Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib Jennifer E

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. 2016 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 2016 American Association for Cancer Research. sensitivity to exposure of a specific drug, then such pathways

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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 demonstrated 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 (3 105 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. Following current manuscript further substantiates this link as a potential JC-1 exposure, the JC-1 emission was read on a GloMax mechanism in models of DLBCL, MCL, and MZL. We believe (Promega) plate reader once every 5 minutes for 180 minutes fi these ndings add credence to the idea that understanding mecha- to generate a kinetic trace of mitochondrial membrane poten- nisms of acquired drug resistance could lead to the development tial over time. of rational drug-drug combinations with novel agents now emerging in the clinic. The findings of these series of experiments lay the foundation for clinical studies of ACY-1215 and ibrutinib Flow cytometry/JC-1 fl in B-cell lymphoid malignancies. FACSCalibur System was used to acquire the uorescence signals (1 105 events/sample); data was analyzed using Flowjo 8.8.6. Cells (3 105/mL) were quantitated for apopto- Materials and Methods sis using Alex Fluor 488/Annexin V (Dead cell apoptosis kit Drugs and reagents Invitrogen #V13240) or for the determination of the trans- ACY-1215 was provided by Acetylon Pharmaceuticals, Inc.. membrane mitochondrial membrane potential (Dym) via Bortezomib, ibrutinib, romidepsin, verapamil, and vorinostat staining with JC-1 1.3 ug/mL JC-1 dye (Invitrogen) as described were obtained from Selleck Chemicals. All drugs were diluted previously (14).

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Western blotting tant or parental) in 50% Matrigel (BD Biosciences) were subcu- Western blotting was performed as described previously (14). taneously injected into the flanks of 5- to 7-week-old beige/SCID Antibodies used were as follows: anti-acetylated lysine, anti- mice (Taconic Farms, Inc). Treatment was initiated when tumor GRP78/BiP, anti-HDAC6, anti-HDAC9, anti-PERK and p-PERK, volume measured 80 mm3. Tumor volume was assessed using the anti-p-eif2a, anti-IRE1a,, anti-FYN, anti-eif2a, anti-p-eif2a, two largest perpendicular axes (l ¼ length; w ¼ width) and anti-BTK, anti-p-BTK, anti-PCL-g2, anti-p-PCL-g2, anti-CARD11, calculated using the formula v ¼ 0.5(l2 w). Mice were divided anti-ATF4, anti-AKT, anti-p-AKT, anti-CHOP, anti-Helios, anti- into 4 cohorts of 8–10 mice per cohort as follows: (i) untreated b-actin, anti-BIM (C23C5), anti-Bcl2 (Cell Signaling Technolo- control; (ii) ACY-1215: 50 mg/kg days 1–5, 8–12, 15–19; (iii) gy); anti-MAPK10, anti-p-IRE1a and anti-SH3BP5 (Abcam); ibrutinib: 3 mg/kg days 1–20; (iv) ACY-1215 plus ibrutinib. Drugs anti-XBP-1 (Santa Cruz Biotechnology). Densitometry analysis were diluted in sterile dextrose 5% in water and were administered was performed on scanned images using ImageJ software (NIH, via the intraperitoneal (IP) route (20). Mice were assessed for Bethesda, MD). weight loss and tumor volume 3/week. Animals were sacrificed when the tumor volume exceeded 2,000 mm3 or after sustain- Gene expression profiling ed loss of >10% body weight in accordance with institutional RNA was extracted from cells using the RNeasy minikit guidelines. (Qiagen). RNA was quantified and assessed for integrity via Bioanalyzer 2100 (Agilent) for an RNA Integrity Number great- Pharmacokinetic/pharmacodynamic in vivo studies er than 8 for RNA sequencing by the Columbia Genome Center. Mice were studied for pharmacokinetic and pharmacodynamic mRNA was enriched from total RNA samples using poly-A pull effects of ACY-1215 and ibrutinib. Approximately 250 mLof down and libraries were prepared using the TruSeq RNA prep blood was collected by submandibular vein bleed at 0.5, 1, and kit (Illumina). Sample-derived libraries were then sequenced 2 hours after treatment. Mice were sacrificed at 4, 6, and 8 hours using the HiSeq2500 platform (Illumina). after treatment; blood and tumor tissues were collected for mea- Base calling was performed using Real Time Analysis (Illumina) surement of drug concentration and Western blot analysis. and bcl2fastq(v 1.8.4) for converting BCL to fastq format. Reads were then mapped to a reference genome (Human:NCBI/build LC/MS-MS analysis for quantitation of ACY-1215 and ibrutinib 37.2) using TopHat (v2.0.4). Relative abundance was estimated ACY1215 and ibrutinib were measured in mouse serum and fl using Cuf inks (version 2.0.2). Differentially expressed genes tumor tissues using ultra performance LC/MS-MS after liquid– were tested for using the R package DESeq2. liquid extraction using deuterated ibrutinib as internal stan- Gene set enrichment analysis was performed using a preranked dard. Serum samples (100 mL) or aqueous tissue homogenate tool from the Broad Institute's GSEA software (17). The metric of containing 10 mg wet tissue disrupted by a Tissue Tearer P log10 of the value sign of the log fold change was used to rank homogenizer were spiked with deuterated internal standard at the genes. Pathway analysis on this preranked list of genes was a level of 50 ng/mL. This was mixed with 100 mL of acetonitrile, then performed using Gene Ontology and Lymphoid biology vortexed, and incubated for 10 minutes followed by addition of gene sets from the Staudt lab (18). 750 mL of methyl tert-butyl ether. The mixture was vortexed for 10 minutes, centrifuged and the organic layer was transferred Semi-quantitative PCR to a LC-MS vial, evaporated under nitrogen stream and resus- Semi-quantitative Multiplex RT-PCR was performed as pended in 50% methanol. Calibration standards and quality described previously (19). Reverse transcription (RT) was per- control (QC) samples for both the compounds were prepared formed in a 20 mL reaction system with a total of 2 mg of RNase spanning a range of 0.5 ng/mL to 1,000 ng/mL and extracted free DNase treated RNA (Omniscript RT kit, Qiagen) and oligo-d same as the samples. (T). Multiplex PCR reactions were run in 30 cycles. Primers LC/MS-MS analysis was performed on a platform compris- (Thermo Fisher Scientific) were designed to span multiple exons ing Agilent 1290 Infinity UHPLC integrated to Agilent 6410 of the target genes. Primer sequences can be found in Table 1. triple quad mass spectrometer controlled by MassHunter v 3.1 Images were analyzed using the ImageJ software (NIH, Bethesda, (Agilent Technologies). Chromatographic separation was per- MD) and band densities were normalized to GAPDH. formed on an Agilent Poroshell C18 column (50 2.1 mm, 2.7u, 100A) maintained at 40C. The flow rate was maintained In vivo studies at 500 mL/minute. The initial flow conditions were 50% solvent Animals were housed and maintained in accordance with an A (water containing 0.1% formic acid) and 50% solvent B IUCAC-approved protocol. OCI-Ly10 cells (1 107; either resis- (methanol with 0.1% formic acid). Solvent B was raised to

