VIB-KU Leuven Center for Cancer Biology

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM6 cells, and its expression is reduced across human cancers. In various human cancer cell lines and mouse models, ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilisation but paradoxically increases bioenergetics and accelerates neoplastic growth via activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerates neoplastic growth.

Human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) has emerged as a biologically distinct entity, typically affecting younger, non-smoking patients and showing improved survival compared to HPV-negative tumors. Accurate HPV status determination is essential for correct staging, prognostic assessment, and treatment de-escalation. Despite advances, substantial variability persists among diagnostic methods and clinical workflows. A narrative review of PubMed, Scopus, and Web of Science databases was conducted up to July 2025. Studies addressing HPV detection techniques in OPSCC-including p16^INK4a^ immunohistochemistry (IHC), HPV DNA and RNA assays, liquid biopsy approaches, and computational surrogates-were critically analyzed regarding diagnostic accuracy, clinical applicability, and emerging innovations. Tissue-based assays remain the diagnostic reference standard. p16 IHC provides high sensitivity but limited specificity and should be confirmed with nucleic acid-based methods such as DNA PCR, in situ hybridization (ISH), or E6/E7 mRNA detection. Combined or "orthogonal" testing minimizes discordance and refines risk stratification. Liquid biopsy detection of circulating HPV DNA using droplet digital PCR or next-generation sequencing has shown high sensitivity and specificity in cohorts of patients with HPV-associated OPSCC, supporting its potential role as a complementary biomarker for treatment monitoring and surveillance. However, circulating HPV DNA alone does not unequivocally identify the anatomic source of HPV DNA and should be interpreted together with clinical, radiologic, and tissue-based findings. Oral rinse and saliva assays show moderate diagnostic performance, while artificial intelligence-based radiomic and histopathologic models are emerging as complementary tools. Reliable HPV attribution in OPSCC requires a multimodal diagnostic strategy integrating p16 IHC, molecular confirmation, and ctHPV-DNA monitoring. Methodological standardization and prospective validation are essential to implement precision-guided, cost-effective workflows in routine clinical practice.

Targeted therapies are important for invasive breast cancer (IBC) treatment but are generally not standard of care in the neoadjuvant setting. To identify therapies with the potential to improve neoadjuvant treatment response, it is essential to develop patient-derived preclinical models that faithfully reflect the diversity of primary IBC subtypes in patients. Here, we generated and characterized a large-scale cohort of 60 mouse-intraductal patient-derived xenograft (MIND-PDX) models representing all subtypes of primary IBC, as well as seven matched PDX-derived organoids. We showed that our IBC-MIND cohort can serve as a platform for preclinical evaluation of experimental neoadjuvant treatments. For triple-negative IBC, we demonstrated that neoadjuvant treatment does not benefit from addition of a PARP inhibitor, whereas for estrogen receptor-positive IBC, the combination of a CDK4/6 inhibitor and fulvestrant improved neoadjuvant treatment response. Our work provides a valuable resource of primary IBC models to study breast cancer biology and develop neoadjuvant treatments.

Reprogrammed lipid metabolism with massive upregulation of tumor cell-autonomous synthesis of saturated fatty acids is a hallmark of prostate cancer (PCa) and is driven in part by aberrations in androgen receptor (AR) signaling. While lipid alterations are well described in primary PCa, the extent to which the circulating lipidome reflects tumor-associated metabolic changes in metastatic disease, and its role in therapy response, remains to be determined. This study aims to assess whether plasma lipid profiling captures tumor metabolic rewiring, and whether this reflects response to AR-targeting therapy, in metastatic castration-resistant PCa (mCRPC). Quantitative plasma lipidomics was performed on plasma samples collected from patients with mCRPC (n = 50) and cancer-free subjects (C-FS, n = 14). Samples from patients with mCRPC were collected longitudinally at the time of progression on androgen deprivation therapy prior to initiation of first-line enzalutamide (Enza), after the start of treatment with Enza, before progression on Enza. Compared to C-FS, patients with mCRPC showed distinct lipidomic signatures, characterized by increased levels of monounsaturated lipids and altered composition of the phospholipid and sphingolipid pool, mimicking the aberrations known to occur in primary PCa tissue. Enza treatment markedly reduced total lipid levels, decreased major phospholipid classes and ceramides, while increasing sphingomyelins. Notably, quantitative differences in specific sphingolipid species occurring after Enza treatment correlated with survival outcomes. Plasma lipidomics reflects key metabolic features of PCa and is profoundly impacted by AR inhibition, with prognostic relevance in patients with mCRPC. These findings support its potential as a non-invasive tool for monitoring disease activity and treatment response, and lay the groundwork for lipid-based biomarkers in mCRPC, while indicating that the lipidomic alterations observed may help inform ongoing and forthcoming research on metabolic targeting.

