vivo, groups of TRAF2DN/Bcl-2 mice that had developed leukemia were injected i.v. with liposomes alone or liposomes containing either CDDO or CDDO-Im, at a dose of 20 mg/kg/day. Each mouse received a total of nine injections administered over a period of 22 days. The concentration of B cells in the blood of these mice was monitored daily after each injection, using a mini-FACS (Guava Technologies, Inc.). CDDO-treated mice showed a steady reduction in the number of leukemic cells in blood during the treatment and this tendency was maintained 10 days after the last treatment. In contrast, CDDO-Im treated mice showed a striking increase in the concentration of B cells in blood (B220+ events) immediately after the first inoculation. One mouse of this group died after the first injection, and 2 more mice died after 5 injections. Only 2 mice treated with CDDO-Im survived the full treatment, showing a striking reduction of leukemic cells in blood by the end of the treatment. Administration of empty liposomes had no inhibitory effect on the leukemia, and mice in this control group had massive splenomegaly (1431±323 mg; n=3) and severe disseminated lymphadenopathy. In contrast, CDDO-treated mice had less severe splenomegaly (938±234; n=4) but still had severe lymphadenopathy. CDDO-Im treated mice showed a dramatic reduction in the spleen size that was evident also in those mice that died after 5 injections (474±185 mg; n=4) and had no signs of lymphadenopathy. Although preliminary, these results indicate that in vivo administration of CDDO and CDDO-Im reduced the tumor burden in a transgenic model of CLL, and illustrate the potential of triterpenoids as single agents for the treatment of CLL.

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3665-3665
Author(s):  
Feng Guo ◽  
Peng Zhou ◽  
Liang Ma
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Vector ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Activity Assay ◽  
Lymphoma Cells ◽  
Aberrant Expression ◽  
Wide Range ◽  
P100 Processing

Abstract Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the Protein Tyrosine Phosphatase Lyp Reduces the Responsiveness of Chronic Lymphocytic Leukemia B-Cells to B-Cell Receptor Ligation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 800-800
Author(s):  
Roberto Negro ◽  
Pablo G Longo ◽  
Michela Tarnani ◽  
Stefania Gobessi ◽  
Luca Laurenti ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Signaling Molecules ◽  
Negative Regulator ◽  
Nucleotide Sequence Analysis ◽  
Aberrant Expression ◽  
Bcr Signaling

Abstract Abstract 800 CLL B cells display many features that suggest a role for antigen stimulation in the development and progression of the disease. These include the expression of stereotyped B-cell receptors (BCRs), the association between IgVH gene mutation status and prognosis, and the gene-expression profile of antigen-stimulated B cells. In addition, CLL B cells have other BCR-related features that distinguish them from normal B lymphocytes, such as lower levels of surface Ig, less efficient BCR signal transduction and increased basal activity of the proximal BCR signaling molecules Lyn and Syk. We have now investigated whether any of these features are related to aberrant expression or function of the phosphatases SHP-1, SHP-2 and Lyp (PTPN22), which regulate the amplitude and duration of the BCR signal by dephosphorylating various components of the BCR signal transduction unit. These phosphatases are also interesting because mutated or polymorphic variants have been linked to various malignant or autoimmune diseases. We started our study by performing nucleotide sequence analysis of the complete coding region of SHP1, SHP2 and Lyp in 8, 21 and 29 CLL B cell samples, respectively. Overall, only two mutations were identified (an R527C substitution in SHP2 and a Q456E substitution in Lyp, each in a single patient), suggesting that these phosphatases are infrequently mutated in CLL. The previously reported Lyp polymorphisms R620W and R263Q were observed in 2 additional cases. We next investigated expression of these phosphatases in purified CLL and normal B cells by immunoblotting. Expression of SHP1 and SHP2 was relatively uniform in the different CLL B-cells samples (n=42) and was not different from normal B cells (n=4). In contrast, expression of Lyp was markedly higher in most CLL samples, with 35 of the 49 investigated cases exhibiting 2 to more than 10 fold higher levels than normal B cells (n=5) (CLL, mean Lyp levels 4.7, SD +/−3.7; normal B cells, mean Lyp levels 0.9, SD +/−0.1, P=0.022). The mean Lyp levels were somewhat higher in U-CLL than M-CLL (6.0 vs. 3.9) and ZAP-70-positive than ZAP-70-negative cases (5.6 vs. 4.7), but these differences were not statistically significant. Analysis of Lyp expression in various lymphoma B-cell lines (n=9) also did not reveal significant differences with respect to normal B-cells, suggesting that Lyp overexpression is a specific feature of CLL. To determine what are the consequences of Lyp overexpression on BCR signaling, we downregulated Lyp in primary CLL B-cells by RNA interference and investigated activation of BCR signaling molecules following sIgM crosslinking. Downregulation of Lyp resulted in a substantial increase in BCR-induced phosphorylation of Lyn (Y397), Syk (Y352), BLNK (Y84) and ERK (T202/Y204), suggesting that overexpression of this phosphatase may be at least partially responsible for the lower BCR signaling capacity of CLL B-cells. Since Lyp expression can be induced in resting T cells by activation with anti-CD3, we investigated whether BCR stimulation will have a similar effect on CLL B-cells. A two-fold increase in Lyp levels was observed after 24 hours of sustained BCR stimulation with immobilized anti-IgM, whereas transient stimulation with soluble anti-IgM resulted in a 20% decrease in Lyp levels. These effects were specific for Lyp, since no such changes were observed in the expression of SHP1 and SHP2. In summary, this study shows that CLL B-cells specifically overexpress the phosphatase Lyp, and important negative regulator of BCR signaling that has been implicated in the pathogenesis of several common autoimmune diseases. Given the observation that Lyp can be induced by sustained BCR engagement and in view of recent findings that Lyp is also overexpressed in anergic B cells, these data further support the notion that CLL cells are continuously exposed to (auto)antigen in vivo. Disclosures: No relevant conflicts of interest to declare.

