AbstractTargeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2.

摘要靶向蛋白质降解（TPD）是一种新兴的治疗策略，它将受益于新的化学实体，可以募集更多种类的泛素E3连接酶来靶向蛋白质进行蛋白酶体降解。在这里，我们描述了一种TPD策略，涉及募集FBXO22以诱导组蛋白甲基转移酶和癌基因NSD2的降解。

UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex.

UNC8732促进FBXO22介导的NSD2在具有NSD2功能获得突变p.E1099K的急性淋巴细胞白血病细胞中的降解，从而导致生长抑制，细胞凋亡和耐药性逆转。UNC8732的伯胺被代谢成醛物质，该醛物质与FBXO22的C326结合以募集SCFFBXO22-Cullin复合物。

We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD..

我们进一步证明，先前报道的靶向XIAP的含烷基胺降解剂类似地依赖于SCFFBXO22。总体而言，我们提出了一种有效的NSD2降解剂，用于探索NSD2疾病表型，并为TPD提供了一种新的FBXO22募集策略。。

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Fig. 1: UNC8732 inhibits cell growth and restores GC sensitivity of NSD2 p.E1099K mutant ALL cells.Fig. 2: UNC8732 metabolism to the corresponding aldehyde drives NSD2 degradation.Fig. 3: FBXO22 is responsible for compound-mediated degradation of NSD2.Fig. 4: Aldehyde degraders bind FBXO22 in a cysteine 326-dependent manner.Fig.

图1:UNC8732抑制细胞生长并恢复NSD2 p.E1099K突变体ALL细胞的GC敏感性。图2:UNC8732代谢为相应的醛驱动NSD2降解。图3:FBXO22负责化合物介导的NSD2降解。图4：醛降解物以半胱氨酸326依赖性方式结合FBXO22。图。

5: A primary amine-containing degrader recruits the SCFFBXO22 complex for degradation of XIAP..

5： 含有伯胺的降解剂募集SCFFBXO22复合物用于降解XIAP。。

Data availability

数据可用性

All supporting data for this study can be found within the article, its extended data figures and Supplementary Information documents. All mass spectrometry data are available at massive.ucsd.edu—accession MSV000093206. The sequence of the pCDF-BirA vector is available at GenBank (accession JF914075.1).

这项研究的所有支持数据都可以在文章，其扩展数据和补充信息文件中找到。所有质谱数据均可从massive.ucsd.edu-accession MSV000093206获得。pCDF-BirA载体的序列可在GenBank（登录号JF914075.1）上获得。

Source data are provided with this paper..

本文提供了源数据。。

Code availability

代码可用性

Analysis code for further BioID data processing and visualization is available via Zenodo at https://doi.org/10.5281/zenodo.10930672 (ref. 52).

用于进一步生物数据处理和可视化的分析代码可通过Zenodo获得https://doi.org/10.5281/zenodo.10930672（参考文献52）。

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d0minicO/NSD2_BioID: NSD2 BioID. Zenodo https://doi.org/10.5281/zenodo.10930672 (2024).Download referencesAcknowledgementsThe authors thank the members of the James laboratory and Stephen Frye for helpful discussions and input throughout the project. The authors thank P. H. Buttery and J.

d0minicO/NSD2\u BioID：NSD2 BioID。泽诺多https://doi.org/10.5281/zenodo.10930672（2024年）。下载参考文献致谢作者感谢詹姆斯实验室（JamesLaboratory）和斯蒂芬·弗莱（StephenFrye）的成员在整个项目中的有益讨论和投入。作者感谢P.H.Buttery和J。

L. R. Sanchez for the review of experimental data. This work is supported by grants from the Canadian Institutes of Health Research (CIHR) (FDN154328, OGB190363) and the Princess Margaret Cancer Foundation to C.H.A., from the National Institutes of Health (NCI) (R01CA242305) to L.I.J., a Leukemia and Lymphoma Society Specialized Center for Research and Florida Department of Health Grant 22L03 to J.D.L., and grants from the CIHR (PJT156093) and Princess Margaret Cancer Foundation to B.R.

