The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
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Ignacio García-Tuñón1,*, María Hernández-Sánchez1,*, José Luis Ordoñez1, Veronica Alonso-Pérez1, Miguel Álamo-Quijada1, Rocio Benito1, Carmen Guerrero2,5,6, Jesús María Hernández-Rivas1,2,3,#, Manuel Sánchez-Martín2,4,6,#
1Unidad de Diagnóstico Molecular y Celular del Cáncer, Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), Salamanca, Spain
2IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain
3Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain
4Servicio de Transgénesis, Nucleus, Universidad de Salamanca, Salamanca, Spain
5Instituto Biología Molecular y Celular del Cáncer (USAL/CSIC), Salamanca, Spain
6Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain
*Co-first authors, these authors contributed equally to the work
#These authors have contributed equally and share senior authorship
Manuel Sánchez-Martín, email: [email protected]
Jesús María Hernández-Rivas, email: [email protected]
Keywords: CRISPR/Cas9, genome edition, BCR/ABL, leukemia
Received: December 28, 2016 Accepted: January 27, 2017 Published: February 09, 2017
CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.
CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.
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