MHH-CALL-3

Bortezomib Induces Apoptosis in Prednisone-Resistant Childhood ALL Cells

The ubiquitin-proteasome degradation pathway (UPDP) is possibly altered and may be involved in multi-agent chemotherapy resistance in childhood ALL patients. The proteasome inhibitor bortezomib blocks proteasomal degradation and hence the UPDP. To investigate whether treatment of ALL with the proteasome inhibitor bortezomib acts synergistically with glucocorticoid treatment, human B-cell precursor leukemic cell lines MHH cALL 2 (PPR) and MHH cALL 3 (PGR) were treated with prednisone (6.2 μM) and various concentrations of bortezomib (1.5-12 nM) for up to 96 hours.

Both cell lines showed a dose-dependent increase in apoptosis after bortezomib single treatment (Fig. 1). Single therapy with 12 nM bortezomib-induced in PGR a 42-fold and in PPR cells a 29-fold higher caspase activity compared to treatment with prednisone alone. However, only the PGR cells showed an additive effect on apoptosis induction after combined treatment with prednisone and bortezomib. In the PPR cell line, the amount of PI-positive, lethal cells increased within 48 hours to 20-fold after combined treatment, using 7 nM bortezomib, compared to single prednisone, and 3-fold compared to single bortezomib treatment. In contrast, apoptosis was reduced compared to bortezomib single treatment.

Fig. 1 Increase of PI-positive cells in human B-cell precursor ALL cell lines (MHH cALL 2 and MHH cALL 3). (Junk S, et al., 2009)

Expression of Mer in ALL Cell Lines Correlates with a Quiescent Phenotype in CNS Cocultures

Patients with t(1;19)-positive ALL are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. High Mer expression was detected in MHH-CALL3 and 697 cells with the t(1;19) translocation and REH and UoCB6 cells with the t(12;21) translocation. Low or absent Mer expression was found in SUP-B15 cells carrying the t(9;22) translocation, RS4;11 cells with t(4;11), and in the promyelocytic AML cell line HL-60 (Fig. 2A).

Hypothesizing a role for CNS glia in supporting the survival of Mer-expressing ALL cells, a coculture model with U343 cells was established derived from a low-grade mixed astrocytoma/oligodendroglioma. Coculture of ALL cell lines with U343 for 48 hours resulted in a reduction of viable cells in all cell lines analyzed (Fig. 2B). The observed effect was more pronounced in Mer^high cells and less appreciable in Mer^low cells (Fig. 2B). U343 coculture induced a quiescent phenotype in ALL cells characterized by an arrest in G0/G1 and a drop in S-phase (Fig. 2C-H).

Fig. 2 U343 coculture induces quiescence in Mer-expressing ALL cell lines (MHH-CALL3, 697, REH, UoCB6, SUP-B15, and RS4;11). (Krause S, et al., 2015)