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Definitions

• This invention relates to the use of inhibiting colony stimulating factor (CSF)-l receptor signaling in the treatment of human diseases. In one embodiment, this invention relates to the use of inhibitor of colony stimulating factor (CSF)-l receptor for the treatment of brain cancer.
• Noncancerous stromal cells represent genetically stable therapeutic targets that can play critical roles in tumor development and progression.
• Macrophages are one such cell type that is associated with poor patient prognosis and treatment response in many cancers, including gliomas.
• CSF colony stimulating factor
• CSF-1R colony stimulating factor-l receptor
• GBM glioblastoma multiforme
• Glioma-associated macrophages could originate from microglia, the resident macrophage population in the brain, and/or be recruited from the periphery. The relative contributions of resident microglia versus recruited macrophages to gliomagenesis have not been extensively addressed. Both of these macrophages will be referred collectively herein as tumor-associated macrophages (TAMs). It is currently not known whether therapeutic targeting of TAMs in glioblastoma multiforme (GBM) represents a viable strategy.
• TAMs tumor-associated macrophages
• Glioblastoma multiforme the most common and aggressive primary brain tumor, is renowned for its terminal prognosis, emphasizing the urgency of developing new effective therapies. Hence, there is a need for investigating therapeutic targeting of TAMs and the use of CSF-1R inhibitor for the treatment of brain cancer.
• Macrophages are dependent upon colony stimulating factor (CSF)-l for differentiation and survival; therefore, an inhibitor of its receptor, CSF-1R, was used to target macrophages in a mouse glioma model, the RCAS-PDGF-B-HA/Nestin-Tv-a;Ink4a/Arf / ⁇ mouse model of gliomagenesis.
• CSF-1R colony stimulating factor
• CSF-1R inhibition dramatically increased survival in mice and regressed established GBMs. Tumor cell apoptosis was significantly increased, and proliferation and tumor grade markedly decreased. Surprisingly, TAMs were not depleted in the CSF-1R inhibitor-treated tumors. However analysis of gene expression in TAMs isolated from treated tumors revealed a decrease in alternatively activated/M2 macrophage polarization markers, consistent with impaired tumor-promoting functions. These gene signatures were also associated with improved survival specifically in the proneural subtype of patient gliomas. Collectively, these results establish macrophages as valid therapeutic targets in gliomas, and highlight the clinical potential for CSF-1R inhibitors in GBM. BRIEF DESCRIPTION OF THE FIGURES
• Figure 1 shows CSF-1R inhibition specifically targets macrophages in the PDG model, significantly improves survival and decreases glioma malignancy.
• Figure IB shows BLZ945 blocks macrophage survival in culture as determined by MTT assay, with a comparable effect to CSF-1 deprivation.
• Figure 1C shows BLZ945 was tested against independent PDG tumor cell lines and the PDGFR-dependent human U-87 MG glioma cell line using MTT assay. Concentrations of BLZ945 up to 6700 nM had no effect. The results depict triplicate wells from one of 3 representative experiments.
• Figure IE shows symptom-free survival curves.
• Figure 2A shows experimental design: PDG mice underwent MRI scans to assess tumor volume and were randomly assigned to vehicle or BLZ945 groups, with follow-up MRI as depicted.
• Figure 2D shows representative images of T2- weighted MRI scans from beginning and endpoint of the trial. Dashed line indicates region of interest used to calculate tumor volume.
• Figure 2E shows waterfall plots depicting change in tumor volume at endpoint relative to starting tumor volume for each individual mouse. Horizontal dashed lines indicate 30% decrease in tumor volume. P values were obtained using unpaired two-tailed Student's t-test. Data are presented as mean + SEM. ** P ⁇ 0.01, *** P ⁇ 0.001.
• Figure 3 shows short-term BLZ945 treatment results in reduced tumor grade and proliferation, and increased apoptosis.
• Figure 3A shows representative H&E images from the 7-day trial depicting grade IV/GBM (vehicle) and tumor response (BLZ945).
• Figure 3B shows representative images from 7-day trial stained for 01ig2 (tumor cells) BrdU, cleaved caspase-3 (CC3), and DAPI.
• White arrows indicate rare BrdU + 01ig2 + cells in BLZ945 groups.
• Figure 3C shows quantitation of total DAPI + cells per tumor.
• Figure 3D shows percentage of 01ig2 + cells relative to total DAPI + cells.
• Figure 3E shows percentage of proliferating BrdU + 01ig2 + cells.
• Figure 4 shows CSF-1R inhibition signature reveals changes in macrophage polarization and predicts survival advantage in proneural GBM patients.
• Figure 4B shows a lasso logistic regression model was trained on expression data in Figure 4A, identifying 5 genes differentiating BLZ945 and vehicle. BLZ945 downregulates expression of Mrcl/CD206 in BMDMs in vitro, determined by flow cytometry ( Figure 4C) and qRT-PCR ( Figure 4D).
• Figure 4F shows glioma cells were co-cultured with BMDMs that were either unstimulated or pre-conditioned with GCM. Co-cultures were treated +/- BLZ945 and tumor cell cycle entry evaluated, revealing an increase when cultured with GCM- preconditioned BMDMs, which was blocked by BLZ945.
• BMDMs were cultured in non- conditioned media supplemented with CSF-1.
• Figure 4H shows The Cancer Genome Atlas (TCGA) proneural patients were classified into "BLZ945-like” and "Vehicle-like” classes using the lasso signature shown in Figure 4B. "BLZ945-like” classified patients show increased median survival of 10 months.
• GCM glioma cell- conditioned media
• BMDM primary bone marrow-derived macrophages
• Figure 41 shows hazard ratios (HR) and confidence intervals (CI) for the lasso regression signature determined for each subtype of TCGA and Combined (Murat, Phillips, Freije, and Rembrandt) datasets.
• HR means are plotted with associated 95% CI: HRs with a CI that does not cross 1.0 are considered significant.
• the proneural subtype alone showed significant association with survival in both TCGA and Combined datasets.
