FAHD1 a new regulator of mitochondria and cell proliferation

The goal of the project FAHD1, a new regulator of mitochondrial function and cell proliferation is to analyze the role of FAHD domain-containing protein 1, a member of the fumarylacetoacetate hydrolase (FAH) superfamily, in the regulation of mitochondrial function and cell proliferation. FAHD1 is a mitochondrial enzyme with acylpyruvate hydrolase (ApH) and oxaloacetate (OAA) decarboxylase (ODx) activity. According to our hyptohesis, FAHD1, the first eukaryotic ODx identified so far, is a novel regulator of mitochondrial function which, by regulating the OAA concentration in the mitochondrial matrix, modulates flux through the tricarboxylic acid (TCA) cycle and thereby supports mitochondrial electron transport. Based on recently finished structural analyses of the FAHD1 active site, we will test the recently proposed mechanism by which FAHD1 catalyzes the decarboxylation of OAA through mutation of specific amino acids, and to investigate whether the catalytic activity of FAHD1 is required for its beneficial effects on mitochondrial function. In a second part of the project we will test the hypothesis that pharmacological inhibition of FAHD1 by small molecules will induce mitochondrial dysfunction and inhibit proliferation of human cancer cells, in particular breast cancer cells. Based on the available structural information on the FAHD1 catalytic domain and available synthetic FAHD1 inhibitors active in the low micromolar range, a new generation of small molecules will be synthesized and analyzed for their efficacy as inhibitors of FAHD1 in vitro. For the most efficient inhibitors, co-crystals with FAHD1 will be prepared and by X-ray analysis. The resulting optimized compounds will again be evaluated in enzyme inhibition assays and ligand binding assays. Nanomolar inhibitors emerging from these experiments will be tested for their ability to modulate the proliferation and survival of human cells and for their effects on mitochondrial function in these cells. The project will link the enzymatic function of FAHD1 to its reported role as regulator of mitochondrial function and will allow a dissection of the role of ApH versus ODx activity as relevant for mitochondrial effects. The identification of nanomolar inhibitors of FAHD1 and their use as novel agents to inhibit proliferation and survival of cancer cells is another major innovative aspect of the project.

In the project, structure and function of the protein FAHD1, a novel oxaloacetate decarboxylase, was studied. In particular, X-ray structure analysis was used to establish the structure of the active site of the enzyme; targeted mutations of single amino acid residues in the active site were introduced and their influence on enzymatic activity was studied. We found that the mechanisms for decarboxylation of oxaloacetate differs from the mechanism underlying the hydrolysis of acylpyruvate, representing the two known functions of this bifunctional mitochondrial enzyme. Instructed by this analysis, we developed and validated a first generation of pharmacological FAHD1 inhibitors. Based on preliminary results indicating upregulation of FAHD1 in various human malignancies, we demonstrated in a collaboration with the University of Porto (Portugal) that FAHD1 is overexpressed in breast cancer patient samples. Breast cancer cells can be divided, according to their cellular origin, into the luminal and basal type; the basal type, also referred to as "triple-negative" type, is more difficult to treat compared to the luminal type. We tested the hypothesis that expression of FAHD1 plays a role for proliferation and survival of breast cancer cells. We found that both luminal and basal cell types express FAHD1; genetic inactivation by shRNA-mediated inactivation of FAHD1 influenced the proliferation of both luminal and basal cell types. Of interest, FAHD1 depletion in luminal cells led to inhibition of cell proliferation, dependent on available carbon sources in the culture medium, in particular glutamine. On the other hand, depletion of FAHD1 induced cell death in basal cells (e.g., BT-20). We also identified molecular mechanisms by which FAHD1 influences the proliferation of breast cancer cells. The primary effect of FAHD1 inactivation was shown to consist of a partial inactivation of complex II of the mitochondrial electron transport chain, based on the accumulation of oxaloacetate in the mitochondrial matrix. Through mechanisms that are still not fully understood, this leads to a reduction in the protein content of the enzyme glutaminase, which is essential for glutamine-dependent breast cancer cells. These findings enable a new therapeutic option for targeted elimination of tumor cells, in particular triple negative breast cancer cells, which are notoriously difficult to treat. Finally, we developed and synthesized small molecules which can inhibit the activity of FAHD1, albeit still in the low micromolar range. In our view, the further development of these molecules will allow a new therapeutic strategy for patients with breast cancer, which will be studied in future experiments.