B-Cell Receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones.
Previous studies have suggested that in a sizable fraction of CLL patients the B cell clone is functionally anergic and characterized by constitutive phosphorylation of ERK1/2 kinases.
We have analyzed all the described anergy-related features in CLL cells and we observed that constitutive ERK1/2 phosphorylation (used as surrogate marker for anergy) together with NF-ATc1 nuclear translocation correlates with the in vitro inability to mobilize intracellular calcium upon anti-IgM stimulation and with reduced expression of surface IgM. The latter features are reminiscent of B cells where chronic Ag-stimulation favours continuous recycling of B cell receptor. Of interest the cells of the pERK(+) subset of patients have higher expression levels of CD22 and CD5, two receptors that are required for the negative regulation of BCR signalling.
The activation of the biochemical program that culminates in the induction of B-cell anergy is transient and requires chronic BCR triggering. We showed that Ag removal, obtained by culturing CLL cells in vitro, causes the loss of the anergic signature (reduction of pERK and nuclear NF-ATc1 levels) and, together with the re-expression of surface IgM, restores the ability of CLL cells to fully respond to BCR triggering.
These results led us to explore the possibility to interfere with the molecular features of anergy in a therapeutic perspective, using two classes of MAPK inhibitors (targeting MEK1/2 or ERK1/2) and one specific peptide (VIVIT) that blocks NF-AT nuclear translocation.
Constitutive ERK1/2 activation was efficiently inhibited after 1 hour treatment with two MEK inhibitors (U0126 and CI1040), and 48 hours treatment with both compounds induced selective apoptosis in the restricted group of pERK(+) patients. In details, pERK(+) samples (n=21) treated with 10μM of U0126 showed a mean survival of 36.58 ± 5.242 (compared to 60.63 ± 6.557 survival of the pERK(-) subset, p=0,01), while pERK(+) samples treated with 10μM of CI1040 (n=17) experienced a mean survival of 36.59 ± 4.145 (compared to 59.17 ± 6.313 survival of the pERK(-) subset; p=0,01). The same results were obtained when we directly inhibited ERK1/2 activity with a new generation compound. By screening 16 samples treated with 10μM of ERK inhibitor, we observed that pERK(+) patients are very sensitive to treatment (29.38 ± 8.064 mean survival) compared to pERK(-) samples (68.12 ± 8.81; p=0,03).
Taken together, these data suggest that MEK1/2 can be efficiently targeted in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 represents a good biomarker to predict and monitor treatment response.
In parallel, inhibition of NF-ATc1 activation with a cell permeable version of VIVIT peptide, reduced cell viability in the group of NF-AT(+) patients (29.47 ± 7.509 mean survival in the NF-AT(+) group and 53.63 ± 6.044 in the NF-AT(-) subset, p=0,02), As expected, the killing activity also correlated with ERK activation (21.30 ± 4.191 mean survival for the pERK(+) group and 42.95 ± 5.465 mean survival for the pERK(-) subset; p=0,01).
The apoptotic effect observed in vitro after the use of ERK1/2 and NF-AT inhibitors is preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation (together with the reduction of surface levels of CD22), and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program is responsible for the survival of leukemic lymphocytes.
Combination studies with different inhibitors revealed that the concomitant inhibition of MAPK and NF-AT signalling did not enhance apoptosis, thus confirming that both pathways are directly interconnected. A possible link between ERK and NF-AT is the activation of the SHIP1/CD5 axis whose phosphorylation is lost after inhibitors treatment and re-acquired upon BCR triggering.
In conclusion, our results demonstrate that one subset of CLL patients is characterized by the abnormal expansion of B cells with anergic features. In these cells the constitutive phosphorylation of ERK and NF-ATc1 activation can be efficiently targeted for therapeutic purpose, thus opening new clinical perspectives.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.