Sensitivity to Wnt Pathway Inhibition in CLL Is Associated with Specific Gene Expression Signatures

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 801-801
Author(s):  
Lili Wang ◽  
Alex K Shalek ◽  
Jellert Gaublomme ◽  
Nir Yosef ◽  
Jennifer R Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Wnt Pathway ◽  
Differentially Expressed ◽  
Differential Sensitivity ◽  
Specific Gene ◽  
Disease Heterogeneity

Abstract Abstract 801 We have recently found that the Wnt/b-catenin signaling pathway plays a key role in chronic lymphocytic leukemia (CLL). We were, however, intrigued by the question of whether this aberrant pathway may function differently in independent leukemias, and contribute to disease heterogeneity. To assess differential activity of the Wnt pathway across patients, we tested the effects of blocking Wnt activation on CLL cell survival. We knocked down a key downstream gene, LEF1, which is the most differentially expressed gene in CLL compared to normal B cells (based on gene expression microarrays). Addressing this question requires genetic manipulation of primary normal and malignant human B cells, and yet these cells are notoriously difficult to transfect. We therefore focused on developing a method for introducing siRNAs into normal and malignant B cells. We adapted a novel delivery system consisting of vertical silicon nanowires (SiNWs, Shalek et al PNAS 2010) that penetrate the plasma membrane in a minimally invasive fashion and deliver biomolecular cargo directly into the cytoplasm. We achieved consistent and reliable delivery of fluorescently labeled siRNAs (at 50–200 pmol) into normal and CLL B cells. siRNA was delivered to >90% of cells with >85% cell viability remaining after 48 hours. We used this platform to knockdown LEF1 in 20 CLL-B and 5 normal CD19+ B cell samples, and examined cell survival 48 hours after siRNA delivery using an ATP-based CellTiter-Glo assay. Indeed, our studies revealed a heterogeneous response among CLL-B cells to LEF1 inhibition. As a group, CLL-B cells were significantly more sensitive to LEF1 knockdown with a survival rate of 77% (12% s.e.m) compared to 97% (13% s.e.m) in normal B cells. CLL B cells from different patients showed differential sensitivity to LEF1 knockdown, with 8 non-responders, 8 intermediate responders and 4 strong responders (i.e. significant death). Sensitivity to LEF1 inhibition did not correlate with known CLL cytogenetic prognostic factors. To determine if the differential response to LEF1 knockdown was associated with specific gene signatures, we examined gene expression data generated from CLL-B cells from 12 (4 strong, 3 intermediate, and 5 non-responders) of the 20 CLLs tested (using the Affymetrix U133 Plus 2 Array). To increase statistical power, we used each CLL's expression profile (using only genes that showed variability across samples) to create clusters of ∼19 CLLs that showed similar expression profiles (using microarray data from our compendium of 177 additional CLLs). We further reduced the number of genes to ∼4000 genes by retaining only those whose expression levels were significantly different in at least one associated cluster relative to normal CD19+ B cell controls (T-test, FDR<10−4; p-values converted using the Benjamini-Hochberg method). These analyses led to the identification of several hundred genes whose expression correlated significantly with LEF1 knockdown's effect on cell viability. Analysis of these differentially expressed genes identified several potentially important pathways. Ongoing analyses include the identification and validation of a molecular signature for this effect. This signature could enable rapid identification of patients who would be most responsive to therapy with LEF1 inhibitors, which are under development along with other Wnt pathway inhibitors. Disclosures: No relevant conflicts of interest to declare.

