CYB561A3 is the key lysosomal iron reductase required for Burkitt B-cell growth and survival

Abstract

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.

Graphical abstract

Figure 1.

CYB561A3 is essential for the proliferation of Burkitt lymphoma cells. (A) Scatterplots showing the statistical significance of selected top hits that were selectively important for P3HR-1 Burkitt vs GM12878 LCLs. Statistical significance was quantitated by the STARS algorithm, using 2 biological replicates for each axis. (B) Distribution of Avana human genome-wide CRISPR screen sgRNA log₂ fold-change values at day 21 vs input in Cas9⁺ P3HR-1 Burkitt lymphoma (left) or GM12878 LCL (right). Values for CYB561A3 and CYB561 targeting sgRNAs (red lines) are overlaid on gray gradients depicting all Avana sgRNA library values. Average day 21 vs input values from 4 screen biological replicates are shown. (C) DepMap CRISPR screen dependency scores for sgRNAs targeting CYB561A3 across cell lines from the indicated cancer cells of origin. Circles represent DepMap screen values for cell lines from the tissue of origin indicated at right. Negative values indicate selection against sgRNAs targeting CYB561A3 over a 21-day growth and survival screen. Values less than −1 (red vertical line) indicate the DepMap cutoff for CYB561A3 dependency. (D) Growth curve analysis of Cas9⁺ P3HR-1 (top) and GM12878 (bottom) with control or independent CYB561A3 sgRNAs, as indicated. (E) Indel-sequencing analysis of Cas9 activity at the CYB561A3 locus. Shown are the percentage of indels in P3HR-1 or GM12878 cells expressing the indicated control or CYB561A3 sgRNAs. (F) Growth curve analysis of Cas9⁺ P3HR-1 after expression of the indicated green fluorescent protein (GFP) control or CYB561A3^R cDNAs and control or CYB561A3 sgRNA, as indicated. (G) Fold change live cell number of Mutu Burkitt cells with EBV latency I or III (Mutu I and III) from days 4 to 8 postexpression of CYB561A3 vs control sgRNAs. (H) Fold change live cell numbers of Kem I EBV latency I Burkitt and Kem III EBV latency III LCLs from days 4 to 8 postexpression of CYB561A3 vs control sgRNAs. (I) Fold-change live cell numbers from EBV⁺ Akata Burkitt cells with EBV latency I vs EBV^– Akata from days 4 to 8 postexpression of CYB561A3 vs control sgRNAs. Mean ± standard deviation fold-change values from n = 3 biological replicates (E-I). *P < .05; **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).

Figure 2.

CYB561A3 KO causes Burkitt cell cycle arrest and lysosomal and mitochondrial damage. (A) Cell cycle analysis of P3HR-1 and GM12878 nine days after control or CYB561A3 sgRNA expression. Lower panel, mean ± standard deviation (SD) values from n = 3 replicates are shown. (B) Fluorescence-activated cell sorter forward scatter (FSC; cell size) and side scatter (SSC; internal complexity) mean ± SD values from n = 3 replicates obtained 12 days after P3HR-1 control or CYB561A3 sgRNA expression. (C) Fluorescence-activated cell sorter analysis of mitochondrial reactive oxygen species signals detected by mitoSOX in P3HR-1 cells 12 days after control or CYB561A3 sgRNA expression, with mean ± SD values from n = 3 replicates. (D) Immunofluorescence analysis of P3HR-1 transfected with a green fluorescent protein (GFP)-tagged CYB561A3 expression vector and stained with LysoTracker to visualize lysosomes. Shown below is the merge, together with Hoechst-stained nucleus. Image is representative of n = 3 replicates. (E) Acridine Orange (1 μg/mL) staining of lysosomes in P3HR-1 cells 12 days after control or CYB561A3 sgRNA expression. Scale bar indicates 10 μm. Representative images from n = 3 replicates are shown. (F) EM images of P3HR-1 twelve days after control or CYB561A3 sgRNA expression. Arrows indicate representative mitochondria (red), lysosomes (white), and autophagosomes (blue). White scale bar sizes are noted at bottom right. (G) Seahorse analysis of P3HR-1 and GM12878 cell oxygen consumption rate (OCR) 12 days after control (black) or CYB561A3 (red) sgRNA expression. Time points of oligomycin (3.5 µM), CCCP (2 µM), and piericidin A (100 nM) addition are indicated. Mean ± SD from n = 3 replicates are shown. **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).

Figure 3.

