Research team Prokesch and Krstic

TIMO is a doctoral program unifying clinical and preclinical know-how, methods, and supervision at our university. Find more details about the consortium and the 15 PhD projects funded by the Austrian Science Fund (FWF) and the Medical University of Graz at: https://www.medunigraz.at/doktoratsstudien/phd-program/timo

What: Our prior work established p53 as crucial factor coordinating the response to HCC therapy in combination with nutrient restriction (Krstic J et al., Science Advances, 2022). In the current project, we investigate the impact of p53 hotspot mutations on the tumour microenvironment (TME) upon combination of dietary interventions with immune checkpoint therapy.

How: Within the doctoral program TIMO, we work with CRISPR-modified HCC cell lines harbouring combinations of p53 mutant and wildtype alleles. Those are characterized and used for orthotopic, syngeneic transplantation models with different (combination) treatments. Moreover, we investigate the TME in precision cut-liver slices from clinical biopsies.

Who: A PhD student (to be recruited) will be supervised by a translational team of PIs. Funding by the FWF doc.funds program (A. Prokesch as PI and speaker of TIMO, J. Krstic as co-PI).

What: Chemotherapy can induce patient-specific metabolic reprogramming in the tumor microenvironment (TME) and anthracyclines can trigger immunogenic cell death, potentially shaping anti-tumor immunity. How these metabolic adaptations influence responsiveness to immune checkpoint inhibition (ICI) is unclear. We hypothesize that anthracycline-induced metabolic rewiring differs across patients and modulates TME features that determine the response to ICI. Our aim is to predict patient-specific response to chemotherapy/ICI combinations by defining the metabolic changes that govern TME composition and immune activation.

How: Within the doctoral program TIMO, we will establish patient-derived breast cancer organoids, treat them with an anthracycline, and quantify metabolic rewiring via energetic profiling (Seahorse™), metabolomics and RNA sequencing of metabolic pathways-relevant genes. To assess responsiveness to ICI, we will perform co-cultures with patient-matched immune cells and analyse T-cell migration and activation. Complementary in vivo studies will use a syngeneic mouse model to measure anthracycline-driven changes in the TME and subsequent response to ICI.

Who: A PhD student (to be recruited) will be supervised by a translational team of PIs. Funding by the FWF doc.funds program (J. Krstic as PI, A. Prokesch as TIMO speaker).

What: Nutrient deprivation (ND) can sensitize subsets of cancer cells to standard-of-care (SOC) therapies and reduce tumor growth in vivo, but its benefits are not universal. We hypothesize that ND acts pleiotropically by constraining extrinsic cues and by targeting intrinsic metabolic pathways. We aim to define “metabotypes” that distinguish responders (R) from non-responders (NR) to ND plus SOC therapies and to use these signatures for predictive patient stratification. 

How: To improve the settings for ND studies performed in vitro, we first developed a cell culture medium that mimics the composition of human plasma upon prolonged fasting. We will next perform a high-throughput viability screen across 20 breast cancer cell lines exposed to over 20 SOC agents in control and fasting-like media to classify R and NR. Metabotypes in matched R/NR pairs will be profiled by untargeted mass spectrometry, while bioenergetic states will be quantified via measurements of oxidative phosphorylation and glycolysis. Selected cell lines will be tested in orthotopic xenograft models to evaluate in vivo responses to adjunct ND. The role of systemic factors will be interrogated both in situ and in vitro to link circulating factors, cellular metabolism, and therapeutic outcomes.

Who: This project is spearheaded by Theresia Weiermair, a PhD student supported by a scholarship from the Austrian Academy of Sciences (ÖAW Doc stipend). The project is funded by FWF, PI: J. Krstic.

What: Due to a lack of understanding of how immune checkpoints are regulated during the development of lymphomas, only a small percentage of patients (10-20%) respond favourably to checkpoint therapy (CBT). We observed significant decrease in the levels of NR4A1 and p53 alongside high content of checkpoint components in animal models and patients with diffuse large B cell lymphomas (DLBCL). How these transcription factors and tumour suppressors regulate immune evasion and response to checkpoint therapy is investigated in the consortium project LYMPHOCHECK.

How: In our project part, we will unravel the regulatory landscape of NR4A1 and p53 in DLBCL using novel in vivo models with B cell-specific, inducible deletion of NR4A1 and/or p53 in combination with high-end next-generation sequencing methods (PRO-seq and ChIP-seq).

Who: Our project part is driven by Helene Michenthaler (PostDoc) and Lilian Lamprecht (PreDoc). Funding by the FWF Research Groups program (A. Prokesch, program lead: Alexander Deutsch).

 What: This project investigates the mechanisms of cell death in hepatocellular carcinoma (HCC) to improve therapeutic outcomes by leveraging innovative preclinical models and methodologies. By combining HCC-derived cell lines and patient-derived organoids, we aim to elucidate how p53 status and metabolic conditions influence cell fate upon treatment. The study integrates dynamic, real-time viability monitoring with molecular profiling to characterize cell death pathways, including apoptosis and ferroptosis. 

How: Central to the project is the concept of cell death priming, which assesses the susceptibility of cancer cells to nutrient deprivation (ND) as an adjunct therapy. Novel assays for apoptotic and ferroptotic priming will be developed to evaluate the effects of treatments and NR on cellular responses. 

Who: Lilian Lamprecht (PreDoc) is working on developing and characterizing patient-derived organoids and a master’s student (to be recruited) will continue in her steps. The project is funded by MEFOgraz. PI: J. Krstic.