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) is implicated in the regulation of central carbon metabolism, but conflicting findings across experimental systems limit mechanistic insight. Here, we apply a multi-omics approach in cells ectopically expressing the ISGylation machinery independent of immune stimuli, to generate a systematic view of ISGylation in metabolic control. ISGylation preferentially targets metabolic enzymes, with marked enrichment among glycolytic proteins, suppressing the energy-yielding phase of glycolysis. Tracer metabolomics reveals a bottleneck at glyceraldehyde-3-phosphate dehydrogenase (GAPDH), reflected by accumulation of upstream intermediates and depletion of downstream metabolites. This arises from multisite ISGylation of lysines near its catalytic and regulatory regions, which reduces enzymatic activity without disrupting tetramer assembly. These findings identify GAPDH as a central metabolic checkpoint regulated by ISGylation and uncover a direct post-translational mechanism by which ISG15 controls energy metabolism.

The novel hypoxia-activated prodrug CP-506 selectively targets the hypoxic, treatment-resistant tumor microenvironment. Given the alkylating effector metabolites of CP-506, we hypothesized that defects in interstrand crosslink (ICL) and double-strand break repair influence treatment efficacy. In vitro and in vivo isogenic cancer models proficient or deficient in the Fanconi anemia (FA), homologous recombination (HR), or non-homologous end joining (NHEJ) pathway were used to assess CP-506-induced cytotoxicity and DNA damage. Viability and clonogenic assays demonstrated enhanced sensitivity to CP-506 in FA- or HR-deficient cells compared to parental cells, which was confirmed by spheroid growth inhibition studies. In vivo, CP-506 caused greater enhancement ratios in FA- and HR-deficient xenografts versus parental controls (p < 0.0001) but not in NHEJ-deficient xenografts (p = 0.18). Mechanistically, CP-506 increased γH2AX expression (1.9- to 9.3-fold) in FA- and HR-deficient cells and xenografts, whereas NHEJ-deficient models showed a 0.5-fold reduction. Alkaline comet assays confirmed CP-506-induced ICLs and DNA strand breaks but did not explain the differential therapeutic responses among isogenic cancer cells. These data indicate that deficiencies within FA or HR, but not NHEJ or nucleotide excision repair (NER), determine CP-506 sensitivity, consistent with a synthetic-lethal interaction. Therefore, tumor hypoxia and DNA repair status are key biomarkers for stratifying patients in CP-506 clinical trials.

While primary invasive lobular carcinoma (ILC) is well characterized, metastatic ILC remains understudied. Within the post-mortem tissue donation programs, UPTIDER (Belgium) and Hope for Others (USA), we first aimed to explore intra-patient heterogeneity of key prognostic and predictive markers (stromal tumor-infiltrating lymphocytes (sTIL), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and KI67). Secondly, we compared detection of the metastases by pathology on autopsy samples versus pre-mortem imaging. In total, 306 metastases from 12 patients were collected at autopsy (median: 27 per patient). Both primary tumors (n = 15) and metastases (n = 232) had low sTIL levels, with a median of 2% (range: 0.67-6.67%) and 0.67% (range: 0-13.33%), respectively. Regression models showed lower ER- and PR-expression in metastases (respectively, n = 265 and n = 64) compared to primary tumors (both p < 0.01). KI67 was significantly higher in metastases (n = 262, p = 0.02). HER2-low metastases were found in all but one patient although in varying proportion of metastases (range: 7.5-100%). Central radiology and pathology review had a median concordance of 78% at organ level (range: 33.33-100%) and 71% at patient level (range: 55.88-85.29%). Our findings suggest that a single metastatic biopsy has great limitations to guide treatment and that more adequate methods are needed to detect and monitor ILC metastases.

