Histone Acetyltransferase KAT6A Upregulates PI3K/AKT Signaling Through TRIM24 Binding Deguan Lv1,2, Feng Jia3, Yanli Hou4, Youzhou Sang2, Angel A

Published OnlineFirst October 11, 2017; DOI: 10.1158/0008-5472.CAN-17-1388

Cancer Tumor and Stem Cell Biology Research

Histone Acetyltransferase KAT6A Upregulates PI3K/AKT Signaling through TRIM24 Binding Deguan Lv1,2, Feng Jia3, Yanli Hou4, Youzhou Sang2, Angel A. Alvarez5, Weiwei Zhang2, Wei-Qiang Gao2,BoHu5, Shi-Yuan Cheng2,5, Jianwei Ge3, Yanxin Li6, and Haizhong Feng2

Abstract

Lysine acetyltransferase KAT6A is a chromatin regulator that PI3K/AKT signaling and tumorigenesis. Overexpressing activated contributes to histone modification and cancer, but the basis of its AKT or PIK3CA rescued the growth inhibition due to KAT6A actions are not well understood. Here, we identify a KAT6A silencing. Conversely, the pan-PI3K inhibitor LY294002 abrogat- signaling pathway that facilitates glioblastoma (GBM), where it ed the growth-promoting effect of KAT6A. Overexpression is upregulated. KAT6A expression was associated with GBM of KAT6A or TRIM24, but not KAT6A acetyltransferase activity– patient survival. KAT6A silencing suppressed cell proliferation, deficient mutants or TRIM24 mutants lacking H3K23ac-bind- cell migration, colony formation, and tumor development in an ing sites, promoted PIK3CA expression, AKT phosphorylation, orthotopic mouse xenograft model system. Mechanistic investi- and cell proliferation. Taken together, our results define gations demonstrated that KAT6A acetylates lysine 23 of histone an essential role of KAT6A in glioma formation, rationalizing H3 (H3K23), which recruits the nuclear receptor binding protein its candidacy as a therapeutic target for GBM treatment. TRIM24 to activate PIK3CA transcription, thereby enhancing Cancer Res; 77(22); 6190–201. 2017 AACR.

Introduction deregulated in cancer, including glioma, partially due to aberrant histone modifications and mutations (2–4). However, the Glioblastoma (GBM) is the most common primary malignant mechanisms by which histone modifiers regulate gliomagenesis cancer of the central nervous system with a grim median survival are still being elucidated. of less than 1.5 years upon diagnosis (1). Recent global genomic Lysine acetyltransferase 6A (KAT6A, also known as MYST3 and and transcriptome analyses reveal that the epigenetic landscape is MOZ) is a MYST-family histone acetyltransferase, which controls fundamental cellular processes, including gene transcription (5), cellular senescence (6), cardiac septum development (7), memory 1State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical T-cell diversity (8), and maintenance of normal hematopoietic Stem Cell Research Center, Ren Ji Hospital, School of Biomedical Engineering, stem cells (9). Dysregulation of the KAT6A histone acetyltransfer- Shanghai Jiao Tong University, Shanghai, China. 2Renji-Med X Clinical Stem Cell 3 ase activity or aberrant expression of KAT6A has been associated Research Center, Ren Ji Hospital, Shanghai, China. Department of Neurosur- – gery, Ren Ji Hospital, Shanghai, China. 4Department of Radiotherapy, Ren Ji with oncogenesis in leukemia (10 13) and breast cancer (14). fi fi Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. KAT6A's oncogenic activity was rst identi ed as a recurrent 5Department of Neurology, Northwestern Brain Tumor Institute, The Robert H. fusion partner of the CREB binding protein (CBP) as a conse- Lurie Comprehensive Cancer Center, Northwestern University Feinberg School quence of gene translocation, t(8;16)(p11;p13) in the FAB 6 of Medicine, Chicago, Illinois. Key Laboratory of Pediatric Hematology and M4/M5 subtype of acute myeloid leukemia (AML; ref. 13). Then, Oncology Ministry of Health, Pediatric Translational Medicine Institute, Shanghai the KAT6A-TIF2 fusion was identified as a consequence of another Children's Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. translocation, inv(8)(p11;p13) in AML. This AML fusion event requires the KAT6A nucleosome binding motif and TIF2-medi- Note: Supplementary data for this article are available at Cancer Research ated recruitment of CBP (10). KAT6A is composed of tandem Online (http://cancerres.aacrjournals.org/). PHD fingers, a MYST domain, an acidic region, and a Ser/Met D. Lv, F. Jia, and Y. Hou contributed equally to this article. (SM)-rich domain (15). KAT6A binds and acetylates histones via Corresponding Authors: Haizhong Feng, State Key Laboratory of Oncogenes its MYST domain (15). In addition, KAT6A and the related factor and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Lysine acetyltransferase 6B (KAT6B, also known as MORF) form a Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, tetrameric complex with ING5 (Inhibitor of growth 5), EAF6 China; Phone: 8621-6838-3921; Fax: 8621-6838-3916; E-mail: [email protected]; Yanxin Li, Pediatric Translational Medicine Institute, (Esa1-associated factor 6 ortholog), and the bromodomain-PHD fi Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong nger protein BRPF1 (15). Increasing evidence suggests that University, Shanghai 200127, China. Phone: 8621-3862-6295; E-mail: KAT6A is implicated in regulating tumor progression (16), but [email protected]; and Jianwei Ge, Department of Neurosurgery, Ren Ji mechanisms that facilitate its oncogenic activity independent of Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, gene fusion remain elusive. China. Phone: 8621-6838-3768; E-mail: [email protected] In this study, we firstly examined KAT6A expression in glioma doi: 10.1158/0008-5472.CAN-17-1388 cells and clinical GBM specimens. We then assessed the role of 2017 American Association for Cancer Research. KAT6A in cell proliferation, cell migration, and tumor growth in

