CDK Family Research Articles

A new insight into potential roles of Spfoxl-2 in the testicular development of Scylla paramamosain by RNAi and transcriptome analysis

In our previous study, we found that the Spfoxl-2 transcript was highly expressed in gonads and explored its potential target genes in the ovary of Scylla paramamosain. In the current study, we primally analyzed its potential target genes in the testis through RNAi and RNA-Seq technology and compared with that in the ovary. The results showed that a total of 7892 unigenes were differentially expressed after Spfoxl-2 silencing in the testis, including plenty of conserved genes involved in testicular development, such as Dmrt family genes, Sox family genes, Caspase family genes, Cdk family genes, Kinesin family genes, Fox family genes and other genes. Further analysis revealed that these differentially expressed genes (DEGs) were enriched in crucial pathways involved in spermatogenesis, such as DNA replication, Cell cycle, Spliceosome, Homologous recombination, Meiosis and Apoptosis. The comparison results of potential target genes in the ovary and testis reveal 135 common potential target genes, including some genes involved in the immune response. According to our knowledge, the present work was the first to disclose the functions of foxl-2 in the testis of crustacean species using transcriptome analysis. It not only identifies key genes and pathways involved in the testicular development of S. paramamosain, but also reveals a new molecular-level understanding of the function of foxl-2 in testicular development.

Binding selectivity-dependent molecular mechanism of inhibitors towards CDK2 and CDK6 investigated by multiple short molecular dynamics and free energy landscapes

Understanding selectivity-dependent molecular mechanism of inhibitors towards CDK2 over CDK6 is prominent for improving drug design towards the CDK family. Multiple short molecular dynamics (MD) simulations combined with MM-GBSA approach are adopted to investigate molecular mechanism on binding selectivity of inhibitors X64, X3A, and 4 AU to CDK2 and CDK6. The RMSF analysis and calculations of molecular surface areas indicate that local structural and global flexibility of CDK6 are stronger than that of CDK2. Based on dynamics cross-correlation maps (DCCMs), motion modes of CDK2 and CDK6 produce difference due to associations of X64, X3A, and 4 AU. The calculated binding free energies (BFEs) demonstrate that the compensation between binding enthalpy and entropy of X64, X34, and 4 AU is a key force driving selectivity of inhibitors towards CDK2 over CDK6. This work provides valuable information for designing highly selective inhibitors towards CDK2 and CDK6 and further promotes identification of efficient anticancer drugs in the future.

Discovery of 5,7-Dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-ones as Highly Selective CDK2 Inhibitors.

A series of exceptionally selective CDK2 inhibitors are described. Starting from an HTS hit, we successfully scaffold hopped to a 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one core structure, which imparted a promising initial selectivity within the CDK family. Extensive further SAR identified additional factors that drove selectivity to above 200× for CDKs 1/4/6/7/9. General kinome selectivity was also greatly improved. Finally, use of in vivo metabolite identification allowed us to pinpoint sulfonamide dealkylation as the primary metabolite, which was ameliorated through the deuterium effect.

Comprehensive proteomic analysis reveals dynamic phospho-profiling in human early erythropoiesis.

Normal early erythropoiesis depends on the precise regulation of protein expression and phosphorylation modification. Dysregulation of protein levels or modification contributes to erythroid disorders. To date, the dynamics of protein phosphorylation profiling across human erythroid development is not fully understood. Here, we characterized quantitative proteomic and phosphoproteomic profiling by tandem mass-tagging technology. We systemically built phospho-expression profiling and expression clusters of 11 414 phosphopeptides for human early erythropoiesis. The standardization methods for multitier integrative analyses revealed multiple functional modules of phosphoproteins (e.g., regulation of the G2/M transition) and active phosphorylated signalling (e.g., cell cycle-related pathways). Our further analysis revealed that CDK family members were the main kinases that phosphorylate substrates in erythroid progenitors and identified that CDK9 played an important role in the proliferation of erythroid progenitors. Collectively, our phosphoproteomic profiling, integrative network analysis and functional studies define landscapes of the phosphoproteome and reveal signalling pathways that are involved in human early erythropoiesis. This study will serve as a valuable resource for further investigations of phosphatase and kinase functions in human erythropoiesis and erythroid-related diseases.

