Abundant anti‐apoptotic BCL‐2 is a molecular target in leukaemias with t(4;11) translocation

Abstract

Summary Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti‐apoptotic BCL‐2 expression and target modulation were characterized in cell lines with t(4;11) and BCL‐2 expression was examined in MLL and non‐MLL infant/paediatric leukaemia cases by Western blot analysis and/or real‐time polymerase chain reaction. Cytotoxicity of Genasense™ (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3‐4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single‐agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4‐11 cells to select anti‐leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S‐phase progression. The abundant BCL‐2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4‐11 cells, and these effects occur through apoptosis.

Potent obatoclax cytotoxicity and activation of triple death mode killing across infant acute lymphoblastic leukemia

“…Molecular-cytogenetic classification by MLL status and partner genes was described. 5,16 An apheresis sample from a 6.5-year-old boy (WBC, 408 3 10 3 /mL) with MLL-AF4 ALL was obtained from the Children's Hospital of Philadelphia. Mononuclear cells were enriched by Ficoll-Paque (Amersham, Pittsburgh, PA) centrifugation before cryopreservation of diagnostic specimens.…”

“…5 Propidium iodide (PI) uptake, activated caspase 3, and terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay data were analyzed using CellQuest Pro version 5.2 (BD Biosciences). Cell cycle was modeled in live cells, using ModFit LT (Verity Software House, Topsham, ME).…”

“…Three-dimensional scatter plots were generated using the modeling software CombiTool (version 2.001; Bäretswil, Switzerland) to graph CIs of actual drug interactions in MTT assays and predicted Loewe additivity zero-interaction response surfaces. 5 A sigmoid E max model (top down to bottom, variable slope) within Prism was used to calculate EC 50 s for all cell line assays. Obatoclax-chemical inhibitor combinations were compared in Excel (Microsoft, Redmond, Washington) by t-test with 2-tailed distribution.…”

Aberrant Proliferative and Apoptotic Pathways in Acute Lymphoblastic Leukemia (ALL): Molecular Therapies to Overcome Chemo-Resistance

“…They have shown that Bcl-2 elimination yielded rapid loss of leukemic cells and significantly prolonged mouse survival, formally validating Bcl-2 as a rational target for a targeted cancer therapy (Letai et al, 2004). Furthermore, high Bcl-2 levels are observed also in ALL characterized by t(4;11) (Robinson et al, 2008). It has been demonstrated that the equilibrium in the formation of Bcl-2:Bax heterodimers (suppressors of death) and Bax:Bax homodimers (activators of death) appears to be central in the molecular regulation of apoptosis (Coustan-Smith et al, 1996).…”

Cotargeting BCL-2 and BCL-XL for maximal efficacy in ALL

“…The one exception identified, where targeting BCL-2 alone appeared to be sufficient, was MLLr-ALL. Previous studies have demonstrated high expression of BCL-2 in MLLr pediatric ALL 8 and shown activity of the 1st generation BCL-2 antagonists in MLLr-ALL, supporting the findings by Khaw et al 8 It has been recently shown that MLL/ AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79 methylation at the BCL-2 locus, and this may explain the higher sensitivity of MLLr-ALL xenografts to venetoclax. 9 These data suggest that venetoclax alone may be the agent of choice in children with MLLr-ALL, whereas navitoclax may be more efficacious in non-MLL pediatric ALL (see figure).…”