• P values were obtained using unpaired two-tailed Student's t-test in Figures 4C-G, Chi-squared test in Figure 4H, and Wald's test in Figure 41. Data are presented as mean + SEM. *P ⁇ 0.05, **P ⁇ 0.01, *** P ⁇ 0.001.
• Figure 5 shows macrophage numbers are increased in a mouse model of gliomagenesis compared to normal brain.
• CDl lb + myeloid cells/macrophages accounted for the overwhelming majority of leukocytes (89.9-98.5% of CD45 + cells), with a 3.8-fold increase in CD45 + CDl lb + cells in the tumors (12.7+ 2.0%) compared to normal brain (3.3 + 0.5%), and no differences in the populations of CD45 + CD1 lb " cells.
• Figure 5B shows normal brain or GBM tissue sections from symptomatic PDG mice were immunofluorescently co-stained for CSF-IR, CD68 (macrophages), and DAPI.
• FIG. 5D shows normal brain or GBM tissue sections from symptomatic PDG mice were stained for CSF- 1R in combination with the macrophage markers F4/80 and CD l ib. F4/80, CD l ib, and CD68 were also examined in combination with Iba-1 (macrophages/ microglia). DAPI was used for the nuclear counterstain. Scale bar, 50 ⁇ . Data are presented as mean + SEM. P values were obtained using unpaired two-tailed Student's t-test; *P ⁇ 0.05; **P ⁇ 0.01. [0019] Figure 6 shows BLZ945 significantly decreases the viability of macrophages in culture but has no effect on glioma cell line proliferation or neuro sphere formation.
• Figure 6A shows chemical structure of the CSF-1R inhibitor BLZ945.
• Figure 6B shows Western blot analysis of primary BMDMs, which were cultured in the absence of CSF-1 for 12 hours prior to stimulation, followed by CSF-1 addition for the time points indicated. This results in a progressive increase in CSF-1R phosphorylation that is effectively inhibited by 67 nM BLZ945.
• BMDMs were continuously cultured with CSF-1.
• the same dose of BLZ945 (67nM) blocks wild-type C57BL/6 BMDMs as shown in Figure IB.
• Figure 6C shows Nestm-Tv-a.;Ink4a/Arf l ⁇ BMDM survival, as determined by the MTT assay, which is comparable to the effects of CSF-1 deprivation from the culture.
• Figure 6D shows BLZ945 blocks survival of the CRL-2467 microglia cell line.
• Figure 6E shows the potency of BLZ945 was tested against multiple PDG tumor cell lines derived from independent mice in culture using the MTT assay. There was no effect of BLZ945, even at concentrations up to 6700 nM, which is lOOx the IC50 for CSF-1R inhibition in cell-based assays. The results depict mean + SEM of triplicate wells from one of 3 representative experiments in Figures 6C-E.
• Figure 6F shows BLZ945 does not affect the number or size of secondary neurospheres (NS) derived from 3 independent mice bearing PDG tumors, which were seeded in duplicate wells for each condition. Data are presented as mean + SEM. P values were obtained by comparing each concentration of BLZ945 to the untreated control at the end of the experiment using unpaired two-tailed Student's t-test; **P ⁇ 0.01, ***P ⁇ 0.001 in Figures 6C-D; for all the comparisons in Figure 6E and Figure 6F, there were no significant differences. [0021]
• Figure 7 shows BLZ945 crosses the blood-brain barrier, is well tolerated for long-term treatments, and significantly reduces tumor grade.
• FIG. 7A tests whether BLZ945 could cross the blood-brain barrier.
• Tumor-bearing PDG mice were treated with a single dose of 200 mg/kg BLZ945 by oral gavage and sacrificed at different time points post-dosing as indicated to determine BLZ945 pharmacokinetics (PK).
• the concentration of the drug in the brain was similar to levels in the plasma and decreased thereafter.
• BLZ945 concentration in the contralateral non-tumor bearing hemisphere of the brain was comparable to the level achieved in the tumor at all time points tested, indicating that the drug is able to effectively cross the blood-brain barrier and was not solely due to the potentially selective disruption of this barrier within the tumor.
• Data are presented as mean + SEM.
• Figure 7B shows BLZ945 is well-tolerated for up to 26 weeks. Mean weight for female and male mice over the 26-week time course of the long-term survival trial depicted in Figure IE. Mice were divided by treatment group: vehicle and BLZ945.
• Figure 8 shows tumor growth is inhibited in individual mice in response to BLZ945. PDG mice underwent MRI scans to assess tumor volume between 4-5 weeks post-injection and were randomly assigned to vehicle (20% captisol) or BLZ945 (200 mg/kg) treatment groups. Figure 8A-C shows tumor volume over the time course for individual mice (from Figure 2).
• 3 3 group ranged from 48.7- 132.3 mm .
• a vehicle cohort with tumor volume > 40 mm was not included for comparison because those mice would not have survived to the trial endpoint.
• Figure 9 shows BLZ945 treatment inhibits intratumoral CSF-1R phosphorylation.
• Tumors were harvested from mice after 3 days of treatment with either BLZ945 or vehicle. Samples were biochemically fractionated as described, and CSF-1R phosphorylation was assessed by western blotting (Figure 9A).
• Figure 9B shows a significant reduction in CSF- 1R phosphorylation, but no significant change in total receptor levels, as determined by quantitation of the phosphorylated and total CSF- 1 receptor bands using ImageJ software.
• n 5 mice per group. Data are presented as mean + SEM. P values were obtained using unpaired two-tailed Student's t-test; **P ⁇ 0.01, ns, not significant.
• Figure 10 shows decreased angiogenesis and evidence of pronounced tumor response in BLZ945-treated tumors.
• Figure 10B shows representative images of tumors from 7 day BLZ945 trial stained for CD31 (endothelial cells, red), smooth muscle actin (SMA, pericytes, green), and DAPI (blue).
• Figure IOC shows quantitation of the microvessel density (CD31 count relative to the total tumor area).
