Biology subjects

Herold, M. J.

Publications and source records attributed to Herold, M. J..

14 recordsLinked to original sources

bioRxiv2026

Complement receptor-mediated uptake into renal mononuclear phagocytes promotes intracellular UPEC persistence and limits β-lactam efficacy in pyelonephritis

IntroductionAcute pyelonephritis remains a major clinical problem. Relapses occur despite apparently appropriate antibiotic therapy, suggesting that uropathogenic Escherichia coli (UPEC) persist in intrarenal niches. Intracellular bacterial reservoirs are a plausible explanation, but the relevant host cells, entry mechanisms and therapeutic implications in the kidney remain undefined. In principle, such reservoirs should favor choosing intracellularly active antibiotics, but increasing resistance to many of these agents leaves {beta}-lactams widely used in clinical practice, despite their predominantly extracellular activity. MethodsWe analyzed murine pyelonephritis to identify the cellular reservoir of persistent UPEC. We generated mice genetically deficient for complement receptors CR3 and CR4 and tested their role in bacterial entry and persistence in vivo. Pharmacological complement receptor inhibition was applied to assess whether blocking bacterial re-entry into host cells improves antibiotic efficacy. ResultsRenal MNP were identified as the major intracellular reservoir for UPEC in mice. Complement opsonization enabled bacterial entry into these cells through CR3 and CR4, allowing UPEC to evade neutrophil-mediated killing and extracellularly active antibiotics. Genetic deletion of CR3 and CR4 abolished intracellular bacterial persistence and reduced renal bacterial burden. Because MNP undergo physiological turnover, intracellular UPEC must periodically exit host cells and infect new ones. Pharmacological inhibition of complement receptors prevented such bacterial re-entry and enhanced the efficacy of {beta}-lactam antibiotics which cannot penetrate cell membranes. ConclusionsComplement receptor-mediated entry into renal MNP establishes an intracellular UPEC reservoir that promotes persistence during experimental pyelonephritis. Blocking these receptors prevents renewal of the intracellular niche and improves {beta}-lactam efficacy in mice.

microbiology↗

bioRxiv2025

Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

microbiology↗

bioRxiv2025

A novel, high-density CRISPR activation platform for mapping cancer dependencies and resistance pathways ex vivo and in vivo

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.

cancer biology↗

bioRxiv2025

Thioredoxin reductase 3 inhibition sensitizes triple-negative breast cancer cells to EGFR inhibitors

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis and limited treatments. Although 40-70% of TNBC cases overexpress EGFR, anti-EGFR therapies show minimal clinical benefit. This may result from inherent resistance, inactive EGFR, or its absence on the plasma membrane. Here, we used genome wide CRISPR knock out library screening to identify factors that mediate resistance to EGFR inhibitor. We discovered that depletion of a redox protein, thioredoxin reductase 3 (TXNRD3), in MDA-MB-231 cells reduces cell survival after Erlotinib treatment suggesting that loss of TXNRD3 may sensitize TNBC cells to EGFR inhibitors. siRNA-induced knockdown or pharmacological inhibition of TXNRD3 using an FDA-approved drug Auranofin significantly sensitized EGFR-high TNBC cells to EGFR inhibitors. Mechanistically, TXNRD3 knockdown or inhibition using Auranofin increased oxidative stress-mediated accumulation of phosphorylated EGFR at Y1068 and increased surface accumulation. Interestingly, combination of Auranofin with EGFR inhibition markedly induced antibody-dependent cell mediated cytotoxicity (ADCC) and exerted a significant anti-cancer activity in vivo model. Overall, our findings indicate that targeting TXNRD3 with Auranofin can activate EGFR and enhance its surface localization, thereby sensitizing TNBC cells to anti-EGFR therapies. This approach offers a promising strategy for treating TNBC patients who are resistant to current EGFR-targeted treatments.

cancer biology↗

bioRxiv2024

Transcriptomic alterations including p53 pathway dysregulation prime DNMT3A mutant cells for transformation

