Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells In

Published OnlineFirst August 23, 2018; DOI: 10.1158/0008-5472.CAN-18-1229

Cancer Tumor Biology and Immunology Research

Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efﬁcacy of Immunotherapy in Mice Ayumi Hashimoto1, Chan Gao2, Jerome Mastio1, Andrew Kossenkov1, Scott I. Abrams3, Ashok V. Purandare2, Heshani Desilva2, Susan Wee2, John Hunt, Maria Jure-Kunkel2, and Dmitry I. Gabrilovich1

Abstract

The role of myeloid cells as regulators of tumor progression CCAAT-enhancer binding protein-a in this effect. Although that significantly impact the efficacy of cancer immunothera- CK2 inhibition did not directly affect tumor cells, it dramat- pies makes them an attractive target for inhibition. Here we ically enhanced the antitumor activity of immune checkpoint explore the effect of a novel, potent, and selective inhibitor of receptor blockade using anti-CTLA-4 antibody. These results serine/threonine protein kinase casein kinase 2 (CK2) on suggest a potential role of CK2 inhibitors in combination modulating myeloid cells in the tumor microenvironment. therapies against cancer. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced Significance: These findings demonstrate the modulatory the amount of polymorphonuclear myeloid-derived suppres- effects of casein kinase 2 inhibitors on myeloid cell differen- sor cells and tumor-associated macrophages. This effect was tiation in the tumor microenvironment, which subsequently not caused by the induction of apoptosis, but rather by a block synergize with the antitumor effects of checkpoint inhibitor of differentiation. Our results implicated downregulation of CTLA4. Cancer Res; 78(19); 5644–55. 2018 AACR.

Introduction abundant (<20%) population are pathologically activated monocytes, termed monocytic MDSC (M-MDSC). It is now evident that the ultimate success of cancer immuno- We have previously demonstrated that defective DC differen- therapy depends on its ability to overcome limitations imposed by tiation in cancer and the accumulation of PMN-MDSC was tumors. Moreover, the same is probably true for other types of mediated by downregulation of Notch signaling (5). These results oncologic therapies. Myeloid cells are critical components of the were later confirmed by others (6). Inhibition of Notch signaling tumor microenvironment widely implicated in suppressing antitu- was partially caused by phosphorylation of Notch by casein kinase morimmunity (1–3). These cells are composed of tumor-associated 2 (CK2; ref. 5). This suggested that targeting CK2 could be macrophages (TAM), dendritic cells (DC), neutrophils, and path- potentially beneficial for improving immune responses in cancer. ologically activated immature myeloid cells with the potent ability CK2 is a serine/threonine protein kinase with numerous intra- to suppress immune responses, termed myeloid-derived suppressor cellular protein substrates. CK2 phosphorylates its substrates by cells (MDSC). In recent years, it has become clear that MDSC are not utilizing either ATP or GTP as a phosphate donor. CK2 expression only animportant negative regulator of immune responses, but also and activity are elevated in many types of cancer. Functionally, it is contribute to other aspects of tumor growth, namely tumor angio- has been observed that activation of CK2 is associated with genesis, tumor cell invasion, and formation of pre-metastatic niches suppression of apoptosis of cancer cells (7–9). Conversely, down- (4). In tumor-bearing (TB) mice, the total population of MDSC regulation of CK2 expression or inhibition of its activity facilitates consists of two large groups of cells; the most abundant (>75%) cell death triggered upon drug exposure, ligation of death recep- population are immature, pathologically activated neutrophils, tors, and ionizing radiation (8, 10). Inhibition of CK2 sensitized termed polymorphonuclear MDSC (PMN-MDSC) and the less T-lymphoblastic cells to drug-induced apoptosis by increasing cellular uptake of the drug (11). CK2 inhibition also increased the sensitivity of rhabdomyosarcoma and colon carcinoma cells to 1Wistar Institute, Philadelphia, Pennsylvania. 2Bristol-Myers Squibb, Princeton, TRAIL-induced apoptosis (12), and induced ROS-mediated apo- New Jersey. 3Roswell Park Comprehensive Cancer Center, Department of Immunology, Buffalo, New York, Medimmune, Gaithersburg, Maryland. ptosis of leukemia cells (8). CK2 regulates a number of signaling pathways involved in tumor progression and also is critical for cell Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). differentiation. Speci cally, the phosphatase, PTEN, regulates cell survival by blocking the PI3K–AKT axis and has been shown to be Current address for M. Jure-Kunkel: Medimmune, Gaithersburg, Maryland. phosphorylated by CK2 (13), which facilitates its proteasomal Corresponding Author: Dmitry I. Gabrilovich, The Wistar Institute, 3601 Spruce degradation (14). As a result of the inhibition of PTEN by CK2, the St., Philadelphia, PA 19104. Phone: 215-495-6955; Fax: 215-573-2456; E-mail: AKT survival signal is prolonged (15). In addition, CK2 can [email protected] directly phosphorylate AKT at Ser129 (16). CK2 has also been doi: 10.1158/0008-5472.CAN-18-1229 implicated in direct targeting of the NF-kB and Wnt signaling 2018 American Association for Cancer Research. pathways (16–18).

