Page 68 - 2022_01-Haematologica-web

B.Z. Carter et al.
CyTOF mass cytometry
Cells were stained with metal-tagged antibodies (Online Supplementary Table S2) against cell surface markers, barcoded, pooled, then stained with metal-tagged antibodies against intra- cellular proteins and subjected to cytometry by time of flight (CyTOF) analysis as described previously.34,36,37 Briefly, viable sin- gle cells (cisplatin-low) were gated with FlowJo (software v10.7, FlowJo LLC) and exported as flow cytometry standard (FCS) data for subsequent analysis in Cytofkit.38 Cell populations identified and embedded by RPhenoGraph in the “Cytofkit_analyzedFCS” files were gated in FlowJo to quantify marker expression. ArcSinh-transformed counts for each protein expression in desired cell populations were visualized with heat maps.
Statistical analysis
Cell line experiments were performed in triplicates. Results were expressed as the mean ± standard error of the mean (SEM). The Student t-text was used to assess differences between groups; P-values ≤0.05 were considered statistically significant. The combination index (CI),39 determined using Calcusyn soft- ware, was expressed as mean CI values at effective dose (ED)50, ED75, and ED90. A CI<1 was considered synergistic; CI=1, additive; and CI>1, antagonistic. EC50 and half maximal inhibitory concen- tration (IC50) values were calculated using Calcusyn. Mouse sur- vival was estimated using the Kaplan-Meier method. Survival data were analyzed using the log-rank test.
Results
MCL-1 regulates metabolic functions in acute myeloid leukemia (AML) cell lines and its inhibition decreases AML metabolism
MCL-1-KD or -OE did not alter viability of AML cell line Molm13, MV4-11, or OCI-AML3 in vitro (Online Supplementary Figure S1A). However, NSGS mice harboring MCL-1-OE Molm13 cells exhibited higher leukemia bur- den and succumbed sooner to AML, while mice harboring MCL-1-KD OCI-AML3 cells had decreased leukemia bur- den and survived longer compared with their respective controls (Online Supplementary Figure S1B), suggesting that MCL-1 levels affect leukemia expansion. In order to deter- mine whether this effect requires the modulation of bioen- ergetic/metabolic activity, we assayed mitochondrial respi- ration and found that MCL-1-OE increased, whereas MCL-1-KD or inhibition with AZD5991 (at doses not affecting cell viability) decreased, OCR and ATP produc- tion in OCI-AML3 cells (Figure 1A). Compared with parental MV4-11 (MV4-11P), the venetoclax-resistant MV4-11 (MV4-11R) cells that expressed increased MCL-127 had higher OCR and ATP production. In both cell types, AZD5991 inhibited OCR and ATP generation (Online Supplementary Figure S1C). In addition, compared with con- trols, MCL-1-OE OCI-AML3 cells exhibited higher levels of cellular and mitochondrial ROS, lower GSH levels, and lower GSH/GSSG ratios, whereas MCL-1-KD OCI-AML3 cells displayed lower ROS, higher GSH levels, and higher GSH/GSSG ratios (Figure 1B and C).
In order to further explore metabolic functions of MCL- 1, we traced 13C incorporation from 13C2-1,2-glucose and 13C5-glutamine into downstream metabolites40 using an MCL-1-KD clone (shC16) with an approximately 65% MCL-1 reduction (Online Supplementary Figure S1D), selected by limiting dilution of MCL-1-KD OCI-AML3 cells and OCI-AML3 cells treated with 50 nM AZD5991
(Online Supplementary Figure S1E). Metabolomic analysis revealed markedly lower overall incorporations of 13C from both glucose and glutamine into key tricarboxylic acid (TCA) cycle intermediates including citrate, fumarate, and malate, in MCL-1-KD and AZD5991-treated OCI- AML3 cells compared to their respective controls (Figure 1D). An examination of the patterns of fractional 13C enrichment from 13C5-glutamine into citrate, malate, and fumarate demonstrated increased M+4, but decreased M+3 fractional enrichment, in both AZD5991-treated and MCL-1-KD OCI-AML3 cells (Figure 1E), suggesting decreased glutamine entry into the TCA cycle accompa- nied by a smaller fraction of glutamine-derived carbon being used for anaplerotic reactions that fuel biosynthesis.
In both 13C2-1,2-glucose and 13C5-glutamine tracing exper- iments, the total amount of lactate secreted by AZD5991- treated or MCL-1-KD cells was statistically significantly lower than that secreted by their respective controls (Figure 1F). Notably, statistically significant 13C enrichment (M>0) in the secreted lactate of both the AZD5991-treated and MCL-1-KD OCI-AML3 cells, was observed from 13C2- 1,2-glucose, but not 13C5-glutamine (Figure 1G). Glycolysis of 13C2-1,2-glucose generates lactate with two 13C labels (M+2 lactate). Thus, a reduction in M+2 lactate secretion suggests MCL-1 inhibition decreases glycolytic flux.
Next, we assessed whether the decrease in glycolytic flux was accompanied by a decrease in flux through the oxidative branch of the pentose phosphate pathway (oxPPP), a major pathway involved in the generation of the reducing equivalent NADPH that is used for ROS neutralization. When 13C2-1,2-glucose is metabolized through the oxPPP and re-enters glycolysis via the non- oxPPP, the elimination of one 13C carbon through decar- boxylation yields lactate containing only one 13C. The relative oxPPP flux, which we calculated by multiplying the ratio of [M+1]/([M+1]+[M+2]) from extracellular lac- tate with lactate secretion rate during 13C2-1,2-glucose tracing, was lower in both AZD5991-treated and MCL- 1-KD OCI-AML3 cells than in their respective controls (Figure 1H).
6-Phosphogluconic acid levels were also greatly decreased under MCL-1 inhibition (Figure 1H), in accor- dance with oxPPP modulation.41 Consistent with the aforementioned extracellular flux assay results, AZD5991- treated, and MCL-1-KD OCI-AML3 cells, exhibited decreased ATP levels (Figure 1I). These findings indicated that MCL-1 inhibition downregulates key pathways that support cellular energetics in AML cells as summarized in Figure 1J. Furthermore, MCL-1 modulated the level of c- MYC in OCI-AML3 cells (Online Supplementary Figure S1F), which is a well-known metabolic regulator in malig- nant cells.
MCL-1 regulates adhesion molecule expression in acute myeloid leukemia (AML) cell lines and AML-stroma interactions
Since MSC in the BM microenvironment upregulate MCL-1, we investigated whether MCL-1 had a role in leukemia-stroma interactions. MCL-1-OE OCI-AML3 cells exhibited increased surface expression of CXCR4 and CD44, both of which participate in leukemia-MSC interac- tions and drug resistance, and increased OCI-AML3 migra- tion towards, and adhesion to, MSC (Figure 2A). Conversely, MCL-1-KD OCI-AML3 exhibited decreased surface expression of CXCR4 and CD44 and decreased
60
haematologica | 2022; 107(1)