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bioRxiv2020

Exploring epitope and functional diversity of anti-SARS-CoV2 antibodies using AI-based methods

Since the beginning of the COVID19 pandemics, an unprecedented research effort has been conducted to analyze the antibody responses in patients, and many trials based on passive immunotherapy -- notably monoclonal antibodies -- are ongoing. Twenty-one antibodies have entered clinical trials, 6 having reached phase 2/3, phase 3 or having received emergency authorization. These represent only the tip of the iceberg, since many more antibodies have been discovered and represent opportunities either for diagnosis purposes or as drug candidates. The main problem facing laboratories willing to develop such antibodies is the huge task of analyzing them and choosing the best candidate for exhaustive experimental validation. In this work we show how artificial intelligence-based methods can help in analyzing large sets of antibodies in order to determine in a few hours the best candidates in few hours. The MAbCluster method, which only requires knowledge of the amino acid sequences of the antibodies, allows to group the antibodies having the same epitope, considering only their amino acid sequences and their 3D structures (actual or predicted), and to infer some of their functional properties. We then use MAbTope to predict the epitopes for all antibodies for which they are not already known. This allows an exhaustive comparison of the available epitopes, but also gives a synthetic view of the possible combinations. Finally, we show how these results can be used to predict which antibodies might be affected by the different mutations arising in the circulating strains of the virus, such as the N501Y mutation that has started to spread in Great-Britain.

bioinformatics↗

bioRxiv2022

Efficacy and safety of a patch containing adipose-derived stem cells for skin wound healing. Results form a comprehensive pre-clinical evaluation program

Mesenchymal stem cell-based therapies are emerging as innovative approaches to treat chronic wounds. A common administration route used in clinical trials consists of local injections leading to uncontrolled/sub-optimal delivery. This study reports a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates Adipose-derived Stem Cells (ASCs) in a clinical-grade sponge of porcine crosslinked-gelatin. ASCs were prepared from the fat of ischemic patients. Transcriptome and proteome of ASC-patches and ASC monolayers were assessed by microarrays, bio-arrays and mass spectrometry. Tumorigenesis was investigated in immunosuppressed mice according to the European Pharmacopeia. Angiogenesis was assessed in vivo in the chick chorioallantoic membrane model. Efficacy of the ASC-patch was tested in a rat model of ischemic full-thickness skin defect. Cell stability was assessed by luminescence using ASC-patches generated from ASCs stably transduced with firefly luciferase. ASCs from ischemic patients upregulated the transcription of multiple genes involved in skin wound healing when cultured within the ASC-patch formulation. The patch was not only a concentrator, but also a reservoir of both ASC-derived regenerative factors and sponge-derived soluble fragments having healing capacity. The secretome of the ASC-patch promoted dermal fibroblast survival and epidermal epithelialization. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Transplanted patches were early invaded by new vessels in vivo, and a marked angiogenesis was confirmed in two independent animal models. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a rat animal model. Of note, ASCs were viable and locally stable for at least two weeks in vivo. We provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.

bioengineering↗

bioRxiv2023

Single-nucleus RNA sequencing provides insights into the GL261-GSC syngeneic mouse model of glioblastoma

