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Tissue sections of spleen and liver were stained with the F4/80 macrophage marker. These photos are representative of the whole organ of two tested mice.

Supplementary Table 1. Crystallographic Data and Refinement statistics. Supplementary Table 2. Comparing Target Inhibition by Quizartinib and PLX3397 in Engineered Ba/F3 cells Expressing FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y. Supplementary Table 3. Inhibitory Concentration (IC50) for Proliferation of Human Leukemia Cell Lines in PLX3397. Supplementary Table 4. 48 Hour Inhibitory Concentration (IC50) for Proliferation of Ba/F3 Cells Expressing Quizartinib and PLX3397-Resistant FLT3-ITD Mutant Isoforms. Supplementary Table 5. Patient Characteristics. Supplementary Table 6. Low Frequency FLT3 Kinase Domain Mutations of Uncertain Significance Observed at the Time of Resistance in Patient 1.14. Supplementary Figure 1. Composite omit map contoured at 1sigma level for the bound quizartinib and a structural water molecule. Supplementary Figure 2. Comparing the actual and previously predicted binding modes of quizartinib. Supplementary Figure 3. Structural model showing that D835 induces the DFG-in conformation of the activation loop, resulting in an orientation of F830 that precludes quizartinib binding. Supplementary Figure 4. Structural superposition of quizartinib (green) and PLX3397 (orange) highlighting the key structural difference responsible for their different susceptibilities to L691. Supplementary Figure 5. PLX3397 Inhibits FLT3 Signaling In Vitro. Supplementary Figure 6. Molm14 F691L Cells Demonstrate Resistance to Quizartinib. Supplementary Figure 7. Activity of PLX3397Against Quizartinib Resistance-Causing FLT3-ITD Kinase Domain Mutations.

Supplementary Methods, Figures 1-10, Tables 1-8 from Leukocyte Complexity Predicts Breast Cancer Survival and Functionally Regulates Response to Chemotherapy

Abundant macrophage infiltration of solid cancers commonly correlates with poor prognosis. Tumor-promoting functions of macrophages include angiogenesis, metastasis formation, and suppression of Th1-type immune responses. Here, we show that successful treatment of cervical carcinoma in mouse models with synthetic long peptide (SLP) vaccines induced influx of cytokine-producing CD8 T cells that strongly altered the numbers and phenotype of intratumoral macrophages. On the basis of the expression of CD11b, CD11c, F4/80, Ly6C, Ly6G, and MHC II, we identified four myeloid subpopulations that increased in numbers from 2.0-fold to 8.7-fold in regressing tumors. These changes of the intratumoral myeloid composition coincided with macrophage recruitment by chemokines, including CCL2 and CCL5, and were completely dependent on a vaccine-induced influx of tumor-specific CD8 T cells. CD4 T cells were dispensable. Incubation of tumor cells with T cell–derived IFNγ and TNFα recapitulated the chemokine profile observed in vivo, confirming the capacity of antitumor CD8 T cells to mediate macrophage infiltration of tumors. Strikingly, complete regressions of large established tumors depended on the tumor-infiltrating macrophages that were induced by this immunotherapy, because a small-molecule drug inhibitor targeting CSF-1R diminished the number of intratumoral macrophages and abrogated the complete remissions. Survival rates after therapeutic SLP vaccination deteriorated in the presence of CSF-1R blockers. Together, these results show that therapeutic peptide vaccination could induce cytokine-producing T cells with strong macrophage-skewing capacity necessary for tumor shrinkage, and suggest that the development of macrophage-polarizing, rather than macrophage-depleting, agents is warranted. Cancer Immunol Res; 3(9); 1042–51. ©2015 AACR.