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CK2 inhibitor and MDSC

In this study we tested the effects of novel potent and selective the CD8 T cell depletion study, LLC tumor bearing mice were CK2 inhibitors and found that in TB, but not in tumor-free mice, intraperitoneally injected with 100 mg of anti-CD8 alpha antibody they caused a substantial decrease in PMN-MDSC and macro- (BioXcell, clone 53–6.7) on day 3, 0, 3, 7, 10, and 14. phage differentiation, and dramatically enhanced the effect of anti-CTLA-4 immune-based therapy. In vitro cytotoxic activity The tumor cell lines were plated at 2 103 cells/well into 96-well Materials and Methods plates and cultured 16 hours. BMS-595 was dissolved with DMSO and diluted with media, and added into the wells and incubated for Human samples 3 days. The cell viability was measured using MTS assay (Promega). Cord blood samples were collected at Helen F. Graham Cancer IC values were calculated using linear regression analysis. Center. All patients signed written informed consent form. The 50 studies were conducted in accordance with recognized ethical Flow cytometry guidelines and were approved by Institutional Review Board of Single-cell suspensions were prepared from spleens and bone the Christiana Care Health System at the Helen F. Graham Cancer marrow. Tumortissues were cut intosmall pieces, and digested with Center and The Wistar Institute. the Mouse Tumor Dissociation Kit (Miltenyi Biotec). Red blood cells in the cell suspensions were lysed using ammonium chloride Mice lysis buffer. The list of antibodies is provided in Supplemental Table All procedures were performed accordance with the NIH Guide S1. Flow cytometry data were acquired using a BD LSR II flow for the Care and Use of Laboratory Animal guidelines and cytometer and analyzed using FlowJo software (Tree Star). approved by the Institutional Animal Care and Use Committee at The Wistar Institute. Female 5- to 6-week-old C57BL/6 mice and Mouse hematopoietic progenitor cells culture Balb/c mice (Charles River Labs and Envigo) were maintained in a Lineage negative cells were purified from C57BL/6 na€ve bone temperature controlled room with a 12/12-hour light/dark sched- marrow using the Lineage Cell Depletion Kit (Miltenyi). Lineage ule and food provided ad libitum. negative cells were cultured in RPMI (Corning Incorporated) supplemented with 10% FBS, 1% antibiotics, and 50 mmol/L CK2 inhibitors 2-mercaptoethanol (Thermo Fisher Scientific Inc.) with 20 ng/mL BMS-211 (FW: 825.24) is a prodrug of the parent pan-CK2 of recombinant GM-CSF (Invitrogen), at 50,000 cells/well in inhibitor, BMS-699 (Details to be published). Suspension for- 24-well plates. Tumor explant supernatants (TES) were obtained mulations for BMS-211 were prepared in 0.5% methocel-A4m, by culturing small pieces of EL4 tumors with complete RPMI 0.1% TWEEN-80. Particle size of the suspension was typically media for 24 hours. On day 1, TES or media with DMSO or m 10 m (D50). Lower concentration formulations were prepared BMS-595 solution was added into the wells at 10% to get the final by appropriate dilution (v/v) of the highest concentration for- concentration of 10 or 100 nmol/L BMS-595. On day 3, half of mulation. BMS-595 (FW: 532.44) is also pan-CK2 inhibitor and the culture supernatant was exchanged with fresh media supple- ¼ was solved with vehicle (Kolliphor TPGS:EtOH:PEG300 1:1:8) mented with 20 ng/mL of GM-CSF, with or without 10% TES. On in vivo for experiments. day 6, the cells were collected and analyzed by flow cytometry. To assess proliferation, hematopoietic progenitor cells (HPC) were Combination therapy cultured with 10 mmol/L of BrdUrd for 24 hours, and then Lewis lung carcinoma (LLC), CT26 colon carcinoma, 4T1 breast analyzed by flow cytometry using the BrdUrd Detection Kit carcinoma, and EL4 lymphoma were obtained from ATCC and (BD Pharmingen). cultured in DMEM (Corning Incorporated) supplemented with 10% FBS (Atlanta Biologicals, Inc.) and 1% antibiotics (Thermo Human HPCs culture fi þ Fisher Scienti c Inc.). Cells were incubated in a 37 Cand5%CO2. CD34 HPCs were purified from human cord blood by Ficoll- 70% to 80% confluent cells were harvested using 0.25% Trypsin based density gradient centrifugation followed by CD34 magnetic (Thermo Fisher Scientific Inc.) and passaged or used for experiments. beads selection (Miltenyi Biotec). OP9 mouse bone marrow Cells were tested on mycoplasma contamination every 3 months. stromal cells were cultured in the six-well plate at 104 cells/well 6 þ þ Balb/c mice were subcutaneously implanted with 1 10 4T1 or the day before CD34 purification. On day 0, CD34 cells were 6 CT26 cells in 0.2 mL PBS on day 0 and C57BL/6 mice with 1 10 cocultured with OP9 cells at 105 cells/well in Iscove's modified 5 MC38 or 5 10 LLC cells in 0.2 mL PBS on day 0. Six or seven days Dulbecco's medium supplemented with 20% FBS, 1% antibiotics, post-implantation, TB mice were randomly divided into four groups 20 ng/mL human GM-CSF, and 100 ng/mL human G-CSF (Pepro- (n ¼ 7–10). Mice were treated with BMS-211 or BMS-595 solution Tech). After overnight culture, tumor-conditioned media (TCM) orally daily for 21 days at 20 mg/kg or 60, respectively. In the 4T1 from RPMI8226 human myeloma cell lines was added at 10% model, anti-CTLA-4 (clone 9D9) mIgG2a antibody or the isotype with DMSO or 100 nmol/L BMS-595. On day 3 and 6, fresh media control was intraperitoneally injected into the mice at 20 mg/mouse containing 20 ng/mL GM-CSF, 100 ng/mL G-CSF, 10% TCM, and on days 6, 13, and 20. In MC38 model, anti-CTLA-4 mIgG2a DMSO or 100 nmol/L BMS-595 was added in the wells. On day 9, antibody or the isotype control was injected intraperitoneally into the cells were collected and analyzed by flow cytometry. themiceat200mg/mouse on days 6, 13, and 20. In CT26 model, anti-CTLA-4 mIgG2a antibody was intraperitoneally injected at 5 Suppression assay þ mg/mouse on days 7, 14, and 21, and anti-CTLA-4 mIgG2b Ly6G cells were purified from spleen cells or tumor cells by antibody was intraperitoneally injected at 200 mg/mouse on days positive selection using biotinylated Ly6G antibody and strepta- 7, 14, and 21. In LLC model, anti-CTLA-4 mIgG2a antibody was vidin microbeads (Miltenyi). The purity of the cell populations þ intraperitoneally injected at 200 mg/mouse on day 7 and 14. For was >95%. CD11b Ly6ChighLy6G cells were isolated from