Phosphoproteome profiling of rice tissues provides new insights into responsive mechanisms and kinase activity upon salt stress

Rice is a model monocot plant and a major food crop worldwide that is known as the most salt-sensitive cereal. Phosphorylation of proteins regulates the stimulus perception and triggers responses; however, remains largely unknown in crops, particularly in rice under salinity. Here, we report in-depth physiological, proteomic, and phosphoproteomic kinetics of rice shoot and root tissues in response to short-term salt stress. We identified 8160 proteins, 5684 phosphopeptides that assigned to 3142 ‎phosphoproteins. In the root, hypophosphorylation of KAT1, BORs, HAKs, NHX2, and NHX8 transporters, RBOHs and nitrate reductase implied less efficiency in ion, minerals, and ROS haemostasis, thus likely resulting in membrane damages and sensitivity of rice to salinity. Predominantly, the PKA, CDK, MAPK, and PP2A families modulated plant response through interaction with densely interconnected protein modules. We identified 17 phosphomotifs reflecting the identities of the upstream kinases. Furthermore, phylogenetic mapping of 245 identified kinases together with the kinase of known target motifs and function, expanded the biological characters of kinases in rice. We also assigned target motifs for rice kinase families, including PDK1, PKA, CAMKL, CK1, CDK, GSK, and MAPK. This study expands the set of kinase substrates and their signaling networks. Our data also assigns new stress-responsive functions to a large number of novel phosphoproteins, thus will serve as a resource for further studies of salt stress signaling in plants. • We represent the largest profiling of 5422 phosphopeptides in rice upon salinity. • Critical transporters hypophosphorylated like KAT1, BORs, HAKs, NHX2/8, NCX1, CAX1. • CAMTA1/3/4, CDPK12/17/20/24, CBL23, RLKs, and PP2C likely caused salt sensitivity. • Phospho-regulation by PKA, CDK, MAPK, and PP2A families modulated plant response. • This study assigns stress-responsive functions to numerous novel phosphoproteins.

Cyclin-Dependent Kinase 1 Inhibition Potentiates the Proliferation of Tonsil-Derived Mesenchymal Stem Cells by Delaying Cellular Senescence.

Mesenchymal stem cells (MSCs) have been widely used in tissue regeneration and stem cell therapy and are currently being tested in numerous clinical trials. Senescence-related changes in MSC properties have attracted considerable attention. Senescent MSCs exhibit a compromised potential for proliferation; senescence acts as a stress response that prevents the proliferation of dysfunctional cells by inducing an irreversible cell cycle arrest. Here, we established a senescent MSC model using senescence-associated β-galactosidase, proliferation, and cell cycle assays. We further identified novel biomarker candidates for old, senescent tonsil-derived MSCs (TMSCs) using transcriptomics. A plot of the cellular senescence pathway showed cyclin-dependent kinase 1 (CDK1; +8-fold) and CDK2 (+2-fold), and transforming growth factor beta 2 (TGFB2; +2-fold) showed significantly higher expression in old TMSCs than in young TMSCs. The CDK family was shown to be related to cell cycle and proliferation, as confirmed by quantitative RT-PCR. As replicative senescence of TMSCs, the gene and protein expression of CDK1 was significantly increased, which was further validated by inhibiting CDK1 using an inhibitor and siRNA. Taken together, we suggest that the CDK1 can be used as a selective senescence biomarker of MSCs and broaden the research criteria for senescent mechanisms.

The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis.

SummaryBone marrow mesenchymal stem cells (MSCs) are critical regulators of postnatal bone homeostasis. Osteoporosis is characterized by bone volume and strength deterioration, partly due to MSC dysfunction. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Here, CDK8 in MSCs was identified as important for bone homeostasis. CDK8 level was increased in aged MSCs along with the association with aging-related signals. Mouse genetic studies revealed that CDK8 in MSCs plays a crucial role in bone resorption and homeostasis. Mechanistically, CDK8 in MSCs extrinsically controls osteoclastogenesis through the signal transducer and transcription 1 (STAT1)-receptor activator of the nuclear factor κ Β ligand (RANKL) axis. Moreover, aged MSCs have high osteoclastogenesis-supporting activity, partly through a CDK8-dependent manner. Finally, pharmacological inhibition of CDK8 effectively repressed MSC-dependent osteoclastogenesis and prevented ovariectomy-induced osteoclastic activation and bone loss. These findings highlight that the CDK8-STAT1-RANKL axis in MSCs could play a crucial role in bone resorption and homeostasis.

