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Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi). BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression. When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy. Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy. Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL. Disclosures Patten: Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or γ radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or γ radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

B-cell chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a variable clinical course. The disease is characterised by the proliferation in the bone marrow and lymph node of a clonal population of CD5+ve cells that accumulates in the peripheral blood. Therefore, the characteristics of the proliferative compartment are important in determining the kinetics of disease progression in CLL and the sensitivity of the malignant cells to cytotoxic drugs. However, laboratory studies on drug sensitivity of CLL have been performed exclusively on resting circulating peripheral blood cells since it is not feasible to obtain cells from the proliferating pool in sufficient numbers for in vitro analysis. CLL cells can be stimulated to proliferate in vitro using CpG oligonucleotides (ODN) and other factors. The aim of the present study was to generate and validate an in vitro model using malignant cells from the peripheral blood of patients with CLL. The expression pattern of proteins eg., survivin in this model should mimic that in proliferating CLL cells in the bone marrow and lymph nodes. Survivin is a member of the family of inhibitor of apoptosis (IAP) proteins with an additional role in cell cycle progression. Survivin has been shown to be expressed in proliferating bone marrow and lymphoid cells. Cells from patients with CLL were activated for 72h with a combination of ODN (1μM), IL-2 (100u/ml) and CD40L (0.5μg/ml) (ODN*). Activated cells retained their characteristic CLL immunophenotype as determined by the continued expression of CD5, CD19, CD23 and CD25 (n=5). Cell proliferation was confirmed by increased incorporation of 3H-thymidine into DNA in activated cells (n=12). Novel findings in the ODN* activated CLL cells were significant increases in expression of CD38 (n=7, p=0.0001) and of T-cell zeta associated protein (ZAP-70) tyrosine kinase (n=14, p=0.0005). The increased expression of both these proteins in circulating peripheral blood CLL cells has been associated with poor prognosis. All six ODN* activated CLL isolates analysed by western blotting showed increased survivin expression with no constitutive expression in the controls. Drug sensitivity was studied in cells from eight patients using the MTT assay. Activated cells showed significantly greater resistance to chlorambucil (median IC50=164.4±28.18μM) compared to control cells (median IC50=93.63±14.96μM, p=0.044). Figure 1 shows representative IC50 curves. The increased resistance of the activated cells to chlorambucil may be a consequence of the upregulation of survivin. In summary, the in vitro model replicates several key features of authentic proliferating CLL cells found in bone marrow and lymph nodes. It also shows increased resistance to the conventional drug chlorambucil. This model may be of value in evaluating novel drugs and drug combinations which may be more effective in killing the proliferating population that maintain the malignant cell population in CLL. Figure Figure

A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgVH) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC50 (μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgVH mutational status by sequence analysis of FR1/JH polymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70+ve and the remaining 33/46 (72%) were ZAP-70−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70+ve and ZAP-70−ve groups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70+ve and ZAP-70−ve patients. The IC50 levels for CdA, Chl and FdR between ZAP+ve (median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP−ve groups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70+ve and 15 ZAP-70−ve patients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70−ve group. IgVH mutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70+ve/IgVH unmutated) and in 16 with good prognosis (ZAP-70−ve/IgVH mutated). Statistical analysis of MDR protein expression and IC50 in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70+ve/IgVH unmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP−ve group is unclear.

B-Chronic lymphocytic leukemia (B-CLL) patients whose malignant cells harbour unmutated immunoglobulin heavy chain variable region (IgVH) genes or express the zeta-associated protein tyrosine kinase ZAP-70 show a worse prognosis than do patients with mutated IgVH genes or ZAP-70−ve expression. The inability of malignant cells to activate the pro-apoptotic p53 pathway in response to ionizing radiation (IR) also correlates with a poor prognosis. We studied ZAP-70 expression and IgVH mutation status in 161 patients with B-CLL in order to determine the degree of concordance between these two prognostic criteria (M104/F57, wbc 2.44–576x109/l lymphocytes 0.56–287x109/l). We also studied the functional status of the p53 pathway and the apoptotic response to ionizing radiation in cells from a subset of patients from both prognostic categories. A human ZAP-70 antibody (clone 2F3-2) was conjugated to the Alexa Fluor 488 dye using a zenon mouse IgG labelling kit and used for a FACS based assay. FACS results were expressed as a ratio of B-cell mean cell fluorescence to T-cell mean cell fluorescence with a cut off at > 0.75 identifying a ZAP-70+ve sub-group. IgVH mutational status was studied by sequence analysis of FR1/JH polymerase chain reaction products. The ability of 5Gy ionizing radiation to augment levels of p53 and its transcriptional target p21CIP1 was quantified by western blot analysis. Cleavage of the caspase 3 target poly(ADP ribose) polymerase (PARP) was used as a measure of apoptosis induction. ZAP-70+ve expression was observed in 25% (41/161) of the samples with a median ratio of 0.85 (range 0.76–1.46) while the remaining 120 samples were ZAP-70−ve, with a median ratio of 0.56 (range 0.19–0.73). IgVH mutation status was analysed in 92 of these patients. Assignment of prognostic category by both criteria was concordant in 72/92 (78.2%) of the cases of which 54/92 (58.6%) were ZAP−ve/IgVH mutated (good prognosis) and 18/92 (19.5%) were ZAP+ve/IgVH unmutated (poor prognosis) patients. The remaining 21.7% were discordant, ie., either ZAP+ve/IgVH mutated (5.4%) or ZAP−ve/IgVH unmutated (16.3%). Isolates from 5/6 ZAP+ve/IgVH unmutated patients upregulated p53 in response to IR but nevertheless failed to initiate PARP cleavage, suggestive of a block in the apoptotic pathway distal to p53 induction. In 9 ZAP−ve/IgVH mutated isolates studied, 7 induced p53, p21 and PARP cleavage following IR. In conclusion, this large cohort of CLL patients demonstrated a good correlation between ZAP-70 expression and IgVH mutational status in identifying a poor prognosis sub-group. However, this prognostic category, as defined by both IgVH mutation status and ZAP-70 expression failed in some cases to predict the ability of B-CLL cells to induce an apoptotic response to DNA damage in vitro. Induction of the p53 pathway was not always sufficient to secure an apoptotic response, especially in the poor prognosis group. A combination of ZAP-70 and IgVH analysis with a functional assay for DNA damage-induced apoptosis will identify individuals in either prognostic category who are unlikely to respond to conventional cytotoxic drugs. Alternative therapeutic strategies independent of DNA damage-inducing agents may be of value in the treatment of these patients.