The synergism of MCL1 and glycolysis on pediatric acute lymphoblastic leukemia cell survival and prednisolone resistance

Abstract

In vitro and in vivo resistance to prednisolone are predictive for an adverse prognosis in pediatric precursor B-acute lymphoblastic leukemia. Causes of resistance are still poorly understood. In this study, we observed that prednisolone exposure of prednisolone-sensitive patients' leukemic cells decreased anti-apoptotic MCL1 protein levels by 2.9-fold, while MCL1 protein expression in prednisolone-resistant leukemic patients' cells was unaffected (P<0.01). Locked nucleic acid oligonucleotides directed against MCL1 reduced MCL1 protein levels by 82±16% (P<0.05) in leukemic cells, decreased proliferation by 9-fold and sensitized to prednisolone up to 80.8-fold, compared to a non-silencingcontrol locked nucleic acid (P<0.05). Remarkably, we discovered that MCL1-silencing up-regulated the glucose consumption of leukemic cells by 2.5-fold (P<0.05), suggesting a potential rescue mechanism mediated by glycolysis. Targeting glycolysis by 2-deoxyglucose synergistically inhibited leukemic survival by 23.2-fold in MCL1-silenced cells (P<0.05). Moreover, 2-deoxyglucose and MCL1 locked nucleic acid concomitantly sensitized leukemic cells to prednisolone compared to MCL1 locked nucleic acid or 2-deoxyglucose alone (P<0.05). In conclusion, these results indicate the need to target both MCL1 and glycolysis simultaneously to inhibit leukemic survival and sensitize acute leukemia patients towards prednisolone. ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3patients as approved by the local institutional review board. Only samples with 90% or over leukemic cells upon processing were used in the present study. Reh, 697, Sem, Jurkat, Loucy and HEK293T cells were obtained from DMSZ. The leukemic cell lines were cultured in RPMI+Glutamax (Gibco) and HEK293T cells in DMEM+Glutamax (Gibco) at 37°C in humidified air containing 5% CO 2 . Cell viability and cell count were determined by a trypan blue exclusion staining assay and analyzed by MACSQuant. LNA transfection and 2-deoxyglucose treatmentCell lines were cultured in the presence of either 10 mM locked nucleotide acid oligonucleotides directed against MCL1 (MCL1 LNA), i.e. SPC4120 (MCL1 LNA-a), SPC4342 (MCL1 LNA-b), SPC4343 (MCL1 LNA-c), or a non-silencing control oligonucleotide LNA, i.e. SPC3088. Twenty-four hours after LNA transfection, cells were cultured with and without 0.5 mM 2-deoxyglucose (Sigma). Online Supplementary Figure S1A and B illustrates that 0.5mM 2-deoxyglucose only modestly hampers cell count, while it has a quantifiable effect on glucose consumption. After 96 h, culture medium was replaced by fresh medium containing fresh LNA +/-2-DG. Apoptosis measurementAnnexinV/PI double positive and AnnexinV single positive cells were measured on the BD FACS Calibur flow cytometer. Quantitative RT-PCRMCL1 mRNA levels were quantified by incorporation of SYBR Green (Thermo Scientific) by quantitative real-time PCR (Applied Biosystems 7900HT). Primers for MCL1 were; 5'-GGAGGAG-GACGAGTTGTAC-3' (forward) and 5'-AAG GCA CCA AAA GAA ATG-3' (reverse). Reverse phase...

SLC2A5 overexpression in childhood philadelphia chromosome‐positive acute lymphoblastic leukaemia

“…Glycolysis is critical in childhood ALL. Several pre‐clinical studies showed that aerobic glycolysis inhibitors (Hulleman et al , ; Aries et al , ; Leni et al , ), as well as other metabolic pathway inhibitors (Hermanova et al , ), showed direct anti‐cancer cytotoxicity or synergised with other anti‐leukaemia agents in childhood leukaemia. Therefore, we performed microarray‐based analyses to screen glycolysis/glycogenesis‐related gene expression in childhood B‐ALL and found that SLC2A5 was overexpressed in Ph+ALL patients.…”

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress

“…Recently, the antiapoptotic protein Mcl-1 was shown to be required for cell survival in BCR-ABL+ leukemia [11], and to play a role in the survival of ALL [12, 13], CML [15], hematopoietic stem cells [35], T-lymphocytes [36], and plasma cells [37]. Herein, we demonstrate that Mcl-1 plays a crucial role in the survival of BCR-ABL+ ALL cells undergoing energy/ER-stress.…”

Enhanced Fructose Utilization Mediated by SLC2A5 Is a Unique Metabolic Feature of Acute Myeloid Leukemia with Therapeutic Potential

“…Inhibition of glycolysis using a glucose analog, 2-deoxyglucose (2-DG) has already been shown to re-sensitize acute lymphoblastic leukemia (ALL) cells to prednisolone therapy (Hulleman et al, 2009). The combination of 2-DG and an MCL1 anti-apoptotic protein inhibitor is proved to be synergistic in overcoming prednisolone resistance in ALL cells (Aries et al, 2013). Our recent study of AML cell lines revealed enhanced expression of genes relating to both glycolysis and the TCA cycle (Chen et al, 2014).…”