B-cell Chronic Lymphocytic Leukemia Samples Research Articles

Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry.

BET Bromodomain Inhibition Reduces Leukemic Burden and Prolongs Survival In The Eμ-TCL1 Transgenic Mouse Model Of Chronic Lymphocytic Leukemia (CLL) Independent Of TP53 Mutation Status

IntroductionInhibition of Brd4, a bromodomain and extra-terminal (BET) protein, results in antiproliferative effects and terminal differentiation in the MYC-driven B-cell malignancies multiple myeloma and Burkitt's lymphoma by selectively repressing MYC. Elevated MYC expression correlates with progression of B-cell chronic lymphocytic leukemia (B-CLL) marking Brd4 as a potential therapeutic target. 17p deletion or somatic TP53 mutations are the poorest prognostic factors in B-CLL, resulting refractoriness to conventional therapy. The recent identification of a Brd4-interacting protein as a validated target in CLL led us to evaluate the potential of BET inhibition, using jq1, for the treatment of B-CLL. ResultsJq1 remarkably reduced proliferation of the immortalized human B-CLL cell lines, Mec-1, Mec-2, and WaC3 (IC50 values: 1.12, 0.68, and 0.64 μM, respectively). These results were validated in 13 frozen and 7 fresh primary whole peripheral blood B-CLL samples (median EC50 0.66 μM [range, 0.051 to 2.169 μM]) using a FACS-based proprietary platform (Vivia Biothech). Eμ-TCL1 (n=13) & Eμ-TCL1:p53R172H/+ (n=9) mice treated with intraperitoneal (IP) injections of jq1 at 50 mg/kg/day for 21 consecutive days had a significant reduction in lymphocytes and white blood cells (WBC) (with excellent tolerance and no off-target effects on hemoglobin level or platelet counts) compared to vehicle-treated mice. The median lymphocyte count at baseline and after 2 weeks of jq1 therapy was 13.4 & 5.92 x10^3/μL (p=0.008) for Eμ-TCL1 mice and 13.2 & 7.2 x10^3/μL (p=0.009) for Eμ-TCL1:p53R172H/+ mice. jq1 therapy significantly prolonged survival in both Eμ-TCL1 (428 vs 377 days, p=0.05) and Eμ-TCL1:p53R172H/+ (382.5 vs 300 days, p=0.0003) mice compared to mice receiving vehicle. FACs analysis was performed on blood taken from Eμ-TCL1 (n=5) and Eμ-TCL1:p53R172H/+ (n=6) mice at baseline and after 7 daily IP jq1 doses. Eμ-TCL1:p53R172H/+ mice showed a significant reduction in CD5+/CD19+ lymphocytes (mean difference, 14.37 [95% CI, 2.4 to 26.3]; p=0.03), which was not observed in Eμ-TCL1 mice. Cell cycle analysis of cultured Eμ-TCL1 splenocytes treated with jq1 showed a significant increase (p<0.0005) in the number of sub-G1 cells compared to untreated splenocytes, regardless of p53 status. Sensitivity against fludarabine was also tested in these samples and we show that 10μM fludarabine did not elicit significant changes in the number of cells in sub-G1 compared to untreated cells but a significant difference (p=0.009) was observed between splenocytes treated with 10μM fludarabine and 1μM jq1. In depth analyses of RNA sequencing data of splenocytes from 10 matched-pairs samples (5 Eμ-TCL1 & 5 Eμ-TCL1:p53R172H/+) treated in culture with 0 or 1μM jq1 for 24 hours are currently underway to identify BET targets in B-CLL, but preliminary gene expression analysis reveal that the sensitivity of B-CLL to BET inhibition is not Myc-dependent despite elevated Myc levels. Notably, c-Fos was one of the most significant differentially expressed genes, regardless of p53 status. Interestingly, AP-1 complex, a heterodimer of c-Fos and c-Jun, is inhibited by direct interaction with Tcl-1. We are currently validating these findings in additional samples and performing functional assays to gain a better understanding of BET targets in B-CLL. ConclusionBET bromodomain inhibition markedly reduced proliferation in both human and mouse B-CLL samples. Furthermore, the loss of wild-type p53 activity did not desensitize CLL cells to jq1, which was also effective at inducing massive cell death in fludarabine resistant CLL cells, regardless of p53 status. Mice receiving jq1 exhibited a significant reduction in leukemic burden and prolonged survival compared to control mice, with a more pronounced effect observed in mice with mutant p53. These data demonstrate the potent effect of BET inhibition in B-CLL and more importantly, provide evidence as an effective treatment for aggressive forms of B-CLL with 17p deletion or TP53 mutation or chemoresistant refractory disease. [Display omitted] [Display omitted] Disclosures:Primo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Martinez:Vivia Biotech: Employment. Bradner:Tensha Therapeutics: Dr. Bradner is a scientific founder of Tensha Therapeutics, which is developing drug-like derivatives of the JQ1 bromodomain inhibitor as cancer therapeutics, through a license from the Dana-Farber Cancer Institute. Other. Ballesteros:Vivia Biotech: Employment.

