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. 2023 Sep 6;13(9):2072-2089.
doi: 10.1158/2159-8290.CD-22-0874.

FH Variant Pathogenicity Promotes Purine Salvage Pathway Dependence in Kidney Cancer

Blake R Wilde  1   2 , Nishma Chakraborty #  1 , Nedas Matulionis #  1 , Stephanie Hernandez #  1 , Daiki Ueno  3 , Michayla E Gee  1 , Edward D Esplin  4 , Karen Ouyang  4 , Keith Nykamp  4 , Brian Shuch  2   3 , Heather R Christofk  1   4   5
Affiliations

FH Variant Pathogenicity Promotes Purine Salvage Pathway Dependence in Kidney Cancer

Blake R Wilde et al. Cancer Discov. .

Abstract

Fumarate accumulation due to loss of fumarate hydratase (FH) drives cellular transformation. Germline FH alterations lead to hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to an aggressive form of kidney cancer. There is an unmet need to classify FH variants by cancer-associated risk. We quantified catalytic efficiencies of 74 variants of uncertain significance. Over half were enzymatically inactive, which is strong evidence of pathogenicity. We next generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks de novo purine biosynthesis, rendering FH-deficient cells reliant on purine salvage for proliferation. Genetic or pharmacologic inhibition of the purine salvage pathway reduced HLRCC tumor growth in vivo. These findings suggest the pathogenicity of patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors.

Significance: This study functionally characterizes patient-associated FH variants with unknown significance for pathogenicity. This study also reveals nucleotide salvage pathways as a targetable feature of FH-deficient cancers, which are shown to be sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. This presents a new rapidly translatable treatment strategy for FH-deficient cancers. This article is featured in Selected Articles from This Issue, p. 1949.

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Conflict of interest statement

Conflict of Interest: The authors declare no potential conflicts of interest.

