Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer

Abstract

Objective: Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited.

Design: To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets.

Results: By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer.

Conclusions: BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453).

Trial registration number: NCT02884453; Pre-results.

Keywords: Molecular Biology; Oesophageal Cancer.

Conflict of interest statement

Competing interests: IYC has received research funding from Janssen and Pharmacyclics as part of a sponsored research agreement. IC accepts research funding from Janssen-Cilag.

© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Figures

Figure 1

Identifying candidate genetic dependencies in oesophageal tumour cell lines. (A) Schematic of compilation of genomic profiles of 17 oesophageal tumour-derived cell lines using array comparative genomic hybridisation and exome sequencing. Oesophageal tumour cell lines were classified according to histology (SCC vs EAC) and cancer driver alterations. The 17 oesophageal cancer cell lines were screened in 384-well plate format using a small molecule drug library of compounds in late drug development or clinical trials. These drug data and high-throughput siRNA screening data (714 siRNA targeting kinase and kinase related genes26) from the same cell lines were interrogated and integrated with histological and cancer driver alterations to identify drug or siRNA sensitivities associated with SCC or EAC histology and cancer driver mutations. (B) Graph annotating predicted DNA coding mutations and copy number alterations present in two or more of 17 oesophageal cancer cell lines (8 EAC and 9 SCC) that are also to be found recurrently mutated in primary oesophageal tumours. aCGH, array comparative genomic hybridisation; EAC, oesophageal adenocarcinoma; SCC, squamous cell carcinoma.

Figure 2

Small molecule drug sensitivity profiling and integration with genomic profiles of oesophageal tumour cell lines. (A) Heatmap displaying AUC measurements for each of the 80 small molecule inhibitors in the high-throughput drug screen and for each of the oesophageal cancer cell lines that passed quality control. Box and whisker plots of AUC values illustrating preferential sensitivity of SCC oesophageal cell lines to (B) dasatinib (p=0.032, MP test), (C) BI-2536 (p=0.033, MP test), (D) gefitinib (p=0.034, MP test) and (E) canertinib (p=0.038, MP test). (F) Box and whisker plots of AUC values illustrating preferential sensitivity of ERBB2 and EGFR amplified oesophageal cancer cell lines to lapatinib (p=0.038, Mann-Whitney U test). (G) Box and whisker plots of AUC values illustrating preferential sensitivity of oesophageal tumour cell lines harbouring PIK3CA mutations and BEZ235 (p=0.018, MP test) and (H) vinorelbine (p=0.032, MP test). AUC, area under the curve; MP, median permutation; SCC, squamous cell carcinoma.

Figure 3

Integration of genomic profiles with siRNA data. (A) Radar plot showing key genetic dependencies associated with oesophageal tumour cell lines compared with tumour cell lines from other histologies. The concentric circles indicate the degree of statistical significance and depth of colour indicates separation of Z scores. Radar plots showing key genetic dependencies associated with (B) oesophageal SCC histology and (C) EAC histology. (D) Radar plot showing key genetic dependencies associated with ERBB2 amplification. Box and whisker plots of Z score values showing that targeting of (E) ERBB2 (p=0.019, MP test) and (F) MAP2K3 (p=0.0082, MP test) is selectively lethal in ERBB2 amplified oesophageal cancer cell lines. (G) Radar plot showing key genetic dependencies associated with MYC amplification. (H) Box and whisker plots of Z score values showing that targeting of BTK (p=0.017, MP test) and (I) ALK (p=0.008, MP test) is selectively lethal in MYC amplified oesophageal cell lines. (J) Radar plot showing key genetic dependencies associated with copy number deletions in SMARCA4 in oesophageal cancer cell lines. (K) Box and whisker plots of Z score values showing that targeting BRD4 (p=0.022, MP test) is selectively lethal in oesophageal tumour cell lines harbouring SMARCA4 copy number loss. (L) Radar plot showing key genetic dependencies associated with CCND1 amplification. (M) Box and whisker plots of Z score values showing that targeting ROCK1 (p=0.0056, MP test) is selectively lethal in CCND1 amplified oesophageal cancer cell lines. EAC, oesophageal adenocarcinoma; MP, median permutation; SCC, squamous cell carcinoma.

