Inhibiting signaling in a BCAN-TRK1 brain tumor

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. The use of  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 is covered on Trkmutations.

An overview of use of a TrkA small molecule inhibitor to treat a mouse model of BCAN-TRK1 driven gliomas is covered on trkfusions.

The   BCAN-TRK1 rearrangement has been introduced on this site. What is new and notable is how genetically well defined the model is.

Review of TrkA signaling

Cartoon showing TrkA signaling from receptor to mucleus

Figure 1. Signaling pathways of the TrkA receptor

Small molecule inhibition of brevican-TrkA signaling

When Cook and coworkers used CRISPR to create BCAN-TRK1 rearrangements in mouse neural stem cells (aNSC) they obtained three different populations (1) pure deletions that can form tumors in mouse brains (2) pure inversion that do nothing, and (3) a combination of both in each cell.  “Cell lines” were isolated from tumors formed from CRISPR mixes.  The authors used a Trk treatment to ask if these cells had become addicted to Trk signaling.

A drug called entrectinib reduces cell viability in BCAN-REK1 driven tumors and not in PDGF driven tumors

Figure 2 from Cook 2017 a. entrectinib reduces cell viability in BCAN-TRK1 driven tumors but not in PDGF driven tumors. b. Entrectinib inhibits down stream  signaling.

The PDGFb cell line that is rendered metastatic by ectopic expression of PDGFb.  PDGF receptor signaling activates many of the pathways in TrkA signaling.  Entectinib does not act on the PDGF receptor.  The BNN2 cell line contains only the BCAN-TRK1 fusion by deletion.  The BNN4 cell line contains both the BCAN-TRK1 fusion by deletion as well as the non carcinogenic fusion by inversion.

Past the blood brain barrier, small molecule inhibition of brevican signaling in gliomas in mice.

Tumors containing a clone of BCAN-TRK1 via deletion (tumour 1″) and a combination of inversion and deletion (“tumour 2”) were injected into mouse brains. After 14 days of growing in the hosts’ brains, entrectinib was or vehicle were given for two days.  Immunostaining for pAkt1  within tumors is shown

Immunostaining of brain tumors induced by BCAN-TRK1 and treated with entrectinib

Fig 3 Entrectinib influence on pAKt1 in BCAN-TRK1 tumors in mouse brains.

Unlike cultured cells (figure 2b), entrectinib appears to inhibit pAkt1 autophosphorylation.

Entrectinib does not induce apoptosis in a p53 null background

Another extremely interesting aspect of these tumors is that even though entrectinib reduces cell viability, it does not appear to do so by activating the caspase system as has been shown for the SCYL3-TRK1 fusion.

Western blots done using anti cleaved Casp3 antibodies show absence of cleaved Casp3 bands in all constructs except PDGFb. Comparison was done againts actin antibodies as contol for all lanes. This suggests that Entectinib does not appear to kill BCAN-TRK1 transformed aNSC via activation of apoptosis/caspase

Figure 4. Entectinib does not appear to kill BCAN-TRK1 transformed aNSC via activation of apoptosis/caspase a. from supplemental data of Cook (2017) b. typical Western blot showing caspase 3 cleavage (AbCam)

In a continued effort to test the hypothesis that BCAN-TRK1 (del) causes cells to become addicted to TrkA signaling, BNN4 and  control PDGFb tumor cells were treated with entrectinib or vehicle after 24h growth factor withdrawal.  Apoptosis associated   Casp-3 cleavage was assessed after two hours PDGFb cells treated with 10 Gy ionizing radiation (IR) as a positive control.  IR causes DNA damage pushing the cell into undergoing apoptosis.  Panel 4b came from the website of a commercially available caspase 3 antibody (AbCam).  Like many proteases, caspase 3 is produced in an inactive “pro-” form.  Proteolytic cleavage by an upstream protease releases  the auto-inhibitory peptide.

The TUNEL assay is a means of detecting the attempted repair of DNA breaks.  TUNEL staining was performed on BNN4 tumors from mice sacrificed on treatment day 14. Scale bar=0.1mm. Error bars=mean +/- SEM..

A cartoon showing how Tunnel assay works. Tunnel assay showing entrectinib decreases DNA fragmentation

Figure 5. Entrectinib, if anything decreases DNA fragmentation. top general overview of how the TUNEL assay works. bottom, from Figure supp7d, Cook 2017

One thing that needs to be remembered is that these studies were performed in a backdrop of dysfunctional  p53.   p53 participates in DNA repair pathways as  well as apoptosis.  In a way it is not surprising that entrectinib did not induce cell demise by apoptosis in this particular background.

Entrectinib induces cell cycle arrest

The Ki67 antigen is used as a marker for proliferating cells.  After two days of entrectinib treatment, 26 days post implantation, tumors were collected and stained with Ki67 antibody.

Immunostains showing tissues stained with Ki67 antigen (a marker for cell proliferation) after two days of entrectinib treatment and 26 days post implantation. Entrectinib reduces Ki67.

Figure 6 Entrectinib reduces mitosis marker Ki67.  error bars are +/- SEM

Important Information

Like any good study, this study has raised more questions than it has answered.  One cannot assume that a mutated TP53 is the sole driver of any cancer.  This study has also shown that an unregulated  TrkA kinase requires p53 to be dysfunctional.  The good news is that a small molecule inhibitor can kill the tumors, albeit via a means other than apoptosis.  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.

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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