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Hashimoto et al.

spleen cells by cell sorting on a FACSAria cell sorter (BD was used to estimate raw read counts and FPKM values using þ Biosciences). CD8 T cells from PMEL mice that recognize the transcriptome information from Ensemble v84 set. DESeq2 (21) gp100-derived peptide were used as responders. Splenocytes from was used to estimate significance of differential expression PMEL mice were mixed with splenocytes from na€ve mice at 1:4 between five replicates of untreated and BMS-595–treated sam- ratio in complete RPMI media and plated into 96-well U-bottom ples. Gene expression changes were considered significant if þ þ plates at 105 cells/well. Ly6G or CD11b Ly6ChighLy6G cells passed FDR <5% threshold and had expression for all replicates were added to the wells at 1:16–1:1 ratios. Murine gp100 peptide from one group higher or lower than any replicates of another (25–33) EGSRNQDWL (AnaSpec, Inc.) was added into the wells group. Gene set enrichment analysis of gene sets was done using at the final concentration of 0.1 mg/mL. After 48 hours, cells were QIAGEN's Ingenuity Pathway Analysis software (IPA; QIAGEN pulsed with 3H-thymidine (1 mCi/well; GE healthcare) for 16 Redwood City, www.qiagen.com/ingenuity) using "Canonical hours. 3H-thymidine uptake was counted using a liquid scintil- Pathways," "Diseases & Functions," and "Upstream Analysis" lation counter as counts per minute (cpm) and calculated the options. Only significantly enriched at P < 0.05 functions percentage of proliferation to the positive control (the wells with and regulators with significantly predicted activation state responder cells and peptide but without suppressive cells). (Z-score |Z| > 2) were considered. Pathways significantly affected at FDR <5% with predicted activation stated Z-score of at least 1 Apoptosis were reported. The data were submitted to the Gene Expression þ þ þ Ly6G cells and CD11b Ly6G F4/80 Ly6Clow macrophages Omnibus (GEO) database and can be accessed using accession were treated with BMS-595 for 24 to 72 hours. The cells were stained number: GSE117712. with FITC-Annexin V (BD Pharmingen) in Annexin V binding buffer (BD Pharmingen) for 30 minutes at room temperature, and then Statistical analysis suspended with DAPI containing MACS buffer and analyzed by flow Statistical analyses were performed using two-tailed Student cytometry. HPCs were cultured under the same condition as previ- t test or Mann–Whitney U test and GraphPad Prism 5 software ously described with BMS-595 and 12.5 and 25 mmol/L of Z-VAD- (GraphPad Software Inc.). Paired t test was used since data were FMK (Calbiochem), and then analyzed using flow cytometry. normally distributed. All the data are presented as mean SD and P value less than 0.05 was considered significant. Quantitative RT-PCR RNA was extracted with a total RNA Extraction Kit (Omega Bio-tek). cDNA was synthesized (cDNA Reverse Transcriptase Kit; Results Applied Biosystems), and PCR was performed in triplicate for CK2 inhibitor has potent antitumor effects in combination with each sample. To detect expression of Notch-, IRF8-, CCAAT- anti-CTLA4 antibody enhancer binding protein-a (C/EBPa-), or C/EBPb-regulated Potent and selective, ATP-competitive CK2 inhibitors genes, qRT-PCR was performed with 10 mL SYBR Master Mixture (BMS-595, BMS-699, and BMS-211) were developed by Bristol- (Applied Biosystems) and the list of the primers is provided in Myers Squibb (Supplementary Fig. S1A). Inhibition of prolifer- Supplemental Table S2. Expressions of the different genes were ation of various mouse cell lines (LLC, CT26 colon carcinoma, normalized to b-actin. Relative expression was calculated using EL4 lymphoma) were at relatively high IC50 values between –DD the 2 Ct method. 250 nmol/L and 1 mmol/L, and the 4T1 breast adenocarcinoma cells were even less sensitive (Supplementary Fig. S1B). The Western blot analysis treatment of immunocompetent C57BL/6 mice bearing LLC Nuclear and cytoplasm extracts were obtained from HPC tumors (Fig. 1A) or immunodeficient NSG mice bearing 4T1 or treated with DMSO or 100 nmol/L of BMS-595 for 24 hours. MC38 tumors with CK2 inhibitors demonstrated modest direct Nuclear protein was extracted using CelLytic NuCLEAR Extraction antitumor effects of the compounds (Supplementary Fig. S1C and Kit (Sigma) with phosphatase inhibitor cocktail (Sigma). To S1D). BMS-595 inhibited the in vitro proliferation of human retrieve the cytoplasm lysate, the cells were lysed with RIPA buffer colorectal and lung cancer cell lines with an IC50 ranging from (Sigma) supplemented with a protease inhibitor cocktail (Sigma) 10 nmol/L to greater than 1 mmol/L (Supplementary Fig. S2). In and a phosphatase inhibitor cocktail. Denatured protein samples sensitive cell lines, antiproliferative effects of BMS-595 and its were separated by electrophoresis with 25 mg/lane and transferred structurally related analog strongly correlated with CK2 kinase onto PVDF membrane. The list of antibodies provided in Sup- inhibition. plemental Table S1. The detected proteins were visualized by the However, although as a single-agent neither the CK2 inhibitor ECL system (GE Healthcare Life Sciences). nor the immune check-point inhibitor, anti-CTLA-4-mIgG2a antibody at low dose, potently impacted antitumor activity, in RNA-seq combination, they showed remarkable antitumor effects in all LLC-TB mice were treated with vehicle or 60 mg/kg of BMS-595 tested tumor models (LLC, 4T1, MC38, and CT26; Fig. 1A). for 2 weeks. HPC was isolated from the bone marrow of the Complete rejection (CR) was observed in 60% to 90% of mice vehicle- or BMS-595-treated mice. Total RNA was extracted from treated with the combination therapy (Fig. 1A). Because anti- the HPC using the Qiagen's Rneasy Mini Kit and RNA quality was CTLA-4-mIgG2a has the potential to deplete Treg cells in tumors, assessed using the Agilent RNA ScreenTape Assay on Agilent's in a separate set of experiments, we tested anti-CTLA-4-mIgG2b 2200 TapeStation. RNA-seq library was prepared using TruSeq antibody, which was reported not to deplete intratumoral Treg Stranded Total RNA Kit (Illumina) and ran in a 75bp paired end cells in the CT26 model (22). In 4T1 model, anti-CTLA-4-mIgG2b run, 40 million reads on Illumina NextSeq instrument. did not deplete Treg cells in the tumors (Fig. 1B). Combination of RNA-seq data was aligned using bowtie2 (19) against mm10 the CK2 inhibitor with anti-CTLA-4-mIgG2b resulted in complete version of the human genome and RSEM v1.2.31 software (20) tumor rejection in all 10 mice used in the experiment (Fig. 1C),

