ETV6-NTRK3, produces a kinase polymer from a transcription factor

The ETV6-NTRK3 fusion is diagnostic for both secretory carcinoma in breast, and mammary analog secretory carcinoma or MASC. The ETV6 gene codes for a transcription factor in the ETS (E26 transformation specific) family. The protein product of this gene is often referred to as TEL. The C-terminal end of TEL binds DNA while the N-terminal pointed (PNT) domain is involved in dimerization, and ultimately, polymerization. The PNT domain is also called the sterile alpha motif, or SAM.  The ETV6 gene resides in a site of chromosome 12 (12p13) described by Huret and coworkers (1986) as an aberrant breakpoint in chronic myelogenous leukemia. It is prone to fusion with other genes. Many of these aberrant protein products drive hematologic malignancies.

Diseases caused by variations in the ETV6  gene

(UniProt unless otherwise noted)

  • acute myeloid leukemia (AML). t(12;22)(p13;q11)  w/MN1
  • t(4;12)(q12;p13)  w/CHIC2
  • childhood acute lymphoblastic leukemia t(12;21)(p12;q22)
  • t(12;21)(p13;q22) w/RUNX1/AML1.
  • pre-B acute lymphoid leukemia. t(9;12)(p24;p13) w/ JAK2.
  • ACM leukemia. t(9;15;12 (p24;q15;p13) w/JAK2
  • myelodysplastic syndrome (MDS)  t(5;12)(q31;p13) w/ACSL6.
  • acute eosinophilic leukemia (AEL). t(5;12)(q31;p13) w/ACSL6.
  • myelodysplastic syndrome t(1;12)(p36.1;p13) w/MDS2
  • acute lymphoblastic leukemia. t(9;12)(p13;p13) w/PAX5.
  • mammary analogy secretory carcinoma or MASC w/ETV6.

Notation: t (1st chromosome;2nd chromosome)(1st band;2nd band) w/partner

Function of TEL, the ETV6  gene product

(UniProt and Genecards unless otherwise noted)

The ETV6 gene is found in every nucleated cell.  Where TEL (the protein) is expressed is interesting in terms of cancer biology:

  • monocytes, B cells, CD4 T cells but not in other immune cells
  • colon
  • salivary gland
Estimated TEL protein expression in various human tissues

TEL tissue expression.

 

The ETV6 and NTRK3 genes

Ensembl chromosome 12 and 15 showing location of ETV6 and NTRK3 genes respectively.

ETV6 and NTRK3 chromosome location

 

TEL/ETV6 and TrkC, the proteins

Most of the the ETS DNA binding domain is removed in the TEL-TrkC fusion protein.  Likewise, little but the kinase domain of TrkC remains in the fusion protein.  Most view the TEL transcription factor as a dimer like many other transcription factor.  TEL is also a multi-meric scaffolding protein.

Picture showing proetin domains of ETV6 and TrkC. Inset shows x-ray crystalograph of ETV6-NTRK3 interactions.

Properties of the fusion partners TEL and TrkC.

 

Tognon (2002) took our understanding of how these fusion proteins drive cancer to a new level.

  • They replaced the SAM domain of TEL in the TEL-TrkC fusion  with FK506 binding protein (FKBP) that forms dimers in the presence of FK506.  This is like the original TrkC only instead of NT3 inducing dimerization, FK506 induces dimerization.
  • FKBP-TrkC fusion proteins failed to transform NIH 3T3 cells even though the TrkC kinase domain was functional after the addition of FK506.
  • Two binding surfaces on the SAM/PNT region of TEL were manipulated. Tognon and coworkers hypothesized that it was multimers that transformed cells.
  • They  mutated each TEL-TrkC SAM/PNT binding interface in a manner shown previously to abolish self-association of wild-type TEL.
  • Each mutation completely blocked the ability of TEL-TRKC to polymerize, to autophosphorylate, and to transform NIH3T3 cells.

A follow up publication from the Sorensen Laboratory addressed how downstream signaling molecules are recruited to The TEL-TrkC complex.  Lannon (2008) found that a C-terminal 19 amino acids of the TrkC portion of the fusion protein is capable of binding to the insulin receptor substrate 1 (IRS-1).   The insulin receptor itself appears to be necessary for suppression of anoikis, programmed cell death, in ETV6-NTRK3 transformed mouse embryonic fibroblasts.  Expression of the insulin receptor along with ETV6-NTRK3 (the gene) was required for full transformation and localization of IRS-1 to the membrane.  (Martin, 2006).  This same laboratory explored the use of pharmacological inhibitors of the insulin growth factor receptor kinase in ETV6-NTRK3 transformed breast epithelial cells (Tognon, 2011).  The size of the Tel/TrkC (the protein) complexes also seems to be dependent on IGF receptor kinase activity (Tognon, 2012).  The possibility of use of IGFR and Trk kinase inhibitors to treat ETV6-NTRK3 driven breast cancers is exciting simply because of the tendency for secondary mutations to render kinase inhibitors ineffective.  The use of entrectinib, a highly potent pan-Trk inhibitor, caused an 89% reduction in tumor burden in a patient harboring a ETV6-NTRK3 fusion. (Drilon, 2016).