Table 1. Primers used for semiquantitative RT-PCR Gene Forward Reverse MAPK10 5'-CGGACTCCGAGCACAATAAA-3' 5'-GGAGCAGGTAGATGCAAGATAG-3' SH3BP5 5'-ATGAGGGTAGGAGGGACTTATAG-3' 5'-CGTCACCACCTCGTCTTTATC-3' FYN 5'-GGATTGGCCCGATTGATAGA-3' 5'-GGTTTCACTCTCGCGGATAAG-3' HDAC9 5'-GAGGCAAGAACAGGAAGTAGAG-3' 5'-GGATGGAGATGTTCCACTAAGG-3' IKZF2 5'-CACCCAACCACTCCCAAATA-3' 5'-CTGAGCCATATCCACCTCTTTC-3' HDAC6 5'-AGTGGCCGCATTATCCTTATC-3' 5'-GAGGGTCCTTCTCTGTCTTCTA-3' LCK 5'-CTACGGGACATTCACCATCAA-3' 5'-CTCTCTGTGGTCTCAGGAAATG-3' GAPDH 5'- - 3' 5' - - 3'

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70% over 1.75 minutes and to 95% by 1.95 minutes, held until and was not overcome by inhibition of efflux pumps as deter- 3.5 minutes and back to initial conditions by 4 minutes with a mined via verapamil co-exposure (Fig. 1A; ref. 22). Interest- total run time of 6 minutes. The retention time for ACY-1215 ingly, the resistant line was cross resistant to vorinostat which and ibrutinib was 1.34 and 2.08 minutes, respectively. The strongly inhibits HDAC6 (R10 IC50 ¼ not reached vs. P IC50 ¼ mass spectrometer was operated under multiple reaction mon- 0.6 mmol/L). Treatment of the resistant line with bortezomib itoring (MRM) mode with positive electrospray ionization. For demonstrated intermediate activity underscoring the notion MRM, following transitions were utilized for quantitation: that although their molecular targets are vastly different (i.e., ACY1215 434.2>274.0; ibrutinib 441.2>304.1 and ibruti- proteasome vs. HDAC6), both drug mechanisms converge on nib-d5 446.2>309.1. Mass spectrometer was operated using the processing of misfolded proteins (R10 IC50 ¼ 8nmol/L the following parameters: gas temperature, 300 C; gas flow, vs. P IC50 ¼ 3.3 nmol/L). In contrast, the resistant line was 13 L/minute; nebulizer, 30 psi; capillary 3 Kv; desolvation gas sensitive to romidepsin which is known to predominantly in- flow, 500 L/hour; cone gas flow, 50 L/hour and collision hibit HDAC1, 2, and 3 with minimal activity against HDAC6 energy, 30 v. (R10 IC50 ¼ 3 mmol/L vs. P IC50 ¼ 2.25 mmol/L; Fig. 1B; ref. 23). Lower limit of quantification (LLOQ), defined as the lowest Both the resistant and parental LY10 cell lines were xenografted concentration with an accuracy and precision of <20% was into the flanks of SCID beige mice at 107 cells. Mice were divided determined to be 0.5 ng/mL for ACY1215 and 2.5 ng/mL for into 4 cohorts, resistant (R) control, parental (P) control, (R) ACY- ibrutinib in serum. In tumor samples, the LLOQ was determined 1215, (P) ACY-1215. Mice were treated with ACY-1215 50 mg/kg to be 1 ng/mL for both ACY1215 and 2.5 ng/mL for ibrutinib. The days 1–5, 8–12, 15–19 by intraperitoneal route. The tumor intra-assay accuracy and precision for ACY1215 was 96.1% and volume doubling time was calculated for each cohort using 1.32%, respectively, while for ibrutinib, the intra-assay accuracy GraphPad Prism software. The resistant cohorts exhibited accel- was 99.6% with a precision of 1.72%. The assay showed an inter- erated tumor growth and decreased survival demonstrating that assay precision for ACY1215 0.90% and for ibrutinib 1.52%. the resistant line maintains a highly aggressive phenotype over time in vivo. Treating the resistant mouse with ACY-1215 had Statistical analysis no impact on the tumor doubling time as compared with the For determination of the inhibitory concentration of 50% of untreated resistant control mice (6.857 vs. 7.004 days). In addi- cells (IC50) and synergy, all experiments were run in triplicate tion, the rate of growth was greater in the resistant control mouse and repeated at least twice. IC50 was calculated with Calcusyn cohort as compared with the parental control cohort (7.004 vs. software (Biosoft). Relative risk ratio (RRR) was used as a 8.039 days to doubling). The volumetric doubling time of (P) model for establishing synergy between 2 drugs (21). RRR is ACY-1215 treated tumors was more than twice as long compared based on calculating the ratio between the actual value and with those of the (R) line treated with ACY-1215 (15.62 vs. 6.857 expected value (EV). In the case of two cytotoxic compounds, days; Fig. 1C). This translated into a shorter survival for both the EV is calculated by the formula: EV ¼ (NA XNB)/100, where NA resistant cohorts as compared with parental cohorts. Median represents the percentage of viable cells treated with drug A and survival in days was as follows for each cohort: resistant control NB represents the percentage of viable cells treated with drug B. (26.5) < resistant ACY-1215 (38) < parental control (43.5) < RRR<1 represents a synergistic effect; values equal to 1 indicate parental ACY-1215 (54; Fig. 1D). the mean additive effect; and values>1 represent an antagonis- tic effect. Flow cytometry assays were performed in duplicate, The IRE-1/XBP-1 pathway is upregulated in cells resistant to repeated at least twice, and reportedasthemeanwithassoci- ACY-1215 ated SDs. To evaluate for intrinsic drug resistance, basal levels of Bcl2 In vivo statistical analysis was performed using Prism Graph- and Bim were correlated with the IC50 of a panel of lymphoma Pad's two-way ANOVA Analysis. Overall survival (OS) was esti- cell lines. It has been demonstrated by others that decreased mated using the Kaplan–Meier method, and presented as the levels of Bim and increased levels of Bcl2 correlate with drug mean OS with 95% confidence intervals. Area under the serum resistance (24, 25). The Bcl2-Bim ratio correlated to sensitivity concentration time curve (AUC) and half-life were determined to ACY-1215 and cell lines with relatively high levels of Bcl2 noncompartmentally using Phoenix Winnonlin software version and low levels of Bim were relatively resistant to ACY-1215 6.3 (Certara). All drug concentrations are represented as the mean (Fig. 2A). Next we investigated whether resistance to apopto- with the SD where applicable. sis in the resistant cell line was due to alterations in the Bcl2 family proteins, which are known to play critical roles in regulating apoptosis. Mitochondrial membrane depolarization Results was evaluated following treatment with ACY-1215 in parental The development of an ACY-1215–resistant cell line is stable and resistant cells to in an effort to study changes following and not dependent on efflux pumps acquired drug resistance. Compared with the parental line, the The diffuse large B-cell lymphoma cell line OCI-Ly10 was resistant line did not depolarize the mitochondrial membrane exposed to increasing concentrations of ACY-1215 over the following treatment with ACY-1215 2.5 mmol/L, confirming course of 1 year. Systematic incremental increases in drug resistance to induction of apoptosis (Fig. 2B). BH3 profiling is a exposure led to the development of a distinct cell line with functional assay which informs the cellular dependence on an IC50 value 10- to 20-fold greater than that of the parental antiapoptotic proteins for evasion of cell death (26). There line. The resistant R10-OCI-LY10 (R10) exhibited an IC50 of was no difference in the BH3 profilesoftheresistantand 10 mmol/L as compared with parental IC50 of 0.9 mmol/L after parental cell lines (Fig. 2C). Taken together, these findings 48 hours of exposure (Fig. 1A). Resistance was maintained conclude that functional alterations in the Bcl2 family of after repeated passages for 1 month in the absence of drug proteins do not contribute to resistance to ACY-1215.

