HBO1 promotes replication stress response through ATR-dependent phosphorylation of Ser50/53

HBO1 promotes replication stress response through ATR-dependent phosphorylation of Ser50/53 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HBO1 promotes replication stress response through ATR-dependent phosphorylation of Ser50/53 Jianfeng Shen, Chunyan Zong, Zhe Zhang, Yiran Wang, Yan Fang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6875450/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mounting evidence has shown that histone acetyltransferase binding to ORC1 (HBO1) serves as an oncoprotein, warranting the use of small molecule inhibitor WM-3835 for cancer therapy. However, HBO1 is ubiquitously expressed in both tumor and normal tissues, with potential to increase the risk of systematic toxicity. This unmet need highlights the importance of identifying suitable biomarkers to predict the sensitivity to HBO1 inhibitor. Here, we show that ATR, a key regulator of DNA replication stress, is a novel interacting partner of HBO1. Additionally, we reveal the regulatory function of HBO1 in DNA replication stress responses, in an ATR-dependent manner. Mechanistically, ATR mediated HBO1 Ser50/53 phosphorylation interferes with genomic binding of HBO1 and regulates gene expression. Notably, the overexpression of HBO1 mutated at the ATR phosphorylation site (S50/53A) dampens the expression of DNA repair related genes and suppresses tumor colony formation, consistent with the observations of WM-3835 treatment. Inhibition of ATR significantly antagonized the treatment sensitivity of WM-3835. Collectively, our findings uncovered a previously unidentified role of HBO1 in the regulation of replication stress and discovered ATR as a potential biomarker for WM-3835 treatment. Biological sciences/Cancer/Cancer therapy Biological sciences/Molecular biology/DNA damage and repair/DNA damage response HBO1 replication stress ATR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Significance (1) IP-MS identified ATR as a novel HBO1-binding protein. (2) Phosphorylation of HBO1 at Ser50/53 by ATR is essential for the regulation of HBO1 on gene expression. (3) Intact ATR is required for the treatment efficacy of HBO1 inhibitor WM-3835 in cancer cells. Introduction The histone acetyltransferase binding to ORC1 (HBO1, also termed KAT7 or MYST2) is a key member of the MYST family of acetyltransferases. Functioning as the core catalytic subunit of the HBO1 complex, which incorporates various cofactors and co-proteins, HBO1 specifically acetylates histones H3 at lysine 14 (H3K14ac) and H4 at lysine 5, 8, and 12 (H4K5, H4K8, H4K12ac) [ 1 – 4 ]. By modulating histone acetylation, HBO1 induces chromatin relaxation, facilitating the recruitment of transcription factors and thereby regulating transcriptional initiation [ 5 – 7 ]. Consequently, HBO1 plays pivotal roles in diverse biological processes, including chromatin remodeling [ 4 ], DNA replication [ 8 , 9 ], transcriptional regulation [ 10 ], protein ubiquitination [ 11 ], immune response modulation [ 12 ], stem cell pluripotency, self-renewal maintenance [ 13 ], and embryonic development [ 1 ]. Emerging evidence highlights the oncogenic role of HBO1 in multiple malignancies, where its overexpression promotes cancer cell proliferation, cell cycle progression, and drug resistance [ 14 – 17 ]. For instance, HBO1-mediated H3K14ac enhances the processivity of RNA polymerase II, sustaining the expression of oncogenes (e.g., Hoxa9 and Hoxa10) in leukemia stem cells [ 18 ]. Additionally, HBO1 facilitates gastric cancer progression by activating YAP1 [ 19 ] and drives bladder cancer proliferation through the Wnt/β-catenin pathway [ 20 ]. Despite these findings, the precise mechanisms underlying the role of HBO1 in tumorigenesis remain incompletely understood, warranting further investigation. Given its oncogenic properties, targeting HBO1 has emerged as a promising therapeutic strategy. Genetic knockdown or pharmacological inhibition of HBO1 effectively suppresses tumor growth [ 21 , 22 ]. In 2019, MacPherson et al. developed WM-3835, a potent small-molecule inhibitor that competitively binds the acetyl-CoA site of HBO1, selectively depleting H3K14ac levels [ 18 ]. Preclinical studies demonstrate that WM-3835 exhibits robust anti-tumor activity in non-small cell lung cancer [ 23 ], prostate cancer [ 24 ], osteosarcoma [ 25 ], and acute lymphoblastic leukemia [ 26 ], inducing G1/S cell cycle arrest and apoptosis. However, since HBO1 is ubiquitously expressed in both normal and malignant cells, the potential toxicity of WM-3835 necessitates the identification of predictive biomarkers and patient stratification strategies to optimize its clinical translation. In this study, we employed immunoprecipitation-mass spectrometry (IP-MS) to identify HBO1-interacting proteins, revealing ATR, a critical kinase involved in DNA damage response and replication stress, as a novel binding partner. Mechanistically, we demonstrate that ATR phosphorylates HBO1, modulating its function in gene expression regulation and replication stress management. Our findings elucidate an ATR-HBO1 signaling axis, providing new insights into HBO1 function in cancer progression, and highlighting ATR as a potential predictive biomarker for the use of HBO1 inhibitor. Results 1. Proteomic analysis indicates that ATR is a key HBO1 interacting protein. We here used melanoma as a model to elucidate the biological function of HBO1. To investigate the function of HBO1 in tumor pathogenesis, we firstly identified the interacting proteins involved. We immunoprecipitated endogenous HBO1 binding proteins from UM cell line OMM2.3 using anti-HBO1 antibodies, and analyzed the co-precipitated proteins by mass spectrometry (Fig. 1 A, Table S1 ). Among 993 identified binding partners (iBAQ intensity > 0 in IP and input), we detected known HBO1-associated proteins (e.g., MCMs) and novel interactors (Fig. 1 B). Gene Ontology (GO) analysis revealed enrichment in DNA damage repair, replication, and translation pathways (Fig. 1 C). Strikingly, ATR, a central kinase in replication stress response, emerged as a HBO1 interactor. We validated this interaction endogenously in OMM2.3 and 92.1 UM cells via reciprocal Co-IP with HBO1- and ATR-specific antibodies (Fig. 1 D) and exogenously in 293T cells overexpressing Flag-ATR and myc-HBO1 (Fig. 1 E). Intriguingly, pharmacological inhibition of the acetyltransferase activity of HBO1 enhanced its binding to ATR (Fig. 1 F), suggesting a regulatory crosstalk between HBO1 acetylation and ATR interaction. 2. ATR interacts with HBO1 and phosphorylates HBO1 at Ser50 and Ser53. ATR is a critical regulator of replication stress response; however, the molecular mechanism underlying its interaction with HBO1 remains poorly understood. Structurally, HBO1 consists of an N-terminal domain (NTD) harboring a zinc finger motif and a C-terminal MYST catalytic domain [ 27 ]. To map the ATR interaction domain, we generated lentiviral expression vectors encoding myc-tagged HBO1 truncation mutants (Fig. 2 A). Intriguingly, co-immunoprecipitation assays revealed that the N-terminal D1 domain of HBO1 mediates ATR binding (Fig. 2 B). Previous studies have established that UV-induced replication stress triggers ATR-dependent phosphorylation of HBO1, facilitating DNA damage repair [ 28 , 29 ]. This prompted us to investigate whether HBO1 serves as a phosphorylation substrate of ATR. Using OMM2.3 cells treated with either DMSO or an ATR inhibitor, we immunoprecipitated HBO1 and probed for phospho-ATR substrates with an anti-phospho-S/TQ antibody. Notably, HBO1 exhibited robust ATR-dependent phosphorylation, which was markedly attenuated upon ATR inhibition (Fig. 2 C). Moreover, pharmacological suppression of acetyltransferase activity of HBO1 elevated S/TQ phosphorylation levels (Fig. 2 D), corroborating our findings in Fig. 1 F. These results collectively demonstrate that HBO1 is an ATR substrate and warrant further investigation of its phosphorylation sites. Previous studies have identified critical serine (Ser) and threonine (Thr) phosphorylation sites within the NTD of HBO1 [ 30 , 31 ]. PhosphoSitePlus database annotation highlighted Ser50, Ser53, Ser57, and Thr88 as the most probable phosphorylation sites on HBO1 (Fig. 2 E). While Ser57 is a known PLK1 target [ 31 ] and Thr88 is phosphorylated by CDK1 [ 32 ], we hypothesized that ATR preferentially targets Ser50/Ser53. To test this, we generated a polyclonal antibody against phospho-Ser50/53 (HBO1 pS50/53) and validated its specificity under UV irradiation. HBO1 pS50/53 signals increased within 0.5 hours post-UV exposure (Fig. 2 F), confirming antibody efficacy. We subsequently introduced alanine substitutions at Ser50/Ser53 (HBO1 SA) to abrogate phosphorylation and constructed Flag-tagged wild-type (F_HBO1) and mutant (F_HBO1 SA) expression plasmids (Fig. 2 G). Western blot analysis of transfected OMM2.3 cells confirmed equivalent expression of both constructs, while HBO1 pS50/53 signals were selectively abolished in the mutant (Fig. 2 H). Immunofluorescence (IF) further validated the antibody specificity and the phospho-deficient phenotype of HBO1 SA-expressing cells (Fig. 2 I-L). Interestingly, IF revealed that HBO1 acetyltransferase inhibition dramatically reduced HBO1 pS50/53 foci formation—a contrast to Western blot data—suggesting that the S/TQ motif detected in Fig. 2 D encompasses additional phosphorylation sites beyond Ser50/53. 3. ATR mediated HBO1 Ser50/53 phosphorylation regulates gene expression. To elucidate the biological significance of HBO1 phosphorylation at Ser50/53, we investigated how these modifications influence the genomic occupancy of HBO1, given its established role in transcriptional regulation via histone acetylation. Using CUT&Tag-seq with an anti-Flag antibody, we profiled genome-wide binding patterns in OMM2.3 cells expressing Flag-tagged empty vector (control), wild-type HBO1 (F_HBO1), or the phospho-deficient mutant (F_HBO1 SA, Ser50/53Ala) ( Fig. S1 A ). Strikingly, the F_HBO1 SA mutant exhibited markedly enhanced chromatin binding compared to wild-type HBO1, as evidenced by increased sequencing read intensities and larger DNA fragment sizes ( Fig. S1 B, C ). While wild-type HBO1 occupied 5,998 genes, the SA mutant bound to 23,539 genes—a ~ 4-fold increase ( Fig. S1 D ). These findings demonstrate that ablation of Ser50/53 phosphorylation substantially augments the chromatin affinity of HBO1. Peak distribution analysis revealed that both F_HBO1 and F_HBO1 SA were predominantly localized to transcription start sites (TSS), consistent with HBO1’s role in transcriptional regulation (Fig. 3 A, B). Quantitatively, 31.78% of F_HBO1 peaks and 34.29% of F_HBO1 SA peaks mapped to promoter regions (Fig. 3 C), further supporting HBO1’s functional association with gene regulatory. GO analysis demonstrated that genes bound by HBO1 were significantly enriched in chromatin remodeling-related biological processes (Fig. 3 D). Comparative genomic localization revealed substantial overlap between wild-type HBO1 and HBO1 SA binding sites, with 93.7% of HBO1-bound genes also occupied by the SA mutant (Fig. 3 E). Strikingly, the HBO1 SA mutant uniquely bound to 17,918 additional genes, predominantly involved in chromatin remodeling and DNA repair pathways (Fig. 3 F), suggesting that phospho-deficient HBO1 Ser50/53 substantially expands the genomic binding capacity of HBO1. Both wild-type and mutant HBO1 consistently occupied key chromatin regulatory genes including DDX17 , EIF4A2 , AKT1 , and ARAF (Fig. 3 G). However, the SA mutant specifically enriched at DNA repair genes (e.g., BACH1 , CHK1 , TOPBP1 , RPA2 ), indicating a phosphorylation-dependent shift in target preference (Fig. 3 G). Motif analysis uncovered differential binding patterns: while wild-type HBO1 preferentially associated with KLF10 motifs (consistent with prior reports [ 33 ]), the SA mutant