CDK2 In Complex Research Articles

Artepillin C Inhibits Cell Proliferation by Cell Cycle Arrest at G0/G1 Phase Accompanied by Up-Regulation of p27Kip1 in Human Hepatoma HepG2 Cells

Artepillin C, 3, 5-diprenyl-4-hydroxycinnamic acid, is one of the bioactive constituents in Brazilian propolis. In the present study, the anticarcinogenic activity of this compound was investigated in human hepatoma HepG2 cells. Artepillin C inhibited the cell proliferation in a dose- and time-dependent manner accompanied by G0/G1 phase arrest in the cell cycle. This compound caused a decrease in the phosphorylation levels of the retinoblastoma protein at Ser780 and Ser807/811 and a decrease in the kinase activity of the cyclinD and CDK4 complex without any change in these protein levels. Artepillin C increased the protein level of p27Kip1, known as a CDK inhibitor. This up-regulation was regulated by both the transcriptional and post-transcriptional levels, i.e., the treatment increased the mRNA of p27Kip1 and decreased the proteosome activity. Thus, artepillin C induces cell cycle arrest at G0/G1 phase accompanied by up-regulation of p27Kip1, resulting in the inhibition of cell proliferation in HepG2 cells. This study suggested that artepillin C will be a promising anti-cancer agent against hepatoma cancer.

Inhibitory Effects of Periplocin on Lymphoma Cells: A Network Pharmacology Approach and Experimental Validation.

PurposeLymphoma is considered to be one of the most pressing health problems worldwide owing to its high incidence and mortality. Previous studies have shown that periplocin, a naturally occurring compound, inhibits growth and induces apoptosis in several cancers. However, the effects of periplocin on lymphoma and the underlying mechanisms of action remain unclear.MethodsThe PharmMapper database was used to predict the potential targets of periplocin. The GeneCard database was used to identify lymphoma-related genes. A few intersecting genes were obtained, and the protein-protein interaction network was visualized using STRING Gene ontology analysis. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using R project. MTS assay, flow cytometry, real-time quantitative polymerase chain reaction (qPCR), and Western blotting were used to verify whether periplocin possesses anti-lymphoma activity.ResultsA total of 216 intersecting genes were identified. Numerous cancer-related signaling pathways were visualized using Cytoscape software, with the PI3K-Akt signaling pathway being the highest-ranked pathway related to cell proliferation, apoptosis, and cell cycle progression. HuT 78 and Jurkat cell lines were used to verify the predictions. Periplocin significantly inhibited their proliferation in a dose- and time-dependent manner, but had no effect on the viability of peripheral blood lymphocytes. Flow cytometry revealed that treatment with periplocin increased the apoptotic rate and ratio of HuT 78 and Jurkat cells in the G2/M phase. CDK1 and cyclin B1 complex formation is a key gatekeeper to mitotic division in the G2/M phase. Western blot analysis revealed that periplocin significantly decreased the protein levels of CDK1 and cyclin B1; however, real-time qPCR revealed no effect on gene expression.ConclusionPeriplocin showed anti-tumor effects in lymphoma cells through multiple targets and signaling pathways, and could be a novel therapeutic agent for the treatment of lymphoma.

Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders.

CDK4/6 inhibitors (CDK4/6i) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function, and are exquisitely sensitive to CDK4/6i. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6i and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90/CDC37 complex. In contrast, CDK4/6i and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6i and CDK4/6i-derived degraders and the need for novel inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.

Development of isatin-thiazolo[3,2-a]benzimidazole hybrids as novel CDK2 inhibitors with potent in vitro apoptotic anti-proliferative activity: Synthesis, biological and molecular dynamics investigations

