CDK9 inhibition activates innate immune response through viral mimicry.

Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition-induced cancer cell-selective anti-proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics, and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB-driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late-stage prostate cancer, a currently lethal disease.

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50 = 3.94 nM) with high efficacy (Dmax = 96 %) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

Inhibition of CDK9 exhibits anticancer activity in hepatocellular carcinoma cells via targeting ribonucleotide reductase.

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC.

Application of the CDK9 inhibitor FIT-039 for the treatment of KSHV-associated malignancy.

Chronic infection with Kaposi's sarcoma-associated herpes virus (KSHV) in B lymphocytes causes primary effusion lymphoma (PEL), the most aggressive form of KSHV-related cancer, which is resistant to conventional chemotherapy. In this study, we report that the BCBL-1 KSHV+ PEL cell line does not harbor oncogenic mutations responsible for its aggressive malignancy. Assuming that KSHV viral oncogenes play crucial roles in PEL proliferation, we examined the effect of cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on KSHV viral gene expression and KSHV+ PEL proliferation. We found that FIT-039 treatment impaired the proliferation of KSHV+ PEL cells and the expression of KSHV viral genes in vitro. The effects of FIT-039 treatment on PEL cells were further evaluated in the PEL xenograft model that retains a more physiological environment for the growth of PEL growth and KSHV propagation, and we confirmed that FIT-039 administration drastically inhibited PEL growth in vivo. Our current study indicates that FIT-039 is a potential new anticancer drug targeting KSHV for PEL patients.

Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9.

PURPOSE: To investigate the role of cyclin-dependent kinase 9 (CDK9) and the therapeutic potential of a CDK9 inhibitor (flavopiridol) in monocrotaline (MCT)-induced pulmonary hypertension (PH). METHODS: For the in vivo experiments, rats with PH were established by a single intraperitoneal injection of MCT (60 mg/kg). After 2 weeks of MCT injection, rats were then treated with flavopiridol (5 mg/kg, i.p., twice a week) or vehicle for 2 weeks. For the in vitro experiments, human pulmonary artery smooth muscle cells (HPASMCs) were treated with flavopiridol (0.025-1 µM) or vehicle under hypoxic conditions. Hemodynamic recording, right ventricle histology, lung histology, and pulmonary arterial tissue isolation were performed. The expression levels of CDK9, RNA polymerase II, c-Myc, Mcl-1, and survivin were determined by qRT-PCR and western blotting, and the proliferation and apoptosis of rat pulmonary arterial tissues and/or HPASMCs were also assayed. RESULTS: Compared to the control group, CDK9 was upregulated in pulmonary arterial tissues from MCT-induced PH rats and hypoxic cultured HPASMCs. Upregulation of CDK9 was associated with enhanced phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at serine-2 (Ser-2), promoting the expression of prosurvival and antiapoptotic proteins (c-Myc, Mcl-1, and survivin). Furthermore, treatment with flavopiridol (5 mg/kg) significantly alleviated pulmonary artery remodeling and partially reversed the progression of MCT-induced PH. Consistently, flavopiridol (0.5 µM) treatment decreased the proliferation and induced the apoptosis of cultured HPASMCs under hypoxic conditions. As a result of CDK9 inhibition and subsequent inhibition of RNA pol II CTD phosphorylation at Ser-2, flavopiridol decreased c-Myc, Mcl-1, and survivin expression in isolated pulmonary small arteries, leading to cell growth inhibition and apoptosis. CONCLUSION: Flavopiridol mitigates the progression of MCT-induced PH in rats by targeting CDK9.

CDK9 Inhibitor Induces Apoptosis, Autophagy, and Suppression of Tumor Growth in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a hematopoietic malignancy with a poor prognosis that develops in approximately 5% of human T-cell leukemia virus type 1 (HTLV-1) carriers. Cyclin-dependent kinase 9 (CDK9), together with Cyclin T, forms a transcription elongation factor, positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the second serine (Ser2) of the seven amino acid repeat sequence in the C-terminal domain of RNA polymerase II (RNAP II). CDK9 inhibitors suppress cell proliferation by inducing apoptosis in chronic lymphocytic leukemia and breast cancer but there are no reports on autophagy of CDK9 inhibitors. Here, we investigated the effect of LY2857785, a novel CDK9 selective inhibitor, on cell death in ATL-related cell lines in vitro, freshly isolated cells from ATL patients ex vivo, and on ATL tumor xenografts in NOD/SCID mice in vivo. LY2857785 significantly reduced cell viability and induced apoptosis, as shown by annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, and suppressed the levels of anti-apoptotic protein myeloid cell leukemia-1 (MCL-1). LY2857785 decreased RNAP II Ser2 phosphorylation and downstream c-Myc protein levels. Interestingly, LY2857785 also increased microtubule-associated proteins 1A/1B light chain 3B (LC3)-II binding to autophagosome membranes. Furthermore, LY2857785 decreased the viability of freshly isolated ATL cells and induced apoptosis. Finally, LY2857785 significantly decreased the growth of ATL tumor xenografts. These results suggest that LY2857785 induces cell death of ATL cells by MCL-1-dependent apoptosis and autophagy and has anti-tumor activity.

