Enhancing sensitivity of triple-negative breast cancer to DNA-damaging therapy through chemical inhibition of the m6A methyltransferase METTL3

IF 20.1 1区 医学 Q1 ONCOLOGY
Bianca Cesaro, Alessia Iaiza, Fabio Piscopo, Marco Tarullo, Eleonora Cesari, Dante Rotili, Antonello Mai, Alberto Diana, Michela Londero, Luca Del Giacco, Riccardo Masetti, Alba Di Leone, Chiara Naro, Silvia Masciarelli, Giulia Fontemaggi, Claudio Sette, Francesco Fazi, Alessandro Fatica
{"title":"Enhancing sensitivity of triple-negative breast cancer to DNA-damaging therapy through chemical inhibition of the m6A methyltransferase METTL3","authors":"Bianca Cesaro,&nbsp;Alessia Iaiza,&nbsp;Fabio Piscopo,&nbsp;Marco Tarullo,&nbsp;Eleonora Cesari,&nbsp;Dante Rotili,&nbsp;Antonello Mai,&nbsp;Alberto Diana,&nbsp;Michela Londero,&nbsp;Luca Del Giacco,&nbsp;Riccardo Masetti,&nbsp;Alba Di Leone,&nbsp;Chiara Naro,&nbsp;Silvia Masciarelli,&nbsp;Giulia Fontemaggi,&nbsp;Claudio Sette,&nbsp;Francesco Fazi,&nbsp;Alessandro Fatica","doi":"10.1002/cac2.12509","DOIUrl":null,"url":null,"abstract":"<p>Dear Editor,</p><p>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is a critical mRNA modification catalyzed by the enzyme methyltransferase-like 3 (METTL3), with implications in RNA metabolism. METTL3 upregulation is associated with cancer progression, metastasis, and drug resistance, making it a potential therapeutic target [<span>1</span>]. The small-molecule METTL3 inhibitor, STM2457, has shown promise in treating acute myeloid leukemia (AML) and has demonstrated good tolerance in mice [<span>2, 3</span>]. However, the specific cancer types where METTL3 inhibitors are most effective remain unknown.</p><p>In breast cancer, <i>METTL3</i> knockdown markedly suppresses proliferation, invasiveness, and metastasis [<span>4</span>]. Therefore, METTL3 inhibition is proposed as a therapeutic approach for breast cancer. Triple-negative breast cancer (TNBC), the most aggressive subtype, lacks targeted therapies, and its primary treatments involve conventional chemotherapy and DNA-damaging agents [<span>5</span>]. Homologous recombination deficiency, such as mutations in the breast cancer gene 1 (<i>BRCA1</i>) and <i>BRCA2</i>, serves as a predictive biomarker for identifying patients who would benefit from genotoxic chemotherapy and poly(ADP-ribose) polymerase (<i>PARP</i>) inhibitors. Notably, METTL3 is recruited to DNA-damaged sites and is crucial for subsequent DNA repair [<span>6, 7</span>]. Consequently, <i>METTL3</i> knockdown reduces DNA repair activity and sensitizes cancer cells to genotoxic drugs [<span>7, 8</span>]. However, while TNBC exhibits elevated METTL3 levels, and its nuclear catalytic activity associates with invasiveness and metastasis [<span>9</span>], it remains uncertain whether METTL3 inhibition enhances chemotherapy response in TNBC.</p><p>Here, we aimed to explore the potential of METTL3 catalytic inhibition by STM2457 as a valuable treatment option for TNBC. Furthermore, we assessed the impact of STM2457 on the sensitivity of TNBC cells and a TNBC patient-derived organoid line to clinical DNA-damaging therapies, like platinum-based chemotherapy and the PARP inhibitor olaparib (Supplementary file of methods).</p><p>STM2457 significantly reduced the proliferation and viability of TNBC cells, including both <i>BRCA1/2</i> wild-type (MDA-MB-231 and MDA-MB-468) and <i>BRCA1</i>-mutated (MDA-MB-436, HCC1395, and HCC1937) cell lines. STM2457 exhibited negligible effects on the proliferation of non-tumoral breast epithelial cells (MCF-10A), with significant reduction observed only at the highest concentration tested (100 μmol/L) (Figure 1A, Supplementary Figure S1A-B). The treatment with 10 μmol/L STM2457 for 48 h decreased the global m<sup>6</sup>A levels in mRNA by approximately 50% in both MDA-MB-231 and MCF-10A cells (Supplementary Figure S1C). Colony formation assays further confirmed the anti-proliferative impact of STM2457 on TNBC cell lines (Figure 1B, Supplementary Figure S2). Moreover, wound healing assays indicated that the inhibition of METTL3 activity reduced TNBC cell migration (Figure 1C). The specificity of the effects exerted by STM2457 were confirmed by METTL3 knockdown (Supplementary Figure S3). To identify the molecular pathways regulated by METTL3 inhibition, we investigated its effect on MDA-MB-231 cells, one of the most aggressive TNBC cell line. Cells were treated with 10 μmol/L STM2457 for 48 h and subjected to RNA-sequencing. Bioinformatics analysis revealed 3,182 genes downregulated upon STM2457 treatment, with 3,237 genes upregulated by the drug (Supplementary Figure S4A, Supplementary Table S1). Gene ontology (GO) analysis of differentially expressed genes showed a significant downregulation of biological processes involved in cell proliferation, translation, and DNA repair (Supplementary Figure S4B). This result is consistent with previous analyses performed in breast cancer cells knocked down for METTL3 expression [<span>8, 9</span>] and in AML cell lines treated with STM2457 [<span>2</span>]. Comparison of our RNA-sequencing dataset with a methylated RNA immunoprecipitation (MeRIP)-sequencing dataset performed in MDA-MB-231 cells [<span>9</span>] showed a substantial overlap between the transcripts regulated by STM2457 treatment and those that are m<sup>6</sup>A methylated (Supplementary Figure S5A). GO analysis of the m<sup>6</sup>A-methylated genes, which exhibit differential expression upon METLL3 inhibition, revealed a significant downregulation of biological processes related to the cell cycle and DNA damage repair (Supplementary Figure S5B). Consistent with global m<sup>6</sup>A level observations, individual MeRIP-qPCR analysis on highly methylated transcripts confirmed around a 50% reduction due to STM2457 treatment (Supplementary Figure S6). We focused on the DNA damage repair pathway because of its relevance for TNBC therapy [<span>8</span>].