Abstract Hypoxia-inducible factor 2α (HIF-2α) is a central regulator of cellular homeostasis and a known oncogenic driver in multiple cancers. Although HIF-2α is canonically defined as a nuclear transcription factor, its cytoplasmic presence and non-canonical functions remain poorly understood. Here, we performed a structural survey of HIF-2α to determine the mechanisms underlying subcellular localization, protein abundance, and activity using a deletion-construct library, transcriptional assays, and in vivo xenograft models. We found that the oxygen-dependent degradation domain (ODD), the N-terminal intrinsically disordered region (n-IDR) and the N-terminal transactivation domain (NTAD) promote cytoplasmic localization, whereas the C-terminal IDR drives nuclear accumulation. Surprisingly, we found that HIF-2α nuclear localization occurs also in the absence of PAS A and B, the domains required for ARNT (HIF-1β) dimerization, resolving the long-standing question in the field. These data suggest a dominant role for non-canonical cytoplasmic mechanisms in HIF-2α-driven tumorigenesis. Strikingly, neither NTAD nor the C-terminal CTAD was required for tumor growth in vivo , in coherence with our transcriptional assays indicating that CTAD is dispensable for transactivation and NTAD functions as a suppressor rather than an activator of transcription. Proteomic analyses reveal HIF-2α interactions with regulators of mitochondrial function, translation initiation, RNA splicing, vesicular transport, and DNA replication. Together, these findings uncover previously unrecognized structural and functional complexity of HIF-2α compartmentalization and expand its role beyond canonical transcriptional regulation.

Caenorhabditis elegans naturally inhabits low-oxygen environments but is typically studied at 21% oxygen. We cultured adult C. elegans at 2.5%, 5%, and 21% oxygen during the fertile period, quantifying daily output of hatched larvae, unfertilized oocytes, and abnormal progeny (unhatched embryos, dead or deformed larvae). While we found no evidence for increased fertility at low oxygen levels compared to 21%, we observed a trend towards fewer unfertilized oocytes after peak fertility at 5% oxygen and significantly fewer at 2.5%. Our results indicate that reproductive output differs between laboratory conditions (high oxygen) and moderately low oxygen levels (closer to natural environments).

BackgroundPheochromocytomas and paragangliomas (PPGLs) are rare and mostly non-metastatic tumors originating from adrenal medulla and paraganglia. Metastatic PGLs have a worse prognosis, but currently, there are no established criteria to determine PPGL metastatic potential. AimThe aim of this study was to investigate whether SDHB and CDK1 expression is associated with metastatic capacity in PPGL. Material and methodsA tissue microarray (TMA) was constructed from 175 tumors from a total of 149 unique PPGL patients treated at Sahlgrenska University Hospital. SDHB, CDK1, and proliferation index Ki-67 were assessed and correlated to metastatic capacity defined as a confirmed metastatic event. ResultsNegative SDHB expression was more common in patients with metastatic PPGL and displayed a trend toward lower overall survival and correlation with higher Ki-67%. High CDK1 expression was not associated with any of the parameters mentioned above. ConclusionNegative SDHB expression but not high CDK1 expression is associated with metastatic capacity in PPGL.

Cancer cells survive treatment through mechanisms that remain unclear. This study investigates the chemical changes that occur in cancer cells after treatment, focusing on lipid metabolism as a potential marker for survival and resistance. Using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and advanced multivariate statistical analysis, we compared the chemical profiles of untreated and surviving cancer cells. Region-of-Interest (ROI) analysis revealed distinct differences in the lipid compartments, with surviving cancer cells showing significant accumulation of lipid droplets. While Principal Component Analysis (PCA) was able to differentiate the chemistry of untreated and surviving cancer cells as well as their cellular components, Multivariate Curve Resolution (MCR) provided a clearer and more detailed distinction, enabling the identification of specific cellular features such as the cytoplasm, nucleus, and lipid droplets within the surviving cells. The separation of the chemistry in nucleus and lipid droplets emphasizes the effectiveness in complex spectral analysis. Furthermore, the ability to map the distribution of lipid droplets in surviving cells can advance our understanding of how these structures contribute to cancer cell survival during treatment. The study highlights the importance of lipid droplets as potential biomarkers for cancer cell adaptation and survival post-treatment, with implications for developing new therapeutic strategies.