• Figure 10D shows the average vessel length (CD31 length relative to the CD31 count), and
• Figure 11 shows increased phagocytosis in BLZ945-treated tumors. Representative images of tumors from the short-term BLZ945 trial stained for CDl lb (macrophages), cleaved caspase-3 (CC3), 01ig2 and DAPI were shown in Figure 11A. White arrows indicate apoptotic tumor cells (CC3 + 01ig2 + ) that have been engulfed/ phagocytosed by CDl lb + macrophages.
• Gray arrowheads indicate apoptotic tumor cells (CC3 + 01ig2 + ) that are in close contact with but have not been phagocytosed by CDl lb + macrophages; these types of interactions were not counted for phagocytic index or capacity.
• Figure 11B shows phagocytic index calculated as the mean percentage of CC3 + 01ig2 + cells that had been engulfed by CDl lb + macrophages per mouse.
• FIG. 12 shows CSF-1R inhibition depletes normal microglia but does not affect the number of TAMs in treated gliomas.
• Figure 12A shows representative flow cytometry plots and Figure 12B shows the quantitation of CDl lb + Ly6G " microglia and CDl lb + Ly6G + myeloid cells. Data are presented as mean + SEM.
• Figure 12C shows representative images of tumors (upper panel) and adjacent hippocampus (lower panel) from the short-term BLZ945 trial stained for CD68 (macrophages, red) and DAPI (blue).
• Figure 13 shows gene expression profiling of BLZ945-treated TAMs, demonstrating a downregulation of alternatively activated/M2 polarization markers with no change in classically activated/Ml polarization markers.
• Figure 13A shows representative flow cytometry plots and gating strategy for sorting CD1 lb + Gr-l " TAMs from tumors treated with vehicle or BLZ945 for 7 days.
• SVM Support Vector Machine
• Figure 13C show Gene set enrichment analysis (GSEA), revealing that targets of Egr2, a transcription factor downstream of CSF- 1R signaling, were downregulated in BLZ945 treated TAMs.
• GSEA Gene set enrichment analysis
• targets of Egr2, a transcription factor downstream of CSF- 1R signaling were downregulated in BLZ945 treated TAMs.
• Figure 14 shows analysis of immune cell infiltration in BLZ945-treated tumors.
• Figure 15 shows primary glioma cultures are composed of heterogeneous cell types.
• Primary glioma cell cultures were prepared from GBM as described in methods. At P3, cells were stained for Nestin to reveal the presence of tumor cells in combination with the macrophage marker CD l ib as well as the astrocyte marker GFAP. DAPI was used for the nuclear counterstain. Scale bar, 50 ⁇ .
• Figure 16 shows "BLZ945-like” lasso and support vector machine classes demonstrate a survival advantage in a Proneural specific, G-CIMP independent manner.
• Figure 16A shows lasso logistic regression signature identified in Figure 4B was used to classify TCGA GBM patients into "BLZ945-like” and "Vehicle-like” classes. The regression model was trained on the mouse TAM expression data, restricting to genes with known human homologues that were present in TCGA total tumor expression data.
• Figure 16B shows GBM patients from the Murat, Freije, Phillips, and Rembrandt databases were subtyped as described and binned into one dataset.
• the lasso logistic regression signature was used to classify patients in the Combined datasets.
• "BLZ945-like" classified patients demonstrated a survival advantage in only the proneural subtype (6.5 months). While there was a significant decrease in median survival using the "BLZ945-like" class in the Neural subtype from the TCGA dataset in Figure 16 A, this effect was not replicated in the Combined datasets for Neural patients.
• Figure 16C shows a support vector machine (SVM) was trained as described and used to classify TCGA patients into "BLZ945-like” and “Vehicle-like” classification. "BLZ945-like” classified TCGA patients showed a Proneural specific survival advantage (7.6 months).
• Figure 16D shows an SVM was trained and used as in Figure 16C to classify patients in the Combined datasets into "BLZ945-like” and "Vehicle-like” classification. "BLZ945-like” classified patients showed a Proneural specific survival advantage (31.5 months).
• Figure 16E sought to determine if the survival advantage offered by the "BLZ945-like" signature was due to an enrichment of Glioma CpG Island Methylator Phenotype (G-CIMP) patients, which have previously been shown to be associated with improved overall survival.
• G-CIMP Glioma CpG Island Methylator Phenotype
• TAMs tumor-associated macrophages
• BMDM bone marrow-derived macrophages
• CSF colony stimulating factor
• CSF-1R colony stimulating factor- 1 receptor
• GBM glioblastoma multiforme
• GCM glioma cell-conditioned media.
• PDG refers to PDGF-driven gliomas, using the RCAS-PDGF- B/Nestin-Tv-aJ/iHa/Ar 7" mouse model of gliomagenesis.
• TCGA refers to The Cancer Genome Atlas.
• therapeutic reagent or “regimen” is meant any type of treatment employed in the treatment of cancers, including, without limitation, chemo therapeutic pharmaceuticals, biological response modifiers, radiation, diet, vitamin therapy, hormone therapies, gene therapy, surgery etc.
• c-FMS is the cellular receptor for CSF-1 (M-CSF).
• the extracellular domains of the receptor are characterized by the presence of five immunoglobulin-like domains and a single transmembrane segment. Inside the cell, the transmembrane domain is joined to the tyrosine kinase domain by a juxtamembrane domain, which bears a number of regulatory phosphorylation sites.
• the structure of the c-FMS tyrosine kinase domain has been determined in Apo form and co-liganded with small molecule inhibitors of different chemo types.
• c-FMS is an attractive target for drug discovery because it appears to play a pivotal role in the regulation of macrophage function. Both the extracellular (and in particular the purported CSF-1 -binding site) and the intracellular tyrosine kinase domains have been targeted in the generation of therapeutics.
• the present invention has shown that the CSF-1R inhibition is a potent strategy to block malignant progression, regress established GBMs and dramatically enhance survival in a preclinical model of gliomagenesis.
• CSF-1R inhibition is a potent strategy to block malignant progression, regress established GBMs and dramatically enhance survival in a preclinical model of gliomagenesis.
• increased macrophage infiltration correlates with malignancy in human gliomas, as shown here in the PDG model, supporting therapeutic targeting of TAMs in patients.