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gliomas using cell culture and orthotopic xenograft models. Colony formation assay Finally, we determined the mechanisms by which KAT6A regu- Soft agar colony formation assays were performed as we lates glioma tumorigenesis. described previously (21). Briefly, 1 Â 104 cells were seeded in a 0.4% noble agar top layer with a bottom layer of 0.8% noble agar Materials and Methods in each of the triplicate wells of a 12-well plate. Cell culture media Cell lines was changed every 3 days thereafter. Colonies were scored after – LN428, LN340, U87, LN229, D54, T98G, U251, and LN444 2 3 weeks and data were analyzed. GBM cells were from ATCC, and were cultured in 10% FBS/ DMEM. All cell lines in this study were authenticated using STR shRNA knockdown and transfection DNA fingerprinting, most recently in March 2017 by Shanghai Lentiviruses were produced by cotransfecting various Biowing Applied Biotechnology Co., Ltd (Shanghai, China), and cDNA and packaging plasmids into HEK293T cells using mycoplasma infection was detected using LookOut Mycoplasma Lipofectamine 2000 reagent according to manufacturer's PCR Detection Kit (Sigma-Aldrich). Only lower passage cell lines instruction (#52758, Invitrogen). Forty-eight hours after trans- were used for the study. fection, the viral-containing supernatants were filtered and added into the culture media supplemented with 8 mg/mL polybrene. Transduced human GBM cells were harvested and Plasmids expression of exogenous proteins was validated by Western Flag-TRIM24 was a gift from Michelle Barton (University of blot analysis. Texas M.D. Anderson Cancer Center, Houston, TX; Addgene plasmid # 28138; ref. 17), pCIG-PIK3CA was a gift from Joseph RNA isolation and qRT-PCR Gleeson (Rockefeller University, New York, NY; Addgene plasmid Total RNA was isolated from cells with TRIzol Plus RNA # 73056; ref. 18), and pLNCX-Myr-AKT was a gift from Joan Purification Kit (Thermo Fisher Scientific) for reverse transcrip- Brugge (Harvard Medical School, Boston, MA; Addgene plasmid # tion with Reverse Transcription Kit (Takara) according to the 17245; ref. 19). Wild-type KAT6A cDNA was amplified from U87 manufacturer's instructions. qRT-PCR was performed with the cells, and then inserted into a pcDNA3 vector (Invitrogen). Power SYBR Green Master Mix (Life Technologies) on the Applied KAT6AG657E, KAT6AC543G/G657E, and TRIM24F979A/N980A point Biosystems StepOne Plus Real-Time Thermal Cycling Block. mutations were generated using a site-directed mutagenesis kit ÀDDC Results were analyzed using the 2 t method. Primers are listed (Invitrogen) following the manufacturer's protocol. KAT6A in Supplementary Table S1. shRNAs were purchased from Genechem, Inc.

Chromatin immunoprecipitation–qPCR Immunoprecipitation and Western blotting assays Chromatin immunoprecipitation (ChIP) was performed using Cells were lysed in an immunoprecipitation (IP) buffer the Chromatin Immunoprecipitation Kit (Millipore-Upstate) (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L according to the manufacturer's instructions. Immunoprecipi- EDTA, 2 mmol/L Na3VO4, 5 mmol/L NaF, 1%Triton X-100, and fi tated DNA was puri ed after phenol extraction and was used for protease inhibitor cocktail) at 4 C for 30 minutes. The lysates were qRT-PCR. Primers are listed in Supplementary Table S1. centrifuged, and then protein concentrations were determined. Equal amounts of cell lysates were immunoprecipitated with Tumorigenesis studies specific antibodies and protein G-agarose beads (Invitrogen). All animal experiments were performed in accordance to a Standard Western blotting was done with antibody against b-actin protocol approved by Shanghai Jiao Tong University Institutional (I-19), Histone H3 (FL-136; Santa Cruz Biotechnology); Flag Animal Care and Use Committee. Athymic (Ncr nu/nu) female (Sigma-Aldrich); acetyl-Histone H3 (Lys23; #07-355, Millipore- mice at an age of 6–8 weeks (SLAC) were randomly divided into 5 Upstate); TRIM24 (#14208-1-AP, ProteinTech); KAT6A per group. A total of 5 Â 105 U87 GBM cells were stereotactically (H00007994-M07, Abnova); AKT (#4060S), phospho-AKT implanted into the brains to generate orthotopic xenografts. Mice (#9272S), acetyl-Histone H3 (Lys14)(D4B9), and acetyl-Histone were euthanized when neuropathologic symptoms developed. H3 (Lys9; C5B11; Cell Signaling Technology); BRPF1 Tumor volumes were measured and estimated as (W2 Â L)/2, W < (PA5-27783), KAT6B (PA5-37101), and PIK3CA (4F3) (Thermo L (22). Fisher Scientific); KAT5/Tip60 (C-7, sc-166323, Santa Cruz Biotechnology). IHC staining of human glioma specimens All the work related to human tissues were performed under the Cell proliferation and migration assays Institutional Review Board approved protocols approved at Cell proliferation analysis was performed using a WST-1 assay Shanghai Jiao Tong University, according to the Declaration of kit (Roche) and cell migration analysis was performed using a Helsinki, and the investigators obtained informed written consent Boyden chamber as described previously (20). Briefly, cells were from the subjects (wherever necessary). Human tissues including seeded in medium, split, and detected with a WST-1 assay kit. For 13 WHO Grade II, 22 WHO Grade III, and 111 primary human cell migration analysis, cells were serum-starved for 24 hours, GBM specimens were collected from Ren Ji Hospital, School of washed with PBS, and resuspended in DMEM plus 0.1% FBS. Medicine, Shanghai Jiao Tong University (Shanghai, China). The Then, cells were placed into the top compartment of a Boyden tissue sections from paraffin-embedded deidentified human chamber and the bottom chamber was added with 10% glioma specimens were stained with antibodies against KAT6A FBS/DMEM. After 16–18 hours, the membrane was fixed, stained, (1:50). IHC staining was scored as 0–3 according to the percentage and analyzed. of positive cells, as described previously (23) Two separate