Flow cytometric analysis of Notch1 and Jagged1 expression in normal blood cells and leukemia cells

Notch1 and its ligand Jagged1 are proteins with important roles in the growth of leukemia cells. Although the detection of Notch1 protein in acute lymphoblastic leukemia cells using immunoblot analysis has been previously reported, the expression patterns of Notch1 and Jagged1 detected by flow cytometry (FCM) in normal blood cells and various leukemia cells have not been well-characterised. In the present study, we examined the expression patterns of Notch1 and Jagged1 in 10 normal blood samples, 8 bone marrow samples, 11 leukemia/lymphoma cell lines and leukemia cells from 22 patients with acute myeloid leukemia (AML), mature T-cell neoplasms or B-cell chronic lymphocytic leukemia (B-CLL) using FCM. The results showed that Notch1 expression is relatively strong in monocytes and granulocytes but weak in lymphocytes. The expression of Notch1 is stronger in bone marrow cells than in the equivalent cells in blood. All the cell lines examined strongly expressed Notch1, and eight cell lines expressed Jagged1. In leukemia cells from patients, four AML samples expressed Notch1 and/or Jagged1. However, three samples expressed neither Notch1 and/or Jagged1 and none of the mature T-cell neoplasm samples expressed either protein. However, all B-CLL samples expressed high levels of both Notch1 and Jagged1. We found that the expression of Notch1 and Jagged1 is detected in various hematological malignancies by FCM. The examination of these proteins is likely to be useful in the characterisation of diseases and individual cases. Examination of these proteins may also be useful in the selection of patients most likely to benefit from novel molecular-targeted therapies using Notch inhibitors in the future.

RelB, Together with Rela, Maintains the Cellular Survival In Chronic Lymphocytic Leukemia

AbstractAbstract 3583RelA, a classical NF-κB subunit, has been shown to function critically in cellular survival and drug-resistance of B-cell chronic lymphocytic leukemia (B-CLL). The significance of RelB, contributing to the alternative NF-κB activity, has not been addressed in B-CLL yet. The protein expression levels of NF-κB subunits in 60 B-CLL samples were examined in this study. According to the RelA and RelB expression status in the nuclear extracts of peripheral blood and bone marrow, the B-CLL samples were classified as RelA+/RelB−, RelA+/RelB+, and RelA−/RelB− subgroups. The frequencies of the individual group were 30%, 30%, and 40%. In RelA+/RelB+ subgroup, both the RelA DNA-binding complex and the RelB DNA-binding complex were further detected in the nuclear extracts of B-CLL cells by electrophoretic mobility super-shift assay. Fresh CD19+ B-CLL cells from different subgroups were cultured for 48 hours ex vivo, the spontaneous apoptosis were quantified by Annexin-V/PI staining and the frequencies of apoptosis were 48±4.5% (RelA+/RelB−), 24±8.6% (RelA+/RelB+), and 65±5.0% (RelA−/RelB−) individually. In addition, when fresh B-CLL cells were incubated with MG-132, a proteosome inhibitor, a significant reduction of cellular survival in RelA+/RelB− and RelA+/RelB+ group was observed in a dose and time-dependent manner. B-CLL cells from RelA+/RelB+ group were cultured for 0, 6 and 24 hours in the presence of CD40L (250μ g/ml), the cellular survival was not affected and the protein expression levels of NF-κB family members stayed unchanged. While the CD19+ B-CLL cells from RelA+/RelB− group were cultured with CD40L, a modest decrease in apoptosis and a slightly increased RelB expression in cytoplasm and nucleus after 24-hour treatment were identified. The induction of RelA and RelB expression, companied by an evident decrease in apoptosis, was detected in cells from RelA−/RelB− group. The alternative NF-κB signaling pathway, in terms of RelB/p52 nuclear translocation, functions specifically in lymphoid organogenesis and B-cell development. In this study, we show that RelB expression is detected in B-CLL cells and partially maintain the basal survival. CD40L triggers the expression of RelB, correlating with the enhanced survival. Taken together, not only RelA but also RelB subunit of NF-κB family plays an important role in the cellular behavior of B-CLL cells. Disclosures:No relevant conflicts of interest to declare.

SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity.

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway via c-jun-NH2 Terminal Kinase Activation and PTEN Upregulation

Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors. B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses. After 48 hours, 3 mumol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-kappaB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased the apoptotic effect of either agent alone. ATO also induced c-jun-NH(2) terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-kappaB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production. We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapies.

A new method to detect loss of heterozygosity using cohort heterozygosity comparisons

BackgroundLoss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort.MethodsDNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes.ResultsUsing a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions.ConclusionsWe have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.

CD8+ T Cells Engineered to Express a ROR1-Specific Chimeric Antigen Receptor Specifically Recognize ROR1 Positive B Cell Tumors.

Abstract 930The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells. Disclosures:No relevant conflicts of interest to declare.

Impaired up-regulation of polo-like kinase 2 in B-cell chronic lymphocytic leukaemia lymphocytes resistant to fludarabine and 2-chlorodeoxyadenosine: a potential marker of defective damage response

The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs.

Exposure of B Cell Chronic Lymphocytic Leukemia (B-CLL) Cells to Nutlin-3 Induces a Characteristic Gene Expression Profile, which Correlates with Nutlin-3-Mediated Cytotoxicity (Supplementry Table)

By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.

Apoptosis of B-cell chronic lymphocytic leukemia cells induced by a novel BH3 peptidomimetic

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in human adults of the Western world and no definitive cure is yet available. The disease is characterized by accumulation of clonal malignant B lymphocytes resistant to apoptosis. Strategies to hit the anti-apoptotic drift of the Bcl-2 family in B-CLL cells are being explored. A novel peptidomimetic based on the BH3 domain of the pro-apoptotic protein Bim and recently shown to exert significant apoptotic activity on acute myeloid leukemia cells, both in vitro and in vivo, was assayed on ex-vivo derived leukemic cells from untreated B-CLL patients (n=7). We found that this peptide, named 072RB, induced apoptosis of B-CLL samples at a concentration that does not affect viability of peripheral and bone marrow derived lymphocytes from healthy donors. Apoptosis was demonstrated by activation of Bak and Bax, externalization of plasma membranes phosphadydilserines, appearance of hypodiploid events in DNA flow cytometry histograms and was accompanied by dissipation of the mitochondrial transmembrane potential. Before the onset of marked apoptotic signs a progressive decline of the relevant anti-apoptotic proteins Bcl-XL and Mcl-1 could be observed. The negative control peptide 072RBL94A was ineffective for B-CLL cells, supporting the sequence specificity of 072RB activity. No relationship was found between responsiveness to 072RB and Mcl-1/Bcl-XL basal levels or decrease magnitude, possibly because of the limited sample size of the study. Altogether, we demonstrate that 072RB induces significant apoptosis of B-CLL cells subsequent to Bcl-XL and Mcl-1 down-regulation.

Tcl1 functions as a transcriptional regulator and is directly involved in the pathogenesis of CLL

B cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B cells causes a CD5-positive leukemia similar to aggressive human B-CLLs. To examine the mechanisms by which Tcl1 protein exerts oncogenic activity in B cells, we investigated the effect of Tcl1 expression on NF-kappaB and activator protein 1 (AP-1) activity. We found that Tcl1 physically interacts with c-Jun, JunB, and c-Fos and inhibits AP-1 transcriptional activity. Additionally, Tcl1 activates NF-kappaB by physically interacting with p300/CREB binding protein. We then sequenced the TCL1 gene in 600 B-CLL samples and found 2 heterozygous mutations: T38I and R52H. Importantly, both mutants showed gain of function as AP-1 inhibitors. The results indicate that Tcl1 overexpression causes B-CLL by directly enhancing NF-kappaB activity and inhibiting AP-1.