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CK2 inhibitor and MDSC

Figure 1. BMS-211 and BMS-595 enhanced the effect of immunotherapy in combination with anti-CTLA-4 antibody. A, Individual tumor growth curves in four subcutaneous murine syngeneic tumor models treated with CK2 inhibitor, anti-CTLA-4 mIgG2a antibody, or the combination as indicated. CR, complete response. B, 4T1 tumor-bearing mice were treated with anti-CTLA-4 mIgG2b, anti-PD1 antibody, or control mIgG at 20 mg/mouse on day 6 and 10. On day 12, the tumor cells were analyzed by flow cytometry. C, CT26 tumor bearing mice were treated with anti-CTLA-4 mIgG2b alone or in combination with CK2 inhibitor. Doses and treatment schedule are described in Materials and Methods. D, Individual tumor growth curve, followed by CT26 or MC38 rechallenge. Mice from indicated groups, who rejected CT26 or MC38 tumors, were rechallenged subcutaneously 90 days later in the opposite site with 1 106 CT26 or MC38 tumor cells, respectively. As control, naive mice were also inoculated with the same cells (n ¼ 10). E, LLC tumor-bearing mice were treated with BMS-595 and anti-CD8 antibody (n ¼ 7). suggesting that Treg depletion is not involved in the effects PMN-MDSC was not significantly changed, whereas populations mediated by CK2 inhibition. Mice that completely rejected tumor of TAMs and DC were decreased (Fig. 2A). CK2 inhibition caused in the combination treatment groups were rechallenged with the significant, albeit a modest decrease in granulocytic cells in the same tumor cells after 90 days. All of these mice rejected a bone marrow (BM) (Fig. 2A). The discrepancy between a secondary tumor challenge, indicating that immune memory was decreased presence of granulocytic cells in the BM and spleen generated in the initial treatment (Fig. 1D). Anti-CD8 antibody after CK2 inhibitor treatment and the lack of changes in the tumor injection of LLC TB mice completely cancelled the antitumor site could be explained by the fact that the decrease of granulocytic þ efficacy of CK2 inhibitor, which suggested that CD8 T cells were cells in the periphery may not be sufficient to prevent the migra- major effector cells in CK2 inhibitor-induced antitumor effect tion of substantial numbers of PMN-MDSC to the tumor site. To (Fig. 1E). Thus, although the CK2 inhibitor had limited antitumor verify this possibility, we depleted MDSC from spleens and blood effects, it has potent antitumor activity in combination with using agonistic DR5 (TRAIL-R) antibody previously shown to CTLA-4 immune check-point blockade and this effect was medi- selectively deplete a substantial portion of PMN-MDSC in TB mice ated by an immunological mechanism. (23). Consistent with previous observations, treatment with DR5 antibody alone had minor antitumor activity (Fig. 2B), while Effect of CK2 inhibition on host cells in the tumor causing a significant reduction in PMN-MDSC in the spleens of microenvironment mice (Fig. 2C). However, treatment with DR5 antibody did not To better understand the mechanism of action of the CK2 change the presence of PMN-MDSC in the tumor site (Fig. 2C). inhibitor, we investigated the presence of myeloid cells and Because the CK2 inhibitor affected populations of granulocytic lymphocytes in LLC TB mice treated for 2 weeks with BMS-595. cells and M in TB mice, we tested its effect on these cells in na€ve, The treatment did not affect the presence of T, B, or NK cells in tumor-free mice. Two-week treatment with BMS-595 did not spleens or tumors of TB mice. It also did not affect populations of change the proportion of neutrophils in the BM or the absolute þ þ CD11b Ly6G Ly6Chi M-MDSC and CD11c I-Abhi DC. Howev- number of these cells in the spleen. Similarly, no changes were þ þ er, substantial decreases in the presence of CD11b Ly6G Ly6Clo seen in the presence of splenic M (Fig. 2D). þ PMN-MDSC and CD11b Ly6G F4/80hi macrophages (M) were We verified that the populations of granulocytic and monocytes observed in the spleen (Fig. 2A). In tumors, the frequency of cells in the spleen and tumor of TB mice were immune suppressive

