CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders

CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Cheng , Libing Xiang , Dongxin Zhao , Yaoyang Zhang , Ying Li , Xia Yin , Wen Di , Peiye Shen , Guanglei Zhuang , View ORCID Profile Li Tan doi: https://doi.org/10.1101/2025.03.31.646293 Kaiyan Ye 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qinyang He 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China 4 University of Chinese Academy of Sciences , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yan Cheng 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meiying Zhang 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Huiling Mu 5 School of Chinese Materia Medica, Nanjing University of Chinese Medicine , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mei-Chun Cai 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shaoqing Zhou 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China 4 University of Chinese Academy of Sciences , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Huaijiang Xiang 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China 4 University of Chinese Academy of Sciences , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yuqiao Hou 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yunxia Li 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yinuo Zang 6 Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, Faculty of Life Sciences, University College London , London, United Kindom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jingxin Lv 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lin Cheng 7 Obstetrics and Gynecology Department, Second Hospital of Jilin University , Changchun, Jilin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Libing Xiang 8 Ovarian Cancer Program, Department of Gynecologic Oncology, Zhongshan Hospital, Fudan University , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dongxin Zhao 4 University of Chinese Academy of Sciences , Beijing, China 5 School of Chinese Materia Medica, Nanjing University of Chinese Medicine , Nanjing, China 9 Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yaoyang Zhang 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ying Li 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xia Yin 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wen Di 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peiye Shen 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: tanli{at}sioc.ac.cn zhuangguanglei{at}gmail.com shenpeiye{at}renji.com Guanglei Zhuang 1 State Key Laboratory of Systems Medicine for Cancer, Department of Obstetrics and Gynecology, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine , Shanghai, China 2 Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: tanli{at}sioc.ac.cn zhuangguanglei{at}gmail.com shenpeiye{at}renji.com Li Tan 3 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China 10 State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Li Tan For correspondence: tanli{at}sioc.ac.cn zhuangguanglei{at}gmail.com shenpeiye{at}renji.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Transcriptional machinery often operates in the configuration of biomolecular condensates, presenting emerging opportunities to develop therapies targeting transcriptionally addicted malignancies. In this study, we discovered that cyclin-dependent kinase 12 (CDK12), a key regulator of transcription elongation, formed dynamic liquid-like droplets within nuclear speckles, a process enhanced by high CDK12 expression and CDK12 inhibitor treatment. Recognizing this unique property, we rationally designed ZSQ253, a modular cyclin K molecular glue degrader that coupled binding moieties for both CDK12 and DDB1. By promoting phase separation-driven assembly and subsequent proteasomal degradation of the CDK12-cyclin K complex, ZSQ253 interfered with oncogenic transcription and demonstrated potent antitumor capability. Further structure-guided optimization yielded HQY1428, a derivative with a multi-site gluing effect and improved drug activity. Given the frequent genomic co-amplification of CDK12 and ERBB2 across diverse human cancers, we showed that combining HQY1428 with HER2 inhibitors provided synergistic therapeutic benefits. Collectively, our findings establish CDK12 condensation within nuclear speckles as an exploitable vulnerability, and introduce a novel mechanism of action underlying cyclin K molecular glue degraders. Introduction Cumulative evidence implicates dysregulated transcription cycles in human cancer and underscores the enormous opportunities of therapeutically targeting core components of transcriptional machinery ( 1 , 2 ). Many of them, including RNA polymerase II (RNAPII), transcription factors, mediators, and coactivators, form biomolecular condensates within the nucleus to orchestrate gene expression ( 3 - 6 ). Previous research has primarily focused on the dynamic behaviors and regulatory mechanisms underlying these membraneless compartments assembled through liquid-liquid phase separation (LLPS) ( 7 , 8 ). For example, the intrinsically disordered sequence at the carboxy-terminal domain (CTD) of RNAPII facilitates its clustering and phase separation ( 9 , 10 ). CTD phosphorylation disrupts the RNAPII liquid droplets, allowing multivalent interactions with other elements to drive the transition from transcription initiation to co-transcriptional splicing ( 11 , 12 ). Recent studies have begun to reveal the functional role of LLPS in partitioning cancer therapeutics, as exemplified by selective accumulation of antineoplastic compounds in specific transcriptional condensates that influences target engagement ( 13 - 15 ). The potential of harnessing protein condensation to modulate drug activity represents an exciting frontier for further investigation ( 16 , 17 ). CDK12 is a pivotal cyclin-dependent kinase that regulates transcriptional cycles in both physiological and pathological contexts ( 18 ). CDK12 partners with cyclin K and phosphorylates the serine 2 (S2) residues of tandem heptapeptide repeats (YSPTSPS) in RNAPII CTD to promote transcription elongation ( 19 , 20 ). Genetically, CDK12 resides on a chromosome 17q locus proximal to ERBB2 (encoding HER2), leading to concurrent amplification of the two oncogenes across various cancer types ( 21 - 26 ). Therefore, CDK12 is not only an emerging therapeutic target by itself, but also points to a promising avenue for combination therapy in HER2-positive patients ( 27 , 28 ). At the subcellular level, CDK12 is mainly localized to nuclear speckles ( 29 ), specialized ribonucleoprotein granules that organize via phase separation and serve as central hubs for nascent RNA processing ( 30 - 32 ). A significant proportion of nuclear speckle constituents contain low-complexity intrinsically disordered regions (IDRs) ( 33 , 34 ). These observations raise the important question of whether CDK12 forms liquid-like condensates to exert its function within nuclear speckles. CDK12 as a serine/threonine kinase has been successfully blocked by covalent inhibitors developed by our group and others. However, the clinical utility of these compounds is often limited by poor pharmacokinetic properties and unacceptable systemic toxicities ( 35 - 39 ). An alternative and possibly more advantageous strategy is provided by the recent introduction of targeted protein degradation mediated by proteolysis-targeting chimeras (PROTACs) or molecular glue degraders (MGDs) ( 40 - 43 ). Compared with heterodimeric PROTACs, which consist of two separate ligands and a conjugating linker, monomeric MGDs with smaller and more rigid scaffolds have garnered considerable interest owing to their drug-like properties ( 44 ). Notably, a spectrum of unexpectedly diverse cyclin K MGDs, e.g., CR8 and HQ461, are serendipitously discovered and act as chemical inducers of proximity between the CDK12-cyclin K complex and DDB1-CUL4-RBX1 E3 ubiquitin ligase ( 43 , 45 - 47 ). Here, we adopted a different approach and rationally designed a new class of monovalent degraders by incorporating the binding modules of CDK12 and DDB1. The resulting lead molecule promoted CDK12 condensation in nuclear speckles and accelerated proteasomal degradation of CDK12-cyclin K, thereby conferring potent single-agent antitumor activity and synergistic efficacy with HER2 inhibitors. Our findings uncovered the phase separation capacity of CDK12, which could be leveraged to broaden the application of cyclin K molecular glue degraders. Results Formation of CDK12 condensates in nuclear speckles Bioinformatics analysis using the Predictor of Natural Disordered Regions (PONDR) ( Figure 1A ) and AlphaFold2 ( Figure 1B ) predicted that both CDK12 and cyclin K contain extensive intrinsically disordered regions (IDRs), suggesting their potential to undergo phase separation. To test this possibility experimentally, we stably expressed monomeric enhanced green fluorescent protein (mEGFP)-tagged CDK12 (mEGFP-CDK12) and cyclin K (mEGFP-cyclin K) in SKOV3 cells. Live-cell fluorescence microscopy revealed that only mEGFP-CDK12 formed discrete nuclear puncta, whereas mEGFP-cyclin K exhibited diffuse nuclear distribution ( Figure 1C ). Treatment with 1,6-hexanediol (1,6-hex), a chemical disruptor of hydrophobic interactions, significantly reduced the number of mEGFP-CDK12 puncta ( Figure 1D ). Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that mEGFP-CDK12 puncta rapidly recovered fluorescence within seconds ( Figure 1E ; Supplementary Figure 1A), confirming that CDK12 formed dynamic liquid-like nuclear condensates. In line with previous reports ( 29 ), mEGFP-CDK12 puncta prominently colocalized with nuclear speckles ( Figure 1F ). To assess the endogenous CDK12 distribution, we analyzed its expression in a panel of ovarian cancer cell lines (Supplementary Figure 1B). Immunofluorescence staining revealed that endogenous CDK12 also formed distinct nuclear puncta in CDK12-high cell lines (SKOV3, COV362, HEY, OVCA420) ( Figure 1G ), but not in CDK12-low cell lines (TOV-21G, OVCAR3, DOV13, OVCA429) (Supplementary Figure 1C), implying a correlation between CDK12 expression levels and condensate formation. Given the emerging data highlighting the interplay between phase-separated condensates and small-molecule compounds ( 13 - 15 ), SKOV3 cells overexpressing mCherry-cyclin K alone or co-expressing mEGFP-CDK12 and mCherry-cyclin K were treated with THZ531, a CDK12 covalent inhibitor ( 36 ). Notably, both CDK12 overexpression and THZ531 treatment increased CDK12 puncta accumulation in nuclear speckles, with synergistic effects observed when these two conditions were combined ( Figure 1H ). Moreover, the enrichment of CDK12 puncta facilitated the recruitment of cyclin K into these condensates, indicating enhanced CDK12-cyclin K complex formation. These results provide compelling evidence that CDK12 aggregates into liquid-like condensates within nuclear speckles, and that their physicochemical properties can be altered by CDK12 expression levels or CDK12 inhibitor treatment. Download figure Open in new tab Figure 1. Formation of CDK12 condensates in nuclear speckles A. Graphical depiction of the intrinsically disordered regions (IDRs) of CDK12 and cyclin K, as predicted using the Predictor of Natural Disordered Regions (PONDR) VSL2. The orange bar designates IDR. The kinase domain of CDK12 and the cyclin-like domain of cyclin K are highlighted with blue and green bars, respectively. B. The protein structures of CDK12 and cyclin K predicted by AlphaFold2. C. Representative images of SKOV3 cells overexpressing mEGFP, mEGFP-CDK12, or mEGFP-cyclin K. Cell nuclei are outlined with dashed white lines. Scale bars, 5 μm. D. Representative images of SKOV3 cells overexpressing mEGFP-CDK12 before and after treatment with 3% 1,6-hexanediol (1,6-hex) for 15 s. Box plot shows fold change in the number of mEGFP-CDK12 