O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition.

Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase cyclin-dependent kinase 9 (CDK9) is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics, and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity/flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.

Cyclin-Dependent Kinase 9 Inhibition as a Potential Treatment for Hepatocellular Carcinoma.

PURPOSE: Composite cyclin-dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. METHODS: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. RESULTS: CDK9 inhibitors exhibited potent antiproliferative activities in HCC cells regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1 and survivin. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited significantly slower tumor growth than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving a CDK9 inhibitor also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Knockdown of CDK9 expression reduced the proliferative activities of HCC cells. CONCLUSION: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

O-GlcNAc transferase couples MRE11 to transcriptionally active chromatin to suppress DNA damage.

BACKGROUND: Transcription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects. METHODS: We used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples. RESULTS: Inhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors. CONCLUSIONS: Both OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments.

CDK9 inhibition improves diabetic nephropathy by reducing inflammation in the kidneys.

Diabetic nephropathy (DN) is a chronic inflammatory renal disease induced by hyperglycemia. Recent studies have implicated cyclin-dependent kinase 9 (CDK9) in inflammatory responses and renal fibrosis. In this study, we explored a potential role of CDK9 in DN by using cultured mouse mesangial cell line SV40 MES-13 and streptozotocin-induced type 1 mouse model of diabetes. We inhibited CDK9 in mice and in cultured cells by a highly selective CDK9 inhibitor, LDC000067 (LDC), and evaluated inflammatory and fibrogenic outcome by mRNA and protein analyses. Our studies show that treatment of diabetic mice with LDC significantly inhibits the levels of inflammatory cytokines and fibrogenic genes in kidney specimens. These reductions were associated with improved renal function. We also found that LDC treatment suppressed MAPK-AP1 activation. We then confirmed the involvement of CDK9 in cultured SV40 MES-13 cells and showed that deficiency in CDK9 prevents glucose-induced inflammatory and fibrogenic proteins. This protection was also afforded by suppression of MAPK-AP1. Taken together, our results how that hyperglycemia activates CDK9-MAPK-AP1 axis in kidneys to induce inflammation and fibrosis, leading to renal dysfunction. Our findings also suggest that CDK9 may serve as a potential therapeutic target for DN.

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells.

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

MiR-224 ameliorates inflammation and symptoms in mouse model of allergic rhinitis by targeting CDK9.

OBJECTIVES: To explore the regulatory effects of microRNA (miRNA)-224 and its potential target gene, cyclin dependent kinase 9 (CDK9), in the pathological process of allergic rhinitis (AR). METHODS: To investigate the role of miR-224 and CDK9, it was screened by bioinformatics prediction software and verified by dual-luciferase reporter assay. The mouse model of AR was established by ovalbumin (OVA).The animal models were intervened with miR-224 agomir, negative control agomir, and saline respectively. The symptoms of sneezing and nasal rubbing were recorded. The expressions of miR224, CDK9, and cytokines in the nasal mucosa of different groups were analyzed by rt-PCR or western blotting. Enzyme-linked immunoassay (ELISA) was used to evaluate the levels of IgE and Histamine (HA) in the serum. The infiltration of inflammatory cells in the nasal mucosa was studied by immunohistochemistry. The expression and distribution of CDK9 in the nasal mucosa of mice were revealed by immunofluorescence. RESULTS: In the nasal mucosa of the animal models, the level of miR-224 was downregulated, while that of CDK9 was upregulated. The upregulation of miR-224 by miR-224 agomir reduced the frequencies of nasal rubbing and sneezing, the expression of CDK9, the levels of cytokines, and the concentrations of IgE and HA. Moreover, miR-224 appeared to attenuate the infiltration of inflammatory cells and hypersecretion of glands in the nasal mucosa. The expression of CDK9, which was distributed under the mucosa, especially in the submucosa interstitial tissue, was significantly reduced. CONCLUSION: MiR-224 affected the pathogenesis of AR by targeting CDK9. It proves that miR-224 could be a novel potential therapeutic target for AR.