Supplementary Data from Results from Phase I Extension Study Assessing Pexidartinib Treatment in Six Cohorts with Solid Tumors including TGCT, and Abnormal CSF1 Transcripts in TGCT

Purpose:To assess the response to pexidartinib treatment in six cohorts of adult patients with advanced, incurable solid tumors associated with colony-stimulating factor 1 receptor (CSF1R) and/or KIT proto-oncogene receptor tyrosine kinase activity.Patients and Methods:From this two-part phase I, multicenter study, pexidartinib, a small-molecule tyrosine kinase inhibitor that targets CSF1R, KIT, and FMS-like tyrosine kinase 3 (FLT3), was evaluated in six adult patient cohorts (part 2, extension) with advanced solid tumors associated with dysregulated CSF1R. Adverse events, pharmacokinetics, and tumor responses were assessed for all patients; patients with tenosynovial giant cell tumor (TGCT) were also evaluated for tumor volume score (TVS) and patient-reported outcomes (PRO). CSF1 transcripts and gene expression were explored in TGCT biopsies.Results:Ninety-one patients were treated: TGCT patients (n = 39) had a median treatment duration of 511 days, while other solid tumor patients (n = 52) had a median treatment duration of 56 days. TGCT patients had response rates of 62% (RECIST 1.1) and 56% (TVS) for the full analysis set. PRO assessments for pain showed improvement in patient symptoms, and 76% (19/25) of TGCT tissue biopsy specimens showed evidence of abnormal CSF1 transcripts. Pexidartinib treatment of TGCT resulted in tumor regression and symptomatic benefit in most patients. Pexidartinib toxicity was manageable over the entire study.Conclusions:These results offer insight into outcome patterns in cancers whose biology suggests use of a CSF1R inhibitor. Pexidartinib results in tumor regression in TGCT patients, providing prolonged control with an acceptable safety profile.

PDF file - 32K, Expression of CSF-1 by SM1.

Growing evidence suggests that tumor-associated macrophages (TAM) promote cancer progression and therapeutic resistance by enhancing angiogenesis, matrix-remodeling, and immunosuppression. In this study, prostate cancer under androgen blockade therapy (ABT) was investigated, demonstrating that TAMs contribute to prostate cancer disease recurrence through paracrine signaling processes. ABT induced the tumor cells to express macrophage colony-stimulating factor 1 (M-CSF1 or CSF1) and other cytokines that recruit and modulate macrophages, causing a significant increase in TAM infiltration. Inhibitors of CSF1 signaling through its receptor, CSF1R, were tested in combination with ABT, demonstrating that blockade of TAM influx in this setting disrupts tumor promotion and sustains a more durable therapeutic response compared with ABT alone. Cancer Res; 75(6); 950–62. ©2015 AACR.

Time course of treatment response to ADT in CWR22Rv1 orthotopic tumors.

Figure S1. Flow cytometric analysis of leukocyte infiltration in orthotopic PDAC tumors. Figure S2. CSF1 blockade depletes CD206+ Mo-MDSCs and upregulates MHCII expression in Lymphoid DCs. Figure S3. A-B) Mice bearing orthotopic KC. Table S1. Gene lists from array analysis are depicted. Lists were selected by fold change > or < 1.5, pvalue

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Supplemental Figure Legends from CSF1 Receptor Targeting in Prostate Cancer Reverses Macrophage-Mediated Resistance to Androgen Blockade Therapy

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ABT also induced an immunosuppressive phenotype in TAMs.

The impact of CSF-1/CSF1R blockade is not tumor-intrinsic.

Chemical structure of PLX3397.

The prominent presence of macrophages in Myc-CaP tumors.

Splenic macrophages expand upon castration.

PDF file - 177K, Targeting TAMs decreases TICs. Macrophages directly promote chemoresistance. Efficacy of targeting CSF1R in combination with chemotherapy.

PDF file - 59K, Combined anti-tumor activity of adoptive cell transfer (ACT) immunotherapy and PLX3397 in SM1 and B16 murine melanoma.

PDF file - 68K, Gating strategy for the studies of myeloid cells.

PDF file - 490K, Effects of PLX3397 on the distribution of adoptively transferred lymphocytes in the OT-1 ACT model.

