LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma

Abstract

Background & aims: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity.

Methods: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy.

Results: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth.

Conclusions: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models.

Impact and implications: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Keywords: Hippo pathway; Src family kinase; bile duct tumors; tyrosine kinase inhibitor.

Fig. 1.. NTRC is a novel TKI that preferentially inhibits LCK.

(A) Chemical structure of NTRC 0652–0. (B) An in vitro screen of 150 kinases identified 12 potential substrates. (C) Kinetics, affinity, and selectivity of top substrates. (D) Global changes in tyrosine phosphorylation (pY) were assessed in HuCCT-1 cells following pharmacologic LCK inhibition (NTRC, y-axis) or genetic deletion (sgLCK, x-axis) versus vehicle control. Changes in pY with NTRC and sgLCK were highly correlated (Spearman correlation, p<1e-15). (E) MS spectra for representative significantly hypophosphorylated proteins in both NTRC-treated and sgLCK conditions.

Fig. 2.. NTRC 0652–0 inhibits YAP tyrosine phosphorylation and activity and induces apoptotic cell death.

(A) Immunoblot analysis of HuCCT-1 cells showed decreased YAP ^Y357 phosphorylation following NTRC 0652–0 treatment. (B) Immunofluorescence in HuCCT-1 cells showed nuclear to cytoplasmic redistribution of YAP following NTRC 0652–0 treatment, scale 10 μm. (C) Global TEAD-dependent transcription was decreased in HuCCT-1 reporter cells treated with Verteporfin or NTRC 0652–0. (D) Expression of canonical YAP target genes by qRT-PCR was significantly decreased following NTRC 0652–0 treatment. (E) Viability dose-response curve and calculated IC₅₀ of HuCCT-1 cells treated with NTRC 0652–0. (F) Cell death and (G) caspase 3/7 activity were increased in HuCCT-1 cells treated with NTRC 0652–0. (All panels represent N≥3 replicate experiments, significance by unpaired t-test: * p<0.05).

Fig. 3.. NTRC is cytotoxic in a subset of patient-derived organoids (PDO) with basal YAP tyrosine phosphorylation and drug-induced YAP inhibition.

(A) Derivation of PDO from primary human CCA (schema). PDO histology and phenotypic marker immunofluorescence, with expected CCA pattern (CK19⁺/HNF4a⁻/α-SMA⁻, positive controls for HNF4a and α-SMA staining included, original magnification 200x.) (B) Photomicrographs and viability dose-response curve of a representative NTRC-sensitive organoid, PDO389 (scale bar 500 μm). (C) Calculated IC₅₀ of NTRC 0652–0 in a panel of PDO models. (D) Immunoblot showed hypophosphorylation of YAP ^Y357 in NTRC-resistant PDOs. (E) YAP target gene expression was reduced in the NTRC-sensitive PDO442 following NTRC treatment (2 μM, 24 hr; N=3, unpaired t-tests, p< 0.05). (F) No change in YAP target gene expression was seen in the NTRC-resistant PDO484 (4 μM, 24 hr; N=3, unpaired t-tests, p = ns).

Fig. 4.. NTRC 0652–0 is therapeutic in an in vivo model of FGFR2-fusion CCA.

(A) Pharmacokinetic analysis of NTRC 0652–0 drug level in plasma and tumor from PDX283 mice (N=2 per dose, per time point). (B) PDX283 tumor volume was decreased in mice treated with NTRC 0652–0 at 30 or 45 mg/kg/day for 15 days versus vehicle control (N=8 per group, 2way ANOVA, multiple comparisons for each dose versus vehicle. adjusted p <0.0001 [30 mg/kg], <0.0006 [45 mg/kg]). (C) Mouse body weight (2way ANOVA, multiple comparisons, adjusted p= ns). (D) Serum liver and renal chemistries. Elevation of ALP and ALT remained below the upper limit of normal, with no change in BUN (2way ANOVA, multiple comparisons, * P <0.05). (E) Mice bearing Liv31 tumors were treated with vehicle or 30 mg/kg of NTRC 0652–0 for 21 days (N=5 per group). NTRC 0652–0 treatment significantly decreased tumor growth. (Mixed effects analysis due to missing data. One vehicle-treated mouse was sacrificed on day 16 for excess tumor growth per animal care protocols. Adjusted P-value = 0.0002). (F) Immunoblot of phospho-YAP^Y357, total YAP, and actin in PDX283 and Liv31 tumors from representative mice treated with vehicle or NTRC 0652–0 at 30 mg/kg/day. (G) YAP immunohistochemistry in Liv31 tumors (40x images, 20 μm scale bar). (H) Ki-67 and cleaved caspase 3 immunohistochemistry in Liv31 tumors (40x images, 20 μm scale bar. Positive cells per 20x field, unpaired t-test, P <0.05.)