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Vorinostat ACY-1215 100 A 100 48 h LY10R B 72 h LY10R 80

80 48 h LY10 RP

60 72 h LY10P 60 48 h LY10R 40 40 72 h LY10R 20 20 48 h LY10P 72 h LY10P Cell viability (%) 0 0 0 5 10 15 20 0 0.5 1 1.5 Concentraon (mmol/L) Concentraon (μmol/L)

R10 – OCI-LY10 Bortezomib 100 100 48 h LY10R 80 80 72 h LY10R 48 h LY10P 60 60 - 72 h LY10P 40 R10 40 - 20 R10 passaged 1 month 20 Cell viability (%) without drug 0 0 0 5 10 15 20 0 5 10 15 Concentraon (mmol/L) Concentraon (nmol/L)

R10 – OCI-LY10 Romidepsin 100 100 - ACY-1215 80 80 - ACY-1215 plus Verapamil 60 60 48 h LY10R 40 40 72 h LY10R 20 48 h LY10P 20 72 h LY10P Cell viability (%) 0 0 0 2 4 0 5 10 15 20 Concentraon (nmol/L) Concentraon (mmol/L)

C OCI-LY-10 Parental vs. resistant D OCI-LY-10 Parental vs. resistant 2,000 (R) Control 100

) (R) ACY-1215 3 1,500 (P) Control (R) Control (P) ACY-1215 (R) ACY-1215 * 1,000 (P) Control 50 ** (P) ACY-1215

500 Percent survival P = 0.0383

Tumor volume (mm Tumor P = 0.0046

0 0 0 10 20 30 40 0 20 40 60 80 Day Day

Figure 1. Development and characterization of selective HDAC6 inhibitor resistant cell line. The DLBCL cell line, OCI-LY10, was exposed to increasing concentrations of ACY-1215 over time. A, Concentration–effect relationships were established for resistant and parental Ly10 at 48 and 72 hours following exposure to ACY-1215. Forty-eight–hour concentration–effect relationship of increasing concentrations of ACY-1215 in the resistant line after immediate exposure and after a wash-out period of 1 month. Concentration–effect relationship was determined with ACY-1215 alone or in combination with verapamil 20 mmol/L to inhibit efﬂux pumps. B, Concentration–effect relationships were established for resistant and parental Ly10 at 48 and 72 hours following exposure to vorinostat, bortezomib, and romidepsin. C, SCID-beige mice were injected with LY10 107 in their ﬂanks and treated with ACY-1215 50 mg/kg days 1–5, 8–12, 15–19 via the intraperitoneal route. D, Kaplan–Meier Curve was calculated for the resistant control mice as compared with the resistant ACY-1215 mice, parental control, and treatment cohorts.

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DLBCL-ABC MCL TCL A D Parental Resistant HDAC6

OCI-LY10 Hbl1 Jym2 Maver H9 HH Ac-Tubulin

Bcl2 GRP78

Bim PERK

β-Acn p-EIF-2α 1,000 EIF-2α 100 ATF4

10 PERK Pathway MAPK10 1

0.1 p-IRE-1

0.01 Rao Bcl2 : BIM IRE-1 XBP-1 0.001

IC50 (mmol/L): 0.9 1.6 4.0 0.9 1.2 2.5 p-AKT (S473) IRE-1 Pathway Parental Resistance AKT B β-Acn

Untreated

(%) P- P- R- R- Untreated Treated Untreated Treated Live 74 35 69 65 Apoptoc 26 65 28 26 Treated*

C Ly10 Parental Ly10 Resistant 300,000 300,000

200,000 200,000

100,000 100,000 Intensity (RFU) Intensity (RFU) 0 0

Figure 2. The IRE-1/XBP-1 pathway is upregulated in the cells resistant to ACY-1215. A, Western blot analysis of a panel of lymphoma cell lines for Bcl2 and Bim

was compared with the IC50 in these cell lines. B, Mitochondrial membrane potential was measured following 48-hour exposure of cells to ACY-1215 2.5 mmol/L via ﬂow cytometry. C, BH3 proﬁling was performed on the parental and resistant cell lines. D, Baseline characteristics of the resistant line was compared with the parental line with respect to the unfolded protein response (UPR).