Glioblastoma (GBM) is an aggressive tumor with very bad prognosis. The urgent need to find new effective therapies is challenged by the unique characteristics of GBM, including high intra and intertumoral heterogeneity. Using single-nucleus transcriptomics (snRNA-seq), we characterized the panorama of a preclinical immunocompetent murine model based in the implantation of mouse glioblastoma stem cells (GL261-GSCs) into the brain parenchyma. Additionally, we performed Visium spatial transcriptomics in the in vivo model to confirm the location of annotated cells. To understand the technical bias of this approach, we performed two scRNA-seq methods in GBM cells. We thoroughly characterized the tumor microenvironment (TME) at early and late stages of tumor development and upon treatment with temozolomide (TMZ), the standard of care for patients with GBM, and with Tat-Cx43266-283, a promising experimental treatment. We identified prominent GBM targets that can be addressed using this preclinical model, such as Grik2, Nlgn3, Gap43 or Kcnn4, which are involved in electrical and synaptic integration of GBM cells into neural circuits, as well as the expression of Nt5e, Cd274 or Irf8, which indicates the development of immune evasive properties in these GBM cells. In agreement, snRNA-seq unveiled high expression of several immunosuppressive-associated molecules in immune cells, such as Csf1r, Arg1, Mrc1 and Tgfb1, suggesting the development of an immunosuppressive microenvironment. We also show the landscape of cytokines, cytokine receptors, checkpoint ligands and receptors in tumor and TME cells, which are crucial data for a rational design of immunotherapy studies. Thus, Mrc1, PD-L1, TIM-3 or B7-H3 are among the immunotherapy targets that can be addressed in this model. Finally, the comparison of the preclinical GL261-GSC GBM model with human GBM subtypes unveiled important similarities with the recently identified TMEmed human GBM, indicating that preclinical data obtained in GL261-GSC GBM model might be applied to TMEmed human GBM, improving patient stratification in clinical trials. In conclusion, this work provides crucial information for future preclinical studies in GBM improving their clinical application.

neuroscience↗

bioRxiv2024

Self-Supervised Learning Reveals Clinically Relevant Histomorphological Patterns for Therapeutic Strategies in Colon Cancer

Self-supervised learning (SSL) automates the extraction and interpretation of histopathology features on unannotated hematoxylin-and-eosin-stained whole-slide images (WSIs). We trained an SSL Barlow Twins-encoder on 435 TCGA colon adenocarcinoma WSIs to extract features from small image patches. Leiden community detection then grouped tiles into histomorphological phenotype clusters (HPCs). HPC reproducibility and predictive ability for overall survival was confirmed in an independent clinical trial cohort (N=1213 WSIs). This unbiased atlas resulted in 47 HPCs displaying unique and sharing clinically significant histomorphological traits, highlighting tissue type, quantity, and architecture, especially in the context of tumor stroma. Through in-depth analysis of these HPCs, including immune landscape and gene set enrichment analysis, and association to clinical outcomes, we shed light on the factors influencing survival and responses to treatments like standard adjuvant chemotherapy and experimental therapies. Further exploration of HPCs may unveil new insights and aid decision-making and personalized treatments for colon cancer patients.

bioinformatics↗

bioRxiv2025

Synthetic peptide hydrogels as a model of the bone marrow niche demonstrate efficacy of a combined CRISPR-CAR T-cell therapy for acute myeloid leukaemia

Leukaemias, driven by mutations in hematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other hematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments efficacy. Here, we present a bioengineered human-cell containing BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissues mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.

bioengineering↗

bioRxiv2018

An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in humans and dogs. Currently, no standard treatment is available but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs1395) was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium. The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.\n\nAuthor summaryStargardt disease (STGD) is the most common inherited retinal disease causing visual impairment and blindness in children and young adults, affecting 1 in 8-10 thousand people. For other inherited retinal diseases, the dog has become an established comparative animal model, both for identifying the underlying genetic causes and for developing new treatment methods.\n\nTo date, there is no standard treatment for STGD and the mouse model is the only available animal model to study the disease. As a nocturnal animal, the morphology of the mouse eye differs from humans and therefore the mouse model is not ideal for developing methods for treatment. We have studied a novel form of retinal degeneration in Labrador retrievers showing clinical signs similar to human STGD. To investigate the genetic cause of the disease, we used whole-genome sequencing of a family quartet including two affected offspring and their unaffected parents. This led to the identification of a loss-of-function mutation in the ABCA4 gene. The findings of this study may enable the development of a canine model for human STGD.

genetics↗

bioRxiv2020

Selective germline genome edited pigs and their long immune tolerance in Non Human Primates