showed strong affinity for CTCF binding sites (Fig. 3 H). Given the established role of CTCF in chromatin architecture maintenance [ 34 ], we propose that phospho-deficient HBO1 may modulate chromatin three-dimensional organization, potentially facilitating its expanded genomic occupancy. To elucidate the functional consequences of HBO1 Ser50/53 phosphorylation on transcriptional regulation, we performed RNA sequencing analysis in OMM2.3 cells overexpressing either wild-type F_HBO1 or the phospho-deficient F_HBO1 SA mutant. Comparative transcriptome profiling identified 585 significantly upregulated and 562 downregulated genes in F_HBO1 SA-expressing cells compared to wild-type controls (|log 2 FC| ≥ 0.585, P value<0.05; Fig. 3 I). GO analysis revealed that the downregulated gene set was remarkably enriched for biological processes related to DNA damage repair and chromatin remodeling (Fig. 3 J). Intriguingly, this transcriptional repression pattern correlated with the expanded chromatin binding capacity of the SA mutant observed in our CUT&Tag analysis (Fig. 3 F). These findings suggest that the phospho-deficient HBO1 SA mutant may function as a transcriptional repressor of DNA repair genes through its enhanced chromatin occupancy at these loci. To investigate the functional significance of HBO1 Ser50/53 phosphorylation in WM-3835-mediated H3K14ac inhibition and tumor suppression, we first conducted transcriptome profiling of control and WM-3835-treated OMM2.3 cells (Fig. 3 I, K). RNA-seq analysis revealed significant transcriptional changes, with 1,368 genes upregulated and 1,212 genes downregulated following WM-3835 treatment (Fig. 3 K). Notably, upregulated genes were enriched in apoptosis and growth inhibition pathways, consistent with the tumoricidal effect of WM-3835, while downregulated genes were associated with DNA damage repair and cell cycle progression, mirroring the transcriptional profile observed with Ser50/53 mutation. Comparative analysis of differentially expressed genes (DEGs) identified 1,147 and 2,580 genes altered by HBO1 SA mutation and WM-3835 treatment, respectively, with 377 co-regulated genes exhibiting largely concordant expression patterns ( Fig. S1 E, F ). To determine whether these transcriptional changes were directly mediated by HBO1 binding, we integrated our CUT&Tag and RNA-seq datasets (Fig. 3 L). Among 5,621 genes bound by both wild-type and mutant HBO1, 631 showed WM-3835-responsive expression changes, while 319 were affected by Ser50/53 mutation ( Fig. S2 A, B ). Strikingly, genes specifically bound by HBO1 SA exhibited more pronounced transcriptional alterations following WM-3835 treatment, with upregulation of apoptosis-related genes and suppression of cell division, migration, and DNA repair pathways ( Fig. S2 C ). Importantly, HBO1 SA preferentially bound and repressed genes involved in DNA damage response and cell cycle regulation ( Fig. S2 D ). Collectively, these findings demonstrate that ATR-mediated phosphorylation of HBO1 at Ser50/53 modulates H3K14ac dynamics and gene expression programs. The convergence of WM-3835-induced transcriptional changes with HBO1 SA-mediated regulation suggests that pharmacological inhibition of H3K14ac may functionally mimic the effects of phospho-deficient HBO1, providing mechanistic insight into the anticancer activity of WM-3835. 4. HBO1 regulates replication stress via Ser50/53 phosphorylation We then examined the expression of HBO1 and found the universal expression of HBO1 in both normal (PIG1 and ARPE-19), and melanoma cell lines ( Fig. S3A ). Importantly, higher level of HBO1 expression was associated with poor overall survival in UM patients (Fig. 4 A, Table S2 ). To study the function, we constructed HBO1 knockdown and overexpression OMM2.3 and 92.1 stable cell lines ( Fig. S3B, C ). Compared with control cells, HBO1-depleted cells showed a remarkable decrease by about 3/4 in OMM2.3 and 1/2 in 92.1 in colony area. Consistently, upon HBO1-overexpression, OMM2.3 (P = 0.0006) and 92.1 (P = 0.0076) exhibited significantly increased colony formation ability (Fig. 4 B, C). These results suggested the oncogenic role of HBO1 in UM, warranting further tests of cell cycle and replication stress. In accordance with earlier reports [ 24 , 26 ], flow cytometry analysis also showed the block of cell cycle at G0/G1 phase by WM-3835 (Fig. 4 D, S4A). Given the interaction between HBO1 and ATR, we then examined the role of HBO1 in replication stress response. We calculated the replication stress response score (RSR score) using the characteristic gene set of replication stress response [ 35 ]. Pearson correlation analysis showed that HBO1 expression was significantly positively correlated with RSR score in various tissues ( Fig. S5A, Table S3 ). ATR, CHK1, and RPA32 phosphorylation are markers of ATR pathway activation, while phosphorylated RPA32 (phospho-RPA32) and γH2AX are defined as replication stress markers [ 36 ]. Under the treatment of hydroxyurea (HU), phospho-RPA32 and γH2AX were increased, confirming the activation of replication stress. Importantly, replication stress was significantly suppressed when HBO1 was knocked-down or inhibited (Fig. 4 F-I, S5B), demonstrating the crucial role of HBO1 in replication stress. We next examined the clinical relevance of HBO1 phosphorylation at Ser50/53. Immunohistochemistry (IHC) staining of melanoma tissue array revealed that HBO1 pS50/53 intensity was markedly elevated in melanoma, compared with that of adjacent and normal skin tissues (Fig. 4 J). Key genes involved in melanoma progression are often associated with cancer stages (Clark level) and Breslow thickness [ 37 ]. However, in our data, patients diagnosed with advanced stages, greater Breslow thickness, and lymph node metastasis did not show higher HBO1 pS50/53 level ( Fig. S6A ). Interestingly, inhibition of HBO1 pSer50/53 significantly reduced the number of OMM2.3 colonies (Fig. 4 K), with no regard to cell cycle arrest ( Fig. S6B ). Both IF staining and WB showed remarkable reduction of phospho-RPA32 and γH2AX when the HBO1 Ser50/53 phosphorylation sites were mutated, suggesting the requirement of such phosphorylation sites of HBO1 by ATR signaling activation (Fig. 4 L, M). These findings were also consistent with the transcriptomic data in Fig. 3 I-K and Fig. S1 F , showing the alteration of gene expression under WM-3835 treatment in cells reconstituted with HBO1 SA. 5. Inhibition of ATR activity alters WM-3835 sensitivity. Building on above results, we then speculated that ATR activity may affect the treatment efficacy of WM-3835. We treated OMM2.3 and 92.1 cells with WM-3835 and BAY-1895344, an ATR inhibitor currently under clinical trial evaluation for the treatment of advanced solid tumors [ 38 ]. Synergy score demonstrated that the effects of WM-3835 was antagonized by the addition of BAY-1895344 (Fig. 5 A). Consistent to these observations, knockdown of ATR impaired the sensitivity of 92.1 cells to WM-3835 treatment (Fig. 5 B, C). We also examined the level of phospho-RPA32 and γH2AX, to evaluate the impact on replication stress by BAY-1895344 and WM-3835. We found that the inhibition of phospho-RPA32 and γH2AX by WM-3835 was significantly attenuated under BAY-1895344 treatment (Fig. 5 D, E). These data support the role of ATR as a potential predictive biomarker to WM-3835 sensitivity. Discussion The histone acetyltransferase HBO1 has been predominantly characterized as exerting its biological functions through its catalytic acetylation activity [ 39 – 41 ]. While previous studies have established that HBO1 targeting induces tumor cell death via cell cycle arrest [ 26 ], our work significantly expands this understanding by identifying ATR as a novel binding partner and critical regulator of HBO1. This discovery functionally links replication stress regulation function of ATR with chromatin regulation function of HBO1. The 611-amino acid HBO1 protein features two principal domains: an N-terminal DNA-binding domain containing a zinc finger motif (zf-C2HC, 184-212aa) that interacts with replication initiation factors MCM2 and ORC1 [ 42 ], and a C-terminal MYST acetyltransferase domain (340-607aa) responsible for catalytic activity [ 27 ]. Despite its functional importance, the NTD remains poorly characterized due to the lack of structural information. Our findings substantially advance the understanding of HBO1 NTD. Emerging evidence indicates that post-translational modifications (PTMs) of HBO1 NTD serve as molecular switches modulating its activity. For instance, CDK1-mediated phosphorylation at Thr85/Thr88 primes subsequent PLK1-dependent phosphorylation at Ser57, facilitating pre-replicative complex formation [ 32 , 43 ]. Additionally, CDK11-mediated HBO1 phosphorylation enhances its acetyltransferase activity [ 30 ]. In line with these findings, our study demonstrates that prevention of Ser50/53 phosphorylation dramatically alter the genomic binding landscape of HBO1 (Fig. 3 ). The phospho-deficient mutant (HBO1 SA) exhibited substantially increased genomic occupancy (23,539 binding sites vs. 5,998 in wild-type), suggesting that phosphorylation at these sites normally restricts widespread DNA binding. Notably, motif analysis of HBO1 SA binding regions revealed strong enrichment for CTCF motifs. Given the established role of CTCF in maintaining chromatin accessibility [ 34 ], this observation may explain the expanded genomic binding of the phospho-deficient mutant. Furthermore, while wild-type HBO1 preferentially associates with transcription start sites and promoter regions, consistent with reports in hESCs [ 33 ] and HeLa cells [ 44 ], the SA mutant showed increased binding at replication origins, suggesting phosphorylation-dependent regulation of replication licensing. Our findings uncover a novel ATR-HBO1 axis in replication stress management. We demonstrate that ATR-mediated phosphorylation of HBO1 Ser50/53 modulates its binding to DNA damage repair genes, independent of its acetyltransferase activity. Intriguingly, the transcriptional effects of HBO1 SA mutation paralleled those of WM-3835 treatment, both downregulating DNA repair and cell cycle-related genes. Mechanistically, we found that HBO1 SA directly binds to and represses TOPBP1 and RPA2 , which are critical components of the ATR activation cascade [ 45 , 46 ]. This observation explains how ATR-dependent HBO1 phosphorylation promotes replication stress response: 1) RPA-coated ssDNA recruits ATR-ATRIP complexes; 2) TOPBP1 mediates ATR activation; 3) Activated ATR phosphorylates HBO1 Ser50/53; 4) Phospho-HBO1 upregulates DNA repair genes through targeted genomic binding. This feedforward mechanism ensures appropriate cellular responses to replication stress. While WM-3835 (a first-in-class HBO1 inhibitor) shows promise in inducing G0/G1 arrest in several tumor types [ 24 , 47 ], our work reveals its functional interplay with ATR signaling. Surprisingly, ATR inhibition attenuated WM-3835 efficacy, suggesting ATR expression may serve as a predictive biomarker for HBO1-targeted therapy. This finding has important clinical implications, as combining WM-3835 with drugs targeting DNA damage response and cell cycle checkpoint kinases (including ATR, CHK1, WEE1, and MYT1) [ 48 ], common strategies in DDR-targeted therapy, may require careful patient stratification. Taken together, our study establishes ATR as a key regulator of HBO1 through phosphorylation-dependent modulation of its genomic binding. This ATR-HBO1 axis represents a novel mechanism coordinating chromatin regulation with replication stress response. From a translational perspective, we propose ATR expression as a potential biomarker for HBO1 inhibitor sensitivity, providing a framework for precision medicine approaches targeting this pathway. Materials and Methods Cell culture Human renal epithelial cell line HEK293T and human cutaneous melanoma cell lines A375 and A2058 cells was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, #8122522). Human cutaneous melanocyte cell PIG1 and retinal pigment epithelium cell ARPE-19 were cultured in RPMI1640 medium (Gibco, #8122322). Human cervical cancer cell HeLa and Human UM cell lines (OMM1, OMM2.3, MEL202, MEL270, MEL290, 92.1, MUM2B) were cultured in RPMI1640 medium (Gibco, #8122322). All mediums are supplemented with 10% fetal bovine serum (FBS, Gibco, #16140071) and 1% antibiotics (Penicillin-streptomycin, Gibco, #10378016). All cells were cultured in a 37℃ humidified incubator containing 5% CO 2 . Western blotting Total cell lysates were prepared in urea lysis buffer supplemented with protease inhibitor and phosphatase inhibitor. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, #ISEQ00010). After blocking with 5% non-fat milk for 1 hour at room temperature, the membranes were incubated with anti-HBO1 (Proteintech, 13751-1-AP, 1:2000), anti-ATR (Abclonal, A21253,1:1000), anti-myc (Santa cruz, sc-40, 1:1000), anti-Flag (Proteintech, 66008-4-Ig, 1:10000), anti-Phospho-ATM/ATR Substrate (S/TQ) (CST, 9607S, 1:1000), anti-HBO1 pS50/53 (1:500), anti-phospho-RPA32 (BETHYL, A300-245A, 1:2000), anti-γH2AX (SIGMA, 05-636-I, 1:1000), anti-RPA32 (CST, 35869S, 1:1000), anti-H3K14Ac (Abclonal, A7254, 1:1000), anti-phospho-ATR(CST, #58014, 1:1000), anti- RPA70 (CST, #2267S, 1:1000) antibody overnight at 4℃ and then with the appropriate secondary antibodies conjugated to a fluorescent tag (CST, #5151 & #5470, 1:10000) for 1 hour at room temperature. Anti-β-Actin (Proteintech, 66009-1-Ig, 1:20000), anti-β-tubulin (Abclonal, A12289, 1:10000) and anti-GAPDH (Proteintech, 60004, 1:20000) antibody served as the loading control. The immunoblots were recorded with the Odyssey infrared imaging system (LI-COR Biosciences). Full length uncropped original western blots are provided in supplementary material. Co-immunoprecipitation (Co-IP) Cells were pelleted and washed with PBS, and then lysates were prepared in 500 µL lysis buffer containing 120 mM NaCl, 20 mM Tris-Cl, 2 mM EDTA, 1% NP-40 and 5% Glycerol supplemented with 1×protease inhibitor. Anti-HBO1 (Proteintech, 13751-1-AP, 4 µL), anti-ATR (CST, 13934S, 2.5 µL), anti-myc (Santa cruz, sc-40, 5 µL), anti-Flag (Proteintech, 66008-4-Ig, 5 µL) antibody or normal mouse IgG (Santa cruz, sc-2025, 1:10000) antibody was incubated with the cell lysates overnight at 4℃, after which 30 µL protein A magnetic beads (CST, #73778) were added and incubated for 2 additional hours. Then, the magnetic beads were washed three times with lysis buffer. For IP-MS analysis, 100 µL glycine solution was used for each tube to elute the protein complexes from the beads, while 1×SDS loading buffer (NCM, WB2001) was used per sample for SDS-PAGE analysis. Transfection and virus packaging Two shRNA sequences targeting HBO1 and ATR were cloned into the pLKO.1_TRC vector. The HBO1 targeting sequences were: 5’-CCTCTCAAGTAGCTGGGATTA-3’(shHBO1_1); 5’-CCTCTCAAGTAGCTGGGATTA − 3’ (shHBO1_2). The ATR targeting sequences were: 5’-CCGGATACTTACAGATGTAAA-3’(shATR_1); 5’-GTAATGCATTTGGTATGAATC-3’ (shATR_2). The full length of human HBO1 (isoform NM_007067), myc_HBO1 D1, myc_HBO1 D2 and myc_HBO1 D3 were cloned into the pHAGE-myc-puro vector respectively for overexpression of HBO1 and HBO1 D1/2/3. Primers used for cloning are shown in Table S4 . CMV_Flag_ATR (#41909) was purchased from addgene. PolyJet DNA In Vitro Transfection Reagent (SignaGen, SL100688) was used for plasmid transfection following the manufacturer’s instructions. After lentiviral packaging with HEK293T cells, cells were infected with lentiviruses and selected by incubation with 2 µg/mL puromycin for 3 days. Immunofluorescence (IF) Cells adhering to a glass slide were fixed with 4% paraformaldehyde (Biosharp, #70071800) for 15 min, permeabilized with 0.1% Triton X-100 (Sigma, T9284) for 15 min and then blocked with 2% BSA solution for 1 h at room temperature. After incubation with primary antibody against HBO1 pS50/53 (1:200), phospho-RPA32 (BETHYL, A300-245A, 1:2000) and γH2AX (SIGMA, 05-636-I, 1:500) overnight, the cells were washed three times with PBS and subsequently incubated with Alexa Fluor 594 secondary antibody (Invitrogen, A11012, 1:1000) or Alexa Fluor 488 secondary antibody (Invitrogen, A11029, 1:1000) for 1 h. The coverslips were then mounted with ProLong Gold mounting medium with DAPI (Invitrogen, P36931) and observed under an inverted fluorescence microscope (Nikon, Japan). Foci number was quantified for each condition. The results presented were obtained from three independent biological replicates; at least 50 cells were measured per replicate. RNA extraction and quantification RNA purification was performed using EZ-press RNA purification kit (EZBioscience, #B0004DP) according to the manufacturer’s guidelines. Then, cDNA synthesis was achieved using the HiScript III RT SuperMix (Vazyme, #R323). Finally, real-time quantitative PCR (RT-qPCR) was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q711). mRNA expression values were calculated using the ΔΔCt method and human β-Actin gene as a control. A detailed list with primers used in the present study is provided in Table S5 . RNA-seq RNA sequencing was performed by Novogene (Beijing, China). Total RNA was harvested using Trizol. The integrity of the RNA was assessed by Agilent 2100 bioanalyzer (Thermo Fisher Scientific). Approximately 1 µg mRNA from each sample was used for RNA sequencing (Illumina HiSeq PE150 platform). GO analysis was performed by DAVID ( https://davidbioinformatics.nih.gov/summary.jsp ). CUT&Tag CUT&Tag assay was performed using Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme, TD904) following the manufacturer’s protocol. Briefly, 1×10 5 OMM2.3 cells transfected with F_HBO1 or F_HBO1 SA were collected. After binding to concanavalin A–coated magnetic beads (ConA beads), bead-bound cells were incubated with anti-Flag antibody (CST, #14793, 1:50) or normal rabbit IgG (CST, 2729, 1:100) for 2 h at room temperature. After brief wash with dig-wash buffer, cells were then incubated with goat anti rabbit secondary antibody (Abcam, ab6702, 1:100) for 1h at room temperature. When antibody binding procedures were finished, the bead-bound cells were then mixed with hyperactive pA-Tn5 transposon and tagmentated with tagmentation buffer. Tagmentated DNA was then extracted and amplified to form the sequencing-ready libraries. After the PCR reaction, libraries were purified with the DNA clean beads (Vazyme, N411) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). The library preparations were sequenced on Illumina Novaseq platform at Novogene (Beijing, China). GO analysis was implemented by DAVID. In Venn diagrams, numbers represent genes co-occurring between conditions. Colony formation A volume of 3 mL of complete medium containing 2000 cells was seeded in each well of a 6-well plate and incubated for about 2 weeks. Once colonies formed in control conditions, plates were fixed with 4% paraformaldehyde, stained with crystal violet. Colony formation efficiency is indicated by colony area. The quantifications presented were obtained from three independent biological replicates. Cell viability assay Cells were seeded at a density of 1,000 cells per well in triplicate into 96-well plates. For drug sensitivity, cells were treated with WM-3835 (Selleck, S9805) and BAY-1895344 (Selleck, S8666) for 48 hours. The absorbance at 450 nm was then measured. The data were recorded and analyzed. The quantifications presented were obtained from three independent biological replicates. The synergy score was calculated by SynergyFinder ( https://synergyfinder.aittokallio.group/20250411123730439716/ ). When synergy score is less than − 10, the interaction between two drugs is likely to be antagonistic. Cell cycle FACS 2×10 5 melanoma cells were collected and centrifuged to remove the medium and washed twice with PBS. After resuspension with 75% ethanol and incubation at 4℃ overnight, ethanol was removed by centrifugation and washed twice with PBS. Finally, cells were stained using 0.5 mL PI/RNase Staining Buffer (Multi sciences, CCS012) and incubated at room temperature for 30 min followed by FACS analysis. Immunohistochemistry (IHC) Tissue microarrays (ZL-MEL361 and ZL-MEL963) for IHC staining were bought from Weiaobio (Shanghai, China). The information of tissue microarrays is provided in Table S6 . IHC staining of tissue microarrays was performed by Servicebio (Wuhan, China). Tissues were deparaffinized and rehydrated through an alcohol series, followed by antigen retrieval with sodium citrate buffer. Then tissue sections were blocked with 3% bovine serum albumin (BSA) 30 min at room temperature and then incubated with anti-HBO1 pS50/53 (1:200) antibody at 4℃ overnight. Phospho Ser50 and Ser53 HBO1 rabbit polyclonal antibodies were generated by HUABIO (Hangzhou, China). Finally, the tissues were covered with horseradish peroxidase (HRP) labeled secondary antibody and incubated at room temperature for 50 min. All immunostained slides were scanned on 3D Histech Quant Center (3D Histech), and computerized image analysis was performed by Halo (Indica labs). Immunostaining for HBO1 pS50/53 was analyzed in melanoma tissues, adjacent normal tissues and normal skin tissues using percentage of positive cells and histochemistry score (H-score). H-score was determined based on the proportion of positive cells and the intensity of nuclear staining as previously described [ 49 ]. H-score=∑(pi×i)= (percentage of cells of weak intensity ×1) + (percentage of cells of moderate intensity ×2)+ (percentage of cells of strong intensity ×3). In the formula, pi represents the percentage of positive cells in the slide; i represents the intensity of HBO1 pS50/53 staining. Statistical analysis GraphPad Prism 9.0 was used for statistical analyses. Descriptive values are presented as mean + standard error of the mean (SEM) unless stated elsewhere. Differences between two groups were analyzed by unpaired Student’s t-test while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) with post-hoc intergroup comparisons with Turkey’s test. When data did not meet the normal distribution, the Mann–Whitney U-test was performed. Test details were indicated in the figure legends. Results were considered statistically significant when p < 0.05. TCGA overall survival analysis and Pearson correlation analysis (HBO1 vs RSR signature score) was performed on GEPIA ( http://gepia.cancer-pku.cn/ ). RSR signature gene set was shown in Table S7 . Declarations Conflict of interest The authors declare that they have no conflict of interest. Author contributions C.Z. designed and performed the experiments, analyzed the experimental results, and drafted the manuscript. Z.Z. conducted bioinformatics analysis of the sequencing data. Y.W. and G.Z. contributed to the generation of expression constructs and establishment of stable cell lines. Y.F. and Q.L. assisted in drug sensitivity assays and offered scientific input for project development. J.S. conceived and supervised the study, interpreted the data, and revised the manuscript. Acknowledgement This work was supported by General Program of National Natural Science Foundation of China (No. 82472752 and 81972667 to J.S.), National Key R&D Program of China (2021YFC2701103 to J.S.), and the Center for High Performance Computing at Shanghai Jiao Tong University. Data availability The data that support the findings of this study are available in Gene Expression Omnibus (GEO) with accession number GSE299365 and GSE299366. References Lan R, Wang Q. Deciphering structure, function and mechanism of lysine acetyltransferase HBO1 in protein acetylation, transcription regulation, DNA replication and its oncogenic properties in cancer. Cell Mol Life Sci. 2020;77:637-649. 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Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair. Nat Commun. 2017;8:16102. Matsunuma R, Niida H, Ohhata T, Kitagawa K, Sakai S, Uchida C, et al. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation. Mol Cell Biol. 2016;36:394-406. Zong H, Li Z, Liu L, Hong Y, Yun X, Jiang J, et al. Cyclin-dependent kinase 11(p58) interacts with HBO1 and enhances its histone acetyltransferase activity. FEBS Lett. 2005;579:3579-88. Song B, Liu XS, Rice SJ, Kuang S, Elzey BD, Konieczny SF, et al. Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer. Mol Cancer Ther. 2013;12:58-68. Wu ZQ, Liu X. Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing. Proc Natl Acad Sci U S A. 2008;105:1919-24. Zhang C, Shan Y, Lin H, Zhang Y, Xing Q, Zhu J, et al. HBO1 determines SMAD action in pluripotency and mesendoderm specification. Nucleic Acids Res. 2024;52:4935-4949. Yang X, Cheng L, Xin Y, Zhang J, Chen X, Xu J, et al. CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis. Genome Biol. 2025;26:44. Dreyer SB, Upstill-Goddard R, Paulus-Hock V, Paris C, Lampraki EM, Dray E, et al. Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer. Gastroenterology. 2021;160:362-377.e13. Dobbelstein M, Sørensen CS. Exploiting replicative stress to treat cancer. Nat Rev Drug Discov. 2015;14:405-23. Andtbacka RH, Gershenwald JE. Role of sentinel lymph node biopsy in patients with thin melanoma. J Natl Compr Canc Netw. 2009;7:308-17. Yap TA, Tan DSP, Terbuch A, Caldwell R, Guo C, Goh BC, et al. First-in-Human Trial of the Oral Ataxia Telangiectasia and RAD3-Related (ATR) Inhibitor BAY 1895344 in Patients with Advanced Solid Tumors. Cancer Discov. 2021;11:80-91. Su Z, Zhang Y, Tang J, Zhou Y, Long C. Multifunctional acyltransferase HBO1: a key regulatory factor for cellular functions. Cell Mol Biol Lett. 2024;29:141. Avvakumov N, Lalonde ME, Saksouk N, Paquet E, Glass KC, Landry AJ, et al. Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation. Mol Cell Biol. 2012;32:689-703. Miotto B, Struhl K. HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by Geminin. Mol Cell. 2010;37:57-66. Burke TW, Cook JG, Asano M, Nevins JR. Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J Biol Chem. 2001;276:15397-408. Iizuka M, Sarmento OF, Sekiya T, Scrable H, Allis CD, Smith MM. Hbo1 Links p53-dependent stress signaling to DNA replication licensing. Mol Cell Biol. 2008;28:140-53. Niu Z, Chen C, Wang S, Lu C, Wu Z, Wang A, et al. HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription. Nat Commun. 2024;15:3561. Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nat Cell Biol. 2014;16:2-9. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol. 2017;18:622-636. Wang H, Guan T, Hu R, Huang Z, Liang Z, Lin X, et al. Targeting KAT7 inhibits the progression of colorectal cancer. Theranostics. 2025;15:1478-1495. Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer. 2018;18:586-595. Dogan S, Vasudevaraja V, Xu B, Serrano J, Ptashkin RN, Jung HJ, et al. DNA methylation-based classification of sinonasal undifferentiated carcinoma. Mod Pathol. 2019;32:1447-1459. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6875450","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475522737,"identity":"450801b2-0e41-461e-87af-421c5b1a7442","order_by":0,"name":"Jianfeng Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACxuYDbEDKhsEAwmcmQktbAkhLGglaGNjAWg6ToIW5jf3Zg487ztub859Ok2CosE5sYD97gIDDeMwNZ565nbhzRu42CYYz6YkNPHkJ+LXM72GT5m27nWBwg3ebBGPb4cQGCR4DArawP5P+23bO3uD8WaCWf0RpYTCTZmw7wLjhANBhjA1EaeExk+xtS07ccCN3s0XCsXTjNp4c/FoMgQ6T+NlmB3LYxhsfaqxl+9nPENDSgMxLAGI2vOqBQJ6QglEwCkbBKBgFDADHpEQNV7kdhgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9779-6225","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jianfeng","middleName":"","lastName":"Shen","suffix":""},{"id":475522738,"identity":"8e761268-5b81-4320-9ce3-22f4747753d5","order_by":1,"name":"Chunyan Zong","email":"","orcid":"https://orcid.org/0000-0001-7013-8740","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Zong","suffix":""},{"id":475522739,"identity":"a43573e0-2f12-4d88-bbbe-bc0a1c59faa6","order_by":2,"name":"Zhe Zhang","email":"","orcid":"","institution":"Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhang","suffix":""},{"id":475522740,"identity":"9eec38d8-a50e-4e3b-a029-9cf9ce125362","order_by":3,"name":"Yiran Wang","email":"","orcid":"","institution":"Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yiran","middleName":"","lastName":"Wang","suffix":""},{"id":475522741,"identity":"208a0f01-6381-4edf-8dc0-371594ec1bc1","order_by":4,"name":"Yan Fang","email":"","orcid":"","institution":"Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Fang","suffix":""},{"id":475522742,"identity":"b55f0f26-60cd-443e-bedb-9b83ee828ece","order_by":5,"name":"Guopei Zheng","email":"","orcid":"","institution":"Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Guopei","middleName":"","lastName":"Zheng","suffix":""},{"id":475522743,"identity":"516579fe-b674-4190-980c-49852992539b","order_by":6,"name":"Qian Li","email":"","orcid":"","institution":"Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-12 01:25:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6875450/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6875450/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85475476,"identity":"9cafde2e-8222-48c9-a661-0dadd4ba40da","added_by":"auto","created_at":"2025-06-26 09:58:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1791589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIP-MS identifies ATR as a HBO1 interacting protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the IP-MS experimental workflow. \u003cstrong\u003e(B)\u003c/strong\u003e Representative list of proteins identified through HBO1 IP-MS analysis. \u003cstrong\u003e(C) \u003c/strong\u003eGO enrichment analysis of proteins co-immunoprecipitated with HBO1. \u003cstrong\u003e(D)\u003c/strong\u003e Co-IP analysis of endogenous HBO1 and ATR interaction. Proteins were detected by western blotting, with 1% cell lysate loaded as input control. Molecular weight markers (kDa) are indicated.\u003cstrong\u003e (E) \u003c/strong\u003eCo-IP of exogenously expressed HBO1 and ATR. Myc-tagged and Flag-tagged proteins were detected by western blotting, with 1% cell lysate loaded as input control. \u003cstrong\u003e(F)\u003c/strong\u003e Co-IP analysis of HBO1-ATR interaction in OMM2.3 cells treated with either DMSO (vehicle control) or WM-3835 (20 μM) for 48 hours. Endogenous proteins were detected by western blotting, with 1% cell lysate loaded as input control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/d5d279d8a2b14833473c884d.png"},{"id":85475475,"identity":"717b9bae-48f6-42de-8315-39aee02151ab","added_by":"auto","created_at":"2025-06-26 09:58:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4471416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATR interacts with HBO1 and phosphorylates HBO1 at Ser50/53.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic of Myc-tagged HBO1 domain constructs. \u003cstrong\u003e(B)\u003c/strong\u003e Co-IP analysis of Myc-tagged HBO1 domains or Flag-tagged ATR in 293T cells. \u003cstrong\u003e(C)\u003c/strong\u003e HBO1 immunoprecipitation from OMM2.3 cells pretreated with DMSO or BAY-1895344 (ATR inhibitor). Input (3% lysate) was probed; ATR phosphorylation (S/TQ motif) was detected by immunoblot after IP. \u003cstrong\u003e(D)\u003c/strong\u003e Time-course analysis of HBO1 immunoprecipitation from OMM2.3 cells treated with WM-3835 for 0, 24, or 48 hours. \u003cstrong\u003e(E)\u003c/strong\u003e In silico prediction of HBO1 post-translational modification (PTM) sites. \u003cstrong\u003e(F)\u003c/strong\u003e Validation of HBO1 pS50/53 antibody specificity in HeLa cells post-UV (4 mJ/cm\u003csup\u003e2\u003c/sup\u003e). Lysates were collected at indicated timepoints (0–6 h). \u003cstrong\u003e(G)\u003c/strong\u003e Schematic of HBO1 Ser50/Ser53-to-Ala (SA) mutant construct. (H) Immunoblot confirmation of Flag-tagged wild-type HBO1 (F_HBO1) and SA mutant overexpression in OMM2.3 cells. \u003cstrong\u003e(I)\u003c/strong\u003e IF of HBO1 pS50/53 in OMM2.3 cells expressing F_HBO1 or F_HBO1 SA, with/without UV (6 mJ/cm\u003csup\u003e2\u003c/sup\u003e). Cells were fixed 1 h post-irradiation. Scale bar: 10 μm. \u003cstrong\u003e(J)\u003c/strong\u003e Quantification of HBO1 pS50/53 foci from \u003cstrong\u003e(I)\u003c/strong\u003e. The center line shows the mean (n = 147, 157, 154 and 141). Significance by two-tailed unpaired t-test. Significance assessed using two-tailed unpaired t test. \u003cstrong\u003e(K)\u003c/strong\u003e IF of HBO1 pS50/53 in OMM2.3 cells treated with DMSO, BAY-1895344, or WM-3835. \u003cstrong\u003e(L)\u003c/strong\u003e Quantification of HBO1 pS50/53 foci from \u003cstrong\u003e(K)\u003c/strong\u003e. The center line shows the mean (n = 215, 145, 216 and 132). Significance assessed using two-tailed unpaired t test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/cddd0d76ddecb7c0489c8d60.png"},{"id":85476005,"identity":"9e567635-941c-4fb5-b065-b925a57c3c56","added_by":"auto","created_at":"2025-06-26 10:06:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5478899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBO1 regulates gene expression through Ser50/53 in an ATR dependent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eCUT\u0026amp;Tag signal densities (top) and heatmap (bottom) of Flag-tagged HBO1 and HBO1 SA binding at transcription start sites (TSS ± 3 kb). \u003cstrong\u003e(B)\u003c/strong\u003eDistribution profile of CUT\u0026amp;Tag peaks relative to TSS positions. Color-coding indicates genomic regions as shown in the labels on the right. \u003cstrong\u003e(C)\u003c/strong\u003eGenomic annotation of Flag-tagged peaks identified by CUT\u0026amp;Tag. UTR: untranslated region. \u003cstrong\u003e(D)\u003c/strong\u003e GO analysis of biological processes associated with F_HBO1-bound genes from CUT\u0026amp;Tag data. \u003cstrong\u003e(E)\u003c/strong\u003eVenn diagram comparing F_HBO1 (red) and F_HBO1 SA (blue) binding genes.\u003cstrong\u003e (F)\u003c/strong\u003eGO analysis of biological processes specifically associated with F_HBO1 SA-bound genes. \u003cstrong\u003e(G)\u003c/strong\u003e Genome browser tracks showing: (Top) Co-regulated genes by F_HBO1 and F_HBO1 SA, enriched in chromatin remodeling; (Bottom) F_HBO1 SA-specific target genes involved in DNA repair. \u003cstrong\u003e(H)\u003c/strong\u003e Motif enrichment analysis of peak sequences from F_HBO1 and F_HBO1 SA cells. \u003cstrong\u003e(I)\u003c/strong\u003eScatter plots of differentially expressed genes (DEGs): (Top) F_HBO1 SA vs F_HBO1; (Bottom) WM-3835 (20 μM) vs DMSO-treated cells. DEG thresholds: |log2FC| ≥ 0.585, P \u0026lt; 0.05. \u003cstrong\u003e(J)\u003c/strong\u003eGO analysis of biological processes for DEGs (F_HBO1 SA vs F_HBO1; |log2FC| ≥ 0.585, P \u0026lt; 0.05). \u003cstrong\u003e(K)\u003c/strong\u003e GO analysis of biological processes for DEGs after 48-hour WM-3835 (20 μM) treatment (vs DMSO; |log2FC| ≥ 0.585, P \u0026lt; 0.05). \u003cstrong\u003e(L)\u003c/strong\u003e Overlap between CUT\u0026amp;Tag-identified binding genes and RNA-seq DEGs from \u003cstrong\u003e(I)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/34d29852811415d0e07c54a2.png"},{"id":85475483,"identity":"540bf69a-218b-4ffe-93c8-5d87e39cadd4","added_by":"auto","created_at":"2025-06-26 09:58:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9289251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBO1 regulates replication stress via Ser50/53 phosphorylation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eKaplan-Meier survival analysis of melanoma patients from TCGA cohort stratified by HBO1 expression. Hazard ratios (HR, Cox proportional hazards model) are color-coded (red: HR \u0026gt;1; blue: HR \u0026lt;1). \u003cstrong\u003e(B)\u003c/strong\u003e Representative colony formation assays in OMM2.3 and 92.1 uveal melanoma cell lines. \u003cstrong\u003e(C)\u003c/strong\u003eQuantitative analysis of colony formation capacity (n=3 biological replicates; mean ± SEM; unpaired t-test). \u003cstrong\u003e(D, E)\u003c/strong\u003eCell cycle distribution analyzed by flow cytometry in OMM2.3 \u003cstrong\u003e(D)\u003c/strong\u003e and 92.1 \u003cstrong\u003e(E)\u003c/strong\u003e cells treated with 20 μM WM-3835 or vehicle control for 48 hours. \u003cstrong\u003e(F)\u003c/strong\u003e Immunoblot analysis of replication stress response (RSR) proteins in HBO1-knockdown vs control OMM2.3 cells treated with 2 mM HU for indicated durations. \u003cstrong\u003e(G)\u003c/strong\u003e Immunoblots of RSR pathway proteins in OMM2.3 cells treated with HU (2 mM) and/or WM-3835 (20 μM for 48 h). \u003cstrong\u003e(H)\u003c/strong\u003e Immunofluorescence staining of phospho-RPA32 (S4/S8) in OMM2.3 cells treated with WM-3835 (0-20 μM for 48 h). Scale bar: 10 μm. \u003cstrong\u003e(I)\u003c/strong\u003eQuantification of phospho-RPA32 foci per cell (n= 142, 195 and 203). The center line shows the mean. P values calculated using two-tailed unpaired t test. \u003cstrong\u003e(J)\u003c/strong\u003eLeft: Representative immunohistochemistry of HBO1 pS50/53 in melanoma specimens, adjacent tissue, and normal skin (scale: 50 μm; insets: 2× magnification). Right: Scatter plot of HBO1 pS50/53 positivity and H-scores for indicated tissues. \u003cstrong\u003e(K) \u003c/strong\u003eLeft: Colony formation assays in OMM2.3 cells overexpressing Flag-tagged HBO1 or HBO1 S50/53A mutant. Right: Quantified colony areas (n=3; mean + SEM; unpaired t-test). \u003cstrong\u003e(L)\u003c/strong\u003eImmunoblot analysis of RSR proteins in OMM2.3 cells expressing F_HBO1 or F_HBO1 SA treated with HU (2 mM) ± WM-3835 (20 μM for 48 h). \u003cstrong\u003e(M)\u003c/strong\u003e Left: Representative immunofluorescence images of phospho-RPA32 and γH2AX in OMM2.3 cells expressing F_HBO1 or F_HBO1 SA (scale bar: 10 μm). Right: Quantification of DNA damage foci (phospho-RPA32: n=156 and 203 cells; γH2AX: n=186 and 191 cells). The center line shows the mean. P values calculated using two-tailed unpaired t test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/c38a44dab391de5dfcdb9ae3.png"},{"id":85477456,"identity":"f82a2ef1-41c9-4f17-8706-3f7aba9721a6","added_by":"auto","created_at":"2025-06-26 10:22:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3047796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ATR activity alters WM-3835 mediated phenotype.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSynergy analysis of WM-3835 (HBO1 inhibitor) and BAY-1895344 (ATR inhibitor) combination treatment in OMM2.3 and 92.1 cells. Heatmap shows combination inhibition rate.\u003cstrong\u003e (B)\u003c/strong\u003e qRT-PCR validation of ATR knockdown efficiency in 92.1 cells. Gene expression was normalized to β-Actin (n=3 technical replicates; mean + SEM). \u003cstrong\u003e(C)\u003c/strong\u003e Cell viability assessed by CCK-8 assay in 92.1 cells with or without ATR knockdown treated with increasing concentrations of WM-3835 for 72 hours (n=3 biological replicates; mean ± SEM). \u003cstrong\u003e(D)\u003c/strong\u003e Immunoblot analysis of RSR proteins in OMM2.3 cells treated with: hydroxyurea (HU, 2 mM, 0-24 h), WM-3835 (20 μM, 48 h), and/or BAY-1895344 (0.1 μM, 48 h). \u003cstrong\u003e(E)\u003c/strong\u003eDNA damage response analysis. Left: Representative immunofluorescence images of phospho-RPA32 (S4/S8) and γH2AX in OMM2.3 cells treated with HU (2 mM, 24 h), WM-3835 (20 μM, 48 h), or BAY-1895344 (0.1 μM, 48 h). Scale bar: 20 μm. Right: Quantification of cells with \u0026gt;10 phospho-RPA32 or γH2AX foci (n=3 independent experiments; mean + SEM; two-tailed unpaired t-test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/6c5d8465c142c45d22e2d0bc.png"},{"id":88786793,"identity":"20bcd9be-8a5b-4fb1-a98c-43e69720a159","added_by":"auto","created_at":"2025-08-11 12:04:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23092147,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/b8b0023b-c174-41a8-bd52-d80007e9ae3a.pdf"},{"id":85476009,"identity":"f5df2d3e-c6c9-4db2-b74c-8c754371c95a","added_by":"auto","created_at":"2025-06-26 10:06:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9225255,"visible":true,"origin":"","legend":"Raw data","description":"","filename":"Rawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/96f860656b3a8ebf1e75578e.pdf"},{"id":85477015,"identity":"a1de3af7-15a0-4c0f-8063-23f3046e3fbf","added_by":"auto","created_at":"2025-06-26 10:14:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5683357,"visible":true,"origin":"","legend":"Supplementary Figures and Tables","description":"","filename":"SupplementaryFiguresandTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6875450/v1/6537605cab1b0498ecb302a7.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"HBO1 promotes replication stress response through ATR-dependent phosphorylation of Ser50/53","fulltext":[{"header":"Significance","content":"\u003cp\u003e(1) IP-MS identified ATR as a novel HBO1-binding protein.\u003c/p\u003e\n\u003cp\u003e(2) Phosphorylation of HBO1 at Ser50/53 by ATR is essential for the regulation of HBO1 on gene expression.\u003c/p\u003e\n\u003cp\u003e(3) Intact ATR is required for the treatment efficacy of HBO1 inhibitor WM-3835 in cancer cells.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe histone acetyltransferase binding to ORC1 (HBO1, also termed KAT7 or MYST2) is a key member of the MYST family of acetyltransferases. Functioning as the core catalytic subunit of the HBO1 complex, which incorporates various cofactors and co-proteins, HBO1 specifically acetylates histones H3 at lysine 14 (H3K14ac) and H4 at lysine 5, 8, and 12 (H4K5, H4K8, H4K12ac) [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. By modulating histone acetylation, HBO1 induces chromatin relaxation, facilitating the recruitment of transcription factors and thereby regulating transcriptional initiation [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, HBO1 plays pivotal roles in diverse biological processes, including chromatin remodeling [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], DNA replication [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], transcriptional regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], protein ubiquitination [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], immune response modulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], stem cell pluripotency, self-renewal maintenance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and embryonic development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence highlights the oncogenic role of HBO1 in multiple malignancies, where its overexpression promotes cancer cell proliferation, cell cycle progression, and drug resistance [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For instance, HBO1-mediated H3K14ac enhances the processivity of RNA polymerase II, sustaining the expression of oncogenes (e.g., Hoxa9 and Hoxa10) in leukemia stem cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, HBO1 facilitates gastric cancer progression by activating YAP1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and drives bladder cancer proliferation through the Wnt/β-catenin pathway [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite these findings, the precise mechanisms underlying the role of HBO1 in tumorigenesis remain incompletely understood, warranting further investigation.\u003c/p\u003e \u003cp\u003eGiven its oncogenic properties, targeting HBO1 has emerged as a promising therapeutic strategy. Genetic knockdown or pharmacological inhibition of HBO1 effectively suppresses tumor growth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In 2019, MacPherson et al. developed WM-3835, a potent small-molecule inhibitor that competitively binds the acetyl-CoA site of HBO1, selectively depleting H3K14ac levels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Preclinical studies demonstrate that WM-3835 exhibits robust anti-tumor activity in non-small cell lung cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], osteosarcoma [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and acute lymphoblastic leukemia [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], inducing G1/S cell cycle arrest and apoptosis. However, since HBO1 is ubiquitously expressed in both normal and malignant cells, the potential toxicity of WM-3835 necessitates the identification of predictive biomarkers and patient stratification strategies to optimize its clinical translation.\u003c/p\u003e \u003cp\u003eIn this study, we employed immunoprecipitation-mass spectrometry (IP-MS) to identify HBO1-interacting proteins, revealing ATR, a critical kinase involved in DNA damage response and replication stress, as a novel binding partner. Mechanistically, we demonstrate that ATR phosphorylates HBO1, modulating its function in gene expression regulation and replication stress management. Our findings elucidate an ATR-HBO1 signaling axis, providing new insights into HBO1 function in cancer progression, and highlighting ATR as a potential predictive biomarker for the use of HBO1 inhibitor.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. Proteomic analysis indicates that ATR is a key HBO1 interacting protein.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe here used melanoma as a model to elucidate the biological function of HBO1. To investigate the function of HBO1 in tumor pathogenesis, we firstly identified the interacting proteins involved. We immunoprecipitated endogenous HBO1 binding proteins from UM cell line OMM2.3 using anti-HBO1 antibodies, and analyzed the co-precipitated proteins by mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Among 993 identified binding partners (iBAQ intensity\u0026thinsp;\u0026gt;\u0026thinsp;0 in IP and input), we detected known HBO1-associated proteins (e.g., MCMs) and novel interactors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Gene Ontology (GO) analysis revealed enrichment in DNA damage repair, replication, and translation pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Strikingly, ATR, a central kinase in replication stress response, emerged as a HBO1 interactor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe validated this interaction endogenously in OMM2.3 and 92.1 UM cells via reciprocal Co-IP with HBO1- and ATR-specific antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and exogenously in 293T cells overexpressing Flag-ATR and myc-HBO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Intriguingly, pharmacological inhibition of the acetyltransferase activity of HBO1 enhanced its binding to ATR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting a regulatory crosstalk between HBO1 acetylation and ATR interaction.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. ATR interacts with HBO1 and phosphorylates HBO1 at Ser50 and Ser53.