ATF3 Coordinates Antitumor Synergy Between Epigenetic Drugs and Protein Disulﬁde Isomerase Inhibitors Ravyn M

Published OnlineFirst June 19, 2020; DOI: 10.1158/0008-5472.CAN-19-4046

CANCER RESEARCH | MOLECULAR CELL BIOLOGY

ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulﬁde Isomerase Inhibitors Ravyn M. Duncan1, Leticia Reyes1, Katelyn Moats1, Reeder M. Robinson1, Sara A. Murphy2, Balveen Kaur2, Holly A.F. Stessman3, and Nathan G. Dolloff1

ABSTRACT ◥ Histone deacetylase inhibitors (HDACi) are largely ineffective in response in cancer cells. The HSP40/HSP70 family genes DNAJB1 the treatment of solid tumors. In this study, we describe a new class and HSPA6 were found to be critical ATF3-dependent genes that of protein disulfide isomerase (PDI) inhibitors that significantly and elicited the antitumor response after PDI and HDAC inhibition. In synergistically enhance the antitumor activity of HDACi in glio- summary, this study presents a synergistic antitumor combination blastoma and pancreatic cancer preclinical models. RNA- of PDI and HDAC inhibitors and demonstrates a mechanistic and sequencing screening coupled with gene silencing studies identified tumor suppressive role of ATF3. Combined treatment with PDI and ATF3 as the driver of this antitumor synergy. ATF3 was highly HDACi offers a dual therapeutic strategy in solid tumors and the induced by combined PDI and HDACi treatment as a result of opportunity to achieve previously unrealized activity of HDACi in increased acetylation of key histone lysine residues (acetylated oncology. histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with Significance: This study uses a first-in-class PDI inhibitor enter- increased RNA polymerase II recruitment to the ATF3 promoter, ing clinical development to enhance the effects of epigenetic drugs a synergistic upregulation of ATF3, and a subsequent apoptotic in some of the deadliest forms of cancer.

Introduction most successful to date with four drugs (panobinostat/FARYDAK, vorinostat/ZOLINZA, romidepsin/ISTODAX, and belinostat/BELEODAQ) Changes in the epigenetic landscape are underlying hallmarks of receiving approval for the treatment of cutaneous and peripheral T-cell cancer. Silencing of tumor suppressor genes via global changes in lymphoma (CTCL and PTCL) and multiple myeloma (6–8). Aside from their histone acetylation and DNA methylation are examples of epigenetic efficacy in CTCL, PTCL, and multiple myeloma, which account for less than alterations that are observed early in tumorigenesis (1–4). Epigenetic 1% of all cancers (9), HDACi remain largely ineffective in other cancer types, regulation involves a complex interplay between multiple classes of particularly solid tumors. enzymes, DNA-binding elements, transcriptional cofactors, and post- Therapeutic resistance has been implicated in the broad lack of translational modifications that affect chromatin structure. Therapeu- clinical activity that is observed with HDACi monotherapy, and a tic platforms targeting epigenetic regulators have evolved in parallel variety of molecular mechanisms that confer resistance have been with our increased understanding of this field. Emerging drug classes reported. These include hyperactivation of the LIFR-JAK1-STAT3 include DNA methyltransferase inhibitors, histone deacetylase inhi- antiapoptotic cascade (10), dysregulation of HDAC proteins (11), bitors (HDACi), BET/bromodomain inhibitors, lysine-specific protective NF-kB signaling (12), and disruptions in redox homeosta- demethylase 1 inhibitors, and others. Epigenetic targets have proven sis (13). The most effective use of HDACi appears to be in combination druggable with potent, specific, and isoform selective small mole- therapy (11, 14, 15). However, it is not clear which combinations will cules (5). Some of these agents are FDA approved, while others are be most effective, nor is there a precise molecular understanding for in clinical trials for cancer and other diseases. However, despite the how to potentiate the effects of HDACi. emergence of promising epigenetic drug candidates, few have trans- Protein disulfide isomerase (PDI) inhibitors are an emerging class of lated into successful clinical programs. HDACi are perhaps the antitumor agent. The overall function of PDI family members is to coordinate oxidative protein folding in the endoplasmic reticulum (ER). PDI isoforms share a common thioredoxin-like catalytic redox 1 Department of Cellular and Molecular Pharmacology and Experimental center consisting of pairs of reactive cysteines that catalyze disulfide Therapeutics, Medical University of South Carolina, Charleston, South Carolina. bridge formation and isomerization between thiol groups of newly 2Department of Neurosurgery, Health Science Center at Houston, McGovern Medical School, University of Texas, Houston, Texas. 3Department of Pharma- synthesized polypeptides. As PDI utilizes redox chemistry to regulate cology, Creighton University School of Medicine, Omaha, Nebraska. protein folding, it serves a bifunctional role as mediator of both protein and redox homeostasis. Proteotoxic and oxidative stress are recognized Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). as phenotypic hallmarks of cancer cells and are heightened in a wide range of cancer types (16). Therefore, the unique dual role PDI plays, as Corresponding Author: Nathan G. Dolloff, Medical University of South Carolina, 173 Ashley Ave MSC509, Charleston, SC 29425. Phone: 843-876-2204; Fax: 843- a mediator of both proteotoxic and oxidative stress coupled with the 876-2343; E-mail: [email protected] fact that PDI isoforms have been linked to poor prognosis and resistance to therapy in several cancer types, make this family an Cancer Res 2020;80:1–13 appealing oncology drug target (17, 18). doi: 10.1158/0008-5472.CAN-19-4046 In this study we used combination drug library screening and 2020 American Association for Cancer Research. uncovered a highly synergistic combination between an IND-stage

