Research ArticleHEALTH AND MEDICINE

Dietary thiamine influences l-asparaginase sensitivity in a subset of leukemia cells

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Science Advances  09 Oct 2020:
Vol. 6, no. 41, eabc7120
DOI: 10.1126/sciadv.abc7120
  • Fig. 1 Functional genomics identifies metabolic determinants of proliferation under ASNase treatment.

    (A) Right: Schematic outlining cell line competition assay. Left: Log2 fold change in abundance from initial pool, of barcodes (n = 3) representing indicated cell lines in the competition assay, for ASNase-treated tumors (n = 10) relative to mean of vehicle-treated tumors (n = 10). Boxes represent the median and first and third quartiles, and whiskers represent the minimum and maximum of all data points. Statistics: false discovery rate (FDR)–adjusted P, by two-tailed unpaired t test for unequal variances, of ASNase tumor group versus vehicle tumor group. Individual CCLE RNA-seq ASNS expression levels of cell lines are also shown (x indicates no data available). (B) Schematic depicting pooled CRISPR screen under ASNase treatment (0.25 U/ml) using a metabolism-focused single-guide RNA (sgRNA) library. (C) Left: The top 25 genes differentially required under ASNase treatment are shown. Right: Gene scores for Jurkat cells grown in untreated versus ASNase-treated vessels. Most genes, as well as nontargeting control sgRNAs, gave similar scores in untreated and treated vessels. AA, amino acid. (D) Log2 fold change in the abundance of individual sgRNAs in untreated (black) or ASNase-treated (gray) for top-scoring genes, ASNS, TPK1, GOT2, and MDH2. (E) Schematic demonstrating that top-scoring genes in the CRISPR screen highlight a specific route from glutamine to asparagine as essential under ASNase treatment.

  • Fig. 2 TPP enables de novo asparagine synthesis and proliferation under ASNase treatment.

    (A) Immunoblot analysis of vector control and two clonal TPK1 KOs made from Jurkat cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Fold change in cell number (log2) of vector control, TPK1_KO1, and TPK1_KO2 Jurkat cells, after untreated or 0.0005 U/ml ASNase conditions for 5 days (mean ± SD, n = 3). Statistics: P < 0.05 by two-tailed unpaired t test for equal variances, for all 15 untreated-treated pairs. (C) Schematic depicting a metabolic route of asparagine synthesis from glutamine. Filled circles represent 13C atoms derived from [U-13C]-glutamine. (D to H) Total abundance [a.u. (arbitrary units)] of indicated metabolites derived from labeled glutamine in vector control, TPK1_KO1, and cDNA-rescued TPK1_KO1 Jurkat cells. Cells were incubated for 24 hours in medium containing [U-13C]-glutamine (2 mM) in the presence or absence of asparagine (378 μM) and TPP (3 μM). Colors indicate mass isotopologs (mean ± SD, n = 3).

  • Fig. 3 SLC19A2 expression is a determinant of growth at physiologically relevant thiamine concentrations.

    (A) Log2 fold change in abundance since initial pool, of DNA barcodes representing indicated cell lines in a competition assay, for cells grown in the absence of thiamine (−thiamine), relative to growth responses in the presence of 3 μM thiamine (+thiamine) (mean ± SD, n = 3 independent barcodes). Media were supplemented with 10% dialyzed FBS (dFBS), which contributes ~1 nM thiamine. (B) Low-thiamine cell competition responses were correlated with CCLE mRNA expression data of metabolic genes, and thiamine transporter 1 (SLC19A2), but not thiamine transporter 2 (SLC19A3), had one of the top-scoring Pearson’s correlation coefficients. TPM, transcripts per million. (C) Left: Gene scores for a CRISPR screen done with Jurkat cells in the presence (+thiamine) or absence (−thiamine) of 3 μM thiamine. Media were supplemented with 10% dialyzed FBS, which contributes ~1 nM thiamine. Most genes gave similar scores in the two conditions. Right: The top 10 genes differentially required in low thiamine are shown. (D) Log2 fold change in the abundance of individual sgRNAs in our CRISPR screen, in the presence (+thiamine) or absence (−thiamine) of 3 μM thiamine, for SLC19A2 (top) and SLC19A3 (bottom). (E) Baseline mRNA levels by real-time quantitative polymerase chain reaction (RT-qPCR) of indicated genes for cell lines representing the following cancers: T-ALL (JURKAT), B-ALL (RCH-ACV, KOPN8, REH, and NALM6), diffuse large B cell lymphoma (SUDHL4), and Burkitt lymphoma (ST486). ND, not detected (mean ± SD, n = 3).

