Biology subjects

Jonkers, J.

Publications and source records attributed to Jonkers, J..

At least 19 recordsLinked to original sources

bioRxiv2026

SMIM1 drives an alternative iron uptake pathway

Iron is essential for numerous biological processes, and its abundance is critical in human cells to prevent anemia or toxic overload. Although transferrin receptor-mediated iron uptake is the canonical route, human cells also possess an unstudied alternative pathway. To reveal this missing mechanism, we developed a large-scale, gain-of-function approach to assign genes to a catalogue of quantitative cellular traits. Applying this genetic strategy, we identified SMIM1, the antigen responsible for the rare Vel blood group and a transmembrane protein with unknown function, as a key regulator of iron homeostasis. We found that SMIM1 expression completely suppresses the iron deficiency caused by transferrin receptor loss. Mechanistically, SMIM1 mediates iron uptake through clathrin-dependent endocytosis of hemoglobin (Hb). SMIM1 directly binds Hb via its extracellular HKCK motif, leading to its transport to lysosomes and subsequent iron release. In the human population, genetic variants in SMIM1 influence Hb levels and erythrocyte traits. SMIM1-deficient mice display iron deficiency in early erythroid precursors in response to erythropoietic stress, underscoring the physiological relevance of this process.

cell biology↗

bioRxiv2026

CIP2A tetramerization is required for mitotic DNA repair

DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.

cell biology↗

bioRxiv2026

Defective BRCA1-mediated DNA end resection drives tandem duplication formation and FANCM synthetic lethality

AbstractBRCA1-linked cancer genomes contain abundant genome-wide [~]10 kb Group 1 tandem duplications (TDs) that are drivers of tumorigenesis. Group 1 TD formation is recapitulated at a chromosomal Tus/Ter site-specific replication fork barrier in DNA end resection-defective mouse embryonic stem (mES) cells lacking Brca1 exon 11. To explore relationships between DNA end resection and Group 1 TD formation, we analyzed Brca1 coiled coil (CC) domain mutants--separation-of-function alleles that are impaired for homologous recombination but competent for DNA end resection. Notably, Brca1 CC mutants retain the ability to suppress Group 1 TDs in the Tus/Ter system and in a mouse model of Brca1-linked tumorigenesis. These data show that Brca1 CC domain mutant cancers follow a path of tumorigenesis distinct from that of other pathogenic Brca1 alleles. FANCM is a TD co-suppressor, the loss of which is synthetic lethal/sick in combination with Brca1 exon 11 mutation. In contrast, Fancm deletion is well-tolerated by Brca1 CC mutant mES cells. Thus, Group 1 TD formation and Fancm synthetic lethality are linked phenotypes related to defective BRCA1-mediated DNA end resection.

molecular biology↗

bioRxiv2025

TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

BRCA1/2-deficient cells are hypersensitive to replication stress- and DNA-damage-inducing agents such as poly(ADP-ribose) polymerase inhibitors (PARPi), platinum drugs, and ionizing radiation (IR), largely due to impaired homologous recombination repair and replication fork (RF) protection. The precise mechanisms underlying RF vulnerability in the absence of BRCA1/2 remain incompletely understood, however. Here, we identify Thousand And One Amino Acid Kinase 1 (TAOK1) as a novel regulator of RF dynamics that sensitizes BRCA1/2-deficient cells to both PARPi and IR. Unlike established DNA repair factors, such as PARG or the 53BP1-RIF1-shieldin-CST pathway, which mediate PARPi sensitivity while protecting cells against IR, TAOK1 promotes RF degradation and suppresses post-replicative damage repair. TAOK1 depletion stabilizes RFs and reduces replication-associated DNA damage in BRCA1/2-deficient cells, thereby conferring therapy resistance. Interestingly, we found that TAOK1 functions in RF regulation to be independent of its kinase activity. Instead, it interacts with PCNA and regulates ISG15 levels and thereby affects RF stability. Our findings reveal TAOK1 as a non-canonical mediator of therapy sensitivity in BRCA1/2-deficient tumors. TeaserTAOK1 controls replication fork stability through PCNA and ISG15, affecting therapy response in BRCA1/2-deficient cancers.

cancer biology↗

bioRxiv2024

TRIP12 governs DNA Polymerase β involvement in DNA damage response and repair

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific repair controllers that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase {beta} (Pol{beta}), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Pol{beta}. Required for Pol{beta} foci formation, TRIP12 regulates Pol{beta} involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Pol{beta} affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Pol{beta}-directed BER, optimizes DNA repair pathway choice at complex lesion sites. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/588474v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@70c5bdorg.highwire.dtl.DTLVardef@1c60dfborg.highwire.dtl.DTLVardef@1c00ef6org.highwire.dtl.DTLVardef@1677f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Chemotherapy-driven de novo Wnt pathway activation dictates a dynamic shift to a drug-tolerant state in breast cancer cells