五十、 R.Sanchez用于审查实验数据。这项工作得到了加拿大卫生研究院（CIHR）（FDN154328，OGB190363）和玛格丽特公主癌症基金会（Princess Margaret Cancer Foundation）向C.H.A.的资助，美国国立卫生研究院（NCI）（R01CA242305）向L.I.J.的资助，白血病和淋巴瘤学会专门研究中心（Leukemia and Lymphoma Society Specialized Center for Research）和佛罗里达州卫生部（Florida Department of Health）向J.D.L.的资助22L03，以及CIHR（PJT156093）和玛格丽特公主癌症基金会向B.R.的资助。

D.Y.N. is supported by a Canada Graduate Scholarship – Doctoral Research Award from CIHR (494204) and a Doctoral Training Scholarship from Fonds de recherche du Québec – Santé (320128). J.R.T. is supported by the UNC Lineberger Comprehensive Cancer Center Cancer Epigenetics Training Program (5T32CA217824-05).

D、 Y.N.获得了加拿大研究生奖学金（CIHR授予的博士研究奖）（494204）和魁北克研究基金会（Fonds de recherche du Québec–Santé）授予的博士培训奖学金（320128）。J、 R.T.得到了UNC Lineberger综合癌症中心癌症表观遗传学培训计划（5T32CA217824-05）的支持。

D.B.-L. is supported by CRS grant 25418. D.W. is supported by the NSERC Discovery (RGPIN-480432) and NSERC Collaborative Research and Development (CRDPJ-504037) grants. Portions of this work have been supported by certain funds managed by Deerfield Management Company, L.P. Deerfield Management Company is a healthcare-focused investment management firm.

D、 B.-L.得到CRS grant 25418的支持。D、 W.得到了NSERC Discovery（RGPIN-480432）和NSERC Collaborative Research and Development（CRDPJ-504037）的资助。这项工作的一部分得到了迪尔菲尔德管理公司（L.P.）管理的某些基金的支持。迪尔菲尔德管理公司是一家专注于医疗保健的投资管理公司。

The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, BristolMyersSquibb, Genentech, Genome Canada through Ontario Genomics Institute (OGI-196), EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPENGrant875510), Janssen,.

结构基因组学协会是一家注册慈善机构（编号1097737），通过安大略基因组学研究所（OGI-196），欧盟/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Association（EUbOPENGrant875510），詹森，从拜耳公司，勃林格殷格翰，百时美施贵宝，Genentech，Genome Canada获得资金，。

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PubMed Google ScholarContributionsD.Y.N., J.L., A.P., M.S., A.J.L. and M.M. designed, performed and analyzed cellular experiments. J.R.T., R.P.H. and L.I.J. designed and synthesized the compounds. S.S., M.K., T.M.G.K., F.L. and D.Y.N. designed, performed and analyzed biophysical experiments.

PubMed谷歌学术贡献SD。Y、 N.，J.L.，A.P.，M.S.，A.J.L.和M.M.设计，执行和分析了细胞实验。J、 R.T.，R.P.H.和L.I.J.设计并合成了这些化合物。S、 S.，M.K.，T.M.G.K.，F.L.和D.Y.N.设计，执行和分析了生物物理实验。

S.D. and M.K. performed cloning and protein purification. J.S.-G., M.E.R.M., D.D.G.O. and D.Y.N. designed, performed and analyzed BioID and global proteomics experiments. E.W. and M.K. designed, performed and analyzed HDX-MS experiments. E.P. performed in vitro ubiquitination experiments. L.Z.P., D.B.-L., N.G.B., A.M.B., A.W.S., J.L.C., D.J.W., B.R., J.D.L., L.I.J.

S、 D.和M.K.进行了克隆和蛋白质纯化。J、 S.G.、M.E.R.M.、D.D.G.O.和D.Y.N.设计、执行和分析了BioID和全球蛋白质组学实验。E、 W.和M.K.设计，执行和分析了HDX-MS实验。E、 P.进行了体外泛素化实验。五十、 Z.P.，D.B.-L.，N.G.B.，A.M.B.，A.W.S.，J.L.C.，D.J.W.，B.R.，J.D.L.，L.I.J。

and C.H.A. provided supervision and/or funding. D.Y.N., J.R.T., L.I.J. and C.H.A. wrote the manuscript.Corresponding authorsCorrespondence to.