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Purpose: Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397). Experimental Design: We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg. Results: Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis. Conclusions: Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

BACKGROUND Although observational studies have shown percutaneous patent foramen ovale (PFO) closure to be a safe means of reducing the frequency and duration of migraine, randomized clinical trials have not met their primary efficacy endpoints. OBJECTIVES The authors report the results of a pooled analysis of individual participant data from the 2 randomized trials using the Amplatzer PFO Occluder to assess the efficacy and safety of percutaneous device closure as a therapy for episodic migraine with or without aura. METHODS The authors analyzed individual patient-level data from 2 randomized migraine trials (the PRIMA [Percutaneous Closure of Patent Foramen Ovale in Migraine With Aura] and PREMIUM [Prospective Randomized Investigation to Evaluate Incidence of Headache Reduction in Subjects with Migraine and PFO Using the Amplatzer PFO Occluder Compared to Medical Management] studies). Efficacy endpoints were mean reduction in monthly migraine days, responder rate (defined as ≥50% reduction in monthly migraine attacks), mean reduction in monthly migraine attacks, and percentage of patients who experienced complete cessation of migraine. The safety endpoint was major procedure- and device-related adverse events. RESULTS Among 337 subjects, 176 were randomized by blocks to device closure and 161 to medical treatment only. At 12-month follow-up, the analysis met 3 of the 4 efficacy endpoints: mean reduction of monthly migraine days (-3.1 days vs. -1.9 days; p = 0.02), mean reduction of monthly migraine attacks (-2.0 vs. -1.4; p = 0.01), and number of subjects who experienced complete cessation of migraine (14 [9%] vs. 1 [0.7%]; p

The mechanisms of brain metastasis are incompletely understood. Circulating tumor cells travel to the right heart and through the pulmonary circulation, where they may become lung metastases, and can circulate further to the left heart and brain. In patients who develop brain metastases without lung involvement, we hypothesized that cancer cells may travel directly from the right atrium to left atrium via a patent foramen ovale (PFO), akin to paradoxical embolism. If the prevalence of PFO is greater in these individuals compared to the general population (20–30%), PFO may play a role in brain metastasis, and prophylactic closure may provide benefit. Accordingly, we investigated the prevalence of PFO in patients with brain metastases without prior lung involvement. We prospectively identified patients with brain metastases from a non-lung primary cancer with no preceding or concurrent lung involvement. Nine eligible participants underwent a transcranial Doppler study with intravenous agitated saline to assess for PFO. Among nine participants, primary cancers were breast (n = 6), upper gastrointestinal (n = 2), and thyroid (n = 1). A positive bubble study was identified in 2/9 (22.2%) participants: one female with breast cancer and one male with duodenal adenocarcinoma. No participants developed lung metastases on subsequent chest imaging. In this prospective pilot study, we found a similar prevalence of PFO in patients who developed brain metastases without preceding lung involvement compared to estimates for the general population. Through a larger study is needed, the development of brain metastases in these individuals may primarily reflect tumor-specific biological factors diecting metastasis organotropism.

International audience; Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

Purpose: To assess the response to pexidartinib treatment in six cohorts of adult patients with advanced, incurable solid tumors associated with colony-stimulating factor 1 receptor (CSF1R) and/or KIT proto-oncogene receptor tyrosine kinase activity. Patients and Methods: From this two-part phase I, multicenter study, pexidartinib, a small-molecule tyrosine kinase inhibitor that targets CSF1R, KIT, and FMS-like tyrosine kinase 3 (FLT3), was evaluated in six adult patient cohorts (part 2, extension) with advanced solid tumors associated with dysregulated CSF1R. Adverse events, pharmacokinetics, and tumor responses were assessed for all patients; patients with tenosynovial giant cell tumor (TGCT) were also evaluated for tumor volume score (TVS) and patient-reported outcomes (PRO). CSF1 transcripts and gene expression were explored in TGCT biopsies. Results: Ninety-one patients were treated: TGCT patients (n = 39) had a median treatment duration of 511 days, while other solid tumor patients (n = 52) had a median treatment duration of 56 days. TGCT patients had response rates of 62% (RECIST 1.1) and 56% (TVS) for the full analysis set. PRO assessments for pain showed improvement in patient symptoms, and 76% (19/25) of TGCT tissue biopsy specimens showed evidence of abnormal CSF1 transcripts. Pexidartinib treatment of TGCT resulted in tumor regression and symptomatic benefit in most patients. Pexidartinib toxicity was manageable over the entire study. Conclusions: These results offer insight into outcome patterns in cancers whose biology suggests use of a CSF1R inhibitor. Pexidartinib results in tumor regression in TGCT patients, providing prolonged control with an acceptable safety profile.

