TPM3-NTRK1, an actin targeted kinase?

Tropomyosins bind to the cytoskeleton protein actin. They also modulate how other actin binding proteins bind to actin. Nemaline myopathy and Rod myopathy are diseases caused by variations in the TPM3 gene. Several splice variants of tropomyosin are coded for by the TPM3 gene. Unless otherwise noted, information is compiled from Genecards and uniprot.org.

Functions of products of the TPM3 gene product

  • Binds to actin filaments in muscle and non-muscle cells.
  • Plays a central role, in association with the troponin complex, that regulates the calcium dependent
    contraction of vertebrate striated muscle.
  • Smooth muscle contraction is regulated by interaction with caldesmon and tropomyosin.
  • In non-muscle cells, tropomyosin is implicated in stabilizing cytoskeleton actin filaments.
Tropomyosin 3 protein levels in various tissues.

Tropomyosin 3 is a ubiquitously a expressed housekeeping protein.

TPM3 and NTRK1 the genes

TPM3 and NTRK1 are located on the same general region of chromosome 1. See the history page of this website for more information on the work of Marin-Zanca (1986), Ardini and coworkers (2015), and more.

Ensembl chromosome 1 showing TPM3 and NTRK1 location.

Chromosome location of TPM3 and NTRK1

Tropomyosin 3 and TrkA, the proteins

Figure showing protein domains of TPM3 and TrkA fusions. Inset shows crystal structure of TrkA kinase interaction with actin.

Protein domains of native TrkA and splice variants of the TPM3 gene.

Splice sites were determined by Ardini et al (2014). The image of tropomyosin on filamentous actin (F-actin) is a combination X-rayscrystal and cryo electron microscope structure from van der Ecken (2015).

What this could mean

PI3 kinase is well known as a regulator of actin polymerization. Actin polymerization is necessary for neurite outgrowth (Zhou, 2006).

Diagram A shows NGF signalling pathway and B shows the neuronal interactions.

TPM3-NTRK1 fusions A. NGF signaling pathways involving the TrkA protein product of NTRK1 gene, adapted from Zhou, 2006. B. The final product in the neuron

  1. F-actin, presumably with tropomyosin, can be recruited to the cell membrane to focal adhesions. Can the tropolyosin-TrkA fusion protein activate PI3K in this location in any cell type?
  2. Does the tropomyosin-TrkA fusion protein preferentially associate with any particular pool of F-actin in the cell?
  3. Is F-actin formation less organized in the fusion protein expressing cell?

The tropomyosin-TrkA fusion protein in a colonic cell line

Key Findings

  1. Enzymes downstream of TrkA are phosphorylated at activation sites in the absence of TrkA ligands.
  2. The protein product of the TPM3-NTRK1 fusion gene is localized to the cytosol or perimembrane space.
  3. Phosphorylation of enzymes downstream of TrkA is sensitive to a pan-TrkA inhibitor.

Ardini and coworkers used a cell line (KM12) isolated from a primary human colorectal carcinoma (HCC) classified as Dukes’ B2 (Morikawa , 1988.)

  • Dukes Stage B2: Penetrating through muscularis propria; nodes not involved
  • The original KM12 tumor was classified as a poorly differentiated adenocarcinoma, a morphology maintained in different organs of nude mice (Moridawa, 1988)
  • The KM12 cell line was grown in nude mice to simulate and actual tumor in a human patient.

The goal was to further characterize the KM12 cell line

  • Molecular screening revealed wild-type RAS and BRAF, typically mutated in cancer.
  • The NTRK1 gene was targeted for characterizing fusion partners, particularly with TMP3.
  • Antibodies against TrkA were used to immunoprecipitate both TrkA and the tropomyoxin-TrkA fusion protein from the KM12 cells and a control. These precipitates were subjected to Western blotting to confirm the presence of TrkA and tropomyosin reactive material at the expected mass of the fusion protein.
Picture of western blots showing TPM3-TrkA fusion products. Fig C shows Tropomyosin 3 control blot using sigma antibody.

