Genetically modified mice with BCAN-TRK1 driven gliomas

This post is all about the use of a small molecule Trk inhibitor to improve the survival of genetically modified mice harboring a BCAN-TRK1 fusion in their brains.  The study of Cook and coworkers (2017) is very extensive.

The use of the CRISPR system to create these mutations is covered in a sister site Trkmutations.

The complex cell signaling in response to the small molecule “Trk treatment” is also covered in a separate BCAN-TRK1 signaling post.

This post is cutting to the chase. Can a small molecule Trk inhibitor improve the survival of gliomas growing in the brains of mice? What makes this study truly unique and exciting is a combination of two things: (1)  The tumors were either produced from adult neural stem cells (aNSC) and injected  in the brains of the mice, and to make things more clinical, the gene altering CRISPR assemble was injected directly into mouse brains. (2) In order to be effective, the small molecule inhibitor has to actually cross the blood brain barrier.

Very brief comment on the model

Cook and coworkers used CRISPR to delete a portion of chromosome 3 in their mouse model (Figure 1a). In humans, it would be chromosome 1. In this fusion by deletion, the kinase domain of of TrkA would be under the control of the BCAN promoter. Brevican expression is restricted to the brain (Figure 1b). Brevican is a neuronal extracellular matrix protein. According to immunohistochemistry assessment available on ProteinAtlas.org, TrkA is expressed in a broad range of tissues though it is medium to high in regions of the brain.

Picture of Ntrk1- Bcan artificial gene construct with graph showing expression in mouse brain tissue.

Figure 1. The BCAN-TRK1 fusion by deletion a. diagram of the mouse chromosome 3 deletion used to create the BCAN-TRK1 fusion b. Brevican, the BCAN gene product is only expressed in the brain. c. TrkA is also expressed in the brain.

Two slightly different fusion sites, both induce gliomas

We have covered slightly different fusion junctions of the same gene fusion set both being found in tumors. Cook and coworkers produced a gene fusion at TRK1 intron 11 (BN1, Figure 2a,b) and intron 10 (Figure 2c,d) when injected in the brains of nude mice.

Two different Ntrk1-Bcan fusions products induce similar kind of gliomas in mice brains.

Figure 2. Two different TRK1 fusion sites both produce gliomas a. BN1 fusion sites b. aNSC with this fusion produces a glioma but control aNSC do not. c. The fusion at TRK1 intron 10 also produces d. gliomas in the brains of nude mice.

Genetically engineered gliomas grow more virulent with passage

In the Cook 2017 study clones were isolated from populations of CRISPR produced BCAN-TRK1 gene rearrangements. Cell lines are clones injected in mouse brains, grown into gliomas, and recultured. The hypothesis is that they would become more aggressive with each passage.

Survival curves showing stronger brains gliomas with each passage in mice.

Figure 3. BCAN-TRK1 gliomas grow stronger with passage. Survival curves are as follows a. Original CRISPR mutants before and after “purifying” them into clones. b. A pure deletion clone was grown in the brain in one mice and then injected into another. c.  An inv/del clone grown as a glioma in one mouse brain and injected into more.

Note that none of the control mice and BCAN-TRK1 via inversion mice died during the course of this experiment (Fig 3a).  In less than 50 days one of the BCAN-TRK1 (del) mice had died (red trace, Fig 3a). One of these mice remained at the end of 100 days. The BCAN-TRK (del/inv) mice (green trace, Fig 3a) died off more quickly. When gliomas were isolated from mice and injected into healthy nude mice, death came quicker (Fig 3b/c, **, p<0.01).

The small molecule entrectinib kills gliomas

Graph showing relative cell viability after Entrectinib treatment in tumor cell lines.

Figure 4. From Cook(2017) fig 4. a. Entrectinib reduces viability of aNSC cells with oncogenic BCAN-TRK1 gene fusions. c. Entectinib enhancs survival of nude mice whose brains were injected with these aNSC derived gliomas

As an aside Cook and coworkers monitored weigh changes during the course of treatment with entrectinib or the vehicle (Figure 5) starting of treatment, which was 12 days after implantation. The percentage of weight change being calculated each day for each animal relative to the starting weight.  Red lines indicate animals sacrificed during the 20 day weight monitoring period.

Graph showing weight changes in lab mice treatment with entrectinib and vehicle control.

Figure 5. Weight changes during entrectinib and vehicle treatment

MRI images showing brains of mice treated with entrectinib and vehicle control.

Figure 6. Representative magnetic resonance imaging analysis performed on entrectinib- or vehicle-treated mice on day 13 and 20 post-implantation.

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

Important Information

Perhaps one of the most important things shown in this study is the ability of entrectinib to cross what is presumably the blood brain barrier (BBB). The BBB is a major barrier to small molecule delivery. To grow as they did, the tumors would have to have been vascularized. 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 in section Ignyta authored publications.

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