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individuals who were blinded to the slides examined and scored tumorigenesis were assessed. Compared with the control xeno- each sample. graft models, knockdown of KAT6A significantly inhibited glioma tumor growth (Fig. 2H and I). These data support that KAT6A is Statistical analysis critical for cell proliferation, cell migration, and tumor growth in GraphPad Prism version 5.0 for Windows (GraphPad Software gliomas. Inc.) was used to perform one-way ANOVA with Newman–Keuls post hoc test or an unpaired two-tailed Student t test. Kaplan–Meier KAT6A regulates AKT activity survival analysis was carried out using log-rank tests. A Spearman As AKT activation is important for histone acetyltransferase – rank correlation analysis was used to investigate the correlation of function (27 30) and glioma cell proliferation (26), we deter- KAT6A gene expression levels in human clinical GBM specimens. A mined the effect of knockdown on phosphorylation of P value of less than 0.05 was considered significant. AKT (p-AKT) in U87 and LN229 GBM cells. As shown in Fig. 3A, knockdown of KAT6A markedly decreased AKT phosphorylation Results (Fig. 3A), suggesting that KAT6A regulates Akt activity in gliomas. To support that KAT6A mediates AKT activity, we overexpressed KAT6A expression is prognostic for clinical GBM a constitutively activated (CA) AKT (Myr-AKT) mutant in LN229 To identify whether KAT6A is important for glioma progres- GBM cell with KAT6A shRNAs or a control shRNA. As shown sion, we first assessed expression of KAT6A in clinical specimens of in Fig. 3B–D, compared with the control, overexpression of the CA patients. We downloaded two GBM microarray datasets, AKT mutant in LN229/shC cells promotes AKT phosphorylation GDS1962 (24) and GSE7696 (25), and examined KAT6A mRNA (Fig. 3B), cell proliferation (Fig. 3C), and colony formation (Fig. expression in clinical GBM samples and normal brain tissue. As 3D). Moreover, overexpression of the CA AKT mutant in LN229/ shown in Fig. 1A and B, KAT6A was expressed at higher levels in shKAT6A cells markedly restored AKT phosphorylation (Fig. 3B), GBM compared with normal brain tissue. cell proliferation (Fig. 3C), and colony formation in soft agar (Fig. Then, we performed IHC staining assays in the total 146 clinical 3D) inhibited by KAT6A knockdown. To rule out nonspecific brain tumors, including 13 WHO grade II, 22 WHO grade III, and changes or off-target effects that may account for our results, we 111 GBM. As shown in Fig. 1C, KAT6A staining was negative or employed direct genetic manipulations using CRISPR technology weak in WHO grade II tumors. KAT6A was expressed in the to directly validate the functions of KAT6A. We constructed majority of high-grade gliomas (WHO grade III and GBM KAT6A-knockout U87 and LN229 GBM cells using two different tumors), and was particularly high in GBM (Fig. 1C and D), sgRNAs, and found that KAT6A knockout markedly impaired AKT suggesting that the levels of KAT6A expression are correlated with phosphorylation in both GBM cells compared with the controls glioma grade. (Fig. 3E). Overexpression of the CA AKT mutant also restored Finally, we examined the relationship of KAT6A expression and KAT6A knockout–inhibited AKT phosphorylation (Fig. 3F), cell GBM patient survival by Kaplan–Meier survival analysis. As proliferation (Fig. 3G), and colony formation in soft agar (Fig. – shown in Fig. 1E, Kaplan Meier survival analysis revealed a 3H) in LN229 cells. These data demonstrate that KAT6A regulates statistically significant worse prognosis for GBM patients with AKT activity to promote glioma cell proliferation. high KAT6A protein levels compared with those with low. The median patient survival times of these patients were 9.97 and KAT6A upregulates PIK3CA expression in gliomas to activate P < 13.53 months, respectively ( 0.05). Taken together, these PI3K/AKT signaling observations strongly indicate that upregulation of KAT6A was As PI3K signaling pathway has a critical role in driving tumor closely associated with progression and poor prognosis in GBM initiation and progression and AKT is the dominant effector of patients. PI3K signaling (31), we assessed the effects of KAT6A knockdown on expression of phosphatidylinositol-4,5-bisphosphate 3-kinase Knockdown of KAT6A inhibits glioma cell proliferation, catalytic subunit alpha (PIK3CA) in U87 and LN229 GBM cells. As migration, colony formation, and tumor growth shown in Fig. 4A and B, KAT6A knockdown markedly inhibited To demonstrate the role of KAT6A in glioma tumorigenesis, we expression levels of PIK3CA protein and mRNA in both GBM cells. detected the levels of KAT6A protein and mRNA expression in We also detected mRNA expression levels of KAT6A homologous LN428, LN444, LN340, T98G, LN229, U87, and D54 GBM cells. KAT6B and KAT5 in U87 and LN229 GBM cells, and found that As shown in Fig. 2A and Supplementary Fig. S1A, KAT6A was their expressions are relatively lower than that of KAT6A in U87 ubiquitously expressed in all GBM cells, and expressed relatively and LN229 GBM cells (Supplementary Fig. S2A). Knockdown of higher in LN229 and U87 GBM cells compared with other cells. KAT6B or KAT5 did not impair PI3KCA protein (Supplementary Then, we used lentivirus-mediated short hairpin RNAs (shRNA) Fig. S2B and S2D) and mRNA (Supplementary Fig. S2C and S2E) of KAT6A or a control shRNA to deplete KAT6A in U87 and LN229 expression but increased AKT phosphorylation (Supplementary GBM cells (Fig. 2B; Supplementary Fig. S1B). As shown in Fig. 2C– Fig. S2B and S2D). Moreover, ChIP-qPCR assays using antibodies G, knockdown of endogenous KAT6A significantly inhibited cell against KAT6A or its partner BRPF1 (Fig. 4C and D) demonstrated proliferation, cell migration, and colony formation in soft agar in that KAT6A and BRPF1 bind to the PIK3CA promoter. These data both GBM cells. suggest that KAT6A mediates PIK3CA expression to regulate To further determine whether KAT6A is critical for glioma glioma cell proliferation. tumorigenesis, we employed an orthotopic GBM model as To validate that KAT6A regulates PIK3CA expression in glioma described previously (26). U87 GBM cells transduced with cells, we overexpressed PIK3CA in LN229 GBM cells with KAT6A shKAT6A-1, shKAT6A-2, or shControl were separately implanted shRNAs or a control shRNA. As shown in Fig. 4E–G, compared intracranially to generate orthotopic xenografts in immunocom- with the control, overexpression of PIK3CA in LN229/shC cells promised mice. Then, the effects of KAT6A depletion on glioma promotes AKT phosphorylation (Fig. 4E), cell proliferation (Fig.

