Engineered BCAN-TRK1 drives glioma formation

This post only covers a small portion of a very in depth study of Peter Cook and coworkers of Memorial Sloan Kettering Cancer Center in New York City. This group used a new genetic engineering technique called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to introduce the BCAN-TRK1 gene rearrangement in adult neural stem cells of mice lacking two functional copies of the TP53 tumor suppressor gene. These altered cells were injected into the brains of nude mice. This study followed a previous report of a glial neuronal patient with a BCAN-TRK1 rearrangement responding to the small molecule Trk inhibitor entectinib. The Cook study advanced our understanding by using a more genetically defined model to prove that the BCAN-TRK1 gene rearrangement could drive cancer in a genetically defined background.

Making Trk Mutations using CRISPR.

  • CAS-9 (CRISPR associated protein 9) is a bacterial endonuclease that recognizes viral DNA sequences not found in the bacterial geneome.
  • PAM , protospacer adjacent motif,   is the “non-self” sequence recognized by CAS-9 enzyme. The canonical PAM sequence is 5’-NGG-3’ where N is any nucleotide. Efforts are underway to alter the CAS-9 protein to bind to other PAM sequences.
  • The guide RNA portion of the CAS-9 enzyme complex targets it to more specific regions of the gene to be modified.
  • Donor DNA may be added to the repair process.
Cartoon showing how Trk mutations are generated in vitro using Cas9 enzyme complex

Figure 1. Gene engineering using CRISPR-CAS9

For additional reading, Origene has some excellent overviews on the subject.

Cook and coworkers isolated adult neural stem cells (aNSC) from the brains of mice that lacked both alleles of the TP53 gene that codes for the tumor suppressor p53. This particular model was chosen because p53 is inactivated in 85% of human glioblastomas. Neural stem cells may differentiate into neurons or a variety of glial cells that help maintain the health of the neurons.

cartoon showing differentiation of neural stem to other cell types, like glia, neurons, astrocytes and oligodendrocytes

Figure 2. Neural stem cells can differentiate into a variety of different neural cells.

CRISPR was found to create a variety of chromosome  rearrangements between the BCAN and TRK1 genes. The first was the intended deletion (outlined in green in Figure 3). The second was an inversion that resulted in rearrangements that contained the 5’ ends of the TRK1 and BCAN genes fused to anti-sense versions of the BCAN and TRK1 genes, respectively. These are outlined in red in Figure 3. In reading in the anti-sense direction, surely, there must be a stop codon present somewhere.

The CRISPR treatment resulted in a mixed population of aNSC that the authors called “nonclonal.” It is assumed that CRISPR acted on both alleles of mouse chromosome 3 containing the BCAN and TRK1 genes. Some cells contained two inversions, others two deletions, and a third mixed population (yellow outline). Selecting one cell from this population and allowing it to divide results in a clone.  By definition, a clone is a group of cells derived from the same cell, and initially genetically identical.

Diagram showimg 2 different types of BCAN-TRK1 gene rearrangements. Only deletion in DNA sequence was able to make glial tumors in lab mice brains

Figure 3. CRISPR was used to create two different types of BCAN-TRK1 gene rearrangements (right). The mixed population of cells were separated and propagated as clones (left). Only the rearrangement by deletion as able to drive glial tumors in the brains of nude mice (bottom).

 

Figure 3 also contains part of the supplemental table 1 from Cook 2017.

The brains of four- to six-week-old female nude mice were injected with aNSC at 1mm to 0.1 mm from bregma, a cranial landmark, at a depth of 2 mm.

  • Rows 1-2 Two different amounts of “non-clonal”  aNSC  were able to form tumors in the brains of mice.
  • Row 3. Presence of the wild type version of the tumor suppressor p53 prevented tumor formation.
  • Row 4. A clone carrying both the deletion and the inversion of the BCAN-NTRK1 gene rearrangement was able to form tumors in mouse brains five out of five times.
  • Row 5 A clone containing only the BCAN-NTRK1 fusion by deletion formed tumors in the brains of mice five out of five times.
  • Row 6. The BCAN-NTRK1 fusion by inversion was unable to form tumors in mouse brains.
Pictures showing control mice brains and mice brains with tumor formation

Figure 4. Visualization of Cook 2017 supplemental Table 1 row 1.

 

Creating mutations in mouse brains that resemble real tumors

The authors were of the opinion that manipulation of aNSC might in some ways not fully represent natural gliomas. They generated an all-in-one adenoviral vector engineered to create the BCAN-TRK1 rearrangement directly in the brain of adult mice.

  • FLAG-spCas9

  • the sgRNA-BN1 pair (Ad-BN)

  • p53f/f mice with Ad-Cre system to knock out p53.

Three things were successfully generated using this system, and shown in Figure 5 of Cook (2017) as well as Figure 5 of this post.

Genomic rtPCR showing expression of BCAN-TRK1 deletion expression in various tumor samples. Comparison with actin expression and disfunctional p53.

Figure 5. Creating a glioma directly in a non immunocompromised p53f/f mouse. e. genomic PCR reveals the BCAN-TRK1 rearrangement by deltion f. rtPCR reveals transcription of this gene rearrangement g. Genomic PCR reveals creation of a tumor with dysfunctional p53  suppressor.

  • Use of a small molecule to inhibit glioma cell signaling in these mouse models is covered in a BCAN-TRK1 signaling post.
  • Another separate post addresses the important matter of how this small molecule improves the survival of mice with BCAN-TRK1 gliomas growing in their brains

Important Information

The purpose of this study was to generate an animal to test a small molecule inhibitor or Trk, ROS1, and ALK kinases.  An open clinical trial is testing a Trk inhibitor for solid tumors driven by Trk, ROS1, or ALK gene rearrangements.  A Phase 1 summary of this   Trk treatment  is available online.

Reference

Cook PJ, Thomas R, Kannan R, de Leon ES, Drilon A, Rosenblum MK, Scaltriti M, Benezra R, Ventura A.(2017) Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target. Nat Commun. 2017 Jul 11;8:15987.  PubMed

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