Organ transplantation is the only curative treatment for patients with terminal organ failure, however, there is a worldwide organ shortage. Genetically modified pig organs and tissues have become an attractive and practical alternative solution for the severe organ shortage, which has been made possible by significant progress in xenotransplantation in recent years. The past several decades witnessed an expanding list of genetically engineered pigs due to technology advancements, however, the necessary combination of genetic modifications in pig for human organ xenotransplantation has not been determined. In the current study, we created a selective germline genome edited pig (SGGEP). The first triple xenoantigens (GGTA, B4GAL, and CAMH) knockout somatic cells were generated to serve as a prototype cells and then human proteins were expressed in the xenoantigen knockout cells, which include human complement system negative regulatory proteins (CD46, CD55, and CD59); human coagulation system negative regulatory proteins thrombomodulin (THBD); tissue factor pathway inhibitor (TFPI); CD39; macrophage negative regulatory proteins (human CD47); and natural killer cell negative regulatory human HLA-E. After the successful establishment of SGGEP by the nuclear tranfer, we engrafted SGGEP skin to NHP, up to 25 days graft survival without immunosuppressive drugs was observed. Because a pig skin graft does not impact the success of a subsequent allograft or autograft or vice versa, thus our SGGEP could have a great potential for clinical value to save severe and large area burn patients and the other human organ failure. Therefore, this combination of specific gene modifications is a major milestone and provides proof of concept to initiate investigator-initiated clinical trials (IITs) in severe burn patients with defined processes and governance measures in place and the other clinical application.

synthetic biology↗

bioRxiv2020

Validation of lipid-related therapeutic targets for coronary heart disease prevention using human genetics

Drug target Mendelian randomization (MR) studies use DNA sequence variants in or near a gene encoding a drug target, that alter its expression or function, as a tool to anticipate the effect of drug action on the same target. Here, we applied MR to prioritize drug targets for their causal relevance for coronary heart disease (CHD). The targets were further prioritized using genetic co-localization, protein expression profiles from the Human Protein Atlas and, for targets with a licensed drug or an agent in clinical development, by sourcing data from the British National Formulary and clinicaltrials.gov. Out of the 341 drug targets identified through their association with circulating blood lipids (HDL-C, LDL-C and triglycerides), we were able to robustly prioritize 30 targets that might elicit beneficial treatment effects in the prevention or treatment of CHD. The prioritized list included NPC1L1 and PCSK9, the targets of licensed drugs whose efficacy has been already proven in clinical trials. To conclude, we discuss how this approach can be generalized to other targets, disease biomarkers and clinical end-points to help prioritize and validate targets during the drug development process. One Sentence SummaryWe provide genetic support for lipid-modifying drug targets for coronary heart disease prevention using drug target Mendelian randomization and further prioritization based on clinical and biological evidence.

genetics↗

bioRxiv2021

Non-Viral Engineering of CAR-NK and CAR-T cells using the Tc Buster Transposon System™

Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

bioengineering↗

bioRxiv2023

A kinome-wide synthetic lethal CRISPR/Cas9 screen reveals that mTOR inhibition prevents adaptive resistance to CDK4/CDK6 blockade in HNSCC

The comprehensive genomic analysis of the head and neck cancer (HNSCC) oncogenome revealed frequent loss of p16INK4A (CDKN2A) in most HPV negative HNSCC lesions, often concomitant with amplification of the cyclin D1 (CCND1) gene locus. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors as single agents have shown modest effect in the clinic, even when combined with cetuximab. The aberrant activation of PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials targeting mTOR showed promising results in terms of objective responses and progression free survival. However, the clinical efficacy of mTOR inhibitors (mTORi) for advanced HNSCC patients may be limited due to intrinsic or acquired resistance. By a kinome-wide CRISPR/Cas9 screen, we identified cell cycle inhibition as a synthetic lethal target for mTORi. Combination of mTORi and palbociclib, a CDK4/6 specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor in HNSCC. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that concomitant mTOR blockade reverts the adaptive resistance to palbociclib, thereby providing a novel multimodal therapeutic option for HNSCC patients by co-targeting mTOR and CDK4/6. Our findings may have broad implications to halt the emergence of palbociclib resistance.