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A B GDS1962 GSE7696 6 300 *** * 5 200 4 100 3 2 0 0 Relative KAT6A mRNA expression Figure 1. Relative KAT6A mRNA expression = 80) KAT6A expression is correlated with n Normal( brain = 4) glioma progression. A and B, GBM (n (n = 23) Expression levels of KAT6A mRNA are Normal brainGBM (n = 81) signiﬁcantly higher in GBM samples compared with normal brain tissues. C edarG II edarG III MBG Expression data of KAT6A mRNA were downloaded from the GDS1962 dataset (24) and the GSE7696 dataset (25) and analyzed. C, IHC staining of KAT6A in clinical glioma specimens. Scale bars, 50 mm. Arrows, positive staining. Data are representative of two independent experiments with similar results. D, Quantitative analysis of KAT6A protein expression in C. E, Kaplan–Meier analysis of patients with high KAT6A protein-expressing GBM KAT6A versus low KAT6A protein-expressing GBM. Statistical analysis was performed by log-rank test in a GraphPad Prism version 5.0 for Windows. Median survival (in months) for low KAT6A (13.53) and high KAT6A (9.97) protein expression. Black bars, censored data. Error bars, ÆSD. Ã, P < 0.05; ÃÃÃ, P < 0.05. Data are representative of two independent D E experiments with similar results. High KAT6A (n = 82) 5 120 * Low KAT6A (n = 29) 4 80 3 * 2 P < 0.05 40

1 Percent survival

0 0 Grade II Grade III GBM 030 06 09 Relative KAT6A protein expression n n n ( = 13) ( = 22) ( = 111) Months

4F), and colony formation (Fig. 4G). Furthermore, PIK3CA overexpression to activate PI3K/AKT signaling pathway, resulting in expression in LN229/shKAT6A cells markedly restored AKT phos- enhancing glioma tumorigenesis. phorylation (Fig. 4E), cell proliferation (Fig. 4F), and colony formation in soft agar (Fig. 4G) inhibited by KAT6A knockdown. KAT6A acetyltransferase activity is required for activating We also downloaded expression data of PIK3CA and KAT6A in PI3K/AKT signaling 547 GBM samples from The Cancer Genome Atlas (TCGA), and To determine potential mechanisms by which KAT6A regulates found that PIK3CA expression is correlated with KAT6A expres- PI3K/AKT signaling, we detected histone H3 acetylation using sion (Fig. 4H). These data suggest that KAT6A upregulates PIK3CA Western blot assays. H3K9, H3K14, and H3K23 were reported as

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A B C shC shKAT6A-1 1.5 shKAT6A-2

* * LN428LN444 LN340T98G LN229 U87 D54 shC shKAT6A-1shKAT6A-2shC shKAT6A-1shKAT6A-2 1.0 KAT6A KAT6A β-Actin β-Actin 0.5 U87 LN229 0 Relative cell proliferation D E U87 LN229 shC shKAT6A-1 shKAT6A-2 shC shKAT6A-1 1.5 shKAT6A-2

* * U87 1.0

0.5 Relative cell migration LN229 0 U87 LN229 F G shC shKAT6A-1 shKAT6A-2 shC shKAT6A-1 1.5 shKAT6A-2

U87 * * 1.0

0.5 LN229

Relative soft agar colony 0 U87 LN229

H I 60 ** U87 3 shC shKAT6A-1 shKAT6A-2 40

E&H 20 Tumor size (mm ) 0

shC

shKAT6A-1shKAT6A-2

Figure 2. Knockdown of KAT6A inhibits glioma cell proliferation, migration, colony formation in soft agar, and tumor growth. A, Western blotting analysis of KAT6A protein expression in GBM cells. b-Actin was used as a control. B, Western blot analysis of KAT6A knockdown using two different shRNAs (shKAT6A-1 and shKAT6A-2) or a control shRNA (shC). C, Effects of KAT6A knockdown on GBM cell proliferation. D, Representative cell migration images using a Boyden chamber assay of U87 and LN229 GBM cells with shC, shKAT6A-1, or shKAT6A-2. Scale bars, 400 mm. E, Quantification of GBM cell migration in D. F, Representative soft agar colony formation images. Scale bars, 300 mm. G, Quantification of soft agar colonies in F. H, Representative hematoxylin and eosin (H&E) staining images of mouse brain sections with U87/shC, U87/shKAT6A-1, or U87/shKAT6A-2 tumors. Brains were harvested at 6–7 weeks after transplantation. Scale bars, 1 mm. Data are from two independent experiments with 5 mice per group with similar results. I, Quantification of tumor size in H. Data are representative of two independent experiments with similar results. Error bars, ÆSD. Ã, P < 0.05; ÃÃ, P < 0.01.

