Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene

Trk (NTRK) receptor and NTRK gene fusions are oncogenic drivers of a wide variety of tumors. Although Trk receptors are typically activated at the cell surface, signaling of constitutive active Trk and diverse intracellular NTRK fusion oncogenes is barely investigated. Here, we show that a high intracellular abundance is sufficient for neurotrophin-independent, constitutive activation of TrkB kinase domains. In HEK293 cells, constitutive active TrkB kinase and an intracellular NTRK2-fusion oncogene (SQSTM1-NTRK2) reduced actin filopodia dynamics, phosphorylated FAK, and altered the cell morphology. Atypical cellular responses could be mimicked with the intracellular kinase domain, which did not activate the Trk-associated MAPK/ERK pathway. In glioblastoma-like U87MG cells, expression of TrkB or SQSTM1-NTRK2 reduced cell motility and caused drastic changes in the transcriptome. Clinically approved Trk inhibitors or mutating Y705 in the kinase domain, blocked the cellular effects and transcriptome changes. Atypical signaling was also seen for TrkA and TrkC. Moreover, hallmarks of atypical pTrk kinase were found in biopsies of Nestin-positive glioblastoma. Therefore, we suggest Western blot-like immunoassay screening of NTRK-related (brain) tumor biopsies to identify patients with atypical panTrk or phosphoTrk signals. Such patients could be candidates for treatment with NTRK inhibitors such as Larotrectinhib or Entrectinhib.

. Differences in stem cell marker and osteopontin expression in primary and recurrent glioblastoma.Cancer Cell Int.22:87.Abbreviations: le -left; ri -right; WB -Western blot.

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Figures, Videos, and tables table S1.Properties of anti-Trk antibodies

Video 1 .
Fig. S1.TrkB model and construct.A. Deduced amino acid sequence of Ntrk2 (trkB full-length -trkB.FL) encoded by Mus musculus (reference: NM_001025074; NP_001020245).In the depicted amino acid sequence, in blue is the initiating methionine and signal peptide, in orange is the transmembrane domain, in purple the lysine residue of the ATP binding site and in green are the important serine or tyrosine residues of the kinase domain.Putative Nglycosylation sequons are indicated in red and yellow.

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Fig. S1.B.Table explaining the various TrkB constructs generated for use in this study.

Fig. S2 .
Fig. S2.Constitutive activation of TrkB by overexpression and immunoreactivity profiles of anti-Trk, anti-pTrk-kin, anti-pTrk-PLCγ and anti-pTrk-Shc for various TrkB mutants.A. Immunostaining of HEK293 cells expressing either TrkB wildtype or indicated TrkB mutants.Cells were co-transfected with GFP.Immunofluorescence of GFP (green) and panTrk (red, anti-panTrk (C-term) -A7H6R), together with DAPI as nuclear counterstain (blue).Cells were immunostained after an expression time of 48 h.panTrk binds to the C-terminus of the receptor and shows there the integrity of the open reading frame of all mutants.HEK293 themselves do not express TrkB.Confocal images; scale bar: 100 µm.B. Immunofluorescence of GFP (green) and pTrk-kin (red, anti-pY674/675-TrkA (anti-pY706/707-TrkB) C50F3), together with DAPI (blue).pTrk-kin antibody binds to the phosphorylated 2 nd and 3 rd Y residues in the YxxxYY motif of the Trk kinase domain.When these sites are mutated (Y 705/706 ), there is no fluorescence seen as the antibody no longer recognizes them (i.e.-YYF, YFY, YEY, YDY).When the ATP site is mutated (K 571 N), lack of ATP prevents TrkB autophosphorylation and the Y residues in the kinase domain remain unphosphorylated (ATP, YD-ATP).All missense mutations in Y 705 for D, E, or F, interrupted the anti-pTrk-kin immunoreactivity TrkB.Mutations in the Shc (Y 515 F) and

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Fig. S5.A-D: Filopodia formation is preserved in the kinase deficient TrkB-T1 expressing cells, but not in Trk kinase expressing cells.A, B. TrkB overexpression causes changes in actin morphology of HEK293 cells.This effect can acutely be reversed by the Trk inhibitor K252a.HEK293 cells expressing the kinase-deficient, truncated slice variant

Fig. S7 .
Fig. S7.U87MG cells expressing Trk-kinase constructs.A-C.Constitutive active TrkB localizes to the perinuclear Golgilike region and accumulates in actin-rich protrusions of U87MG cells.Immunofluorescence of TrkB receptor (green), pTrk-kin (magenta), and Acti-stain-670 phalloidin (cyan).(in A) In absence of Doxycyline, TrkB expression is not detectable.(in B) Induction of TrkB expression with Doxycycline leads to constitutive activation of TrkB.pTrk signals are pronounced at the perinuclear, Golgi apparatus-like region (cyan arrows).Confocal image; scale bar: 50 µm.(in C) Confocal stack.pTrk-kin localizes also to F-actin rich protrusions (arrows in magenta).Scale bar: 10 µm.D.Migration of U87MG cells.Representative phase contrast microscopy images.Indicated in magenta are cells that were automatically counted by unbiased cell counting with ImageJ (see Material and methods).E. U87MG cells expressing TrkB-wt or SQSTM1-NTRK2 were not dying within the indicated time span of 96 hours, albeit the cells express a rather high amount of intracellular Trk kinase activity (Figure5).

Fig. S8
Fig. S8 TrkB in U87MG -RNA seq controls.A-C.RNA-seq analysis: Volcano plots showing gene expression patterns for various RNA-seq controls.Log2-fold-change values are plotted to P-adjusted values obtained from DESeq2 analysis of the transcriptome.(in A) TrkB-wt uninduced control (w/o) is compared to TrkB-YFF uninduced control (w/o), showing no change in the transcriptome patterns and no leaky TrkB expression.(in B) TrkB-YFF induced (Dox)when compared to uninduced control (w/o) shows predominantly an upregulation of the three NTRK genes as they share sequence homologies.Doxycycline induction causes expression of only NTRK and none of the immune response genes (as seen in Figure6).Thus, lentiviral constructs themselves do not cause an anti-viral/immune responses in the cell line.(in C) TrkB-YFF uninduced control (w/o) is compared to the U87MG cell line, in order to eliminate possible underlying effects arising from the cell line or from the lentiviral infection itself (see also Figure6F).

Fig. S9 .
Fig. S9.NIH-3T3 cells expressing Trk-kinase constructs. A. Western blot analysis of cell lysates from NIH-3T3 cell lines expressing either TrkB-wt or SQSTM1-NTRK2.DOX was applied for 72 h, BDNF (20 ng/ml) was applied for 15 min.Cells were not serum-depleted.Antibodies are indicated.The figure shows that SQSTM1-NTRK2, but not TrkB-wt, becomes constitutive active in stable NIH-3T3 cells.TrkB-wt runs exclusively at 130 kDa in this cell model.Note the increase in the anti-panTrk signal by BDNF stimulation.B. Morphological cell transformation of NIH-3T3 cells by SQSTM1-NTRK2.Upper layer: Giemsa staining of confluent cell cultures expressing either TrkB-wt or SQSTM1-NTRK2.Note the change in culture morphology in SQSTM1-NTRK2-expressing cells.Lower panels: Brightfield image of indicated conditions, 72 days after expression induction with DOX. C. Immunofluorescence of TrkB-wt or SQSTM1-

kin age / sex comment tissue samples (Fig. 1): Nestin+ in histological pre-examination (Polat et al., 2022)
Table explaining the various TrkB constructs generated for use in this study.