Biology subjects

van Tellingen, O.

Publications and source records attributed to van Tellingen, O..

9 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↗

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↗

bioRxiv2021

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

cancer biology↗

bioRxiv2020

Reanalysis of in vivo drug synergy validation study rules out synergy in most cases

In a recent paper in Nature Communications, Narayan et al 1 described the development of an in silico method to predict synergy of drug combinations, validated in vivo using 5 different models for 5 different drug pairs. The published analysis of the in vivo experimental data used the Chou and Talalay Combination Index, which ignores important aspects of the study design, including, but not limited to the variability between individual mice and the longitudinal nature of the data. Furthermore, the original publication reported the Combination Index as static numbers without properly accounting for experimental variability. When 95% confidence intervals are recalculated using bootstrapping methods, 4 out of 5 drug pairs do not show a statistically significant synergistic effect. Three models (originally figures 5A, B and E) are subject to severe inaccuracies in the data handling and reporting. In one model, (originally figure 5D), the data better supports an additive effect between drugs rather than synergy. As the Chou and Talalay Combination Index is poorly suited to this data, we applied mixed-effects models fitted to the longitudinal tumor growth data as an alternative means of estimating synergy. The results support the findings from the bootstrapping analysis, namely that the majority of the drug pairs do not achieve a synergistic effect. We conclude that the in vivo validation of the in silico method to predict synergy of drug combinations as proposed by Narayan et al has a negative outcome when analyzed with appropriate statistical methods.

pharmacology and toxicology↗

bioRxiv2019

Multiple Low Dose therapy: an effective strategy to treat EGFR inhibitor-resistant NSCLC tumours

Targeted cancer drugs often elicit powerful initial responses, but generally fail to deliver long-term benefit due to the emergence of resistant cells1,2. This is thought to be the consequence of strong selective pressure exerted on the cancer drug target by a Maximum Tolerated Dose (MTD) of a drug3-5. We hypothesized that partial inhibition of multiple components in the same oncogenic signalling pathway might add up to complete pathway inhibition, while at the same time decreasing the selective pressure on each individual component to acquire a resistance mutation. We report here testing of this Multiple Low Dose (MLD) model of drug administration in Epidermal Growth Factor Receptor (EGFR) mutant non-small cell lung cancer (NSCLC). We show that as little as 20% of the individual drug doses required for full inhibition of cell viability is sufficient to completely block MAPK signalling and proliferation when used in 3D (RAF+MEK+ERK inhibitors) or 4D (EGFR+RAF+MEK+ERK inhibitors) combinations. Importantly, EGFR mutant NSCLC cells treated with EGFR inhibitors at a high dose rapidly developed resistance in vitro, but the cells treated with 3D or 4D MLD therapy did not. Moreover, NSCLC cells that had gained resistance to high dose anti-EGFR therapies were still sensitive to MLD therapy. Using several animal models, including patient derived xenografts of NSCLC tumours that are resistant to EGFR inhibitors erlotinib and osimertinib6,7, we found durable responses to MLD therapy without associated toxicity. These data support the notion that partial inhibition of multiple components of cancer-activated signalling pathways is difficult to circumvent and suggest that MLD therapy could deliver clinical benefit. We propose that MLD strategy could be an effective treatment option for EGFR mutant NSCLC patients, especially those having acquired resistance to even third generation EGFR inhibitor therapy.

cancer biology↗