Meet the authors: Yuzhi Wang, Conner Traugot, and Mingyi Xie.

We talk to authors Yuzhi Wang, Conner Traugot, and Mingyi Xie about their paper "N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation" (this issue of Molecular Cell), their path to research science, and the interesting findings that keep bringing them back to the bench.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation.

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins.

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

Conversion to Radical Nephrectomy From Robotic Partial Nephrectomy Is Most Commonly Due to Anatomic and Oncologic Complexity.

PURPOSE: Partial nephrectomy is standard-of-care treatment for small renal masses. As utilization of partial nephrectomy increases and includes larger and complex tumors, the risk of conversion to radical nephrectomy likely increases. We evaluated incidence and reason for conversion to radical nephrectomy in patients scheduled for partial nephrectomy by surgeons participating in MUSIC (the Michigan Urologic Surgery Improvement Collaborative). MATERIALS AND METHODS: All patients in whom robotic partial nephrectomy was planned were stratified by completed procedure (robotic partial nephrectomy vs radical nephrectomy). Preoperative and intraoperative records were reviewed for preoperative assessment of difficulty and reason for conversion. Patient, tumor, pathologic, and practice variables were compared between cohorts. RESULTS: Of 650 patients scheduled for robotic partial nephrectomy, conversion to radical nephrectomy occurred in 27 (4.2%) patients. No conversions to open were reported. Preoperative documentation indicated a plan for possible conversion in 18 (67%) patients including partial with possible radical (n = 8), partial vs radical (n = 6), or likely radical nephrectomy (n = 4). Intraoperative documentation indicated that only 5 (19%) conversions were secondary to bleeding, with the remaining conversions due to tumor complexity and/or oncologic concerns. Patients undergoing conversion had larger (4.7 vs 2.8 cm, P < .001) and higher-complexity tumors (64% vs 6%, P < .001) with R.E.N.A.L. (for radius, exophytic/endophytic, nearness of tumor to collecting system, anterior/posterior, location relative to polar line) nephrometry score ≥ 10. The converted cases had a higher rate of ≥ pT3 (27% vs 8.4%, P = .008). CONCLUSIONS: There was a low rate of conversion from robotic partial to radical nephrectomy in the MUSIC-KIDNEY (Kidney mass: Identifying and Defining Necessary Evaluation and therapY) collaborative, and an even lower risk of conversion due to uncontrolled bleeding. Targeted review of each conversion identified appropriate decision-making based on oncologic risk in most cases.

Clinical efficacy of blood derivatives on wound healing: A systematic review and network meta-analysis.

This study aims to evaluate the clinical effects of different blood derivatives on wound healing using network meta-analysis. PubMed, Embase, OVID, Web of Science, SCOPUS and Cochrane Central were searched to obtain studies about blood derivatives on wound healing until October 2023. R 4.2.0 and Stata 15.0 softwares were used for data analysis. Forty-four studies comprising 5164 patients were included. The results of network meta-analysis showed that the healing area from high to low was GF + ORCCB, ORCCB, GF, PRF, Unnas paste dressing, APG, PRP injection, PRP, PRP + thrombin gel, PPP, HPL, CT. The healing time from low to high was PRP + thrombin gel, GF, PRP, PC + K, PC, APG, PRF, CT, Silver sulfadiazine ointment. The number of patients cured from high to low was APG, PRP injection, PRP, Aurix, PRF, Leucopatch, HPL, Antimicrobial Ointment Dressing, CT, 60 µg/cm2 repifermin, 120 µg/cm2 repifermin, AFG, PPP. The order of analgesic effect from high to low was AFG, Aminogam gel, PRF, PRP, Oxidised oil, APG, GF, CT. The order of the number of wound infection cases from low to high is APG, 20 µg/cm2 repifermin, 60 µg/cm2 repifermin, PRP, LeucoPatch, CT, PPP, Antiseptic ointment dressing. Healing area: GF + ORCCB had the best effect; Healing time: PRP + thrombin gel took the shortest time. The number of cured patients and the reduction of wound infection: APG has the best effect. Analgesic effect: AFG has the best effect. More studies with large sample sizes are needed to confirm the above findings.

TRIM21 undergoes phase separation dependent CC domain to regulate autophagy.