The efficacy of chemotherapy is often hindered by the enrichment of a population of cancer cells that enter a drug-tolerant persister (DTP) state, mimicking embryonic diapause, yet the underlying mechanisms of this transition remain poorly understood. This study demonstrates that both parental and chemotherapy-induced Wnt-active (WntHigh) cells in Triple-negative breast cancer exhibit transcriptional and functional properties characteristic of DTP cells, including a diapause transcriptional signature, reduced MYC expression, reversible restricted proliferation, and pronounced chemoresistance. Our findings reveal that the de novo activation of the Wnt signaling pathway, triggered by the transcriptional upregulation of components essential for canonical Wnt ligand-secretion and -activation, is critical for enriching the diapause-DTP (DTPDiap) population across various chemotherapy regimens. The diapause-DTP/WntHigh population can be selectively ablated by concomitant, rather than sequential, pharmacological inhibition of Wnt ligand-secretion alongside chemotherapy, highlighting new vulnerabilities in DTPDiap cell-emergence and potentially yielding a therapeutic opportunity against DTPs. This study shows that activation of Wnt signaling pathway is sufficient and necessary for the induction of a DTPDiap state and enhances our understanding of the introductory mechanisms driving DTP cell-enrichment upon chemotherapy.

cancer biology↗

bioRxiv2024

Transcriptomic analysis of pubertal and adult virgin mouse mammary epithelial and stromal cell populations

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and other stromal cells. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

developmental biology↗

bioRxiv2023

H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs)1,2. In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as {gamma}H2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA13-6. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours7,8, we identify a novel function for {gamma}H2AX in orchestrating drug-induced replication fork degradation. Mechanistically, {gamma}H2AX-dependent replication fork degradation is elicited by the inhibition of CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection of stalled forks. In summary, our results demonstrate a novel role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

cancer biology↗

bioRxiv2022

CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice we determined that CAFs in both invasive lobular breast cancer and triple negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo tracing and in vitro studies revealed the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. In vitro functional assays showed that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data show that CD26+ and CD26- NFs transform into distinct CAF subpopulations in breast cancer.

cancer biology↗

bioRxiv2022

Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

To maintain genome integrity, cells must avoid DNA damage by ensuring the accurate duplication of the genome and by having efficient repair and signaling systems that counteract the genome-destabilizing potential of DNA lesions. To uncover genes and pathways that suppress DNA damage in human cells, we undertook genome-scale CRISPR/Cas9 screens that monitored the levels of DNA damage in the absence or presence of DNA replication stress. We identified 160 genes in RKO cells whose mutation caused high levels of DNA damage in the absence of exogenous genotoxic treatment. This list was highly enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. Furthermore, the majority of these 160 genes are involved in a limited set of biological processes related to DNA replication and repair, nucleotide biosynthesis, RNA metabolism and iron sulfur cluster biogenesis, suggesting that genome integrity may be insulated from a wide range of cellular processes. Among the many genes identified and validated in this study, we discovered that GNB1L, a schizophrenia/autism-susceptibility gene implicated in 22q11.2 syndrome, protects cells from replication catastrophe promoted by mild DNA replication stress. We show that GNB1L is involved in the biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs) through its interaction with the TTT co-chaperone complex. These results implicate PIKK biogenesis as a potential root cause for the neuropsychiatric phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage in human cells therefore provides a powerful approach to probe genome maintenance mechanisms.

molecular biology↗

bioRxiv2022

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR-defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPi) but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combined molecular profiling with functional analysis of the HR status of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent functional restoration of BRCA1/2. We observed restoration of HR in 64% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we found that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by the loss of PARG. Our combined multi-omics analysis catalogued additional genes and pathways potentially involved in modulating PARPi response.