The absence of CYB561A3 will cause a P3HR-1 iron starvation response. (A) Volcano plot of differentially expressed genes in P3HR-1 twelve days after control or CYB561A3 sgRNA expression (n = 3 replicates). Selected genes upregulated (red) or downregulated (blue) by CYB561A3 depletion are highlighted. (B) Gene Ontology enrichment analysis of significantly upregulated cellular components in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (C) Gene Ontology enrichment analysis of significantly downregulated biological processes in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (D) Volcano plot analysis of log₂ fold-change messenger RNA abundances in Mutu III vs Mutu I (y-axis) and in P3HR-1 with CYB561A3 vs control sgRNA expression (x-axis). (E) Immunoblot analysis of whole-cell lysates (WCL) from P3HR-1 (top) and GM12878 (bottom) at 10 days’ post-control or CYB561A3 sgRNA expression for the indicated proteins. (F) Mean ± SD values of quantitative polymerase chain reaction analysis of the indicated messenger RNA expression in P3HR-1 cells 10 days after control or CYB561A3 sgRNA expression from n = 3 replicates. (G) Immunofluorescence analysis of TFRC expression in P3HR-1 ten days after control or CYB561A3 sgRNA expression. Scale bar, 10 μm. Representative images from n = 2 replicates are shown. (H) Immunoblot analysis of WCL from P3HR-1 with control green fluorescent protein (GFP) or CYB561A3^R (CYB^R) cDNA 7 days’ post-sgRNA expression. (I) Mean ± SD values from P3HR-1 cells with control (black) or CYB561A3 (gray) sgRNA and cultured in the indicated concentration of ferrous citrate. Ferric citrate or phosphate-buffered saline was added 4 days’ post-sgRNA expression, and fold change was measured over the next 3 days. (J) Immunoblot analysis of WCL from P3HR-1 that expressed control or CYB561A3 sgRNA for 7 days and cultured in phosphate-buffered saline or ferrous citrate for days 4 to 7. Panels E, H, and J show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant (unpaired Student t test).

Figure 4.

CYB561A3 ferrireductase activity is critical for Burkitt B-cell iron acquisition. (A) Mean ± standard deviation (SD) fold-change live cell values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 sgRNA (gray) expression and cultured in the indicated concentration of ascorbic acid. Phosphate-buffered saline or ascorbic acid was added at days 4 to 9 post-sgRNA expression. Fold change was measured from days 4 to 9. (B) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing control or CYB561A3 sgRNAs and cultured in the indicated ascorbic acid concentrations, as in panel A. (C) Fluorescence-activated cell sorter analysis of plasma membrane TFRC expression in P3HR-1 expressing control (pink) or CYB561A3 (blue) sgRNAs. Phosphate-buffered saline or ascorbic acid was added from days 4 to 9 post-sgRNA expression, at which time fluorescence-activated cell sorter was done. Representative histograms from n = 3 replicates are shown. (D) Schematic model of CYB561A3. Red stars represent selected amino acids indicated in CYB561A3 electron transport. (E) Confocal microscopy analysis of P3HR-1 lysosomal marker LAMP1, antihemagglutinin (HA) tagged CYB561A3 WT, or indicated alanine point mutant stained for HA, and Hoechst-stained nuclei merge image. Scale bars, 10 μm. Representative images from n = 3 replicates are shown. (F) Mean ± SD values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 (gray) sgRNA and the, cDNA encoding control green fluorescent protein (GFP) or CYB561A3 WT or alanine point mutants. Fold change was measured from days 4 to 8 post-sgRNA expression. (G) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing the indicated sgRNA and cDNA, as in panel F, at day 8 post-sgRNA expression. (H) CYB561 overexpression does not compensate for loss of CYB561A3. Stable expression of HA-epitope–tagged CYB561 vs CYB561A3^R cDNAs was validated by confocal analysis of P3HR-1 stained with anti-HA antibody (left). Control or CYB561A3 sgRNAs were then expressed, as indicated. Mean ± SD fold-change live cell values from n = 3 replicates of cells with the indicated sgRNA and cDNA expression. Panels B and G show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).

Figure 5.