Given the known inﬂuence of ACY-1215 on the unfolded comitant activation of XBP-1 and AKT as compared with protein response (UPR; refs. 6, 7), evaluation of PERK and IRE- the parental line perhaps driving accelerated growth in these 1 pathways were evaluated by immunoblot (Fig. 2D). The resis- ACY-1215–resistant cells. This corresponds to previously tant line displayed relatively stable GRP78, the master regulator of published preclinical studies of CLL. Kriss and colleagues dem- the UPR. The GRP78–PERK pathway also remained relatively onstrated that the Em-TCL1 mouse model was found to have stable. The IRE-1 pathway, however, was upregulated with con- upregulated levels of the ER stress response pathway, in particular

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IRE-1 and XBP-1 (12). In the Em-TCL1 model, the tonic IRE-1/ calculated by the relative risk ration (RRR) where a RRR < 1 XBP-1 pathway led to a constitutively active B-cell receptor path- connotes synergy. KD way. In addition, in an XBP-1 /Em-TCL1 mouse model, mice To confirm the clinical relevance of this observation, three with knocked-down XBP-1 had significantly slower progression to primary patient samples of leukemic phase lymphoma were WT B-cell leukemia than XBP-1 /Em-TCL1 mice (13). Together, collected under an IRB-approved protocol. The patient samples these findings suggest a functional link between the IRE-1/XBP- spanned three subtypes of lymphoma including chronic lympho- 1 and B-cell receptor pathways. cytic lymphoma (CLL), lymphoplasmacytic B-cell lymphoma (LPL), and marginal zone lymphoma (MZL) harboring a 17p Gene expression profiling reveals distinct modulation of the deletion. These three diseases are known for their sensitivity to B-cell receptor pathway in the resistant cell line as compared BTK inhibitors. Cells were treated with increasing concentrations with the parental cell line of ibrutinib in combination with ACY-1215 and viability and Gene expression profiling was performed on both the resistant synergy were measured after 24, 48, 72, and 96 hours. All three of and parental cell lines. The parental and resistant cell lines had the lymphoma subtypes achieved marked synergy with RRR of distinct gene expression signatures as represented by principal 0.54, 0.43, and 0.22, respectively (Fig. 4B). component analysis and as can be visualized on the heatmap The molecular effects of the combination treatment was eval- (Fig. 3A). The gene expression data were analyzed by GSEA and uated in three lymphoma cell lines, 2 ABC-DLBCL (LY-10 and gene cluster analysis. There were 1,363 genes from resistant line 2- HBL-1) and one MCL line (JEKO1) as well as the primary human log fold upregulated as compared with the parental and 1825 MZL 17p- patient sample by immunoblot (Fig. 4C). The combi- genes 2-log fold downregulated as compared with the parental nation led to a decrease in p-IRE1- a which has been shown to (P < 0.05). GSEA revealed increased expression of pathways interplay with the BTK pathway (12, 13). This corresponded to known to drive lymphomagenesis of activated B-cell (ABC) a decrease p-BTK, total BTK, and its downstream targets such as lymphoma such as the BTK pathway (Fig. 3b). In addition to p-PLC2-g and CARD11. There was decreased expression of changes in the UPR, differentially expressed genes in the resistant SH3BP5, the negative regulator of the BTK in all cell lines. This line included upregulation of MAPK10, HELIOS, HDAC9, and is expected as its function is to inhibit autophosphorylation of FYN, as well as downregulation of SH3BP5 (a negative regulator BTK and the cells treated with the combination exhibited com- of BTK) and LCK. The change in expression was confirmed by plete inhibition of p-BTK (27). These findings show that targeting PCR (Fig. 3C) and Western blot analysis (Fig. 3D). Given the the IRE1 pathway of the UPR and the BTK pathway with a selective upregulation of FYN, a tyrosine kinase in the B-cell receptor HDAC6 inhibitor and ibrutinib are synergistic at both the cyto- pathway, and the downregulation of SH3BP5 a negative regulator toxic and biologic level. of the Bruton tyrosine kinase, as well as findings generated by GSEA, the resistant line was treated with ibrutinib. Cell viability of The combination of ACY-1215 and ibrutinib led to marked the resistant and parental lines was evaluated over time. Ibrutinib tumor growth delay and prolonged overall survival in a 2 mmol/L was able to overcome resistance with a 70% viability xenograft model of lymphoma in the resistant line versus 64% viability in the parental line The parental LY10 cell line was xenografted into the flanks of at 72 hours (Fig. 3E). These data reveal that cells exposed to SCID beige mice at 107 cells. Mice were divided into 4 cohorts, ACY-1215, a selective HDAC6 inhibitor, rely heavily on the B-cell control, ACY-1215, ibrutinib, and the combination. Mice were receptor pathway for expansion. It also suggests that treatment treated with ACY-1215 50 mg/kg days 1–5, 8–12, 15–19, and with the combination of ACY-1215 and ibrutinib may lead to a ibrutinib 3 mg/kg once daily on days 1–20. Both drugs were given synergistic interaction. by intraperitoneal route (Fig. 5A). The combination of ACY-1215 and ibrutinib was well tolerated with weight loss observed in both ACY-1215 plus ibrutinib is highly synergistic in lymphoma cell the ACY-1215 and combination cohorts that returned to baseline lines and primary human lymphoma samples by day 20 (Fig. 5B). This finding is similar to that seen in prior On the basis of the findings generated from the resistant cell mouse studies of single agent and combinations with ACY-1215 line, a panel of lymphoma cell lines were treated with ACY-1215, (6, 7). Mice demonstrated significant tumor growth delay fol- ibrutinib, or the combination and viability and synergy were lowing treatment with the combination as compared with either measured over 24, 48, and 72 hours (Fig. 4A). Cell lines included ACY-1215, ibrutinib, or untreated mice (P ¼ 0.0003, P ¼ 0.0006, the GCB-DLBCL OCI-Ly7, the ABC-DLBCL cell lines HBL1, OCI- and P < 0.0001, respectively; Fig. 5C). Mice treated with the Ly10, RIVA, mantle cell lymphoma (MCL) lines HBL2, JEKO1, combination had a prolonged overall survival compared with REC1, and the T-cell lymphoma line H9. As expected, synergy was the control (Fig. 5D). Of the mice that completed one cycle of most pronounced in the ABC-DLBCL and MCL cell lines with therapy, the mean survival in days was as follows: combination synergy coefficients as low as 0.10 (Ly10) and 0.15(JEKO1), (66.5) > ibrutinib (57.6) > ACY-1215 (56.6) > control (40.1) > respectively (where RRR<1 connotes synergy). Both of these resistant control (26.5). disease entities are known to be driven by tonic signaling of the B-cell receptor (BCR) pathway and are highly sensitive to BTK Pharmacokinetic and pharmacodynamic effects of ACY-1215 inhibitors. There was little-to-no synergy in GCB-DLBCL and in combination with ibrutinib T-cell lymphoma, respectively, as these cell lines do not rely on The concentration of ACY-1215 and ibrutinib was measured the BCR pathway for growth. The heatmaps represent the viability at sequential time points in serum and tumor tissue after a or synergy of a panel of cell lines following treatment with single intraperitoneal injection of ACY-1215 (50 mg/kg) or ACY-1215, ibrutinib or the combination at 24, 48, and 72 hours. ibrutinib (3 mg/kg) or a combination of both. The concentra- Red boxes indicate lower viability and higher synergy, whereas tions achieved for both drugs are similar or greater than the blue boxes represent high viability and no synergy. Synergy was concentrations found to induce cytotoxicity in the in vitro