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Figure 2. BMS-595 induced the changes in myeloid cell populations. A, LLC TB mice were orally treated with vehicle or 60 mg/kg of BMS-595 for 2 weeks (n ¼ 8–10). Single cell suspensions were prepared from spleen, tumor, and bone marrow and analyzed by ﬂow cytometry. , P < 0.05 in two-tailed Student t test. B and C, LLC TB mice were treated for 10 days with DR5 antibody. Tumor growth (B) and the proportion of spleen and tumor PMN-MDSC (C) was measured. Each group included four mice. , P < 0.05 in two-tailed Student t test. D, Tumor-free na€ve C57BL/6 mice were orally treated with vehicle or 60 mg/kg of BMS-595 for 2 weeks (n ¼ 7). Then spleen and bone marrow were processed to single cells and analyzed by ﬂow cytometry.

and can be indeed identified as PMN-MDSC and M-MDSC, days of culture, BMS-595 decreased the proportion and the respectively (Supplementary Fig. S3). Treatment with the CK2 absolute number of granulocytic cells (Fig. 3D). In contrast, the inhibitor did not abrogate suppressive activity of these cells proportion and the number of monocytic cells were increased (Supplementary Fig. S3). Thus, the CK2 inhibitor caused a sig- by BMS-595. nificant reduction in the presence of PMN-MDSC in the BM and Such a dramatic decrease in granulocytic cells could be the spleens of TB mice and decreased the population of splenic M result of BMS-595 induced apoptosis, a block of proliferation of þ and TAMs. It did not, however, affect these cells in tumor-free the precursors, or inhibition of differentiation. Ly6G cells were mice. isolated after 3-day culture of HPC with GM-CSF and were treated, in the presence of GM-CSF, with BMS-595 for 24 hours. This time Mechanisms regulating the effect of CK2 inhibition on frame was selected because a 24-hour incubation of granulocytes PMN-MDSC and macrophages with GM-CSF sustained viability of granulocytes above 70%. After We then evaluated the effect of CK2 inhibition on the gener- 48 hours, the viability dropped below 40% and any effect of CK2 ation of myeloid cells. Treatment of LLC TB mice with BMS-595 inhibition on apoptosis is difficult to establish. The CK2 inhibitor did not affect common myeloid (CMP) and megakaryocyte– caused a very small decrease in the total number of cells recovered erythroid (MEP) progenitor cells in the BM, but slightly increased after incubation (Fig. 4A), which was associated with a modest the proportion of granulocyte-macrophage progenitors (GMP) (5%) upregulation of apoptosis (Fig. 4B). No difference in cell (Fig. 3A). This increase in GMP likely reflects compensatory proliferation was observed (Fig. 4C). changes resulting from the decreased presence of granulocytes To address the potential role of apoptosis in the substantial loss and macrophages in tissues. In the na€ve mice, BMS-595 treatment of granulocytic cells during HPC differentiation, experiments increased the proportion of GMP but decreased that of MEP were repeated in the presence of the apoptosis inhibitor z-VAD. (Fig. 3A). We assessed the differentiation of myeloid cells in vitro z-VAD at optimal doses (12–25 mmol/L) did not prevent inhibi- from enriched BM-derived HPCs with and without tumor explant tion of granulocytic cell differentiation by BMS-595 (Fig. 4D). supernatant (TES). BMS-595 had a modest effect on the total To clarify the effect of the CK2 inhibitor on differentiated number of cells generated from HPC during 6-day culture with PMN-MDSC, these cells were isolated from BM and spleens of GM-CSF (Fig. 3B). However, it caused a dramatic (almost three- LLC TB mice. BMS-595 caused a small decrease in the number of fold) decrease in the proportion of granulocytic cells (Fig. 3C). We cells recovered after a 24-hour culture of BM PMN-MDSC, and no evaluated the effect of BMS-595 on the myeloid cell differentia- increase in apoptosis was observed (Fig. 5A). Similar results were þ tion from CD34 progenitor cells from human cord blood. After 9 observed with treatment of splenic PMN-MDSC with the CK2