Abstract 2924: Target identification, selectivity profiling and mechanistic insights of a Cdk9 inhibitor using complementary proteomics methods

Abstract In an effort to map the selectivity and understand the mode of action of a CDK9 inhibitor (compound 1), we employed several orthogonal proteomics methods. Our results show a clear selectivity against the CDK family of kinases, with the highest specific affinity towards CDK9. In addition, our multi-method approach allows us to also map the protein binding sites of the inhibitor, identify non-kinase (off-) targets as well as detect the cellular molecular responses to the added inhibitor. Here we describe the methods used, their strengths and weaknesses, how they can be used in the drug discovery pipeline and how they synergize to provide mechanistic insights of compounds of interest. We have used chemoproteomics, kinase affinity tools (kinobeads), Cellular Thermal Shift Assay (CETSA) and Limited Proteolysis (LiP). The results obtained clearly show that CDK9 is the primary target of Compound 1, with affinity curves highly correlated between the different target deconvolution techniques. The chemoproteomic approach rely on a compound derivate, able to bind to a sepharose bead. Subsequently, the binding competition assays are performed on lysed cell material. The choice of a mild lysis buffer allowed us to identify, not only CDK9, but also it’s molecular partners in the p-Tefb complex (Cyclin T1, Cyclin T2 and Aff4) with similar concentration response behavior. The results for the kinases identified in the study were strikingly similar when also profiling the compound without the chemical modification using the kinobeads assay. In the CETSA experiments, where both lysed cells and intact cells were profiled, the lysate experiment most closely resembles that of the previous pull-downs. Here, only direct binders of Compound 1 show a thermal shift, for example several of the pulled down kinases but not the p-Tefb complex partners that were co-competed previously. In the intact cell version of CETSA, not only the direct binders of the compound show stability shifts, but also downstream events and other secondary modulatory effects leave thermal traces in the cell. For example, Compound 1 also binds to GSK3A/B, causing their melting temperature to increase. Inhibition of GSK3 further affects the phosphorylation state and cellular location of FOXK1, which in turn is identified as a destabilized protein. Finally, Limited Proteolysis was used to identify target protein and using the LiP-Quant approach their LiP scores were assigned. Further, out of the identified CDK targets, mapping of peptide cleavage pattern was performed for the members of the CDK family for which structural data is published. The result identified the peptides to be directly adjacent to the ATP binding pocket of CDK9 or regions of high homology. The use of complementary techniques, based on unique biological and biochemical processes, allow robust and confident characterization of inhibitor compounds. Citation Format: Adam Hendricks, Nigel Beaton, Alexey Chernobrovkin, Eric Miele, Ghaith Hamza, Piero Ricchiuto, Ronald Tomlinson, Tomas Friman, Cassandra Borenstein, Bernard Barlaam, Sudhir Hande, Chris De Savi, Rick Davies, Martin Main, Joakim Hellner, Kristina Beeler, Yuehan Feng, Roland Bruderer, Lukas Reiter, Daniel Martinez Molina, Maria Paola Castaldi. Target identification, selectivity profiling and mechanistic insights of a Cdk9 inhibitor using complementary proteomics methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2924.

Abstract 2568: ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification

Abstract Cyclin E1 amplification is prevalent in cancers with high unmet medical need such as high grade serous ovarian cancer, stomach cancer and esophageal cancer. Cancer cell lines with high expression level of cyclin E1 gene exhibited profound sensitivity to CDK2 gene depletion, suggesting CDK2 selective inhibitors have the potential to treat patients harboring Cyclin E1 alterations. Here we report the development and preclinical characterization of ARTS-021, an orally bioavailable small molecule CDK2 selective inhibitor. In enzymatic assay against a panel of different CDKs, ARTS-021 potently inhibits the activity of CDK2/CyclinE1 complex with 50% inhibitory concentration (IC50) in the sub-nanomolar range, displaying superior selectivity against other CDKs (CDK1,4,6,7,9). Very low hit rate (S(10)=0.022, at 1uM) is observed when ARTS-021 is assessed in whole Kinome profiling, further revealing the selectivity of this compound. ARTS-021 also demonstrates potent CDK2 inhibition and selectivity against other CDK family members (CDK1,4,6,9) in a series of cellular assays. Consistent with genetic CDK2 dependence data, cell lines with CCNE1 amplification are particularly sensitive to ARTS-021. Double nanomolar ARTS-021 inhibits Rb phosphorylation and blocks G1/S transition, leading to cell growth arrest specifically in CCNE1 amplified cell lines. This CDK2 dependent anti-tumor activity is further validated in vivo, where daily ARTS-021 administration leads to tumor stasis in CCNE1 amplified but not wild type xenograft models. Taken together, these data demonstrate ARTS-021 as a promising CDK2 selective inhibitor with strong potential towards the development of effective therapies for CCNE1 altered cancer. Citation Format: jiaqi Liang, Dai Cheng, Yali Guo, xiaobin zhang, Xipan Liu, Jia Fu, Qiang Ding, Wen Xiong, Fang Li, Yaoyu Chen, Qing Sheng. ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2568.