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A R P B Parental Resistant

10 5 0 –5 –10 –15 –500 50 PC1

CDGene expression values Parental Resistant Log2 fold (FPKM) (FPKM) change Parental Resistant Parental Resistant 1087 1506 0.5 HDAC6 FYN HDAC9 2 4437 10.8 Helios MAPK10 17 99 2.5

LCK 28 9 –1.6 SH3BP5

FYN 15 1169 6.2 P-BTK (Y223) IKZF2 195 831 2 BTK SH3BP5 17080 2336 –2.9

GAPDH 56269 63070 0.16 P-PLCG2 (Y759)

1.5 PLCG2 1 β-Acn 0.5 Parental

0 Resistant Relave to GAPDH to Relave

E LY10 Ibrunib 100 90 80 70 60 50 48 h Resistant 40 30 72 h Resistant 20 48 h Parental Cell survival (% of control) 10 72 h Parental 0 1234567 Drug concentraon (mmol/L)

Figure 3. Resistant cell line has differentially expressed gene profile as compared with parental line and identifies that the BCR pathway is upregulated in resistant cells. A, Resistant (R) cells and parental cells (P) were evaluated by RNA Seq for gene expression. The two cells lines were distinct as demonstrated by principal component analysis. The heatmap represents the top 100 genes with significant overall 2-log fold change between the resistant and parental lines. B, Gene set enrichment analysis (GSEA) was performed to compare enrichment of pathways in resistant verses parental lines. C, Differentially expressed genes of interest were confirmed via PCR for the resistant line as compared with the parental line. D, Protein expression of genes of interest was confirmed with immunoblot analysis. E, Concentration–effect relationships of ibrutinib in resistant and parental lines was evaluated at 48 and 72 hours.

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A Ibrunib (mmol/L) Ibrunib (mmol/L) 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

ACY- ACY- 1215 24 Hours 48 Hours 72 Hours 1215 24 Hours 48 Hours 72 Hours (μmol/L) (μmol/L) 0 84.9 63.2 39.6 86.0 52.3 17.4 96.6 76.2 17.5 OCI- 1 1.03 1.09 1.04 1.06 0.99 0.96 1.07 0.82 0.67 1 83.1 63.8 40.3 91.2 50.4 13.9 101.1 73.0 11.7 OCI-

(GC) LY7 (GC)

DLBCL LY7 1.5 76.4 62.0 42.3 85.6 48.8 9.4 88.0 62.2 11.1 DLBCL 1.5 0.96 1.03 1.08 1.05 0.96 0.97 1.14 0.56 0.75 0 99.0 91.6 84.1 72.5 74.0 58.5 67.2 64.4 40.5 1 0.91 0.91 0.73 0.93 0.80 0.57 0.76 0.61 0.52 HBL1 1 90.5 85.2 63.7 52.8 47.9 28.9 46.5 34.9 19.9 HBL1 1.5 83.3 68.3 53.6 48.2 38.5 14.4 32.4 23.8 9.2 1.5 0.87 0.87 0.62 0.77 0.68 0.48 0.66 0.32 0.29 0 75.1 63.0 37.6 43.8 24.7 10.8 25.9 9.6 2.7 OCI- OCI- 0.5 0.94 0.79 0.46 0.49 0.31 0.21 0.21 0.11 0.39 1 56.7 40.1 13.9 16.4 5.8 1.7 4.8 0.9 0.9 LY10 LY10 1.5 34.8 22.4 6.0 5.4 1.3 0.7 0.8 0.5 0.6 1 1.01 0.77 0.35 0.43 0.18 0.24 0.10 0.16 0.68 DLBCL (ABC) DLBCL (ABC) 0 88.6 87.9 72.8 78.7 76.3 68.1 72.3 67.5 59.2 1 1.00 1.07 1.06 0.86 0.99 0.98 0.37 0.42 0.39 RIVA 1 82.5 70.4 25.1 69.9 63.0 23.7 62.9 54.0 19.1 RIVA 1.5 0.89 1.05 1.05 0.71 0.81 0.82 0.26 0.29 0.27 1.5 71.5 56.1 17.1 66.3 49.7 15.7 56.0 41.0 11.8

0 107.0 103.3 56.5 108.1 93.9 49.4 94.0 79.8 43.8 RRR 1 1.06 1.00 1.09 1.27 1.17 0.99 0.66 1.14 0.93 HBL2 1 105.5 100.2 50.1 99.2 91.9 41.9 98.8 81.0 38.9 HBL2 1.5 1.01 0.92 1.04 1.18 0.87 0.91 0.68 0.85 0.77 1.5 108.0 100.3 72.9 101.3 96.4 62.4 91.8 88.5 62.2 0 99.6 110.5 69.6 84.0 58.0 20.1 142.5 75.2 0.9 1 0.81 0.86 0.94 0.62 0.67 0.29 0.29 0.22 0.18

