Fig 2: Generation of hES cell lines with conditional LMNA::NTRK1 fusion expression.A Strategy for conditional LMNA::NTRK1 fusion gene expression. A donor template with homology arms (shaded boxes) and a promoter-less selectable marker (hygromycin or puromycin) is introduced at the DSB sites (scissors) by HDR. A splice acceptor (SA) sequence allows the expression of the marker from the LMNA promoter. Induction of the fusion occurs after removal of the selectable marker by expression of the CRE recombinase. B Dual color FISH analysis showing the NTRK1 locus and the rearranged allele in clones 1 (hygromycin positive; red, centromeric; green, telomeric) and 3 (puromycin positive; green, centromeric; red, telomeric) after loss of the centromeric probe either by t(1;1) or ID. C RT-PCR analysis in hES cells showing the LMNA::NTRK1 fusion level over a period of 30 days after expression of the CRE recombinase in clones 1 and 3. D Dual color FISH analysis on clone 13, (from NHEJ strategy, see S1D) showing loss of 5′ NTRK1 probe (red, centromeric) and RT-PCR confirming the presence of the LMNA::NTRK1 fusion in two distinct clones. E Western blot analysis showing the LMNA::TRKA fusion protein in clones isolated after removal of the marker (clones 1.14 and 3.3) and clone 13. Phosphorylation of TRKA is not detected.

Fig 3: Expression of LMNA::NTRK1 fusion in human mesenchymal progenitors (hES-MP).A Diagram of LMNA::NTRK1 transcript with primers position for RT-PCR. Time course (days) experiment for LMNA::NTRK1 fusion and NTRK1 expression in hES-MP (clones 1 and 3) after Cre expression. B Western blot showing LMNA::TRKA product and phosphorylation of TRKA in hES-MP cells expressing the fusion transcript. C Histogram plot from RNAseq analysis showing NTRK1, NESTIN, S100A and SOX10 RNA levels in hES and hES-MP expressing the LMNA::NTRK1 fusion compared to wild-type isogenic cell line and colorectal cancer cell line KM12 expressing the TPM3::NTRK1 fusion. D GSEA analysis for cell lines expressing the LMNA::NTRK1 fusion (clones 13 and 1.14 (hES-MP) and 13 (hES)). GSEA shows an enrichment of pathways involved in Ewing sarcoma and soft tissue sarcoma.

Fig 4: Activity of zurletrectinib against solvent front and xDFG TRK mutant kinases in vitro.a Molecular docking and b in vitro kinase assays showing binding and activity of zurletrectinib against TRKA mutant kinases. Data were analyzed using GraphPad Prism 9 and are presented as % of inhibition normalized to untreated samples (mean ± STDEV). Groups were compared using a paired Student’s T-Test. P < 0.05 was considered statistically significant. Proliferation and clonogenic assays, and western blot analyses following treatment of two human primary (c) and two engineered mouse (d) NTRK fusion-positive cancer cell lines harboring TRK resistance mutations with zurletrectinib. The activity of zurletrectinib was compared against the activity of other 1st- (larotrectinib) or next-generation (selitrectinib and repotrectinib) TRK inhibitors (b–d).

Fig 5: Generation of the ETV6::NTRK3 chromosomal translocation in hES cells and differentiation to hES-MP.A Breakpoints within ETV6 on chromosome 12 and NTRK3 on chromosome 15 create the ETV6::NTRK3 t(12;15)(p13;q25). B A donor template containing a puromycin selectable marker under the control of a PGK promoter is inserted by HDR at the ETV6 and NTRK3 participating loci after induction of DSBs by sgRNA (scissors). The selectable marker is removed by Cre recombinase allowing the expression of the oncogenic fusion. C Dual color FISH analysis with telomeric (red) and centromeric (green) probes on ETV6 showing the ETV6::NTRK3 translocation on clone G2. D RT-PCR time course (days) for ETV6::NTRK3 fusion after Cre expression in hES and hES-MP cells. The fusion product is detectable in cells before Cre recombination indicating a partial splicing of PGK-Puro sequence. E Western blot analysis showing the ETV6::TRKC fusion protein and the TRKC phosphorylation in hES-MP clone G2-6 and G2 after expression of Cre recombinase. The total ETV6::TRKC protein was detected by pan-TRK antibodies and the TRKC phosphorylation by phospho TRKA/B antibodies (same used for detection of LMNA::pTRKA).