Published OnlineFirst December 19, 2016; DOI: 10.1158/1078-0432.CCR-16-2022 Cancer Therapy: Preclinical Clinical Cancer Research Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib Jennifer E. Amengual1, Sathyen A. Prabhu1, Maximilian Lombardo1, Kelly Zullo1, Paul M. Johannet2, Yulissa Gonzalez1, Luigi Scotto1, Xavier Jirau Serrano1, Ying Wei3, Jimmy Duong3, Renu Nandakumar4, Serge Cremers4, Akanksha Verma5, Olivier Elemento5, and Owen A. O'Connor1 Abstract Purpose: Pan-class I/II histone deacetylase (HDAC) inhi- Results: Systematic incremental increases in drug exposure bitors are effective treatments for select lymphomas. led to the development of distinct resistant cell lines with IC50 Isoform-selective HDAC inhibitors are emerging as poten- values 10- to 20-fold greater than that for parental lines. GEP tially more targeted agents. ACY-1215 (ricolinostat) is a revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, first-in-class selective HDAC6 inhibitor. To better under- as well as downregulation of SH3BP5 and LCK. Gene-set stand the discrete function of HDAC6 and its role in lym- enrichment analysis (GSEA) revealed modulation of the BTK phoma, we developed a lymphoma cell line resistant to pathway.IbrutinibwasfoundtobesynergisticwithACY-1215 ACY-1215. in cell lines as well as in 3 primary patient samples of lym- Experimental Design: The diffuse large B-cell lymphoma cell phoma. In vivo confirmation of antitumor synergy was dem- line OCI-Ly10 was exposed to increasing concentrations of onstratedwithaxenograftofDLBCL. ACY-1215 over an extended period of time, leading to the Conclusions: The development of this ACY-1215–resistant development of a resistant cell line. Gene expression profiling cell line has provided valuable insights into the mechanistic (GEP) was performed to investigate differentially expressed role of HDAC6 in lymphoma and offered a novel method to genes. Combination studies of ACY-1215 and ibrutinib were identify rational synergistic drug combinations. Translation of performed in cell lines, primary human lymphoma tissue, and these findings to the clinic is underway. Clin Cancer Res; 1–13. a xenograft mouse model. Ó2016 AACR. Introduction belongs to the class 2b family of HDACs and differs from other HDACs in that it resides predominantly in the cytoplasm. Pan-class I/II histone deacetylase (HDAC) inhibitors have It is known to play a role in protein homeostasis and the proven to be successful agents for the treatment of lymphoma, unfolded protein response (UPR; refs. 6, 7). HDAC6 inhibition though their clinical application has been restricted predom- has demonstrated activity in preclinical models of lymphoma inantly to the T-cell lymphomas (1–5). While the precise role of and multiple myeloma and is presently being studied in clinical individual HDAC isoforms in lymphoma has remained an area studies both as a single agent and in combination. Although of active research, isoform-selective HDAC inhibitors have very well tolerated clinically, activity as a single agent has been begun to emerge. The first example of this is ACY-1215 (rico- limited and combination strategies have proven more effica- linostat), an isoform-selective HDAC6 inhibitor. HDAC6 cious thus far. Combinations of ACY-1215 with lenalidomide, pomalidomide, and bortezomib are presently in clinical study – 1Center for Lymphoid Malignancies, Columbia University Medical Center, New for patients with multiple myeloma (8 11). York, New York. 2Stanford University School of Medicine, Stanford, California. In an effort to gain insights into the role of HDAC6 in lym- 3Department of Biostatistics, Mailman School of Public Health, Columbia Uni- phoma and to identify novel pathways that may be synergistic versity, New York, New York. 4Division of Clinical Pathology, Department of with ACY-1215, a lymphoma cell line was developed to be Pathology and Cell Biology, Columbia University Medical Center, New York, New 5 resistant to the HDAC6-selective inhibitor ACY-1215. Drug resis- York. Institute for Computational Biomedicine, Weill Cornell Medical College, tance can be defined as intrinsic or acquired. Intrinsic drug New York, New York. resistance is often difficult to demonstrate in tissue culture, and Note: Supplementary data for this article are available at Clinical Cancer is defined as cells that harbor preexisting conditions which render Research Online (http://clincancerres.aacrjournals.org/). them unresponsive to a particular drug or drug combination. Corresponding Author: Jennifer E. Amengual, Columbia University, 51 W 51st Acquired drug resistance typically emerges in stages, and at least Street, Suite 200, New York, NY 10019. Phone: 212-326-5720; Fax: 212-326-5725; theoretically is attributed to the emergence of pathways that E-mail: [email protected] bypass the inhibition posed by a particular drug. We reasoned doi: 10.1158/1078-0432.CCR-16-2022 that if the emergence of compensatory pathways could mitigate Ó2016 American Association for Cancer Research. sensitivity