Tripartite motif (TRIM) family proteins as E3-ligases participate in various biological processes. TRIM21, as the first autoantibody protein, has been found to be associated with autophagy. However, the role of TRIM21 engaging in autophagy is still unclear. In this study, TRIM21 forms significate puncta in the cytoplasm and undergoes liquid-liquid phase separation in vitro. Furthermore, we identify phase separation of the coiled-coil (CC) domain is essential for autophagosome to mediate autophagy-related protein recruited. These findings show that phase separation of the CC domain of TRIM21 promotes autophagosome to impact cell fate.

Biomass-derived magnetic nanocomposites modified by choline chloride/citric acid based natural deep eutectic solvents for the magnetic solid phase extraction of trypsin.

A novel biomass-derived magnetic nanocomposite of Fe3O4-Chitin@NADES-CC composed of a natural deep eutectic solvent (NADES), biological polysaccharide (Chitin) and magnetic Fe3O4 was synthesized. After being systematically characterized by Fourier transform infrared spectrometry, thermogravimetry, vibrating sample magnetometry, X-ray diffraction, transmission electron microscopy and dynamic light scattering, Fe3O4-Chitin@NADES-CC was used as an extractant to separate trypsin (Tryp) on the basis of magnetic solid phase extraction. Simultaneously, the extraction conditions of Fe3O4-Chitin@NADES-CC for Tryp were investigated in turn by single-factor experiments, including screening the types of extractants, the initial concentration of Tryp, the pH value of the solution, the influence of ionic strength, extraction time and temperature, etc. Under the optimal conditions, the extraction capacity of Fe3O4-Chitin@NADES-CC for Tryp could reach up to 1082.67 mg g-1. Adsorption isotherm tests certified that the Langmuir adsorption equilibrium fitted well with the extraction model in this study, which showed that the extraction of Fe3O4-Chitin@NADES-CC for Tryp was monolayer adsorption. In addition, in the sections on the regeneration-reuse, selectivity and methodological studies, all the results exhibited the superiority of the Fe3O4-Chitin@NADES-CC and Tryp separation strategy which has been established in this work. Finally, Fe3O4-Chitin@NADES-CC was ultimately applied to the separation of Tryp from a real bovine pancreas crude extract by the analysis of SDS-PAGE. All the above results highlight that the proposed Fe3O4-Chitin@NADES-CC biomass-derived magnetic nanocomposite can be applied in the field of protein purification.

Effects of honokiol protects against chronic kidney disease via BNIP3/NIX and FUNDC1-mediated mitophagy and AMPK pathways.

BACKGROUND: Chronic kidney disease (CKD) is a serious health threat worldwide. Defective mitophagy has been reported to induce mitochondrial dysfunction, which is closely associated with CKD pathogenesis. Honokiol (HKL) is a bioactive component of Magnolia officinalis that has multiple efficacies. Our study aimed to investigate the effect of HKL on a CKD rat model and explore the possible mechanisms of mitophagy mediated by Bcl-2 interacting protein 3 and BNIP3-like (NIX) (also known as the BNIP3/NIX pathway) and FUN14 domain-containing 1 (the FUNDC1 pathway) and the role of the AMP-activated protein kinase (AMPK) pathway. METHODS: A CKD rat model was established by feeding the animals dietary adenine (0.75% w/w, 3 weeks). Simultaneously, the treatment group was given HKL (5 mg/kg/day, 4 weeks) by gavage. Renal function was assessed by measuring serum creatinine (Scr) and blood urea nitrogen (BUN) levels. Pathological changes were analyzed by periodic acid-Schiff (PAS) and Masson's trichrome staining. Protein expression was evaluated by Western blotting and immunohistochemistry. RESULTS: HKL treatment ameliorated the decline in renal function and reduced tubular lesions and interstitial fibrosis in CKD rats. Accordingly, the renal fibrosis markers Col-IV and α-SMA were decreased by HKL. Moreover, HKL suppressed the upregulation of the proapoptotic proteins Bad and Bax and Cleaved caspase-3 expression in CKD rats. Furthermore, HKL suppressed BNIP3, NIX and FUNDC1 expression, leading to the reduction of excessive mitophagy in CKD rats. Additionally, AMPK was activated by adenine, and HKL reversed this change and significantly decreased the level of activated AMPK (phosphorylated AMPK, P-AMPK). CONCLUSION: HKL exerted a renoprotective effect on CKD rats, which was possibly associated with BNIP3/NIX and FUNDC1-mediated mitophagy and the AMPK pathway.