cancer biology↗

bioRxiv2022

MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

MDC1 is a key protein in DNA damage signaling. When DNA double-strand breaks (DSBs) occur, MDC1 localizes to sites of damage to promote the recruitment of other factors, including the 53BP1-mediated DSB repair pathway. By studying mechanisms of poly(ADP-ribose) polymerase inhibitor (PARPi) resistance in BRCA2;p53-deficient mouse mammary tumors, we identified a thus far unknown role of MDC1 in replication fork biology. MDC1 localizes at active replication forks during normal fork replication and its loss reduces fork speed. We show that MDC1 contributes to the restart of replication forks and thereby promotes sensitivity to PARPi and cisplatin. Loss of MDC1 causes MRE11-mediated resection, resulting in delayed fork restart. This improves DNA damage tolerance and causes chemoresistance in BRCA1/2-deficient cells. Hence, our results show a role for MDC1 in replication fork progression that mediates PARPi- and cisplatin-induced DNA damage, in addition to its role in DSB repair.

molecular biology↗

bioRxiv2021

Hyperactive WNT/CTNNB1 signaling induces a competing cell proliferation and epidermal differentiation response in the mouse mammary epithelium

In the past forty years, the WNT/CTNNB1 signaling pathway has emerged as a key player in mammary gland development and homeostasis. While also evidently involved in breast cancer, much unclarity continues to surround its precise role in mammary tumor formation and progression. This is largely due to the fact that the specific and direct effects of hyperactive WNT/CTNNB1 signaling on the mammary epithelium remain unknown. Here we use a primary mouse mammary organoid culture system to close this fundamental knowledge gap. We show that hyperactive WNT/CTNNB1 signaling induces competing cell proliferation and differentiation responses. While proliferation is dominant at lower levels of WNT/CTNNB1 signaling activity, higher levels cause reprogramming towards an epidermal cell fate. We show that this involves de novo activation of the epidermal differentiation cluster (EDC) locus and we identify master regulatory transcription factors that likely control the process. This is the first time that the molecular and cellular dose-response effects of WNT/CTNNB1 signaling in the mammary epithelium have been dissected in such detail. Our analyses reveal that the mammary epithelium is exquisitely sensitive to small changes in WNT/CTNNB1 signaling and offer a mechanistic explanation for the squamous differentiation that is observed in some WNT/CTNNB1 driven tumors.

cancer biology↗

bioRxiv2021

Loss of Nuclear DNA ligase III Can Revert PARP Inhibitor Resistance in BRCA1-deficient Cells by Increasing DNA Replication Stress

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

cancer biology↗

bioRxiv2021

MYC promotes immune-suppression in TNBC via inhibition of IFN signaling

Immune checkpoint inhibitor (ICI) treatment has thus far shown limited efficacy in triple-negative breast cancer (TNBC) patients, presumably due to sparse or unresponsive tumor-infiltrating lymphocytes. We reveal a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of BRCA1-proficient and -deficient TNBC, MYC overexpression dramatically decreased lymphocyte infiltration in tumors, along with immune signature loss. Likewise, MYC overexpression suppressed inflammatory signaling induced by BRCA1/2 inactivation in human TNBC cell lines. Moreover, MYC overexpression prevented the recruitment and activation of lymphocytes in co-cultures with human and mouse TNBC models. Chromatin immunoprecipitation (ChIP)-sequencing revealed that MYC directly binds promoters of multiple interferon-signaling genes, which were downregulated upon MYC expression. Finally, MYC overexpression suppressed induction of interferon signaling and tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist. Together, our data reveal that MYC suppresses innate immunity and facilitates immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs. Statement of SignificanceMYC suppresses recruitment and activation of immune cells in TNBC by repressing the transcription of interferon genes. These findings provide a mechanistic rationale for the association of high MYC expression levels with immune exclusion in human TNBCs, which might underlie the relatively poor response of many TNBCs to ICI.

cancer biology↗

bioRxiv2020

SMARCAD1 Mediated Active Replication Fork Stability Maintains Genome Integrity

Stalled fork protection pathway mediated by BRCA1/2 proteins is critical for replication fork stability that has implications in tumorigenesis. However, it is unclear if additional mechanisms are required to maintain replication fork stability. We describe a novel mechanism by which the chromatin remodeler SMARCAD1 stabilizes active replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and tolerance to replication poisons; this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naive- or PARPi-resistant tumors require SMARCAD1 mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

cell biology↗

bioRxiv2020

Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

In daily practice, different types of biomolecules are usually extracted for large-scale omics analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. While lysis of cells and tissues with urea is the accepted standard for phosphoproteomic applications, DNA, RNA and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry (MS)-based phosphoproteomics has been reported, but not benchmarked. Here we compared urea-with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g. 94% shared class 1 identifications) and deduced kinase activities (e.g. ATM, ATR, CHEK1/2, PRKDC). AGPC-based sample extraction can thus replace standard cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.

molecular biology↗

bioRxiv2019

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

cancer biology↗