</p><p>First, we investigated the effect of METTL3 inhibition in combination with DNA-damaging agents. Platinum-based chemotherapeutic agents and PARP inhibitors, such as olaparib, showed mostly favourable responses in TNBC harboring <i>BRCA1/2</i> mutations [<span>5</span>]. Since STM2457 downregulated genes involved in the DNA repair pathway, we hypothesized that it may reduce homologous recombination (HR) proficiency also in TNBC cells that are wild-type for <i>BRCA1/2</i>. To test this hypothesis, we examined the effect of METTL3 inhibition on the response to cisplatin and olaparib in MDA-MB-231 cells, which are <i>BRCA1/2</i> wild-type and characterized by lower sensitivity to DNA-damaging agents. MDA-MB-231 cells were treated with doses of STM2457, cisplatin, and olaparib that were below their half maximal inhibitory concentration (IC<sub>50</sub>) either as single agents or in combination. Combined treatment with STM2457 and either cisplatin or olaparib significantly reduced MDA-MB-231 cell proliferation and increased apoptosis compared to single-drug treatments (Figure 1D-G). Notably, treatment with STM2457 alone was sufficient to induce DNA damage, as indicated by the elevated phosphorylation of the H2A histone family member X (γH2AX) (Supplementary Figure S7A-B). Additionally, we noticed a global reduction in RecA-related protein 51 (RAD51) protein levels and RAD51 foci upon DNA damage (Supplementary Figure S7C-D). These findings are in line with those obtained in cells knocked down for METTL3 expression [<span>7, 8</span>]. Moreover, we observed substantial and consistent increase in DNA damage in response to combined treatments (Supplementary Figure S7A-B). Together, these data indicate that STM2457 strongly sensitizes HR-proficient TNBC cells with wild-type <i>BRCA1/2</i> to DNA damage induced by genotoxic chemotherapy or PARP inhibitors.</p><p>As STM2457 treatment reduced TNBC cell migration, we further explored its impact on cancer cell metastasis. We chose the metastatic TNBC cell line MDA-MB-231 for injection into zebrafish larvae, a well-established in vivo xenograft model for studying metastasis [<span>10</span>]. MDA-MB-231 cells expressing green fluorescent protein (GFP) were microinjected into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Embryos were then exposed to compounds, either independently or in combinations, as employed in the in vitro assays. We observed no toxicity during the treatment with STM2457 in the larvae. While some extravasated cells were scattered throughout the larva, GFP-positive clusters were enriched in the caudal hematopoietic tissue region. We quantified micrometastases in this area (Figure 1H). Intriguingly, while each drug alone reduced cell metastasis, the combined STM2457 + cisplatin and STM2457 + olaparib treatments exhibited a notable mutually reinforcing trend, leading to a substantial reduction in both the number and size of micrometastases (Figure 1H).</p><p>Finally, we assessed the effect of STM2457 in a patient-derived organoid (BCO-21) from a TNBC patient with wild-type <i>BRCA1/2</i> genes (Figure 1I). This patient was ineligible for olaparib treatment. Initially, we determined the IC<sub>50</sub> for single-drug treatments with STM2457, olaparib, and carboplatin, a platinum-based chemotherapy used in TNBC. In line with the results from MDA-MB-231 cells, STM2457 decreased BCO-21 cell viability (Figure 1J, Supplementary Figure S8). We then investigated whether STM2457 enhances the BCO-21 sensitivity to DNA-damaging therapy. Notably, combination treatment with both optimal (10 μmol/L) and suboptimal (5 μmol/L) doses of STM2457 synergistically increased the cytotoxic effect of carboplatin and olaparib (combination index &lt; 1; Figure 1K, Supplementary Figure S8).</p><p>In conclusion, this study showed that STM2457 displayed anti-tumor activity in TNBC and enhanced chemotherapy response while sensitizing <i>BRCA1/2</i> wild-type patients to olaparib. Given the observed upregulation of METTL3 activity across various cancer types, METTL3 inhibitors may offer a novel approach to improve the efficacy of DNA-damaging agents, overcome inherent resistance, and mitigate genotoxic drug adverse effects.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":null,"pages":null},"PeriodicalIF":20.1000,"publicationDate":"2023-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12509","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12509","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Dear Editor,

