genome-engineering-of-primary-human-b-cells-using-crisprcas9

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

University of Minnesota

Semaphorins, specifically type IV, are important regulators of axonal guidance and have been increasingly implicated in poor prognoses in a number of different solid cancers. In conjunction with their cognate PLXNB family receptors, type IV members have been increasingly shown to mediate oncogenic functions necessary for tumor development and malignant spread. In this study, we investigated the role of semaphorin 4C (SEMA4C) in osteosarcoma growth, progression, and metastasis. We investigated the expression and localization of SEMA4C in primary osteosarcoma patient tissues and its tumorigenic functions in these malignancies. We demonstrate that overexpression of SEMA4C promotes properties of cellular transformation, while RNAi knockdown of SEMA4C promotes adhesion and reduces cellular proliferation, colony formation, migration, wound healing, tumor growth, and lung metastasis. These phenotypic changes were accompanied by reductions in activated AKT signaling, G1 cell cycle delay, and decreases in expression of mesenchymal marker genes SNAI1, SNAI2, and TWIST1. Lastly, monoclonal antibody blockade of SEMA4C in vitro mirrored that of the genetic studies. Together, our results indicate a multi-dimensional oncogenic role for SEMA4C in metastatic osteosarcoma and more importantly that SEMA4C has actionable clinical potential.

SEMA4C is a novel target to limit osteosarcoma growth, progression, and metastasis.

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.

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

We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas (OSAs) using the Sleeping Beauty (SB) transposon system. Here, we followed up investigating the genetic role of ZNF217 in OSA initiation and progression through establishment of a novel genetically engineered mouse model (GEMM), in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of OSA transformation including proliferation, cell motility, anchorage independent growth, and ultimately promoting OSA growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacological blockade of AKT signaling with nucleoside analogue triciribine (TCN) in ZNF217+ orthotopically-injected OSA cell lines reduced tumor growth and metastasis. Our data demonstrate that TCN treatment may be a relevant and efficacious therapeutic strategy for OSA patients with ZNF217+ and p-AKT rich tumors. With the recent revitalization of TCN for clinical studies in other solid cancers (PTX-200, Prescient Therapeutics), our study provides rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ OSA.

Implication of ZNF217 in accelerating tumor development and therapeutically targeting ZNF217-induced PI3K-AKT signaling for the treatment of metastatic osteosarcoma.

Joseph J. Peterson

Department of Pediatrics,
Center for Genomic Engineering,
Masonic Cancer Center

University of Minnesota

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Joseph J. Peterson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics,Center for Genomic Engineering,Masonic Cancer Center

University of Minnesota

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Department of Pediatrics,
Center for Genomic Engineering,
Masonic Cancer Center