puncta. * P < 0.05, two-sided Student’s t -test. Scale bar, 5 μm. E. Representative images of the FRAP experiment in SKOV3 cells overexpressing mEGFP-CDK12. The yellow square highlights the puncta undergoing targeted bleaching. The fluorescence intensities of the bleached area and the unbleached control are quantified and plotted. Data are presented as mean ± standard deviation (n = 3). Scale bar, 5 μm. F. Representative immunofluorescence images of CDK12 (green) and SC35 (red) in SKOV3 cells overexpressing mEGFP-CDK12. Cell nuclei were counterstained with DAPI (blue). Quantification of fluorescence intensity of CDK12 and SC35 along the line indicated in images is shown on the right. Scale bar, 5 μm. G. Representative immunofluorescence images of CDK12 (green) and SC35 (red) in SKOV3, COV362, HEY, and OVCA420 cells. Cell nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. H. Representative immunofluorescence images of CDK12 (green), cyclin K (red), and SC35 (purple) in THZ531-treated SKOV3 cells overexpressing mCherry-cyclin K or co-expressing mEGFP-CDK12 and mCherry-cyclin K. Cell nuclei were counterstained with DAPI (blue). Box plot shows the area and intensity of CDK12 and cyclin K puncta in each group. Each point represents a single cell. * P < 0.05, Kruskal-Wallis test with Benjamini-Hochberg correction. Scale bars, 5 μm. Development of a cyclin K molecular glue degrader When CDK12 was overexpressed, SKOV3 cells were counterintuitively sensitized to CR8 ( 43 ), a cyclin K molecular glue degrader (Supplementary Figure 2A), which was attributable to the increased degradation efficiency (Supplementary Figure 2B). Therefore, CDK12 phase separation might enrich the CDK12-cyclin K complex in nuclear speckles and confer a unique susceptibility to cyclin K molecular glue degraders. To explore whether pharmacologically enhancing CDK12 condensation could also offer such an innovative therapeutic opportunity, we exploited a modular design approach and ultimately synthesized ZSQ253 (Supplementary Figure 2C). The CDK12-binding module of ZSQ253, derived from TL12-186, a pan-kinase PROTAC known to degrade CDK12 ( 48 ), and THZ531( 36 ), functioned as a CDK12 inhibitor to promote the formation of CDK12 condensates. Simultaneously, the terminal phenyl ring of UB-007, a CR8 analog ( 49 ), was adopted as the DDB1-binding module to enable degradation of the CDK12-cyclin K complex ( Figure 2A ). Molecular docking based on the CDK12-DDB1-CR8 co-crystal structure (PDB: 6TD3) predicted that ZSQ253 could occupy the ATP-binding pocket of CDK12, with its terminal phenyl group protruding outside to interact with the interfacial residues of DDB1, allowing the formation of a ternary complex ( Figure 2B ). The phenyl to methyl replacement yielded ZSQ2549, a control compound of ZSQ253. To assess the degradation effect, we treated SKOV3 cells with ZSQ253 or ZSQ2549 and conducted global proteomic profiling using mass spectrometry (Supplementary Table 1). This revealed that cyclin K was the most significantly downregulated protein ( Figure 2C ). Western blot analysis confirmed that ZSQ253 induced depletion of cyclin K and CDK12 without affecting CDK7 or CDK9 ( Figure 2D ). Additionally, pre-treatment with the proteasomal inhibitor PS341, but not the lysosomal inhibitor chloroquine, hindered protein degradation, indicating that ZSQ253 activity was dependent on the ubiquitin-proteasome system. We further knocked out individual core components of DDB1-CUL4-RBX1 E3 ubiquitin ligase in SKOV3 cells and observed that DDB1 or RBX1 deletion abrogated cyclin K degradation ( Figure 2E ; Supplementary Figure 2D) and mitigated the cytotoxicity ( Figure 2F ; Supplementary Figure 2E) elicited by ZSQ253 or CR8, but not by ZSQ2549 or THZ531. Genetic knockout of CUL4A or CUL4B did not produce the same results, likely due to their functional redundancy ( 50 ). Collectively, we have successfully developed a novel class of modular cyclin K molecular glue degraders. Download figure Open in new tab Figure 2. Development of a cyclin K molecular glue degrader A. Schematic illustration of the design rationale for ZSQ253. The CDK12-binding module was created by leveraging TL12-186 (pan-kinase PROTAC) and THZ531 (CDK12/13 inhibitor). The DDB1-binding module was derived from UB-007, a CR8 analog. The chemical structures of its control compound ZSQ2549 and the reported cyclin K molecular glue degrader CR8 are also shown. B. Molecular docking showed the binding mode of ZSQ253 based on the CDK12-DDB1-CR8 co-crystal structure (PDB: 6TD3). C. Whole-proteome quantification of SKOV3 cells treated with 0.5 μM ZSQ253 or ZSQ2549 for 4 h. D. Immunoblotting analysis of cyclin K and CDK12 degradation in Jurkat cells that were pretreated with 0.1 μM PS341 or 10 μM chloroquine for 2 h, followed by indicated compound treatment for an additional 6 h. β-Actin was used as the loading control. E. Immunoblotting analysis of the indicated proteins in SKOV3 cells with DDB1 , RBX1 , CUL4A , or CUL4B knockout after treatment with 0.3 μM ZSQ253 for 6 h. GAPDH was used as the loading control. F. SKOV3 cells with DDB1 , RBX1 , CUL4A , or CUL4B knockout were treated with various concentrations of ZSQ253 for 72 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Disruption of CDK12 condensates by ZSQ253 Initially, we verified that ZSQ2549, containing only the CDK12-binding module, acted as a CDK12 inhibitor and facilitated CDK12 puncta formation as well as cyclin K recruitment in nuclear speckles, especially upon CDK12 overexpression (Supplementary Figure 3A). ZSQ253, which included the additional DDB1-binding module, evidently disrupted CDK12 condensates and degraded cyclin K protein ( Figure 3A ). Consequently, when ZSQ253 was benchmarked against ZSQ2549 and CR8 at equivalent concentrations, superior antitumor potency was observed in both SKOV3 and COV362 cells ( Figure 3B ; Supplementary Figure 3B). EdU (5-ethynyl-2’-deoxyuridine) incorporation