Impaired up-Regulation of Polo-Like Kinase 2 (PLK2) during Fludarabine- and 2-Chlorodeoxyadenosine (CdA)-Induced Cell Killing in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes: A Potential Marker of Defective Damage Response

Patients with B-cell chronic lymphocytic leukemia (B-CLL) carrying alterations in the p53 pathway respond poorly to most of the existing conventional treatments, presumably because an intact p53 pathway is required for cell killing induced by nucleoside analogs (NA) and alkylators. It is thus critical to identify these patients in order to orient them toward alternative non-genotoxic approaches. One promising way to select these patients is to use p53 functional assays which rely on the detection of an impaired up-regulation of p21 (a transcriptional target of p53) in response to ionizing radiations (IR). Since IR is not used therapeutically in B-CLL, we hypothesized that it would be sensible to investigate p53 dysfunction after incubation with NA, which are standard first line approach in B-CLL patients. We firstly performed microarrays in B-CLL samples (n=4) incubated with clinically achievable concentrations of fludarabine and CdA, and selected for sensitivity to these drugs (i.e. low half maximal inhibitory concentration (IC50) by MTT assay). We observed thatNA increased expression of genes which were predominantly p53- dependent,among the latter genes, PLK2 was the most significantly activated at earliest time points (8h) after NA exposure, andthere was no major difference between fludarabine and CdA at equitoxic concentration.Conversely, in highly resistant samples (i.e. high IC50 by MTT assay, n=3), p53 and PLK2 response analyzed by microarray was abolished. We confirmed a significant dose- and time-dependent PLK2 mRNA upregulation after CdA and fludarabine using quantitative real-time PCR (qPCR), and also showed that PLK2 was increased in response to other DNA damaging conditions (UV light), and in response to p53 activation by Nutlin-3. Based on these results, we sought to compare the levels of PLK2 mRNA induction with in vitro chemosensitivity to NA in a large number of individual B-CLL patient's samples (n=18). We observed that cytotoxicity induced by a fixed concentration of fludarabine (3μM) or CdA (0.3μM), measured by MTT at 96h, was well correlated (P<0.01) with PLK2 mRNA induction, measured by qPCR at 24h. In particular, in 12 patients sensitive to fludarabine (median IC50=0.7 μM, range 0.12–2.21), increase in PLK2 mRNA (after normalization with an housekeeping gene) was 10.3 fold (arbitrary units, range 3.6–22.4, P<0.0001) as compared to untreated control. In comparison, in 6 patients with high level of in vitro refractoriness to fludarabine (median IC50=24.6 μM, range 13.6->100), PLK2 increase was only 1.4 fold above untreated control. In parallel experiments, we had verified that chemoresistance to NA was not due to deficiency of deoxycytidine kinase, the limiting enzyme in the conversion of NA into phosphorylated derivatives. Interestingly, lack of PLK2 activation and chemoresistance to NA were observed in patient with normal 17p by FISH, showing that TP53 deletion assessment is only partially overlapping with p53 functional tests. In conclusion, our results show that PLK2 RNA activation after a 24-h incubation with NA is a potential tool to investigate p53 functional integrity in B-CLL cells, and might represent an alternative to p21 protein measurement after IR. Because of the tight correlation with in vitro toxicity of NA, PLK2 testing might constitute a predictor of clinical sensitivity to these drugs which are broadly used in B-CLL

Correlation of clinical activity of lumiliximab in combination with FCR and ZAP 70 expression on B-cell chronic lymphocytic leukemia cells