• depletion is not strictly necessary for effective macrophage-targeted therapy as it is shown that alteration of TAM tumor-promoting functions can significantly affect malignancy.
• proneural gliomas in particular are dependent on TAMs, as indicated by the preclinical data presented herein and suggested by the prognostic advantage associated with the gene signatures found specifically in patients of this subtype. As such, it is reasonable to predict that models of other GBM subtypes may also respond similarly to CSF-IR inhibition.
• myeloid cells including macrophages, have been implicated in blunting chemotherapeutic response in breast cancer models and in promoting re-vascularization and tumor growth following irradiation in GBM xenograft models. Thus, it would be logical to consider CSF-IR inhibitors in combination with therapies directed against the cancer cells in gliomas.
• CSF-IR inhibitors include, but are not limited to, CYC10268, a pyrazine series (Cytopia); AZ683, 3-amido-4- anilinocinnolines, Cinnoline, pyridyl and thiazolyl bisamide series, anilide series (all developed by AstraZeneca); ABT-869 (Abbott Laboratories); ARRY-382 (Array BioPharma); JNJ- 28312141, heteroaryl amides, quinolinone series, pyrido-pyrimide series (all developed by Johnson and Johnson); GW2580 (Glaxo Smith Kline); quinoline derivatives including ⁇ 20227 (Kirin Brewery); 7-azaindole series, PLX3397 (Plexxikon); 1,4-disubstituted pyrrolo-[3,2- c]pyridine derivative (Korea Institute of Science and Technology); and a benzothiazole series (Novartis
• anti-CSF-1 or anti-CSF-lR antibodies include anti-CSF-1 or anti-CSF-lR antibodies.
• Antibodies may include, but are not limited to, isolated antibodies, monoclonal antibodies, and fragments of antibodies.
• Representative examples of anti-CSF-1 antibodies include, but are not limited to, IMC-CSF (ImClone), 7H5.2G10 (Deposit No. DSM ACC2922; Hoffmann-La Roche), and MCS IOO (Novartis). See Sherr et al., 1989; Ashmun et al., 1989; Kitaura et al., 2008; WO 2011/107553; and WO 2009/112245.
• Another method of CSF-1 signaling inhibition is by antisense oligonucleotide or small interfering RNA (siRNA) directed against CSF-1 or CSF-1R.
• siRNA small interfering RNA
• a method of identifying or monitoring the effects of a therapeutic agent or regimen on a brain cancer patient According to this method, a selected therapeutic agent or treatment regimen is administered to the patient.
• the therapeutic agent or regimen comprises or results in signaling inhibition of CSF-1 and/or CSF-1R.
• the therapeutic agent or regimen comprises the use of CSF-1 signaling inhibition and another cancer treatment generally known in the art.
• a sample containing myeloid cells of the subject is examined for expression of genes that show differential expression as shown herein.
• a method of determining whether a brain cancer patient would be responsive to treatment with a therapeutic agent or regimen comprising inhibition of CSF-1 signaling comprises the steps of treating said patient with the therapeutic agent or regimen; isolating myeloid cells from said patient; and determining expression of one or more genes in said myeloid cells, said genes include adrenomeduUin (ADM), arginase 1 (ARG1), clotting factor F13A1, mannose receptor C type 1 (MRC1ICD206), and protease inhibitor SERPINB2, wherein differential gene expression in said myeloid cells from treated patient as compared to myeloid cells from a patient treated with control reagent or regimen would indicate that said patient would be responsive to treatment with the therapeutic agent or regimen.
• ADM adrenomeduUin
• ARG1 arginase 1
• clotting factor F13A1A1 clotting factor F13A1
• MRC1ICD206 mannose receptor C type 1
• SERPINB2 protease inhibitor
• CSF-1 signaling can be accomplished by one of the methods discussed above for targeting CSF-1 or CSF-1R.
• CSF-1 signaling inhibition is accomplished by the use of a CSF-1R inhibitor.
• the therapeutic regimen comprises method of CSF-1 signaling inhibition and another generally known method of cancer treatment, such as chemotherapeutic pharmaceuticals, biological response modifiers, radiation, diet, vitamin therapy, hormone therapies, gene therapy, surgery etc.
• the present invention also provides uses of the differential gene expression disclosed herein to determine whether a brain cancer patient would be responsive to treatment with a therapeutic agent or regimen comprising inhibition of CSF-1 signaling.
• gene expression can be determined by any method generally known in the art, such as PCR or microarray.
• gene expression in said myeloid cells further includes expression of one or more genes such as CD163, Cadherin 1 (CDH1), Heme oxygenase 1 (HMOXl), Interleukin 1 receptor type II (IL1R2), and Stabilin 1 (STAB1).
• gene expression in said myeloid cells further includes expression of one or more genes as listed in Table 2.
• gene expression for ADM, ARG1, F13A1, and MRC1ICD206 are downregulated in the myeloid cells from the treated patient.
• gene expression for SERPINB2 is upregulated in the myeloid cells from the treated patient.
• the gene signature(s) described herein are expected to have an important role in patient stratification and management prior to, and during treatment (for example, CSF-1R inhibitor therapy).
• patients can be biopsied prior to CSF-1R inhibitor treatment, and then monitored for treatment efficacy as determined by changes in the aforementioned gene signature(s).
• the complete gene signature provided the most robust separation between patient groups.
• either ADM or F13A1 as single gene is also capable of stratifying patient groups by survival. Patients with lower expression of either ADM or F13A1 had better survival outcome compared to patients with high levels of either gene.
• the gene signature can be reduced to analysis of either ADM or F13A1, and important predictive value can still be attained.
• the myeloid cells are macrophages, for example, tumor-associated macrophages, bone marrow-derived macrophages, or peripheral macrophage precursors/monocytes.
• the brain cancer is primary brain cancer such as astrocytoma, oligodendroglioma, neuroblastoma, medulloblastoma or ependydoma.
• the brain cancer is a mixed glioma, for example, a malignant tumor that contains astrocytes and oligodendrocytes.
• the brain cancer is glioma, including high-grade glioblastoma multiforme.
• the glioma molecular subtype is proneural.