BRAFV600E Mutations Occur In The Hematopoietic Stem Cell Compartment In Hairy Cell Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 816-816
Author(s):  
Stephen S. Chung ◽  
Jae H. Park ◽  
Eunhee Kim ◽  
Young Rock Chung ◽  
Wenhuo Hu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Mutant Braf

Abstract Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder recently found to be characterized by somatic BRAFV600E mutations. The malignant cell in HCL exhibits features consistent with a mature B-lymphocyte, including cell-surface expression of the pan-B-cell marker CD19 and monotypic surface immunoglobulins with clonal rearrangements of immunoglobulin heavy and light chains. Despite possessing these stereotypic features, the cell of origin of HCL has been long debated, and no cell type along the continuum of developing B-lymphocytes has been definitively identified as the normal counterpart of HCL cells. We hypothesized that HCL may originate from immature hematopoietic cells, and therefore investigated the hematopoietic-stem/progenitor cell (HSPC) compartment in HCL patients. We found that HCL patients exhibited a significantly increased frequency of immunophenotypically defined long-term hematopoietic stem cells (LT-HSCs; lineage-negative (Lin-neg) CD34+CD38-CD90+CD45RA- cells), pro-B cells (Lin-neg CD10+ cells), and CD34-CD38+ CD10+CD19+ hematogones, as well as a decreased frequency of granulocyte-macrophage progenitor cells (Lin-neg CD34+CD38+CD45RA+CD123+) relative to age-matched normal controls. Sequencing of cDNA from highly pure FACS-sorted cell populations from the bone marrow of HCL patients revealed the presence of the BRAFV600E allele in LT-HSCs and in pro-B cells (Figure). Transplantation of LT-HSCs from the pretreatment bone marrow of HCL patients into NOD/SCID/IL2r-gnull mice resulted in stable human grafts characterized by an expanded B-progenitor population and development of a clonal population of hCD19+hCD103+hCD25+ B cells characteristic of HCL 6 months after transplantation. Together, these data suggest that HCL arises from HSCs that then differentiate into committed B-cells which ultimately give rise to the characteristic clonal B-cell proliferation of HCL. Given the human HSC genetic and functional cell data, we conditionally expressed BRafV600E from its endogenous locus at different stages of hematopoiesis, including in HSPCs and committed B cells. Mice with conditional expression of BRafV600E in Mx1Cre+ BRafV600E knock-in mice died of a lethal hematopoietic malignancy characterized by features of human HCL including splenomegaly, anemia, thrombocytopenia, increased circulating sCD25, and increased clonogenic capacity of B-lineage cells (evidenced by infinite serial replating in the presence of IL-7) (Figure). This disorder was transplantable into lethally-irradiated recipient mice. In contrast, mice with expression of BRafV600E restricted to the B-cell lineage with Cd19 Cre manifested no overt malignant phenotype up to one year of age. Stimulation of these mice with alloantigen through injections of sheep red blood cells resulted in germinal center B-cell hyperplasia, but still did not result in development of a clonal B-cell proliferation. Recent case reports have noted that refractory HCL patients respond to mutant BRAF inhibition with vemurafenib. We investigated the effect of vemurafenib on HSPCs and hematopoiesis in patients treated on a phase II study of the mutant BRAF inhibitor vemurafenib for relapsed/refractory HCL as well as in our in vivo murine models. Flow cytometric analysis of bone marrow cells from vemurafenib treated HCL patients revealed normalization of HSPC frequencies within three months of starting therapy, concomitant with an improvement in peripheral blood counts. Consistent with this, evaluation of the in vitro clonogenic capacity of sorted LT-HSC's from the bone marrow of HCL patients revealed a significant increase in myeloid/erythroid colony formation in HCL patients treated for 3 months with vemurafenib compared to their pretreatment marrows. Likewise, treatment of wildtype mice transplanted with Mx1Cre+ BRafV600E mutant bone marrow cells revealed improvement in anemia and hepatosplenomegaly with in vivo therapy. Overall, these findings link the pathogenesis of HCL to a specific somatic genetic abnormality present in HSCs and provide evidence that mature B-cell malignancies can initiate in the HSC compartment. Moreover, these data suggest that the use of therapies targeting MAP kinase signaling in HCL may lead to durable remissions not only by eliminating the mature leukemic cells but also through targeted inhibition of signaling and survival in HCL initiating cells. Disclosures: No relevant conflicts of interest to declare.

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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