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CK2 inhibitor and MDSC

Figure 3. BMS-595 decreased differentiation of granulocytic cells from mouse and human progenitor cells. A, LLC TB mice or na€ve mice were treated with vehicle or 60 mg/kg of BMS-595 for 2 weeks (n ¼ 8–10). Bone marrow cells were processed and analyzed by flow cytometry. , P < 0.05 in two-tailed Student t test. B and C, HPCs were isolated from na€ve bone marrow and cultured with or without TES and 10 or 100 nmol/L BMS-595 for 6 days. B, Total number of cells. C, Proportion of granulocytic and monocytes cells. D, Human CD34þ progenitor cells were isolated from cord blood and cultured with TCM and 100 nmol/L BMS-595 for 9 days. Three independent experiments were performed in triplicates. Representative data are shown. , P < 0.05; , P < 0.01 from DMSO control in two-tailed Student t test; n ¼ 3. inhibitor (Fig. 5B). Altogether, these results indicate that cell death of granulocyte and macrophage differentiation is a novel finding. plays a relatively minor role in the decrease of PMN-MDSC by To elucidate this mechanism, LLC TB mice were treated for 2 CK2 inhibition in TB mice. It raised the possibility that the CK2 weeks with BMS-595. HPCs were isolated from the BM and inhibitor may interfere with granulocytic differentiation. transcriptome was evaluated by RNA-seq. CK2 inhibitors caused In parallel, we evaluated the effect of the CK2 inhibitor on M. changes in 2633 genes with FDR <5%. Most significantly changed First, we tested the hypothesis that the CK2 inhibitor causes TAM genes are presented in Fig. 6A. Although no changes in apoptosis þ cell death. CD11b Ly6CloLy6G F4/80hi M were sorted from the were determined in these cells, genes associated with apoptosis spleens and tumors of LLC TB mice and then were treated with were upregulated in HPC in mice treated with the CK2 inhibitor the CK2 inhibitor. BMS-595 did not affect the total number of M whereas downregulated pathways included oxidation of lipids (Fig. 5C) or cause apoptosis in these cells (Fig. 5D). Next, we (Fig. 6B). The CK2 inhibitor caused increased expression of genes evaluated the effect of the CK2 inhibitor on M differentiation. regulated by proinflammatory mediators: LPS, IL1b, TNF, NF-kB, We tested the possibility that the CK2 inhibitor can block differ- and IL6. It also included E1A-binding protein P400, which is entiation of M-MDSC to M. Monocytic cells were isolated from involved in transcriptional activation of E2F1, which is regulated bone marrow of naive mice and were cultured for 3 days with by the retinoblastoma (Rb) protein. Not surprisingly, Rb1 was BMS-595 in the presence of TES. The CK2 inhibitor significantly one of the regulators that were prominently inhibited by BMS-595 reduced the proportion of macrophages generated from mono- in HPC (Fig. 6). Consistent with previous results, the CK2 inhib- cytic cells. The proportion of DC generated from monocytic cells itor caused increased expression of genes regulated by Notch was not changed (Fig. 5E). These results indicate that BMS-595 signaling (Supplementary Fig. S4). However, it did not prevent inhibited differentiation of PMN-MDSC and macrophages from the substantial decrease in PMN-MDSC. Therefore, we further their precursors without causing apoptosis of these cells. studied genes regulated by transcription factors that were previously directly implicated in PMN-MDSC differentiation: IRF8, Molecular mechanisms regulating the effect of the CK2 C/EBPa, and C/EBPb. BM-derived HPC from TB mice were treated inhibitor on myeloid cells with GM-CSF and BMS-595 for 24 and 48 hours. CK2 inhibition Although the pro-apoptotic effects of CK2 inhibition on tumor caused a significant increase in the expression of only few genes cells is well established (7, 8, 24), the role of CK2 in the inhibition regulated by IRF8 (Fig. 7A). To test whether this upregulation was

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Figure 4. BMS-595 did not induce apoptosis in granulocytes. A and B, HPCs were cultured with 20 ng/mL of GM-CSF for 1 day and treated with 10% of TES. On day 3, Ly6Gþ cells were purified and treated with 100 nmol/L of BMS-595 for 24 hours. A, Total number of cells collected from the culture. B, Apoptosis in the cells was analyzed by Annexin V staining using flow cytometry. C, Cell cycle of cells was analyzed after 24-hour culture of HPCs with 20 ng/mL of GM-CSF in the presence of 10% of TES, 100 nmol/L of BMS-595, and 10 mmol/L of BrdUrd. Anti-BrdUrd antibody and 7AAD were used in flow cytometry to evaluate cell cycle (n ¼ 3). D, HPCs were cultured with 20 ng/mL of GM-CSF, 10% of TES, 100 nmol/L of BMS-595, and 12.5 or 25 mmol/L of Z-VAD-FMK. On day 6, the cells were collected and analyzed by flow cytometry.

Figure 5. BMS-595 did not induce apoptosis of macrophage but inhibited their differentiation from monocytes. A and B, Ly6Gþ cells were purified from bone marrow (A) and spleen (B) of LLC TB mice and cultured with 10 or 100 nmol/L of BMS-595 for 24 hour. The cells were collected, and total number of cells was counted (left). In parallel, apoptosis was measured by Annexin V staining using flow cytometry. C and D, Macrophages (CD11bþLy6GF4/80þLy6Clow) were sorted from tumors of LLC TB mice and cultured for 1 or 3 days with 100 nmol/L BMS-595. C, Total number of cells. D, Proportion of apoptotic cells measured by Annexin V on day 3 (n ¼ 3). E, Monocytic cells (CD11bþLy6ChighLy6G) were sorted from na€ve bone marrow cells and cultured with 20 ng/mL GM-CSF, TES, and 100 nmol/L BMS-595 for 3 days. The cells were collected, and proportion of macrophages and DC were evaluated by flow cytometry (n ¼ 3).