• the brain cancer could include metastatic brain cancer.
• the present invention provides a screening method for identifying a cancer therapeutic agent or regimen useful for the treatment of brain cancer.
• This method can be employed to screen or select from among many pharmaceutical reagents or therapies for the treatment of individual or groups of brain cancers.
• a selected therapeutic agent or treatment regimen is administered to a mammalian test subject having a cancer.
• the test subject is desirably a research animal, e.g., a laboratory mouse or other.
• a sample containing cells of the test subject is examined and a gene expression profile is generated.
• the present invention provides a method of screening for a therapeutic reagent or regimen that is useful for treating brain cancer, wherein the therapeutic reagent or regimen comprises inhibition of CSF-1 signaling.
• the method comprises the steps of treating a subject with the therapeutic reagent or regimen; and determining expression of one or more genes in myeloid cells obtained from such subject, said genes include adrenomedullin (ADM), arginase 1 (ARG1), clotting factor F13A1, mannose receptor C type 1 (MRC1/CD206), and protease inhibitor SERPINB2, wherein differential gene expression in myeloid cells from subject treated with the therapeutic reagent or regimen as compared to myeloid cells from subject that is treated with a control reagent or regimen would indicate that said therapeutic reagent or regimen is useful for treating brain cancer.
• ADM adrenomedullin
• ARG1 arginase 1
• clotting factor F13A1A1 clotting factor F13A1
• MRC1/CD206 mannose receptor C type
• CSF-1 signaling can be accomplished by any method discussed above for targeting CSF-1 or CSF-1R.
• CSF-1 signaling inhibition is accomplished by the use of a CSF-1R inhibitor.
• the therapeutic regimen comprises method of CSF-1 signaling inhibition and another generally known method of cancer treatment, such as chemotherapeutic pharmaceuticals, biological response modifiers, radiation, diet, vitamin therapy, hormone therapies, gene therapy, surgery etc.
• the present invention also provides uses of the differential gene expression disclosed herein to screen for a therapeutic reagent or regimen for treating brain cancer, wherein the therapeutic reagent or regimen comprises inhibition of CSF-1 signaling.
• gene expression can be determined by any method generally known in the art, such as PCR or microarray.
• gene expression in said myeloid cells further includes expression of one or more genes such as CD163, Cadherin 1 (CDH1), Heme oxygenase 1 (HMOXl), Interleukin 1 receptor type II (IL1R2), and Stabilin 1 (STABl).
• gene expression in said myeloid cells further includes expression of one or more genes as listed in Table 2.
• gene expression for ADM, ARG1, F13A1, and MRC1ICD206 are downregulated in the myeloid cells from the treated patient.
• gene expression for SERPINB2 is upregulated in the myeloid cells from the treated patient.
• the myeloid cells are macrophages, for example, tumor-associated macrophages, bone marrow-derived macrophages, or peripheral macrophage precursors/monocytes.
• the brain cancer is glioma, including high-grade glioblastoma multiforme.
• the glioma molecular subtype is proneural.
• the brain cancer could include metastatic brain cancer, or primary brain cancers such as astrocytoma, oligodendroglioma, neuroblastoma, medulloblastoma or ependydoma.
• the brain cancer is a mixed glioma, for example, a malignant tumor that contains astrocytes and oligodendrocytes.
• the present invention also provides kits that can be used to detect the expression of genes that show differential expression as shown herein.
• kits are provided that can be used in the monitoring or screening assays disclosed herein.
• the kit may include a microarray or nucleic acid primers and probes for the detection of one or more genes that show differential expression as shown herein.
• the kits can include instructional materials disclosing means of use of the compositions in the kit.
• the instructional materials can be written, in an electronic form (such as a computer diskette or compact disk) or can be visual (such as video files).
• the kits can further include other agents to facilitate the particular application for which the kit is designed.
• mice were fully anesthetized with 10 mg/ml ketamine/1 mg/ml xylazine and were subcutaneously injected with 50 ⁇ of the local anesthetic 0.25% bupivacaine at the surgical site.
• Mice were intracranially injected with 1 ⁇ containing 2 x 10 5 DF-1:RCAS-PDGF- B-HA cells between 5-6 weeks of age using a fixed stereotactic apparatus (Stoelting). Injections were made to the right frontal cortex, approximately 1.5 mm lateral and 1 mm caudal from bregma, and at a depth of 2 mm.
• the CSF-IR inhibitor was obtained from the Novartis Institutes for Biomedical Research (Emeryville, CA). The drug was formulated in 20% captisol at a concentration of 12.5 mg/ml. The vehicle control, 20% captisol, was processed in the same manner.
• mice were dosed with 200 mg/kg BLZ945 or vehicle (20% captisol) by oral gavage once per day. To determine if the drug was able to cross the blood-brain barrier, tumor-bearing mice were treated with a single dose of the CSF-IR inhibitor and sacrificed at different time points post-treatment.
• Plasma, and the left (contralateral) and right (tumor-bearing) hemispheres of the brain were snap frozen in liquid nitrogen for subsequent analysis of CSF-IR inhibitor concentrations in the tissue.
• dosing begun at 17 days/2.5 weeks post-injection of RCAS-PDGF-B-HA.
• mice underwent MRI scans at 4-5 weeks post-injection of RCAS-PDGF-B-HA, as previously described (5).
• ROI regions of interest
• the total tumor volume is the sum of the ROI volume in each slice, and the volume for the first and last slice in which the tumor appears is halved to approximate the volume of a trapezoid.
• tumor volume was in the range of 4.5-40 mm
• animals were randomly assigned to treatment groups.
• a third cohort of mice with tumors larger than 40 mm was also treated with the CSF-IR inhibitor (denoted as BLZ945 Large).
• a size-matched vehicle treated cohort was not included for this larger starting tumor burden because these mice would not have been able to survive to the trial endpoint.
• mice were euthanized at defined time points as described in the figure legends or when they became symptomatic from their tumors, which included signs of poor grooming, lethargy, weight loss, hunching, macrocephaly, or seizures.
