DOI: 10.5061/dryad.66t1g1k7s
title: Supplemental information for: Inhibition of CSF1R and KIT with pexidartinib reduces inflammatory signaling and cell viability in endometriosis

description: Endometriosis is a common and debilitating disease, affecting ~170 million
 women worldwide. Affected patients have limited therapeutic options such
 as hormonal suppression or surgical excision of the lesions, though
 therapies are often not completely curative. Targeting receptor
 tyrosine-kinases (RTKs) could provide a nonhormonal treatment option for
 endometriosis. We determined that two RTKs, Macrophage colony stimulating
 factor receptor (CSF1R) and Mast/stem cell growth factor receptor KIT
 (KIT), are overexpressed in endometriotic lesions and could be novel
 nonhormonal therapeutic targets for endometriosis. The kinase activity of
 CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor
 that was recently approved by the US Food and Drug Administration (FDA).
 Using immunohistochemistry, we detected CSF1R and KIT in endometriotic
 tissues obtained from peritoneal lesions, colorectal lesions, and
 endometriomas. Specifically, we show that KIT is localized to the
 epithelium of the lesions, while CSF1R is expressed in the stroma and
 macrophages of the endometriotic lesions. Given the high epithelial
 expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used
 to evaluate the efficacy of dual CSF1R and KIT inhibition with
 pexidartinib. We found that pexidartinib suppressed activation in 12Z
 cells of JNK, STAT3 and AKT signaling pathways, which control key
 pro-inflammatory and survival networks within the cell. Using quantitative
 real time PCR, we determined that pexidartinib suppressed interleukin 8
 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that
 pexidartinib decreased cell growth and viability. Overall, these results
 indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces
 pro-inflammatory signaling and cell viability in endometriosis.
description: # Supplemental Information for "Inhibition of CSF1R and KIT with
 pexidartinib reduces inflammatory signaling and cell viability in
 endometriosis" ##
 Description of the data and file structure The data provided here is
 supplemental information derived from the investigative work that led to
 the primary manuscript. First, work with immunohistochemistry shows the
 relationship of expression patterns in the protein CSF1R and the
 macrophage marker, CD68. The following western blots describe
 dose-response assays using protein phosphorylation in immortalized
 endometriosis cells (12Z) to assess for upregulation of CSF1R and KIT
 signaling when exposed to their respective ligands. Next, western blots of
 control experiments are shown evaluating if pexidartinib is able to reduce
 protein phosphorylation independent of cell exposure to ligand
 stimulation. Additional control experiments show the results of gene
 expression when 12Z cells were exposed to pexidartinib without stimulatory
 ligands to assess for a ligand-independent effect. Lastly, proof of the
 12Z cell line used is shown along with uncropped images of all western
 blots for quality assessment. Data is presented as images of
 immunohistochemistry, western blots, and then results of protein
 phosphorylation and gene expression comparisons in various experiments. ##
 Figure Legends for Supplemental Figures and Supplemental Information
 **Supplemental Figure 1. Colocalized expression of CSF1R and the
 macrophage marker CD68 in endometriosis lesions.** Pathology-confirmed
 endometriosis lesions representing various lesion types were stained with
 CSF1R antibody (A,C,E) or CD68 antibody (B,D,F). CSF1R was expressed by
 both stromal and inflammatory cell types and overlapped with CD68+ cells
 (signifying macrophages). Colocalization appeared more consistent in
 advanced-stage endometriosis lesions (endometrioma and colorectal
 lesions). Some regions of epithelium stained for CSF1R, with these tending
 to occur in regions of tubal metaplasia (indicated by arrowheads in panel
 A) Scale bar: 100mm in all images. Images represent experiments performed
 in 3-4 patients per lesion subtype.  **Supplemental Figure 2. Dose
 response assay evaluating JNK activation at 30 and 60 minutes.** At 60
 minutes after MCSF stimulation there was evidence of a dose-response (A).
 After 30 minutes of KITL stimulation, there was a suggestion of a dose
 response curve, though no clear relationship existed after 60 minutes (B).
 Ratios shown on the phosphorylated blot reflects the normalized ratio of
 phosphorylated protein levels against total protein and GAPDH levels.
 Abbreviations: L-ladder. pJNK-phosphorylated JNK **Supplemental Figure 3.