Suppressing the activation of protein kinase A as a DNA damage-independent mechanistic lead for dihydromethysticin prophylaxis of NNK-induced lung carcinogenesis.

Our earlier work demonstrated varying potency of dihydromethysticin (DHM) as the active kava phytochemical for prophylaxis of tobacco carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung carcinogenesis. Efficacy was dependent on timing of DHM gavage ahead of NNK insult. In addition to DNA adducts in the lung tissues mitigated by DHM in a time-dependent manner, our in vivo data strongly implicated the existence of DNA damage-independent mechanism(s) in NNK-induced lung carcinogenesis targeted by DHM to fully exert its anti-initiation efficacy. In the present work, RNA seq transcriptomic profiling of NNK-exposed (2 h) lung tissues with/without a DHM (8 h) pretreatment revealed a snap shot of canonical acute phase tissue damage and stress response signaling pathways as well as an activation of protein kinase A (PKA) pathway induced by NNK and the restraining effects of DHM. The activation of the PKA pathway by NNK active metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) at a concentration incapable of promoting DNA adduct was confirmed in a lung cancer cell culture model, potentially through NNAL binding to and activation of the ß-adrenergic receptor. Our in vitro and in vivo data overall support the hypothesis that DHM suppresses PKA activation as a key DNA damage-independent mechanistic lead, contributing to its effective prophylaxis of NNK-induced lung carcinogenesis. Systems biology approaches with a detailed temporal dissection of timing of DHM intake versus NNK exposure are warranted to fill the knowledge gaps concerning the DNA damage-driven mechanisms and DNA damage-independent mechanisms to optimize the implementation strategy for DHM to achieve maximal lung cancer chemoprevention.

Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice.

The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.

Electrochemical Synthesis of Polysubstituted Sulfonated Pyrazoles via Cascade Intermolecular Condensation, Radical-Radical Cross Coupling Sulfonylation, and Pyrazole Annulation.

Electrochemical synthesis of polysubstituted sulfonated pyrazoles from enaminones and sulfonyl hydrazides was established under metal-free, exogenous-oxidant-free, and mild conditions. By judicious choice of different electrochemical reaction conditions, NH2-functionalized enaminones or N,N-disubstituted enaminones can react with aryl/alkyl sulfonyl hydrazides to afford tetra- or trisubstituted sulfonated pyrazoles in moderate to good yields, respectively. The gram-scale electrochemical transformation demonstrated the efficiency and practicability of this synthetic strategy. In addition, the sulfonated NH-pyrazole can be obtained via the dissociation of the N-tosyl group. Mechanistic studies reveal that the electrochemical cascade reaction synthesis of polysubstituted sulfonated pyrazoles proceeded via the sequence of intermolecular condensation, radical-radical cross coupling sulfonylation, and pyrazole annulation.

Alzheimer's disease brain-derived extracellular vesicles spread tau pathology in interneurons.

Extracellular vesicles are highly transmissible and play critical roles in the propagation of tau pathology, although the underlying mechanism remains elusive. Here, for the first time, we comprehensively characterized the physicochemical structure and pathogenic function of human brain-derived extracellular vesicles isolated from Alzheimer's disease, prodromal Alzheimer's disease, and non-demented control cases. Alzheimer's disease extracellular vesicles were significantly enriched in epitope-specific tau oligomers in comparison to prodromal Alzheimer's disease or control extracellular vesicles as determined by dot blot and atomic force microscopy. Alzheimer's disease extracellular vesicles were more efficiently internalized by murine cortical neurons, as well as more efficient in transferring and misfolding tau, than prodromal Alzheimer's disease and control extracellular vesicles in vitro. Strikingly, the inoculation of Alzheimer's disease or prodromal Alzheimer's disease extracellular vesicles containing only 300 pg of tau into the outer molecular layer of the dentate gyrus of 18-month-old C57BL/6 mice resulted in the accumulation of abnormally phosphorylated tau throughout the hippocampus by 4.5 months, whereas inoculation of an equal amount of tau from control extracellular vesicles, isolated tau oligomers, or fibrils from the same Alzheimer's disease donor showed little tau pathology. Furthermore, Alzheimer's disease extracellular vesicles induced misfolding of endogenous tau in both oligomeric and sarkosyl-insoluble forms in the hippocampal region. Unexpectedly, phosphorylated tau was primarily accumulated in glutamic acid decarboxylase 67 (GAD67) GABAergic interneurons and, to a lesser extent, glutamate receptor 2/3-positive excitatory mossy cells, showing preferential extracellular vesicle-mediated GABAergic interneuronal tau propagation. Whole-cell patch clamp recordings of CA1 pyramidal cells showed significant reduction in the amplitude of spontaneous inhibitory post-synaptic currents. This was accompanied by reductions in c-fos+ GAD67+ neurons and GAD67+ neuronal puncta surrounding pyramidal neurons in the CA1 region, confirming reduced GABAergic transmission in this region. Our study posits a novel mechanism for the spread of tau in hippocampal GABAergic interneurons via brain-derived extracellular vesicles and their subsequent neuronal dysfunction.