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A B C shC shKAT6A-1 shKAT6A-2 * 2.0 * shC shKAT6A-1 shKAT6A-2 * shC shKAT6A-1shKAT6A-2shC shKAT6A-1shKAT6A-2 Myr-Akt - + - + - + * KAT6A 1.5 KAT6A 1.0 p-AKT p-Akt 0.5 AKT Akt

β-Actin Relative cell proliferation 0 β-Actin Myr-Akt - + - + - + U87 LN229 LN229 LN229 shC D shKAT6A-1 E F shKAT6A-2 3 * * KAT6A- EV sgRNA1 sgRNA2 KAT6A- * * EV sgRNA1sgRNA2shC sgRNA1sgRNA2 Myr-Akt - + - + - + 2 KAT6A KAT6A p-AKT p-Akt 1 AKT Akt β-Actin β- Relative soft agar colony 0 Actin Myr-Akt - + - + - + U87 LN229 LN229 LN229 G H EV KAT6A-sgRNA1 EV KAT6A-sgRNA2 KAT6A-sgRNA1 * KAT6A-sgRNA2 * 3 2.0 * * * * * * 1.5 2 1.0 1 0.5 Relative soft agar colony Relative cell proliferation 0 0 Myr-Akt - + - + - + Myr-Akt - + - + - + LN229 LN229

Figure 3. KAT6A depletion inhibits AKT activation. A, Western blot analysis of effects of KAT6A knockdown on AKT activation. B, Effects of overexpression of a constitutively activated Akt (Myr-AKT) mutant on KAT6A knockdown–inhibited Akt activation. C and D, Effects of overexpression of Myr-AKT on KAT6A knockdown– inhibited glioma cell proliferation (C) and soft agar colony formation (D). E, Western blot analysis of effects of KAT6A knockout using two different sgRNAs on Akt phosphorylation. EV, empty vector. F, Effects of overexpression of a constitutively activated AKT (Myr-AKT) mutant on KAT6A knockout-inhibited Akt activation. G and H, Effects of overexpression of Myr-AKT on KAT6A depletion–inhibited glioma cell proliferation (G) and soft agar colony formation (H). Error bars, ÆSD. Ã, P < 0.05. Data are representative of two independent experiments with similar results.

targets of KAT6A/KAT6B (16). As shown in Fig. 5A, compared with As the functions of H3K23 acetylation (H3K23ac) mediated by the control, histone H3, knockdown of KAT6A decreased the KAT6A in solid tumors is not well established, we focused to acetylation levels of H3K23, H3K9, and H3K14. Compared with determine H3K23ac roles in KAT6A-regulated glioma cell prolif- H3K9ac and H3K14ac, the acetylation levels of H3K23 were eration. To examine whether KAT6A acetyltransferase activity is particularly suppressed by KAT6A knockdown in LN229 and required for activating PI3K/AKT signaling, we generated two U87 GBM cells. KAT6A acetyltransferase activity–deﬁcient mutants, KAT6AG657E

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A B C shC KAT6A ChIP-qPCR shKAT6A-1 3 1.5 shKAT6A-2 ** shC shKAT6A-1shKAT6A-2shC shKAT6A-1shKAT6A-2 mRNA * * 2 KAT6A 1.0 promoter PIK3CA

PIK3CA levels 1 0.5 (% of input)

β-Actin PIK3CA 0

Relative 0 U87 LN229 U87 LN229 -884/-637-548/-251-264/-56 -2000/-1741-1127/-973 D E F shC BRPF1 ChIP-qPCR shKAT6A-1 3 shKAT6A-2 shC shKAT6A-1 shKAT6A-2 * 2.0 * ** PIK3CA - + - + - + 2 * * PIK3CA 1.5

1 p-Akt 1.0 (% of input)

PIK3CA promoter Akt 0.5 0 KAT6A Relative cell proliferation 0 -264/-56 PIK3CA - + - + - + -1127/-973-884/-637-548/-251 -2000/-1741 β-Actin LN229 LN229 G H n shC TCGA ( = 547) shKAT6A-1 15 shKAT6A-2 3 * * * 10 2 * 5 1 r = 0.4 P < 0.001

PIK3CA mRNA levles 0

Relative soft agar colony 0 467589 PIK3CA - + - + - + KAT6A mRNA levels LN229

Figure 4. KAT6A upregulates PIK3CA expression in glioma cells to activate PI3K/AKT signal. A, Effects of KAT6A knockdown on PIK3CA protein expression in U87 and LN229 GBM cells. B, qRT-PCR analysis of effects of KAT6A knockdown on PIK3CA mRNA expression. C and D, ChIP-qPCR assays of the binding of KAT6A (C) and BRPF1 (D) with the PIK3CA promoter. After ChIP using antibodies against KAT6A, BRPF1, or control IgG, qPCR assays were performed using primers corresponding to ﬁve different loci of the PIK3CA promoter. E–G, Overexpression of PIK3CA rescues KAT6A knockdown–inhibited AKT activation (E), glioma cell proliferation (F), and soft agar colony formation (G). H, Correlation of expression between PIK3CA and KAT6A in 547 GBM samples from TCGA dataset. Data are representative of two independent experiments with similar results. Error bars, Æ SD. Ã, P < 0.05. ÃÃ, P < 0.01.

and KAT6AC543G/G657E (10, 14). As shown in Fig. 5B–D, over- KAT6AG657E or KAT6AC543G/G657E mutant had no effects on expression of KAT6A wild-type (WT) in LN229 GBM cells pro- PIK3CA expression, H3K23 acetylation, AKT phosphorylation, moted PIK3CA expression, H3K23 acetylation, Akt phosphory- cell proliferation, and colony formation (Fig. 5B–D). These data lation, cell proliferation, and colony formation compared with demonstrate that KAT6A acetyltransferase activity is important for the control, empty vector (EV). However, overexpression of activating PI3K/AKT signaling.