cancer biology↗

bioRxiv2024

The polyamine transporter ATP13A3 mediates DFMO-induced polyamine uptake in neuroblastoma

High-risk neuroblastomas, often associated with MYCN oncogene amplification, are addicted to polyamines, small polycations vital for cellular functioning. We have shown that neuroblastoma cells increase polyamine uptake when exposed to the polyamine biosynthesis inhibitor DFMO, currently in clinical trial, and that this mechanism limits the efficacy of the drug. While this finding resulted in the clinical development of polyamine transport inhibitors including AMXT 1501, presently under clinical investigation in combination with DFMO, the mechanisms and transporters involved in DFMO-induced polyamine uptake are unknown. Knockdown of ATP13A3, a member of the P5B-ATPase family, limited basal and DFMO-induced polyamine uptake, attenuated MYCN-amplified and non-MYCN-amplified neuroblastoma cell growth and potentiated the inhibitory effects of DFMO. Overexpression of ATP13A3 in neuroblastoma cells increased polyamine uptake, which was inhibited by AMXT 1501, highlighting ATP13A3 as a key target of the drug. The association between high ATP13A3 expression and poorer survival in neuroblastoma further supports a role of this transporter in neuroblastoma progression. Thus, this study identified ATP13A3 as a critical regulator of basal and DFMO-induced polyamine uptake and a novel therapeutic target for neuroblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/581161v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@11bef78org.highwire.dtl.DTLVardef@106c250org.highwire.dtl.DTLVardef@bf39b1org.highwire.dtl.DTLVardef@64d05f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

DNA damage triggers cell signalling cascades that mediate repair. This signalling is frequently dysregulated in cancers. The proteins that mediate this signalling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small molecule inhibitors already in clinical trials. Here we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established, tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.

biochemistry↗

bioRxiv2019

Human Umbilical Cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

neuroscience↗

bioRxiv2018

Comparing the efficacy of cancer therapies between subgroups in basket trials

An increase in the number of targeted anti-cancer drugs and growing genomic stratification of patients has led to the development of basket clinical trials in which a single drug is tested simultaneously in multiple tumor subtypes under a master protocol. Basket trials typically involve few patients per type, making it difficult to rigorously compare responses across types. We describe the use of permutation testing to analyze tumor volume changes and Progression Free Survival across subtypes in basket trials for neratinib, larotrectinib, pembrolizumab, and imatinib. Permutation testing is a complement to the standard Simons two-stage binomial approach and can test for differences among subgroups using empirical null distributions while controlling for multiple hypothesis testing. This approach uncovers examples of therapeutic benefit missed by a binomial test; in the case of the SUMMIT trial, our analysis identifies an overlooked opportunity for use of neratinib in lung cancers carrying ERBB2 Exon 20 mutations.

cancer biology↗

bioRxiv2020

Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro, Evidencing S-Hydroxychloroquine as a Potentially Superior Drug for COVID-19

In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R- and S-enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S-chloroquine (S-CQ) and S-hydroxychloroquine (S-HCQ) significantly more active against SARS-CoV-2, as compared to R-CQ and R-HCQ, respectively. In addition, Mpro, as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S-enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC50 value of S-HCQ was higher than 20 M against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S-HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S-HCQ, substantially free of the R-enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.

biochemistry↗

bioRxiv2023

Improved CAR-T cell activity associated with increased mitochondrial function primed by galactose

CD19 CAR-T cells have led to durable remissions in patients with refractory B-cell malignancies; nevertheless, most patients eventually relapse in the long term. Many interventions aimed at improving current products have been reported, with a subset of them focusing on a direct or indirect link to the metabolic state of the CAR-T cells. We assessed clinical products from an ongoing clinical trial utilizing CD19-28z CAR-T cells from patients with acute lymphoblastic leukemia. CAR-T clinical products leading to a complete response had significantly higher mitochondrial function (by oxygen consumption rate) irrespective of mitochondrial content. Next, we replaced the carbon source of the media from glucose to galactose to impact cellular metabolism. Galactose-containing media increased mitochondrial activity in CAR-T cells, and improved in vitro efficacy, without any consistent phenotypic change in memory profile. Finally, CAR-T cells produced in galactose-based glucose-free media resulted in increased mitochondrial activity. Using an in vivo model of Nalm6 injected mice, galactose-primed CAR-T cells significantly improved leukemia-free survival compared to standard glucose-cultured CAR-T cells. Our results prove the significance of mitochondrial metabolism on CAR-T cell efficacy and suggest a translational pathway to improve clinical products.