CDK9 attenuation exerts protective effects on catabolism and hypertrophy in chondrocytes and ameliorates osteoarthritis development.

Osteoarthritis (OA) is generally considered to be characterized by progressive articular cartilage destruction. Increasing evidence demonstrates that CDK9, which is a member of cyclin-dependent kinase family, plays a significant role in the regulation of acute and chronic inflammatory diseases. IL-1ß, a major proinflammatory cytokine, was used to establish a model of OA in vitro after stimulating chondrocytes. We found that CDK9 was highly expressed in in vitro and in vivo models of inflammation. The role of LDC000067 (abbreviated as LDC067), a specific inhibitor of CDK9, in protecting articular cartilage from immune response has not been fully clarified. Intriguingly, in this study, we demonstrated that LDC067 prevented IL-1ß-induced production of metalloproteinases (MMPs) and inflammatory cytokines, including MMP3, MMP9, MMP13, IL-6, IL-8 and TNF-ɑ. Furthermore, we revealed that LDC067 inhibited IL-1ß-induced NF-κB signaling pathway activation in chondrocytes. The inhibition of CDK9 could also delay cartilage degeneration in an anterior cruciate ligament transection (ACLT) mouse model in vivo. Taken together, these results highlighted the significance of this CDK9 inhibitor in preventing cartilage destruction and indicated that LDC067 might serve as a potential therapeutic agent for OA.

MALAT1 induces osteosarcoma progression by targeting miR-206/CDK9 axis.

Long noncoding RNAs (LncRNAs) have been reported to participate in cancer development, including osteosarcoma. Here, in our study, we observed that lncRNA metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) was remarkably overexpressed in osteosarcoma. However, the role it plays in osteosarcoma proliferation mediated by miR-206/cyclin-dependent kinase 9 (CDK9) axis remains uninvestigated. It was found that miR-206 was decreased and CDK9 was elevated in human osteosarcoma cells including MG63, Saos-2, U2OS, and KHOS compared with human osteoblast cell line hFOB 1.19. In addition, it was exhibited that knockdown of MALAT1 was able to inhibit osteosarcoma cell proliferation, which suggested that MALAT1 played an oncogenic role in osteosarcoma development. Bioinformatics analysis indicated that MALAT1 can function as a competing endogenous RNA by sponging miR-206. Because miR-206 has been identified as a significant tumor suppressive gene in multiple cancers, we validated that mimics of miR-206 can restrain osteosarcoma progression. Furthermore, dual-luciferase reporter assay, RNA binding protein immunoprecipitation, and RNA pull-down assay demonstrated the correlation between miR-206 and MALAT1. Besides these, CDK9 was predicted as a downstream gene of miR-206, and we observed that MALAT1 can regulate osteosarcoma progress by modulating CDK9 expression via sponging miR-206. In conclusion, our study implied that MALAT1/miR-206/CDK9 axis can provide novel insights into the biological mechanism of osteosarcoma progression.

Regulation of the circadian rhythmic expression of Sglt1 in the mouse small intestine through histone acetylation and the mRNA elongation factor, BRD4-P-TEFb.

Jejunal sodium/glucose co-transporter (Sglt1) displays circadian expression. The jejunum was collected every 4 h from mice, and we examined histone acetylation and binding of bromodomain-containing protein-4 (BRD4) around of the gene. Histone acetylation increased in the transcribed region of Sglt1 prior to induction of the gene. Furthermore, the binding of mRNA elongation factor around the gene showed circadian rhythm.

Discovery of novel indirubin-3'-monoxime derivatives as potent inhibitors against CDK2 and CDK9.

Indirubin-3'-monoxime (IM) is a potent cyclin-dependent kinase (CDK) inhibitor. Twenty novel IM derivatives were prepared to investigate the structure-activity relationships (SAR) of this compound class. Six compounds showed significant inhibition against both CDK2/cyclin E1 and CDK9/cyclin T1. The most potent compound 7t exhibited IC50 values at submicromolar level. Preliminary SAR trends were suggested and cytotoxicity of these compounds was investigated. Molecular docking studies on compounds 7l and 7t provided conducive clues for further structural optimization.