Microglia, the main immunocompetent cells of the brain regulate neuronal function in health and disease, but their contribution to cerebral blood flow (CBF) remained elusive. Here we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. We show that microglia establish direct purinergic contacts with cells in the neurovascular unit that shape cerebral perfusion in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in the barrel cortex after whisker stimulation. We also reveal that hypercapnia, which is associated with acidification, induces microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Furthermore, the absence or dysfunction of microglia markedly impairs adaptation to hypoperfusion via P2Y12R after transient unilateral common carotid artery occlusion, which is also influenced by CX3CR1-mediated actions. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation with broad implications for common neurological diseases.

Microgliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto-immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF) - a secreted cytokine that regulates microglia development and survival - is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP-damaged myelin sheaths are lost and robustly phagocytosed upon-repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP-mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.

Background Cosmic radiation exposures have been found to elicit cognitive impairments involving a wide-range of underlying neuropathology including elevated oxidative stress, neural stem cell loss, and compromised neuronal architecture. Cognitive impairments have also been associated with sustained microglia activation following low dose exposure to helium ions. Space-relevant charged particles elicit neuroinflammation that persists long-term post-irradiation. Here, we investigated the potential neurocognitive benefits of microglia depletion following low dose whole body exposure to helium ions. Methods Adult mice were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia 2 weeks after whole body helium irradiation (4He, 30 cGy, 400 MeV/n). Cohorts of mice maintained on a normal and PLX5622 diet were tested for cognitive function using seven independent behavioral tasks, microglial activation, hippocampal neuronal morphology, spine density, and electrophysiology properties 4–6 weeks later. Results PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiated animals on normal diet exhibited a range of behavioral deficits involving the medial pre-frontal cortex and hippocampus and increased microglial activation. Animals on PLX5622 diet exhibited no radiation-induced cognitive deficits, and expression of resting and activated microglia were almost completely abolished, without any effects on the oligodendrocyte progenitors, throughout the brain. While PLX5622 treatment was found to attenuate radiation-induced increases in post-synaptic density protein 95 (PSD-95) puncta and to preserve mushroom type spine densities, other morphologic features of neurons and electrophysiologic measures of intrinsic excitability were relatively unaffected. Conclusions Our data suggest that microglia play a critical role in cosmic radiation-induced cognitive deficits in mice and, that approaches targeting microglial function are poised to provide considerable benefit to the brain exposed to charged particles.

For men with castration-resistant prostate cancer (CRPC), androgen-deprivation therapy (ADT) often becomes ineffective requiring the addition of docetaxel, a proven effective chemotherapy option. Tumor-associated macrophages (TAMs) are known to provide protumorigenic influences that contribute to treatment failure. In this study, we examined the contribution of TAMs to docetaxel treatment. An increased infiltration of macrophages in CRPC tumors was observed after treatment with docetaxel. Prostate cancer cells treated with docetaxel released more macrophage colony-stimulating factor (M-CSF-1 or CSF-1), IL-10 and other factors, which can recruit and modulate circulating monocytes to promote their protumorigenic functions. Inhibition of CSF-1 receptor kinase signaling with a small molecule antagonist (PLX3397) in CRPC models significantly reduces the infiltration of TAMs and their influences. As such, the addition of PLX3397 to docetaxel treatment resulted in a more durable tumor growth suppression than docetaxel alone. This study reveals a rational strategy to abrogate the influences of TAMs and extend the treatment response to docetaxel in CRPC.

FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have activity in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (ITD) mutations, but efficacy is limited by resistance-conferring kinase domain mutations. This phase 1/2 study evaluated the safety, tolerability, and efficacy of the oral FLT3 inhibitor PLX3397 (pexidartinib), which has activity against the FLT3 TKI–resistant F691L gatekeeper mutation in relapsed/refractory FLT3-ITD–mutant AML. Ninety patients were treated: 34 in dose escalation (part 1) and 56 in dose expansion (part 2). Doses of 800 to 5000 mg per day in divided doses were tested. No maximally tolerated dose was reached. Plasma inhibitory assay demonstrated that patients dosed with ≥3000 mg had sufficient levels of active drug in their trough plasma samples to achieve 95% inhibition of FLT3 phosphorylation in an FLT3-ITD AML cell line. Based on a plateau in drug exposure, the 3000-mg dose was chosen as the recommended phase 2 dose. The most frequently reported treatment-emergent adverse events were diarrhea (50%), fatigue (47%), and nausea (46%). Based on modified response criteria, the overall response rate to pexidartinib among all patients was 21%. Twenty-three percent of patients treated at ≥2000 mg responded. The overall composite complete response rate for the study was 11%. Six patients were successfully bridged to transplantation. Median overall survival (OS) of patients treated in dose expansion was 112 days (90% confidence interval [CI], 77-150 days), and median OS of responders with complete remission with or without recovery of blood counts was 265 days (90% CI, 170-422 days). This trial was registered at www.clinicaltrials.gov as #NCT01349049.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by progressive neuronal demyelination and degeneration. Much of this damage can be attributed to microglia, the resident innate immune cells of the CNS, as well as monocyte-derived macrophages, which breach the blood-brain barrier in this inflammatory state. Upon activation, both microglia and macrophages release a variety of factors that greatly contribute to disease progression, and thus therapeutic approaches in MS focus on diminishing their activity. We use the CSF1R inhibitor PLX5622, administered in mouse chow, to ablate microglia and macrophages during the course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we show that ablation of these cells significantly improves animal mobility and weight gain in EAE. Further, we show that this treatment addresses the pathological hallmarks of MS, as it reduces demyelination and immune activation. White matter lesion areas in microglia/macrophage-depleted animals show substantial preservation of mature, myelinating oligodendrocytes in comparison to control animals. Taken together, these findings suggest that ablation of microglia/macrophages during the symptomatic phase of EAE reduces CNS inflammation and may also promote a more permissive environment for remyelination and recovery. This microglia and macrophage-targeted therapy could be a promising avenue for treatment of MS.

Aging is known as a major risk factor for the structure and function of the nervous system. There is urgent need to overcome such deleterious effects of age-related neurodegeneration. Here we show that peripheral nerves of 24-month-old aging C57BL/6 mice of either sex show similar pathological alterations as nerves from aging human individuals, whereas 12-month-old adult mice lack such alterations. Specifically, nerve fibers showed demyelination, remyelination and axonal lesion. Moreover, in the aging mice, neuromuscular junctions showed features typical for dying-back neuropathies, as revealed by a decline of presynaptic markers, associated with α-bungarotoxin-positive postsynapses. In line with these observations were reduced muscle strengths. These alterations were accompanied by elevated numbers of endoneurial macrophages, partially comprising the features of phagocytosing macrophages. Comparable profiles of macrophages could be identified in peripheral nerve biopsies of aging persons. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by applying an orally administered CSF-1R specific kinase (c-FMS) inhibitor. The 6-month-lasting treatment started before development of degenerative changes at 18 months and reduced macrophage numbers in mice by ∼70%, without side effects. Strikingly, nerve structure was ameliorated and muscle strength preserved. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may pave the way for treating degeneration in the aging peripheral nervous system by targeting macrophages, leading to reduced weakness, improved mobility, and eventually increased quality of life in the elderly.SIGNIFICANCE STATEMENTAging is a major risk factor for the structure and function of the nervous system. Here we show that peripheral nerves of 24-month-old aging mice show similar degenerative alterations as nerves from aging human individuals. Both in mice and humans, these alterations were accompanied by endoneurial macrophages. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by blocking a cytokine receptor, essential for macrophage survival. The treatment strongly reduced macrophage numbers and substantially improved nerve structure and muscle strength. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may be helpful for treatment weakness and reduced mobility in the elderly.