assays showed that ZSQ253 impaired cell proliferation to a significantly greater extent than ZSQ2549 and CR8 ( Figure 3C ; Supplementary Figure 3C). Moreover, ZSQ253 treatment induced more obvious G2/M arrest ( Figure 3D ; Supplementary Figure 3D) and cell apoptosis ( Figure 3E ; Supplementary Figure 3E). Given that CDK12 inactivation is known to downregulate DNA damage response (DDR) genes ( 51 , 52 ), Western blot detected elevated γH2AX and reduced RAD51 levels ( Figure 3F ; Supplementary Figure 3F). Consistently, immunofluorescence staining confirmed a marked appearance of γH2AX foci within the nuclei of ovarian cancer cells treated with ZSQ253, indicating the accumulation of DNA double-strand breaks ( Figure 3G ; Supplementary Figure 3G). These findings demonstrate that ZSQ253 causes disruption of CDK12 condensates and exerts pronounced antiproliferative and cytotoxic effects in ovarian cancer. Download figure Open in new tab Figure 3. Disruption of CDK12 condensates by ZSQ253 A. Representative immunofluorescence images of CDK12 (green), cyclin K (red), and SC35 (purple) in SKOV3 cells co-expressing mEGFP-CDK12 and mCherry-cyclin K after treatment with 1 μM ZSQ2549, ZSQ253, or CR8 for 24 h. Cell nuclei were counterstained with DAPI (blue). Box plot shows the area and intensity of CDK12 and cyclin K puncta in each group. Each point represents a single cell. * P < 0.05, Kruskal-Wallis test with Benjamini-Hochberg correction. Scale bars, 5 μm. B. SKOV3 cells were treated with various concentrations of ZSQ2549, ZSQ253, or CR8, and cell viability was determined by crystal violet staining. C. EdU (green) incorporation assay on SKOV3 cells after treatment with 0.3 μM ZSQ2549, ZSQ253, or CR8 for 24 h. Cell nuclei were counterstained with DAPI (blue). Quantification of the percentage of EdU-positive cells is plotted as mean ± standard deviation (n = 3). * P < 0.05, ANOVA followed by Tukey’s post-test. Scale bar, 50 μm. D. Cell cycle analysis by flow cytometry on SKOV3 cells after treatment with 0.3 μM ZSQ2549, ZSQ253, or CR8 for 48 h. E. Flow cytometric analysis of cell death using annexin V/PI double labeling in SKOV3 cells after treatment with 0.3 μM ZSQ2549, ZSQ253, or CR8 for 72 h. F. Immunoblotting analysis of the indicated proteins in SKOV3 cells treated with 0.3 μM ZSQ2549, ZSQ253, or CR8 over a time course. GAPDH was used as the loading control. G. Representative immunofluorescence images of γH2AX (green) in SKOV3 cells treated with 0.3 μM ZSQ2549, ZSQ253, or CR8 for 72 h. Cell nuclei were counterstained with DAPI (blue). Quantification of the percentage of cells containing more than five γH2AX foci is plotted as mean ± standard deviation (n = 3). * P < 0.05, ANOVA followed by Tukey’s post-test. Scale bar, 10 μm. Transcriptional changes upon ZSQ253 treatment To determine whether pharmacological modulation of CDK12 condensates translated into transcriptional alterations, we performed RNA sequencing (RNA-seq) on drug-exposed SKOV3 cells (Supplementary Figure 4A). Following brief treatment with ZSQ2549 (Supplementary Table 2), ZSQ253 (Supplementary Table 3), or CR8 (Supplementary Table 4), we observed that differentially expressed genes (DEGs) were predominantly downregulated ( Figure 4A ). A significant positive correlation of transcriptome-wide changes was found across the three compounds ( Figure 4B ), suggesting their comparable global effects on gene expression. Nonetheless, cyclin K molecular glue degraders, particularly ZSQ253, exhibited a relatively strong impact (Supplementary Figure 4B). Gene set enrichment analysis (GSEA) pinpointed suppressed pathways related to DNA repair, DNA replication, cell cycle, and organelle biogenesis ( Figure 4C ), in keeping with the antitumor activity. Likewise, Cytoscape analysis revealed multiple disrupted oncogenic pathways especially those involved in DNA damage response (Supplementary Figure 4C). Accordingly, the expression of core DDR genes curated by TCGA ( 53 ) was disproportionately suppressed (Supplementary Figure 4D). Considering that nuclear speckles serve as central hubs for splicing regulation, we further conducted alternative splicing analysis using the rMATS pipeline ( 54 ) and identified 1363-7187 events in response to ZSQ2549, ZSQ253, or CR8 ( Figure 4D ). Transcripts displaying skipped exons, as exemplified by EXO5 ( Figure 4E ), comprised the largest fraction. There were 525 differentially spliced genes shared by all three treatments, with ZSQ253 leading to more severe splicing defects ( Figure 4F ). Taken together, these results indicate that ZSQ253 induces dramatic transcriptional changes in both gene expression and splicing. Download figure Open in new tab Figure 4. Transcriptional changes upon ZSQ253 treatment A. Volcano plots showing differentially expressed genes in SKOV3 cells treated with 0.3 µM ZSQ2549, ZSQ253, or CR8 for 6 h. B. Correlation estimation of fold changes induced by drug treatment in SKOV3 cells at the transcriptome-wide scale. Pearson correlation coefficient (R) and P value are presented. C. GSEA plots showing disproportionate downregulation of genes involved in DNA damage response, cell cycle, and nucleotide metabolic processes in SKOV3 cells treated with 0.3 μM ZSQ2549, ZSQ253, or CR8 for 6 h. D. Bar graph showing the number of differential splicing events (FDR ≤ 0.01, dPSI > 0.05) in SKOV3 cells treated with ZSQ2549, ZSQ253, or CR8. A3SS, alternative 3’ splice site; A5SS, alternative 5’ splice site; MXE, mutually exclusive exon; RI, retained intron; SE, skipped exon. E. Sashimi plot showing skipped exon of EXO5 in SKOV3 cells treated with ZSQ2549, ZSQ253, or CR8. F. Venn diagram showing the number of overlapping alternatively spliced genes in SKOV3 cells treated with ZSQ2549, ZSQ253, or CR8. Chemical design and in vivo efficacy of HQY1428 Building on the structure of ZSQ253, we sought to enhance drug potency and optimize the pharmacokinetic properties for in vivo applications. Through