• mice were euthanized by carbon dioxide asphyxiation or fully anesthetized with avertin (2,2,2-tribromoethanol, Sigma) and cervically dislocated prior to tissue harvest.
• mice were fully anesthetized with avertin and transcardially perfused with 20 ml of PBS. The brain was then isolated and the tumor macro-dissected from the surrounding normal tissue.
• mice were injected intraperitoneally with 100 mg/g of bromodeoxyuridine (BrdU; Sigma) 2 hours prior to sacrifice.
• bromodeoxyuridine BrdU; Sigma
• mice were fully anesthetized with avertin, transcardially perfused with 10 ml of PBS, followed by 10 ml of 4% paraformaldehyde in PBS (PFA).
• PFA paraformaldehyde
• the brain was post-fixed in PFA overnight at 4°C while other tissues were cryopreserved in 30% sucrose at 4°C. After post-fixation, the brain was then transferred to 30% sucrose and incubated at 4°C until the brain was fully equilibrated and sank to the bottom of the tube (typically 2 to 3 days). All tissues were then embedded in OCT (Tissue-Tek) and 10 ⁇ cryostat tissue sections were used for all subsequent analysis. Histology, Immunohistochemistry, And Analysis
• H&E hematoxylin and eosin
• tissue sections were counterstained with DAPI (5 mg/ml stock diluted 1:5000 in PBS) for 5 minutes prior to mounting with PROLONG GOLD ANTIFADE mounting media (Invitrogen).
• DAPI 5 mg/ml stock diluted 1:5000 in PBS
• tissue sections were first subjected to citrate buffer-based antigen retrieval by submerging in antigen unmasking solution (0.94% v/v in distilled water; Vector Laboratories) and micro waving for 10 minutes on half power, followed by cooling to room temperature for at least 30 minutes.
• tissue were then washed in PBS and blocked with mouse Ig blocking reagent (Vector Laboratories) according to the manufacturer's instructions for 1 hour at room temperature.
• tissue sections were incubated with 2M HC1 for 15 minutes at room temperature to denature DNA and then in neutralizing 0.1M sodium borate buffer (pH 8.5) for 5 minutes. After PBS washes, the rest of the staining was performed according to standard protocol.
• Tissue sections were then washed 6 times for 5 minutes in PBS and blocked overnight at 4°C in a new buffer of 5% donkey serum, 3% bovine serum albumin, and 0.5% PNB in PBS. The following day, slides were incubated for 2 hours at room temperature with the next set of primary antibodies: rabbit anti-01ig2 (1:200) and rat anti-CD l ib (1:200) diluted in 5% donkey serum, 3% bovine serum albumin, and 0.5% PNB in PBS. Slides were washed 6 times for 5 minutes in PBS prior to incubation with donkey-anti-rabbit Alexa647 (1:500) and donkey- anti-rat Alexa488 (1:500) secondary antibodies in 0.5% PNB for 1 hour at room temperature.
• Tissue sections were then washed 4 times for 5 minutes in PBS prior to staining with DAPI (5 mg/mL stock diluted 1:5000 in PBS) for 5 minutes, washed twice more in PBS for 5 minutes, and mounted with PROLONG GOLD ANTIFADE mounting media (Invitrogen). Co-staining for CSF-1R (first primary antibody) and Ibal (second primary antibody) was also performed in series in the same manner, with the addition of citrate buffer based antigen retrieval at the outset.
• DAPI mg/mL stock diluted 1:5000 in PBS
• Tissue sections were visualized under a Carl Zeiss Axioimager Zl microscope equipped with an Apotome. The analysis of immunofluorescence staining, cell number, proliferation, apoptosis, and colocalization studies were performed using TISSUEQUEST analysis software (TissueGnostics) as previously described (7). Overviews of tissue sections from gliomas stained for angiogenesis analysis were generated by TissueGnostics acquisition software by stitching together individual 200x images. All parameters of angiogenesis were quantitated using METAMORPH (Molecular Devices), as previously described (8).
• TISSUEQUEST analysis software TISSUEQUEST analysis software
• mice analyzed For analysis of phagocytosis, 15 randomly selected fields of view from within the tumor were acquired using the 63x oil immersion objective (total magnification 630x) and the Apotome to ensure cells were in the same optical section. Positive cells were counted manually using VOLOCITY (PerkinElmer) and were discriminated by the presence of a DAPI + nucleus. Apoptotic cells were counted as those that had cytoplasmic cleaved caspase-3 (CC3) + staining and condensed nuclei. A cell was considered to have been engulfed by a macrophage when it was surrounded by a contiguous CDl lb + ring that encircled at least two-thirds of the cell border. The numbers of mice analyzed are specified in the figure legends.
• mice were treated with the CSF-1R inhibitor or vehicle and sacrificed 1 hour following the final dose and tumors were harvested.
• Samples were biochemically fractionated as described previously (9). Synaptosomal membrane fractions were lysed in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl [pH 7.5], 50 mM NaCl, lx complete Mini protease inhibitor cocktail (Roche), lx PHOSSTOP phosphatase inhibitor cocktail (Roche)) and protein was quantified using the BCA assay (Pierce). Protein lysates were loaded (90 ⁇ g/lane) onto SDS-PAGE gels and transferred to PVDF membranes for immunoblotting.
• Membranes were probed with antibodies against phospho-CSF- lR Y721 (1 : 1000; Cell Signaling Technology), CSF-1R (1 : 1000; Santa Cruz Biotechnology), or GAPDH (1 : 1000; Cell Signaling Technology) and detected using HRP- conjugated anti-rabbit (Jackson Immunoresearch) antibodies using chemiluminescence detection (Millipore). Bands from western blots were quantified in the dynamic range using the Gel analysis module in IMAGEJ software.
• BMDMs Primary bone marrow derived macrophages
• the tumor was digested to a single cell suspension by incubation with 5 ml of papain digestion solution (0.94 mg/ml papain [Worthington], 0.48 mM EDTA, 0.18 mg/ml N-Acetyl-L-cysteine [Sigma], 0.06 mg/ml DNase I [Sigma], diluted in Earl's Balanced Salt Solution and allowed to activate at room temperature for at least 30 minutes).