Abstract 3931: TGN-1062 inhibits CDK7 and augments the effects of gemcitabine and nab-paclitaxel in pancreatic cancer models

Abstract Purpose: Overexpression of Cyclin-Dependent Kinase 7 (CDK7) is linked to poor clinical outcomes in a variety of cancer types including AML, breast, ovarian, pancreatic, and lung cancers. CDK7 plays a dual role in tumor progression by regulating the cell cycle via phosphorylation of members of the CDK family and stimulation of transcription via phosphorylation of RNA Polymerase II. CDK7 has also been shown to be associated with super-enhancers which can lead to overexpression of oncogenic driver genes such as BCL2, GFI1, and MYC. The development of a selective inhibitor of CDK7 is a promising strategy to control expression of these oncogenic genes in patients’ tumors. Previously, we described the design and synthesis of a potent and selective small molecule inhibitor of CDK7, TGN-1062, which was designed to bind to the catalytic ATP pocket. We showed efficacy in both AML and ovarian in vitro and in vivo models with correlative biomarker data. Here, we have generated further data on the effect and safety of TGN-1062 in pancreatic cancer cell line (CDX) and patient derived xenograft (PDX) models, both as a single agent and in combination with standard of care (SOC) therapies. Methods: In vivo efficacy studies were conducted in mice engrafted with pancreatic CDX and PDX tumors and treated with TGN-1062 with and without SOC therapies. Tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation levels were measured in both tumors and plasma using mass spectrometry. Immunohistochemistry (IHC) was performed on tumors to determine changes in cleaved Caspase-3, MYC, Ki67, and RNA Pol II. Rats were dosed with TGN-1062 to assess plasma exposures and preliminary tolerability. Results: In a pancreatic CDX model (MIA PaCa-2), TGN-1062 showed 38% TGI as a single agent and 121% TGI when in combination with gemcitabine and nab-paclitaxel, which on its own showed 37% TGI. In a pancreatic PDX model, TGN-1062 did not exhibit single agent effect, but combination with gemcitabine and nab-paclitaxel showed 95% TGI compared to 79% TGI with gemcitabine/nab-paclitaxel alone. Drug accumulation, biomarker analysis, and IHC from CDX and PDX tumors, showed that TGN-1062 treatment decreased RNA Pol II phosphorylation and MYC expression, and induced apoptosis. Rat pharmacokinetics indicated 100% oral bioavailability. Conclusion: Our potent and selective small molecule inhibitor of CDK7, TGN-1062, shows promise in pancreatic cancer models as a single or in combination with SOC. Preliminary data suggests that TGN-1062 can be developed further in multiple cancer indications. Citation Format: Alexis Weston, Trason Thode, Ryan Rodriguez del Villar, Serina Ng, Samuel Sampson, Shelby Rheinschmidt, Tithi Ghosh Halder, Raffaella Soldi, Mohan Kaadige, Srinivas Kasibhatla, Haiyong Han, Justin Moser, Vincent Chung, Joseph Chao, Victoria Villaflor, Daniel Von Hoff, Sunil Sharma. TGN-1062 inhibits CDK7 and augments the effects of gemcitabine and nab-paclitaxel in pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3931.

Abstract 2300: Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models

Abstract Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2300.