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PDI inhibitor candidate (E64FC26; refs. 19, 20) and HDACi. Synergy Production of short hairpin RNA lentiviral particles and infection was evident in hematologic as well as solid tumor cells and was HEK-293T cells were infected with 5 mg DNA (Supplementary remarkably high in some cases, potentiating the effects of HDACi by Table S2), 2.5 mg pCMV-dR8.91, and 0.5 mg pCMV-VSV-G diluted in >200-fold. We demonstrate the preclinical potential of this novel Opti-MEM and Lipofectamine 2000. Seventy-two hours after trans- combination in multiple tumor types, including glioblastoma and fection, supernatant containing viral particles was collected and stored pancreatic cancer, which are two of the deadliest forms of cancer, at 80C or used immediately to infect cell lines. Viral particles were carrying dismal 5-year survival rates of less than 10% (21). We propose titered using the qPCR Lentivirus Titration (Titer) Kit (Applied this new strategy as a means to maximize the potential of PDI Biological Materials Inc.) as per the manufacturer's protocol. Cells inhibitors in future translational clinical studies and to rescue were infected with viral particles at a multiplicity of infection of 10 with previously unrealized antitumor activity of HDACi. We delineate the 8 mg/mL polybrene. Cells were seeded for experiments after stable molecular mechanism guiding this combination regimen, critical to knockdown was confirmed. Cells used for transient overexpression which is the transcriptional induction of the ATF3 gene. ATF3 experiments were transfected with 2.5 mg DNA (Supplementary upregulation is initiated by PDI inhibitor–induced ER stress and Table S2) diluted in Opti-MEM and Lipofectamine 2000. Cells were then further potentiated by HDACi-induced chromatin remodeling. seeded for experiments 96 hours after transfection. Thus, we establish ATF3 and its proapoptotic downstream transcriptional program as a mechanistic pathway to enhancing epigenetic RT-qPCR therapy. Total RNA was isolated and purified using the RNeasy Plus Mini Kit (Qiagen). RNA was reversed transcribed and quantified using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) as per the Materials and Methods manufacturer's protocol. Each reaction contained 56 ng RNA and was Cell lines, reagents, and antibodies amplified with 500 nmol/L forward and reverse primers. Gene expres- Cell lines were purchased from the ATCC and were confirmed to be sion was normalized to GAPDH, which was constitutively expressed Mycoplasma free at the time of experimentation using the LookOut and did not change based on treatment conditions. Fold change was DD Mycoplasma Detection Kit (Sigma). T cells were expanded from quantified following the 2 Ct method. Primers are listed in Supple- human peripheral blood monoleukocytes purchased from AllCells mentary Table S3. LLC (PB006F). All cells were maintained at 37 C and 5% CO2. Experiments were performed in a 1:1 mixture of the recommended RNA sequencing supplemented medium and RPMI1640 (HyClone SH30027.01) with Cells were treated as indicated for 16 hours. RNA was extracted and cells between passage 2–15. Antibodies used for Western blotting, purified using the RNeasy Plus Mini Kit (Qiagen). Samples were chromatin immunoprecipitation (ChIP) assays and reverse tran- sequenced at the Medical University of South Carolina. Hollings scriptase qPCR (RT-qPCR) are listed in Supplementary Table S1. Cancer Center Genomics Shared Resource (Charleston, SC) on the E64FC26 was synthesized as described previously (19, 20). E64FC26 Illumina HiSeq2500. A TruSeq mRNA library preparation was used on was >95% pure as determined by high-performance liquid chro- extracted mRNA, and a rapid mode single-end 1 50 cycle protocol matography (HPLC), and target product identity was confirmed by was used for transcript expression quantification. Biological replicates nuclear magnetic resonance and LC/MS. The NCI-Approved were run in triplicate. Sample data were analyzed at Creighton Univer- Oncology Drug Set was provided by the NCI Developmental sity (Omaha, NE) using the Tuxedo tools suite (23). In brief, samples Therapeutics Program. Primary patient glioblastoma neurosphere were mapped to the hg19 human reference genome (ftp://igenome: cultures were maintained as described previously (22). Cell viability [email protected]/Homo_sapiens/UCSC/hg19/Homo_ was measured in 96-well culture plates using CellTiter Glo Lumi- sapiens_UCSC_hg19.tar.gz; downloaded September 7, 2018) using nescent Cell Viability Assay (Promega) according to the manufac- TopHat v2.1.0 (Bowtie2 v2.2.6) followed by transcript assembly using turer's instructions. The EC50 and potentiation were calculated as cufflinks v2.2.1. Transcript differences were called using cuffdiff on described previously (19). merged transcriptome replicates by condition/treatment. Data visualiza- tion and mining were performed using the cummeRbund program for R Confocal microscopy (v3.5). Gene Expression Omnibus accession number GSE142210. Cells were seeded in an 8-well Nunc LabTek II Chamber Slide (Thermo Fisher Scientific) and treated with the indicated drug for Chromatin immunoprecipitation 16 hours. Cells were fixed using BD Cytofix/Cytoperm solution as per Cells were treated as indicated and then cross-linked with 1% the manufacturer's protocol. Slides were stained with the indicated formaldehyde for 10 minutes. Formaldehyde was quenched with the antibody in BD Perm/Wash (Supplementary Table S1). Primary addition of 125 mmol/L glycine for 5 minutes. Cells were lysed in antibodies were incubated for 45 minutes followed by secondary membrane lysis buffer (10 mmol/L TRIS pH 7.4, 3 mmol/L MgCl2, antibodies or actin stain for 30 minutes. All samples were stained 10 mmol/L NaCl, and 0.1% Igepal) for 20 minutes on ice, and then with Hoechst 33342 nuclear stain for 15 minutes. Slides were mounted the lysate was incubated with 40 U/mL micrococcal nuclease for with ProLong diamond antifade mountant. Confocal microscopy was 20 minutes at 37C to generate DNA fragments of 300–500 base pair performed using the Zeiss LSM 880 NLO Laser Scanning Confocal sizes. Chromatin was extracted using nuclear lysis buffer (50 mmol/L Microscope (Zeiss). Each sample was scanned with a 63 oil objective TRIS pH 8.0, 10 mmol/L EDTA, and 1% SDS) for 20 minutes on ice. to obtain 3–7 fields for analysis. Image analyses were performed Chromatin was precleared for nonspecific binding with Protein A/G using ZEN-blue and ZEN-black Software (Zeiss) and IMARIS agarose beads for 1 hour at 4C. Beads were removed and 10–25 mgof Microscopy Image Analysis Software (Bitplane) was used to create the chromatin was incubated overnight at 4C with the indicated 3D-reconstructions of z-stacked images to calculate the number of antibody (Supplementary Table S1). The next day, Protein A/G ubiquitin aggregates per cell and the total area of the ubiquitin agarose beads were added to each sample and incubated for 1 hour aggregates. at 4C. Immunoprecipitation samples were washed with low salt