N6-methyladenosine (m6A) is a critical mRNA modification catalyzed by the enzyme methyltransferase-like 3 (METTL3), with implications in RNA metabolism. METTL3 upregulation is associated with cancer progression, metastasis, and drug resistance, making it a potential therapeutic target [1]. The small-molecule METTL3 inhibitor, STM2457, has shown promise in treating acute myeloid leukemia (AML) and has demonstrated good tolerance in mice [2, 3]. However, the specific cancer types where METTL3 inhibitors are most effective remain unknown.

In breast cancer, METTL3 knockdown markedly suppresses proliferation, invasiveness, and metastasis [4]. Therefore, METTL3 inhibition is proposed as a therapeutic approach for breast cancer. Triple-negative breast cancer (TNBC), the most aggressive subtype, lacks targeted therapies, and its primary treatments involve conventional chemotherapy and DNA-damaging agents [5]. Homologous recombination deficiency, such as mutations in the breast cancer gene 1 (BRCA1) and BRCA2, serves as a predictive biomarker for identifying patients who would benefit from genotoxic chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors. Notably, METTL3 is recruited to DNA-damaged sites and is crucial for subsequent DNA repair [6, 7]. Consequently, METTL3 knockdown reduces DNA repair activity and sensitizes cancer cells to genotoxic drugs [7, 8]. However, while TNBC exhibits elevated METTL3 levels, and its nuclear catalytic activity associates with invasiveness and metastasis [9], it remains uncertain whether METTL3 inhibition enhances chemotherapy response in TNBC.