Viability (percent of control) of Viability (percent JEKO JEKO1 1 85.4 65.4 24.1 57.8 38.1 4.2 98.0 26.3 0.2 1 1.5 0.77 0.69 0.44 0.67 0.50 0.06 0.43 0.16 0.15 1.5 57.8 49.3 23.5 35.8 19.2 2.0 56.6 9.2 0.1 MANTLE CELL MANTLE MANTLE CELL MANTLE 0 108.8 102.5 63.1 85.8 68.0 38.8 110.0 91.6 31.3 1 1.06 0.97 1.00 0.89 0.80 0.76 0.64 0.57 0.47 REC1 REC1 1 121.2 94.7 52.4 77.1 53.4 18.4 104.3 61.3 12.4 1.5 0.96 0.88 1.00 0.73 0.69 0.37 0.57 0.34 0.23 1.5 108.3 72.5 46.6 67.4 35.5 9.5 89.3 31.4 6.7 0 100.7 99.5 92.3 100.2 99.1 85.1 105.2 95.1 85.3 1 1.00 1.05 0.96 1.02 1.01 1.06 0.79 1.04 0.94 H9 H9 1 100.5 101.0 72.2 101.8 96.8 85.3 101.7 101.6 80.7 T-CELL 1.5 1.02 1.02 1.10 1.05 1.04 1.15 1.04 1.01 1.12 T-CELL 1.5 99.0 101.1 93.3 99.2 99.8 83.3 100.1 94.2 82.9

% Viability 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 RRR 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

B Ibrunib (mmol/L) 024802480248024 8 ACY-1215 (μmol/L) 24 Hours 48 Hours 72 Hours 96 Hours 0 100.0 99.1 95.2 83.5 100.0 75.9 68.9 52.8 100.0 58.1 59.1 38.1 100.0 60.0 48.8 27.0 CLL 2 98.2 90.4 87.9 69.1 83.8 57.0 49.6 32.2 81.9 43.4 37.0 18.7 83.8 36.8 29.4 12.1 0 100.0 145.9 152.2 114.3 100.0 125.9 118.5 126.6 100.0 133.9 142.0 97.1 100.0 117.5 116.6 81.5 LPL 2 107.5 118.8 111.1 94.9 88.7 88.5 80.9 48.1 94.7 94.3 81.2 71.9 75.8 51.3 59.8 31.9

of control) MZL 0 100.00 52.29 62.56 81.49 100.00 87.58 76.89 65.84 100.00 103.64 94.49 66.85 100.00 96.74 88.38 50.59

Viability (percent Viability (17p-) 0.5 84.60 94.55 73.24 89.62 60.86 82.47 42.02 66.38 64.64 49.40 13.84 51.28 40.08 35.89 7.81 35.47 Ibrunib (mmol/L) 248248248248

ACY-1215 (μmol/L) 24 Hours 48 Hours 72 Hours 96 Hours CLL 2 0.93 0.94 0.84 0.9 0.86 0.73 0.91 0.77 0.6 0.73 0.72 0.54 LPL 2 0.76 0.68 0.77 0.79 0.77 0.43 0.74 0.6 0.78 0.58 0.68 0.52 RRR MZL (17p-) 0.5 2.14 1.38 1.3 1.55 0.9 1.66 0.76 0.23 0.53 0.93 0.22 1.75

OCI-LY-10 HBL-1 JEKO-1 Primary Human MZL (17p-) C 24 H C A I AI C A I AI C A I AI C A I AI p-IRE1-α IRE1-α XBP-1s

XBP-1u

MAPK10 SH3BP5 p-BTK BTK p-PLC2-γ PLC2-γ CARD11 β-Acn

Figure 4. Ibrutinib plus ACY-1215 is synergistic in cell lines and primary human lymphoma samples. A, Heatmap represents the viability of a panel of cell lines following treatment with ACY-1215, ibrutinib, or the combination at 24, 48, and 72 hours. Red boxes indicate lower viability. Synergy was calculated by the relative risk ratio (RRR). RRR < 1 connotes synergy and is represented by red boxes. B, Primary human lymphoma samples, chronic lymphocytic leukemia (CLL), lymphoplasmacytic lymphoma (LPL), and 17p deleted marginal zone lymphoma (MZL), were treated with ACY-1215, ibrutinib, or the combination over 24 to 96 hours. Viability was measured and synergy was calculated by RRR and represented in the heatmaps. C, The IRE-1 and BTK pathways were evaluated following treatment with the combination of ibrutinib and ACY-1215 by immunoblot analysis.

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A B 23 ACY-1215 (50 mg/kg) 22.5

Ibrutinib (3 mg/kg) 22 21.5 Control 21 ACY-1215 20.5 Ibrutinib 5 10 15 20 (g) Weight 20 Combo 19.5 0 5 10 15 20 25 Day

C D OCI-LY10 Parental engrafted mice Kaplan–Meier plot

2,000 * P = 0.0003 100 (R) Control

) Control 3 ** P = 0.0006 (P) Control ACY-1215 1,500 ***P < 0.0001 (P) ACY-1215 Ibrutinib (P) Ibrutinib Combo (P) Combo 1,000 *** 50 * **

500 survival Percent Tumor volume (mm volume Tumor 0 0 0 20 40 60 0 20 40 60 80 100 Day Day

Figure 5. The combination of ACY-1215 and ibrutinib leads to statistically signiﬁcant tumor growth delay compared with single-agent treatment in a xenograft mouse model of lymphoma. A, SCID-beige mice were injected with LY10 107 in their ﬂanks and treated with ACY-1215 50 mg/kg days 1–5, 8–12, 15–19, ibrutinib 3 mg/kg days 1–20, or the combination via the intraperitoneal route. B, Mice were weighed every 3 to 4 days as a measurement for toxicity. C, Tumor volume was measured over time as a function of treatment group. D, Kaplan–Meier curve was calculated for the resistant control mice as compared with the parental control and treatment cohorts.