 Dose response assay evaluating STAT3 activation at 30 and 60 minutes.**
 There was no strong evidence of a dose-response curve after MCSF
 stimulation (A). At both 30 and 60 minutes after KITL stimulation there
 was a suggestion of an escalating increase in phosphorylation ratios (B).
 Ratios shown on the phosphorylated blot reflect the normalized ratio of
 phosphorylated protein levels against total protein and GAPDH levels.
 Abbreviations: L-ladder. pSTAT3-phosphorylated STAT3. **Supplemental
 Figure 4. Dose response assay evaluating AKT activation at 30 and 60
 minutes.** While there was not a linear increase in AKT activation, MCSF
 stimulation at a dose of 25ng/mL increased AKT phosphorylation ~1.3-fold
 at 60 minutes (A). Similarly, there was not a consistent dose response
 after KITL stimulation, but a dose of 25ng/mL did increase phosphorylation
 ~1.3-fold at 30 minutes (B). Ratios shown on the phosphorylated blot
 reflect the normalized ratio of phosphorylated protein levels against
 total protein and GAPDH levels. Abbreviations: L-ladder.
 pAKT-phosphorylated AKT. **Supplemental Figure 5. Histograms showing
 assessment for changes in phosphorylation in the presence of
 pexidartinib.** Quantification assessment for pJNK 60 minutes after MCSF
 stimulation (A) and 60 minutes after KITL (B) in the presence of
 pexidartinib. Quantification of pSTAT3 changes 30 minutes after MCSF (C)
 and 30 minutes after KITL (D) in the presence of pexidartinib. Histograms
 represent Mean +/- SEM. Analyzed by One-Way ANOVA. * p&lt;0.05. **
 p&lt;0.01. *** p&lt;0.001. Abbreviations: AU-arbitrary units.
 pJNK-phosphorylated JNK. pSTAT3-phosphorylated STAT3. T-time. Min-minutes.
 **Supplemental Figure 6. Analysis of pJNK, pSTAT3, and pAKT activity in
 presence of pexidartinib without ligand stimulation.** Western blots
 assessing for changes in pJNK (A), pSTAT3 (B), and pAKT (C) activity were
 performed without ligand stimulation to see if pexidartinib inhibition
 occurred independent of ligand activity. Time points of T=60 and T=90min
 used to reflect total drug exposure duration in ligand experiments.
 Abbreviations: Pex-pexidartinib; pJNK-phosphorylated JNK;
 pSTAT3-phosphorylated STAT3; pAKT-phosphorylated AKT; T-time; Min-minutes.
 Experiments were replicated using three biological replicates. Displayed
 western blots representative of overall results. **Supplemental Figure 7.
 Quantification and comparison of western blot analysis of pJNK, pSTAT3,
 and pAKT activity in presence of pexidartinib without ligand
 stimulation.** Phosphorylated activity was unchanged across the majority
 of signaling pathways when cells were exposed to pexidartinib without
 ligand. Only pSTAT3 (D) was reduced at 90 minutes of exposure at both the
 3 and 10mM condition. Time points of T=60 and T=90min used to reflect
 total drug exposure duration in ligand experiments. Histograms represent
 Mean +/- SEM. Analyzed by One-Way ANOVA. * p&lt;0.05. ** p&lt;0.01. ***
 p&lt;0.001. Abbreviations: Pex-pexidartinib; AU-arbitrary units; T-time;
 Min-minutes. Experiments were replicated using three biological replicates
 and three technical replicates. **Supplemental Figure 8. Gene expression
 of *IL8*, *CCND1 *and GAPDH in the presence of pexidartinib inhibition
 without ligand stimulation.** There were no statistically significant
 differences between mRNA expressions of *IL8* (A) and *CCND1* (B) when
 comparing vehicle to 3 or 10mM pexidartinib doses. The absolute gene
 expression of *GAPDH* (C) was similar across all conditions, suggesting no
 evidence of general suppression of gene expression. Histograms represent
 Mean +/- SEM. Analyzed by One-Way ANOVA. Abbreviations: Pex-pexidartinib.