In the current medical era, human health is experiencing numerous challenges, particularly the human malignancies. Therefore, the therapeutic arsenal for these malignancies is to be inexorably enhanced with new treatments that target tumor cells in a selective manner. In this regard, the present work aims at developing a new set of small molecules featuring the privileged isatin scaffold conjugated with a thiazolo[3,2-a]benzimidazole (TBI) motif through a cleavable hydrazide linker (7a-e and 10a-i) as potential anticancer CDK2 inhibitors. The large tricyclic TBI motif is anticipated to achieve a plethora of hydrophobic interactions within the CDK2 binding site. The growth of the two examined cell lines was significantly inhibited by most the prepared hybrids with IC50 ranges; (2.60 ± 1.47–20.90 ± 1.17 µM, against MDA-MB-231) and (1.27 ± 0.06–16.83 ± 0.95 µM, against MCF-7). In particular, hybrids 7a, 7d and 10a displayed potent dual activity against the examined cell lines, and thus selected for further investigations. They exerted a significance alteration in the cell cycle progression, in addition to an apoptosis induction within both MDA-MB-231 and MCF-7 cells. Furthermore, 7a, 7d and 10a displayed potent CDK2 inhibitory action (IC50 = 96.46 ± 5.3, 26.24 ± 1.4 and 42.95 ± 2.3 nM, respectively). The docking simulations unveiled, as expected, the ability of the TBI ring to well-accommodate and establish several hydrophobic interactions within a hydrophobic pocket in the CDK2 binding site. Also, the docking simulations highlighted the significance of incorporation of the hydrazide linker and isatin unsubstituted (NH) functionality in the H-bonding interactions. Interestingly, the most potent CDK2 inhibitor 7d achieved the best binding score (-11.2 Kcal/mole) and formed the most stable complex with CDK2 enzyme (RMSD = 1.24 Å) in a 100 ns MD simulation. In addition, the MM-PBSA calculations ascribed the lowest binding free energy to the 7d–CDK2 complex (−323.69 ± 15.17 kJ/mol). This could be attributed to an incorporation of the 5-OCH3 group that was engaged in an extra hydrogen bonding with key THR14 amino acid residue. Finally, these results suggested hybrid 7d as a good candidate for further optimization as promising breast cancer antitumor agent and CDK2 inhibitor.

Tetrahydroindazole inhibitors of CDK2/cyclin complexes

Over 50 tetrahydroindazoles were synthesized after 7-bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-5,6,7,7a-tetrahydro-1H-indazol-4(3aH)-one (3) was identified as a hit compound in a high throughput screen for inhibition of CDK2 in complex with cyclin A. The activity of the most promising analogues was evaluated by inhibition of CDK2 enzyme complexes with various cyclins. Analogues 53 and 59 showed 3-fold better binding affinity for CDK2 and 2- to 10-fold improved inhibitory activity against CDK2/cyclin A1, E, and O compared to screening hit 3. The data from the enzyme and binding assays indicate that the binding of the analogues to a CDK2/cyclin complex is favored over binding to free CDK2. Computational analysis was used to predict a potential binding site at the CDK2/cyclin E1 interface.

IP7-SPX Domain Interaction Controls Fungal Virulence by Stabilizing Phosphate Signaling Machinery.

In the human-pathogenic fungus Cryptococcus neoformans, the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP7 (isomer 5-PP-IP5) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP7 synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81. Pho81 is the cyclin-dependent kinase (CDK) inhibitor of the phosphate signaling (PHO) pathway. We also provide novel mechanistic insight into the role of IP7 in PHO pathway regulation by demonstrating that IP7 functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP7-Pho81 interaction using site-directed mutagenesis led to a dramatic loss of fungal virulence in a mouse infection model, and the effect was similar to that observed following PHO81 gene deletion, highlighting the key importance of Pho81 in fungal virulence. Furthermore, our findings provide additional evidence of evolutionary divergence in PHO pathway regulation in fungi by demonstrating that IP7 isomers have evolved different roles in PHO pathway control in C. neoformans and nonpathogenic yeast.IMPORTANCE Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deaths occurring annually, 180,000 of which are attributable to the AIDS-related pathogen, Cryptococcus neoformans Here, we demonstrate that interaction of the inositol pyrophosphate, IP7, with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP7-Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP7-Pho81 interaction or preventing Pho81 production leads to a dramatic loss in fungal virulence, coupled with Pho81 having no homologue in humans, highlights Pho81 function as a potential target for the development of urgently needed antifungal drugs.

97LBA Late Breaking - Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders

CDK4 and CDK6 kinases are master regulators of cell cycle progression, and they have thus been attractive targets for cancer therapy. CDK4/6 inhibitors (CDK4/6i) showed significant clinical effectiveness in ER+ breast cancers, and three CDK4/6i are now FDA-approved for this indication. However, in several tumor types, CDK4/6i have only been modestly effective, but the underlying mechanism(s) accounting for the discrepancy have remained elusive.