7073 Background: Lumiliximab is an anti-CD23 monoclonal antibody under investigation for the treatment of patients with relapsed B-cell chronic lymphocytic leukemia (BCLL). Lumiliximab binds specifically to human CD23, a glycoprotein expressed on the majority of BCLL cells. As ZAP70 is a poor prognostic factor of CLL, expression of the ZAP70 antigen by malignant B cells was analyzed on samples taken pre-treatment and day 2 post-treatment in a subset of patients with relapsed BCLL enrolled in a clinical study evaluating lumiliximab as monotherapy. The ability of lumiliximab to induce ex-vivo apoptosis was also evaluated in BCLL samples collected in this subset. Subsequently, pre-treatment ZAP70 expression was analyzed in patients treated lumiliximab in combination with fludarabine, cyclophosphamide, and rituximab (L + FCR). Methods: In the lumiliximab monotherapy study, ZAP70 expression was quantified by Western blot assay using lysates from purified BCLL cells and apoptosis was evaluated by flow cytometric assessment of Caspase-3 activation on pre- treatment and day 2 treated samples (ex-vivo). In the L + FCR study, frequencies of BCLL cells and ZAP70 positive cells were estimated by four color flow cytometry using standard commercially available antibodies. BCLL cells were identified by first gating on viable CD45+ cells and subselecting with antibodies to CD5 and CD19 receptors (CD5+CD19+ phenotype). ZAP70 expression was reported as % BCLL cells. Results: Analysis of data from the monotherapy study suggested induction of apoptosis and decrease in CD5+CD19+ cells in patients expressing high levels of ZAP70 on purified B cells. Moreover, apoptosis was also observed in a patient that expressed activated ZAP70. Results from the L + FCR study demonstrated that clinical activity in this combination trial is observed in patients having high or low levels of ZAP70 expression. Conclusions: Lumiliximab potentially provides clinical benefit in BCLL patients with both low and high levels of ZAP70, whose expression is associated with aggressive disease and poor prognosis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Biogen Idec Biogen Idec Biogen Idec

Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression. Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA. ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1 was determined to be in the range of 10(3) to 10(4) molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1 protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. The restricted, uniform, and constitutive cell surface expression of ROR1 protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.

The presence of high‐risk chromosome aberrations in chronic lymphocytic leukaemia does not correlate with centrosome aberrations

Chromosome aberrations are frequently found in B-cell chronic lymphocytic leukaemia (B-CLL), and specific chromosome aberrations identify poor prognostic subgroups. Almost all the aberrations identified in B-CLL involve loci where genes with a role in the regulation of centrosome duplication have been mapped. Centrosome aberrations have been described as a possible cause of numerical chromosome abnormalities in both solid and haematological tumours. However, little is known about the possible role of centrosome aberrations in B-CLL. To investigate whether centrosome aberrations do occur in B-CLL and correlate with cytogenetically defined prognostic subgroups, we examined a set of 64 B-CLL samples by immunofluorescent staining. B-CLL cases differed significantly from controls in the mean frequency of cells with centrosome aberrations, while no difference was found between subgroups with or without specific chromosome aberrations. Our results indicated that although centrosome aberrations were a common feature in B-CLL, they did not represent a reliable prognostic marker.

Regulation and Function of NOTCH2 in B-CLL Cells.

B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI's) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

Identification of somatic hypermutations in the TP53 gene in B-cell chronic lymphocytic leukemia

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P = 0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase ( AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

Analysis of 11q22–q23 deletion target genes in B-cell chronic lymphocytic leukaemia: Evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B

Deletion of 11q22–q23 is associated with an aggressive course of B-cell chronic lymphocytic leukaemia (B-CLL). Since only in a subset of these cases biallelic inactivation of ATM was observed, we sought to identify other disease-associated genes within 11q22–q23 by analysing NPAT (cell-cycle regulation), CUL5 (ubiquitin-dependent apoptosis regulation) and PPP2R1B (component of the cell-cycle and apoptosis regulating PP2A) for point mutations and their expression in B-CLL by single-strand conformation polymorphism/sequence analysis of the transcripts and real-time polymerase chain reaction. Though none of the genes were affected by deleterious mutations, we observed a significant down-regulation of NPAT in B-CLL versus CD19+ B cells and of CUL5 in 11q deletion versus non-deletion B-CLL samples and measured reduced PPP2R1B transcript levels in a subset of B-CLL cases. Alternative splicing of PPP2R1B transcripts (skipping of exons 2/3, 3, 9) was associated with a reduced activity of protein phosphatase 2A. Together, these results implicate deregulation of the cell-cycle and apoptosis regulators NPAT, CUL5 and PPP2R1B and a role for these genes in the pathogenesis of B-CLL.

Regulation of NOTCH2 Signaling in B-CLL: Involvement of PKC-δ.

We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics).Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling.In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.