Here, we aimed to explore the potential of METTL3 catalytic inhibition by STM2457 as a valuable treatment option for TNBC. Furthermore, we assessed the impact of STM2457 on the sensitivity of TNBC cells and a TNBC patient-derived organoid line to clinical DNA-damaging therapies, like platinum-based chemotherapy and the PARP inhibitor olaparib (Supplementary file of methods).

STM2457 significantly reduced the proliferation and viability of TNBC cells, including both BRCA1/2 wild-type (MDA-MB-231 and MDA-MB-468) and BRCA1-mutated (MDA-MB-436, HCC1395, and HCC1937) cell lines. STM2457 exhibited negligible effects on the proliferation of non-tumoral breast epithelial cells (MCF-10A), with significant reduction observed only at the highest concentration tested (100 μmol/L) (Figure 1A, Supplementary Figure S1A-B). The treatment with 10 μmol/L STM2457 for 48 h decreased the global m6A levels in mRNA by approximately 50% in both MDA-MB-231 and MCF-10A cells (Supplementary Figure S1C). Colony formation assays further confirmed the anti-proliferative impact of STM2457 on TNBC cell lines (Figure 1B, Supplementary Figure S2). Moreover, wound healing assays indicated that the inhibition of METTL3 activity reduced TNBC cell migration (Figure 1C). The specificity of the effects exerted by STM2457 were confirmed by METTL3 knockdown (Supplementary Figure S3). To identify the molecular pathways regulated by METTL3 inhibition, we investigated its effect on MDA-MB-231 cells, one of the most aggressive TNBC cell line. Cells were treated with 10 μmol/L STM2457 for 48 h and subjected to RNA-sequencing. Bioinformatics analysis revealed 3,182 genes downregulated upon STM2457 treatment, with 3,237 genes upregulated by the drug (Supplementary Figure S4A, Supplementary Table S1). Gene ontology (GO) analysis of differentially expressed genes showed a significant downregulation of biological processes involved in cell proliferation, translation, and DNA repair (Supplementary Figure S4B). This result is consistent with previous analyses performed in breast cancer cells knocked down for METTL3 expression [8, 9] and in AML cell lines treated with STM2457 [2]. Comparison of our RNA-sequencing dataset with a methylated RNA immunoprecipitation (MeRIP)-sequencing dataset performed in MDA-MB-231 cells [9] showed a substantial overlap between the transcripts regulated by STM2457 treatment and those that are m6A methylated (Supplementary Figure S5A). GO analysis of the m6A-methylated genes, which exhibit differential expression upon METLL3 inhibition, revealed a significant downregulation of biological processes related to the cell cycle and DNA damage repair (Supplementary Figure S5B). Consistent with global m6A level observations, individual MeRIP-qPCR analysis on highly methylated transcripts confirmed around a 50% reduction due to STM2457 treatment (Supplementary Figure S6). We focused on the DNA damage repair pathway because of its relevance for TNBC therapy [8].