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CK2 inhibitor and MDSC

Figure 6. BMS-595 caused changes in gene expressions. HPCs were isolated from the LLC TB mice treated with vehicle or 60 mg/kg of BMS-595 for 2 weeks and used for RNA- seq analysis. A, Most changed genes between the groups. B, Changes in pathways, functions, and regulators between treated and not treated groups. sufﬁcient to block granulocytic differentiation, we used HPC BMS-595 did not affect genes regulated by C/EBPb, however, it isolated from the BM of IRF8 knockout (KO) mice. CK2 inhibitor caused downregulation of many genes controlled by C/EBPa (Fig. caused similar reduced differentiation of granulocytes from WT 7A). C/EBPa is a critical regulator of myeloid differentiation. and IRF8 KO HPC (Fig. 7B), which exclude possible involvement Active form of C/EBPa is C/EBPa-p42 protein. C/EBPa-p30 of IRF8 in CK2 inhibitor-mediated effects. Treatment of HPC with protein is produced by alternative transcription initiation. It

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Figure 7. BMS-595 increased dominant-negative p30 subunit of C/EBPa. A, HPCs were cultured with 20 ng/mL of GM-CSF overnight and then treated with 10% TES and 100 nmol/L BMS-595 for 24 and 48 hours. Total RNA was extracted and gene expression was analyzed by qRT-PCR. n ¼ 3, , P < 0.05; , P < 0.01 in two-tailed Student t test. B, HPCs from WT or IRF-8-KO mice (n ¼ 3) were cultured with TES and 100 nmol/L BMS-595 for 6 days, and analyzed by ﬂow cytometry. , P < 0.01; , P < 0.001 in Student t test. C and D, HPCs were cultured overnight with 20 ng/mL of GM-CSF and then treated with 10% TES and 100 nmol/L BMS-595 for 24 hours. Cells were collected and lysed to obtain cytoplasm and nuclear extracts. C, C/EBPa, b-actin, and Histone H3 were detected by Western blot analysis. D, Phosphorylations on C/EBPa (S21,T222/226,andS193) were detected by Western blot analysis.

retains the DNA-binding domain but lacks the N terminal trans- cies (7, 8, 24). However, its effect on myeloid cells was described activation domain of the longer form of C/EBPa-p42. C/EBPa- in only several studies. CK2 was implicated in the regulation p30 exhibits a dominant negative function over C/EBPa-p42 (25, of phagocytosis by macrophages (27). It was also involved in 26). Treatment of HPC with the CK2 inhibitor reduced the the regulation of p47phox and p40phox components of NADPH presence of the functionally active p42 subunit of C/EBPa in the oxidase in neutrophils and macrophages (28–30). No informa- cytoplasm and increased the dominant negative p30 subunit of C/ tion existed on the effect of CK2 inhibition on myeloid EBPa in both the cytoplasm and nucleus (Fig. 7C). No differences cell differentiation. were found in phosphorylation of three major sites of C/EBPa Based on known mechanisms of action, we expected that the (S21,T222/226,S193; Fig. 7D). These results suggest possible mech- CK2 inhibitors would cause apoptosis of myeloid cells. However, anism regulating function of C/EBPa by CK2 inhibition. this was not the case. Instead, its effect was associated with a block of differentiation from the precursors of granulocytes and macro- Discussion phages. Because M-MDSC differentiate into TAM in tumors and M in the spleens, this may explain the observed substantial This study was based on previous observations that CK2 may be decrease of M after the treatment. PMN-MDSC do not differ- involved in the regulation of MDSC and DC differentiation in entiate from precursors in tumors and accumulate there after cancer via inhibition of Notch signaling (5). We suggested that migration from the circulation. The decrease of PMN-MDSC inhibition of CK2 activity with potent and selective inhibitors caused by CK2 inhibition was observed in the BM and spleen could improve differentiation of DC and potentially antitumor but not in tumors. This can be explained by the fact that despite immune responses. In TB mice, the effect of the CK2 inhibitors on depletion, there was still enough PMN-MDSC in circulation to DC was modest. Unexpectedly, we observed a dramatic decrease migrate to the tumor site. The biological effect of CK2 inhibition in the presence PMN-MDSC and TAM. This effect was not apparently involves a systemic decrease of PMN-MDSC that observed in tumor-free mice. It is possible CK2 activity is more attenuates the suppression of T cells expanded in peripheral critical for abnormal myelopoiesis observed in TB mice. CK2 lymphoid organs and depletion of TAM in tumors that decrease inhibitors dramatically enhanced the antitumor activity of anti- local suppression. CTLA-4 antibody in several mouse tumor models. Since direct In the search for a mechanism of how CK2 inhibition could antitumor effects of CK2 inhibitor are marginal, it suggested that affect myeloid cell differentiation, we found that CK2 inhibition regulation of myeloid cells could be responsible for the observed signiﬁcantly reduced the expression of genes regulated by the synergistic antitumor effect. CK2 inhibition has a known C/EBPa transcription factor, a leucine zipper transcription factor pro-apoptotic effect in various solid and hematologic malignan- mainly involved in monopoiesis and granulopoiesis. It also