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buffer (20 mmol/L TRIS pH 8.0, 2 mmol/L EDTA, 1% Triton X-100, NCI-Approved Oncology Drug Set. We conducted combination and 150 mmol/L NaCl), high salt buffer (20 mmol/L TRIS pH 8.0, 2 screening in cell lines from pancreatic cancer (PANC-1), glioblastoma mmol/L EDTA, 1% Triton X-100, and 500 mmol/L NaCl), and wash (T98G), ovarian cancer (SKOV3), and acute myeloid leukemia (AML, buffer (10 mmol/L TRIS pH 8.0 and 2 mmol/L EDTA). Chromatin was MV4-11) using a phenotypic screening approach similar to the one we eluted from the antibodies with elution buffer (100 mmol/L NaHCO3 reported previously where synergy is calculated using the schema and 1% SDS) for 1 hour at 65C. Cross-link reversal was performed in illustrated in Supplementary Fig. S1 (26). The screening returned seven the presence of 20 mg/mL Proteinase K and 200 mmol/L NaCl for positive hits that were synergistic with 1 mmol/L E64FC26 in all cell 4 hours at 65C. DNA was purified with the Qiagen DNA Purification lines. Notably, these drugs represented only two mechanistic classes, Kit (#27104). Quantitative PCR was performed using PowerUp SYBR proteasome inhibitors and HDACi. Proteasome inhibitors as expected, Green Master Mix (Thermo Fisher Scientific, A25741). Data were given our previous work (19, 20), and all three inhibitors in the set analyzed using the percent input method. The input samples repre- (bortezomib, carfilzomib, and ixazomib) were positive hits (Fig. 1A; sented 10% of the chromatin. All experiments were repeated 2–5 times Supplementary Figs. S2–S4). HDACi demonstrated the greatest syn- with samples separately cross-linked for reproducibility. Primers were ergy, and all four HDACis in the set (vorinostat, belinostat, panobino- designed to flank gene promoters on the basis of published reports or stat, and romidepsin) were positive hits (Fig. 1A; Supplementary Figs. by mapping the genes using the open-access Ensembl database for S2 and S3). vertebrate genomes (Supplementary Table S3; refs. 24, 25). BLAST Validation experiments with expanded dose curves revealed searches were performed to ensure no off-target binding of the primers remarkably high synergy between E64FC26 and HDACi (Fig. 1B; would occur. Supplementary Figs. S5 and S6). For example, in pancreatic cancer cells, E64FC26 reduced the panobinostat EC50 from 1,200 to 5 nmol/L, In vivo studies a 240-fold increase in sensitivity, and potentiated vorinostat by All xenograft studies with SCID Hairless Outbred (SHO) mice 24.5-fold in T98G glioblastoma cells, reducing the EC50 from 13 to were conducted under the approval of the Institutional Animal Care 0.53 mmol/L. Synergy was further demonstrated and quantified using and Use Committee of the Medical University of South Carolina dose matrices and isobologram analyses (Fig. 1C and D). Gemcitabine (Charleston, SC). Two-month-old SHO mice were injected with and temozolomide, which are standard-of-care agents in pancreatic 1 106 human U87 cells (100 mL total volume) in the lower flank cancer and glioblastoma, respectively, were included for comparison of the mouse. Cells were mixed in a 1:1 ratio of RPMI1640 and and showed a general lack of antitumor activity as single agents Matrigel. Mice were randomly assigned to treatment groups and dosed and an absence of synergy with E64FC26. Greater than 10-fold with drugs as indicated once tumors showed growth for three con- synergy was observed with other pan-HDACi (belinostat) and class secutive measurements and reached a tumor volume of 100–200 mm3. I HDAC selective inhibitors (romidepsin, entinostat, and LP-411; Vehicle treatments contained 0.5% (v/v) DMSO and 5% (v/v) Fig. 1E and F; ref. 27). Synergy with HDAC6-selective inhibitors Kolliphor EL (200 mL total in DPBS). Tumor measurements and (tubastatin A and ricolinostat) was also observed, albeit to a lesser analyses were performed by a blinded investigator, and unblinding extent than the other HDACis. We detected significant enhancement occurred after all the experimental data were analyzed. of BET/bromodomain inhibitors (i.e., JQ1, mivebresib, birabresib, and For pharmacokinetic and tissue distribution studies in CD-1 mice, IBET151). However, we did not detect any synergy between E64FC26 animals were fasted overnight before dosing, with food returned after and the demethylating agent, 5-Azacytidine, the EZH2 inhibitor, the 6-hour blood samples were obtained. Three animals were dosed via tazemetostat, or the lysine specific demethylase 1 (LSD1) inhibitor, gavage needle for oral administration at 5 mg/kg or via tail vein GSK-LSD1 (Fig. 1E and F), which shows that indene PDI inhibitors injection for intravenous administration at 2 mg/kg. Blood samples specifically enhance HDACi, but not necessarily all epigenetic drug (30–50 mL) were taken via the submandibular vein at 5 minutes, classes. 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and We broadened the screening to include panels of pancreatic cancer 24 hours after dosing. For tissue distribution studies, plasma or and glioblastoma cell lines, as well as ovarian, colorectal, and neuro- indicated organs were harvested at 10 minutes, 30 minutes, 1 hour, blastoma, and panels of cells of hematologic origin including multiple 2 hours, and 4 hours after dosing. Tissues were homogenized in three myeloma, mantle cell lymphoma, CTCL, and AML. E64FC26 showed volumes of PBS buffer (pH 7.4) to obtain each tissue homogenate single-agent antitumor activity across many tumor types (Supplemen- sample. Subsequently, three volumes of acetonitrile containing inter- tary Fig. S7A) and broad synergy with HDACi in both solid and nal standard were added to one volume of each tissue homogenate, and hematologic cancer cell lines (Fig. 1G and H; Supplementary Fig. S7B). the mixture was vortexed, centrifuged (3,000 g for 10 minutes), and Minimal sensitization was observed in normal cell types, and in some supernatant removed for analysis by LC/MS-MS. Calibration stan- cases the interaction was antagonistic/protective (Fig. 1G). The com- dards were made by preparation of a 1 mg/mL stock solution and bination increased proapoptotic signaling, as activation of caspase-3, subsequently a series of working solutions in methanol:water (1:1, v/v), -8, and -9 were detected (Supplementary Fig. S8). which were spiked into blank tissue homogenate to yield a series of calibration standard samples in the range of 1 ng/mL to 10 mg/mL. Synergy between PDI and HDAC inhibitors is UPR/ER stress LC/MS-MS bioanalysis was performed using a Shimadzu HPLC and dependent AB/MDS Sciex MS/MS system fitted with Phenomenex Kinetex C18 We next investigated the molecular mechanism responsible for column in MRM-negative ion mode. the synergy between PDI inhibitors and HDACi. We first validated the role of PDI by silencing its gene target P4HB (also known as PDIA1). Similar to cotreatment with E64FC26, knockdown of PDI Results expression increased sensitivity to panobinostat (Fig. 2A), and PDI Indene PDI inhibitors potentiate the antitumor effects of HDACi knockdown further enhanced the synergy between E64FC26 and In this study, we screened for synergistic combinations between PDI HDACi. In addition, we found that other PDI inhibitors reported inhibitor, E64FC26, and FDA-approved cancer agents by screening the in the literature (e.g., PACMA31) enhanced HDACi sensitivity