experiments. In addition, these concentrations also recapitulate serum concentration of ibrutinib at 6 and 8 hours was what has been described in human pharmacokinetic studies. 13 (22) and 6 (6) ng/mL, resulting in an average half-life There was no difference in the concentrations of either drug in of 1.1 hours and an average AUC0-8h and AUC0-inf of 752, and resistant or parental mice therefore these data were pooled for 757 h/ng/mL, respectively, for both single administration of analysis (Fig. 6A and B). ibrutinib and in combination with ACY-1215 in both parental The average ACY-1215 serum concentration reached at 0.5 and resistant groups. Ibrutinib tumor concentrations were below and 1 hour after administration was 438 (85) and 327 (133) the lowest limit of quantification in most samples (Fig. 6B). ng/mL, respectively. The average serum concentration of Immunoblot analysis of mouse tumor tissue was evaluated ACY-1215 at 6 and 8 hours was 182 (80) and 200 (89) for modulation of the IRE1 and BTK pathways following ng/mL, resulting in an average half-life of 5.3 hours and an 6 hours of exposure to ACY-1215, ibrutinib, or the combi- average AUC0-6h,AUC0-8h, and AUC0-inf of 1,425, 2,013, and nation. Following the short, single exposure to the drugs there 3,262 hour/ng/mL, respectively, for both single administration was reduction of p-BTK and corresponding reduction of of ACY-1215 and in combination with ibrutinib in both paren- the downstream targets p-PLC2-g and CARD11 (Fig. 6D) tal and resistant groups. There was no substantial difference in which is most pronounced in the combination. These results serum pharmacokinetics of ACY-1215 between groups and confirm those demonstrated in the cell lines treated with the treatments, which was also observed for tumor concentrations combination. of the drug. Mean (þSD) tumor concentrations for single administration of ACY1215 and the combination with ibrutinib in the parental and resistant group at 4, 6, and 8 hours after Discussion injection were 94 (47), 80 (38), and 45 (18) ng/mL, The development of this ACY-1215–resistant cell line has respectively (Fig. 6C). proven to be a powerful tool to identify rational drug partners. The serum pharmacokinetics of ibrutinib exhibited no sub- As treatment strategies for malignancies evolve, targeted stantial differences when the drug was administered alone or in approaches have become exceedingly specific. Although new combination with ACY-1215. The average ibrutinib serum con- highly selective drugs have efficacy against singular targets, what centration reached at 0.5 and 1 hour after administration was collateral effects these new agents have on other pathways is not 778 (85) and 224 (183) ng/mL, respectively. The average immediately apparent. Utilizing gene expression profiling and

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Selective HDAC6 Inhibitor has Synergy with Ibrutinib

AB500 1,000 ACY alone (serum) Ibrutinib alone (serum) ACY in combo (serum) Ibrutinib in combo (serum) 400 ACY alone (tumor) 800 Ibrutinib alone (tumor) ACY in combo (tumor) Ibrutinib in combo (tumor)

300 600

200 400 Ibrutinib (ng/mL) ACY-1215 (ng/mL) ACY-1215 100 200

0 0 0 2 4 6 8 10 0 2 4 6 8 10 Time (hr) Time (hr)

CDC A I AI ACY-1215 Ibrunib p-IRE1-α SERUM IRE1-α (ng/mL) XBP-1s 0.5 h 438 (±83) 778 (±85) 1.0 h 327 (±133) 224 (±183) XBP-1u MAPK10 T1/2 (h) 5.3 1.1 SH3BP5 AUC (h*ng/mL) p-BTK 0−6 h 1,424 --- 0−8 h 2,013 752 BTK 0−inf h 3,262 757 p-PLC2-γ TUMOR PLC2-γ (ng/mL) 4 h 94 (±47) Below the CARD11 6 h 80 (±38) limit β-Acn 8 h 45 (±18) of detecon

Figure 6. Pharmacokinetic and pharmacodynamic effects of ACY-1215 in combination with ibrutinib in mice. Serum and tumor tissue was collected from mice at sequential time points and analyzed for concentration of ACY-1215 and ibrutinib by LC/MS-MS. Mice were treated with ACY-1215 at 50 mg/kg alone and ACY-1215 50 mg/kg with ibrutinib 3 mg/kg. Drug concentrations are represented as mean values. A, Graphical representation of ACY-1215 concentration over time. Mice received one dose of ACY-1215 50 mg/kg or ACY-1215 50 mg/kg plus ibrutinib 3 mg/kg for analysis of serum concentration of ACY-1215. For analysis of drug concentration in tumor tissue, ACY-1215 was administered at 50 mg/kg with or without ibrutinib. B, Graphical representation of ibrutinib concentration over time analyzed in serum and tumor tissue. Mice received one dose of ibrutinib 3 mg/kg via intraperitoneal route. C, Summary of pharmacokinetic data for ACY-1215 and ibrutinib. D, Immunoblot analysis of the IRE1 pathway of the UPR and the BTK pathway from whole-cell lysates of mouse tumor tissue treated with ACY-1215, ibrutinib or the combination. Mice were treated with a single intraperitoneal injection and analyzed at 6 hours. gene set enrichment analysis we identified that the BTK pathway pathway. HDAC inhibition has been shown to restore the expres- circumnavigated resistance to tonic HDAC6 inhibition. sion of BTK-targeting miRNA, leading to decreased signaling of The resistant line displayed upregulation of the IRE1 pathway the BTK pathway and ultimately apoptosis (28). Combinations of the UPR as compared with the parental line. This is in keeping of panobinostat or abexinostat with ibrutinib were synergistic with observations made in models of CLL where inhibition of and overcame BTK mutations known to induce ibrutinib resis- IRE1 led to impaired growth through XBP-1 mediated down- tance. In addition, HDAC6 has been linked to the deacetylation regulation of the BTK pathway. In Em-TCL1 mice knocked down and activation of MYD88, a toll-like receptor kinase known to for XBP-1 mice displayed decreased signaling through the B-cell interplay with the BTK pathway. HDAC6 inhibition with tubacin receptor pathway as revealed by decreased p-BTK. This corre- or vorinostat acetylated and abrogated the effects of MYD88 sponded to slower progression to leukemia relative to age- leading to cell-cycle arrest and cell death (29). These three exam- matched controls (13). By inhibiting IRE1, similar findings were ples point to the interconnectedness of HDAC modulation and replicated establishing a link between the unfolded protein BTK signaling. response and the B-cell receptor pathway. Recent publications Our findings reveal that tonic inhibition of HDAC6 leads to have established other links with HDACs and signaling of the BTK upregulation of the IRE1 pathway of the UPR. This in turn was

