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C. Zhang et al.
metabolism of membrane lipids, amino acids, carbohy- drates and steroids.16 The reaction is NAD(P)+-dependent and generates an important component of the cellular antioxidant system, NAD(P)H, which also acts as an essential coenzyme in several metabolic pathways, such as glycolysis and the tricarboxylic acid (TCA) cycle.14 Retinaldehyde dehydrogenases, a subfamily of ALDH genes, including ALDH1A2, are also capable of converting retinaldehyde into retinoic acid. ALDH activity has also been implicated as a cancer stem cell marker in various solid tumors.17,18 The Aldefluor assay is commonly used to isolate an ALDH-positive population to refine the cancer stem cell population.17,19 Overexpression of ALDH genes confers drug resistance to cancer cells.20 These findings suggest that high levels of ALDH activity may be advan- tageous and, even required, for cancer cell maintenance.
In this study, we elucidated the molecular function and role of ALDH1A2 in the pathogenesis of T-ALL. We demon- strated that ALDH1A2 is directly activated by TAL1 and protects against intracellular stress, supporting leukemia cell metabolism and promoting leukemia cell survival.
Methods
Cell samples
Human leukemia cell lines were cultured in RPMI-1640 medi- um (BioWest) supplemented with 10% fetal bovine serum (BioWest). A T-ALL patient-derived xenograft sample was provid- ed by Alejandro Gutierrez (Boston Children’s Hospital) and expanded in NSG mice. Mouse protocols were approved by the Institutional Animal Care and Use Committee.
Knockdown and overexpression experiments
For knockdown experiments, short-hairpin RNA (shRNA) was inserted into a pLKO.1-puro lentivirus vector. For overexpression experiments, ALDH1A2 cDNA cloned from Jurkat cells was insert- ed into a MSCV-IRES-GFP retrovirus vector. For inhibition of the regulatory element, single-guide RNA (sgRNA) was inserted into a FgH1tUTG lentivirus vector (Addgene plasmid #70183). Virus was produced by co-transfecting the construct with the packaging and envelope plasmids into 293T cells using FuGENE6 (Roche).
Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from cells using a NucleoSpin RNA kit (Macherey-Nagel) and reverse-transcribed using a QuantiTect kit (Qiagen). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on a QuantStudio3 real-time PCR system (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix (Roche). The primer sequences are described in the Online Supplementary Information.
Western blot analysis
Equivalent amounts of protein were resolved on a sodium dodecylsulfate polyacrylamide gel electrophoresis gel, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and subject- ed to immunoblot analysis with specific antibodies against ALDH1A2 (Abcam), TAL1 (Millipore), PARP, caspase-3, α-tubulin, and b-actin (Cell Signaling Technology). The proteins were detect- ed using an enhanced chemiluminescence reagent (Thermo).
Confocal microscopic imaging
Images of EGFP-fused ALDH1A2 protein localization in live cells were captured using an Olympus FV1000 TIRF confocal
microscope (C4). Colocalization of signals was analyzed using IMARIS 9.5 software after staining with Mito Tracker Red CMXRos (Thermo) and Hoechst.
Intracellular reactive oxygen species assay
The cells were treated with freshly prepared CellROX Deep Red reagent (Thermo Fisher Scientific). Fluorescence signals were analyzed with a BD SR II flow cytometer using BD FACSDivaTM software and FlowJo software.
Metabolomic profiling
The extracted metabolites were used for a capillary elec- trophoresis time-of-flight mass spectrometry basic scan, conduct- ed by Human Metabolome Technologies (see details in the Online Supplementary Information). The number of metabolites was nor- malized to cell volume detected by a Scepter 2.0 cell counter (Millipore).
Extracellular flux analysis
The cells were resuspended in Seahorse assay medium (Agilent) and seeded into XF24 plates. Extracellular acidification rate and the oxygen consumption rate were measured by Seahorse XF24 (Agilent). The details are provided in the Online Supplementary Information.
Chromatin immunoprecipitation sequencing and RNA-sequencing
Chromatin immunoprecipitation (ChIP)-sequencing analysis was done in our previous study.21 For RNA-sequencing, RNA was extracted using an miRNeasy kit (Qiagen) followed by DNase treatment (Ambion). Construction of the strand-specific library and sequencing of the single-end 100-bp-long reads by a BGISEQ sequencer were conducted at BGI Biotech (Hong Kong). The details are described in the Online Supplementary Information.
Zebrafish transgenesis
Zebrafish protocols were approved by the Institutional Animal Care and Use Committee. The human ALDH1A2 or mCherry gene was cloned under the zebrafish rag2 promoter and injected into zebrafish embryos to create Tg(rag2: ALDH1A2) or a control Tg(rag2: mCherry) line. The rag2-myr-mAKT2 construct was kindly provided by Alejandro Gutierrez.22
Results
The ALDH1A2 gene is directly activated by the TAL1 complex in T-cell acute lymphoblastic leukemia
To identify the targets directly regulated by TAL1 in T- ALL cells, we referred to a previously performed ChIP- sequencing dataset for TAL1 and other members of the transcriptional complex in a T-ALL cell line (Jurkat).13 We integrated this result with the RNA-sequencing dataset after knocking down each factor in the same cell line, which was generated in our recent study,21 to select genes that were positively regulated by the TAL1 complex. We further filtered genes that were associated with a high level of an active histone mark (H3K27ac) in T-ALL cells but not in normal thymus cells (Online Supplementary Figures S1A and B), representing high-confidence TAL1 tar- gets that were aberrantly activated in T-ALL cells.
ALDH1A2 was among the top hits. An early study showed that the TAL1/GATA3/LMO complex binds at the intronic region of the ALDH1A2 gene and induces its expression in T-ALL cells.12 Consistently, our ChIP-
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haematologica | 2021; 106(6)