The CDK9-SPT5 Axis in Control of Transcription Elongation by RNAPII.

The RNA polymerase II (RNAPII) transcription cycle is regulated at every stage by a network of cyclin-dependent protein kinases (CDKs) and protein phosphatases. Progression of RNAPII from initiation to termination is marked by changing patterns of phosphorylation on the highly repetitive carboxy-terminal domain (CTD) of RPB1, its largest subunit, suggesting the existence of a CTD code. In parallel, the conserved transcription elongation factor SPT5, large subunit of the DRB sensitivity-inducing factor (DSIF), undergoes spatiotemporally regulated changes in phosphorylation state that may be directly linked to the transitions between transcription-cycle phases. Here we review insights gained from recent structural, biochemical, and genetic analyses of human SPT5, which suggest that two of its phosphorylated regions perform distinct functions at different points in transcription. Phosphorylation within a flexible, RNA-binding linker promotes release from the promoter-proximal pause-frequently a rate-limiting step in gene expression-whereas modifications in a repetitive carboxy-terminal region are thought to favor processive elongation, and are removed just prior to termination. Phosphorylations in both motifs depend on CDK9, catalytic subunit of positive transcription elongation factor b (P-TEFb); their different timing of accumulation on chromatin and function during the transcription cycle might reflect their removal by different phosphatases, different kinetics of phosphorylation by CDK9, or both. Perturbations of SPT5 regulation have profound impacts on viability and development in model organisms through largely unknown mechanisms, while enzymes that modify SPT5 have emerged as potential therapeutic targets in cancer; elucidating a putative SPT5 code is therefore a high priority.

Long noncoding RNA small nucleolar RNA host gene 5 facilitates neuropathic pain in spinal nerve injury by promoting SCN9A expression via CDK9.

This study aims to explore the functions and mechanisms of long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) in chronic constriction injury (CCI)-induced neuropathic pain (NP). An NP rat model was established using the CCI method and the NP severity was evaluated by paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). The expression of SNHG5, CDK9, and SCN9A was quantified in rat dorsal root ganglion, in addition to the detections of apoptosis, pathological changes, neuron number, and the co-localization of Nav1.7 and cleaved caspase-3 with NeuN. In ND7/23 cells, the apoptosis and lactate dehydrogenase concentration were assessed, as well as the relationship between SNHG5, CDK9, and SCN9A. In the dorsal root ganglion of CCI-treated rats, SNHG5 and SCN9A were upregulated and downregulation of SNHG5 suppressed SCN9A expression, increased the PWT and PWL, blocked neuroinflammation and neuronal apoptosis, and alleviated NP. Mechanistically, SNHG5 recruited CDK9 to enhance SCN9A-encoded Nav1.7 expression and promoted peripheral neuronal apoptosis and injury. In addition, SCN9A overexpression nullified the alleviative effects of SNHG5 deficiency on NP and neuron loss in CCI rats. In conclusion, SNHG5 promotes SCN9A-encoded Nav1.7 expression by recruiting CDK9, thereby facilitating neuron loss and NP after spinal nerve injury, which may offer a promising target for the management of NP.

CDK9 targeting PROTAC L055 inhibits ERα-positive breast cancer.

Breast cancer is one of the most prevalent malignancies affecting women worldwide, underscoring the urgent need for more effective and specific treatments. Proteolysis-targeting chimeras (PROTACs) have emerged as a promising strategy to develop new lead compounds by selectively targeting oncoproteins for degradation. In this study, we designed, synthesized and evaluated a CRBN-based PROTAC, L055, which targets CDK9. Our findings demonstrate that L055 effectively inhibits the proliferation, induces cell cycle arrest, and decreases the survival of ERα-positive breast cancer cells in vitro. L055 specifically binds to CDK9, facilitating its degradation via the CRBN-dependent proteasomal pathway. Additionally, L055 suppressed the growth of organoids and tumors derived from T47D and MCF7 cells in nude mice. Thus, L055 represents a potential novel therapeutic agent for ERα-positive breast cancer and potentially other malignancies.

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression.

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20-57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20-35 TACTNCYCAKCCFQVC), core-domain (a.a. 36-46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47-57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4-11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.