Abstract Patients with hereditary leiomyomatosis and renal cell carcinoma (HLRCC), characterized by mutations in the fumarate hydratase (FH) gene, are at risk for development of aggressive FH-deficient RCCs. FH-deficient tumor cells undergo a pronounced and irreversible metabolic shift to lactate fermentation due in part to loss and mutation of mitochondrial DNA. Fumarate accumulation in FH-deficient tumor cells leads to increased expression of NAD(P)H-quinone oxidoreductase 1 (NQO1) through activation of the NRF2 transcription factor. Although several therapeutic agents have shown promise in the treatment of FH-deficient RCC, clinical outcomes in patients remain unsatisfactory. In this study, we examined the mechanism and therapeutic efficacy of isobutyl-deoxynyboquinone (IB-DNQ), which undergoes futile redox cycling in the presence of NQO1 and oxygen, leading to sustained generation of the highly reactive and toxic superoxide anion. First, we found that patient-derived FH-deficient tumor cells exhibit minimal oxygen consumption in vitro, and EPR oxygen mapping of FH-deficient tumor xenografts in vivo revealed that tumor oxygen levels were elevated relative to other genetically defined in vivo models of RCC. Infusion of IB-DNQ in tumor-bearing animals resulted in rapid and robust non-mitochondrial oxygen consumption in FH-deficient tumor xenografts as measured by both EPR oxygen imaging and photoacoustic mapping of tumor hemoglobin saturation. Repeated doses of IB-DNQ resulted in reduced tumor growth rates. Metabolomic analyses revealed that IB-DNQ treatment strongly suppressed glycolysis and reduced cellular ATP levels by rapidly depleting NADH and NADPH in FH-deficient tumor cells. Finally, [1-13C]pyruvate hyperpolarized MR spectroscopy revealed decreased conversion of pyruvate to lactate in FH-deficient tumor xenografts following IB-DNQ treatment, providing a direct measurement of the impact of IB-DNQ on lactate fermentation in vivo. The combination of these in vivo imaging techniques and metabolite measurements demonstrate that NQO1-activated quinones can effectively target aerobic glycolysis in FH-deficient tumors which rely heavily on lactate fermentation for growth. Citation Format: Yuki Shibata, Shun Kishimoto, Ye Yang, Ming-Hui Wei, Julia Medina-Velazquez, Burchelle Blackman, Jeeva Munasinghe, Viraj Chegu, Vaishnavi S. Srirama, Tyler A. On, Nallathamby Devasahayam, Chandramouli V. Gadisetti, Jeffrey R. Brender, Murali C. Krishna, Daniel R. Crooks, William Marston Linehan. EPR imaging of oxygen consumption driven by NQO1-activated compounds in FH-deficient renal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7328.

Real-time invasion analysis of UOK262 and UOK365 cells treated with OT-82. Real-time invasion analysis of UOK262 and UOK365 cells demonstrating cell line invasion over 5 days (120 hrs) was performed (A) to show that DMSO (used as the vehicle for OT-82) did not affect invasion in comparison to untreated cells and (B) to demonstrate the effects of OT-82 treatment at either 1 nM or 10 nM in comparison to vehicle alone (DMSO) or the non-invasive control (no serum in the lower chamber). In each case, a representative graph from one of three separate experiments is shown. (C) The invasion inhibition for three repeats at each dose is shown for each cell line. The average invasion inhibition is shown as a percentage compared to the vehicle alone (DMSO) invasion and T-tests were performed comparing each dose to the vehicle alone (DMSO).

Pharmacokinetic profile of OT-82 in nude and NSG mice. (A) Bioanalysis of plasma concentrations of OT-82 were made using a validated LC-MS/MS assay (n=3 nude mice, 50 mg/kg, 7 intervals; n=3 NSG mice, 75mg/kg, 7 intervals). (B) Pharmacokinetic parameters were calculated after a single dose of OT-82. The data from all mice in each group were pooled together to assume one “average” mouse per group.

In vitro analysis of NAMPT inhibition on glycolysis in FH-deficient tumor cells. (A) Effects of the NAMPT inhibitor OT-82 (100 nM) on the extracellular acidification rate (ECAR) in the UOK262 and UOK365 HLRCC cell lines and the RPTEC normal cell line. (B) Representation of the different effects of OT-82 versus DMSO at time point 5 (designated by a star) after the injection of glucose in UOK262, UOK365, and RPTEC. 2-DG, 2-deoxyglucose.