Narazaciclib’s kinase inhibitory activity is differentiated from approved CDK4/6 inhibitors in preclinical models.

e15096 Background: Despite clinical benefit of CDK4/6 inhibitors in HR+Her2-mBC, patients progress. Neutropenia and diarrhea are safety concerns. Narazaciclib (ON 123300) is a multi-targeted kinase inhibitor of CDK4/6, ARK5 (NUAK1), CSF1R, and c-Kit at low nM concentrations designed to enhance efficacy and safety. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; in different regimens. Treatment of tumor cell lines with narazaciclib induces G1; G2 arrest and apoptosis. Mouse models suggest narazaciclib causes less neutropenia than palbociclib (palbo). Since signaling pathways are affected, we explored the activity of narazaciclib in direct comparisons with abemaciclib (abe), palbo and ribociclib (ribo) using in vitro and cell based assays. Methods: Comparison of narazaciclib’s in vitro IC50 profile to abe, palbo and ribo was studied against a panel of 370 kinases (HotSpot). Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Protein specific binding of narazaciclib and palbo was investigated by Cellular Thermal Shift Assay (CETSA). To investigate narazaciclib’s effect on signaling pathways, integrative Inferred Kinase Activity (INKA) analysis was performed. Results: Narazaciclib and abe were found to be the most promiscuous in vitro kinase inhibitors and ribo the most specific. Abe and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abe (0.08 nM), narazaciclib (0.18 nM), palbo (0.75 nM) and ribo (1.3 nM). Narazaciclib and abe displayed nM activity against CDK2/cyclinA. The IC50 values against GSK3β, whose inhibition putatively causes diarrhea, was 374 nM for narazaciclib and 13 nM for abe. Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. CETSA-MS revealed more potential targets engaged by narazaciclib compared to palbo in both lysates and intact cells such as CHEK1, AAK1, BMP2K, GSK3α and GSK3β, but did not identify binding to NUAK1 or CSF1R. INKA analysis demonstrated that narazaciclib induced unique deregulated phosphorylation compared to palbo, including BUB1 (highly expressed in TNBC), NUAK1, CAMK2D, CDK16 and ULK1. Inhibition of autophagy, both at early and late stages, may sensitize cancer cells to narazaciclib and induce irreversible cell proliferation inhibition, providing a novel therapeutic approach. Conclusions: These studies identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement and will guide future studies with narazaciclib targeting specific kinase driven tumors with the potential for improved safety. These preclinical data await confirmation from current and future clinical trials.

Discovery of novel inhibitors of CDK2 using docking and physics-based binding free energy calculation.

Cyclin-dependent kinase (CDK) is a serine/threonine protein kinase family that cooperates with cyclin and plays an important role in the regulation of cell cycle. Cyclin-dependent kinase 2 is an important member of the CDK family and holds great promise as an anti-cancer drug target. In this study, we used molecular docking and physics-based binding free energy calculation method AS-IE that explicitly calculated protein-ligand binding entropy to discover novel inhibitors of CDK2. A total of 17 inhibitors were discovered with the best IC50 reaching ~2μM. Decomposition of the binding free energy using AS-IE reveals key protein-ligand interactions that determines the activity. These results provided a good example of drug design using physics-based free energy calculation method such as AS-IE and the novel compounds offered a good start point for further development of CDK2 inhibitors.

Multi-omics evaluation of SARS-CoV-2 infected mouse lungs reveals dynamics of host responses

SummaryThe outbreak of Coronavirus disease 2019 (COVID-19) throughout the world has caused millions of death, while the dynamics of host responses and the underlying regulation mechanisms during SARS-CoV-2 infection are not well depicted. Lung tissues from a mouse model sensitized to SARS-CoV-2 infection were serially collected at different time points for evaluation of transcriptome, proteome, and phosphoproteome. We showed the ebb and flow of several host responses in the lung across the viral infection. The signaling pathways and kinases regulating networks were alternated at different phases of infection. This multiplex evaluation also revealed that many kinases of the CDK and MAPK family were interactive and served as functional hubs in mediating the signal transduction during SARS-CoV-2 infection. Our study not only revealed the dynamics of lung pathophysiology and their underlying molecular mechanisms during SARS-CoV-2 infection, but also highlighted some molecules and signaling pathways that might guide future investigations on COVID-19 therapies.

Discovery and Optimization of Highly Selective Inhibitors of CDK5.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including GFB-12811, that are highly selective over the other tested kinases. In cellular assays, our compounds demonstrate CDK5 target engagement while avoiding anti-proliferative effects associated with inhibiting other CDKs. In addition, we show that the compounds in this series exhibit promising in vivo PK profiles, enabling their use as tool compounds for interrogating the role of CDK5 in ADPKD and other diseases.

