Functional Characterization of BTK C481S Mutation That Confers
Total Page：16
File Type：pdf, Size：1020Kb
Leukemia (2015) 29, 895–900 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Functional characterization of BTKC481S mutation that confers ibrutinib resistance: exploration of alternative kinase inhibitors S Cheng1, A Guo2,PLu2,JMa1, M Coleman3 and YL Wang2 The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, has produced remarkable clinical response in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. We previously reported the identification of BTKC481S mutation in a CLL patient who progressed following 21-month ibrutinib therapy. Initial characterization at structural and biochemical levels revealed that the mutation disrupts the covalent binding of ibrutinib to BTK, reduces its binding affinity and diminishes its ability to inhibit the BTK enzymatic activity. Herein, we further characterized the functional consequences of BTKC481S in terms of molecular signaling, gene expression and cellular behavior in the patient, as well as in lymphoma cells transfected with either the wild-type or the mutant BTK constructs. Further, using an in vitro CLL proliferation model, alternative kinase inhibitors that have the potential to overcome ibrutinib resistance were explored. Leukemia (2015) 29, 895–900; doi:10.1038/leu.2014.263 INTRODUCTION MATERIALS AND METHODS Bruton tyrosine kinase (BTK) is an essential component of the early Patient, sample collection and cell isolation BCR signaling pathway1 (Supplementary Figure 1). BCR activation The patient was a 49-year-old woman when she was diagnosed with CLL in triggers the formation of a signalosome, which includes protein 2000. In October 2010, after failing multiple therapeutic regimens including γ fludarabine/cyclophosphamide/rutuximab, she was enrolled in a phase I tyrosine kinases LYN, SYK and BTK, as well as phospholipase C 2 5 and phosphoinositide 3-kinase. Within the signalosome, BTK is dose-escalation study of ibrutinib in recurrent B-cell malignancies. She was treated once daily with 560 mg ibrutinib for 21 months and achieved a phosphorylated by LYN and SYK. SYK and BTK subsequently partial response, and then developed disease progression (see Furman et al.9 phosphorylate phospholipase Cγ2, leading to calcium mobiliza- for a detailed case description). A total of four peripheral blood samples tion, activation of ERK, AKT and nuclear factor-κB, which were collected longitudinally before and after the initiation of ibrutinib: subsequently result in cell survival, cell proliferation, differentia- (1) pre-treatment (Pre-Rx); (2) day 1 of month 4, while patient was tion and antibody production.2–4 responding to ibrutinib (Responding); (3) after 20 months on treatment fi Ibrutinib (PCI-32765), an orally administered inhibitor that binds when progressive disease was rst demonstrated (Relapse s1); and (4) 4 weeks later, before dose escalation of ibrutinib to 840 mg (Relapse s2). covalently to cysteine 481 of BTK, has produced remarkable CD5+/CD19+ CLL cells were isolated from these samples as described clinical response in relapsed and refractory chronic lymphocytic previously.10,11 Briefly, cells were isolated from whole blood by negative 5,6 leukemia (CLL) and mantle cell lymphoma. The drug has been selection using RosetteSep Human B Cell Enrichment cocktail (StemCell recently approved by the US Food and Drug Administration for Technologies, Vancouver, BC, Canada) following the manufacturer's the treatment of these diseases. However, it has been reported instructions. The purity of the enriched CLL B cells was 490% by CD5/ fi that in phase I and II studies of CLL, of 246 patients who received CD19 double staining of all four samples. Aliquots of the puri ed cells were immediately cryopreserved as cell suspension in RPMI1640, 20% fetal ibrutinib, 13 patients (5.3%) have progressed following a median 7 bovine serum plus 10% dimethylsulfoxide. Other aliquots of the cells were observation of 14 months. stored at − 80 °C as cell pellets for immunoblot assays or fixed and stored C481S We and others recently reported the identification of BTK for intracellular Ki-67 staining (see below). mutation in several of these patients.8,9 In our patient, the mutation was identified using RNA-Seq and verified using both fi Immunoblotting Sanger sequencing and allele-speci c PCR. We further demon- 12–14 strated that the BTKC481S mutation disrupts the irreversible Immunoblotting was carried out as described previously. The following antibodies were purchased from Cell Signaling Technology covalent binding between the ibrutinib and BTK, and reduces (Beverly, MA, USA): anti-p-BTK (Y223), anti-pERK1/2 (T202/Y204), anti-total the binding affinity of the drug for the enzyme. The result is loss of ERK, anti-p-AKT (S473) and anti-total AKT. Anti-total BTK was obtained from inhibition of BTK enzymatic activity that ultimately causes ibrutinib BD Bioscience (San Jose, CA, USA) and anti-GAPDH from Thermo Scientific resistance in the patient.9 Herein, we further characterize the (Waltham, MA, USA). functional