a series of modifications, we identified a derivative, HQY1428, by incorporating a fluorine atom and amino group into the terminal phenyl ring of ZSQ253 ( Figure 5A ). Molecular docking analysis unveiled that both ZSQ253 and HQY1428 formed hydrogen bonds with the CDK12 hinge region, while their extended phenyl rings interacted with the Arg928 residue on DDB1 through cation-π interactions ( Figure 5B ). Notably, the amino group of HQY1428 formed additional hydrogen bonds with Ile733 on CDK12 and Phe949 on DDB1, further stabilizing the ternary complex. In addition, the fluorine atom of HQY1428 filled more cavities in the hydrophobic pocket between CDK12 and DDB1, and interacted with proximate residues via van der Waals forces. As a result, HQY1428 treatment achieved cyclin K degradation more rapidly (Supplementary Figure 5A) and demonstrated superior anticancer efficacy compared to ZSQ253 ( Figure 5C ), with a 40-fold decrease of IC 50 in SKOV3 cells (Supplementary Figure 5B). At the cellular level, HQY1428, like ZSQ253, suppressed DNA replication ( Figure 5D ), caused G2/M arrest ( Figure 5E ), and triggered cell apoptosis ( Figure 5F ). Moreover, HQY1428 induced DNA double-strand breaks, as evidenced by increased γH2AX levels in immunoblotting ( Figure 5G ) and immunofluorescence staining ( Figure 5H ). To evaluate in vivo performance, we first assessed the metabolic stability of HQY1428 and ZSQ253 in mouse liver microsomes. HQY1428, with an additional amino group and thereby improved aqueous solubility, had a biological half-life (T 1/2 ) of 50.58 minutes, significantly longer than ZSQ253 (3.78 minutes) and the positive control verapamil (12.95 minutes) (Supplementary Figure 5C). HQY1428 was then administered via gavage at doses of 5 and 10 mg/kg/day to treat the intraperitoneal SKOV3 xenografts in BALB/c nude mice. Treatment with HQY1428 resulted in markedly reduced tumor weight ( Figure 5I ) and abdominal masses ( Figure 5J ) in a dose-dependent manner. Importantly, there was no significant loss of body weight ( Figure 5K ), or histological abnormalities in heart, liver, spleen, lung, and kidney (Supplementary Figure 5D). In agreement with the in vitro data, we observed downregulation of cyclin K and CDK12, reduced cell proliferation as indicated by Ki-67 staining, increased cell apoptosis as marked by cleaved caspase-3 and PARP, and elevated γH2AX-labeled DNA damage ( Figure 5L ). These results underscore the in vivo antitumor efficacy and favorable safety profile of HQY1428 as a promising cyclin K molecular glue degrader for future clinical development. Download figure Open in new tab Figure 5. Chemical design and in vivo efficacy of HQY1428 A. The chemical structure of HQY1428 which was obtained by optimizing the DDB1-binding module based on ZSQ253. B. Molecular docking showed the binding modes of ZSQ253 (pink sticks) and HQY1428 (gray sticks) based on the CDK12-DDB1-CR8 co-crystal structure (PDB: 6TD3). Compared to ZSQ253, HQY1428 was predicted to form two additional hydrogen bonds (yellow dashed line) with Ile733 on CDK12 and Phe949 on DDB1. C. SKOV3 cells were treated with various concentrations of HQY1428, ZSQ253, or ZSQ2549, and cell viability was determined by crystal violet staining. D. EdU (green) incorporation assay on SKOV3 cells after treatment with 0.1 μM HQY1428 for 24 h. Cell nuclei were counterstained with DAPI (blue). Quantification of the percentage of EdU-positive cells is plotted as mean ± standard deviation (n = 3). * P < 0.05, unpaired Student’s t test. Scale bar, 50 μm. E. Cell cycle analysis by flow cytometry on SKOV3 cells after treatment with 0.1 μM HQY1428 for 48 h. F. Flow cytometric analysis of cell death using annexin V/PI double labeling in SKOV3 cells after treatment with 0.1 μM HQY1428 for 72 h. G. Immunoblotting analysis of the indicated proteins in SKOV3 cells treated with 0.1 μM HQY1428 over a time course. GAPDH was used as the loading control. H. Representative immunofluorescence images of γH2AX (green) in SKOV3 cells treated with 0.1 μM HQY1428 for 72 h. Cell nuclei were counterstained with DAPI (blue). Quantification of the percentage of cells containing more than five γH2AX foci is plotted as mean ± standard deviation (n = 3). * P < 0.05, unpaired Student’s t test. Scale bar, 10 μm. I. Quantification of SKOV3 tumor weight following vehicle control (2.5% v/v DMSO and 97.5% v/v 30% SBE-β-CD) or HQY1428 (5 or 10 mg/kg/day) treatment. The tumor weight for each mouse was calculated by adding the weights of all resectable implants. * P < 0.05, ANOVA followed by Tukey’s post-test. J. Representative images of SKOV3 xenografts from BALB/c nude mice treated with vehicle control or HQY1428 via oral gavage for 21 days. The three largest abdominal masses are shown for each mouse. Scale bar, 5 mm. K. Body weight measurements of BALB/c nude mice during vehicle control or HQY1428 treatment. L. Representative images of hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining for cyclin K, CDK12, Ki-67, cleaved caspase-3, cleaved PARP, and γH2AX in SKOV3 tumor slices. Scale bar, 50 μm. Rational combination of HER2 inhibitors and HQY1428 It has been recognized that CDK12 and ERBB2 share a genetic locus and are frequently co-amplified in many cancer types ( 35 - 39 ). Indeed, copy number profiles of the two genes closely resembled each other across 9470 TCGA samples ( Figure 6A ). In fact, CDK12 amplification was almost exclusively restricted to ERBB2 -amplified tumors (Supplementary Figure 6A). Consequently, CDK12 and ERBB2 showed strong transcriptional correlation (Supplementary Figure 6B). To corroborate these observations at the protein level, we evaluated a diverse cohort of primary HER2-positive malignancies, including breast, gastric, biliary tract, colorectal, salivary duct, and urothelial cancers (Supplementary Table 5). Immunofluorescence staining pinpointed that CDK12 was highly abundant and formed distinct droplets within