Shnyder, S. D. | bioRxiv Science

bioRxiv2026

Mitochondrial priming in human germ cell tumors is dependent on MCL1 and BCL2L1

Germ cell tumors (GCTs) are highly sensitized to cell death in response to DNA damaging agents, a property that underlies the success of current chemotherapeutic regimens. To address the molecular basis for this, known as apoptotic priming, we evaluated how different BCL2 family members modulate the heightened sensitivity of GCTs to therapy. Our analysis of human GCTs finds consistently high expression of the pro-survival factors MCL1 and BCL2L1 (BCLX) in a cohort of primary tumors and in their embryonic precursor cells, frequently accompanied by copy number gains of these loci and reciprocal losses of their pro-apoptotic interaction partners and inhibitors, PMAIP1 (NOXA) and BAD. We find that co-inhibition of MCL1 and BCLX using selective BH3 mimetics results in a potent synthetic lethality in multiple GCT embryonal carcinoma cell lines. When these cell lines were cultured with the DNA damaging agents cisplatin or etoposide, inhibition of MCL1 or BCLX potentiated their apoptotic effect in undifferentiated embryonal carcinoma cell lines, but not in retinoic acid-differentiated cells. The inhibition of MCL1 also heightened cisplatin sensitivity in p53-deficient or -mutant cell lines, which is associated with resistance to therapy. Employing an in ovo human xenograft model, we validate that the combination of cisplatin and MCL1 inhibition enhanced the therapeutic response by eliminating tumor cells. Our findings identify MCL1 and BCLX as critical factors to maintain GCT viability and as putative therapeutic targets to further augment GCT responsiveness to DNA damaging agents.

cancer biology↗

bioRxiv2026

Hypoxia-induced ALDH7A1 expression protects colorectal cancer cells from oxidative stress and DNA damage via a HIF-1-independent mechanism

Tumour hypoxia is associated with increased invasiveness, metastasis, and drug resistance; however, its impact on drug-metabolising enzymes remains poorly understood. This study investigated the effect of hypoxia on the expression of selected aldehyde dehydrogenase (ALDH) isoforms (ALDH1A1, 1A2, 1A3, 1B1, 2, 3A1, and 7A1) in colorectal cancer (CRC) cells. CRC cell lines (HT29, DLD-1, SW480, and HCT116) were cultured under normoxic and hypoxic (0.1% O2) conditions, while HT29 and DLD-1 cells were additionally grown as multicellular spheroids (MCS). Expression of ALDH isoforms was assessed at the mRNA and protein levels. Functional studies included siRNA-mediated knockdown of ALDH1A1, ALDH3A1, and ALDH7A1, measurement of reactive oxygen species (ROS), and stable overexpression of ALDH7A1 in H1299 cells. ALDH7A1 was consistently upregulated at both transcript and protein levels in HT29 and DLD-1 cells exposed to hypoxia. Elevated ALDH7A1 expression was also observed in hypoxic regions of MCS and CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). Knockdown of ALDH7A1 in DLD-1 cells reduced proliferation, increased ALDH3A1 expression, and significantly elevated ROS levels, indicating a role in redox homeostasis and suggesting functional crosstalk between these isoforms. Conversely, stable overexpression of ALDH7A1 in H1299 cells markedly reduced ROS levels. Taken together, these findings identify ALDH7A1 as a hypoxia-responsive enzyme that promotes adaptation to oxidative stress and may contribute to CRC cell survival within the hypoxic tumour microenvironment.

cancer biology↗

bioRxiv2021

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction

BackgroundBiopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. MethodsReproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. ResultsReproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal. NOTEPlease see the end of the paper for links to ten files of supplementary information and for an independent peer review. KEY POINTSO_LISynthetic hexapeptides have upregulated and downregulated nematode reproduction, following similar work in other nematode species and in fish and frogs. C_LIO_LIPeptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro and influence circulating levels of ovine pituitary hormones in vivo via intracerebroventricular peptide infusion. C_LIO_LIThe amino acid sequence of the 14mer master peptide arose from a (rat) bioassay- guided fractionation using ovine materials, whose aim was to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. C_LIO_LIThe bioactive peptides mimic the non-contiguous six-residue receptor-binding active face of a polypeptide hormone, which in mammals is a derivative of the secretory vesicle prohormone secretogranin II (SgII), taking the form of a novel, structually complex 70mer dubbed SgII-70. C_LIO_LIThe ability has been gained to reinforce or block a default reproductive and tissue- building OFF signal, using peptide mimetics. C_LI

physiology↗