Full Length ArticlePLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma
Graphical abstract
Introduction
Osteosarcoma (OSA) is a rare and aggressive cancer affecting the growing long bones of adolescents and is characterized by a high propensity to metastasize to the lungs [1]. While the current standard of care improves survival outcomes in patients with localized disease, neoadjuvant chemotherapy fails to provide any substantial survival benefit to patients with lung metastases [1,2]. A hallmark of OSA is widespread genomic instability, and a paucity of obvious activated oncogenes, making it difficult to identify specific drivers of OSA development and fatal metastatic spread [3]. Also, heterogeneity between individual tumors is considered one of the major reasons for the lack of progress in leveraging any targetable drivers to date [4]. Therefore, it is imperative that we improve our understanding of commonly altered OSA-specific signaling pathways known to promote malignancy and tumor maintenance in order to develop effective molecularly targeted therapies for patients with metastatic disease [5]. For these reasons, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen for OSA which identified numerous (>250) previously unknown drivers of OSA development and metastasis [6]. In particular, Csf1r (c-fms proto-oncogene) was identified as a candidate OSA driver oncogene in a subset of our primary OSA samples [6].
CSF1R is a transmembrane, tyrosine kinase receptor known to mediate cellular effects of macrophage colony stimulating factor 1 (M-CSF, also known as CSF1) and is primarily expressed by cells in the mononuclear phagocytic lineage [7]. In normal growing bones, osteoblasts produce CSF1, which stimulates proliferation and differentiation of CSF1R-expressing osteoclast progenitors [8]. We hypothesized that increased CSF1R expression could potentiate CSF1-induced signaling in OSA and may represent a therapeutically exploitable pro-tumorigenic, autocrine/paracrine signaling loop. To test this hypothesis, we characterized CSF1R expression and signaling in OSA and coupled these studies with therapeutic targeting of active signaling using a small molecule inhibitor of CSF1R. Together, our studies demonstrate that CSF1R is highly expressed and constitutively active in a subset of OSA samples, CSF1R-CSF1 signaling is oncogenic in immortalized osteoblasts and OSA cell lines, and CSF1R tyrosine kinase inhibition reduces properties of cellular transformation in vitro and OSA tumor growth and metastatic burden in vivo. Further, we provide rationale for continued preclinical evaluation of PLX3397 for the treatment of OSA.
Section snippets
RNA-sequencing data
CSF1, IL-34, and CSF1R expression levels were examined in three independent and publicly available human OSA patient RNA-sequencing data sets and compared to normal human osteoblast data [6,9,10]. The results published in data set #3 are in whole or part based upon data generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, phs000218, managed by the NCI. Information about TARGET can be found at http://ocg.cancer.gov/programs/target.
Tissue microarray (TMA) samples and scoring
OSA tissue
CSF1R and CSF1 are expressed in human and mouse OSA
We previously identified Csf1r as a significantly, recurrently mutated oncogene in a subset of SB-accelerated OSA [6,16]. RNA-sequencing analysis confirmed that the presence of an SB transposon-Csf1r fusion transcript (R-CIS) [6,16] was associated with greatly increased expression of Csf1r (Fig. 1a, blue dots). These RNA-sequencing findings were further confirmed at the protein level in cell lines generated from SB-fusion positive/negative tumors when stained for CSF1R expression (Fig. 1b).
Discussion
In our previously published Sleeping Beauty (SB) transposon-based screen [6], the Csf1r gene was identified as a common insertion site (CIS) locus in a subset of accelerated OSA samples. Evaluation of RNA-sequencing data from this set of OSA samples [16] revealed high expression levels of Csf1r mRNA in the same tumor samples that had been identified to carry putative Csf1r driving SB transposon insertion mutations. It is well-established that human OSAs are remarkably heterogeneous from a
CRediT authorship contribution statement
Branden A. Smeester:Conceptualization, Data curation, Formal analysis, Writing - original draft, Writing - review & editing, Supervision.Nicholas J. Slipek:Conceptualization, Data curation, Formal analysis, Writing - original draft, Writing - review & editing, Supervision.Emily J. Pomeroy:Data curation, Writing - original draft, Writing - review & editing.Kanut Laoharawee:Data curation, Writing - original draft, Writing - review & editing.Sara H. Osum:Data curation, Writing - original draft,
Declaration of Competing Interest
Dr. Largaespada is the co-founder and co-owner of several biotechnology companies including NeoClone Biotechnologies, Inc., Discovery Genomics, Inc. (recently acquired by Immunsoft, Inc.), and B-MoGen Biotechnologies, Inc. (recently acquired by bio-techne corporation). He consults for Genentech, Inc., which is funding some of his research. Dr. Largaespada holds equity in and serves as the Chief Scientific Officer of Surrogen, a subsidiary of Recombinetics, a genome-editing company. The business
Acknowledgements
The authors would like to thank Plexxikon for their gift of PLX3397 formulated rodent chow. The authors acknowledge the Minnesota Supercomputing Institute (MSI) at the University of Minnesota for providing resources that contributed to the research results reported within this paper. URL: http://www.msi.umn.edu. Author B.A.S. was previously supported by an NIH NIAMS T32 AR050938 Musculoskeletal Training Grant and is currently supported by a Doctoral Dissertation Fellowship (DDF) through the
References (42)
- et al.
Cell signaling by receptor tyrosine kinases
Cell
(2010) - et al.
Colony-stimulating factor 1 potentiates lung cancer bone metastasis
Laboratory Investigation; A Journal of Technical Methods and Pathology
(2014) - et al.
A phase Ib study of pexidartinib (PLX3397) and weekly paclitaxel in patients with advanced solid tumors including an ovarian cancer subset
Gynecol. Oncol.
(2016) - et al.
Osteosarcoma: a comprehensive review
SICOT J
(2018) - et al.
Review of osteosarcoma and current management
Rheumatol Ther
(2016) - et al.
Genome-wide analyses on high-grade osteosarcoma: making sense of a genomically most unstable tumor
Int. J. Cancer
(2013) - et al.
Genome-informed targeted therapy for osteosarcoma
Cancer Discovery
(2019) - et al.
Osteosarcoma: accelerating progress makes for a hopeful future
Front. Oncol.
(2018) - et al.
A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis
Nat. Genet.
(2015) - et al.
CSF-1 receptor signaling in myeloid cells
Cold Spring Harb. Perspect. Biol.
(2014)
Colony-stimulating factor-1 (CSF-1) directly inhibits receptor activator of nuclear factor-{kappa}B ligand (RANKL) expression by osteoblasts
Endocrinology
Complementary genomic approaches highlight the PI3K/mTOR pathway as a common vulnerability in osteosarcoma
Proc. Natl. Acad. Sci.
Comparative transcriptome analysis quantifies immune cell transcript levels, metastatic progression, and survival in osteosarcoma
Cancer Res.
Analyzing real-time PCR data by the comparative C(T) method
Nat. Protoc.
Effects of different electroacupuncture scheduling regimens on murine bone tumor-induced hyperalgesia: sex differences and role of inflammation
Evidence-based Complementary and Alternative Medicine : eCAM
The effect of electroacupuncture on osteosarcoma tumor growth and metastasis: analysis of different treatment regimens
Evidence-based Complementary and Alternative Medicine : eCAM
Estimation of rat mammary tumor volume using caliper and ultrasonography measurements
Lab Animal
RNA sequencing of Sleeping Beauty transposon-induced tumors detects transposon-RNA fusions in forward genetic cancer screens
Genome Res.
Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment
Int. J. Cancer
Development and characterization of a conditionally immortalized human fetal osteoblastic cell line
Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral Research
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