What: LAMs are a recently discovered hallmark of metabolic diseases and the tumour microenvironment. Our previous work established a critical role for adipocyte p53 in the appearance of LAMs in obese adipose tissue during weight cycling (Reinisch I et al., Nature Communications, 2024). In this project, we investigate how the hepatocyte p53 status affects LAM infiltration and polarisation in models of steatohepatitis. 

How: We develop in vitro models allowing us to study signalling pathways and transcriptional regulation in monocyte-to-LAM transition. Those are used to scrutinize mechanisms of cell-cell communication between hepatocyte and monocytes/macrophages. Furthermore, hepatocyte-specific p53 knockout and overexpression in vivo models are investigated at the single cell level using multiplex immunofluorescence and single nuclei RNA-sequencing (snRNA-seq) in collaboration with Charlie Scott at the University of Ghent.

Who: Our PhD student Ruonan Xu is propelling the project forward, generously funded by the PhD faculty Molecular Medicine of the Medical University of Graz and EMBO (travel fellowship). PI: A. Prokesch

What: Our previous work established a critical role for adipocyte p53 in the appearance of LAMs in obese AT during weight cycling. In a small clinical cohort, we found that a common p53 polymorphism (P72R) is associated with metabolic parameters in weight regain after dietary weight loss (Reinisch I et al., Nature Communications, 2024). However, the exact p53-driven mechanisms and LAM recruitment/differentiation dynamics upon weight cycling remain enigmatic.

How: In this project, we adapt Zman-seq (Kirschenbaum D et al., Cell, 2024) to temporally track monocyte infiltration and LAM differentiation in AT under weight cycling and during weight regain. Recruitment mechanisms are unravelled with co-culture/transwell assays in collaboration with Sander Kersten (Cornell). Cooperating with Matthias Blüher (University of Leipzig) we analyse human AT biopsies in a large weight loss/weight regain cohort and relate cellular (immune) landscape remodelling (using snRNA-seq) to the P72R p53 polymorphism.

Who: Two Sarahs (Sarah Enzenhofer, Sarah Rimser) are in charge of different aspects of the project. Funding is provided by FWF (A. Prokesch) and the Austrian Academy of Sciences (ÖAW Doc stipend: S. Enzenhofer).

What: In our first round of funding, we uncovered how p53 acts as a key player in fasting/feeding transitions across adipose tissue, liver, and skeletal muscle. Our new project aims to scrutinize p53's regulatory capability at the next level, by investigating its interaction with another key metabolic regulator, Foxo1. The original consortium, consisting of our lab at the Medical University of Graz and the labs of Micheal Schupp (Charite Berlin) and Tim J Schulz (DiFE Potsdam), is now extended by structural biologist Tobias Madl (Medical University of Graz). Together, we aim to unravel regulatory intricacies of the p53/Foxo1 axis and push for innovation in treatment of metabolic disease.

How: To unravel specific modes and consequences of p53/FOXO1 interaction, we combine sophisticated structural biology tools with various NGS methods in cell-free systems, cell model systems, and transgenic tissue-specific animal models.

Who: Next to two PhD candidates in the German labs, one PhD student in the Madl lab and one (to be recruited, supervised by A. Prokesch) will attend to the project parts at the Medical University of Graz. Funding by the FWF and DFG through the Weave program (lead: A. Prokesch).

What: Obesogenic diets and physical inactivity are associated with impaired NAD+ metabolism, which has emerged as a common driver of cardiovascular and metabolic disorders. Although exercise or NAD+ replenishing therapy can reduce obesity and improve metabolic syndrome, the responses vary and the mechanisms that underlie exercise- and NAD+-associated salutary effects across multiple organs remain unknown. Toward this end, the INTERACD+ consortium undertake a mechanistically oriented translational research program to test whether NAD+ replenishment improves positive effects of endurance exercise training.

How: Within our BioTechMed flagship consortium (involving teams from the Medical University of Graz and the Universty of Graz), our lab focuses on organ communication via extracellular vesicles (EVs). We utilize a metabolic syndrome rat model that undergoes endurance training with and without NAD+ precursor supplementation. In addition, we test the clinical translatability of our experimental findings in a pilot feasibility study involving cardiovascular patients.

Who: The EV-related part and the rat experiments are driven by the PhD student Zina Riahi supervised by A. Prokesch. Funding is provided by BioTechMed Graz (lead: Simon Sedej)

What: During fasting, multiple peripheral organs cooperate to maintain systemic metabolic homeostasis. While numerous studies prove the health benefits of fasting, the molecular mechanisms governing the tissue-specific, dynamic responses are still underexplored. In this project we provide unprecedented insights into a cell’s TCL and transcriptional regulatory dynamics throughout the time course of fasting in mice.

How: Using innovative NGS-based methods (PRO-seq, single nuclei ATAC-seq) we unravel immediate early transcriptional effects and enhancer dynamics in time series samples of mice in adipose tissue and liver, yielding mechanisms that may help to explain the salutary effects of fasting on health span and on amelioration of diseases, such as metabolic disorders and cancer.

Who: Several lab members have been contributing to this project. Currently, Helene Michenthaler (PostDoc) is advancing the project together with our internal bioinformatics collaborators (Anna Shchetsova, PhD in the group of Julia Feichtinger). Funding by the FWF (A. Prokesch) and the Medical Universtiy of Graz.

• Andreas Prokesch
• Jelena Krstic
• Helene Michenthaler
• Zina Riahi
• Ruonan Xu
• Theresia Weiermair
• Sarah Enzenhofer
• Sarah Rimser
• Elisabeth Moyschewitz
• Victoria Zopf
• Lilian Lamprecht