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Roles of ALDH in T-ALL
ally critical residues, such as the NAD binding site and the enzymatic catalytic residues, but lacks 96 amino acids in the N-terminus (Figure 2A) that are a part of the homote- trameric interface (Figure 2B). Hence, we postulated that the short isoform might have attenuated enzymatic activ- ity or a different subcellular localization compared to the long isoform.
To investigate this, we first produced each isoform of ALDH1A2 as a purified recombinant His-tagged protein (Online Supplementary Figure S2A) and performed an in vitro enzymatic assay. We incubated each isoform with a sub- strate (retinaldehyde) and a coenzyme (NAD+) and meas- ured the amount of NADH produced over 20 min (Figure 2C). This analysis revealed that the short isoform had enzymatic ability, although slightly lower than that of the long isoform (Figure 2C, Online Supplementary Figure S2B). Next, we analyzed the effect of ALDH1A2 on the produc- tion of retinoic acid using a reporter system. We cloned a trimerized retinoic acid responsive element, which can be activated by the retinoic acid receptor complex, into the luciferase plasmid and then established Jurkat cells that stably express this construct29 (Online Supplementary Figure S2C). In this setting, the luciferase activity is dependent on the amount of retinoic acid produced internally in the Jurkat cells. We then knocked down the endogenous ALDH1A2 by lentiviral shRNA transduction. The luciferase activity was significantly downregulated after ALDH1A2 had been depleted (Online Supplementary Figure S2C), indicating that the short isoform of ALDH1A2 can mediate the production of retinoic acid in T-ALL cells.
We next analyzed the subcellular localization of the short isoform of the endogenous ALDH1A2 protein. We used the CRISPR/Cas9-mediated method to introduce an EGFP gene fused to the 3’ end of the endogenous ALDH1A2 gene into both Jurkat and K562 cells (Online Supplementary Figures S2D and E). We then analyzed the localization of the EGFP signal in the nucleus (with Hoechst staining), mitochondoria (with Mitotracker stain- ing) or cytoplasm (without staining). The results demon- strated that the short isoform of ALDH1A2 in Jurkat cells was localized in the cytoplasm with no co-localization in mitochondria or the nucleus (Figure 2D, Online Supplementary Figure S2F), which was similar to the pat- tern observed for the K562 cells that expressed the long isoform. These results indicate that the short isoform of ALDH1A2 possesses intact enzymatic functions and the same localization pattern as observed for the long isoform.
ALDH1A2 supports T-cell acute lymphoblastic leukemia cell survival and viability
We next analyzed whether ALDH1A2 expression con- fers any functional advantage to T-ALL cells. We first eval- uated the phenotype after depletion of ALDH1A2 by blocking the regulatory element. We designed two inde- pendent sgRNA (#1 and #2), which targeted the TAL1- bound region (Figure 3A). We transduced each of them under a doxycycline-inducible system together with a cat- alytically inactive Cas9 (dCas9) protein fused with the KRAB repressor, thereby epigenetically silencing the tran- scriptional activity at the TAL1-bound region. We observed successful downregulation of ALDH1A2 protein after the induction of each sgRNA (Figure 3B). Importantly, apoptotic cell death was induced after 72 h of induction, as evidenced by the cleavage of caspase-3 and PARP (markers of apoptosis) (Figure 3B).
To independently validate this result, we utilized a lentiviral shRNA system to knock down ALDH1A2 and analyzed the number of annexin-V-positive cells, which is another means to detect apoptosis. The result confirmed that depletion of ALDH1A2 induced apoptosis in multiple TAL1/ALDH1A2-positive T-ALL cell lines but not in the negative cell lines (Figure 3C). Consistently, cell viability was reduced after the shRNA knockdown of ALDH1A2 in an ALDH1A2-positive cell line (Jurkat) (Figure 3D). Conversely, forced expression of ALDH1A2 in an ALDH1A2-negative T-ALL cell line (DND-41) increased cell viability (Figure 3E).
WIN 18,446 is reported to be a pan-ALDH1A inhibitor.30 It has been shown to strongly inhibit the enzymatic activ- ity of ALDH1A2 both in vitro and in vivo.31,32 Since ALDH1A2 is the only member of the ALDH1A family of genes expressed in T-ALL (Online Supplementary Figure S3A), we tested the effect of WIN 18,446 on cell viability of several T-ALL cell lines. Strikingly, two TAL1/ALDH1A2-positive cell lines (Jurkat and RPMI-8402) were more sensitive to this small-molecule inhibitor than two TAL1/ALDH1A2-negative cell lines (KOPT-K1 and DND-41) (Figure 3F, Online Supplementary Figure S3B). These results indicate that the expression of ALDH1A2 is associated with the viability and survival of T-ALL cells. This phenotype was not rescued by the addition of all- trans retinoic acid (Online Supplementary Figure S3C), thus suggesting that this mechanism is likely independent of the amount of retinoic acid produced.
ALDH1A2 affects metabolic pathways in T-cell acute lymphoblastic leukemia cells
We next investigated the molecular mechanisms by which ALDH1A2 supports cell viability and prevents apoptosis. We performed global gene expression profiling by RNA-sequencing after sgRNA-mediated ALDH1A2 depletion. We selected genes that were significantly downregulated (n=96) or upregulated (n=19) in the ALDH1A2-depleted samples compared to the control sam- ples (Figure 4A, Online Supplementary Figure S4A, Online Supplementary Table S2). Interestingly, the downregulated genes included a number of metabolic enzymes and trans- porters involved in the glycolysis pathway (yellow, Figure 4A and B, Online Supplementary Figure 4B and C). In con- trast, several enzymes involved in amino acid metabolism, such as ASS1 and ASNS, were upregulated after ALDH1A2 depletion (Figure 4A, Online Supplementary Figure S4A). The result was validated for each enzyme individually by qRT-PCR (Online Supplementary Figure S4D).
This finding prompted us to analyze the metabolic state of the T-ALL cells. We performed a capillary electrophore- sis time-of-flight mass spectrometry analysis to measure the relative levels of 200 metabolites involved in the major metabolomics pathways after depletion of ALDH1A2 in Jurkat cells (Figure 4C). Strikingly, ALDH1A2 depletion resulted in a reduction in the intermediate metabolites involved in the glycolysis pathway (pink). Suppressed glu- cose metabolism was accompanied by a reduction in acetyl-CoA (yellow), which is one of the key carbon sources that drive the TCA cycle. We also observed a reduction in citric acid, cis-aconitic acid and isocitric acid (yellow), likely due to the deceased integration of glycoly- sis-derived acetyl-CoA into the TCA cycle.
It is noteworthy that the levels of metabolites derived
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