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Figure 1. E64FC26 potentiates the activity of HDACis. A, Synergistic hits in PANC-1 (left) and T98G cells (right). Cells were treated for 48 hours with 1 mmol/L E64FC26 and the indicated drug. Combination index scores less than 1.0 are indicative of synergistic potential. B, Representative cell viability response curves of PANC-1 cells treated with panobinostat and T98G cells treated with vorinostat alone or in combination with 1 mmol/L E64FC26 for 48 hours. Viability was normalized to the RLU values in the absence of the HDACi to account for cell death induced by E64FC26 alone. Thus, separation of the curves indicates synergistic drug interaction. C and D, Isobolograms of PANC-1 cells treated with E64FC26 in combination with panobinostat (top) or gemcitabine (bottom; C) and isobolograms of T98G cells treated with E64FC26 in combination with panobinostat (top) or temozolomide (bottom; D). Numbers inside the boxes represent cell viability (%). The dotted line represents a

combination index score of 1.0. Curves that fall below the dotted line are indicative of synergy. E and F, EC50 values of PANC-1 cells (E) and T98G cells (F) treated with various epigenetic protein inhibitors alone or in combination with 1 mmol/L E64FC26. Pano, panobinostat; vorino: vorinostat; romi, romidepsin; belino, belinostat; rico,

ricolinostat; tuba, tubastatin; entino, entinostat; 5-Aza, 5-azacytadine; taz, tazemetostat. EC50 values were calculated from three individual dose curves. G and H, Waterfall plot showing the change in panobinostat sensitivity in the presence of 1 mmol/L E64FC26 (G) and 500 nmol/L E64FC26 (H) for the speciﬁed cell lines. The

“Panobinostat Potentiation” score was calculated by dividing the EC50 for panobinostat in the absence of E64FC26 by the EC50 for panobinostat in the presence of E64FC26. EC50s for each cell line were extrapolated from independent 8-dose cell viability curves. On the basis of this equation, a score above one-fold is indicative of synergy.

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Figure 2. PDI inhibition results in ER stress caused by the accumulation of misfolded polyubiquitinated proteins. A, PDIA1 was stably knocked down in PANC-1 cells. Cells were treated with panobinostat (pano) in the presence or absence of 1 mmol/L E64FC26 for 48 hours. A representative cell viability curve (left) and the change in EC50 values are shown (right). Statistical significance was determined by Student t test (n ¼ 6). B, Indicated cell lines were treated with 1 mmol/L E64FC26 alone or in combination with 50 nmol/L panobinostat or 100 nmol/L romidepsin (romi) for 16 hours. Western blots for ubiquitin (Ub), acetylated lysine (Ac-Lysine), and acetylated a-tubulin (Ac-Tubulin) are shown. C, Confocal microscopy images of PANC-1 cells treated with 1 mmol/L E64FC26 alone or in combination with 50 nmol/L panobinostat or 100 nmol/L romidepsin for 16 hours. Cells were stained with ubiquitin-FITC and Hoechst 33342 (nucleus). The total area and the total number of ubiquitinated aggregates per cell were calculated using IMARIS Microscopy Image Analysis Software (Bitplane). Statistical significance was determined by Student t test (n ¼ 12; ns, not significant). D, HDAC6 was overexpressed in PANC-1 cells treated with panobinostat in the presence or absence of 1 mmol/L E64FC26 for 48 hours. Cell viability curves are shown. E, PANC-1 cells were treated with panobinostat alone or in combination with 1 mmol/L E64FC26, 5 mmol/L tunicamycin (tunica), or

5 mmol/L thapsigargin (thapsi) for 48 hours. EC50 values were calculated for the combinations (left) with RLU values normalized to account for the individual effects of the ER stress–inducing agents alone. Statistical signiﬁcance was determined by Student t test (n ¼ 6). PANC-1 cells were treated for 16 hours with 50 nmol/L panobinostat and 1 mmol/L E64FC26, 5 mmol/L tunicamycin, or 5 mmol/L thapsigargin. Western blot analysis is shown (right). F, T98G cells were treated with 1 mmol/L E64FC26 and/or 50 nmol/L panobinostat in the presence or absence of 0.5 mg/mL cycloheximide (CHX) for 16 hours. Western blot analysis is shown. G, PANC-1 (left) and T98G cells (right) were treated with 1 mmol/L E64FC26 (26) and/or 37 nmol/L panobinostat in the presence or absence of 0.5 mg/mL cycloheximide. Cell viability is shown. Signiﬁcance was determined by Student t test (P < 0.001; n ¼ 3). Veh, vehicle.