Proliferation analysis of UOK268 and RPTEC in response to OT-82 and nicotinic acid rescue of NAD+/NADH depletion. (A-B) UOK268 or RPTEC cells were grown in 96 well plates from an initial plating of 2,000 cells and cellular confluency was monitored in real-time by an Incucyte S3 Live-Cell Imaging System. After 24 hours of initial measurements cells were treated with either DMSO, 1 nM OT-82, 5 nM OT-82, 10 nM OT-82, or 10 nM OT-82 plus 1 mM of nicotinamide mononucleotide (NMN) and evaluated for an additional 5 days. (C-D) The effect of NMN on NAD+/NADH depletion by OT-82 in UOK268 and RPTEC cells is shown. (E) Effects on the levels of NAD+ and NADH after 24 hours of treatment with either 10nM OT-82, or 10nM OT-82 in combination with 1 mM nicotinic acid (NA) in UOK262, UOK268, and UOK365.

Validation studies to evaluate inhibition of additional HLRCC cell lines by NAMPT inhibitors. A) Effects of the NAMPT inhibitor GNE-618 on cell viability in FH -/- HLRCC cells lines UOK348, UOK271, UOK350, UOK268, UOK365, FH restored UOK268WT and non-transformed kidney epithelial cells RPTEC. Cell viability was assessed by Cell Titer-Glo assay at 96 h. B) Effects of the NAMPT inhibitor OT-82 on cell viability in FH -/- HLRCC cells lines UOK348, UOK271, UOK350, UOK268, UOK365, FH restored UOK268WT and non-transformed kidney epithelial cells RPTEC.

NAMPT immunohistochemical analysis in HLRCC tumors and normal kidney. (A) In patient #1, both a primary kidney HLRCC tumor (upper panel) and an associated metastatic mass (lower panel) demonstrated strong NAMPT staining. Surrounding non-tumor tissues show little NAMPT staining. (B) In patient #4, a primary kidney HLRCC tumor (upper panel) shows strong NAMPT staining in comparison to normal kidney tissue present on the same slide (lower panel). (C) In patient #5, a metastatic mass shows strong NAMPT staining (upper panel). Material from this mass was used to derive the UOK262 cell line, which shows similar positive staining when grown as a xenograft (lower panel). (D) In patient #6, a primary kidney HLRCC tumor demonstrated strong NAMPT staining. Matching H&E staining is included for all samples (A-D).

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted tricarboxylic acid cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared with controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and poly-ADP-ribose levels, and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide (NMN), confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated RCC.

Expression levels of NAD+ biosynthetic and salvage pathway genes in HLRCC tumors, normal kidney samples and pan-kidney data from TCGA. (A) Overview of NAD+ biosynthetic and salvage pathways. (B-E) Box plots for selected NAD+ biosynthetic genes derived from RNA-seq analysis of HLRCC tumors and normal kidney specimens. (F) Box plots for selected NAD+ biosynthetic genes derived from RNA-seq TCGA pan-kidney data. NAMPT (nicotinamide phosphoribosyltransferase), NAPRT (nicotinate phosphoribosyltransferase ), QPRT (quinolinate phosphoribosyltransferase), NMNAT (nicotinamide mononucleotide adenylyl transferase), KIRP (kidney renal papillary cancer), CIMP (CpG island methylator phenotype), PRCC T1/T2 (papillary renal cell carcinoma type 1/type 2), ccRCC (clear cell renal cell carcinoma), ChRCC (chromophobe renal cell carcinoma).

In vivo effects of NAMPT inhibitors in HLRCC xenograft models. Mean body weights are constant throughout the drug study for animals harboring UOK262 xenografts (A) and UOK365 xenografts (B; study shown in Figure 3A-B, n = 10 mice per arm, 95mg/kg OT-82 for 8 week duration). (C-D) H&E stained sections of retinas obtained from NSG mice treated with vehicle or 95mg/kg OT-82 for 5 days. (E-F) Caspase 3 stained sections of retinas obtained from NSG mice treated with vehicle or 95 mg/kg OT-82 for 5 days. Histological sections were evaluated for signs of retinal toxicity by a trained veterinary pathologist. No overt signs of retinal toxicity were observed (n=3 NSG mice).

NAD+ and NADH analysis of UOK365 cells. Analysis of the relative levels of NAD+ and NADH and their ratio after 24 hour treatment of the UOK365 cell line model with a range of concentrations (0.5-100 nM) of either GNE-618 (A-B) or OT-82 (C-D). All levels were compared to cells treated with the amount of DMSO present in the highest concentration of the utilized drugs.

