Biology subjects

de Gooijer, M. C.

Publications and source records attributed to de Gooijer, M. C..

8 recordsLinked to original sources

bioRxiv2025

CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity

BackgroundCranial radiotherapy (cRT), a common treatment for central nervous system tumors, induces progressive cognitive problems in over half of patients. In mice, microglial depletion via CSF1R inhibition can mitigate this effect, but the underlying cellular mechanisms remain unclear. We hypothesized that CSF1R inhibition-induced microglial ablation and repopulation improves brain health by modulating microglial reactivity to radiotherapy, which attenuates glial responses to radiotherapy. MethodsNine-week-old male C57BL/6JRj mice received either a CSF1R-inhibitor supplemented diet (pexidartinib, PLX3397) or control diet, followed by fractionated CT-guided cRT (30 Gy) or sham treatment. The pexidartinib diet was discontinued 10 days post-radiotherapy. Animals were sacrificed at three intervals post-radiotherapy, allowing the assessment of temporal changes. Multiple brain regions were assessed by immunohistochemistry for markers of microglia, astrocytes, oligodendrocytes and proliferating cells. Microglial morphological changes were assessed using the semi-automated microglia morphology analysis pipeline mGlia. ResultsRadiotherapy alone reduced microglial numbers and induced a progressive reactive morphology; mild at 30 days and pronounced at 6 months post cRT. CSF1R inhibition before cRT markedly decreased microglial markers but increased general macrophage markers at 30 days and 6 months after cRT, consistent with monocyte-derived cell engraftment. Morphometric analysis revealed rapid and severe morphological change towards a reactive morphotype at 30 days that persisted until 6 months. Microglial depletion did not prevent loss of neurogenesis or oligodendrocyte progenitor cells (OPCs) and accelerated reactive astrogliosis, though partial OPC recovery in the hippocampus and thalamus was observed at 6 months. ConclusionCSF1R inhibition combined with cRT accelerates reactive gliosis and monocyte-derived macrophage engraftment without protecting vulnerable neural cell populations, though limited long-term OPC recovery occurred. Thus, with this set-up CSF1R inhibition-induced microglial ablation and repopulation does not improve overall brain health, but is beneficial for OPCs on the long term after cRT. Key pointsO_LIPexidartinib and cranial radiotherapy have synergistic effects on microglial ablation C_LIO_LIInfiltrating monocytes repopulate the irradiated brain once the pexidartinib diet is discontinued C_LIO_LIIn the absence of microglia, astrocytes show an accelerated reactivity to radiotherapy C_LIO_LILong-term after cranial radiotherapy oligodendrocyte progenitor cell repopulation is enhanced in the pexidartinib treated animals C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/681366v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1153d64org.highwire.dtl.DTLVardef@171a100org.highwire.dtl.DTLVardef@1013e1borg.highwire.dtl.DTLVardef@949893_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

CSF1R inhibitors pexidartinib and sotuletinib induce rapid glial ablation despite their limited brain penetrability

BackgroundMicroglial reactivity, a hallmark of many neurodegenerative diseases, is thought to contribute significantly to disease pathology. In experimental models, colony stimulating factor 1 receptor (CSF1R) inhibitors transiently deplete microglia to resolve inflammation, leading to improved neuropathology. In oncology, CSF1R inhibitors modulate tumor-associated macrophages (TAMs) toward a tumor-suppressive phenotype by silencing CSF1-CSF1R signaling. As for any therapeutic, target engagement depends on effective drug delivery. In the brain a major hurdle is the limited drug delivery caused by the presence of the blood brain barrier (BBB) containing drug efflux transporters. However, the affinity to these transporters of most CSF1R inhibitors is unknown. MethodsWe assessed the brain penetrance of two CSF1R inhibitors, pexidartinib (PLX3397) and sotuletinib (BLZ945), in the absence and presence of drug transporters ABCB1 and ABCG2. We further assessed their impact on peripheral immune populations, tissue-resident macrophages, microglia and oligodendrocyte progenitor cells (OPCs). ResultsBoth compounds have a limited brain permeability (brain-to-plasma ratio: 0.1). Sotuletinib was a substrate for both ABCB1 and ABCG2, whereas pexidartinib was transported primarily by ABCB1. Despite low brain exposure, both are able to ablate microglia when given to mice at high doses, accompanied by marked depletion of OPCs and macrophage populations in the liver, intestine, and kidney, as well as non-classical monocytes in blood. Pexidartinib additionally altered splenic immune composition, increasing T cells and neutrophils, and reducing dendritic cells and non-classical monocytes. ConclusionThese findings highlight that high-dose CSF1R inhibition rapidly depletes microglia, but induces substantial off-target effects. Such systemic impacts, as well as the impact on OPCs, should be considered when interpreting experimental outcomes or translating CSF1R inhibition into clinical contexts where brain targeting is required. Key messagesO_LICSF1R inhibitors pexidartinib and sotuletinib show poor brain penetrance (brain-to-plasma ratio 0.1) C_LIO_LISotuletinib is a substrate to ABCB1 and ABCG2, pexidartinib is a substrate to ABCB1. C_LIO_LIBoth drugs rapidly deplete microglia, despite poor brain penetration C_LIO_LIMicroglia depletion is accompanied by loss of OPCs and tissue macrophages C_LI

