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 pathogenicity of patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors.

The tricarboxylic citric acid cycle enzyme fumarate hydratase (FH) is a tumor suppressor. When lost in cells, its substrate fumarate accumulates to mM levels and drives oncogenic signaling and transformation. Germline alterations lead to an autosomal dominant condition known as hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to various benign tumors and an aggressive form of kidney cancer. FH alterations of unclear significance are frequently observed with germline testing; thus, there is an unmet need to classify FH variants by their cancer-associated risk, allowing for screening, early diagnosis and treatment. Here we quantify catalytic efficiency of 74 FH variants of uncertain significance. Over half were enzymatically inactive which is strong evidence of pathogenicity. We 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 purine biosynthesis, rendering FH-deficient cells more sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. Together, these findings suggest pathogenicity of many patient-associated FH variants and reveal nucleotide salvage as a targetable vulnerability in FH-deficient cancer cells.

<div>Abstract<p>Fumarate accumulation due to loss of fumarate hydratase (FH) drives cellular transformation. Germline <i>FH</i> 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 <i>FH</i> 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 <i>de novo</i> 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 <i>in vivo</i>. These findings suggest the pathogenicity of patient-associated <i>FH</i> variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors.</p>Significance:<p>This study functionally characterizes patient-associated <i>FH</i> 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.</p></div>

<p>Supplementary Table S1: FH variants with population frequencies, interpretations of pathogenicity, catalytic efficiencies, and multimerization states</p>

<p>Supplementary Fig. S1: Analysis of FH variants and their associated interpretations of pathogenicity. Supplementary Fig. S2: Kinetic parameters for active/partially-active VUS/CI. Supplementary Fig. S3: Recombinant FH produced for 74 VUS/CI. Supplementary Fig. S4: Michaelis-Menten graphs for VUS/CI incubated with fumarate. Supplementary Fig. S5: Michaelis-Menten graphs for VUS/CI incubated with malate. Supplementary Fig. S6: Broad analysis of recombinant FH. Supplementary Fig. S7: Multimerization status of 74 VUS/CI. Supplementary Fig. S8: Tetramerization correlates with catalytic activity. Supplementary Fig. S9: Expression of FH variants in HLRCC cells. Supplementary Fig. S10: Fumarate accumulation decreases TCA intermediates. Supplementary Fig. S11: FH loss enhances de novo glutathione biosynthesis. Supplementary Fig. S12: Coessentiality of FH and nucleotide biosynthesis genes. Supplementary Fig.S13: Loss of FH activity disrupts de novo purine biosynthesis. Supplementary Fig. S14: Dysregulated purine metabolism is independent of HLRCC cell proliferation rate. Supplementary Fig. S15: FH-deficient cells use the purine salvage pathway to maintain purine nucleotide levels. Supplementary Fig. S16: Pyrimidine metabolism in FH-deficient and FH-expressing cells. Supplementary Fig. S17: Disrupting expression of purine salvage enzymes impairs tumor growth. Supplementary Fig. S18: Purine salvage inhibitor 6-mercaptopurine (6-MP) impairs tumor growth.</p>