Figures

Fig. 1:
Fig. 1:. Patient presentation with germline FH variant of uncertain significance, p.Met151Lys, suggests pathogenicity.
(A) Pie chart showing the proportion of HLRCC-associated FH variant classification as either benign (or likely-benign), pathogenic (or likely-pathogenic), conflicting interpretation of pathogenicity (CI), or variant of uncertain significance (VUS) in June 2022. (B) Family history of RCC patient is suspicious of HLRCC. Proband is indicated by black arrow. Colors indicate HLRCC-associated morbidities – RCC (red) and fibroids/hysterectomy/myomectomy (blue). Age is listed for each individual as well as ages at which morbidities manifested or procedures conducted. The presence of FH p.Met151Lys is also indicated. The probands siblings were not tested for FH alterations. (C) [18F]-fluorodeoxyglucose PET/CT scan of patient indicating right renal mass as indicated by white arrow. (D) H&E staining of renal mass demonstrates a typical HLRCC-associated kidney cancer morphology, including eosinophilic papillary architecture with classical peri-nucleolar halos and prominent organiophilic nucleoli. (E) Immunohistochemistry demonstrates the renal mass is FH positive. (F) 2-succinylcysteine (2-SC) is diffusely positive in the cytoplasm and nuclei.
Fig. 2:
Fig. 2:. Biochemical classification of 74 patient FH variants provides strong evidence of pathogenicity for many VUS/CI.
(A) Recombinant FH was produced and purified from E. coli as shown by SDS-PAGE gels stained by Coomassie. Each variant is colored according to the associated interpretation of pathogenicity: pathogenic (red) and VUS/CI (green). For (B-D), recombinant FH was incubated with increasing concentrations of substrate, fumarate (top) or malate (bottom), and the reaction progress was quantified. Wildtype (WT) FH was run simultaneously for each variant, as indicated by the black symbols/lines. (B) All pathogenic variants could not be modeled by Michaelis-Menten kinetics, indicating that these variants are inactive. Michaelis-Menten plots from wildtype enzyme are indicated by black symbols/lines. (C) VUS/CI have a range of enzymatic activities. (D) FH with annotated domains and graphs indicating the catalytic efficiencies of 74 FH variants for fumarate (top) and malate (bottom). For each graph, WT catalytic efficiency is indicated as a dotted line. No variants in the mitochondrial targeting sequence (MTS) were tested because the recombinant FH lacked the first 44 amino acids to improve solubility and purification. (E) Pie chart showing the proportion of VUS/CI that were inactive or active (including partial activity). (F) Graphs showing that M151K (product of FH p.Met151Lys described in Fig. 1) is enzymatically inactive.
Fig. 3:
Fig. 3:. Tetramerization is necessary but not sufficient for transformation.
(A) Recombinant FH was analyzed by Native-PAGE with Coomassie staining. Enzyme separated out to tetramers, dimers, and monomers, indicated as T, D, and M respectively. (B) Densitometry was used to quantify the percentage of each multimerization species for each variant. (C) Percent tetramerization for all variants that did not fit Michaelis-Menten kinetics in Fig. 2. Wildtype (WT) percent tetramer is indicated as a dotted line. All variants that are within 1 standard deviation of WT percent tetramer are indicated by darker gray bars.
Fig. 4:
Fig. 4:. Loss of FH activity is associated with widespread metabolic changes.
(A) Schematic depicting generation of an HLRCC cell line panel with varying levels of FH activity and fumarate. NCCFH1 cells were engineered to express GFP (control), wildtype FH, or FH variants with differing levels of catalytic activity. Metabolites were harvested and analyzed by liquid chromatography-mass spectrometry (LC-MS). Fumarate/malate ratios were calculated and correlated with other metabolites. (B) Fumarate/malate ratios across the panel of HLRCC cell lines engineered to have varying levels of FH activity. Pathogenic variant is indicated in red, 13 VUS/CI are indicated in green, M151K is indicated by the black arrow. C, Heatmap showing that fumarate/malate ratios (FMR) correlate with metabolites that have previously been linked to fumarate accumulation. This analysis was expanded to 164 targeted metabolites. Those that correlated with FMR with a Pearson r ≥ 0.8 or ≤ −0.8 are shown. (D) Volcano plot showing the dysregulation of metabolites in UOK-262 cells expressing FH. Fumarate-regulated metabolites, de novo IMP biosynthesis intermediates, and TCA intermediates are highlighted. Two of the most highly-altered metabolites, SAICAR and AICAR, which are sequential metabolites in de novo IMP biosynthesis, were regulated in opposite directions. (E) Metabolite set enrichment analysis was performed on HLRCC cells expressing GFP or FH. Pathways that were significant in HLRCC cell lines (p ≤ 0.05) are noted with their respective -log10(p-values).
Fig. 5:
Fig. 5:. Loss of FH activity disrupts de novo purine biosynthesis.
(A) Schematic representing the flow of carbons in the TCA cycle and de novo IMP biosynthesis from U-13C-glutamine in HLRCC cells. Fraction of 13C-labeled isotopomers for (B) fumarate, aspartate, AIR, SAICAR, and AICAR. (C) Relative peak areas for de novo IMP biosynthesis intermediates in UOK-262 cells treated with U-13C-glucose. Bars are colored by the relative amount of individual 13C isotopomers. (D) Normalized peak areas for ATP and GTP. (E) Schematic of the purine salvage pathway. (F) Expression of APRT and HPRT1 in HLRCC patient primary tumors, metastases, and adjacent normal kidney tissue. Data is publicly available at NCBI Gene Expression Omnibus GSE157256. (G) Depiction of how ATP and GTP are labeled by amide-15N-glutamine, 8-13C-adenine, and 8-13C-guanine. UOK-262 cells were treated for 6 hours with 4 mM amide-15N-glutamine, 50 μM 8-13C-adenine, and 50 μM 8-13C-guanine, and ATP and GTP were analyzed by LC-MS for mass shifts corresponding to de novo synthesis (purple) or salvage pathway (orange). In order to determine the capacity for salvage pathway activity, each group was normalized to 15N-glutamine only treated samples. (H) Summary of how FH loss impacts purine biosynthesis. In cells without active FH, fumarate accumulates which drives the succination of AICAR by ADSL to produce SAICAR (reverse reaction). This prevents de novo IMP biosynthesis, leaving the cells reliant on the salvage pathway to support purine synthesis.
Fig 6:
Fig 6:. Purine salvage pathway enzymes promote HLRCC tumor growth.
(A) Immunoblots evaluating APRT and HGPRT expression in NCCFH1 and UOK-262 cells infected with LentiCRISPR-Puro and LentiCRISPR-Blast containing sgRNAs targeting AAVS1 (control), APRT, and HPRT1. (B) Fraction of m+1 purine nucleotide isotopologues resulting from 6 hour treatment with 50 μM 8-13C-adenine and 50 μM 8-13C-guanine. (C) Proliferation rates in cells expressing sgAAVS1 or sgAPRT/sgHPRT1 at treated with human plasma-like media supplemented with 50 μM adenine and 50 μM guanine. (D) Volume of NCCFH1 tumor xenografts expressing sgAAVS1 or sgAPRT/sgHPRT1 as determined by caliper measurements (n = 10). Two-way ANOVA was performed for each time point and -log10(p-value) is plotted below, with significant values (p ≤ 0.05) falling into the gray region. (E) Immunoblots evaluating the expression of APRT and HGPRT in xp-152-cl cells infected with LentiCRISPR-Puro and LentiCRISPR-Blast containing sgRNAs targeting AAVS1 (control), APRT, and HPRT1. (F) Fraction of m+1 purine nucleotide isotopologues resulting from 6 hour treatment with 50 μM 8-13C-adenine and 50 μM 8-13C-guanine. (G) Volume of xp-152-cl tumor xenografts expressing sgAAVS1 or sgAPRT/sgHPRT1 as determined by caliper measurements (n = 8). Two-way ANOVA was performed for each time point and -log10(p-value) is plotted below, with significant values (p ≤ 0.05) falling into the gray region. (H) Immunoblots evaluating the expression of APRT and HGPRT in lysates from NCCFH1 and xp-152-cl cell lines and corresponding tumor xenograft lysates.
Fig. 7:
Fig. 7:. Inhibition of purine salvage by 6-mercaptopurine disrupts HLRCC tumor growth.
(A) Schematic of the purine salvage pathway and inhibition by 6-mercaptopurine (6-MP). (B-C) Proliferation (relative to DMSO treated cells) of NCCFH1 and UOK-262 cells expressing GFP or FH and treated with varying concentrations of 6-MP. Fitted values for EC50 are higher for FH-expressing cells. (D) Volume of NCCFH1 tumor xenografts from mice treated with vehicle or 40 mg/kg 6-MP (n = 10). Student’s T-test was performed for each time point and -log10(p-value) is plotted in the middle, with significant values (p ≤ 0.05) falling into the gray region. Mouse weight for each time point is plotted at the bottom. (E) Volume of xp-152-cl tumor xenografts from mice treated with vehicle, 10 mg/kg 6-MP, or 40 mg/kg 6-MP (n = 10). Student’s T-test was performed for each time point and -log10(p-value) is plotted in the middle, with significant values (p ≤ 0.05) falling into the gray region. Mouse weight for each time point is plotted at the bottom. (F) Schematic illustrating how fumarate accumulation disrupts de novo purine biosynthesis, rendering FH-deficient tumor cells reliant on purine salvage for maintenance of purine nucleotides and tumor growth (Created with BioRender.com).
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