Obesity/Type 2 Diabetes-Associated Liver Tumors Are Sensitive to Cyclin D1 Deficiency.

Type 2 diabetes, which is mainly linked to obesity, is associated with increased incidence of liver cancer. We have previously found that in various models of obesity/diabetes, hyperinsulinemia maintains heightened hepatic expression of cyclin D1, suggesting a plausible mechanism linking diabetes and liver cancer progression. Here we show that cyclin D1 is greatly elevated in human livers with diabetes and is among the most significantly upregulated genes in obese/diabetic liver tumors. Liver-specific cyclin D1 deficiency protected obese/diabetic mice against hepatic tumorigenesis, whereas lean/nondiabetic mice developed tumors irrespective of cyclin D1 status. Cyclin D1 dependency positively correlated with liver cancer sensitivity to palbociclib, an FDA-approved CDK4 inhibitor, which was effective in treating orthotopic liver tumors under obese/diabetic conditions. The antidiabetic drug metformin suppressed insulin-induced hepatic cyclin D1 expression and protected against obese/diabetic hepatocarcinogenesis. These results indicate that the cyclin D1-CDK4 complex represents a potential selective therapeutic vulnerability for liver tumors in obese/diabetic patients. SIGNIFICANCE: Obesity/diabetes-associated liver tumors are specifically vulnerable to cyclin D1 deficiency and CDK4 inhibition, suggesting that the obese/diabetic environment confers cancer-selective dependencies that can be therapeutically exploited.

Identification of novel CDK 9 inhibitors based on virtual screening, molecular dynamics simulation, and biological evaluation

AimsCyclin-dependent kinase 9 (CDK9) is a member of the CDK subfamily and plays a major role in the regulation of transcriptional elongation. It has attracted widespread attention as a therapeutic target for cancer. Here, we aimed to explore novel CDK 9 inhibitors by using a hybrid virtual screening strategy. Main methodsA hybrid virtual screening strategy was constructed with computer-aided drug design (CADD). First, compounds were filtered in accordance with Lipinski's rule of five and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Second, a 3D-QSAR pharmacophore model was built and used as a 3D query to screen the obtained hit compounds. Third, the hit compounds were subjected to molecular docking studies. Fourth, molecular dynamics (MD) simulations were performed on CDK9 in complex with the final hits to examine the structural stability. Finally, CDK9 kinase biochemical assay was performed to identify the biological activity of the hit compounds. Key findingsSeven hit compounds were screened out. These hit compounds showed drug-like properties in accordance with Lipinski's rule of five and ADMET. Complexes involving the six hit compounds bound to CDK9 exhibited good structural stability in the MD simulation. Furthermore, these six hit compounds had strong inhibitory activity against CDK9 kinase. In particular, hit 3 showed the most promising activity with the percentage of 71%. SignificanceThe six hit compounds may be promising novel CDK9 inhibitors, and the hybrid virtual screening strategy designed in this study provides an important reference for the design and synthesis of novel CDK9 inhibitors.

Electron density analysis of CDK complexes using the AlteQ method.

Background: A principle of complementarity is a well-established concept in chemistry and biology. This concept is based on the overlap of electron clouds of the molecules in question. Materials & methods: In this article, one such approach (an in-house developed quantum free-orbital AlteQ method) was used to evaluate the complementarity of 51 CDK-ligand complexes. Results: A significant universally applicable correlation (adjusted R2=0.9749;p < 2.2×10-16) relating the product of ligand and enzyme electron densities to the product of distances between the contacting atomic centers and the type of atoms involved in the interaction was found. Conclusion: The terms calculated in this article can provide a good basis for prognosis of bioactivity and scientificallybased molecular docking.

L ARP7 Is a BRCA1 Ubiquitinase Substrate and Regulates Genome Stability and Tumorigenesis

Attenuated DNA repair leads to genomic instability and tumorigenesis. BRCA1/BARD1 are the best-known tumor suppressors that promote homology recombination (HR) and arrest cell cycle. However, it remains ambiguous whether and how their E3 ligase activity regulates HR. Here, we demonstrate that upon genotoxic stress, BRCA1 together with BARD1 catalyzes the K48 polyubiquitination on LARP7, a 7SK RNA binding protein known to control RNAPII pausing, and thereby degrades it through the 26S ubiquitin-proteasome pathway. Depleting LARP7 suppresses the expression of CDK1 complex, arrests the cell at the G2/M DNA damage checkpoint, and reduces BRCA2 phosphorylation, which thereby facilitates RAD51 recruitment to damaged DNA to enhance HR. Importantly, LARP7 depletion observed in breast cancer patients leads to chemoradiotherapy resistance both invitro and invivo. Altogether, this study unveils a mechanism by which BRCA1/BARD1 control HR and cell cycle, and highlights LARP7 as a potential target for cancer prevention and therapy.

