Retrograde mitochondrial signaling in fumarate hydratase deficient cells

• Applicant: Dr. Peter Gollwitzer
• Subject Area: Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 537538941

Loss of function of the mitochondrial tricarboxylic acid (TCA) enzyme Fumarate Hydratase (FH) predisposes to an aggressive form of renal cell carcinoma characterized by unique metabolic features, which include increased glycolysis and impaired oxidative phosphorylation, increased glutamine and amino acids utilisation, and accumulation of fumarate. Previous work from our group indicates that TCA cycle inhibition and acute loss of FH activity elicit an Integrated Stress Response (ISR) mediated by the transcription factor ATF4, a master regulator of nutrient stress response. Yet, how the cell senses the loss of FH, and how the ensuing metabolic response is orchestrated is not fully understood. In this proposal, we will use novel cellular models of acute FH deficiency to elucidate the mechanisms that link FH loss with ATF4 activation, and investigate the role of ATF4 in the response to FH loss. First, using an ATF4-based fluorescents reporter, we will perform a flow cytometry-based CRISPR-Cas9 knockout screen to identify regulators of ISR induction upon loss of FH. We will investigate the potential connection to the recently described OMA1-DELE1-HRI axis of mitochondrial ISR signalling and assay key mitochondrial parameters, including oxygen consumption, the inner mitochondrial membrane potential and mitochondrial redox couples to characterize this unknown ISR activation mechanism. We will perform the cell-based screening and target validation in physiologic cell culture media to mimic physiological nutrient levels. Then, we will focus on the downstream consequences of ISR activation and investigate the interplay of ATF4 and mTOR signaling, another major regulator of cellular metabolism implicated in FH-deficient tumors. To this aim, we will perform steady-state metabolomics in wild-type and ATF4-deficient cells to investigate the role of the ISR in regulating intracellular amino acid levels. We will also apply phosphoproteomics and check the phosphorylation of previously reported substrates of mTORC1 kinase, to determine its crosstalk with ATF4-dependent responses. Overall, this proposal will shed light on the molecular mechanism behind the activation of ISR upon FH loss, and identify the role of ATF4 in the compensatory metabolic changes required for cell survival in FH-deficient cells, paving the way to new exciting venues for targeting FH-deficient tumours.

• DFG Programme: WBP Position