 Experiments were replicated using four biological replicates and three
 technical replicates. **Proof of validation for 12Z cells.** The
 blocked-out cell lines were used in other, non-related research projects
 in our lab and not part of this research study. **Complete western blots
 for JNK pathway assessment of possible dose response to MCSF and KITL.**
 Complete western blots for JNK pathway assessment after MCSF (left blots)
 and KITL (right blots) to perform dose-response assay (Supplemental Figure
 2). No membrane stripping performed after Total Antibody assessment before
 GAPDH probing. Abbreviations: pJNK-phosphorylated JNK. **Complete western
 blots for STAT3 assessment of possible dose response to MCSF and KITL.**
 Complete western blots for STAT3 pathway assessment after MCSF (left
 blots) and KITL (right blots) to perform dose-response assay (Supplemental
 Figure 3). No membrane stripping performed after Total Antibody assessment
 before GAPDH probing. Abbreviations: pSTAT3-phosphorylated STAT3
 **Complete western blots for AKT assessment of possible dose response to
 MCSF and KITL.** Complete western blots for AKT pathway assessment after
 MCSF (left blots) and KITL (right blots) to perform dose-response assay
 (Supplemental Figure 4). No membrane stripping performed after Total
 Antibody assessment before GAPDH probing. Abbreviations:
 pAKT-phosphorylated AKT **Complete western blots for JNK pathway
 assessment after MCSF and KITL in the presence of pexidartinib.** Complete
 western blots for figures 3A and 4A of the main paper, evaluating changes
 in JNK signaling in the presence of pexidartinib after MCSF (left blots)
 and KITL (right blots). No membrane stripping performed after Total
 Antibody assessment before GAPDH probing. Experiments were performed in
 triplicate, with these western blots serving as representative of the
 other experimental results. Abbreviations: pJNK-phosphorylated JNK.
 **Complete western blots for STAT3 pathway assessment after MCSF and KITL
 in the presence of pexidartinib.** Complete western blots for figures 3C
 and 4C of the main paper, evaluating changes in STAT3 signaling in the
 presence of pexidartinib after MCSF (left blots) and KITL (right blots).
 No membrane stripping performed after Total Antibody assessment before
 GAPDH probing. Experiments were performed in triplicate, with these
 western blots serving as representative of the other experimental results.
 Abbreviations: pSTAT3-phosphorylated STAT3 **Complete western blots for
 AKT pathway assessment after MCSF and KITL in the presence of
 pexidartinib.** Complete western blots for Figures 3E and 4E of the main
 paper, evaluating changes in AKT signaling in the presence of pexidartinib
 after MCSF (left blots) and KITL (right blots). No membrane stripping
 performed after Total Antibody assessment before GAPDH probing.
 Experiments were performed in triplicate, with these western blots serving
 as representative of the other experimental results. Abbreviations:
 pAKT-phosphorylated AKT. **Complete western blots for JNK pathway
 inhibition by pexidartinib in absence of inflammatory ligands.** Complete
 western blots for JNK pathway assessment of pexidartinib inhibition in
 absence of inflammatory ligands (Supplemental Figure 6A). No membrane
 stripping performed after Total Antibody assessment before GAPDH probing.
 Experiments were performed in triplicate, with these western blots serving
 as representative of the other experimental results. Abbreviations:
 pJNK-phosphorylated JNK. **Complete western blots for STAT3 pathway
 inhibition by pexidartinib in absence of inflammatory ligands.** Complete
 western blots for STAT3 pathway assessment of pexidartinib inhibition in
 absence of inflammatory ligands (Supplemental Figure 6B). No membrane
 stripping performed after Total Antibody assessment before GAPDH probing.
 Experiments were performed in triplicate, with these western blots serving
 as representative of the other experimental results. Abbreviations:
 pSTAT3-phosphorylated STAT3 **Complete western blots for AKT pathway
 inhibition by pexidartinib in absence of inflammatory ligands.** Complete
 western blots for AKT pathway assessment of pexidartinib inhibition in
 absence of inflammatory ligands (Supplemental Figure 6C). No membrane
 stripping performed after Total Antibody assessment before GAPDH probing.
 Experiments were performed in triplicate, with these western blots serving
 as representative of the other experimental results. Abbreviations:
 pAKT-phosphorylated AKT. ## **Sharing/Access information** Data was
 derived from the following sources and can be accessed online: *