Numerous clinical studies have established the debilitating neurocognitive side effects of chemotherapy in the treatment of breast cancer, often referred as chemobrain. We hypothesize that cognitive impairments are associated with elevated microglial inflammation in the brain. Thus, either elimination of microglia or restoration of microglial function could ameliorate cognitive dysfunction. Using a rodent model of chronic Adriamycin (ADR) treatment, a commonly used breast cancer chemotherapy, we evaluated two strategies to ameliorate chemobrain: 1) microglia depletion using the colony stimulating factor-1 receptor (CSF1R) inhibitor PLX5622 and 2) human induced pluripotent stem cell-derived microglia (iMG)-derived extracellular vesicle (EV) treatment. In strategy 1 mice received ADR once weekly for 4 weeks and were then administered CSF1R inhibitor (PLX5622) starting 72 h post-ADR treatment. ADR-treated animals given a normal diet exhibited significant behavioral deficits and increased microglial activation 4–6 weeks later. PLX5622-treated mice exhibited no ADR-related cognitive deficits and near complete depletion of IBA-1 and CD68+ microglia in the brain. Cytokine and RNA sequencing analysis for inflammation pathways validated these findings. In strategy 2, 1 week after the last ADR treatment, mice received retro-orbital vein injections of iMG-EV (once weekly for 4 weeks) and 1 week later, mice underwent behavior testing. ADR-treated mice receiving EV showed nearly complete restoration of cognitive function and significant reductions in microglial activation as compared to untreated ADR mice. Our data demonstrate that ADR treatment elevates CNS inflammation that is linked to cognitive dysfunction and that attenuation of neuroinflammation reverses the adverse neurocognitive effects of chemotherapy.

Many risk genes for the development of Alzheimer’s disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis., Genetics implicate microglia in Alzheimer’s disease pathogenesis, but their roles remain unclear. Here, the authors find that microglial depletion in a mouse model of Alzheimer’s disease impairs plaque formation and that Aβ-induced changes in neuronal gene expression are microglia-mediated.

Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long-term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short-term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.

Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4 + T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro . PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs. IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4 + T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4 + T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4 + T cells will likely dictate different approaches in order to achieve their elimination. For CD4 + T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8 + T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