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associated with upregulation of the B-cell receptor pathway as line as a collateral effect to overcome tonic HDAC6 inhibition. evidenced by RNA sequencing, GSEA, and immunoblot analysis. Pan-class HDAC inhibition with romidepsin was able to over- Confirmation of the interplay between the UPR and the BCR come this effect as the resistant line was sensitive to treatment pathway were further established by demonstrating synergy with romidepsin. between ACY-1215, a selective HDAC6 inhibitor, and ibrutinib Developing a drug-resistant cell line is an under-utilized strat- across a panel of cell lines (ABC-DLBCL and MCL) known to be egy to identify rational and synergistic drug-drug combinations. sensitive to BTK inhibitors. Furthermore, this synergy was also This strategy could be applied broadly and may proactively established in three primary human lymphoma samples of sub- identify pathways co-opted by malignant cells upon inhibition types also known to be sensitive to BTK inhibitors: CLL, LPL, and by highly selective targeted agents. The findings of this manuscript MZL. The combination was highly synergistic in a rapidly growing identified the synergistic interaction of ACY-1215, a selective 17p- deleted MZL demonstrating a novel approach to these HDAC6 inhibitor, and ibrutinib the first-in-class BTK inhibitor challenging diseases. via this technique. Translation of these findings to the clinic is now The combination of ACY-1215 and ibrutinib led to marked underway. inhibition of p-IRE1 and p-BTK compared with either of the drugs alone and this corresponded to inhibition of downstream Disclosure of Potential Conflicts of Interest fi targets of the BCR such as p-PLC-g and CARD11. These ndings J.E. Amengual reports receiving a commercial research grant from Acetylon were observed in cell lines, primary human lymphoma samples Pharmaceuticals, Inc. No potential conflicts of interest were disclosed by the and xenograft models of lymphoma. In addition, the combi- other authors. nation was well-tolerated in mice. Ly10-xenografted mice treated with the combination had statistically significant tumor Authors' Contributions growth delay compared with those treated with either drug Conception and design: J.E. Amengual, S.A. Prabhu, P.M. Johannet, S. Cremers, alone. O.A. O'Connor Although it is well established that ibrutinib is effective for Development of methodology: J.E. Amengual, S.A. Prabhu, M. Lombardo, patients with 17p- CLL and MCL, clinical experience has P.M.Johannet,Y.Wei,R.Nandakumar,S.Cremers,O.A.O'Connor Acquisition of data (provided animals, acquired and managed patients, informed us that these responses though durable often are provided facilities, etc.): J.E. Amengual, S.A. Prabhu, K.M. Zullo, P.M. Johannet, not permanent (30, 31). It has been observed that patients Y. Gonzalez, X. Jirau-Serrano, R. Nandakumar, S. Cremers relapsing off of ibrutinib have highly aggressive and often Analysis and interpretation of data (e.g., statistical analysis, biostati- explosive disease. Our findings demonstrate that combining stics, computational analysis): J.E. Amengual, S.A. Prabhu, M. Lombardo, ibrutinib with ACY-1215 has potent synergy and that this K.M.Zullo,Y.We,J.K.Duong,R.Nandakumar,S.Cremers,A.Verma, combination is effective at inhibiting the most aggressive O. Elemento, O.A. O'Connor Writing, review, and/or revision of the manuscript: J.E. Amengual, M. Lom- subtypes of lymphoma. bardo, P.M. Johannet, L. Scotto, R. Nandakumar, S. Cremers, O.A. O'Connor The gene expression data derived from the resistant cell line Administrative, technical, or material support (i.e., reporting or organizing also revealed other potential druggable targets. FYN, a Src data, constructing databases): J.E. Amengual, S.A. Prabhu, M. Lombardo, kinase which plays a crucial role in signaling in the T-cell and K.M. Zullo, Y. Gonzalez, O.A. O'Connor B-cell receptor pathways, was also found to be upregulated in Study supervision: J.E. Amengual, L. Scotto, X. Jirau-Serrano, O.A. O'Connor the resistant cell line (32, 33). This kinase is known to be inhibited by dasatinib and combination therapy of dasatinib Acknowledgments plus ACY-1215 is being explored. Helios was also upregulated We would like to kindly thank Anthony Letai, MD, PhD, for providing in the resistant line. Helios is a member of the Ikaros family of technical support for BH3 profiling functional assays. DNA-binding protiens and is found to be mainly expressed in precursor cells of T-cell lineage. Little is known about Helios' Grant Support effects in B lymphocytes but ectopic overexpression has led to This work was supported by the Amos Medical Faculty Development lymphomagenesis in transgenic mice (34). Most of Helios is Program of the American Society of Hematology and Robert Wood Johnson found to be bound to Ikaros, the effects of which are unknown. Foundations and the Columbia University Provost Award for Junior Faculty, research funding from Acetylon Pharmaceuticals, Inc (J.E. Amengual), the In models of myeloma, Ikaros in known to be selectively American Society of Hematology HONORS Award Program (P.M. Johannet) ubiquitinated and degraded by lenalidomide via cereblon and the National Center for Advancing Translational Sciences, NIH, (UL1 ubiquitin ligase (35). Studies evaluating the combination of TR00004; S.Cremers), and (R01 CA194547; O. Elemento). We would also like lenalidomide and ACY-1215 are currently underway in mye- to acknowledge the Lymphoma Research Fund of Columbia University for its loma and how these findings may be translated to B-cell generous support. lymphoma are presently under study. Finally, HDAC9 was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked found to be upregulated in the resistant cell line. HDAC9 is advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate a member of the class IIa family and resides in both the nucleus this fact. and cytoplasm. Although the function of HDAC9 is not well studied, it is likely that its targets may overlap with HDAC6. Received August 11, 2016; revised November 22, 2016; accepted November Upregulation of HDAC9 may have been induced in resistant 23, 2016; published OnlineFirst December 19, 2016.

References 1. Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, et al. Phase 2 trial of 2. Piekarz RL, Frye R, Prince HM, Kirschbaum MH, Zain J, Allen SL, et al. Phase oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory 2 trial of romidepsin in patients with peripheral T-cell lymphoma. Blood cutaneous T-cell lymphoma (CTCL). Blood 2007;109:31–9. 2011;117:5827–34.

OF12 Clin Cancer Res; 2017 Clinical Cancer Research

Selective HDAC6 Inhibitor has Synergy with Ibrutinib

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www.aacrjournals.org Clin Cancer Res; 2017 OF13

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib

Jennifer E. Amengual, Sathyen A. Prabhu, Maximilian Lombardo, et al.

Clin Cancer Res Published OnlineFirst December 19, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-2022

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