CDK14/β-catenin/TCF4/miR-26b positive feedback regulation modulating pancreatic cancer cell phenotypes in vitro and tumor growth in mice model in vivo.

Chemotherapy and radiotherapy have been reported to be basically ineffective for pancreatic ductal adenocarcinoma patients; thus, gene therapy might provide a novel approach. CDK14, a new oncogenic member of the CDK family involved in the pancreatic cancer cell response to gemcitabine treatment, has been reported to be regulated by microRNAs. In the present study, we aimed to investigate whether miR-26b regulated CDK14 expression to affect the phenotype of pancreatic cancer cells. Overexpression or knockdown of CDK14 or miR-26b was generated in pancreatic cancer cell lines and the function of CDK14 and miR-26b on cell phenotype and the Wnt signaling pathway was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine and transwell assays, as well as a xenograft model and western blotting. The predicted binding site between the 3'-untranslated region of CDK14 and miR-26b, miR-26b promoter and TCF4 was verified by luciferase or chromatin immunoprecipitation assays. CDK14 overexpression inhibited p-GSK3β, whereas it promoted p-LRP6, the nuclear translocation of β-catenin and the transactivation of TCF4 transcription factor, thus promoting pancreatic cancer cell aggressiveness. miR-26b directly targeted CDK14 and inhibited CDK14 expression. In vitro and in vivo, miR-26b overexpression inhibited, and CDK14 overexpression promoted, cancer cell aggressiveness; CDK14 overexpression partially attenuated the miR-26b overexpression effects on cancer cells. The effects of miR-26b overexpression on tumor growth and the Wnt/β-catenin/TCF4 signaling were partially reversed by CDK14 overexpression. TCF4 inhibited the expression of miR-26b by targeting its promoter region. CDK14, β-catenin, TCF4 and miR-26b form a positive feedback regulation for modulating pancreatic cancer cell phenotypes in vitro and tumor growth in vivo.

MiR-142-5p directly targets cyclin-dependent kinase 5-mediated upregulation of the inflammatory process in acquired middle ear cholesteatoma

MicroRNAs (miRNAs) play important roles in the regulation of cell proliferation, differentiation, apoptosis, and inflammatory responses. MiR-142-5p is an important inflammation-associated miRNA, whose abnormal expression has been associated with a variety of inflammation-related diseases. However, the role and signaling pathways targeted by miR-142-5p in acquired middle ear cholesteatoma (AMEC) have not been fully elucidated. Cyclin-dependent kinase 5 (CDK5), a special member of the CDK family compared with classic cyclins that plays a critical role in the inflammatory response. In this study, we investigated the roles of miR-142-5p and CDK5 in inflammatory responses in AMEC. Our results revealed that the expression of miR-142-5p was significantly reduced in AMEC, and was negatively correlated with the expression of CDK5 (r=-0.5451). We also found that miR-142-5p can inhibit CDK5 expression by directly target 3’ untranslated region (UTR) of CDK5. Additionally, our findings indicated that the increased expression of CDK5 induces the secretion of inflammatory cytokines. In order to further confirm the involvement of miR-142-5p in the regulation of the inflammatory response in AMEC through its inhibitory effect on CDK5 expression, we studied the inflammatory response in HaCaT cells transfected with small interfering RNA against CDK5 (si-CDK5) and a miR-142-5p inhibitor. The results confirmed that miR-142-5p regulates the inflammatory response in AMEC by downregulating CDK5. In summary, miR-142-5p directly inhibits the CDK5-mediated upregulation of inflammatory cytokines in AMEC, which makes it a potential therapeutic target in this disease.

Systematic Characterization of the “Resistome” of Tumor Cells Against Panels of Small Molecule Inhibitors — Application in the Context of Inhibitors of Transcriptional Cyclin-Dependent Kinases (tCDKs) and Implications for Future “Individualized” Clinical Applications