Economic and intensity effects of coal consumption in China.

The increase in coal consumption and its impact on the environment has become a bottleneck that hinders sustainable development. This paper discusses the effect of economic growth and coal intensity on China's coal consumption during 2005-2017 using the Laspeyres index decomposition method. The decoupling of coal consumption from economic growth was examined in conjunction with the Tapio elasticity index, and the decoupling contributions of economic growth and coal intensity are further determined. The results indicated that economic growth drives an increase in coal consumption; however, the contribution rate declines gradually with decrease in economic growth rate in each province. Further research showed that the secondary industry is the main contributor to the increment, and the rapid development of tertiary industry increases indirect coal consumption. Coal intensity has a positive impact on curbing coal consumption, but it is not sufficient to offset the increment generated by the economic effect. Moreover, in each province, the curbing effect gradually decreased as the decline in coal intensity weakened in the secondary industry. Furthermore, coal consumption is weakly decoupled from economic growth over the long term, and the secondary industry will determine the future trend of decoupling.

Single-shot imaging through scattering media under strong ambient light interference.

Speckle correlation imaging (SCI) has found tremendous versatility compared with other scattering imaging approaches due to its single-shot data acquisition strategy, relatively simple optical setup, and high-fidelity reconstruction performance. However, this simplicity requires SCI experiments to be performed strictly in a darkroom condition. As background noise increases, the speckle contrast rapidly decreases, making precise interpretation of the data extremely difficult. Here, we demonstrate a method by refining the speckle in the autocorrelation domain to achieve high-performance single-shot imaging. Experiment results prove that our method is adapted to estimate objects in a low signal-to-background ratio (SBR) circumstance even if the SBR is about -23dB. Laboratory and outdoor SCI experiments are performed.

Specific recognition of protein by deep eutectic solvent-based magnetic ß-cyclodextrin molecularly imprinted polymer.

A magnetic ß-cyclodextrin (MCD) surface molecularly imprinted polymer (MIP) based on deep eutectic solvents (DESs) as cross-linker and functional monomer (MCD@DES-MIP) was successfully synthesized for the specific recognition of bovine hemoglobin (BHb). The adsorption behavior of MCD@DES-MIP for BHb was investigated by adsorption thermodynamics, adsorption kinetics, and pH control experiments. The maximum adsorption capacity of MCD@DES-MIP for BHb under the optimized conditions was 195.94 mg g-1 and the imprinting factor was 4.68. In addition, the competitive adsorption experiments demonstrated that MCD@DES-MIP showed excellent selective extraction ability for BHb in the binary mixture of BHb and bovine serum albumin (BSA). The actual sample analysis manifested that MCD@DES-MIP effectively separated BHb from complex samples. The results of circular dichroism spectra proved that the secondary structure of BHb did not change during elution. The result indicated that MCD@DES-MIP can be used as a new imprinting material for the separation and purification of BHb.Graphical abstract Magnetic imprinted microspheres (MCD@DES-MIP) were prepared by free radical polymerization using magnetic ß-cyclodextrin (MCD) as carrier, deep eutectic solvents (DESs) as functional monomer and cross-linker. MCD@DES-MIP show high adsorption capacity and excellent selectivity for BHb.

Broadband microwave photonic frequency translator with a wide frequency shifting range.

A microwave photonic topology for shifting the frequency of an input microwave signal is presented. It operates based on a single sideband frequency mixing approach. The amount of microwave signal frequency shift is determined by a local oscillator frequency. The proposed frequency translator (FT) has a large bandwidth and a wide frequency shifting range. It can be designed to obtain a large spurious signal suppression ratio and a low frequency translation loss. Results are presented for the novel structure, which demonstrates the realization of a 2-18 GHz FT with a 10 kHz to 100 MHz frequency shifting range. The results also show the spurious signals are more than 31 dB below the frequencyshifted signal, and a low loss of only around 4 dB for frequency shifting a 10 GHz microwave signal.

Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues.

INTRODUCTION: Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aß) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. METHODS: EVs were isolated from the cortical gray matter of 20 AD and 18 control brains. Tau and Aß levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning. RESULTS: Levels of pS396 tau and Aß1-42 were significantly elevated in AD EVs. High levels of neuron- and glia-specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A, and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy. DISCUSSION: In addition to Aß and tau, ANXA5, VGF, GPM6A, and ACTZ are new signature proteins in AD EVs.

Transcriptional repressor GATA binding 1-mediated repression of SRY-box 2 expression suppresses cancer stem cell functions and tumor initiation.

Cancer stem cells (CSCs) have been reported in a variety of cancers. SRY-box 2 (SOX2) is a member of the SOX family of transcription factors and has been shown to play a critical role in maintaining the functions of CSCs and promoting tumor initiation. However, the underlying mechanisms for the transcriptional regulation of the SOX2 gene in CSCs are unclear. In this study, using in silico and experimental approaches, we identified transcriptional repressor GATA binding 1 (TRPS1), an atypical GATA-type transcription factor, as a critical transcriptional regulator that represses SOX2 expression and thereby suppresses cancer stemness and tumorigenesis. Mechanistically, TRPS1 repressed SOX2 expression by directly targeting the consensus GATA-binding element in the SOX2 promoter as elucidated by ChIP and luciferase reporter assays. Of note, in vitro mammosphere formation assays in culture and in vivo xenograft tumor initiation experiments in mouse models revealed that TRPS1-mediated repression of SOX2 expression suppresses CSC functions and tumor initiation. Taken together, our study provides detailed mechanistic insights into CSC functions and tumor initiation by the TRPS1-SOX2 axis.

The Role and Mechanism of CRT0066101 as an Effective Drug for Treatment of Triple-Negative Breast Cancer.

BACKGROUND/AIMS: Breast cancer is clinically classified into three main subtypes: estrogen receptor-positive (ER+) breast cancer, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and triple-negative breast cancer (TNBC). Without specific targeted therapies, patients with TNBC have poorer prognosis compared with those with ER+ and HER2+ breast cancer. Protein kinase D (PRKD) family members play crucial roles in cancer progression. CRT0066101, a PRKD inhibitor, has been reported to have anticancer activity in many cancer types. Nevertheless, the role and mechanism of CRT0066101 in TNBC have not been well investigated. METHODS: The expression level of PRKDs was analyzed in breast cancer samples and breast cancer cell lines. The effects of inhibiting PRKD activity with CRT0066101 on TNBC cell proliferation, cell cycle, apoptosis, and tumor growth were studied by Cell Counting Kit8 assay, cell cycle assay, propidium iodide/annexin-V assay, and a xenograft mouse model, respectively. To uncover the molecular mechanism of CRT0066101 in TNBC, comparative phosphoproteomic analysis using iTRAQ was employed. RESULTS: We found that PRKD2 and PRKD3 were preferentially expressed in breast cancers. Immunohistochemistry confirmed the overexpression of PRKD2 and PRKD3 in TNBC. CRT0066101, which inhibited the activity of PRKDs, dramatically inhibited proliferation, increased apoptosis and the G1-phase population of TNBC cells in vitro, and reduced breast tumor volume in vivo. Comparative phosphoproteomic analysis between breast cancer cells with and without CRT0066101 treatment revealed that the anti-breast cancer effects involved regulation of a complex network containing multiple enriched pathways and several hub-nodes contributing to multiple cancer-related processes, thus explaining the described effects of CRT0066101 on TNBC in vitro and in vivo. Finally, we validated several targets of PRKD inhibition by treatment with CRT0066101 and small interfering RNAs against PRKD2 and PRKD3 (siPRKD2 and siPRKD3), including p-MYC(T58/ S62), p-MAPK1/3(T202/Y204), p-AKT(S473), p-YAP(S127), and p-CDC2(T14). CONCLUSION: PRKD inhibitor CRT0066101 exhibits anti-TNBC effects via modulating a phosphor-signaling network and inhibiting the phosphorylation of many cancer-driving factors, including MYC, MAPK1/3, AKT, YAP, and CDC2, providing insight into the important roles as well as the molecular mechanism of CRT0066101 as an effective drug for TNBC.