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(Supplementary Fig. S9; ref. 28), further validating the molecular Fig. S14A. Relative to DMSO-treated controls, the individual single connection between PDI inhibition and HDACi potentiation. agents and the combination of these two drugs induced an RNA-seq In previous studies, inhibition of PDI by E64FC26 induced the profile with varying degrees of overlap (Fig. 3A). PDI inhibition alone accumulation of large amounts of misfolded ubiquitinated proteins induced transcripts principally related to ER stress and oxidative stress and a robust ER stress and oxidative stress response in multiple pathways, including GADD45A, ATF3, DDIT3, and HMOX1 myeloma cells (19, 20). In solid tumor cell lines, single-agent treatment (Fig. 3B). Major repressed transcripts included ATF5 and TNS3. The with E64FC26 led to increases in protein ubiquitination that appeared gene sets affected by single-agent panobinostat varied widely between as high molecular weight smears by Western blotting and as dense the two cell lines and were not associated with any one pathway; perinuclear ubiquitin-positive aggregates by confocal imaging (Fig. 2B however, a common subset of transcripts (i.e., DHRS2, PHOSPHO1, and C; Supplementary Fig. S10A). We hypothesized that HDACi TUBB4A, SLC17A7, and others) were significantly upregulated in both might be potentiating levels of E64FC26-induced proteotoxic stress cell lines, and ATF5, JADE2, and TNS3 were commonly repressed. by further increasing the accumulation of misfolded poly- Most importantly, in the combination, we observed a gene set that was ubiquitinated proteins. This, however, was not the case, as the com- common to both cell lines and discovered that three of those gene bination with HDACi did not significantly increase global ubiquitina- transcripts, ATF3, HERPUD1, and DDIT3 (gene encoding CHOP), tion levels or the size or number of ubiquitinated protein aggregates were upregulated synergistically (Fig. 3B and C; Supplementary Tables beyond what was induced by E64FC26 alone (Fig. 2B and C; Sup- S4 and S5). Results were confirmed by qRT-PCR (Fig. 3D; Supple- plementary Fig. S10B). Synergy was also independent of the HDAC6– mentary Fig. S14B and S14C), Western blotting (Fig. 3E), and aggresome pathway, which channels misfolded proteins to the lyso- fluorescence confocal microscopy (Supplementary Fig. S15). ATF3, somal compartment for degradation as a parallel pathway to the as an example, was induced 16-fold by single-agent E64FC26 and ubiquitin–proteasome system (29). In support of HDAC6 indepen- 3-fold by single-agent panobinostat. A purely additive interaction dence, we noted strong synergy with class I selective HDACi that do would produce an expected fold change of 19-fold; however, we not inhibit HDAC6 (Fig. 1A, E, and F), and HDAC6 overexpression observed a highly synergistic 113-fold induction in the combination. failed to rescue cells from the effects of E64FC26 þ HDACi combina- Synergy at the mRNA level translated into even more dramatic tions (Fig. 2D; Supplementary Fig. S11). Synergy was, however, differences at the protein level in response to PDI inhibition combined dependent on ER stress signaling originating from oxidative stress with both panobinostat and romidepsin (Fig. 3E). These data impli- and the UPR pathway. Other ER stress–inducing agents, including cate ATF3, HERPUD1, and CHOP as potential mediators of the tunicamycin and thapsigargin, enhanced the activity of panobinostat proapoptotic response to PDI and HDAC inhibition. (Fig. 2E; Supplementary Fig. S12A). Treatment with the protein translation inhibitor, cycloheximide, relieved most of the misfolded ATF3 is the key driver of PDI and HDAC inhibitor synergy protein burden in cells and protected against death by the combination We next set out to determine whether ATF3, HERPUD1, and of E64FC26 and panobinostat (Fig. 2F and G), suggesting that CHOP were drivers or passengers of the antitumor synergy between increased protein load was critical for PDI þ HDACi synergy. PDI and HDAC inhibitors. To accomplish this, we used RNAi to Furthermore, consistent with previous reports (19, 20), E64FC26 inhibit their expression and evaluated the impact on the synergistic induced oxidative stress characterized by Nrf2 stabilization and nucle- phenotype. Knockdown of ATF3 nearly completely reversed E64FC26 ar translocation downstream of PDI inhibition (Supplementary potentiation of panobinostat in PANC-1 cells and partially in T98G Fig. S12A and S12B). The addition of reactive oxygen species sca- cells (Fig. 4A–C; Supplementary Fig. S16A). Likewise, ATF3 was vengers, N-acetylcysteine and b-mercaptoethanol, partially rescued critical for the apoptotic response, as knockdown reduced activation cell viability in combination treated T98G and PANC-1 cells of caspase-3 and -8 and PARP cleavage (Fig. 4D). Conversely, (Supplementary Fig. S12C and S12D). Taken together, these data knockdown of HERPUD1 and CHOP had no effect on HDACi show that the potentiation of HDACi by PDI inhibition was not due sensitization by E64FC26 in either cell line (Fig. 4E–H; Supplementary to further disruption of protein folding, but rather a convergence of Fig. S16B and S16C). We also observed robust upregulation of ATF4 ER stress and epigenetic signaling events downstream of protein upon E64FC26 and panobinostat treatment, although this occurred folding errors. predominantly at the protein level and did not meet our RNA-seq hit > criteria of log2 (fold change) |3.5|. However, similar to what we PDI and HDAC inhibitor combinations synergistically induce observed with CHOP and HERPUD1, ATF4 knockdown showed no ATF3, HERPUD1, and CHOP effect on PDI and HDAC inhibitor synergy (Supplementary Fig. S17). HDACi alter chromatin structure and gene expression by increasing ATF4 induction was independent of ATF3 (Supplementary Fig. S18), histone acetylation (30, 31). We hypothesized that HDACi-induced suggesting that ATF4 and ATF3 function in parallel, but independent changes in chromatin topography were enhancing proapoptotic pathways in the context of PDI and HDAC inhibition. Altogether, transcriptional programs set in motion by PDI inhibition. To test these findings demonstrate an essential role of ATF3 in driving this theory, we conducted comparative RNA-sequencing (RNA-seq) the antitumor synergy between PDI and HDAC inhibitors and profiling to identify transcripts and pathways that were induced suggests a dispensable or passenger function for HERPUD1, ATF4, specifically by the combination. We used multiple cancer cell lines and CHOP. (PANC-1 and T98G) to identify critical pathways across cells from Reports of ATF3 in the literature are sparse with some studies different genetic backgrounds and tissues of origin. Cells were treated demonstrating tumor suppressor function and others suggesting as indicated in biological triplicates and sequenced. Sample quality protumorigenic roles (32, 33). The function of ATF3 in the context overall was high as assessed by the RNA transcript integrity (TIN) of cancer therapy, although also limited, points to a proapoptotic score (RSeQC v3.0.1; median TIN among samples ¼ 54), and biolog- function (34–36). Our data show a clear antitumor function of ATF3 ical replicates clustered tightly by transcriptional changes through within the specific therapeutic framework of PDI þ HDACi therapy. principle component analysis (R v3.5; Supplementary Fig. S13). Gene To more completely understand this role, we set out to characterize the sets were screened using the decision tree outlined in Supplementary downstream effects of ATF3. We conducted RNA-seq in parallel with

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Figure 3. RNA-seq reveals ATF3, CHOP, and HERPUD1 as downstream regulators of PDI and HDAC inhibition. A, T98G (top) and PANC-1 cells (bottom) were treated with 1 mmol/L E64FC65, 50 nmol/L panobinostat (pano), the combination (combo), or DMSO vehicle control for 16 hours. RNA-seq was performed and the number of transcripts that significantly changed in each treatment condition compared with DMSO alone is shown in the Venn diagram (Padj < 0.05). B, Transcripts were filtered for log2 (fold change) > |3.5| and fragment per kilobase of transcript per million mapped reads (FPKM) > |10| compared with DMSO alone. The number of genes that fit these criteria after the indicated treatment is shown in the Venn diagrams, with the top five significantly changed transcripts listed for each treatment. C, The most significant transcripts affected in both PANC-1 and T98G cell lines as determined by the criteria in B are shown in the heatmap. D, PANC-1 cells were treated with 1 mmol/L E64FC26 (26), 50 nmol/L panobinostat, the combination, or DMSO vehicle control for 16 hours. RNA was extracted and RT-qPCR was performed. Graphs show the fold change in mRNA expression. Significance was determined by Student t test (n ¼ 9). E, Indicated cell lines were treated with 1 mmol/L E64FC26 in the presence or absence of 50 nmol/L panobinostat or 100 nmol/L romidepsin (romi) for 16 hours. Western blots are shown.