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eATR is a critical regulator of replication stress response; however, the molecular mechanism underlying its interaction with HBO1 remains poorly understood. Structurally, HBO1 consists of an N-terminal domain (NTD) harboring a zinc finger motif and a C-terminal MYST catalytic domain [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To map the ATR interaction domain, we generated lentiviral expression vectors encoding myc-tagged HBO1 truncation mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Intriguingly, co-immunoprecipitation assays revealed that the N-terminal D1 domain of HBO1 mediates ATR binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Previous studies have established that UV-induced replication stress triggers ATR-dependent phosphorylation of HBO1, facilitating DNA damage repair [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This prompted us to investigate whether HBO1 serves as a phosphorylation substrate of ATR. Using OMM2.3 cells treated with either DMSO or an ATR inhibitor, we immunoprecipitated HBO1 and probed for phospho-ATR substrates with an anti-phospho-S/TQ antibody. Notably, HBO1 exhibited robust ATR-dependent phosphorylation, which was markedly attenuated upon ATR inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Moreover, pharmacological suppression of acetyltransferase activity of HBO1 elevated S/TQ phosphorylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), corroborating our findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF. These results collectively demonstrate that HBO1 is an ATR substrate and warrant further investigation of its phosphorylation sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have identified critical serine (Ser) and threonine (Thr) phosphorylation sites within the NTD of HBO1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. PhosphoSitePlus database annotation highlighted Ser50, Ser53, Ser57, and Thr88 as the most probable phosphorylation sites on HBO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). While Ser57 is a known PLK1 target [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Thr88 is phosphorylated by CDK1 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we hypothesized that ATR preferentially targets Ser50/Ser53. To test this, we generated a polyclonal antibody against phospho-Ser50/53 (HBO1 pS50/53) and validated its specificity under UV irradiation. HBO1 pS50/53 signals increased within 0.5 hours post-UV exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), confirming antibody efficacy. We subsequently introduced alanine substitutions at Ser50/Ser53 (HBO1 SA) to abrogate phosphorylation and constructed Flag-tagged wild-type (F_HBO1) and mutant (F_HBO1 SA) expression plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Western blot analysis of transfected OMM2.3 cells confirmed equivalent expression of both constructs, while HBO1 pS50/53 signals were selectively abolished in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Immunofluorescence (IF) further validated the antibody specificity and the phospho-deficient phenotype of HBO1 SA-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-L). Interestingly, IF revealed that HBO1 acetyltransferase inhibition dramatically reduced HBO1 pS50/53 foci formation\u0026mdash;a contrast to Western blot data\u0026mdash;suggesting that the S/TQ motif detected in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD encompasses additional phosphorylation sites beyond Ser50/53.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. ATR mediated HBO1 Ser50/53 phosphorylation regulates gene expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the biological significance of HBO1 phosphorylation at Ser50/53, we investigated how these modifications influence the genomic occupancy of HBO1, given its established role in transcriptional regulation via histone acetylation. Using CUT\u0026amp;Tag-seq with an anti-Flag antibody, we profiled genome-wide binding patterns in OMM2.3 cells expressing Flag-tagged empty vector (control), wild-type HBO1 (F_HBO1), or the phospho-deficient mutant (F_HBO1 SA, Ser50/53Ala) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Strikingly, the F_HBO1 SA mutant exhibited markedly enhanced chromatin binding compared to wild-type HBO1, as evidenced by increased sequencing read intensities and larger DNA fragment sizes (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, C\u003c/b\u003e). While wild-type HBO1 occupied 5,998 genes, the SA mutant bound to 23,539 genes\u0026mdash;a\u0026thinsp;~\u0026thinsp;4-fold increase (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e). These findings demonstrate that ablation of Ser50/53 phosphorylation substantially augments the chromatin affinity of HBO1. Peak distribution analysis revealed that both F_HBO1 and F_HBO1 SA were predominantly localized to transcription start sites (TSS), consistent with HBO1\u0026rsquo;s role in transcriptional regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Quantitatively, 31.78% of F_HBO1 peaks and 34.29% of F_HBO1 SA peaks mapped to promoter regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), further supporting HBO1\u0026rsquo;s functional association with gene regulatory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGO analysis demonstrated that genes bound by HBO1 were significantly enriched in chromatin remodeling-related biological processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Comparative genomic localization revealed substantial overlap between wild-type HBO1 and HBO1 SA binding sites, with 93.7% of HBO1-bound genes also occupied by the SA mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Strikingly, the HBO1 SA mutant uniquely bound to 17,918 additional genes, predominantly involved in chromatin remodeling and DNA repair pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), suggesting that phospho-deficient HBO1 Ser50/53 substantially expands the genomic binding capacity of HBO1. Both wild-type and mutant HBO1 consistently occupied key chromatin regulatory genes including \u003cem\u003eDDX17\u003c/em\u003e, \u003cem\u003eEIF4A2\u003c/em\u003e, \u003cem\u003eAKT1\u003c/em\u003e, and \u003cem\u003eARAF\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). However, the SA mutant specifically enriched at DNA repair genes (e.g., \u003cem\u003eBACH1\u003c/em\u003e, \u003cem\u003eCHK1\u003c/em\u003e, \u003cem\u003eTOPBP1\u003c/em\u003e, \u003cem\u003eRPA2\u003c/em\u003e), indicating a phosphorylation-dependent shift in target preference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Motif analysis uncovered differential binding patterns: while wild-type HBO1 preferentially associated with KLF10 motifs (consistent with prior reports [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]), the SA mutant showed strong affinity for CTCF binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Given the established role of CTCF in chromatin architecture maintenance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we propose that phospho-deficient HBO1 may modulate chromatin three-dimensional organization, potentially facilitating its expanded genomic occupancy.\u003c/p\u003e \u003cp\u003eTo elucidate the functional consequences of HBO1 Ser50/53 phosphorylation on transcriptional regulation, we performed RNA sequencing analysis in OMM2.3 cells overexpressing either wild-type F_HBO1 or the phospho-deficient F_HBO1 SA mutant. Comparative transcriptome profiling identified 585 significantly upregulated and 562 downregulated genes in F_HBO1 SA-expressing cells compared to wild-type controls\u003c/p\u003e \u003cp\u003e(|log\u003csub\u003e2\u003c/sub\u003eFC| \u0026ge; 0.585, P value\u0026lt;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). GO analysis revealed that the downregulated gene set was remarkably enriched for biological processes related to DNA damage repair and chromatin remodeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Intriguingly, this transcriptional repression pattern correlated with the expanded chromatin binding capacity of the SA mutant observed in our CUT\u0026amp;Tag analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These findings suggest that the phospho-deficient HBO1 SA mutant may function as a transcriptional repressor of DNA repair genes through its enhanced chromatin occupancy at these loci.\u003c/p\u003e \u003cp\u003eTo investigate the functional significance of HBO1 Ser50/53 phosphorylation in WM-3835-mediated H3K14ac inhibition and tumor suppression, we first conducted transcriptome profiling of control and WM-3835-treated OMM2.3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, K). RNA-seq analysis revealed significant transcriptional changes, with 1,368 genes upregulated and 1,212 genes downregulated following WM-3835 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Notably, upregulated genes were enriched in apoptosis and growth inhibition pathways, consistent with the tumoricidal effect of WM-3835, while downregulated genes were associated with DNA damage repair and cell cycle progression, mirroring the transcriptional profile observed with Ser50/53 mutation. Comparative analysis of differentially expressed genes (DEGs) identified 1,147 and 2,580 genes altered by HBO1 SA mutation and WM-3835 treatment, respectively, with 377 co-regulated genes exhibiting largely concordant expression patterns (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE, F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo determine whether these transcriptional changes were directly mediated by HBO1 binding, we integrated our CUT\u0026amp;Tag and RNA-seq datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Among 5,621 genes bound by both wild-type and mutant HBO1, 631 showed WM-3835-responsive expression changes, while 319 were affected by Ser50/53 mutation (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, B\u003c/b\u003e). Strikingly, genes specifically bound by HBO1 SA exhibited more pronounced transcriptional alterations following WM-3835 treatment, with upregulation of apoptosis-related genes and suppression of cell division, migration, and DNA repair pathways (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC\u003c/b\u003e). Importantly, HBO1 SA preferentially bound and repressed genes involved in DNA damage response and cell cycle regulation (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD\u003c/b\u003e). Collectively, these findings demonstrate that ATR-mediated phosphorylation of HBO1 at Ser50/53 modulates H3K14ac dynamics and gene expression programs. The convergence of WM-3835-induced transcriptional changes with HBO1 SA-mediated regulation suggests that pharmacological inhibition of H3K14ac may functionally mimic the effects of phospho-deficient HBO1, providing mechanistic insight into the anticancer activity of WM-3835.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e4. HBO1 regulates replication stress via Ser50/53 phosphorylation\u003c/h2\u003e \u003cp\u003eWe then examined the expression of HBO1 and found the universal expression of HBO1 in both normal (PIG1 and ARPE-19), and melanoma cell lines (\u003cb\u003eFig. S3A\u003c/b\u003e). Importantly, higher level of HBO1 expression was associated with poor overall survival in UM patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). To study the function, we constructed HBO1 knockdown and overexpression OMM2.3 and 92.1 stable cell lines (\u003cb\u003eFig. S3B, C\u003c/b\u003e). Compared with control cells, HBO1-depleted cells showed a remarkable decrease by about 3/4 in OMM2.3 and 1/2 in 92.1 in colony area. Consistently, upon HBO1-overexpression, OMM2.3 (P\u0026thinsp;=\u0026thinsp;0.0006) and 92.1 (P\u0026thinsp;=\u0026thinsp;0.0076) exhibited significantly increased colony formation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). These results suggested the oncogenic role of HBO1 in UM, warranting further tests of cell cycle and replication stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn accordance with earlier reports [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], flow cytometry analysis also showed the block of cell cycle at G0/G1 phase by WM-3835 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, S4A). Given the interaction between HBO1 and ATR, we then examined the role of HBO1 in replication stress response. We calculated the replication stress response score (RSR score) using the characteristic gene set of replication stress response [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Pearson correlation analysis showed that HBO1 expression was significantly positively correlated with RSR score in various tissues (\u003cb\u003eFig. S5A, Table S3\u003c/b\u003e). ATR, CHK1, and RPA32 phosphorylation are markers of ATR pathway activation, while phosphorylated RPA32 (phospho-RPA32) and γH2AX are defined as replication stress markers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Under the treatment of hydroxyurea (HU), phospho-RPA32 and γH2AX were increased, confirming the activation of replication stress. Importantly, replication stress was significantly suppressed when HBO1 was knocked-down or inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-I, S5B), demonstrating the crucial role of HBO1 in replication stress.