Proteasome subunit expression analysis and chemosensitivity in relapsed paediatric acute leukaemia patients receiving bortezomib-containing chemotherapy

Abstract

Background: Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL).

Methods: We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children's Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1) and AML (COG-AAML07P1). Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12) obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test.

Results: Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both β5i/β5 and β1i/β1 subunits (p = 0.004 and p < 0.001). These ratios correlated with therapy response in AML patients; β1i/β1 ratios were significantly higher (p = 0.028) between patients who did (n = 4) and did not reach complete remission (CR) (n = 8), although for β5i/β5 ratios, this did not reach significance. For ALL patients, the subunit ratios were also higher for patients who showed a good early response to therapy but this relation was not statistically significant. Overall, for this study, the patients were treated with combination therapy, so response was not only attributed to proteasome inhibition. Moreover, the leukaemic blast cells were not purified for these samples.

Conclusions: These first ex vivo results encourage further studies into relative proteasome subunit expression to improve proteasome inhibition-containing therapy and as a potential indicator of bortezomib response in acute leukaemia.

Keywords: Bortezomib; Immunoproteasome; Pediatric acute leukaemia; Proteasome inhibitor.

Fig. 1

Proteasome subunit protein expression in relapsed childhood ALL and AML. Comparison of a constitutive subunit β1 b and constitutive subunit β5 expression for pre-B ALL (n = 46), T-ALL (n = 14) and AML (n = 12) patient samples and PBMCs from healthy adult volunteers (n = 5). c The ratio of paired subunits β1i/β1. d The ratio of paired subunits β5i/β5, within each patient sample for pre-B ALL (n = 46), T-ALL (n = 14) and AML (n = 12) patient samples. Protein expression was assessed by Western blotting and expressed as relative quantifications of subunit expression (ratio proteasome subunit/β-actin based on loading of 15 μg total protein, normalized to CEM). The line denotes the mean. Significant p values are noted (Mann-Whitney U test)

Fig. 2

Ratio proteasome subunit protein expression in patients who achieved complete remission versus patients who did not, dissected by leukaemia subtype. Ratios of immunoproteasome/constitutive proteasome protein expression in a AML patients, b pre-B ALL patients and c T-ALL patients that achieved CR versus patients who did not achieve CR, determined by Western blot analysis after bortezomib-containing re-induction therapy. The line denotes the mean

Fig. 3

Proteasome subunit catalytic activity in relapsed childhood ALL and AML patients. a Correlation of β5i/β5 protein expression and β5i/β5 catalytic activity ratios. b β5i/β5 catalytic activity ratios in pre-B ALL patients (n = 18) compared to T-ALL patients (n = 4) and AML patients (n = 10), and c β5i/β5 catalytic activity ratios in patients who achieved complete remission (n = 14) versus those who did not (n = 18). Closed circles represent pre-B ALL patients, open triangles T-ALL patients and open squares AML patients. The line denotes the mean

Fig. 4

Baseline NF-kB activity between ALL and AML patients and impact of bortezomib treatment. Pre-treatment NF-kB activity measurements determined by p65-ELISA in PBMC of acute leukaemia patients comparing a NF-kB activity of pre-B ALL (n = 26), T-ALL (n = 10) and AML (n = 12) patients, and b NF-kB activity prior to treatment compared to 6 and 24 h after bortezomib-containing chemotherapy in pre-B ALL patients that reached CR (n = 16), and c pre-B ALL patients that did not reach CR (n = 10). The line denotes the mean