pharmacology and toxicology↗

bioRxiv2024

Mitochondrial damage triggers therapy-induced senescence

Glioblastoma (GBM) is a fatal brain tumor with a critical need for better therapies. It is known that the PI3K, MAPK, and CDK4/6 signaling pathways are hyper-activated in these tumors; however, previous studies have used very high concentration of inhibitors to assess their importance, with mixed results. Here we developed PMCi, a combination approach that targets all three pathways simultaneously, at clinically-relevant doses. PMCi effectively suppresses GBM cell proliferation in vitro and in vivo, and outperforms monotherapies and dual combinations. PMCi acts by inducing cellular senescence, which is mediated solely by the mitochondria, and, unlike other forms of senescence, is independent of nuclear damage. This phenotype is caused by a reactive oxygen species (ROS)\cGAS-STING\senescence-associated secretory phenotype (SASP) signaling cascade, that acts in a paracrine manner to establish and maintain senescence. Our results demonstrate that mitochondrial damage is sufficient to drive senescence, and that this can be leveraged to target GBM cells.

cell biology↗

bioRxiv2024

Disconnect between in vitro and in vivo efficacy of the MPS1 inhibitor NTRC 0066-0 against glioblastoma

PurposeGlioblastoma (GBM) is the most common adult primary brain tumor for which new therapeutic strategies are desperately needed. Monopolar spindle 1 (MPS1) is a mitotic kinase that plays a pivotal role in the spindle assembly checkpoint (SAC). GBM appears to be dependent on SAC fidelity, as MPS1 is overexpressed in many GBM patients. Thus, inhibiting MPS1 seems a viable therapeutic strategy to enhance mitotic cell death by attenuating SAC fidelity. NTRC 0066-0 is an MPS1 inhibitor that combines low nanomolar potency with a relatively long on-target residence time. MethodsWe here investigate the potential of NTRC 0066-0 as monotherapy and in combination with chemo-radiation for treatment of GBM using various in vitro and orthotopic in vivo models. ResultsWe show that NTRC 0066-0 efficiently induces GBM cell death in vitro, following continuous exposure with IC50s in the low nanomolar range. In contrast to previous reports of studies with other MPS1 inhibitors, we did not observe synergy in vitro with anti-microtubule drugs, such as docetaxel and vincristine. We demonstrate that NTRC 0066-0 has a high brain penetration, despite being a substrate of the efflux transporter P-glycoprotein. However, even when using recipient Abcb1a/b;Abcg2-/- mice with superior brain penetration and administering NTRC 0066-0 using a dose-dense regimen, we did not observe antitumor efficacy against an orthotopic GBM mouse model, neither as monotherapy nor in combination with standard-of-care temozolomide chemotherapy and radiotherapy. ConclusionThese data indicate that GBM is probably not a suitable indication for developing MPS1 inhibitors.

cancer biology↗

bioRxiv2024

NAC-mediated ribosome localization regulates cell fate and metabolism in intestinal stem cells

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NAC is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function. TeaserThe location of ribosomes in cells is regulated, and defines the fate of intestinal stem cells.

molecular biology↗

bioRxiv2023

Gemcitabine therapeutically disrupts essential SIRT1-mediated p53 repression in Atypical Teratoid/Rhabdoid Tumors