The development of CRISPR/Cas9-based functional genomic approaches have greatly accelerated the pace of research on characterizing the molecular mechanisms of tumor cell resistance to diverse classes of therapeutics. We reasoned that this technology can be applied to systematically and prospectively identify candidate genes regulating tumor cell sensitivity vs. resistance against a collection of inhibitors with overlapping vs. distinct specificities against different structurally- and functionally-related molecular targets; and that the signature of genes emerging from such genome-wide resistance screens could also exhibit overlapping vs. distinct features consistent with the profile targets of the respective compounds. We applied this hypothesis in the context of a collection of small molecule inhibitors against transcriptional cyclin-dependent kinases (tCDKs). This choice was specifically motivated by our recent observations that a collection of transcription factors selectively drive the biology of multiple myeloma (MM) cells (compared to other neoplasias): while direct and selective small molecule inhibitors against these myeloma-selective transcription factors are not currently available, the expression of some them or their target genes is regulated by tCDKs, such as CDK7. We evaluated the CDK7/12/13 inhibitor THZ-01, the CDK7-selective inhibitor YKL05-124 and the CDK12/13 dual inhibitor MFH2-90-1 (referred herein as YKL and MFH respectively); and confirmed that these inhibitors exhibit in vitro activity against MM cells, with variable potency across cell lines. For our CRISPR/Cas9-based screens, we utilized MM.1S cells engineered to stably express the Cas9 nuclease and lentivirally transduced with the pooled genome-wide sgRNA library Brunello (~70,000 sgRNAs, 4 sgRNAs per gene; plus additional non-targeting control sgRNAs); and exposed the MM.1S-Cas9+ sgRNA+ pools of MM cells to each of these compounds (2 separate screens for THZ-01; 1 for YKL and 1 for MFH). Treatments consistent of successive rounds of treatment (at each compounds IC25 dose for anti-MM activity) for several weeks of culture until treatment-resistant populations of tumor cells emerged. PCR and next-generation sequencing we performed, as in other studies of our group and others, to quantify the relative enrichment or depletion of sgRNAs against different genes in treatment-resistant cells vs. sensitive treatment-naïve cells. We documented concordance of results between the 2 THZ-01 resistance screens; and pronounced overlap, but also distinct differences between the genes associated with resistance or sensitization to THZ vs YKL (consistent with their common effect on CKD7, but the lack of CDK12/13 inhibition by YKL) or between THZ-01 vs. MFH; while more limited overlap was observed between MFH and YKL, consistent with their different specificities. The collection of genes associated with resistance to CDK7 or CDK7/12/13 inhibition contain several recognizable, but previously understudied, members or regulators of the CDK family; as well as a diverse group of other genes with known or putative roles in regulation of cell cycle, cell survival, autophagy, proteostasis and intracellular trafficking. Importantly, both the overlapping and distinct “resistome” signatures identified by CRISPR/Cas9 screens for these compounds were different from “resistome” signatures which we have obtained from similar genomewide CRISPR studies for resistance against other classes of investigational or established therapeutics for MM. Our study exemplifies the feasibility of applying CRISPR/Cas9 gene-editing based treatment resistance screens in order to not only define the molecular networks regulating the sensitivity vs. resistance of tumor cells to the respective treatments, but also to provide insights into known or previously underappreciated aspects of the specificity of these compounds against diverse structurally related targets. We envision that similar applications of this approach can be applied to prospectively identify which molecules mediate the antitumor activity of pharmacological agents for which their targets are not currently defined. DisclosuresMitsiades:Novartis: Research Funding; Abbvie: Research Funding; Ono: Research Funding; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Takeda: Other: Employment of family member.

Targeting CDK9 for Anti-Cancer Therapeutics.

Simple SummaryCDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9’s upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9’s activity holds the potential to be a highly effective anti-cancer therapeutic.Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90.

CDK8 maintains stemness and tumorigenicity of glioma stem cells by regulating the c-MYC pathway.

Glioblastoma (GBM) is the most malignant form of glioma. Glioma stem cells (GSCs) contribute to the initiation, progression, and recurrence of GBM as a result of their self-renewal potential and tumorigenicity. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in gliomagenesis remain largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in GSCs. The genetic inhibition of CDK8 by shRNA or CRISPR interference resulted in an abrogation of the self-renewal potential and tumorigenicity of patient-derived GSCs, which could be significantly rescued by the ectopic expression of c-MYC, a stem cell transcription factor. Moreover, we demonstrated that the pharmacological inhibition of CDK8 significantly attenuated the self-renewal potential and tumorigenicity of GSCs. CDK8 expression was significantly higher in human GBM tissues than in normal brain tissues, and its expression was positively correlated with stem cell markers including c-MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. Collectively, these findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs; these findings also identify the CDK8-c-MYC axis as a potential target for GSC-directed therapy.