nuclear speckles ( Figure 6B ; Supplementary Figure 7). Subsequent analysis of the Cancer Cell Line Encyclopedia (CCLE) demonstrated that CDK12 was also preferentially amplified (Supplementary Figure 8A) and expressed (Supplementary Figure 8B) in cell lines with ERBB2 amplification. Accordingly, CDK12 condensation was ubiquitously detected in a panel of ERBB2 -amplified cell lines derived from breast (AU565, SKBR3, BT474), gastric (NCI-N87), esophageal (TE4), lung (Calu-3, NCI-H2170), and ovarian (SKOV3) cancers ( Figure 6C ; Supplementary Figure 8C). Therefore, we hypothesized that dual-targeting of HER2 and CDK12 could represent a rational treatment regimen in this context. As expected, dose-matrix experiments established a marked synergy between the HER2 inhibitor lapatinib and HQY1428 ( Figure 6D ; Supplementary Figure 8D), corresponding to reduced PI3K and MAPK signaling ( Figure 6E ). We then performed RNA-seq on AU565 (Supplementary Tables 6-8) and NCI-N87 cells (Supplementary Tables 9-11) treated with lapatinib, HQY1428, or both ( Figure 6F ). KEGG enrichment analysis confirmed therapeutic perturbation of HER2-mediated MAPK pathway and CDK12-mediated Fanconi anemia pathway, likely contributing to the enhanced antitumor efficacy ( Figure 6G ). In vivo, lapatinib (30 mg/kg/day) and HQY1428 (10 mg/kg/day) more effectively suppressed NCI-N87 xenograft growth than either agent alone ( Figure 6H ), accompanied by the anticipated cyclin K and CDK12 depletion, along with impaired tumor cell proliferation and survival (Supplementary Figure 9A). Importantly, there was no significant loss of body weight ( Figure 6I ), no histological abnormalities in major organs (Supplementary Figure 9B), and no remarkable hematological and biochemical changes in blood (Supplementary Figure 9C). In conclusion, these data highlight the therapeutic potential of combining HER2 inhibitors with cyclin K molecular glue degraders, offering a promising strategy for treating ERBB2 -amplified cancers. Download figure Open in new tab Figure 6. Rational combination of HER2 inhibitors and HQY1428 A. Copy number profiles of CDK12 and ERBB2 across different tumor types from The Cancer Genome Atlas (TCGA) database. B. Representative images of hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) staining for HER2, and immunofluorescence (IF) staining for CDK12 (green) and SC35 (red) in breast and gastric tumor slices. Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm (H&E and IHC) or 10 μm (IF). C. Representative immunofluorescence images of CDK12 (green) and SC35 (red) in AU565 and NCI-N87 cells. Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. D. Heatmap of bliss synergy scores and crystal violet staining demonstrated the synergistic activities of lapatinib and HQY1428 in AU565 and NCI-N87 cells. E. Immunoblotting analysis of the indicated proteins in AU565 and NCI-N87 cells treated with lapatinib, HQY1428, or their combination. GAPDH was used as the loading control. F. Principal component analysis (PCA) of RNA-seq data in AU565 and NCI-N87 cells treated with lapatinib, HQY1428, or their combination for 6 h. G. KEGG enrichment analysis of the downregulated genes in AU565 and NCI-N87 cells treated with lapatinib, HQY1428, or their combination. H. Tumor growth curves (left) and tumor weight quantification (right) of NCI-N87 xenografts treated with vehicle control (0.5% hydroxy-propyl methylcellulose and 0.1% Tween 80), lapatinib (30 mg/kg/day), HQY1428 (10 mg/kg/day), or their combination. Tumor growth curves are presented as mean ± standard deviation (n = 6). * P < 0.05, ANOVA followed by Tukey’s post-test. I. Body weight measurements of BALB/c nude mice during vehicle control, lapatinib, HQY1428, or combination treatment. Discussion This study presented the first evidence that CDK12 formed functional liquid-like condensates within nuclear speckles, a process that could be further enhanced by CDK12 inhibitor treatment. Building on this insight, we fused a CDK12-binding module with a DDB1-engaging module to rationally design a new class of cyclin K molecular glue degraders. These compounds effectively disrupted CDK12 phase separation, perturbed oncogenic transcription, and demonstrated potent single-agent antitumor activity, as well as synergistic efficacy with HER2 blockade in ERBB2 -amplified malignancies ( Figure 7 ). Our findings pinpoint a promising strategy to exploit target protein condensation as a means of improving sensitivity to cancer therapy. Download figure Open in new tab Figure 7. A schematic summary of the study CDK12 undergoes phase separation within nuclear speckles, a process enhanced by both high CDK12 expression and CDK12 inhibitor treatment. Thus, we rationally designed a modular cyclin K molecular glue degrader to disrupt CDK12 condensates, thereby inhibiting transcription. Furthermore, CDK12 is often co-amplified with ERBB2 (encoding HER2) due to their genomic proximity, which provides a promising synergistic therapeutic strategy to combine HER2 inhibitors and cyclin K molecular glue degraders. Previous work has established that CDK12 interacts with hyperphosphorylated RNAPII in nuclear speckles, where it phosphorylates multiple spliceosomal components within these membraneless organelles ( 12 ). CDK12 is uncommon in the CDK family to contain an arginine/serine-rich (RS) domain, which is critical for its localization ( 29 ). Here, we show that both the N- and C-terminal regions of CDK12, including the RS domain, are extensively disordered, enabling dynamic liquid-like condensate formation in nuclear speckles. Intriguingly, cyclin K alone did not undergo phase separation despite bearing IDR itself. Instead, CDK12 compartmentation seemed to serve as a driving force for recruiting cyclin K into nuclear speckles to assemble active CDK12-cyclin K complex, though the precise mechanisms need to be elucidated. The CDK12 close homolog CDK13 may share similar properties but require