Combination chemo-immunotherapy has improved the survival in previously untreated CLL, but the treatment of relapsed and fludarabine refractory CLL continues to be challenging. Inhibiting the B-cell receptor (BCR) signalling pathway appears to be beneficial in the majority of patients but is not sufficient to eradicate disease and combination approaches are already being investigated. We have previously undertaken large scale protein expression profiling in CLL and identified several molecules involved in neurotransmission that are highly expressed in CLL.
To explore signalling mechanisms in neuronal markers that are aberrantly expressed in CLL, and to determine whether they have therapeutic potential in combination with BCR inhibitors.
RNA expression data (Haslinger et al; Journal of Clinical Oncology. 2004) was used to identify candidate antigens that could be screened for protein level expression. Screening was restricted to antigens expressed on plasma membrane with an extracellular epitope and a commercially available antibody suitable for testing in combination with mouse monoclonal gating antibodies (i.e. fluorochrome-conjugated monoclonal, non-conjugated mouse monoclonal labelled using the zenon technique, or rabbit polyclonal detected using fluorochrome-conjugated secondary goat F(ab')2 anti-rabbit antibody). In vitro viability experiments were performed using either mononuclear cells, separated using lymphoprep, or purified CLL cells isolated with a B-cell isolation kit (Miltenyi Biotec), cultured with or without the presence of CD40L transfected fibroblasts as a supporting stromal layer. Viability was assessed using Annexin-V, 7-AAD and counting beads.
To study pathway interactions, the B-cell receptor was stimulated by crosslinking using goat F(ab')2anti-human IgM or IgD and signalling responses were assessed by Syk phosphorylation status (Syk pY348 PE, BD Phosflow) and calcium flux (Fluo-3/Fura red, Invitrogen).
84 target antigens were assessed of which 11 showed binding on all mononuclear cells and 9/84 showed binding on CLL cells and/or normal B-cells but not other leucocytes. Of the B-cell targets, 4/9 molecules have a role in neurotransmission, such as the nicotinic acetyl choline receptor subunit β4 (CHRNB4) and dopamine receptor D4 (DRD4). CHRNB4 had the highest level of expression on B-cell and CLL surface membranes and therefore other subunits of nicotinic acetyl choline receptor were further evaluated using RT-PCR. Of the 15 subunits tested 10 were expressed on CLL cells suggesting the possibility of an important role in the pathophysiology of CLL. However, in vitro experiments did not show a significant effect on viability of CLL cells with either acetyl choline or its antagonist mecamylamine.
Unlike acetyl choline, dopamine was found to be effective in reducing CLL cell survival in vitro, in a dose dependent manner with an LC50 of 8.2μM (95%CI- 5.2 to 12.9, n=6). Assessment of dopamine receptor agonists and agonists demonstrated that the D2-like family specific antagonist domperidone enhances the activity of dopamine and also has single agent activity in reducing in vitro CLL cell viability.
To determine the effect of dopamine on BCR pathway signalling, syk phosphorylation and calcium flux after IgD crosslinking were measured. Stimulation of the BCR pathway by crosslinking using goat F(ab')2 anti human IgD increased the mean fluorescent intensity of syk phosphorylation by 1.9 fold (p=0.0024) compared to 1.5 fold (p=0.0186) when pre-incubated with dopamine and 1.2 fold (p=0.0059) with a syk inhibitor. Similarly the mean percentage rise in CLL cells fluxing calcium has fallen from 20.1% to 8.2% (p=< 0.0001) when treated with dopamine and to 5.6% (p=< 0.0001) when treated with syk inhibitor. In vitro testing demonstrated a synergistic effect in blocking of the D2 and the BCR pathways as a combination of PI3 kinase δ inhibitor, GS-1101, along with domperidone reduced CLL cell viability more efficiently than either agent alone.
Systematic screening has identified several new targets in CLL, in particular several neuronal markers which are highly expressed and functionally active. Biological investigations suggest that these neuronal receptors could play an important role in the pathophysiology of CLL and show potential synergies with therapeutically active BCR inhibitors.
Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.