Figure 3.

The growth response in CLL cultures after BCR engagement is most strongly predicted by pAKT activation and by TCL1 levels. (A) The growth and apoptotic parameters of 29 CLL cultures from Figure 2B were measured after 48 hours of continuous BCR crosslinking at 3 different α-IgM concentrations, compared with CD40/IL-4 stimulation and unstimulated controls. Cases were defined as having a hyperresponsive BCR pattern (left panels, with growth at low α-IgM and apoptosis at higher doses), dose-dependent BCR response (center panels), or absent growth response (right panels) based on MTT assay (top panels) and immunoblots (middle panels). Increased cyclin D2 levels correlated with MTT-measured growth induction as did expression of the AKT target MCL1, whereas cleaved PARP and caspase 3 levels correlated with the degree of apoptosis observed. The 2-hour AKT phospho-activation pattern after BCR engagement (colored as in Figure 2B as follows: red, strong; blue, moderate; and black, absent) were correlated with the 48-hour growth pattern (bottom panel). Cases with a hyperresponsive growth pattern showed strong pAKT induction (red lines) that paralleled the BCR response pattern seen in tonsillar B cells (lines with square boxes), whereas cases with only moderate BCR induced pAKT activation showed dose-dependent growth patterns (blue lines). (B) The pattern of BCR-inducible growth at 48 hours correlated with TCL1 levels. A hyperresponsive/apoptotic outcome was seen in CLLs with the highest TCL1 expression, whereas a lower level but progressive increase of growth on α-IgM escalation (dose-dependent response) was associated with low to moderate TCL1 levels in most cases. CLL cultures not responding to BCR stimulation included those that were negative/low for TCL1 but also some that highly expressed TCL1 where the growth stimulation may have been missed because of timing of the MTT assay.