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A B C LN229 * * KAT6A- EV WT G657E C543G/G657E 2.0 shC shKAT6A-1shKAT6A-2shC shKAT6A-1shKAT6A-2 KAT6A KAT6A 1.5 PIK3CA H3K23ac p-Akt 1.0 H3K9ac Akt 0.5 H3K14ac H3K23ac H3 Relative cell proliferation 0 H3 KAT6A- EV β-Actin WT β-Actin G657E U87 LN229 C543G/G657E LN229 D E LN229 KAT6A- EV WT G657E LY294002 - + - + - + 3 * * KAT6A

2 PIK3CA p-Akt 1 Akt H3K23ac

Relative soft agar colony 0 KAT6A- H3 EV WT G657E β-Actin

C543G/G657E LN229 F G LN229 LN229 3 * * 2.0 * *

1.5 2 * * 1.0 * * 1 0.5

Relative soft agar colony 0

Relative cell proliferation 0 LY294002 - + - + - + LY294002 - + - + - + KAT6A- EV WT G657E KAT6A- EV KAT6A G657E

Figure 5. KAT6A acetyltransferase activity is required for activating PI3K/AKT signaling. A, Western blot analyses of effects of KAT6A knockdown on the acetylation of H3K23, H3K9, H3K14, and KAT6A expression. Histone H3 and b-actin were used as controls. B, Overexpression of KAT6A WT but not acetyltransferase activity–deﬁcient mutant G657E or C543G/G657E restored KAT6A knockdown-inhibited activation of PI3K/AKT signaling pathway. C and D, Effects of overexpression of KAT6A wild-type, acetyltransferase activity-deﬁcient mutant G657E or C543G/G657E on cell proliferation (C) and colony formation (D)insoft agar in LN229 GBM cells. E–G, Inhibition of PI3K suppressed KAT6A-stimulated AKT phosphorylation (E), cell proliferation (F), and colony formation (G). Data are representative of two independent experiments with similar results. Error bars, ÆSD. Ã, P < 0.05.

Then, we investigated whether treatment with PI3K inhibitor mutation compared with the EV controls, whereas expression LY294002 suppresses KAT6A-promoted cell proliferation. As levels of PIK3CA and H3K23ac were unaffected by LY294002 shown in Fig. 5E, LY294002 signiﬁcantly inhibited AKT phos- treatment. Importantly, compared with the EV control, LY294002 phorylation in LN229 GBM cells transfected KAT6A WT or G657E signiﬁcantly inhibited KAT6A WT, but not KAT6A-G657E