和C.H.A.提供监督和/或资金。D、 Y.N.，J.R.T.，L.I.J.和C.H.A.撰写了手稿。通讯作者通讯。

Lindsey I. James or Cheryl H. Arrowsmith.Ethics declarations

林赛I.詹姆斯或谢丽尔H.阿罗史密斯。道德宣言

Competing interests

相互竞争的利益

D.D.G.O. is an employee of Amphista Therapeutics, a company that is developing TPD therapeutic platforms. A.M.B. and A.W.S. are employees of Deerfield Management Company, a healthcare-focused investment management firm. The remaining authors declare no competing interests.

D、 D.G.O.是Amphista Therapeutics的员工，该公司正在开发TPD治疗平台。A、 M.B.和A.W.S.是Deerfield Management Company的员工，Deerfield Management Company是一家专注于医疗保健的投资管理公司。其余作者声明没有利益冲突。

Peer review

同行评审

Reviewer Recognition

审阅者认可

Nature Chemical Biology thanks Milka Kostic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

《自然化学生物学》感谢Milka Kostic和另一位匿名审稿人为这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 UNC8732 inhibits cell growth and restores glucocorticoid sensitivity of NSD2 p.E1099K mutant ALL cells.(a): Total proteome analysis (label-free quantification) of U2OS cells treated with 2 µM UNC8732 or an equivalent volume of DMSO for 3 hours.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1 UNC8732抑制细胞生长并恢复NSD2 p.E1099K突变ALL细胞的糖皮质激素敏感性。（a） ：用2µM UNC8732或等体积的DMSO处理3小时的U2OS细胞的总蛋白质组分析（无标记定量）。

Volcano plot of all quantified proteins (8240) with log2 fold change shown on the x-axis (dashed line equivalent to a 2-fold change) and -log10 adjusted p values shown on the y-axis (dashed line equivalent to p.adjusted = 0.05). Adjusted p-values were derived from Limma and DEP packages (see methods) and were adjusted using the Benjamini-Hochberg procedure.

所有定量蛋白质（8240）的火山图，x轴上显示log2倍变化（虚线相当于2倍变化）和y轴上显示的log10调整后的p值（虚线相当于p.adjusted=0.05）。调整后的p值来自Limma和DEP包（请参见方法），并使用Benjamini-Hochberg程序进行调整。

N=5 independent experiments. (b-d): Viability of isogenic RCH-ACV determined by CellTiter-Glo assay after treatment with varying concentrations of UNC8732 and UNC8884 for (b) 12 days, (c) 15 days, or (d) 21 days. (e-f): Apoptosis of isogenic RCH-ACV detected using annexin V/PI staining by flow cytometry after treatment with varying concentrations of UNC8732 and UNC8884 for (e) 15 days or (f) 21 days.

N=5个独立实验。。（e-f）：在用不同浓度的UNC8732和UNC8884处理（e）15天或（f）21天后，通过流式细胞术使用膜联蛋白V/PI染色检测同基因RCH-ACV的凋亡。

(g-h): Viability of NSD2 mutant RCH-ACV cells determined by CellTiter-Glo after the pretreatment of varying concentrations of UNC8732 and UNC8884 for (g) 12 days or (h) 15 days followed by dexamethasone (1 µM) for 72 hours. (i-j): Apoptosis of NSD2 mutant RCH-ACV cell line detected using annexin V/PI staining by flow cytometry after the pretreatment of varying concentrations of UNC8732 and UNC8884 for (i) 12 days or (j) 15 days followed by dexamethasone (1 µM) for 72 hours.

（g-h）：在不同浓度的UNC8732和UNC8884预处理（g）12天或（h）15天后，然后用地塞米松（1μM）预处理72小时后，通过CellTiter-Glo测定NSD2突变RCH-ACV细胞的活力。（i-j）：在不同浓度的UNC8732和UNC8884预处理（i）12天或（j）15天后，通过流式细胞术使用膜联蛋白V/PI染色检测NSD2突变RCH-ACV细胞系的凋亡，然后是地塞米松（1μM）72小时。

Data represents the mean ± SEM from three biological replicates. The statistical significance was evaluated using the Two.

数据代表来自三个生物学重复的平均值±SEM。使用两者评估统计学显着性。

Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01660-yDownload citationReceived: 31 October 2023Accepted: 31 May 2024Published: 04 July 2024DOI: https://doi.org/10.1038/s41589-024-01660-yShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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