DNMT3A mutations are prevalent in haematologic malignancies. Our mouse model introduced the murine homologue (R878H) of the human hotspot R882H mutation into the mouse Dnmt3a locus, resulting in globally reduced DNA methylation in all tissues. Mice with heterozygous R878H mutations developed {gamma}-radiation induced thymic lymphoma more rapidly than control mice, suggesting a vulnerability to stress stimuli in Dnmt3aR878H/+ cells. In competitive transplantations, Dnmt3aR878H/+ Lin-Sca-1+Kit+ (LSK) cells had a competitive advantage over wt cells, indicating a self-renewal phenotype at the expense of differentiation. RNA-sequencing of Dnmt3aR878H/+ LSKs exposed to low dose {gamma}-radiation showed downregulation of the p53 pathway. Accordingly, reduced PUMA expression was observed by flow cytometry in the bone marrow of {gamma}-irradiated Dnmt3aR878H/+ mice due to altered p53 signalling. These findings provide new insights as to how DNMT3A mutations cause subtle changes in the transcriptome of LSK cells which contribute to their increased self-renewal and propensity for malignant transformation. SIGNIFICANCEHotspot DNMT3A R882H mutations are overrepresented in leukaemia suggesting that they confer a susceptibility to disease if further mutations are acquired. To advance therapies for DNMT3A mutant disease, it is essential to understand the mechanisms by which this mutation primes cells for malignant transformation.

cancer biology↗

bioRxiv2023

Relative importance of MCL-1's Anti-Apoptotic versus Non-Apoptotic Functions in vivo

MCL-1 is an anti-apoptotic member of the BCL-2 protein family that ensures cell survival by blocking the intrinsic apoptotic cell death pathway1. MCL-1 is unique in being essential for early embryonic development and the survival of many cell types, including many cancer cells, which are not affected by the loss of the other anti-apoptotic BCL-2 family members1-4. Non-apoptotic functions of MCL-1 controlling mitochondrial ATP production and dynamics have been proposed to underlie this unique requirement for MCL-15-9. The relative contributions of the anti-apoptotic versus the non-apoptotic functions of MCL-1 in normal physiology have not been addressed. Here we replaced the coding sequence for MCL-1 with those for the anti-apoptotic proteins BCL-XL, BCL-2 or A1. We hypothesised that BCL-XL, BCL-2 and A1 may substitute for MCL-1 in the inhibition of apoptosis, but that they will not be able to replace MCL-1s non-apoptotic function. Strikingly, Mcl-1Bcl-xL/Bcl-xL and Mcl-1Bcl-2/Bcl-2 embryos survived to embryonic day 14.5, greatly surpassing the pre-implantation lethality of Mcl-1-/- embryos at E3.5. This demonstrates that the non-apoptotic functions of MCL-1 are dispensable for early development. However, at later stages of development and life after birth many cell types, particularly ones with high energy demand, were found to require both the anti-apoptotic and the non-apoptotic functions of MCL-1. These findings reveal the relative importance of these distinct functions of MCL-1 in physiology, providing important information for basic biology and the advancement of MCL-1 inhibitors in cancer therapy.

cell biology↗

bioRxiv2022

An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularised CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labour-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This was accomplished through improved viability of the transfected cells, paralleled with provision of enriched environment for the robust establishment and proliferation of singularised iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50-65% for NHEJ and 10% for HDR, indicative of the methods utility in stem cell disease modelling.

genetics↗

bioRxiv2022

Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC (which is abnormally highly expressed in [~]70% of human cancers) we have used a genome-wide CRISPR knockout screen in E{micro}-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discovered that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice. Low expression of the GATOR1 complex genes correlated with poor survival outcomes for human patients with high MYC- expressing cancers. Murine lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors as a single agent therapy, both in vitro and in vivo. These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition. Key PointsO_LIIn vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor of c-MYC-driven lymphomagenesis. C_LIO_LIGATOR1 deficiency in MYC-driven lymphomas confers sensitivity to mTOR inhibition, suggesting a novel therapeutic approach. C_LI

cancer biology↗

bioRxiv2021

The inflammasome-activated cytokine IL-1 is targeted for ubiquitylation and proteasomal degradation to limit its inflammatory potential

Interleukin-1{beta} (IL-1{beta}) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in wide-spread diseases. Therefore, IL-1{beta} activity must be fine-tuned to enable antimicrobial responses whilst limiting collateral damage. Here we report that precursor IL-1{beta} is rapidly turned over by the proteasome and this correlates with its decoration by K11-, K63- and K48-linked ubiquitin chains. The ubiquitylation of IL-1{beta} is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1{beta} cleavage by caspase-1. We further demonstrate that IL-1{beta} K133 is modified by ubiquitin and forms a salt bridge with IL-1{beta} D129. Loss of IL-1{beta} K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1{beta}. Accordingly, IL-1{beta}K133R/K133R mice display increased precursor IL-1{beta} upon inflammasome priming and increased bioactive IL-1{beta}, both in vitro and following LPS injection in vivo. These findings reveal new mechanisms for limiting IL-1{beta} activity and safeguarding against damaging inflammation.