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mutation, overexpression-promoted AKT phosphorylation in while overexpression of Flag-TRIM24-F979A/N980A mutant did LN229 GBM cells (Fig. 5E). We further examined the impact of not alter these growth characteristics (Fig. 6E–F). Taken together, LY294002 on in vitro KAT6A-promoted cell proliferation and our data demonstrate that KAT6A promotes glioma cell prolifer- colony formation, and found that LY294002 markedly sup- ation and tumor growth through H3K23ac/TRIM24–mediated pressed glioma cell proliferation and colony formation of KAT6A PI3K/AKT signaling pathway. WT–overexpressing, but not KAT6A-G657E mutation–overex- – pressing, GBM cells compared with the EV control (Fig. 5F G). Discussion These data further support that KAT6A acetyltransferase activity is required for the PI3K/AKT signaling pathway activation. In this study, we described a novel mechanism by which KAT6A-upregulated PI3K/AKT signaling through TRIM24 binding KAT6A promotes H3K23ac association with TRIM24 to mediate is critical for cell proliferation and tumor growth in gliomas PI3K/AKT signaling (Fig. 7). KAT6A promotes H3K23 acetylation and association Tripartite motif–containing 24 (TRIM24, also known as TIF 1a) with TRIM24, leading to increased PIK3CA expression and is a member of the transcription intermediary factor family (32), PI3K/Akt signaling activation, resulting in enhanced glioma and was reported to bind with H3K23ac to regulate the develop- tumorigenesis. ment of breast cancer (33) and prostate cancer (34). Moreover, Our data demonstrate that KAT6A functions as an oncogene in TRIM24 as a transcriptional activator upregulates PIK3CA mRNA gliomas. The mammalian KAT6A was firstly identified in AML expression and activates PI3K/AKT signaling pathway in gliomas patients (13). Previous work identified KAT6A interactions with (35). We hypothesized that KAT6A promotes H3K23 acetylation, ING5, EAF6, and BRPF1, -2, and -3 (36, 37) to form a macro- and then enhances the association of TRIM24 and H3K23ac. molecular complex to regulate normal hematopoietic stem cell Consequently, TRIM24 functions as a transcriptional activator to maintenance (9) and AML development (36). Recent KAT6A was upregulate PIK3CA expression, leading to activation of PI3K/AKT revealed to be amplified and overexpressed in breast cancers, and signaling, thereby enhancing glioma tumorigenesis. To test this depletion of KAT6A markedly inhibited cell proliferation and hypothesis, we first performed immunoprecipitation (IP) analysis tumor growth in breast cancer through regulating estrogen recep- in U87 and LN229 GBM cells with a KAT6A shRNA or a control tor a expression (ERa; ref. 14). Here, we show that KAT6A shRNA. As shown in Fig. 6A, TRIM24 interacted with H3K23ac in promotes glioma tumorigenesis. Expression of KAT6A was upre- both GBM cells. However, knockdown of KAT6A markedly atten- gulated in GBM samples. Moreover, evaluation of GBM patient uated the association of TRIM24 and H3K23ac. samples revealed a negative correlation between KAT6A expres- Next, we performed ChIP-qPCR assays on PIK3CA using anti- sion and survival in glioma patients. Knockdown of KAT6A by bodies directed against TRIM24 and H3K23ac to determine shRNAs inhibited glioma cell proliferation, cell migration in vitro, whether TRIM24 and H3K23ac bind to the PIK3CA promoter in and glioma tumorigenicity. These results show that KAT6A is LN229 GBM cells with, or without a KAT6A shRNA. As shown critical for glioma tumorigenesis. in Fig. 6B, both TRIM24 and H3K23ac could bind to the PIK3CA Our results also suggest that KAT6A mediates gliomagenesis promoter at À548 to À251 locus as reported previously (35). through PI3K/AKT signaling pathway. Although KAT6A has long Compared with the control (shC), KAT6A knockdown signifi- been linked to the regulation of cell proliferation in normal and cantly inhibited the binding of TRIM24 and H3K23ac with the cancer cells (14, 38), the mechanisms by which KAT6A regulates PIK3CA promoter. These data show that KAT6A regulates PIK3CA cell proliferation in solid tumors are still unclear. Inactivating Sas3 expression through the TRIM24/H3K23ac complex. (KAT6A homologous in yeast) acetyltransferase activity resulted – To further support that TRIM24 is important for KAT6A- in the accumulation of cells at the G2 M phase (38). Enok (KAT6A Drosophila – regulated PI3K/AKT signaling, we overexpressed Flag-tagged homologous in ) promotes the G1 S transition by TRIM24 WT or F979A/N980A mutant (F979A/N980A), which interacting with the Elg1 proliferating cell nuclear antigen inhibits H3K23ac association with TRIM24 (33), or an EV in (PCNA)-unloader complex (39). Our data show that knockdown LN229 GBM cells with a KAT6A shRNA or a control shRNA. As of KAT6A inhibited glioma cell proliferation and tumor growth shown in Fig. 6C, compared with the EV control, overexpression through PI3K/AKT signaling. Overexpression of PIK3CA or CA of Flag-TRIM24 WT promoted PIK3CA expression and Akt phos- AKT restored KAT6A knockdown-inhibited cell proliferation. phorylation, but not H3K23 acetylation in LN229/shC cells, KAT6A and its partner BRPF1 bind with PIK3CA and upregulate whereas overexpression of Flag-TRIM24 WT did not affect PIK3CA PIK3CA transcription in glioma cells. In contrast to a previous expression, AKT phosphorylation, and H3K23 acetylation in report in prostate cancer (28), we observed that knockdown of LN229/shKAT6A cells (Fig. 6C). Overexpression of Flag- KAT6B or KAT5 inhibited AKT phosphorylation but did not affect TRIM24-F979A/N980A mutant did not impair PIK3CA expres- PIK3CA expression in glioma cells, suggesting that KAT6B and sion, AKT phosphorylation, and H3K23 acetylation in both KAT5 may regulate PI3K/AKT signaling through different path- LN229/shC and LN229/shKAT6A cells compared with the EV ways in gliomas. Taken together, our results demonstrate that controls, respectively (Fig. 6C). Moreover, ChIP-qPCR analyses on histone acetyltransferases regulate cell proliferation through PIK3CA showed that overexpression of Flag-TRIM24 WT promot- PI3K/AKT signaling pathway. ed the binding of TRIM24 and H3K23ac with PIK3CA promoter in Our results further suggest that KAT6A upregulates PI3K/AKT LN229/shC cells but not in LN229/shKAT6A cells (Fig. 6D), signaling pathway through H3K23ac-binding TRIM24 transcrip- whereas overexpression of Flag-TRIM24-F979A/N980A mutant tional activation. KAT6A can acetylate nonhistone and histone had no effects on the binding of TRIM24 and H3K23ac with substrates in mammals, and functions as a positive or negative PIK3CA promoter (Fig. 6D). Consistently, overexpression of Flag- regulator (16). KAT6A interacts with transcriptional factors, such TRIM24 WT promotes cell proliferation and colony formation in as p53 and RUNX1, to activate gene transcriptions by acetylating LN229/shC cells but not in LN229/shKAT6A cells (Fig. 6E–F), histones near target gene promoters (15, 40). KAT6A can interact

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A B TRIM24 ChIP-qPCR H3K23ac ChIP-qPCR 3 * 3 * shC shC shKAT6AshC shKAT6A shC shKAT6AshC shKAT6A shKAT6A * H3K23ac IP KAT6A 2 2 * TRIM24 TRIM24 H3K23ac H3 1 1 TRIM24

IP (% of input)

H3K23ac H3K23ac TRIM24 Lysates PIK3CA promoter 0 0 U87 LN229 β-Actin U87 LN229 U87 LN229 U87 LN229 C D Flag-TRIM24- EV WT F979A/N980A TRIM24 ChIP-qPCR H3K23ac ChIP-qPCR shKAT6A - + - + - + 4 * * 3 EV * * WT Flag F979A/N980A 3 * * PIK3CA * * 2 2 p-Akt 1 (% of input) Akt 1 (% of input) PIK3CA promoter PIK3CA promoter H3K23ac 0 0 shKAT6A - + - + - + shKAT6A - + - + - + H3 LN229 LN229 KAT6A β-Actin LN229 F E EV EV WT WT F979A/N980A F979A/N980A 3 * * 2.0 * *