to exposure of a specific drug, then such pathways www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 19, 2016; DOI: 10.1158/1078-0432.CCR-16-2022 Amengual et al. in DMSO. Deuterated internal standard ibrutinib-d5 was Translational Relevance purchased from TLC Pharmaceutical Standards Ltd. All sol- To better understand the function of HDAC6 and its role in vents and other chemicals for sample extraction were LC/MS lymphoma, we developed lymphoma cell lines resistant to the grade. selective HDAC6 inhibitor ACY-1215. ACY-1215 has demon- strated activity in preclinical models of multiple myeloma and Cell lines and culture lymphoma and is currently being studied clinically for both of Hbl-1, OCI-Ly10, and Riva are activated B-cell (ABC) DLBCL fi these diseases. Gene expression pro ling was performed on cell lines; OCI-Ly1, OCI-Ly7, Su-DHL-6 are germinal center the resistant line and compared with the parental line which (GC) DLBCL cell lines; JVM2, MAVER, Hbl-2, Jeko-1, and Rec-1 pointed to modulation of the BTK pathway revealing a poten- are mantle cell lymphoma (MCL) cell lines; HH and H9 are fi tial drug partner. Synergy was con rmed with the combination T-cell lymphoma (TCL) cell lines. Su-DHL-6, HBL-1, Riva, of ACY-1215 and ibrutinib in lymphoma cell lines, primary HBL2, Jeko-1, Rec-1, MAVER, HH, and H9 were obtained from human lymphoma tissue, and a mouse model of lymphoma. ATCC. OCI-Ly1, OCI-Ly7, and OCI-Ly10 were obtained from – The development and characterization of this ACY-1215 DSMZ. Su-DHL-6, Hbl-1, Hbl-2, HH, H9, Jeko-1, Jvm-2, resistant cell line has proven to be a valuable tool to advance MAVER, and Rec-1 were grown in RPMI (10% FBS). OCI-Ly7, our understanding of the functional role of HDAC6 in lym- OCI-Ly10, and Riva were grown in IMDM (10% FBS). All cell phoma. This strategy is a practical method to identify potential lines were authenticated and screened for mycoplasma using rational and synergistic drug-drug combinations. Translation the ATCC/Promega STR Authentication Testing Kit and Lonza fi of these ndings in lymphoma is now underway. MycoAlert for mycoplasma testing. Primary patient lymphoma samples were collected on an approved institutional review board (IRB) protocol. Peripheral blood was collected and peripheral blood mononuclear cells were extracted by Ficoll- could represent logical targets for rational drug-drug combina- Paque density gradient media and centrifugation. tions, preempting acquired drug resistance, at least in that specific context. This paradigm, if validated, could create a logic informing Cell viability assays the development of "rational" upfront combinations that could Cells (3 Â 105 cells/well) were incubated with 1:100 dilution of improve the efficacy of new drug combinations. drug (listed above) alone or in combination. Cell viability was We employed a strategy of gradual drug acclimation to identify assessed using the CellTiter-Glo Luminescent Cell Viability Assay a resistant cell line to try and capture emerging compensatory (Promega Corporation) and confirmed by Vi-Cell Series Cell pathways of resistance to ACY-1215. We conducted gene expres- Viability Analyzer (14, 15). The ACY-1215–resistant cell line was sion profiling (GEP) of the resistant line which was compared derived from the OCI-Ly10 cell line. OCI-Ly10 was exposed to with the parental line. The GEP data revealed modulation of the increasing concentrations of ACY-1215 over time. Systematic B-cell receptor (BCR) pathway, including downregulation of the incremental increases in drug concentration exposure led to the negative regulator of BTK (SH3BP5), increased FYN, and IKZF2. development of distinct cell lines with IC50 values 10- to 20-fold These observations led to systematic evaluation of the combina- greater than that for parental lines. tion of ACY-1215 with ibrutinib, a first-in-class BTK inhibitor, which demonstrated strong synergy. This synergy was demon- BH3 profiling strated across a large panel of cell lines, including ones represent- BH3 profiling was performed as described previously (16). ing DLBCL and mantle cell lymphoma (MCL), and primary Briefly, whole cells were permeabilized using the detergent human samples including chronic lymphocytic lymphoma digitonin which selectively permeabilizes the cell membrane, (CLL), lymphoplasmacytic lymphoma, and marginal zone lym- but not the mitochondrial membrane. BH3 peptides (synthe- in vivo phoma (MZL). In addition, an murine xenograft model of sized by the Tufts University Peptide Synthesis Core and fi fi DLBCL (OCI-LY10) con rmed the therapeutic bene ts of the Genscript) were administered to the cells in prescribed con- combination over the individual drugs. Interestingly, the UPR centrations. Depolarization of the mitochondrial membrane has been shown to be linked to the BCR pathway through IRE-1 potential was assessed by staining the cells with JC-1 dye in and XBP-1 (12, 13) in models of CLL. Work conducted in the 384-well, black, flat-bottom, nontreated plates.