ATF3 knockdown to identify ATF3-dependent target genes following HDAC inhibitors increase acetylation of histones flanking the PDI and HDACi treatment. The combination induced a RNA-seq ATF3 promoter and enhance RNA polymerase II recruitment to profile with several transcripts uniquely affected in ATF3-knockdown the ATF3 transcriptional start site cells (Fig. 5A). Many of these transcripts are implicated in ER stress, Transcriptionally active gene loci are associated with histone lysine the unfolded protein response, and ER-associated degradation residues with specific acetylation marks, and HDACi are known to pathways (Supplementary Fig. S19A). These include multiple mem- alter these acetylation patterns (10, 37, 38). We hypothesized that bers of the HSP40 family (DNAJB1 and DNAJA4)andtheHSP70 ATF3 gene induction following PDI inhibition was being potentiated family members HSPA6, HSPA1A,andHSPA1B (Fig. 5B;Supple- by increased histone acetylation and transcriptional activation in the mentary Table S6). RNA-seq results for HSPA6 and DNAJB1,the presence of HDACi. To evaluate the acetylation status of histones two most significant ATF3-dependent genes, were confirmed by surrounding the ATF3 promoter, we performed ChIP assays with qRT-PCR and Western blotting (Fig. 5C and D). We then con- antibodies targeted to acetylated histone 3 lysine 27 (H3K27-ac), firmed the functional role of HSPA6 and DNAJB1 in mediating cell histone 3 lysine 18 (H3K18-ac), and histone 3 lysine 9 (H3K9-ac), death following PDI and HDAC inhibition using RNAi (Fig. 5E which are histone marks associated with active chromatin (37, 38). We and F). These data highlight a proapoptotic role for HSP40 and found significant enrichment of H3K27-ac and H3K18-ac marks HSP70 downstream of ATF3 in the context of PDI and HDAC flanking the regulatory regions upstream of the ATF3 transcription inhibition. start site (Fig. 6A), which contains two well-defined promoter sites,

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Figure 4. ATF3 drives the synergy between E64FC26 and panobinostat. A, PANC-1 cells were stably transduced with control shRNA (shCTL) or ATF3 shRNA. Cells were treated with 1 mmol/L E64FC26 and 50 nmol/L panobinostat (pano) or DMSO for 16 hours. The Western blot analysis comparing ATF3 expression is shown. B, Stable PANC-1 ATF3-knockdown and shCTL cells were treated with panobinostat in the presence or absence of 1 mmol/L E64FC26 for 48 hours. Cell viability curves are shown (n ¼ 3). C, Fold change in panobinostat potentiation was calculated from PANC-1 (left) and T98G (right) cell viability curves. Significance was determined by Student t test (n ¼ 9). D, PANC-1 control and ATF3-knockdown cells were treated with 1 mmol/L E64FC26 and 50 nmol/L panobinostat or DMSO for 16 hours. The Western blot analysis is shown. Casp3, caspase-3; Casp8, caspase-8. Arrow, cleaved bands. E, CHOP (also known as DDIT3) was stably knocked down in PANC-1 cells. Cells were treated for Western blot analysis (left) and cell viability (right; n ¼ 3). F, Change in panobinostat potentiation in PANC-1 (left) and T98G (right). Significance was determined by Student t test (n ¼ 6). G, PANC-1 cells were stably transduced with HERPUD1 shRNA or shCTL and treated for Western blot analysis (left) or cell viability (right; n ¼ 3). H, Change in panobinostat potentiation in PANC-1 (left) and T98G (right). Significance was determined by Student t test (n ¼ 6).

“A” and “A1” (24). Interestingly, we observed striking enrichment occupancy at promoter “A,” which closely matches the pattern of of H3K27-ac at promoter “A,” particularly with the combination increased histone acetylation (Fig. 6B). These data suggest that (P ¼ 0.001); whereas H3K18-ac surrounded both promoter regions HDACi alter the acetylation status of key histone residues sur- (P ¼ 0.001). In comparison, none of the treatments, single agents or the rounding the ATF3 promoter region leading to enhanced RNA Pol combination, altered the H3K9-ac status (Supplementary Fig. S20). To II recruitment, ATF3 transcription, and the antitumor synergy of understand the functional consequence of histone acetylation ﬂanking the combination. Taken together, our mechanistic studies led us to the ATF3 promoter regions, we conducted additional ChIP assays to develop the molecular model presented in Fig. 6C. PDI inhibition evaluate the recruitment of RNA polymerase II (RNA Pol II) to the promotes protein folding errors, the accumulation of misfolded ATF3 promoters. We found that single-agent E64FC26 led to RNA Pol polyubiquitinated proteins, and an ER stress response. ATF3 is a II occupancy on both ATF3 promoters (Fig. 6A), which is consistent critical ER stress–responsive transcription factor that is induced with previous reports demonstrating usage of both promoters follow- downstream of PDI inhibition and increased proteotoxic stress. In ing induction of ER stress (24, 39). Consistent with gene and protein the presence of HDACi, the histones surrounding ATF3 promoters expression analyses, panobinostat alone failed to recruit RNA Pol II to are acetylated promoting enhanced RNA Pol II recruitment and the ATF3 promoter. Most notably, the combination of E64FC26 with increased ATF3 transcription. ATF3 then induces a select set of panobinostat led to a selective and signiﬁcant increase in RNA Pol II gene targets, including members of the HSP70 and HSP40 family,

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Figure 5. ATF3 induces the transcription of HSP family members. A, PANC-1 cells were stably transduced with control shRNA (shCTL) or ATF3 shRNA (shATF3). Cells were treated with 1 mmol/L E64FC26 alone or in combination (combo) with 50 nmol/L panobinostat (pano) for 16 hours. RNA was extracted and RNA-seq performed. The

Venn diagram shows the number of transcripts that changed significantly when compared with DMSO-treated cells (Padj < 0.05). B, Transcripts were filtered for log2 (fold change) > |2.5| and fragment per kilobase of transcript per million mapped reads (FPKM) > |10|. The heatmap shows expression (FPKM) of the indicated transcripts. C, Stably transduced PANC-1 cells were treated with 1 mmol/L E64FC26 and 50 nmol/L panobinostat for 16 hours. The Western blot analysis is shown. D, Stable PANC-1 ATF3-knockdown and control cells were treated with 1 mmol/L E64FC26 (26) alone or in combination with 50 nmol/L panobinostat for 16 hours. Graphs show the fold change in mRNA expression. Significance was determined by Student t test (n ¼ 3). E, PANC-1 cells were stably transduced with shCTL or DNAJB1 shRNA. Cells were treated with 1 mmol/L E64FC26 and 50 nmol/L panobinostat for 16 hours and collected for Western blot analysis (left). Arrow, cleaved band. Fold change in panobinostat potentiation was calculated from cell viability curves after treatment for 48 hours (right). Significance was determined by Student t test (n ¼ 6). F, PANC-1 cells were stably transduced with shCTL or HSPA6 shRNA. Cells were treated with 1 mmol/L E64FC26, 50 nmol/L panobinostat, the combination (26þ pano), or DMSO control for 16 hours. RNA was extracted and RT-qPCR performed. Fold change in gene expression is shown (left; n ¼ 3). Fold change in panobinostat potentiation was calculated from cell viability curves after treatment for 48 hours (right). Significance was determined by Student t test (n ¼ 6).