Proteins which may be recruited to the Tel-TrkC polymer by IRS-1

When insulin growth factor binds to the IGFR, adapter proteins IRS1/2 are recruited to the cell membrane.  The C-terminal 19 amino acids of TrkC in the fusion protein polymer bind to IRS-1 which also recruits signaling molecules.  Insulin growth factor and a functional IGF receptor are necessary to recruit The TEL-TrkC complex to the membrane.

Cartoon showing IRS-1/ TrkC signaling via the TEL-TrkC fusion protein

IRS-1/ TrkC signaling via the TEL-TrkC fusion protein.

 

IRS-1 does the following:

UniProt accession 35568, based largely on similarity

  • Binds UBT/Nucleolar transcription factor 1 F
  • Binds phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha
  • , binds phosphatidylinositol 3-kinase regulatory subunit alpha with phosphorylated YXXM motifs
  • Binds cell motility associated rho-associated, coiled-coil-containing protein kinase 1 (ROCK1)
  • Binds the non receptor protein tyrosine kinase FER.
  • Interacts with pleckstrin homology (PH) domain-interacting protein (via PH domain)
  • Binds growth factor receptor-bound protein 2 (GRB2), an adaptor protein in the MAPK/ERK pathway.
  • Interacts with suppressor of cytokine signaling 7 (SOCS7).
  • Interacts (via IRS-type PTB domain)  with the IGF recptor
  • Interacts with the insulin receptor (INSR) via the tyrosine-phosphorylated NPXY motif).
  • Interacts with the anaplastic lymphoma kinaes (ALK).
  • Interacts with an RNA activated protein kinase /PKR.

Clinical Trial information:

There is an open NTRK fusion clinical trial that is actively enrolling any solid tumor patient with NTRK fusions (STARTRK-2).   For more information go to the NTRK trial website.

References:

Drilon A, Li G, Dogan S, Gounder M, Shen R, Arcila M, Wang L, Hyman DM, Hechtman J, Wei G, Cam NR, Christiansen J, Luo D, Maneval EC, Bauer T, Patel M, Liu SV, Ou SH, Farago A, Shaw A, Shoemaker RF, Lim J, Hornby Z, Multani P, Ladanyi M, Berger M, Katabi N, Ghossein R, Ho AL. (2016) What hides behind the MASC: clinical response and acquired resistance to entrectinib after ETV6-NTRK3 identification in a mammary analogue secretory carcinoma (MASC).Ann Oncol.27(5):920-6. PubMed

Huret JL, Tanzer J, Henry-Amar M.(1986) Aberrant breakpoints in chronic myelogenous leukaemia; oncogenes and fragile sites. Hum Genet.74(4):447-8.  PubMed

Lannon CL, Martin MJ, Tognon CE, Jin W, Kim SJ, Sorensen PH. (2004) A highly conserved NTRK3 C-terminal sequence in the ETV6-NTRK3 oncoprotein binds the phosphotyrosine binding domain of insulin receptor substrate-1: an essential interaction for transformation. J Biol Chem.279(8):6225-34. PubMed

Martin MJ, Melnyk N, Pollard M, Bowden M, Leong H, Podor TJ, Gleave M, Sorensen PH. (2006) The insulin-like growth factor I receptor is required for Akt activation and suppression of anoikis in cells transformed by the ETV6-NTRK3 chimeric tyrosine kinase. Mol Cell Biol.26(5):1754-69. PubMed

Sutherland GR, Hinton L. (1981) Heritable fragile sites on human chromosomes. VI. Characterization of the fragile site at 12q13. Hum Genet. 57(2):217-9. PubMed

Tognon CE, Mackereth CD, Somasiri AM, McIntosh LP, Sorensen PH. (2004) Mutations in the SAM domain of the ETV6-NTRK3 chimeric tyrosine kinase block polymerization and transformation activity. Mol Cell Biol.24(11):4636-50.  PubMed

Tognon CE, Somasiri AM, Evdokimova VE, Trigo G, Uy EE, Melnyk N, Carboni JM, Gottardis MM, Roskelley CD, Pollak M, Sorensen PH. (2011) ETV6-NTRK3-mediated breast epithelial cell transformation is blocked by targeting the IGF1R signaling pathway. Cancer Res. 71(3):1060-70. PubMed

Tognon CE, Martin MJ, Moradian A, Trigo G, Rotblat B, Cheng SW, Pollard M, Uy E, Chow C, Carboni JM, Gottardis MM, Pollak M, Morin GB, Sorensen PH.(2012) A tripartite complex composed of ETV6-NTRK3, IRS1 and IGF1R is required for ETV6-NTRK3-mediated membrane localization and transformation. Oncogene.31(10):1334-40. PubMed

Tran, H.H., Kim, C.A. (2002) Native interface of the SAM domain polymer of TEL. BMC STRUCT.BIOL. 2: 5-5.  PubMed

Zhu Y, McAvoy S, Kuhn R, Smith DI. (2006) RORA, a large common fragile site gene, is involved in cellular stress response. Oncogene. 25(20):2901-8. PubMed

 

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