further exploration because of relatively low endogenous expression. Notably, elevated CDK12 abundance resulted in increased nuclear puncta accumulation, suggesting a pivotal role of protein concentration. In addition, kinase inhibitor treatment also caused an enlargement of CDK12-cyclin K aggregates. We speculate that the π-π and cation-π interactions between small-molecules and biomolecules can modify the physicochemical features of phase-separated condensates, as proposed in recent reports ( 8 , 15 , 55 ). It remains to be determined whether myriad CDK12 inhibitors of diverse chemistry display different behaviors, and how the consequent drug partitioning would in turn influence the pharmacodynamics and activity of specific compounds. These unprecedented insights illuminate a distinctive phase separation-driven mechanism underpinning the action of lately described cyclin K molecular glue degraders, which may promote CDK12 condensation in nuclear speckles and thereby facilitate proteolysis of the CDK12-cyclin K complex. We formally evaluated this hypothesis by introducing the concept of modular cyclin K molecular glue degraders. First, a CDK12-binding module was synthesized to generate ZSQ2549, which expectedly escalated CDK12 puncta formation and cyclin K recruitment. Then, ZSQ2549 was integrated with a hydrophobic gluing moiety to engage DDB1 and dissolve CDK12-cyclin K condensates. The resulting ZSQ253 exerted remarkable nanomolar cytotoxicity, outperforming the well-characterized cyclin K molecular glue degrader CR8. Structural optimization of ZSQ253 yielded HQY1428 with superior potency through a multi-site gluing machinery. Importantly, the inter-protein binding pocket for molecular glue degraders is generally hydrophobic, which often raises challenges in balancing the affinity and solubility. Nevertheless, HQY1428 showed favorable pharmacokinetic properties and in vivo antitumor efficacy. Additional pharmacological and preclinical characterization of this lead compound is underway. Given the positive correlation between CDK12 levels and condensate formation, it is appealing to employ cyclin K molecular glue degraders against human malignancies harboring ERBB2 amplification due to its genomic proximity with CDK12 . Indeed, across diverse HER2-positive tumor tissues and cell lines, we observed prominent CDK12 expression and distinct CDK12 droplets in nuclear speckles. Recent studies have uncovered that structurally coupled co-amplification of an oncogene and a passenger gene can create unique cancer dependencies and lead to collateral therapeutic vulnerabilities ( 56 , 57 ). Along the same line, our data indicated that combining HER2 inhibition and CDK12-cyclin K degradation achieved synergistic efficacy in ERBB2 -amplified neoplasms without obvious adverse effects. Although only small-molecule inhibitors were tested in the current study, other approved drug modalities such as HER2-directed antibodies (e.g., trastuzumab, pertuzumab) ( 58 , 59 ) and antibody-drug conjugates (e.g., trastuzumab emtansine, trastuzumab deruxtecan) ( 60 , 61 ) will most likely produce consistent results and warrant future investigations. Moreover, these findings provide a compelling rationale for conjugating HER2 monoclonal antibodies with cyclin K molecular glue degraders as a novel treatment option in this disease setting. In summary, our rational design of a modular cyclin K molecular glue degrader with oral availability and antitumor potency demonstrates the feasibility and effectiveness of leveraging protein condensation for cancer management. While the therapeutic potential of modulating LLPS has generated significant interest in drug discovery, directly targeting biomolecular condensates remains challenging due to the limited manipulability and specificity of intrinsically disordered regions. We offer an alternative approach by pharmacologically inducing phase separation to foster accelerated degradation, opening new avenues to develop innovative therapies for human conditions associated with pathogenic LLPS. Materials and Methods Cell lines and reagents Cell lines used in this study were obtained from the American Type Culture Collection (ATCC) or Japanese Collection of Research Bioresources Cell Bank (JCRB). All cell lines were routinely tested for mycoplasma contamination, and cell identity was verified using short tandem repeat (STR) profiling. Cells were maintained in RPMI1640 (Life Technologies) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Commercial small-molecule inhibitors were purchased from Selleck Chemicals and dissolved in DMSO (Sigma-Aldrich) at a stock concentration of 10 mM. Patient samples Human tissue samples were obtained in accordance with ethical guidelines of U.S. Common Rule. The study was approved by the Ethics Committee of Ren Ji Hospital and written informed consent was acquired from all patients. We assembled a cohort of 22 patients diagnosed with HER2-positive cancer which was confirmed by immunohistochemistry. Immunofluorescence staining For tumor cell lines, cells were grown on 8-well culture dishes (Ibidi), fixed with 4% paraformaldehyde for 15 min at room temperature (RT), and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. After three washes with PBS, cells were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min at RT, followed by incubation with primary antibodies at 4 °C overnight. The following primary antibodies were used: CDK12 (HPA008038, Sigma-Aldrich), SC35 (ab11826, Abcam), and γH2AX (#80312, Cell Signaling Technology). After incubation, cells were washed three times with PBS and incubated with Alexa-Fluor conjugated secondary antibodies (Invitrogen) for 1 h in the dark. After three washes with PBS, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 5 min. For formalin-fixed and paraffin-embedded (FFPE) sections, slides were baked, dewaxed with xylene, rehydrated through graded alcohols, and subjected to antigen