\u003c/p\u003e \u003cp\u003eWe next examined the clinical relevance of HBO1 phosphorylation at Ser50/53. Immunohistochemistry (IHC) staining of melanoma tissue array revealed that HBO1 pS50/53 intensity was markedly elevated in melanoma, compared with that of adjacent and normal skin tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Key genes involved in melanoma progression are often associated with cancer stages (Clark level) and Breslow thickness [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, in our data, patients diagnosed with advanced stages, greater Breslow thickness, and lymph node metastasis did not show higher HBO1 pS50/53 level (\u003cb\u003eFig. S6A\u003c/b\u003e). Interestingly, inhibition of HBO1 pSer50/53 significantly reduced the number of OMM2.3 colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), with no regard to cell cycle arrest (\u003cb\u003eFig. S6B\u003c/b\u003e). Both IF staining and WB showed remarkable reduction of phospho-RPA32 and γH2AX when the HBO1 Ser50/53 phosphorylation sites were mutated, suggesting the requirement of such phosphorylation sites of HBO1 by ATR signaling activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M). These findings were also consistent with the transcriptomic data in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K and \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF\u003c/b\u003e, showing the alteration of gene expression under WM-3835 treatment in cells reconstituted with HBO1 SA.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Inhibition of ATR activity alters WM-3835 sensitivity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBuilding on above results, we then speculated that ATR activity may affect the treatment efficacy of WM-3835. We treated OMM2.3 and 92.1 cells with WM-3835 and BAY-1895344, an ATR inhibitor currently under clinical trial evaluation for the treatment of advanced solid tumors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Synergy score demonstrated that the effects of WM-3835 was antagonized by the addition of BAY-1895344 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Consistent to these observations, knockdown of ATR impaired the sensitivity of 92.1 cells to WM-3835 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). We also examined the level of phospho-RPA32 and γH2AX, to evaluate the impact on replication stress by BAY-1895344 and WM-3835. We found that the inhibition of phospho-RPA32 and γH2AX by WM-3835 was significantly attenuated under BAY-1895344 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). These data support the role of ATR as a potential predictive biomarker to WM-3835 sensitivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe histone acetyltransferase HBO1 has been predominantly characterized as exerting its biological functions through its catalytic acetylation activity [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While previous studies have established that HBO1 targeting induces tumor cell death via cell cycle arrest [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], our work significantly expands this understanding by identifying ATR as a novel binding partner and critical regulator of HBO1. This discovery functionally links replication stress regulation function of ATR with chromatin regulation function of HBO1.\u003c/p\u003e \u003cp\u003eThe 611-amino acid HBO1 protein features two principal domains: an N-terminal DNA-binding domain containing a zinc finger motif (zf-C2HC, 184-212aa) that interacts with replication initiation factors MCM2 and ORC1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and a C-terminal MYST acetyltransferase domain (340-607aa) responsible for catalytic activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Despite its functional importance, the NTD remains poorly characterized due to the lack of structural information. Our findings substantially advance the understanding of HBO1 NTD.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that post-translational modifications (PTMs) of HBO1 NTD serve as molecular switches modulating its activity. For instance, CDK1-mediated phosphorylation at Thr85/Thr88 primes subsequent PLK1-dependent phosphorylation at Ser57, facilitating pre-replicative complex formation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, CDK11-mediated HBO1 phosphorylation enhances its acetyltransferase activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In line with these findings, our study demonstrates that prevention of Ser50/53 phosphorylation dramatically alter the genomic binding landscape of HBO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The phospho-deficient mutant (HBO1 SA) exhibited substantially increased genomic occupancy (23,539 binding sites vs. 5,998 in wild-type), suggesting that phosphorylation at these sites normally restricts widespread DNA binding.\u003c/p\u003e \u003cp\u003eNotably, motif analysis of HBO1 SA binding regions revealed strong enrichment for CTCF motifs. Given the established role of CTCF in maintaining chromatin accessibility [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], this observation may explain the expanded genomic binding of the phospho-deficient mutant. Furthermore, while wild-type HBO1 preferentially associates with transcription start sites and promoter regions, consistent with reports in hESCs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and HeLa cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], the SA mutant showed increased binding at replication origins, suggesting phosphorylation-dependent regulation of replication licensing.\u003c/p\u003e \u003cp\u003eOur findings uncover a novel ATR-HBO1 axis in replication stress management. We demonstrate that ATR-mediated phosphorylation of HBO1 Ser50/53 modulates its binding to DNA damage repair genes, independent of its acetyltransferase activity. Intriguingly, the transcriptional effects of HBO1 SA mutation paralleled those of WM-3835 treatment, both downregulating DNA repair and cell cycle-related genes. Mechanistically, we found that HBO1 SA directly binds to and represses \u003cem\u003eTOPBP1\u003c/em\u003e and \u003cem\u003eRPA2\u003c/em\u003e, which are critical components of the ATR activation cascade [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This observation explains how ATR-dependent HBO1 phosphorylation promotes replication stress response: 1) RPA-coated ssDNA recruits ATR-ATRIP complexes; 2) TOPBP1 mediates ATR activation; 3) Activated ATR phosphorylates HBO1 Ser50/53; 4) Phospho-HBO1 upregulates DNA repair genes through targeted genomic binding. This feedforward mechanism ensures appropriate cellular responses to replication stress.\u003c/p\u003e \u003cp\u003eWhile WM-3835 (a first-in-class HBO1 inhibitor) shows promise in inducing G0/G1 arrest in several tumor types [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], our work reveals its functional interplay with ATR signaling. Surprisingly, ATR inhibition attenuated WM-3835 efficacy, suggesting ATR expression may serve as a predictive biomarker for HBO1-targeted therapy. This finding has important clinical implications, as combining WM-3835 with drugs targeting DNA damage response and cell cycle checkpoint kinases (including ATR, CHK1, WEE1, and MYT1) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], common strategies in DDR-targeted therapy, may require careful patient stratification.\u003c/p\u003e \u003cp\u003eTaken together, our study establishes ATR as a key regulator of HBO1 through phosphorylation-dependent modulation of its genomic binding. This ATR-HBO1 axis represents a novel mechanism coordinating chromatin regulation with replication stress response. From a translational perspective, we propose ATR expression as a potential biomarker for HBO1 inhibitor sensitivity, providing a framework for precision medicine approaches targeting this pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eHuman renal epithelial cell line HEK293T and human cutaneous melanoma cell lines A375 and A2058 cells was cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco, #8122522). Human cutaneous melanocyte cell PIG1 and retinal pigment epithelium cell ARPE-19 were cultured in RPMI1640 medium (Gibco, #8122322). Human cervical cancer cell HeLa and Human UM cell lines (OMM1, OMM2.3, MEL202, MEL270, MEL290, 92.1, MUM2B) were cultured in RPMI1640 medium (Gibco, #8122322). All mediums are supplemented with 10% fetal bovine serum (FBS, Gibco, #16140071) and 1% antibiotics (Penicillin-streptomycin, Gibco, #10378016). All cells were cultured in a 37℃ humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eTotal cell lysates were prepared in urea lysis buffer supplemented with protease inhibitor and phosphatase inhibitor. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, #ISEQ00010). After blocking with 5% non-fat milk for 1 hour at room temperature, the membranes were incubated with anti-HBO1 (Proteintech, 13751-1-AP, 1:2000), anti-ATR (Abclonal, A21253,1:1000), anti-myc (Santa cruz, sc-40, 1:1000), anti-Flag (Proteintech, 66008-4-Ig, 1:10000), anti-Phospho-ATM/ATR Substrate (S/TQ) (CST, 9607S, 1:1000), anti-HBO1 pS50/53 (1:500), anti-phospho-RPA32 (BETHYL, A300-245A, 1:2000), anti-\u0026gamma;H2AX (SIGMA, 05-636-I, 1:1000), anti-RPA32 (CST, 35869S, 1:1000), anti-H3K14Ac (Abclonal, A7254, 1:1000), anti-phospho-ATR(CST, #58014, 1:1000), anti- RPA70 (CST, #2267S, 1:1000) antibody overnight at 4℃ and then with the appropriate secondary antibodies conjugated to a fluorescent tag (CST, #5151 \u0026amp; #5470, 1:10000) for 1 hour at room temperature. Anti-\u0026beta;-Actin (Proteintech, 66009-1-Ig, 1:20000), anti-\u0026beta;-tubulin (Abclonal, A12289, 1:10000) and anti-GAPDH (Proteintech, 60004, 1:20000) antibody served as the loading control. The immunoblots were recorded with the Odyssey infrared imaging system (LI-COR Biosciences). Full length uncropped original western blots are provided in supplementary material.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e\n \u003cp\u003eCells were pelleted and washed with PBS, and then lysates were prepared in 500 \u0026micro;L lysis buffer containing 120 mM NaCl, 20 mM Tris-Cl, 2 mM EDTA, 1% NP-40 and 5% Glycerol supplemented with 1\u0026times;protease inhibitor. Anti-HBO1 (Proteintech, 13751-1-AP, 4 \u0026micro;L), anti-ATR (CST, 13934S, 2.5 \u0026micro;L), anti-myc (Santa cruz, sc-40, 5 \u0026micro;L), anti-Flag (Proteintech, 66008-4-Ig, 5 \u0026micro;L) antibody or normal mouse IgG (Santa cruz, sc-2025, 1:10000) antibody was incubated with the cell lysates overnight at 4℃, after which 30 \u0026micro;L protein A magnetic beads (CST, #73778) were added and incubated for 2 additional hours. Then, the magnetic beads were washed three times with lysis buffer. For IP-MS analysis, 100 \u0026micro;L glycine solution was used for each tube to elute the protein complexes from the beads, while 1\u0026times;SDS loading buffer (NCM, WB2001) was used per sample for SDS-PAGE analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eTransfection and virus packaging\u003c/h3\u003e\n\u003cp\u003eTwo shRNA sequences targeting HBO1 and ATR were cloned into the pLKO.1_TRC vector. The HBO1 targeting sequences were: 5\u0026rsquo;-CCTCTCAAGTAGCTGGGATTA-3\u0026rsquo;(shHBO1_1); 5\u0026rsquo;-CCTCTCAAGTAGCTGGGATTA \u0026minus;\u0026thinsp;3\u0026rsquo; (shHBO1_2). The ATR targeting sequences were: 5\u0026rsquo;-CCGGATACTTACAGATGTAAA-3\u0026rsquo;(shATR_1); 5\u0026rsquo;-GTAATGCATTTGGTATGAATC-3\u0026rsquo; (shATR_2).