1.5 2 * * 1.0 * * 1 0.5

Relative soft agar colony 0 Relative cell proliferation 0 - + - + - + shKAT6A - + - + - + shKAT6A LN229 LN229

Figure 6. KAT6A promotes H3K23ac association with TRIM24 to mediate PI3K/AKT signaling. A, Immunoprecipitation (IP) and Western blot analyses of effects of KAT6A knockdown on H3K23ac association with TRIM24. B, ChIP-qPCR assays of the binding of TRIM24 and H3K23ac with the PIK3CA promoter. C, Overexpression of Flag-TRIM24 WT but not the binding mutant of TRIM24 with H3K23ac, F979A/N980A, promoted PIK3CA expression and AKT activation. D, Effects of overexpression of Flag-TRIM24 wild-type or F979A/N980A mutant on the binding of TRIM24 and H3K23ac with PIK3CA promoter. E and F, Effects of overexpression of Flag-TRIM24 WT or F979A/N980A mutant on cell proliferation (E) and colony formation (F) in soft agar in LN229 GBM cells. Data are representative of two independent experiments with similar results. Error bars, ÆSD. Ã, P < 0.05.

with and promote p53 acetylation, which enhances the p53 breast cancer cells (14). On the other hand, KAT6A is required for activity to drive p21 expression and subsequent senescence the expression of several repressors of the INK4A-ARF pathway by (41, 42). KAT6A also has been shown to regulate the expression maintaining the H3K9ac levels but not the H3K14ac or H3K23ac of ERa by directly binding to its promoter via H3K9 acetylation in levels at those gene loci (6). Here, we found that KAT6A

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through H3K23ac/TRIM24-PI3K/AKT pathway. The newly elucidated roles of KAT6A in glioma proliferation also contribute to better understanding the regulation of KAT6A functions in human diseases.

Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed.

Authors' Contributions Conception and design: D. Lv, Y. Hou, Y. Sang, H. Feng Development of methodology: W. Zhang Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Lv, F. Jia, Y. Hou, Y. Sang, W. Zhang, Y. Li Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Lv, Y. Hou, Y. Sang, A.A. Alvarez, H. Feng Writing, review, and/or revision of the manuscript:D. Lv, A.A. Alvarez, B. Hu, S.-Y. Cheng, Y. Li, H. Feng Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Ge, Y. Li, H. Feng Study supervision: W. Gao, H. Feng

Grant Support This work was supported in part by National Natural Science Foundation of Figure 7. China (no. 81372704, 81572467 to H. Feng; no. 81470315, 81772663 to Y. Li; A working model for KAT6A-regulated glioma tumorigenesis. KAT6A/MOZ 81471855 to F. Jia; 81372705 to J. Ge); the Program for Professor of Special interacts with ING5, EAF6, and BRPF1, and highly promotes H3K23 acetylation, Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (no. and recruits TRIM24 binding with PIK3CA promoter to upregulate its 2014024), Shanghai Municipal Education Commission—Gaofeng Clinical transcription, resulting in PI3K/AKT signaling pathway activation and glioma Medicine Grant Support (no. 20161310), New Hundred Talent Program tumorigenesis. (Outstanding Academic Leader) at Shanghai Municipal Health Bureau (2017BR021), Technology Transfer Project of Science & Technology Dept. at Shanghai Jiao Tong University School of Medicine (ZT201701 to H. Feng); significantly promotes H3K23 acetylation in glioma cells com- Shanghai Jiao Tong University School of Medicine Hospital Fund (no. pared with H3K9 and H3K14 acetylation. KAT6A-induced H3K23 14XJ10069), Collaborative Innovation Center for Translation Medicine at acetylation enhances the association of TRIM24 with chromatin. Shanghai Jiao Tong University School of Medicine (TM201502 to Y. Li); Shanghai Natural Science Foundation (16ZR1420200) and Shanghai Jiao Tong Consistent with the previous report on TRIM24 signaling (35), University Medical Engineering Cross Fund (YG2015QN35 to W. Zhang); US our results reveal that overexpression of KAT6A or TRIM24 upre- NIH grants (CA158911, NS093843, NS95634, and CA209345), a Zell Scholar gulates PIK3CA transcription in glioma cells to activate PI3K/AKT Award from the Zell Family Foundation and funds from Northwestern Brain signaling pathway, whereas overexpression of KAT6A acetyltrans- Tumor Institute at Northwestern University (to S.-Y. Cheng); a Brain Cancer ferase activity–deficient mutants or TRIM24 binding mutant with Research Award from James S. McDonnell Foundation (to B. Hu); an NIH/NCI H3K23ac does not. Our data support that KAT6A-regulated PI3K/ training grant T32 CA070085 (to A. Alvarez). The costs of publication of this article were defrayed in part by the payment of AKT signaling pathway depends on its acetyltransferase activity page charges. This article must therefore be hereby marked advertisement in and H3K23/TRIM24 complex. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In summary, our findings identify KAT6A as a potential target for treatment of highly malignant gliomas. Our data reveal a Received May 10, 2017; revised August 28, 2017; accepted October 3, 2017; mechanism by which KAT6A promotes glioma proliferation published OnlineFirst October 11, 2017.

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Histone Acetyltransferase KAT6A Upregulates PI3K/AKT Signaling through TRIM24 Binding

Deguan Lv, Feng Jia, Yanli Hou, et al.

Cancer Res 2017;77:6190-6201. Published OnlineFirst October 11, 2017.

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