First, we investigated the effect of METTL3 inhibition in combination with DNA-damaging agents. Platinum-based chemotherapeutic agents and PARP inhibitors, such as olaparib, showed mostly favourable responses in TNBC harboring BRCA1/2 mutations [5]. Since STM2457 downregulated genes involved in the DNA repair pathway, we hypothesized that it may reduce homologous recombination (HR) proficiency also in TNBC cells that are wild-type for BRCA1/2. To test this hypothesis, we examined the effect of METTL3 inhibition on the response to cisplatin and olaparib in MDA-MB-231 cells, which are BRCA1/2 wild-type and characterized by lower sensitivity to DNA-damaging agents. MDA-MB-231 cells were treated with doses of STM2457, cisplatin, and olaparib that were below their half maximal inhibitory concentration (IC50) either as single agents or in combination. Combined treatment with STM2457 and either cisplatin or olaparib significantly reduced MDA-MB-231 cell proliferation and increased apoptosis compared to single-drug treatments (Figure 1D-G). Notably, treatment with STM2457 alone was sufficient to induce DNA damage, as indicated by the elevated phosphorylation of the H2A histone family member X (γH2AX) (Supplementary Figure S7A-B). Additionally, we noticed a global reduction in RecA-related protein 51 (RAD51) protein levels and RAD51 foci upon DNA damage (Supplementary Figure S7C-D). These findings are in line with those obtained in cells knocked down for METTL3 expression [7, 8]. Moreover, we observed substantial and consistent increase in DNA damage in response to combined treatments (Supplementary Figure S7A-B). Together, these data indicate that STM2457 strongly sensitizes HR-proficient TNBC cells with wild-type BRCA1/2 to DNA damage induced by genotoxic chemotherapy or PARP inhibitors.

As STM2457 treatment reduced TNBC cell migration, we further explored its impact on cancer cell metastasis. We chose the metastatic TNBC cell line MDA-MB-231 for injection into zebrafish larvae, a well-established in vivo xenograft model for studying metastasis [10]. MDA-MB-231 cells expressing green fluorescent protein (GFP) were microinjected into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Embryos were then exposed to compounds, either independently or in combinations, as employed in the in vitro assays. We observed no toxicity during the treatment with STM2457 in the larvae. While some extravasated cells were scattered throughout the larva, GFP-positive clusters were enriched in the caudal hematopoietic tissue region. We quantified micrometastases in this area (Figure 1H). Intriguingly, while each drug alone reduced cell metastasis, the combined STM2457 + cisplatin and STM2457 + olaparib treatments exhibited a notable mutually reinforcing trend, leading to a substantial reduction in both the number and size of micrometastases (Figure 1H).

Finally, we assessed the effect of STM2457 in a patient-derived organoid (BCO-21) from a TNBC patient with wild-type BRCA1/2 genes (Figure 1I). This patient was ineligible for olaparib treatment. Initially, we determined the IC50 for single-drug treatments with STM2457, olaparib, and carboplatin, a platinum-based chemotherapy used in TNBC. In line with the results from MDA-MB-231 cells, STM2457 decreased BCO-21 cell viability (Figure 1J, Supplementary Figure S8). We then investigated whether STM2457 enhances the BCO-21 sensitivity to DNA-damaging therapy. Notably, combination treatment with both optimal (10 μmol/L) and suboptimal (5 μmol/L) doses of STM2457 synergistically increased the cytotoxic effect of carboplatin and olaparib (combination index < 1; Figure 1K, Supplementary Figure S8).

In conclusion, this study showed that STM2457 displayed anti-tumor activity in TNBC and enhanced chemotherapy response while sensitizing BRCA1/2 wild-type patients to olaparib. Given the observed upregulation of METTL3 activity across various cancer types, METTL3 inhibitors may offer a novel approach to improve the efficacy of DNA-damaging agents, overcome inherent resistance, and mitigate genotoxic drug adverse effects.

