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The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma
Orsi Giricz, … , Jeffrey A. Sosman, Amit K. Verma
Orsi Giricz, … , Jeffrey A. Sosman, Amit K. Verma
Published July 25, 2018
Citation Information: JCI Insight. 2018;3(14):e120422. https://doi.org/10.1172/jci.insight.120422.
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Research Article Oncology

The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

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Abstract

Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

Authors

Orsi Giricz, Yongkai Mo, Kimberly B. Dahlman, Xiomaris M. Cotto-Rios, Chiara Vardabasso, Hoa Nguyen, Bernice Matusow, Matthias Bartenstein, Veronika Polishchuk, Douglas B. Johnson, Tushar D. Bhagat, Rafe Shellooe, Elizabeth Burton, James Tsai, Chao Zhang, Gaston Habets, John M. Greally, Yiting Yu, Paraic A. Kenny, Gregg B. Fields, Kith Pradhan, E. Richard Stanley, Emily Bernstein, Gideon Bollag, Evripidis Gavathiotis, Brian L. West, Jeffrey A. Sosman, Amit K. Verma

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Figure 4

CSF1R expression is activated by an aberrant upstream promoter, and the CSF-1R regulates melanoma growth and invasion.

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CSF1R expression is activated by an aberrant upstream promoter, and the...
(A and B) Five different lentiviral shRNAs were evaluated for CSF1R and RUNX1. Among these, shRUNX1#1, shRUNX1#4, shCSF1R#1, and shCSF1R#5 conferred the strongest knockdown. Therefore, these were used for all subsequent experiments. Gene-expression analysis of CSF1R, RUNX1, and IL-34 in melanoma cell lines M14c#5 (A) and A2058 (B) stably harboring shRNA species against CSF1R or RUNX1 (3 biological replicates). (C–F) Proliferation of the stable knockdown cell lines with the doubling times (hours) in parentheses (C and D); for statistical analysis see Supplemental Figure 5A. Three-dimensional Matrigel cell culture of the stable A2058 cells (E) and the parental A2058 cells cultured with increasing dose of PLX3397 (F). (G) Pictures taken on the fifth day of culture; red line, median colony size in pixels. Quantitative analysis of cells undergoing proliferation and apoptosis in the 3-D cultures (t test, 2-tailed). (H and I) Larger images of all 3-D cultures are provided in Supplemental Figure 5, C and D (representative images, 3-D experiments performed twice). Transwell invasion assay (mean ± SEM) of A2058 cells using the CSF-1R inhibitor PLX3397 (H) and of A2058 stable RUNX1 or CSF1R knockdowns (I); images represent 3 biological replicates. All statistical analyses of 3-D colony size and invasion assays: 1-way ANOVA (statistical significance levels are noted with asterisks,**P ≤ 0.01, ****P ≤ 0.0001).

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