which drive the synergistic antitumor effects of PDI and HDAC antitumor activity. We conducted a combination study with panobi- inhibitor combinations. nostat and E64FC65 (intravenous, 10 mg/kg, 2/week), which, like E64FC26, was highly synergistic with panobinostat in cellular assays In vivo efficacy of PDI and HDAC inhibitor combinations in a (Fig. 7A–C; Supplementary Fig. S14B) and demonstrated a favorable mouse model of glioblastoma pharmacokinetic profile in mice, including high systemic exposure and Given the unmet need for new therapies in glioblastoma and the wide tissue distribution to major organ systems (Fig. 7D). We promising activity of PDI and HDAC inhibitor combinations in panels observed central nervous system (CNS) accumulation of E64FC65, of established human cell lines, we decided to screen glioblastoma suggesting blood–brain barrier penetration, however, the maximum – patient derived neurospheres for similar activity. We observed sig- drug concentration (Cmax) and duration of CNS exposure were on the nificant synergy between panobinostat and two indene PDI inhibitors, low end of the spectrum. Panobinostat was given 3/week at 5 mg/kg E64FC26 and E64FC65 (Fig. 7A and B). We next used the (i.p.), which is considered a low dose based on reports in the literature U87 glioblastoma subcutaneous xenograft model, given its proven using the U87 xenograft model (41). Low dose E64FC65 and pano- utility in published reports (40, 41), to evaluate PDI þ HDACi binostat monotherapies reduced tumor growth, although the effect did

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Figure 6. Hyperacetylation surrounding the ATF3 promoter is associated with greater RNA Pol II occupancy. A, Schematic showing the ATF3 gene. Black boxes represent exons and white boxes represent promoters. Primers are indicated as P1, P2, and P3 (top). PANC-1 cells were treated with 1 mmol/L E64FC26, 50 nmol/L panobinostat (pano), the combination (26 þ pano), or DMSO control for 16 hours. Chromatin was fragmented and incubated with an IgG control antibody or H3K27-ac (n ¼ 12), H3K18-ac (n ¼ 6), or RNA Pol II (n ¼ 9) antibody overnight. ChIP assays were performed. The percent input was calculated for each primer region by comparing the DDC change in expression as determined by the 2 t method between samples that were incubated with primary antibody overnight and fragmented chromatin input samples. Thus, 10% input indicates that 10% of the total amount of fragmented chromatin showed acetylation or RNA Pol II occupancy at the primer site. Statistical signiﬁcance was determined using Student t test. B, Schematic representing the histone acetylation sites and RNA Pol II occupancy along the ATF3 gene. C, Representative schematic explaining the mechanism of synergy between PDI and HDAC inhibitors.

not reach statistical significance (vehicle vs. E64FC65, P ¼ 0.0549 and Discussion vehicle vs. panobinostat, P ¼ 0.0685). The combination, on the other HDACi have been largely ineffective in solid tumors despite the hand, dramatically reduced tumor growth compared with either single widely recognized role that epigenetics play in tumor formation and agent alone, reaching the minimum level of statistical significance as progression. Pairing epigenetic drugs with select agents in combina- early as day 16 (P < 0.05; Fig. 7E). The antitumor effect of the tion regimens has generated antitumor efficacy in hematologic can- combination became increasingly evident throughout the course of cers, although effective combinations in solid tumors have remained treatment. For example, on day 35, these tumors were considerably elusive. Here, we present previously uncharacterized synergy between smaller by statistical measures compared with the vehicle (P ¼ 0.0002), a new class of indene PDI inhibitors and HDACi. This combination E64FC65 alone (P ¼ 0.0069), and panobinostat alone (P ¼ 0.0003). was effective across a broad range of hematologic and solid tumors, Notably, tumors in combination-treated mice showed minimal growth including some of the deadliest cancer types like glioblastoma and during the course of treatment, and tumors only began to progress pancreatic cancer. after treatment was withdrawn on day 42. The antitumor response we A potential challenge facing the clinical translation of this combi- observed with the combination of E64FC65 þ panobinostat is com- nation is that HDACi have known dose-limiting toxicities (14, 45). parable with the effects of standard-of-care agents such as temozolo- Although, pharmacodynamic studies suggest that the current dosing mide and radiotherapy in the U87 model based on recent published level of HDACi at or near the maximum-tolerated dose may not be data (42–44). Notably, we maintained mice on the combination necessary. These studies have shown effective induction of HDACi treatment regimen for 42 days without signs of toxicity, demonstrating pharmacodynamic biomarkers at doses far below current prescribing the tolerability and therapeutic index of the combination regimen. practices (46, 47). In representative glioblastoma and pancreatic cancer

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Figure 7. E64FC26 and panobinostat reduce glioblastoma tumor growth. A, Primary patient glioblastoma neurospheres (GBM12 and GBM28) were treated with panobinostat

(pano) in the presence or absence of 1 mmol/L E64FC26 and 1 mmol/L E64FC65 (65) for 48 hours. A representative cell viability curve (left) and the change in EC50 values are shown (right). Statistical signiﬁcance was determined by Student t test (n ¼ 6). B, Representative images of GBM12 neurospheres treated with 1 mmol/L E64FC65, 50 nmol/L panobinostat, the combination (65 þ pano), or DMSO for 48 hours. C, PANC-1 and T98G cells were treated with a dose range of panobinostat in the presence or absence of 1 mmol/L E64FC65 (65) for 48 hours (n ¼ 6). D, Mice were treated with E64FC26 intravenously (i.v.) or orally (p.o.) for the indicated time.