Figure 3A from Ardini, 2014. A. The Western blot with comments in the text. B. Supplemental data showing full length TrkA in some select cell lines C. The anti-tropomyosin 3 antibody from Sigma, used in this study, on a Western blot.

Inhibition of tropomyosin-TrkA kinase activity inhibits phosphorylation of downstream target

Ardini and coworkers used NMS-P626, a version of entrectinib. A defined tumor driven by another fusion protein and the KM12 cells driven by tropomyosin-TrkA were used to demonstrate the effectiveness of this pan-Trk kinase inhibitor.

Fig A shows Trk receoptor signaling pathway. Fig B is a western blot shwoing Fusion products LMNA-TRKA, TPM3-TRKA, along with PLCgama1, AKT, MAPK and loading control GAPDH.

Figure 3B. from Ardini 2014

 

Nude mice with KM12 xenograph tumors on a pan-Trk inhibitor NMS-P626/entrectinib

  • Nude mice were inoculated subcutaneously with 5 x106 KM12 cells.
  • Mice bearing tumors of a minimal volume range were randomized into vehicle and treatment groups of seven.
  • NMS-P626/entrectinib was dissolved in water and given by oral gavage.
  • Treatment started the day after randomization.
  • Tumor dimensions were measured regularly using calipers.
  • For ex-vivo phosphoprotein analysis, animals bearing established KM12 xenograft tumors were treated with a single orally administered dose and sacrificed at six and 12h.
  • Tumor samples were excised and processed for Western analysis as shown.
Left side graph shows a drastic lowering in xenograft tumor volume in trkA inhibitor treated samples as compared to vehicle control. Right side picture shows a western blot with drastic lowering of phosphorylated TrkA and  down stream target phospholipase Cγ as compared to total TrkA and control GAPDH.

Figure 3C,D Ardini, 2014.

 

Some interesting things to note about Figure 3C,D

  • The higher concentration of the pan-Trk inhibitor drastically slows tumor growth compared to the vehicle control.
  • By 6 hr after the kinase inhibitor, a drastic reduction in autophosphorylated (active) TrkA is seen.
  • A perhaps even greater reduction is seen in the activating phosphorylation of the down stream target phospholipase Cγ (PLCϒ).
  • By 12 hours, activating phosphorlation of TrkA and PLCγ has begun to recover.
  • NGF firing through TrkA is needed for the neuron to escape apoptosis in a complex manner covered elsewhere (Ichim 2012).
  • Other studies covered on this website show tumor reduction, rather than slowed growth, in actual patients.

Important Information:

There is an open NTRK fusion clinical trial that is actively enrolling any solid tumor patient with TRK fusions (STARTRK-2). For more information go to the NTRK trial website. The compound used in this clinical trial was tested in a cell line expressing the TPM3-NTRK1 fusion.

References:

Ardini E, Bosotti R, Borgia AL, De Ponti C, Somaschini A, Cammarota R, Amboldi N, Raddrizzani L, Milani A, Magnaghi P, Ballinari D, Casero D, Gasparri F, Banfi P, Avanzi N, Saccardo MB, Alzani R, Bandiera T, Felder E, Donati D, Pesenti E, Sartore-Bianchi A, Gambacorta M, Pierotti MA, Siena S, Veronese S, Galvani A, Isacchi A. (2014) The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition. Mol Oncol. 8(8):1495-507. PubMed

Ichim G, Tauszig-Delamasure S, Mehlen P. (2012) Neurotrophins and cell death. Exp Cell Res. 318(11):1221-8. PubMed

Martin-Zanca D, Hughes SH, Barbacid M. (1986) A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature. 319(6056):743-8. PubMed

Morikawa K, Walker SM, Nakajima M, Pathak S, Jessup JM, Fidler IJ. (1988) Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res. 48(23):6863-71. PubMed

Von der Ecken J, Müller M, Lehman W, Manstein DJ, Penczek PA, Raunser S. (2015) Structure of the F-actin-tropomyosin complex. Nature. 519(7541):114-7. PubMed

Zhou FQ, Snider WD. (2006) Intracellular control of developmental and regenerative axon growth. Philos Trans R Soc Lond B Biol Sci.361(1473):1575-92. PubMed

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