Pembrolizumab combined with chemotherapy has already become the standard first-line treatment for R/M HNSCC. However, data on pembrolizumab in LA HNSCC remains limited, primarily focusing on monotherapy. This study aimed to analyze and explore the efficacy and safety of pembrolizumab combined with chemotherapy as a neoadjuvant treatment.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is characterized by germline mutations of the FH gene that encodes for the TCA cycle enzyme, fumarate hydratase. HLRCC patients are at risk for the development of an aggressive form of type 2 papillary renal cell carcinoma. By studying the mechanism of action of marizomib, a proteasome inhibitor able to cross the blood-brain barrier, we found that it modulates the metabolism of HLRCC cells. Marizomib decreased glycolysis in vitro and in vivo by downregulating p62 and c-Myc. C-Myc downregulation decreased the expression of lactate dehydrogenase A, the enzyme catalyzing the conversion of pyruvate to lactate. In addition, proteasomal inhibition lowered the expression of the glutaminases GLS and GLS2, which support glutamine metabolism and the maintenance of the redox balance. Thus, in HLRCC cells, proteasome inhibition disrupts glucose and glutamine metabolism, restricting nutrients and lowering the cells' antioxidant response capacity. Although the cytotoxicity induced by proteasome inhibitors is complex, the understanding of their metabolic effects in HLRCC may lead to the development of effective therapeutic strategies or to the development of markers of efficacy.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

About 10% of papillary renal cell carcinomas (PRCC) bear mutations in NF2 or SAV1, two members of the Hippo pathway, which controls cell growth by regulating the transcriptional activity of YES1-Associated Protein YAP1. NF2 or SAV1 mutations lead to aberrant YAP1 activation resulting in cell proliferation and anti-apoptotic signals. The src family kinase YES1 is a known regulator of YAP1 transcriptional activity in the context of β-catenin-dependent tumors. Dasatinib and saracatinib are 2 src inhibitors that have shown to also have potent inhibitory effects on YES1. We hypothesized that inhibition of the Hippo pathway might provide a valuable therapeutic approach for patients with PRCC tumors bearing NF2 mutation, and that inhibiting YES1 might inhibit YAP1 in PRCC. Our data demonstrate that inhibition of the Hippo pathway is lethal to NF2-deficient PRCC cell lines, and that inhibiting YES1 with dasatinib or saracatinib effectively inhibits YAP1 and its downstream targets BIRC5 (survivin), CCND1 (cyclin D1) and CTGF (CTGF). Further, dasatinib proves to have an anti-tumor effect in vivo and was cytotoxic in vivo and in vitro, causing G0-G1 cell cycle arrest in NF2-deficient PRCC cell lines. Thus, inhibiting YES1 and the subsequent transcriptional activity of YAP1 with dasatinib or saracatinib might be a viable therapeutic approach for NF2-deficient PRCC tumors. Citation Format: Carole Sourbier, Penny Liao, Christopher J. Ricketts, Darmood Wei, Youfeng Yang, Sarah M. Baranes, Toshiki Kijima, Louis S. Krane, Myriem Boufraqech, Lernik Ohanjanian, Ben Gibbs, Ming-Hui Wei, Len Neckers, Cathy D. Vocke, W. Marston Linehan. Targeting the Hippo pathway in NF-2 deficient papillary kidney cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1288.

Background: Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.Methods: In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.Results: We found that the SDHBR46Q mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4 +/- 63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1 alpha, but not HIF2 alpha, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.Conclusions: These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

Patients with germline fumarate hydratase (FH) mutation are predisposed to develop aggressive kidney cancer with few treatment options and poor therapeutic outcomes. Activity of the proto-oncogene ABL1 is upregulated in FH-deficient kidney tumors and drives a metabolic and survival signaling network necessary to cope with impaired mitochondrial function and abnormal accumulation of intracellular fumarate. Excess fumarate indirectly stimulates ABL1 activity, while restoration of wild-type FH abrogates both ABL1 activation and the cytotoxicity caused by ABL1 inhibition or knockdown. ABL1 upregulates aerobic glycolysis via the mTOR/HIF1α pathway and neutralizes fumarate-induced proteotoxic stress by promoting nuclear localization of the antioxidant response transcription factor NRF2. Our findings identify ABL1 as a pharmacologically tractable therapeutic target in glycolytically dependent, oxidatively stressed tumors.

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-(13)C]-glucose, [U-(13)C,(15)N]-glutamine, and [1,2- (13)C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α-ketoglutarate through to fumarate. [1,2- (13)C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.