immunology↗

bioRxiv2024

eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer

The majority of human breast cancers are dependent on hormone-stimulated estrogen receptor alpha (ER) and are sensitive to its inhibition. Treatment resistance arises in most advanced cancers due to genetic alterations that promote ligand independent activation of ER itself or ER target genes. Whereas re-targeting of the ER ligand binding domain (LBD) with newer ER antagonists can work in some cases, these drugs are largely ineffective in many genetic backgrounds including ER fusions that lose the LBD or in cancers that hyperactivate ER targets. By identifying the mechanism of ER translation, we herein present an alternative strategy to target ER and difficult to treat ER variants. We find that ER translation is cap-independent and mTOR inhibitor insensitive, but dependent on 5 UTR elements and sensitive to pharmacologic inhibition of the translation initiation factor eIF4A, an mRNA helicase. EIF4A inhibition rapidly reduces expression of ER and short-lived targets of ER such as cyclin D1 and other components of the cyclin D-CDK complex in breast cancer cells. These effects translate into suppression of growth of a variety of ligand-independent breast cancer models including those driven by ER fusion proteins that lack the ligand binding site. The efficacy of eIF4A inhibition is enhanced when it is combined with fulvestrant--an ER degrader. Concomitant inhibition of ER synthesis and induction of its degradation causes synergistic and durable inhibition of ER expression and tumor growth. The clinical importance of these findings is confirmed by results of an early clinical trial (NCT04092673) of the selective eIF4A inhibitor zotatifin in patients with estrogen receptor positive metastatic breast cancer. Multiple clinical responses have been observed on combination therapy including durable regressions. These data suggest that eIF4A inhibition could be a useful new strategy for treating advanced ER+ breast cancer.

cancer biology↗

bioRxiv2025

Endogenous Estrogen-Mimetic Compounds in Cell Culture Media Influence Human Mesenchymal Stromal Cell (hMSC) Processes and Differentiation in a Sex-Biased Manner

Estrogens are global regulators of cellular signaling pathways, impacting fundamental processes and phenotypes that are essential for tissue remodeling and homeostasis. Traditional cell culture media contains estrogen-mimetic compounds, including phenol red and endogenous estrogen in fetal bovine serum (FBS). However, the potential of these compounds to bias in vitro studies, particularly when considering sex as a biological variable, remains unclear. This gap in understanding critically impacts the culture of human mesenchymal stromal cells (hMSCs), whose basic functions and differentiation potential, central to cell therapy and tissue engineering, are sensitive to perturbations in the culture conditions. Despite this, the effect of estrogens from cell culture media on male and female hMSCs is not currently considered in cell processing for clinical trials. As such, a baseline understanding of these estrogen-mimetic media influences on hMSCs is critical for clinical efficacy and adequate study design in research. To this end, we investigated the effects of phenol red and fetal bovine serum on the proliferation, metabolism, senescence, and differentiation capacity of male and female hMSCs. Phenol red, FBS, donor sex, and 17{beta}-estradiol (E2) supplementation all had significant impacts on hMSC health and differentiation potential in culture. Notably, dosing with estrogen at the levels found in FBS did not recover most of the hMSC metrics tested. The only outcomes that were not significantly different based on donor sex were senescence and mRNA transcripts for RUNX2 and PPARG, transcriptional regulators for osteogenesis and adipogenesis. Overall, these findings reveal the sex-biased effects of estrogen and estrogen-mimetic compounds in traditional culture media, underscoring a current gap in considering sex as a biological variable in cell therapy and tissue engineering research and manufacturing.

bioengineering↗