Isocitrate Dehydrogenase Mutations Confer Dasatinib Hypersensitivity and SRC Dependence in Intrahepatic Cholangiocarcinoma

Abstract

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant "gatekeeper" mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.

Significance: IDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727-39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.

Figure 1. Isocitrate dehydrogenase (IDH) mutant intrahepatic cholangiocarcinoma (ICC) cells are hypersensitive to dasatinib

A. Schematic of the high-throughput drug screen protocol. 15 IDH wild-type (WT) biliary tract cancer (BTC) and two IDH mutant (IDHm) ICC cell lines were screened across 122 approved or advanced clinical compounds at nine different doses. Viability was quantified at 72 h and the IC50 estimated for each compound and cell line. B. Heat map illustrating the median-centered Ln(IC50) of 17 BTC cell lines screened across 122 clinically relevant compounds. Note: the two IDH mutant (IDHm) ICC lines segregate together in unbiased hierarchical clustering. C. Relative sensitivity (y-axis natural log scale, 0 = median Ln(IC50) across all BTC tested) of two IDHm ICC lines to 122 individual drugs (ranked by average sensitivity of IDHm ICC, x-axis). Note: Dasatinib demonstrates the greatest selective activity against IDHm ICC among drugs screened. D. Sensitivity of 885 cancer cell lines to dasatinib (x-axis) and saracatinib (y-axis), each represented by an individual dot. IDHm ICC lines, RBE (IDH1 R132S) and SNU-1079 (IDH1 R132C) are the two larger red dots. IDHm non-BTC cell lines, HT1080 (IDH1 R132C) and COR-L105 (IDH1 R132C), are represented by purple dots. Drug response is presented as the natural logarithm of the IC50 in μM.

Figure 2. In vitro and In vivo hypersensitivity of IDHm ICC to dasatinib

A–C. Proliferation curves of established human (A), novel human (B) and murine (C) IDHm (red) and WT (black) ICC lines and MMNK-1 cells treated with increasing doses of the tyrosine kinase inhibitors, dasatinib, saracatinib, bosutinib and ponatinib. D, E. Tumors arising from subcutaneously implanted murine IDHm ICC (SS49) cells were treated with either vehicle control or dasatinib 50 mg/kg daily by oral gavage. D. Serial tumor size measurements. E. Histologic analysis and immunostaining at the indicated time points revealed that dasatinib treatment causes widespread necrosis and activation of apoptotic markers. Top panels: H&E stain; Bottom panels: immunohistochemistry for cleaved-caspase 3; inset: quantification of % necrotic tumor for vehicle (n = 3) or dasatinib treatment (n = 7 at 2 days, n = 5 at 14 days). Scale bars = 50 μm. F. Histologic analysis (H&E) of an IDH1 R132C ICC patient-derived xenograft (PDX) treated with vehicle control or dasatinib 50mg/kg daily by oral gavage for seven days. Right panel: quantification of % necrotic tumor for vehicle (n = 5) and dasatinib treatment (n = 5). Scale bars = 1 mm (low power images) or 20μm (high power images). *p < 0.05, **p < 0.01, ****p<0.0001.

Figure 3. Identification of dasatinib targets in IDHm ICC

A. Immunoblot demonstrating that dasatinib causes loss of phospho-p70 S6 Kinase (Thr389) and phospho-S6 (Ser235/236) specifically in IDHm ICC cells. Neither IDHm nor IDH WT ICC show dasatinib-induced changes in phospho-ERK1/2 (Thr202/Tyr204), phosph-STAT3 (Ser727), BCL2, or MCL1. B. Schematic illustrating methods used to characterize dynamic changes in the “active kinome” of ICC cells treated with dasatinib. IDH WT or IDHm ICC lines were treated with dasatinib (20 nM) or vehicle control for one hour prior to harvest. Whole cell lysates were run over a multiplexed kinase inhibitor bead (MIB) column containing a panel of sepharose beads covalently linked to 12 kinase inhibitors with distinct specificity profiles. Due to their preferential binding to active kinases, inactive kinases are discarded in the flow-through while active kinases were isolated in the eluent. The eluent was then subjected to tryptic digestion and peptide identification through liquid chromatography-tandem mass spectrometry (LC-MS/MS). To identify active kinases potently inhibited by dasatinib in each cell line, the active kinome of dasatinib-treated cells was compared to that of vehicle control. C. Heat map of kinases enriched in the active kinome of dasatinib-treated cells compared to vehicle control cells (log₂ ratio is shown). Kinases that are active at baseline, inhibited >75% by dasatinib, and are common to both IDHm ICC are in blue.

Figure 4. SRC is a critical dasatinib target in IDHm ICC

A. Schematic for the introduction of “gatekeeper” mutants into the endogenous loci encoding dasatinib targets in IDHm ICC cells. Plasmids containing Cas9 and a single guide RNA (sgRNA) targeting each individual kinase were co-transfected with a donor oligonucleotide encoding the gatekeeper mutation for each kinase. The gatekeeper mutation prevents the endogenously-expressed kinase from binding to and being inhibited by dasatinib, thus allowing dasatinib targets to remain active. Cells were then plated at confluency and treated with dasatinib 50 nM for 30 days in a six well plate. B. Crystal violet staining of viable cells treated as in (A), demonstrating that introduction of the SRC T341I gatekeeper mutation rescues SNU-1079 cells from dasatinib-induced cytotoxicity. C. Crystal violet stain of SNU-1079 parental cells and cells harboring an endogenous SRC-T341I mutation following treatment with increasing doses of dasatinib or DMSO control (0 nM) for 24 h. D. Caspase 3/7 activity of HuCCT1 cells, SNU-1079 parental cells and SNU-1079-SRC T341I cells treated with dasatinib 100 nM for 24 h relative to DMSO control. **p<0.01. E. Dasatinib treatment (50 nM) for 2 h causes loss of phospho-SRC (Tyr416) in parental SNU-1079 cells but not in SNU-1079-SRC T341I cells as shown by immunoblot of insoluble fractions. F. Proliferation curves of RBE parental (black) or SRC-T341I (red) treated with increasing doses of dasatinib, saracatinib, bosutinib and ponatinib. G. Immunoblot of lysates from RBE parental and SRC-T341I cells showing that the SRC gatekeeper mutation rescues phospho-p70 S6 Kinase (Thr389) and phospho-S6 (Ser235/236) levels in dasatinib-treated cells.