• papain digestion solution 0.94 mg/ml papain [Worthington], 0.48 mM EDTA, 0.18 mg/ml N-Acetyl-L-cysteine [Sigma], 0.06 mg/ml DNase I [Sigma]
• the enzyme was inactivated by the addition of 2 ml of 0.71 mg/ml ovomucoid (Worthington).
• the cell suspension was then passed through a 40 ⁇ mesh to remove undigested tissue, washed with FACS buffer (1% IgG Free BSA in PBS [Jackson Immunoresearch]), and centrifuged at a low speed of 750 rpm (Sorvall Legend RT), to remove debris and obtain the cell pellet.
• FACS buffer 1% IgG Free BSA in PBS [Jackson Immunoresearch]
• tumors were digested to a single cell suspension by incubation for 10 minutes at 37°C with 5 mL of 1.5 mg/ml collagenase III (Worthington) and 0.06 mg/mL DNase I in lx Hanks Balanced Salt Solution (HBSS) with calcium and magnesium. The cell suspension was then washed with PBS and passed through a 40 ⁇ mesh to remove undigested tissue.
• HBSS Hanks Balanced Salt Solution
• the cell pellet was resuspended at room temperature in 15 ml of 25% Percoll prepared from stock isotonic Percoll (90% Percoll [Sigma], 10% lOx HBSS), and then spun for 15 minutes at 1500 rpm (Sorvall Legend RT) with accelerator and brake set to 1. The cell pellet was then washed with lx HBSS prior to being resuspended in FACS buffer.
• samples were run on a BD LSR II (Becton Dickstein), and all subsequent compensation and gating performed with FLOWJO analysis software (TreeStar).
• FLOWJO analysis software TeStar
• samples were run on a BD FACSAria (Becton Dickstein) cell sorter and cells were collected into FACS buffer. Cells were then centrifuged and resuspended in 500 ⁇ Trizol (Invitrogen) before snap freezing in liquid nitrogen and storage at -80°C.
• Macrodissected tumors were digested to a single cell suspension by incubation for 8-12 minutes at 37°C as described above.
• the cell suspension was washed with Neural Stem Cell (NSC) Basal Media (Stem Cell Technologies), and centrifuged at low speed (750 rpm Sorvall Legend RT), to remove debris.
• NSC Neural Stem Cell
• DMEM fetal bovine serum
• FBS FBS
• glioma cultures were grown for 24 hours on poly-L-lysine coated coverslips (BD Biocoat). Cells were then fixed with 4% PFA in 0.1M phosphate buffer overnight at 4°C, permeabilized with 0.1% Triton-X for 5 minutes and blocked with 0.5% PNB for at least one hour. The presence of macrophages, tumor cells and astrocytes were examined by immunofluorescent staining of CDl lb (1:200), Nestin (1:500) and GFAP (1: 1000), respectively (Table 6).
• neurosphere formation the cell pellet was resuspended in neurosphere media consisting of mouse NSC Basal Media, NSC proliferation supplements, 10 ng/ml EGF, 20 ng/ml basic-FGF and 1 mg/ml Heparin (Stem Cell Technologies). Fresh media was added every 72 hours for 2 weeks. Primary neurospheres were collected, mechanically disaggregated to a single cell suspension and propagated by serial passaging. To generate glioma cell lines, secondary neurospheres were dissociated to single cell suspensions and cultivated in DMEM+10% FBS as a monolayer (10). Multiple glioma cell lines were derived from independent mice, denoted GBM1-4 herein. Glioma cells were infected with a pBabe-H2B-mCherry construct as described previously (11).
• BMDMs Bone Marrow-Derived Macrophages
• U-87 MG (HTB- 14) glioma and CRL-2467 microglia cell lines were purchased from ATCC.
• the U-87 MG cell line was cultured in DMEM+10% FBS.
• the CRL-2467 cell line was cultured in DMEM+10% FBS with 30 ng/ml recombinant mouse CSF- 1.
• GCM Glioma Cell-Conditioned Media
• GCM glioma cell-conditioned media
• differentiated BMDMs were cultivated in GCM containing either DMSO as vehicle, or 67nM BLZ945, 670nM BLZ945, or in regular media containing 10 ng/ml mouse recombinant CSF-1 and 10 ng/ml IL-4 (R&D Systems) for 24 hours or 48 hours prior to experimental analysis.
• BMDMs 1 x 10 6 cells were cultivated in DMEM supplemented with recombinant mouse CSF-1 or GCM in the presence of the CSF-1R inhibitor or DMSO as vehicle. After 48 hours, cells were scraped and washed with FACS buffer. Cells were counted and incubated with 1 ⁇ of Fc Block (BD Pharmingen) per 10 6 cells for at least 15 minutes at 4°C.
• Fc Block BD Pharmingen
• Cells were then stained with CD45 and CD1 lb antibodies (Table 7) for 10 minutes at 4°C and washed with FACS buffer. Cells were fixed and permeabilized using the BD Cytofix/CytopermTM kit (BD Biosciences) according to the manufacturer's instructions. Subsequently cells were stained with anti-CD206 antibody (Table 7). For analysis, samples were run on a BD LSR II (Becton Dickstein), and all subsequent compensation and gating performed with FLOWJO analysis software (TreeStar).
• Control or GCM pre-stimulated macrophages derived from ⁇ -actin-GFP "1" mice were co- cultured in a 1: 1 ratio with 1 x 10 5 serum starved mCherry-positive glioma cells (from the cell lines derived above) for 48 hours in the presence of 670nM BLZ945 or DMSO as vehicle. Following collection of trypsinized co-cultured cells, wells were rinsed in additional media and this volume was collected to ensure harvesting of all macrophages, which adhered tightly to cell culture dishes.