BACKGROUND:TF (transcription factor) Prdm16 (positive regulatory domain–containing protein 16) regulates hematopoietic and neuronal stem cell homeostasis, adipose differentiation, and cardiac development. Its role in the circulatory system extends beyond the heart, as Prdm16 loss in arterial endothelial cells (ECs) impairs arterial reperfusion of ischemic mouse limbs due to endothelial dysfunction, and PRDM16 polymorphisms are associated with coronary artery disease.METHODS:Zebrafish were used to analyze vascular development, arteriovenous endothelial specification, and the emergence of arteriovenous malformations in the absence or presence of Prdm16 or Notch signaling. Lentiviral-mediated Prdm16 overexpression in human endothelial (progenitor) cells was coupled to qRT-PCR (real-time quantitative polymerase chain reaction), Western blot, and transcriptional profiling to document Prdm16’s importance for arterial lineage specification. Coimmunoprecipitation in HEK293 (human embryonic kidney 293) cells was performed to assess physical interaction between Prdm16 and the Notch pathway. Existing mouse and human data sets were reanalyzed to evaluate Prdm16 expression in mammalian arteriovenous malformations.RESULTS:Prdm16 actively promotes arterial EC identity while suppressing venous fate. Like in mice, Prdm16 is expressed by arterial ECs early during vascular development in zebrafish, where it synergistically coordinates arterial development together with canonical notch signaling, as their combined loss in zebrafish leads to arteriovenous malformations. PRDM16’s arterializing effect on human ECs is dependent on canonical Notch activity, as it is blunted in the presence of canonical Notch inhibitors and potentiated in the presence of delta-like ligand 4. Mechanistically, Prdm16 does not increase the protein levels of the cleaved intracellular domain of Notch receptors (notch intracellular domain) but rather potentiates the effect of the latter via physical and functional interaction. Prdm16 further finetunes Notch signaling and arterial development by complexing with Hey2 (Hes-related family bHLH TF with YRPW motif 2), the basic helix-loop-helix TF acting downstream of canonical Notch during arterial lineage specification and development.CONCLUSIONS:Together, our data demonstrate an intricate interplay between Prdm16 and Notch in ECs and indicate that Prdm16 signaling may constitute a novel therapeutic target for arteriovenous malformations.

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

Cerebrospinal fluid (CSF) biomarkers are central to Alzheimer's disease (AD) diagnosis and research. However, CSF composition is shaped not only by neurodegeneration, but also by underlying physiological and pathological processes that remain poorly characterized. By integrating multi-omics data from the deeply characterized memory-clinic ACE CSF cohort (N=1,372), the Global Neurodegeneration Proteomics Consortium (N=1,863), and publicly available quantitative trait loci data, we reveal that 73.2-85.9% of the molecular variance in CSF omics data is driven by two main factors: one reflecting CSF turnover rate, and another representing blood-brain barrier (BBB) integrity. CSF turnover mainly determines brain-derived molecules, while BBB damage leads to increased blood-derived protein abundance. CSF turnover/clearance severely impacted core AD biomarker levels, affecting the classification of subjects in the A/T framework. Adjusting biomarker levels for OPCML, a novel reference marker, improved biomarker-based prediction of AD progression and removed confounded associations, revealing a proteomic signature of sporadic AD pathology that closely resembles that of autosomal dominant AD. Finally, using the ACE CSF cohort as discovery (N=1,221) and Knight ADRC as replication (N=1,073), we report a curated AD signature comprising 446 unique proteins. Our findings identify CSF dynamics as a major source of molecular variation, reshaping the interpretation of CSF biomarkers.