\u003c/p\u003e\n\u003cp\u003eThe full length of human HBO1 (isoform NM_007067), myc_HBO1 D1, myc_HBO1 D2 and myc_HBO1 D3 were cloned into the pHAGE-myc-puro vector respectively for overexpression of HBO1 and HBO1 D1/2/3. Primers used for cloning are shown in \u003cstrong\u003eTable S4\u003c/strong\u003e. CMV_Flag_ATR (#41909) was purchased from addgene. PolyJet DNA In Vitro Transfection Reagent (SignaGen, SL100688) was used for plasmid transfection following the manufacturer\u0026rsquo;s instructions. After lentiviral packaging with HEK293T cells, cells were infected with lentiviruses and selected by incubation with 2 \u0026micro;g/mL puromycin for 3 days.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence (IF)\u003c/h3\u003e\n\u003cp\u003eCells adhering to a glass slide were fixed with 4% paraformaldehyde (Biosharp, #70071800) for 15 min, permeabilized with 0.1% Triton X-100 (Sigma, T9284) for 15 min and then blocked with 2% BSA solution for 1 h at room temperature. After incubation with primary antibody against HBO1 pS50/53 (1:200), phospho-RPA32 (BETHYL, A300-245A, 1:2000) and \u0026gamma;H2AX (SIGMA, 05-636-I, 1:500) overnight, the cells were washed three times with PBS and subsequently incubated with Alexa Fluor 594 secondary antibody (Invitrogen, A11012, 1:1000) or Alexa Fluor 488 secondary antibody (Invitrogen, A11029, 1:1000) for 1 h. The coverslips were then mounted with ProLong Gold mounting medium with DAPI (Invitrogen, P36931) and observed under an inverted fluorescence microscope (Nikon, Japan). Foci number was quantified for each condition. The results presented were obtained from three independent biological replicates; at least 50 cells were measured per replicate.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA extraction and quantification\u003c/h2\u003e\n \u003cp\u003eRNA purification was performed using EZ-press RNA purification kit (EZBioscience, #B0004DP) according to the manufacturer\u0026rsquo;s guidelines. Then, cDNA synthesis was achieved using the HiScript III RT SuperMix (Vazyme, #R323). Finally, real-time quantitative PCR (RT-qPCR) was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q711). mRNA expression values were calculated using the \u0026Delta;\u0026Delta;Ct method and human \u0026beta;-Actin gene as a control. A detailed list with primers used in the present study is provided in \u003cstrong\u003eTable S5\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA-seq\u003c/h2\u003e\n \u003cp\u003eRNA sequencing was performed by Novogene (Beijing, China). Total RNA was harvested using Trizol. The integrity of the RNA was assessed by Agilent 2100 bioanalyzer (Thermo Fisher Scientific). Approximately 1 \u0026micro;g mRNA from each sample was used for RNA sequencing (Illumina HiSeq PE150 platform). GO analysis was performed by DAVID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://davidbioinformatics.nih.gov/summary.jsp\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eCUT\u0026amp;Tag\u003c/h2\u003e\n \u003cp\u003eCUT\u0026amp;Tag assay was performed using Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme, TD904) following the manufacturer\u0026rsquo;s protocol. Briefly, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e OMM2.3 cells transfected with F_HBO1 or F_HBO1 SA were collected. After binding to concanavalin A\u0026ndash;coated magnetic beads (ConA beads), bead-bound cells were incubated with anti-Flag antibody (CST, #14793, 1:50) or normal rabbit IgG (CST, 2729, 1:100) for 2 h at room temperature. After brief wash with dig-wash buffer, cells were then incubated with goat anti rabbit secondary antibody (Abcam, ab6702, 1:100) for 1h at room temperature. When antibody binding procedures were finished, the bead-bound cells were then mixed with hyperactive pA-Tn5 transposon and tagmentated with tagmentation buffer. Tagmentated DNA was then extracted and amplified to form the sequencing-ready libraries. After the PCR reaction, libraries were purified with the DNA clean beads (Vazyme, N411) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). The library preparations were sequenced on Illumina Novaseq platform at Novogene (Beijing, China). GO analysis was implemented by DAVID. In Venn diagrams, numbers represent genes co-occurring between conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eColony formation\u003c/h2\u003e\n \u003cp\u003eA volume of 3 mL of complete medium containing 2000 cells was seeded in each well of a 6-well plate and incubated for about 2 weeks. Once colonies formed in control conditions, plates were fixed with 4% paraformaldehyde, stained with crystal violet. Colony formation efficiency is indicated by colony area. The quantifications presented were obtained from three independent biological replicates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCell viability assay\u003c/h2\u003e\n \u003cp\u003eCells were seeded at a density of 1,000 cells per well in triplicate into 96-well plates. For drug sensitivity, cells were treated with WM-3835 (Selleck, S9805) and BAY-1895344 (Selleck, S8666) for 48 hours. The absorbance at 450 nm was then measured. The data were recorded and analyzed. The quantifications presented were obtained from three independent biological replicates. The synergy score was calculated by SynergyFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://synergyfinder.aittokallio.group/20250411123730439716/\u003c/span\u003e\u003c/span\u003e). When synergy score is less than \u0026minus;\u0026thinsp;10, the interaction between two drugs is likely to be antagonistic.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eCell cycle FACS\u003c/h2\u003e\n \u003cp\u003e2\u0026times;10\u003csup\u003e5\u003c/sup\u003e melanoma cells were collected and centrifuged to remove the medium and washed twice with PBS. After resuspension with 75% ethanol and incubation at 4℃ overnight, ethanol was removed by centrifugation and washed twice with PBS. Finally, cells were stained using 0.5 mL PI/RNase Staining Buffer (Multi sciences, CCS012) and incubated at room temperature for 30 min followed by FACS analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\n \u003cp\u003eTissue microarrays (ZL-MEL361 and ZL-MEL963) for IHC staining were bought from Weiaobio (Shanghai, China). The information of tissue microarrays is provided in \u003cstrong\u003eTable S6\u003c/strong\u003e. IHC staining of tissue microarrays was performed by Servicebio (Wuhan, China). Tissues were deparaffinized and rehydrated through an alcohol series, followed by antigen retrieval with sodium citrate buffer. Then tissue sections were blocked with 3% bovine serum albumin (BSA) 30 min at room temperature and then incubated with anti-HBO1 pS50/53 (1:200) antibody at 4℃ overnight. Phospho Ser50 and Ser53 HBO1 rabbit polyclonal antibodies were generated by HUABIO (Hangzhou, China). Finally, the tissues were covered with horseradish peroxidase (HRP) labeled secondary antibody and incubated at room temperature for 50 min. All immunostained slides were scanned on 3D Histech Quant Center (3D Histech), and computerized image analysis was performed by Halo (Indica labs). Immunostaining for HBO1 pS50/53 was analyzed in melanoma tissues, adjacent normal tissues and normal skin tissues using percentage of positive cells and histochemistry score (H-score). H-score was determined based on the proportion of positive cells and the intensity of nuclear staining as previously described [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. H-score=\u0026sum;(pi\u0026times;i)= (percentage of cells of weak intensity \u0026times;1) + (percentage of cells of moderate intensity \u0026times;2)+ (percentage of cells of strong intensity \u0026times;3). In the formula, pi represents the percentage of positive cells in the slide; i represents the intensity of HBO1 pS50/53 staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 9.0 was used for statistical analyses. Descriptive values are presented as mean\u0026thinsp;+\u0026thinsp;standard error of the mean (SEM) unless stated elsewhere. Differences between two groups were analyzed by unpaired Student\u0026rsquo;s t-test while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) with post-hoc intergroup comparisons with Turkey\u0026rsquo;s test. When data did not meet the normal distribution, the Mann\u0026ndash;Whitney U-test was performed. Test details were indicated in the figure legends. Results were considered statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. TCGA overall survival analysis and Pearson correlation analysis (HBO1 vs RSR signature score) was performed on GEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003c/span\u003e). RSR signature gene set was shown in \u003cstrong\u003eTable S7\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.Z. designed and performed the experiments, analyzed the experimental results, and drafted the manuscript. Z.Z. conducted bioinformatics analysis of the sequencing data. Y.W. and G.Z. contributed to the generation of expression constructs and establishment of stable cell lines. Y.F. and Q.L. assisted in drug sensitivity assays and offered scientific input for project development. J.S. conceived and supervised the study, interpreted the data, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by General Program of National Natural Science Foundation of China (No. 82472752 and 81972667 to J.S.), National Key R\u0026amp;D Program of China (2021YFC2701103 to J.S.), and the Center for High Performance Computing at Shanghai Jiao Tong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available in Gene Expression Omnibus (GEO) with accession number GSE299365 and GSE299366.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLan R, Wang Q. 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Mod Pathol. 2019;32:1447-1459.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HBO1, replication stress, ATR","lastPublishedDoi":"10.21203/rs.3.rs-6875450/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6875450/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMounting evidence has shown that histone acetyltransferase binding to ORC1 (HBO1) serves as an oncoprotein, warranting the use of small molecule inhibitor WM-3835 for cancer therapy. However, HBO1 is ubiquitously expressed in both tumor and normal tissues, with potential to increase the risk of systematic toxicity. This unmet need highlights the importance of identifying suitable biomarkers to predict the sensitivity to HBO1 inhibitor. Here, we show that ATR, a key regulator of DNA replication stress, is a novel interacting partner of HBO1. Additionally, we reveal the regulatory function of HBO1 in DNA replication stress responses, in an ATR-dependent manner. Mechanistically, ATR mediated HBO1 Ser50/53 phosphorylation interferes with genomic binding of HBO1 and regulates gene expression. Notably, the overexpression of HBO1 mutated at the ATR phosphorylation site (S50/53A) dampens the expression of DNA repair related genes and suppresses tumor colony formation, consistent with the observations of WM-3835 treatment. Inhibition of ATR significantly antagonized the treatment sensitivity of WM-3835. Collectively, our findings uncovered a previously unidentified role of HBO1 in the regulation of replication stress and discovered ATR as a potential biomarker for WM-3835 treatment.\u003c/p\u003e","manuscriptTitle":"HBO1 promotes replication stress response through ATR-dependent phosphorylation of Ser50/53","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 09:58:37","doi":"10.21203/rs.3.rs-6875450/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59087c9e-a61f-4fce-9795-03d0eea59313","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50493485,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":50493486,"name":"Biological sciences/Molecular biology/DNA damage and repair/DNA damage response"}],"tags":[],"updatedAt":"2025-08-11T11:55:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 09:58:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6875450","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6875450","identity":"rs-6875450","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}