Abstract Image

通过化学抑制 m6A 甲基转移酶 METTL3 提高三阴性乳腺癌对 DNA 损伤疗法的敏感性
(B) 用 5 μmol/L STM2457(STM)处理 MDA-MB-231 和 MCF-10A 细胞系的细胞集落形成试验。(C) 用伤口愈合试验评估经 20 μmol/L STM2457 处理的 MDA-MB-231 细胞的迁移能力;直方图表示迁移百分比的平均值(± SD),n = 3。(D)MDA-MB-231 细胞经 10 μmol/L STM、10 μmol/L 顺铂或两种药物联合处理 72 小时后的 MTT 检测。(F)MDA-MB-231 细胞经 10 μmol/L STM、20 μmol/L olaparib 或两种药物联合处理 72 小时后的 MTT 检测。(H)48 hpf 斑马鱼胚胎与 MDA-MB-231 GFP 阳性细胞直接移植到 PVS。上图是 CHT 区域的代表性荧光立体显微镜图像,其中包含 24 hpi 外渗的 GFP 阳性细胞,显示了异种移植的 4 个离散等级(高、中、低、无)。侧视图,左前方。下图,评估细胞通过 48 hpf 斑马鱼胚胎异种移植的外渗能力。每个条代表至少两个独立实验计算得出的 24 hpi 幼体的平均值%。分析的胚胎总数为 213 个,划分如下:DMSO(n = 28)、STM(n = 45)、顺铂(n = 37)、顺铂 + STM(n = 53)、奥拉帕利(n = 26)、奥拉帕利 + STM(n = 24)。(I)BCO-21 的代表性明视野图像;下图为苏木精-伊红染色。(J)STM2457 对 BCO-21 的细胞毒性作用。将细胞暴露于不同浓度的药物中 5 天,用 Cell Titer Glo 3D 检测法评估细胞活力。(K)STM2457 和卡铂或奥拉帕利对 BCO 生命力的协同作用。将 BCO-21 暴露于次优剂量(5 μmol/L)和最优剂量(10 μmol/L)的 STM、卡铂(10 μmol/L)和奥拉帕利(10 μmol/L)联合治疗 5 天。计算出药物组合与单个药物相比的 CI 值 &lt; 1(表示协同作用),并显示在图表上方。所有结果均以三重复的平均值 ± SEM 表示。*P &lt; 0.05,**P &lt; 0.01,***P &lt; 0.001。缩写:缩写:BCO,乳腺癌类器官;CHT,尾部造血组织;CI,组合指数;DMSO,二甲基亚砜;GFP,绿色荧光蛋白;Hpf,受精后数小时;Hpi,注射后数小时;MTT,3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑;PVS,绒毛膜周围间隙;SD,标准差;SEM,均值标准误差;STM,STM2457。首先,我们研究了 METTL3 抑制与 DNA 损伤药物联合使用的效果。铂类化疗药物和 PARP 抑制剂(如奥拉帕利)对携带 BRCA1/2 基因突变的 TNBC 大多显示出良好的反应[5]。由于 STM2457 下调了参与 DNA 修复途径的基因,我们假设它也可能降低 BRCA1/2 野生型 TNBC 细胞的同源重组(HR)能力。为了验证这一假设,我们研究了抑制 METTL3 对 MDA-MB-231 细胞对顺铂和奥拉帕利反应的影响,MDA-MB-231 细胞是 BRCA1/2 野生型细胞,对 DNA 损伤药物的敏感性较低。用低于半数最大抑制浓度(IC50)的 STM2457、顺铂和奥拉帕利作为单药或联合用药治疗 MDA-MB-231 细胞。与单药治疗相比,STM2457与顺铂或奥拉帕利联合治疗可显著减少MDA-MB-231细胞的增殖并增加细胞凋亡(图1D-G)。值得注意的是,单用 STM2457 就足以诱导 DNA 损伤,H2A 组蛋白家族成员 X(γH2AX)的磷酸化升高就说明了这一点(补充图 S7A-B)。此外,我们还注意到,DNA 损伤后,RecA 相关蛋白 51 (RAD51) 蛋白水平和 RAD51 病灶全面下降(附图 S7C-D)。这些发现与敲除 METTL3 表达的细胞中的发现一致[7, 8]。此外,我们还观察到 DNA 损伤在联合处理后持续大幅增加(补充图 S7A-B)。这些数据共同表明,STM2457能使具有野生型BRCA1/2的HR-proficient TNBC细胞对基因毒性化疗或PARP抑制剂诱导的DNA损伤产生强烈的敏感性。由于STM2457治疗能减少TNBC细胞的迁移,我们进一步探讨了它对癌细胞转移的影响。我们选择了转移性TNBC细胞系MDA-MB-231注射到斑马鱼幼体中,斑马鱼幼体是研究转移的一种成熟的体内异种移植模型[10]。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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