Left, plasma concentration (Plasma conc), half-life (t1/2), steady state volume of distribution (Vss), plasma clearance (CLp), and bioavailability (%F) were calculated. Right, E64FC65 concentration at the time of intravenous injection was calculated for the indicated tissues. PK, pharmacokinetic. E, U87 subcutaneous xenograft mice were treated with 10 mg/kg E64FC65 intravenously two times per week (n ¼ 10), 5 mg/kg panobinostat intraperitoneally three times per week (n ¼ 9), the combination (65 þ pano; n ¼ 11), or DMSO vehicle control (n ¼ 10). The days of treatment (Rx duration; days 0–42) are highlighted. Statistical significance was determined by Student t test comparing tumor growth in the combination group with vehicle, E64FC65 monotherapy, and panobinostat monotherapy (, P < 0.01). Data points are shown as mean SEM. cell models, we show levels of synergy between PDI and HDAC progression, cellular stress, and apoptosis. The outcome of ATF3 inhibitors ranging from 25- to 250-fold. Because of the potentiation transcriptional regulation appears to be both cell type and stress type observed, we used a low dose and intermittent dosing schedule of dependent. In oncogenesis, reports in the literature are conflicting with panobinostat in our preclinical glioblastoma mouse model and were studies suggesting a protumorigenic role, while others demonstrate a able to demonstrate efficacy and tolerability of the combination with tumor suppressor function (32, 33). There is more agreement on the our lead PDI inhibitor. Our results reinforce the rationale for targeting role of ATF3 within the scope of response to therapy, as studies epigenetics in cancer, but suggest that optimal combination regimens overwhelmingly support an antitumor function (48–53), findings that are required. If successful, these combinations have the potential to are consistent with our observations. We propose the molecular model bring forth previously unrealized antitumor efficacy of HDACi and in Fig. 6C whereby ATF3 is transcriptionally upregulated in response may allow for reduced dosing to alleviate toxicity concerns. to protein folding errors and ER stress that are induced by PDI Mechanistically, we demonstrate that PDI and HDAC inhibitor inhibition. In the presence of HDACi, we detect increased acetylation synergy is driven by ATF3, which is a point of convergence down- of histone lysine residues surrounding the ATF3 promoter region. stream of PDI and HDAC inhibition. ATF3 is a member of the These acetylation events, which mark transcriptionally active chro- CREB/ATF family of transcription factors that acts as an adaptive matin, are associated with increased RNA Pol II occupancy at the ATF3 response protein, regulating genes involved in inflammation, cell-cycle promoters, heightened ATF3 transcription, and a proapoptotic

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signaling cascade that also involves upregulation of HSP40 and inhibition may be an active mechanism in solid tumors, but one that HSP70 gene family members DNAJB1 and HSPA6. To our knowledge, requires amplification through combinations with synergistic-targeted this is the first characterization of an ATF3–HSP40/70 apoptotic therapies. Our findings support the use of PDI inhibitors to enhance signaling axis. Additional studies are warranted to determine whether the antitumor activity of HDACi in glioblastoma, pancreatic cancer, this is a generalized apoptotic mechanism or one that is specific to the and other tumor types. unique combination of PDI and HDAC inhibitors. We identified a relatively small and highly specific set of ATF3- Disclosure of Potential Conflicts of Interest dependent genes belonging to the HSP40 and HSP70 families. This was R.M. Robinson reports a patent for WO/2019/046368 issued. N.G. Dolloff reports somewhat unexpected given that HSPs are thought to play a prosur- grants from Leukogene Therapeutics Inc. during the conduct of the study as well as vival function and maintain cellular proteostasis by stabilizing protein other from Leukogene Therapeutics Inc. (equity interest) outside the submitted work and has a patent for US-2020-0165182-A1 issued. No potential conflicts of interest folding. HSP40 family members recognize and escort misfolded were disclosed by the other authors. polypeptides to HSP70 proteins. This association stimulates the ATPase function of HSP70 to refold proteins into their native con- Authors’ Contributions formations (54). It is, therefore, not clear how HSPs drive an antitumor R.M. Duncan: Conceptualization, formal analysis, investigation, methodology. response. However, a major distinction is that our study is focused L. Reyes: Investigation. K. Moats: Investigation. R.M. Robinson: Investigation. specifically within the context of PDI and HDAC inhibition. It is S.A. Murphy: Investigation. B. Kaur: Supervision. H.A.F. Stessman: possible that HSPs function differently in the face of different forms of Investigation. N.G. Dolloff: Conceptualization, resources, formal analysis, cellular stress. In support of this hypothesis, we observed different supervision, funding acquisition, validation, investigation, methodology, writing- original draft, project administration. regulation of HSPs following heat shock stress compared with combined treatment with PDI and HDACi. We found that HSP70 and Acknowledgments DNAJB1 were induced independently of ATF3 under heat stress This work was supported by grants from the South Carolina Center of Biomedical compared with their ATF3 dependence following PDI and HDAC Research Excellence in Oxidants, Redox Balance and Stress Signaling (P20GM103542 inhibition (Supplementary Fig. S19B). A proapoptotic role for HSP70 to N.G. Dolloff), the American Cancer Society (RSG-14-156-01-CDD to N.G. where NF-B signaling is inhibited following TNFa stimulation has Dolloff), the NIH/NCI (1R41CA213488 and R42CA213488 to N.G. Dolloff), the been reported (55–58), as well as DNAJB1 and HSP70 stabilization of South Carolina Clinical & Translational Research Institute with an academic home at caspase-activated DNase (CAD) downstream of T-cell activation– the Medical University of South Carolina (MUSC; UL1 RR029882 and UL1 TR000062 to N.G. Dolloff), the NIH/NCATS CTSA (TL1 TRF001451 and UL1 TR001450 to dependent apoptosis (59). Future studies are required to determine P.V. Halushka), shared resources of the MUSC Hollings Cancer Center (P30 whether these proapoptotic mechanisms are recruited by HSP70 and CA138313), and by the Hollings Cancer Center T32 Ruth L. Kirschstein National DNAJB1 and contribute to the antitumor effects we report here. Research Service Award Training Program T32 (CA193201). Funding for this project Altogether, our study provides a novel combination strategy that was also provided by the State of Nebraska Department of Health and Human Services enhances the antitumor efficacy of both HDAC and PDI inhibitors, Cancer and Smoking Disease Research Programs (LB595 and LB692 to H.A.F. classes of drugs that have either performed poorly in the clinic or are Stessman). emerging agents in oncology, respectively. Pancreatic cancer and The costs of publication of this article were defrayed in part by the payment of page glioblastoma have some of the highest mortality rates among all charges. This article must therefore be hereby marked advertisement in accordance fi cancers, and the current standards of care lack ef cacy. Even with with 18 U.S.C. Section 1734 solely to indicate this fact. the number of new agents in development, pancreatic cancer remains in the top five most deadly and prominent cancers, and glioblastoma Received December 30, 2019; revised April 6, 2020; accepted June 16, 2020; survival rate is dismally low. Despite clinical trial failures, HDAC published first June 19, 2020.

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AACRJournals.org Cancer Res; 80(16) August 15, 2020 OF13

ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulfide Isomerase Inhibitors

Ravyn M. Duncan, Leticia Reyes, Katelyn Moats, et al.

Cancer Res Published OnlineFirst June 19, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-4046

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/06/19/0008-5472.CAN-19-4046.DC1

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