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CK2 inhibitor and MDSC

can affect gene expression independent of DNA-binding via however, that CK2 utilizes different sites for phosphorylation. In interaction with E2F1 (31, 32), HDAC1 or HDAC3 (33, 34). A our study, we observed up-regulation of the 30 kDa dominant- þ þ low level of Cebpa RNA expression is detected in Lin Sca-1 c-Kit negative isoform of C/EBPa-p30 by the CK2 inhibitor. It is known (LSK) cells; Cebpa expression increases twofold in CMP and that p30 can compete for C/EBPa specific promoters with the tenfold in GMP (35). Reduced levels of C/EBPa may contribute active p42 isoform (52). Our data suggest that CK2 may affect the to monopoiesis by hetero-dimerizing with AP-1 proteins such as balance of these isoforms and thus inhibit C/EBPa activity. It is c-Jun and c-Fos (36, 37). This may explain the fact that we did not possible that the sensitivity of monocytic cells to CK2 inhibitors is observe changes in monocytic cells generated from HPC in the different at different stages of maturation or CK2 inhibitors may presence of the CK2 inhibitor. C/EBPa induces transcription of affect other cell types (for instance, fibroblasts) that support several regulatory proteins required for subsequent lineage mat- myeloid cell differentiation. These possibilities require further uration. This includes the transcription factors C/EBPe,Gfi-1, elucidation. KLF5 (38). C/EBPa cooperates with PU.1, c-Myb, and RUNX1 Thus, our study demonstrates a novel effect of CK2 inhibition, to activate genes such as myeloperoxidase, neutrophil elastase, resulting in a decrease of immune suppressive and tumor- lysozyme, lactoferrin, CSF1R, CSF2R, CSF3R in immature gran- promoting population of PMN-MDSC and TAM in TB mice. This ulocytic or monocytic cells (39). Although the role of C/EBPa in resulted in a very substantial augmentation of antitumor activity granulocytic differentiation is well established, it is only in recent of immune therapy. Our results suggest a novel mechanism years that evidence has emerged suggesting it has a possible by which CK2 may interfere with myeloid cell differentiation, regulatory role in the differentiation of M. Transient expression leading to the inhibition of C/EBPa activity. Taken together, of C/EBPa and PU.1 in THP-1 cells synergistically promoted small molecule inhibitors targeting CK2 should be considered differentiation of monocytes to M (40). C/EBPa enabled the as a potentially valuable addition to immune-based combination induction of a monocytic cell differentiation program (41). Our therapies. results suggest that activation of CK2 in cancer can contribute to accumulation of PMN-MDSC and M. The specific molecular Disclosure of Potential Conflicts of Interest mechanisms by which CK2 influences their accumulation war- A. Hashimoto is a senior researcher at Daiichi Sankyo Co., Ltd. Heshani rants further investigation. Desilva has ownership interest (including stock, patents, etc.) in BMY. M. IRF8 is an essential transcription factor for the development of Jure-KunkelisadirectoratBristol-MyersSquibbandhasownership interest (including stock, patents, etc.) in Bristol-Myers Squibb. No poten- myeloid cells. It plays an important role in the switch between tial conflicts of interest were disclosed by the other authors. monocytic and granulocytic differentiation (42, 43) and its downregulation was implicated in the accumulation of PMN-MDSC Authors' Contributions (44–46). It was previously shown that IRF8 can physically interact Conception and design: A. Hashimoto, C. Gao, A.V. Purandare, M. Jure-Kunkel, with C/EBPa and prevents its binding to chromatin blocking the D.I. Gabrilovich ability of C/EBPa to stimulate transcription and neutrophil Development of methodology: A. Hashimoto, A.V. Purandare, D.I. Gabrilovich / differentiation. A partial inhibition of C/EBP activity in irf8 Acquisition of data (provided animals, acquired and managed patients, hematopoietic progenitors alleviates the neutrophil overproduc- provided facilities, etc.): A. Hashimoto, C. Gao, J. Mastio, H. Desilva, J. Hunt Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tion in vivo (47). In our experiments, CK2 inhibition did not / computational analysis): A. Hashimoto, C. Gao, J. Mastio, A. Kossenkov, reverse granulocyte hyperproduction from irf8 progenitors S.I. Abrams, S. Wee, M. Jure-Kunkel, D.I. Gabrilovich in vitro, suggesting that its effect was independent of Irf8. C/EBPa Writing, review, and/or revision of the manuscript: A. Hashimoto, C. Gao, is phosphorylated at several sites, indicating a potential role for J. Mastio, A. Kossenkov, S.I. Abrams, A.V. Purandare, H. Desilva, S. Wee, posttranslational modifications in mediating C/EBPa activity. M. Jure-Kunkel, D.I. Gabrilovich Several kinases were implicated in phosphorylation of C/EBPa. Study supervision: D.I. Gabrilovich 21 Other (design and synthesis of novel CK2 inhibitors used in the publication): S phosphorylation is mediated by Erk1/2 and interferes with A.V. Purandare granulocytic differentiation (48). Phosphorylation of C/EBPa at Other (major role in designing the previously undisclosed CK2 inhibitor S248 is mediated by activated Ras, leading to the enhanced ability used in biological experiments): J. Hunt of C/EBPa to upregulate the expression of the granulocyte-colony stimulating factor receptor (49). Phosphorylation at S193 blocks Acknowledgments proliferation of hepatocytes (50). Dephosphorylation of S193 This work was supported in part by grant from Bristol-Myers Squibb and by promotes proliferation by preferentially binding the retinoblas- NIH grant CA 084488 to D. Gabrilovich. toma protein (Rb) in the repressive Rb–E2F complex. A role for 193 The costs of publication of this article were defrayed in part by the payment of S in modulating granulopoiesis has not been reported. Finally, page charges. This article must therefore be hereby marked advertisement in 222 phosphorylation at T was associated with glycogen synthase accordance with 18 U.S.C. Section 1734 solely to indicate this fact. kinase 3 (GSK3 kinase) activity, which regulates preadipocyte differentiation (51). We did not observe changes in phosphory- Received April 23, 2018; revised July 9, 2018; accepted July 31, 2018; lation of C/EBPa caused by the CK2 inhibitor. It is possible, published first August 23, 2018.

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Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efficacy of Immunotherapy in Mice

Ayumi Hashimoto, Chan Gao, Jerome Mastio, et al.

Cancer Res 2018;78:5644-5655. Published OnlineFirst August 23, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1229

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