• Cell growth rate was determined using the MTT cell proliferation kit (Roche). Briefly, cells were plated in triplicate in 96- well plates (1 x 10 cells/well for glioma cell lines and 5 x 10 3 cells/well for BMDM and CRL-2467 cells) in the presence or absence of 6.7-6700 nM of BLZ945. Media was changed every 48 hours. BMDM and CRL-2467 cells were supplemented with 10 ng/ml and 30 ng/ml recombinant mouse CSF-1 respectively unless otherwise indicated. Ten ⁇ of MTT labeling reagent was added to each well and then incubated for 4 hours at 37°C, followed by the addition of 100 ⁇ MTT solubilization reagent overnight. The mixture was gently resuspended and absorbance was measured at 595 nm and 750 nm on a SPECTRAMAX 340pc plate reader (Molecular Devices).
• TAQMAN probes (Applied Biosystems) for Cdl lb (Mm00434455_ml), Cd68 (Mm03047343_ml), Csf-1 (Mm00432688_ml), Csf-lr (Mm00432689_ml), 1134 (Mm00712774_ml), Mrcl (Mm00485148_ml), and Tv-a (custom), were used for qPCR. Assays were run in triplicate and expression was normalized to ubiquitin C (Mm01201237_ml) for each sample. Microarrays And Gene Expression Profiling
• TCGA expression data was downloaded from the TCGA data portal and all clinical data was downloaded from the data portal (17).
• Clinical and expression data for the Rembrandt data set was downloaded from the NCI website.
• the Freije (GSE4412), Murat (GSE7696), and Phillips (GSE4271) datasets were downloaded from the NCBI website (18-20).
• For the Freije datasets only samples that were run on the HGU133A platform were considered as samples on the HGU133B platform contained minimal overlap with the remaining datasets. Datasets were individually processed and normalized as described above. Within each dataset, genes were mean centered across patients.
• the CSF-IR inhibitor used herein is a potent, highly selective, brain penetrant CSF-IR inhibitor that blocks CSF-IR phosphorylation and kinase activity ( Figures 6 and 7A).
• the inhibitor inhibits CSF-IR at 1 nM, and CSFl-dependent cell proliferation at 67 nM.
• biochemical IC50 values for >200 kinases tested, including PDGFRa (the receptor for PDGF-B) were >10 ⁇ , with the exception of cKIT and PDGFRP (3500 nM and 3300 nM respectively) (data not shown).
• the inhibitor inhibits CSF-IR phosphorylation and significantly decreases the viability of primary bone marrow-derived macrophages (BMDMs) in culture, similar to CSF-1 withdrawal ( Figures IB and 6).
• the inhibitor also blocked the survival of Ink4a/Arf -/-BMDMs, the genetic background of the PDG model, and reduced viability of the microglia cell line CRL-2467 ( Figure 6).
• concentrations up to 6700 nM BLZ945 had no effect on the proliferation of 4 different tumor cell lines derived from PDG mice, tumor neurosphere formation or U-87 MG human glioma cell proliferation ( Figures 1C and 6).
• mice were treated with either the CSF-IR inhibitor or the vehicle control, and evaluated for symptom- free survival (Figure ID).
• Median survival in the vehicle-treated cohort was 5.7 weeks, with no animals surviving past 8 weeks post- injection.
• 64.3% of the CSF-IR inhibitor-treated cohort was still alive at the trial endpoint of 26 weeks post-injection ( Figure IE). This endpoint was chosen because mice in the Ink4a/Arf-/-background start developing spontaneous tumors around 30 weeks of age. Over this extended time period, the PDG mice did not exhibit any visible side effects and the CSF-IR inhibitor was well-tolerated (Figure 7B).
• TAMs have been found to be more M2 polarized, which has been linked to their immunosuppressive and pro-tumorigenic functions. Furthermore, macrophages in human gliomas exhibit an M2-like phenotype, determined by increased levels of the scavenger receptors CD 163 and CD204, which are associated with higher tumor grade. Given the striking enrichment for M2 genes in the restricted 5-gene signature, the 257-gene list was examined to determine if there were additional M2-associated markers altered following CSF-IR inhibitor treatment. This revealed 10 more genes, the majority of which were downregulated (Figure 13, Table 2). Classically activated/Ml polarization genes were not correspondingly upregulated ( Figure 13). These data suggest that in response to CSF-IR inhibition, TAMs lose their M2 polarization and may gain anti-tumorigenic functions.
• Mrcl was selected as it is a well-established cell surface M2 marker, facilitating the use of flow cytometry to examine levels of macrophages in co-culture assays. Mrcl increased in response to GCM, and was downregulated following CSF-IR inhibitor addition ( Figures 4C-D). Similarly, in freshly isolated mouse primary glioma cultures containing TAMs ( Figure 15), Mrcl was also decreased in response to the CSF-1R inhibitor ( Figure 4E).
• Kif20a kinesin family member 20A - 3 ⁇ 4 - ( .. S 2SI -Us il22 laiuih iin-nik-i 22 -2.14 2.421 - ( 14 lirbla killer cell lectin-like receptor subfamily 4 3 ⁇ 4 ' ) (1(11 -us
• Lifr leukemia inhibitory factor receptor -2 10 I 121 - ( . ) 3 ⁇ 4 l igl li.uasc 1. I ) ⁇ ⁇ . ⁇ I I'-ik'ivikk-nl -2.i,f. 221 -Id Lox lysyl oxidase 2 ⁇ 2 0 l')
• Mrcl * mannose receptor, C type 1 (CD206) 440E-07 subfamily A, member 6B
• Psmb7 proteasome proteasome (prosome, macropain) -2. ⁇ 7 4.27E-03 subunit, beta type 7
• Serpinb6b serine (or cysteine) peptidase inhibitor, 2 J3 1.22E-03 clade B, member 6b
• i. ) is. ) iiiciase (D ⁇ ⁇ 11 alpha -2.11 2.13H-05 lopbpl topoisomerase (DNA) II binding protein -2.37 9.9()i:-()()
• G-CIMP corresponds to Glioma CpG Island Methylator Phenotype. P values were obtained using Wald' s test. *Set of proneural patients with methylation data that are definitively not G-CIMP positive (67/133 total Proneural TCGA patients). ** Multivariate cox proportional hazard model using both G-CPMP and 'BLZ945' classification as strata. TABLE 5
• Warnes et al R package version 2.10.1 (2011).

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