Despite the advent of advanced molecular prognostic tools, it is still difficult to predict the course of disease for cancer patients at the individual level. This lack of predictability is also reflected in many experimental cancer model systems, begging the question of whether certain biological aspects of cancer (eg. growth, evolution etc.) can ever be anticipated or if there remains an inherent unpredictability to cancer, similar to other complex biological systems. We demonstrate by a combination of agent-based mathematical modelling, analysis of patient-derived xenograft model systems from multiple cancer types, and in-vitro culture that certain conditions increase stochasticity of the clonal landscape of cancer growth. Our findings indicate that under those conditions, the cancer genome may behave as a complex dynamic system, making its long-term evolution inherently unpredictable.

Neuroblastoma (NB) is a heterogeneous childhood cancer, characterized by the amplification of the MYCN oncogene in 40% of the high-risk cases. Our previous work demonstrated that MYCN drives metabolic reprogramming in NB, including upregulation of antioxidant enzymes. Here, we identify peroxiredoxin 6 (PRDX6) as a promising therapeutic target in NB. Pharmacological inhibition of PRDX6 reduces MYCN levels, induces apoptosis, and promotes neuronal differentiation accompanied by lipid droplet accumulation, essential for the phenotypic reprogramming. Moreover, combined inhibition of PRDX6 and glutathione S-transferase Pi 1 (GSTP1), a key antioxidant enzyme needed for PRDX6 activation, demonstrated synergistic effects both in vitro and in vivo. This strategy results in neuronal maturation as well as activity and initiates downstream pathways distinct from the ones triggered by retinoic acid, the differentiation-inducing agent currently used in clinical practice for NB. Notably, both PRDX6 and GSTP1 are highly expressed in the developing murine adrenal gland, as well as in high-risk, MYCN-amplified NB, correlating with an undifferentiated state and poor prognosis. Together, our results provide insights into the potential of PRDX6 and GSTP1 as therapeutic targets for differentiation induction for children with NB.

Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.

Table S4: Well counts for single cell colonies two months after cisplatin treatment.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest–enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Figure S12: Survival of polyploid cells is reduced by the addition of NOTCH inhibitor during cisplatin treatment.

Figure S9: Transcription factors regulating downregulated genes in HCC1806 cells surviving 10 DPT as quantified using RNAseq and CHEA3 analysis.

Figure S4: Distribution fitting and cell population identification for cell line HCC1806.

Summary Neuroblastoma is a pediatric cancer that exhibits two cellular phenotypes: adrenergic (ADRN) and mesenchymal (MES). ADRN is differentiated and therapy-sensitive, while MES is less differentiated with elevated therapy resistance. To understand neuroblastoma and its treatment response, it is important to elucidate how these phenotypes impact the eco-evolutionary dynamics of cancer cell populations and whether they represent distinct cell types or dynamic cell states. Here, we show that neuroblastoma cells undergo an ADRN to a MES phenotypic switch under chemotherapy treatment. We use a strong inference approach to generate four hypotheses on how this switch may occur: cell types without resistance, cell types with resistance, cell states without resistance, and cell states with resistance. For each of these hypotheses, we create theoretical models to make qualitative predictions about their resulting eco-evolutionary dynamics. Our results provide a framework to further experimentally determine whether ADRN and MES phenotypes are distinct cell types or dynamic cell states.

Figure S8: Pathways downregulated in HCC1806 cells surviving 10 DPT, as quantified with RNAseq and Reactome analysis.

Figure S7: Drug-resilient 786-0 cells exhibit 1-2 whole genome duplications with high fidelity

Figure S11: Survival of polyploid cells is reduced by the addition of NOTCH inhibitor during cisplatin treatment.

Figure S1: Dose response curves for cell lines after 72h cisplatin treatment

Drug-resilient cells exhibit one to two whole-genome duplications with high fidelity. A, Copy numbers in untreated and treated surviving HCC1806 cells 5 DPT, as visualized with AneuFinder (reads per 10 Mb over total amount of reads) from scWGS each row representing a single nucleus. B, Ratio of DNA content within each cell in untreated and surviving HCC1806 cells 5 DPT. The heat maps show the normalized read depth (reads per 10 Mb bins over total amount of reads in the cell) of scWGS, where blue areas show a lower number of reads, and red areas show a higher number of reads. The blocks R1, R2, and R3 in the left represent replicates 1, 2, and 3, respectively. C, Copy number of chromosome X in untreated (CTL), surviving HCC1806 cells at 5 DPT and their progeny, as visualized with chromosomal FISH of cells in interphase.

Table S1: LD50 values for cell lines after 72h cisplatin treatment.

Figure S13: Effects of T2445 treatment on cellular proliferation at 10nM.