BackgroundAtypical Teratoid/Rhabdoid Tumors (ATRT) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. Despite recent advances in understanding the molecular characteristics and subclasses of these tumors, effective therapeutic options remain scarce. MethodsIn this study, we developed and validated a novel patient-derived ATRT culture and xenograft model, which we used alongside a panel of other primary ATRT models for large-scale drug discovery assays. The identified hits were mechanistically and therapeutically investigated using an array of molecular assays and two orthotopic xenograft murine models. ResultsWe found that ATRT are selectively sensitive to the nucleoside analogue gemcitabine, with additional efficacy in Sonic Hedgehog (SHH)-subtype ATRT. Gene expression profiles and protein analyses indicated that gemcitabine treatment causes degradation of Sirtuin 1 (SIRT1), resulting in cell death through activation of NF-kB and p53. Furthermore, we discovered that gemcitabine-induced loss of SIRT1 results in a nucleus-to-cytoplasm translocation of the SHH signaling activator GLI2, explaining the additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of SHH-subgroup ATRT xenograft-bearing mice with gemcitabine resulted in a >30% increase in median survival (p<0.005, log-rank test) and yielded long-term survivors in two independent patient-derived xenograft models. ConclusionsThese findings demonstrate that ATRT are highly sensitive to gemcitabine treatment, and we propose that gemcitabine may form part of a future multimodal treatment strategy for ATRT. Key points- ATRT are specifically sensitive to gemcitabine treatment - SIRT1 may serve as a novel therapeutic target in ATRT - Gemcitabine should be considered for clinical use in ATRT patients Importance of the studyAtypical Teratoid/Rhabdoid Tumors (ATRT) are highly malignant pediatric brain tumors with a 5-year survival of merely 30%, for which effective treatment options are limited. In this study, we propose a potential novel treatment strategy for ATRT patients. We show that ATRT are highly sensitive to the chemotherapeutic gemcitabine, that takes advantage of ATRT-specific SIRT1 overexpression and disrupts p53 suppression and hedgehog signaling. Importantly, we show that gemcitabine significantly prolongs survival of ATRT patient-derived xenograft models, prolonging survival by over 30%. This effect was achieved using gemcitabine concentrations that are achievable in human brain and well-tolerated in pediatric patients. As such, gemcitabine could be readily incorporated into clinical treatment protocols and expand the still very limited therapeutic options for ATRT-patients.

cancer biology↗

bioRxiv2022

Acquired and intrinsic resistance to vemurafenib in BRAFV600E-driven melanoma brain metastases

PurposeBRAFV600-mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but responses are generally less durable than those of extracranial metastases. We here tested the hypothesis that the drug efflux transporters P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP;ABCG2) expressed at the blood-brain barrier (BBB) offer MBMs protection from therapy. MethodsWe intracranially implanted A375 tumor cells in wild-type and Abcb1a/b;Abcg2-/- mice. We characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib administration and determined the efficacy against brain metastases and subcutaneous lesions. ResultsAlthough contrast-enhanced MRI demonstrated that the integrity of the BBB is disrupted in A375 MBMs, vemurafenib achieved greater antitumor efficacy against MBMs in Abcb1a/b;Abcg2-/- mice compared to wild-type mice. Concordantly, P-gp and BCRP are expressed in MBM-associated brain endothelium both in patients and in A375 xenografts and limited vemurafenib penetration into A375 MBMs. Confirming the BBB-specific context of this protection, vemurafenib was equally effective against subcutaneous A375 tumors in WT and Abcb1a/b;Abcg2-/- mice. Intriguingly, although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2-/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Rather, MBMs likely resorted to noncanonical growth signaling, as target inhibition of canonical MAPK pathway signaling components was maintained in resistant intracranial A375 tumors. ConclusionsWe demonstrate that BRAFV600E-driven MBMs are partly intrinsically protected from vemurafenib by the BBB. Intriguingly, MBMs can also rapidly acquire resistance in situ, likely by resorting to non-canonical growth signaling.

pharmacology and toxicology↗

bioRxiv2022

Fractionated brain irradiation profoundly reduces hippocampal immature neuron numbers without affecting spontaneous behavior and cognition in mice

Whole brain radiotherapy (WBRT) is used to treat patients with primary brain tumors, or brain metastasis from various primary tumors to improve tumor control. However, WBRT can lead to cognitive decline. We treated mice with fractionated WBRT (fWBRT) to establish a model system to study the mechanisms underlying cognitive decline. Besides a series of traditional cognitive tests, we also assessed the effect on spontaneous behavior as measured in automated home cages. Male C57Bl/6j mice (n=11 per group) received bi-lateral fWBRT at a dosage of 4 Gy/day on 5 consecutive days. In line with previous reports, immunohistochemical analysis of doublecortin (DCX) positive cells in the dentate gyrus showed a profound reduction in immature neurons at 4 weeks after fWBRT. Surprisingly, spontaneous behavior as measured in automated home cages was not affected. Moreover, learning and memory measured with traditional tasks - including the novel object recognition task, novel location recognition task, Barnes maze, and fear conditioning - was also not affected at 4-6 weeks after fWBRT. At 10-11 weeks after fWBRT a difference in escape latency during the learning phase, but not in the probe phase of the Barnes maze was observed. In conclusion, although we confirmed the effect of fWBRT on neurogenesis at 4 weeks after fWBRT, we did not find clear effects on spontaneous behavior in the automated home cage nor on learning abilities as measured by traditional cognitive tasks. The relationship between the neurobiological effects of fWBRT and cognition seems more complex than often assumed and the choice of animal model, cognitive tasks, neurobiological parameters, and experimental set-up might be important factors in these types of experiments.

animal behavior and cognition↗