impact of the mutation from the perspectives of cell signaling, gene expression and cellular phenotypes. In addition, RNA-Seq and data analysis attempts were made to identify alternative kinase inhibitors to Total RNA was isolated from the purified CLL cells using QIAamp RNA help overcome ibrutinib resistance. Blood Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg of RNA was used for 1Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA; 2Department of Pathology, University of Chicago, Chicago, IL, USA and 3Department of Medicine, Weill Cornell Medical College, New York, NY, USA. Correspondence: Dr YL Wang, Department of Pathology, University of Chicago, MC1089, N316, 5841South Maryland Avenue, Chicago, IL 60637, USA. E-mail: [email protected] Received 8 April 2014; revised 27 August 2014; accepted 29 August 2014; accepted article preview online 5 September 2014; advance online publication, 10 October 2014 Functional characterization of BTKC481S mutation S Cheng et al 896 poly-A RNA selection with oligo (dT) beads. The enriched mRNA was used variety of drugs. After 10 days of culture, the CLL cells were harvested and for the construction of cDNA libraries. RNA sequencing was carried out in BrdU incorporation was determined with FITC BrdU Flow Kit following the an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA). Paired- manufacturer’s instructions (BD). The stained CLL cells were counted in an end sequencing was conducted with each sample running in a separate LSR2 cytometer and data were analyzed with FlowJo software (Tree Star, sequencing lane. More than 100 millions reads were yielded with an Ashland, OR, USA). average of 88x coverage per sample. The reads were aligned to the HG19 genome assembly using TopHat package with Bowtie2 aligner. Reads not mapped to the genome or siRNA-mediated BTK knockdown fl potential PCR duplicates were excluded with samtools. Cuf inks and The small interfering RNA (siRNA) against human BTK was customized and Cuffmerge were used to quantify levels of transcript expression. Reads per purchased from Thermo Scientific. The sense strand sequence of BTK kilobase per million mapped reads were computed: the number of reads siRNA was: 5′-GUAUGAGUAUGACUUUGAAUU-3′, and the antisense mapped to each transcript sequence was normalized by the template sequence was: 5′-UUCAAAGUCAUACUCAUAGUU-3′. A non-targeting siRNA length in kbs and divided by the number of reads mapping to the whole transcriptome. Hierarchical cluster analysis for BCR signature genes was with scrambled sequence was included as the negative control. siRNA transfection were performed with the Amaxa Nucleofector II device performed using Cluster 3.0 software, and heat map was generated using 7 TreeView. The 27-gene BCR signature was previously established by (Amaxa), and 1 × 10 CLL cells were resuspended in Amaxa Solution V Herishanu et al.15 containing 1 μM nontarget control siRNA or siBTK (Life Technologies, Carlsbad, CA, USA). Nucleofection was performed using the Program U-015. The knockdown efficiency was assessed by western blots and Cell transfection shown in Supplementary Figure 2. After transfection, cells were plated in a HBL1 cells were seeded overnight and transfected with a pCMV6 vector 24-well plate for 8 days in 1 ml of RPMI/10% fetal calf serum, CpG C481S that expresses either WT BTK or BTK mutant using the Amaxa oligodeoxynucleotides (dsp30), CD40L and BrdU before cells were Nucleofection technology according to the manufacturer's protocols collected for the BrdU incorporation assay. (Amaxa, Cologne, Germany; Program U-13). The cells were incubated for 12 h, and ibrutinib or vehicle was then added. Cells were continuously cultured for 12 h before cell harvest. Numbers of viable HBL1 cells were Statistical analysis counted with propidium iodide exclusion following periods of time One-way analysis of variance was applied to determine the differences in indicated in Figure 1d. the levels of the 27-gene BCR signature15 among four samples. CLL proliferation assays To measure Ki-67 positivity, the fixed and cryopreserved cells were thawed RESULTS at room temperature, washed with 1x PBS once and permeabilized with C481S 50% methanol. The cells were then stained with PE-Cy5.5-conjugated anti- Functional consequences of the BTK mutation on BCR Ki-67 (BD). The Ki-67 gate was set based on the study of 14 pre-treatment molecular signaling samples collected from patients who participated in the trial. Ki-67 9 Using the serial samples collected from the patient, we analyzed positivity in these 14 patients ranged from 0.33 to 14%. This range was the consequences of the BTKC481S mutation on BCR molecular made consistent with that of Ki-67 positivity (0.1–13.8%) reported previously in the peripheral bloods of 95 CLL patients.16 signaling. As BTK autophosophorylation at Y223 results in kinase For bromodeoxyuridine (BrdU) incorporation assay, 4 × 104 NK-tert, a activation, the level of p-BTK (Y223) was measured by immuno- human bone marrow-derived stroma cell line, was seeded for 6 h in blotting as a surrogate marker for the BTK enzymatic activity.