retrieval in 10 mM citric sodium buffer (pH 6.0) using a steam pressure cooker for 20 min. Slides were then treated with 3% hydrogen peroxide (H 2 O 2 ) in methanol for 10 min to quench endogenous peroxidase activity, permeabilized with 0.5% Triton-X100 in PBS for 15 min, blocked with 10% normal goat serum in PBS for 1 h at RT, and incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: CDK12 (HPA008038, Sigma-Aldrich) and SC35 (ab11826, Abcam). After three washes in PBST (PBS containing 0.1% Tween-20), slides were incubated with Alexa-Fluor conjugated secondary antibodies (Invitrogen) for 1 h in the dark. Finally, slides were washed three times in PBST, cover-slipped with mounting medium containing DAPI (Beyotime), and sealed with nail polish. Fluorescent images were acquired using Leica TCS SP8 confocal microscopy system with a 63× oil-immersion objective or Leica STELLARIS 5 confocal microscopy system with a 100× oil-immersion objective. Image analysis and quantification were performed using LAS X and Fiji (ImageJ) software. Live-cell imaging Cells were grown on 8-well culture dishes (Ibidi) and imaged using Leica TCS SP8 confocal microscopy system equipped with a 63× oil objective. Imaging was performed in an incubation chamber maintained at 37 °C with 5% CO to ensure optimal conditions for live-cell observation. Fluorescence recovery after photobleaching (FRAP) assays were conducted using the FRAP module of the Leica TCS SP8 confocal microscopy system. mEGFP-CDK12 puncta was bleached using a 488-nm laser set to 100% laser power. Time series for FRAP analysis were captured over eight cycles at 1-second intervals. Fluorescence intensity was measured in three independent regions from separate nuclei and quantified using LAS X software. In vivo studies For single-agent treatment, SKOV3 cells (3 × 10 6 ) were intraperitoneally injected into female BALB/c nude mice (5 weeks of age). Upon tumor formation, mice were randomly assigned to the vehicle or treatment group. HQY1428 was administered orally at doses of 5 mg/kg/day or 10 mg/kg/day, with vehicle (2.5% v/v DMSO and 97.5% v/v 30% SBE-β-CD) as the negative control. For combination treatment, NCI-N87 cells (5 × 10 6 ) were mixed with Matrigel (BD Biosciences) and injected subcutaneously into the dorsal flank of female BALB/c nude mice (5 weeks of age). When the mean tumor volume reached 120 mm 3 , mice were randomly assigned to the vehicle or treatment group. HQY1428 was administered orally at a dose of 10 mg/kg/day, while Lapatinib was given at 30 mg/kg/day in 0.5% hydroxy-propyl methylcellulose and 0.1% Tween 80. Mice were monitored and weighed every other day. Tumor volumes were measured with a caliper and calculated as length × width 2 × 0.5. After three weeks, mice were sacrificed, and xenografts were harvested and imaged. Tumor tissues were fixed in 4% paraformaldehyde for immunohistochemistry analysis. The institutional animal care and use committee of Ren Ji Hospital approved animal protocols and all experiments were performed in accordance with Ren Ji Hospital policies regarding the care, welfare, and treatment of laboratory animals. Statistical analysis The sequencing data were deposited in the NCBI BioProject database under the accession number PRJNA1238237. The RNA-seq and copy number datasets for 33 TCGA cancer types were obtained from the Pan-Cancer Atlas and downloaded from the UCSC Xena Explorer (cohort: TCGA Pan-Cancer). The RNA-seq and copy number datasets for cancer cell lines were retrieved from the Cancer Cell Line Encyclopedia (CCLE). Pearson’s correlation coefficient was used to measure the linear correlation between two variables. For continuous variables, differences between two groups were assessed using the two-sided Student’s t -test, whereas one-way analysis of variance (ANOVA) or the Kruskal-Wallis test was used for comparisons among multiple groups. All graphics and statistics were performed with GraphPad Prism (v9.0) or R (v4.1.0). P values of < 0.05 were considered statistically significant. Acknowledgements This work was supported by the National Natural Science Foundation of China (82151212 to L Tan; 82172596 and 82373351 to G Zhuang; 82203271 to P Shen; 82173077 to X Yin; 82173111 to M Zhang; 82403676 to L Cheng; 32171447 and 82373972 to D Zhao), the Strategic Priority Research Program of the Chinese Academy of Sciences Grant (XDB1060000 to L Tan; XDB0830300 to D Zhao), Shanghai Municipal Health Commission (202140049 to MC Cai), Natural Science Foundation of Jilin province (YDZJ202301ZYTS437 to L Cheng), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161313 to G Zhuang), innovative research team of high-level local universities in Shanghai (SHSMU-ZLCX20210200 to G Zhuang), 111 project (no. 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Share CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders Kaiyan Ye , Qinyang He , Yan Cheng , Meiying Zhang , Huiling Mu , Mei-Chun Cai , Shaoqing Zhou , Huaijiang Xiang , Yuqiao Hou , Yunxia Li , Yinuo Zang , Jingxin Lv , Lin Cheng , Libing Xiang , Dongxin Zhao , Yaoyang Zhang , Ying Li , Xia Yin , Wen Di , Peiye Shen , Guanglei Zhuang , Li Tan bioRxiv 2025.03.31.646293; doi: https://doi.org/10.1101/2025.03.31.646293 Share This Article: Copy Citation Tools CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders Kaiyan Ye , Qinyang He , Yan Cheng , Meiying Zhang , Huiling Mu , Mei-Chun Cai , Shaoqing Zhou , Huaijiang Xiang , Yuqiao Hou , Yunxia Li , Yinuo Zang , Jingxin Lv , Lin Cheng , Libing Xiang , Dongxin Zhao , Yaoyang Zhang , Ying Li , Xia Yin , Wen Di , Peiye Shen , Guanglei Zhuang , Li Tan bioRxiv 2025.03.31.646293; doi: https://doi.org/10.1101/2025.03.31.646293 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cancer Biology Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13888) Bioinformatics (41927) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22494) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)