Elsevier

Bone

Volume 136, July 2020, 115353
Bone

Full Length Article
PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma

https://doi.org/10.1016/j.bone.2020.115353Get rights and content

Highlights

  • Sleeping Beauty mutagenesis identified CSF1R as a potential driver of OSA.

  • Oncogenic CSF1R is expressed and is constitutively active in a subset of OSA.

  • PLX3397 treatment effectively reduced local OSA tumor growth and metastasis.

Abstract

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

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)

  • Y. Wittrant et al.

    Colony-stimulating factor-1 (CSF-1) directly inhibits receptor activator of nuclear factor-{kappa}B ligand (RANKL) expression by osteoblasts

    Endocrinology

    (2009)
  • J.A. Perry et al.

    Complementary genomic approaches highlight the PI3K/mTOR pathway as a common vulnerability in osteosarcoma

    Proc. Natl. Acad. Sci.

    (2014)
  • M.C. Scott et al.

    Comparative transcriptome analysis quantifies immune cell transcript levels, metastatic progression, and survival in osteosarcoma

    Cancer Res.

    (2018)
  • T.D. Schmittgen et al.

    Analyzing real-time PCR data by the comparative C(T) method

    Nat. Protoc.

    (2008)
  • B.A. Smeester, N.J. Slipek, E.J. Pomeroy, H.E. Bomberger, G.A. Shamsan, J.J. Peterson, M.R. Crosby, G.M. Draper, K.L....
  • B.A. Smeester et al.

    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

    (2012)
  • B.A. Smeester et al.

    The effect of electroacupuncture on osteosarcoma tumor growth and metastasis: analysis of different treatment regimens

    Evidence-based Complementary and Alternative Medicine : eCAM

    (2013)
  • A. Faustino-Rocha et al.

    Estimation of rat mammary tumor volume using caliper and ultrasonography measurements

    Lab Animal

    (2013)
  • N.A. Temiz et al.

    RNA sequencing of Sleeping Beauty transposon-induced tumors detects transposon-RNA fusions in forward genetic cancer screens

    Genome Res.

    (2016)
  • A.I. Segaliny et al.

    Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment

    Int. J. Cancer

    (2015)
  • S.A. Harris et al.

    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

    (1995)
  • Cited by (20)

    View all citing articles on Scopus
    1

    Contributed equally.

    View full text