immunology↗

bioRxiv2020

Loss of mutant TP53 does not impair the sustained proliferation, survival ormetastasis of diverse cancer cells

The tumour suppressor TP53 is the most frequently mutated gene in human cancer and these aberrations confer poor chemotherapeutic responses1-3. Point mutations typically cluster in the DNA binding domain, with certain hot-spot residues disproportionally represented1-4 These mutations abrogate binding of the TP53 transcription factor to DNA and thereby prevent upregulation of genes critical for tumour suppression (loss-of-function)1-3. Mutant TP53 is reported to additionally contribute to tumour development, sustained growth and metastasis not only through dominant-negative effects on wild-type TP535 but also through neomorphic gain-of-function (GOF) activities6. Understanding the contributions of these postulated attributes of mutant TP53 to the development and expansion of tumours will facilitate the design of rational therapeutic strategies. Here we used CRISPR/CAS9 to delete mutant TP53 in a panel of diverse human cancer cell lines. The loss of mutant TP53 expression had no impact on the survival, proliferative capacity or metabolic state of the tumour cells, nor did it sensitise them to cellular stresses and chemotherapeutic agents. These data suggest that putative GOF effects of mutant TP53 are not universally required for the sustained survival and proliferation of fully malignant cells. Therefore, therapeutic approaches that abrogate expression or function of mutant TP53 would not be expected to have substantial impact.

cancer biology↗

bioRxiv2019

An Erg driven transcriptional program controls B-lymphopoiesis

Summary/AbstractB-cell development is initiated by the stepwise differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating the mature B-cells that mediate protective immunity. This highly regulated process also generates clonal immunological diversity via recombination of immunoglobulin genes. While several transcription factors that control B-cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for the earliest steps in B-cell differentiation. Erg initiates a transcriptional network involving the B-cell lineage defining genes, Ebf1 and Pax5, that directly promotes the expression of key genes involved in V(D)J recombination and formation of the B-cell receptor. Complementation of the Erg-deficiency with a productively rearranged immunoglobulin gene rescued B-cell development, demonstrating that Erg is an essential and exquisitely stage specific regulator of the gene regulatory network controlling B-lymphopoiesis.

immunology↗

bioRxiv2018

miR17~92 is essential for the survival of hematopoietic stem and progenitor cells by restraining pro-apoptotic BIM

The micro RNA cluster miR17~92, also known as oncomiR-1, impacts diverse cellular processes, such as cell survival and proliferation. Constitutive loss of miR17~92 in mice causes severe defects in skeletal development, organ development and hematopoiesis, resulting in early post-natal lethality. The critical functions of miR17~92 in a fully developed animal have not yet been explored. Here we show that deletion of miR17~92 in adult mice [Formula] had no impact on their lifespan or general well-being. However, detailed analysis of the hematopoietic system in [Formula] mice, revealed a dramatic reduction in all mature hematopoietic lineages, which was due to the loss of early hematopoietic stem/progenitor cells (HSPCs). Strikingly, the concomitant loss of the pro-apoptotic BH3-only protein BIM rescued the loss of the HSPCs and all of their differentiated progeny that was caused by the deletion of miR17~92. These findings demonstrate that miR17~92 is critical for the survival of HSPCs by restraining the activity of the pro-apoptotic BH3-only protein BIM.

cell biology↗

bioRxiv2018

Humanized Mcl-1 mice enable accurate pre-clinical evaluation of MCL-1 inhibitors destined for clinical use

MCL-1 is a pro-survival BCL-2 protein required for the sustained growth of many cancers. Recently a highly specific MCL-1-inhibitor, S63845, showing 6-fold higher affinity to human compared to mouse MCL-1 has been described. To accurately test efficacy and tolerability of this BH3 mimetic drug in pre-clinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse in which MCL-1 was replaced with its human homologue. HuMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to MCL-1 inhibition. Importantly, non-transformed cells and lymphomas from huMcl-1;E-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;E-Myc lymphoma cells are transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide leads to long-term remission in ~60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMCL-1 mouse model to test MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.

cancer biology↗

bioRxiv2018

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

cell biology↗