Background: Tenosynovial giant cell tumors (TGCTs) are characterized by rearrangements of the colony-stimulating factor 1 (CSF1) gene. Dysregulated CSF1 may attract CSF1 receptor (CSF1R)-bearing mononuclear cells that form the bulk of the tumor. CSF1R inhibitors including pexidartinib have been developed and have proven to be effective therapies. Methods: Formalin-fixed paraffin-embedded (FFPE) TGCT specimens from the phase 1 first-in-human study of pexidartinib (NCT01004861) were analyzed using a custom sequencing assay to detect CSF1 genetic alterations. The ArcherDX RNA panel (ArcherDX, Inc., Boulder, CO) consisted of 33 gene-specific primer sets specifically designed to target CSF1, including at least 1 primer set to target each exon/exon boundary as well as primer sets to tile the 3′-untranslated region (3′UTR). Plasma samples from 5 phase 1 trials of pexidartinib across multiple tumor types (NCT02777710, NCT01217229, NCT01525602, NCT01790503, NCT02452424) were assayed for CSF1 protein by solid-phase ELISA (Quantikine® Human MCSF, R&D Systems, Inc., Minneapolis, MN) at baseline and following pexidartinib (200-1200 mg). Results: FFPE TGCT specimens (N=25) were successfully isolated, prepared into libraries, and sequenced; 8 showed evidence of gene rearrangements at the junction of CSF1 exons 5/6, and 15 showed alterations of the CSF1 3′UTR (n=23). All 25 TGCT libraries had higher CSF1 expression, with 24 exceeding 2-fold of the RNA control library. The plasma samples from 132 patients were analyzed for CSF1 protein. Pexidartinib treatment (200-1200 mg) led to a dose-dependent increase in plasma CSF1 by day 8, 15, or 29. Significant (>4-fold) CSF1 elevation was observed at 600 mg or higher doses. Discussion: All 25 analyzed TGCT study patients had tumor tissue with elevated CSF1 ligand transcripts, and 23 had CSF1 genomic alterations identified, thus confirming the TGCT etiology for which pexidartinib therapy was recently approved. The initial discovery of gross chromosomal aberrations involving the CSF1 locus used break-apart FISH probes (West et al. Proc Natl Acad Sci USA. 2006;103:690-695). Details of the CSF1 gene aberrations were obtained here using a sequencing assay suitable for FFPE specimens having partially degraded RNA. Abnormal CSF1 transcripts identified in our study are similar to those published by others (Ho et al. Genes Chromosomes Cancer. 2019[Epub]; Tsuda et al. Int J Cancer. 2019;145:3276-3284) after completion of our work, including both predicted fusion proteins and loss of 3′UTR microRNA negative regulatory sites. Increased plasma CSF1 is a useful pharmacodynamics marker of CSF1R inhibition. One potential mechanism for CSF1 plasma elevations could be the inhibition of liver macrophages, known to express CSF1R and thought to play an essential role in the normal clearance of CSF1 from circulation. Citation Format: Paul Severson, Brian L. West, William D. Tap, Zev A. Wainberg, Sandra Tong-Starksen, Henry H. Hsu, Chao Zhang. Identification of abnormal CSF1 transcripts in tenosynovial giant cell tumors and dose-dependent increase in plasma CSF1 levels in response to pexidartinib treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2020.

e14510 Background: Brain metastases can lead to significant morbidity and mortality in patients with advanced cancer. Preventing metastatic disease to the brain could thus substantially improve outcomes. The mechanism of brain metastasis is incompletely understood. Circulating tumor cells drain to the right heart and through the pulmonary circulation, where they may manifest as lung metastases, and can circulate further to the left heart and then to the brain. However, in patients who develop brain metastases without prior lung involvement, metastatic cells may take an alternate route. We hypothesized that cancer cells may pass directly from the right to left heart via a patent foramen ovale (PFO), akin to paradoxical embolism. The prevalence of PFO is approximately 20-30% in the general population; if further elevated in this population, PFO may play a role in brain metastasis development, and ultimately, prophylactic PFO closure may provide benefit. We conducted a pilot study to investigate whether PFO is associated with brain metastases in patients without prior lung involvement. Methods: We prospectively identified patients with brain metastases from a non-lung primary cancer with no preceding lung metastases. Participants underwent a transcranial Doppler study to assess for PFO. Agitated saline was injected intravenously at rest and with Valsalva maneuver. High intensity signals were counted in the middle cerebral arteries for 1 minute after each injection. Spencer grade ≥3 indicated a positive study, consistent with the presence of PFO. Results: We accrued 9 participants who met inclusion criteria. Primary cancers were breast (6 participants), upper gastrointestinal (2 participants), and thyroid (1 participant). A positive study was identified in 2/9 (22.2%) participants. One individual was a female with breast cancer who had no preceding extracranial metastases, and the other individual was a male with duodenal adenocarcinoma whose only prior metastatic disease was distant lymphadenopathy, not active at brain metastasis diagnosis. No participants have developed lung metastases as of their most recent imaging. Conclusions: In this prospective pilot study, we found no increased prevalence of PFO in patients who develop brain metastases without preceding lung involvement compared to estimates for the general population. Though a larger study is needed, the development of brain metastases in these patients may reflect tumors’ biological factors directing metastasis organotropism, rather than a structural pathway.