CYB561A3 KO depletes Burkitt cell ferrous iron. (A) Confocal microscopy analysis of FerroOrange-stained P3HR-1 or GM12878 expressing control or CYB561A3 sgRNAs for the indicated days. Shown at right are mean ± standard deviation (SD) values from n = 3 replicates of fluorescence-activated cell sorter mean FerroOrange signals. Scale bar, 10 μm. (B) Confocal analysis of RPA-stained P3HR-1 expressing control or CYB561A3 sgRNAs for 10 days. Shown at right are mean ± SD values from n = 3 replicates of fluorescence-activated cell sorter mean RPA values. (C) Aconitase activity assay from P3HR-1 cells with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. Bar plots show mean ± SD aconitase activity. (D) Immunoblot analysis of OXPHOS subunits or tubulin load control from whole-cell lysates (WCL) of P3HR-1 with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. (E) Immunoblot analysis of lipoic acid, which is conjugated to PDH-E2 and KGDH-E2 subunits or tubulin load control from WCL of P3HR-1 with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. (F) Fifty percent inhibitory concentration (IC₅₀) analysis of P3HR-1 after control (black) or CYB561A3 sgRNAs #1 (red) or #2 (green) expression and treated with the indicated erastin concentrations from days 9 to 12. Shown are mean ± SD values and calculated IC₅₀ values from n = 3 replicates. (G) Immunoblot analysis of WCL from P3HR-1 and GM12878 at 12 days’ post-control or CYB561A3 sgRNA expression. Panels D, E, and G show representative blots from n = 3 replicates. *P < .05 by 1-way analysis of variance with Bonferroni posttest. **P < .01, ***P < .001 (unpaired Student t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF1α, hypoxia inducible factor 1 subunit α; ns, nonsignificant.

Figure 6.

LCL is more dependent on iron reductase STEAP3 than Burkitt B cells. (A) Distribution of Avana human genome-wide CRISPR screen sgRNA log₂ fold-change values at day 21 vs input in GM12878 (top) and P3HR-1 (bottom). Values for STEAP3 targeting sgRNAs are overlaid on gray gradients depicting all Avana sgRNA library values. Average day 21 vs input values from 4 screen biological replicates are shown. (B) Growth curve analysis of GM12878 (left) and P3HR-1 (right) at the indicated time points after expression of control or independent STEAP3 sgRNAs and puromycin selection. Mean ± standard deviation values from n = 3 replicates are shown. (C) Indel-sequencing analysis of Cas9 activity at the STEAP3 locus. Shown are the percentage of indels in P3HR-1 or GM12878 cells expressing the indicated control or STEAP3 sgRNAs. (D) RNA-sequencing analysis of STEAP family messenger RNAs at the indicated days postprimary human B-cell EBV infection. (E) Chromatin immunoprecipitation–sequencing tracks of the indicated Epstein-Barr nuclear antigens, NF-κB subunits, and H3K27 acetyl (H3K27ac) marks at the STEAP3 locus in LCLs. (F) Immunoblot analysis of WCLs from GM12878 (left) and P3HR-1 (right) 9 days after control or STEAP3 sgRNA expression. (G) Growth curve analysis of STEAP3 cDNA rescue effects on P3HR-1 (left) or GM12878 (right) with control or CYB561A3 sgRNAs. Shown are mean ± standard deviation values from n = 3 replicates. (H) Immunoblot analysis of WCL from GM12878 (left) and P3HR-1 (right) expressing the indicated control or CYB561A3 sgRNAs and cDNAs expressing green fluorescent protein (GFP) vs STEAP3. (I) Log₂ normalized RNA-sequencing STEAP3 (y-axis) vs CYB561A3 (x-axis) values from EBV⁺ (red) and EBV^– (blue) Burkitt lymphoma cell lines profiled by DepMap. Representative blots from n = 3 replicates are shown in panels F and H. *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant (unpaired Student t test).

Figure 7.

Model of CY561A3 and STEAP3 iron metabolism roles in Burkitt cells vs LCLs. Transferrin-bound Fe3⁺ iron is endocytosed by the transferrin receptor TFRC and delivered to lysosomes in both LCLs and Burkitt cells. Iron can also be delivered to B-cell lysosomes by ferritinophagy or by mitophagy. The acidic lysosomal microenvironment facilitates release of Fe3⁺, which is reduced for LCL cytosolic transport by STEAP3 to Fe2⁺, using NADPH as an electron donor. By contrast, Burkitt B cells with the EBV latency I program use the ferrireductase CYB561A3 to reduce Fe3⁺ to Fe2⁺, with ascorbate as the electron donor. The cytosolic labile iron pool then supplies a myriad of cellular functions, including iron-sulfur synthesis and respiration. Ferrous iron is also essential for suppression of hypoxia inducible factor 1 subunit α (HIF1α). The metallothionein MT2A binds ferrous iron, and its Burkitt cell expression is dependent on CYB561A3, presumably through an iron-dependent mechanism. In the absence of CYB561A3, Burkitt cells are rapidly depleted of labile iron; they induce an iron starvation response but nonetheless sustain catastrophic lysosomal and mitochondrial damage. LMPs, latent membrane proteins; NADP⁺, nicotinamide-adenine dinucleotide phosphate, oxidized form; ncRNAs, noncoding RNAs.