Figure 4

BTK expression in oesophageal tumour cell lines. (A) Western blot showing expression of BTK across a panel of oesophageal cancer cell lines. (B) Graph illustrating BTK mRNA expression relative to TE10. (C) Agarose gel electrophoresis of BTK-C PCR products showing presence of the BTK-C splice variant in a panel of oesophageal cancer cell lines. (D) Graph illustrating BTK-C mRNA expression relative to KYSE70. (E) Bar chart showing decreased cell viability in parental TE8 after transfection with individual siBTK oligonucleotides compared with siAllstar. Normalised percentage inhibition = (negative control median − cell viability value) / (negative control median − positive control). (F) Bar chart showing decreased cell viability in TE8 with BTK overexpression after transfection with individual siBTK oligonucleotides compared with siAllstar. (G) Western blot showing decreased BTK protein expression following transfection with individual siBTK in TE8 with BTK overexpression (lentiviral transduction). (H) Bar chart showing decreased cell viability in TE8 after transfection with siBTK-C oligonucleotides. (I) Bar chart showing decreased BTK-C mRNA expression following transfection with siBTK-C oligonucleotides.

Figure 5

Ibrutinib sensitivity in MYC and/or HER2 amplified oesophageal tumour cell lines. (A) Box plot depicting area under the curve values for ibrutinib. Preferential sensitivity to ibrutinib is observed in oesophageal cancer cell lines harbouring MYC and/or ERBB2 amplification. (B) Ibrutinib drug sensitivity curves across the panel of oesophageal tumour cell lines. Cell lines that are not MYC or ERBB2 amplified are depicted in black, MYC and ERBB2 amplified in purple, MYC amplified alone in blue and ERBB2 amplified alone in red. (C) Western blots showing decreased expression of cMYC in MYC amplified KYAE-1 tumour cell line after exposure to 1 µM ibrutinib. (D) Western blot showing decreased expression of cMYC in MYC amplified TE8 tumour cell line after exposure to 1 µM ibrutinib. (E) Bar chart showing decreased cell viability in KYAE-1 (MYC and ERBB2 amplified) following transfection with siMYC oligonucleotides. (F) Western blot showing decreased protein expression of cMYC following transfection of siMYC oligonucleotides in KYAE-1 (MYC and ERBB2 amplified). (G) Cell cycle analysis after 24 and 48 hours of ibrutinib exposure in KYAE-1 (MYC and ERBB2 amplified), (H) TE8 (MYC amplified) and (I) JHEsoAd1 (MYC and ERBB2 non-amplified) tumour cell lines. Prolongation of the G1 phase is observed with increasing doses of ibrutinib in cell line harbouring MYC amplification. G1 arrest was not observed in JHEsoAd1 (MYC and ERBB2 non-amplified) cell line. (J) Western blot showing decreased expression of p-Rb, Rb and cyclin D1 following ibrutinib exposure in KYAE-1 (MYC and ERBB2 amplified). (K) Western blot showing decreased expression of cyclin D1 following ibrutinib exposure in TE8 (MYC amplified). (L) Western blot showing no change in protein expression of p-Rb, Rb and cyclin D1 following ibrutinib exposure in JHEsoAd1 (MYC and ERBB2 non amplified). (M) Bar chart showing the apoptotic cell fraction (percentage of annexin-positive cells) after exposure to increasing doses of ibrutinib (24 and 48 hours). An increase in apoptosis is observed in KYAE-1 (MYC and ERBB2 amplified) that is not seen in JHEsoAd1 (MYC and ERBB2 non-amplified). (N) Western blot showing expression of cleaved PARP1 after ibrutinib exposure in the KYAE-1 (MYC and ERBB2 amplified). Cleaved PARP1 is not seen in JHEsoAd1 after ibrutinib exposure (MYC and ERBB2 non-amplified).

Figure 6

ERK1/2 is downregulated by ibrutinib. (A) Western blot showing decreased p-BTK protein expression in BTK overexpressed (lentiviral transduction) TE8 cell line (MYC amplified) following ibrutinib exposure. (B) Western blot showing decreased p-ERK protein expression in KYAE-1 tumour cell line (MYC and ERBB2 amplified) and (C) TE8 cell line (MYC amplified) following ibrutinib exposure. (D) Western blots showing that ibrutinib exposure results in downregulation of p-MYC S62 and total cMYC in KYAE-1 (MYC and ERBB2 amplified) and (E) TE8 (MYC amplified) oesophageal tumour cell lines.