  • Fig. 4 Physiological thiamine is limiting for ASNase response of SLC19A2-low cells.

    (A) Fold change in cell number (log2) of vector control and SLC19A2 cDNA–expressing REH cells, after untreated or 0.001 U/ml ASNase conditions for 6 days, in the presence (+) or absence (−) of 3 μM thiamine (mean ± SD, n = 3). Media were supplemented with 10% regular FBS, which contributes ~60 nM thiamine. P < 0.05 for all four untreated-treated pairs. (B) Fold change in cell number (log2) of SLC19A2-low (NALM6 and REH) and SLC19A2-high (JURKAT) wild-type cell lines, after untreated or 0.001 U/ml ASNase conditions for 7 to 9 days, at different thiamine concentrations added to thiamine-free RPMI supplemented with 10% double-dialyzed FBS (mean ± SD, n = 3). P < 0.05 for untreated-treated pairs at these thiamine concentrations: JURKAT, all concentrations; NALM6, all above 1.25 nM; REH, all above 0 nM. (C) 13C-thiamine uptake in vector control–, sgSLC19A2_1-, and sgSLC19A2_2-expressing Jurkat cells (mean ± SD, n = 3). (D) Fold change in cell number (log2) of vector control–, sgSLC19A2_1-, and sgSLC19A2_2-expressing Jurkat cells, after untreated or 0.001 U/ml ASNase conditions for 5 days, at different thiamine concentrations added to thiamine-free RPMI supplemented with 10% double-dialyzed FBS (mean ± SD, n = 3). P < 0.05 for all 15 untreated-treated pairs. Statistics for (A), (B), and (D): two-tailed unpaired t test for equal variances.

  • Fig. 5 Dietary thiamine intake influences ASNase sensitivity of SLC19A2-low leukemia cells in vivo.

    (A) Plasma thiamine profiling of mice on conventional chow (n = 3 mice), and mice on a modified AIN-93G purified diet of low thiamine content (n = 8 mice) (mean ± SD). Human serum was profiled simultaneously for relative comparison. Statistics: *P < 0.01 by two-tailed unpaired t test for unequal variances. n.s., not significant. (B) Weights of mice initially on conventional chow, then switched to standard AIN-3G purified diet for 1 week, and lastly switched to a modified AIN-93G purified diet of low thiamine content (n = 8 mice) for indicated times, relative to initial weights on chow (mean ± SD). (C) Left: Kaplan-Meier survival curves of NSG mice on high- or low-thiamine AIN-93G diets engrafted with REH (endogenously low SLC19A2 cell line) by tail vein injection and treated with vehicle or ASNase (1000 U/kg, twice per week). Right: Box-and-whisker plots of survival data. Statistics: Left: n.s., Mantel-Cox P > 0.05/3 (Bonferroni correction); right: n.s., two-tailed unpaired t test for equal variances P > 0.05/3 (Bonferroni correction). For both analyses, **P < 0.005. n = 5 mice for untreated groups and n = 7 mice for ASNase groups. (D) Schematic depicting that environmental thiamine influences ASNase sensitivity of leukemia cells with low SLC19A2 expression.

Supplementary Materials

  • Supplementary Materials

    Dietary thiamine influences L-asparaginase sensitivity in a subset of leukemia cells

    Rohiverth Guarecuco, Robert T. Williams, Lou Baudrier, Konnor La, Maria C. Passarelli, Naz Ekizoglu, Mert Mestanoglu, Hanan Alwaseem, Bety Rostandy, Justine Fidelin, Javier Garcia-Bermudez, Henrik Molina, Kıvanç Birsoy

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