Identifying the Novel Pyrimidine-Based CDK2 Inhibitors as Anticancer Agents Using Text-Mining and Combined Molecular Modeling Approaches

The cycline-dependent kinase (CDK) protein is an important target used in anti-cancer drug designing studies. Pyrimidine is a crucial fragment which is used in many FDA-approved drugs including anti-cancer drugs. In the current study, a small molecule database (Specs SC) that includes more than 210.000 compounds were used in text mining studies and 6668 molecules that carry “pyrimidine” fragments were filtered. These compounds were then screened at the binding pocket of CDK-2 target using molecular docking and molecular dynamics (MD) simulations approaches. Binding free energies of screened compounds were also compared with pyrimidine-based FDA approved anti-cancer drug Abemaciclib which targets CDK. Based on comparison of docking scores of screened compounds, top-7 hits were used in 100 ns MD simulations. Same MD simulations protocol (100 ns) were also applied to Abemaciclib-bound CDK-2 complex structure. Average Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) energies were then calculated. Our MM/GBSA results showed that 6 out of 7 compounds have better MM/GBSA scores than FDA approved compound Abemaciclib. Thus, together with combination of text mining and integrated molecular modeling approaches, we identified novel pyrimidine-based hits against CDK.

Requirement of PP2A-B56Par1 for the Stabilization of the CDK Inhibitor Rum1 and Activation of APC/CSte9 during Pre-Start G1 in S. pombe.

SummaryExit from the cell cycle during the establishment of quiescence and upon cell differentiation requires the sustained inactivation of CDK complexes. Fission yeast cells deprived of nitrogen halt cell cycle progression in pre-Start G1, before becoming quiescent or undergoing sexual differentiation. The CDK inhibitor Rum1 and the APC/C activator Ste9 are fundamental for this arrest, but both are down-regulated by CDK complexes. Here, we show that PP2A-B56Par1 is instrumental for Rum1 stabilization and Ste9 activation. In the absence of PP2A-B56Par1, cells fail to accumulate Rum1, and this results in persistent CDK activity, Ste9 inactivation, retention of the mitotic cyclin Cdc13, and impaired withdrawal from the cell cycle during nitrogen starvation. Importantly, mutation of a putative B56 interacting motif in Rum1 recapitulates these defects. These results underscore the relevance of CDK-counteracting phosphatases in cell differentiation, establishment of the quiescent state, and escape from it in cancer cells.

Ethyl Acetate Fraction of Papua’s Ant Nest Plant (Myrmecodia Pendens) Induces Cell Cycle Arrest and Apoptosis of an Oral Malignant Burkitt Lymphoma Cell through Down Regulation of Cyclin E–CDK2 Complex

Efforts to inhibit the growth of oral Burkitt’s lymphoma cells require an effective strategy, one of that uses Papua’s ant nest plant (Myrmecodia pendens) which are natural medicinal plants. In the present study, induction of cell cycle arrest and apoptosis of an oral Burkitt’s lymphoma cell (Raji cell)-mediated by an ethyl acetate fraction of Papua’s ant nest plant through down regulation of cyclin E-CDK2 complex was examined. For detection of cell cycle arrest was evaluated by flow-cytometer. Induction of apoptosis was detected by colorimetric assay caspase-3, -8 and -9. The level of cyclin E and CDK2 was carried out by Elisa kit. The results revealed the active fraction of the ant nest was markedly induced the cell cycle arrest in the G0-G1 phase according to increased concentration followed bythe induction of apoptosis characterized by elevated of proteolytic activity of caspase-3, -8 and -9. Cyclin E and CDK2 level was significantly decreased in cell treated with ant nest fraction. These results suggest the ethyl acetate fraction of the ant nest plant had a strong potential antitumor activity of oral malignant Burkitt’s lymphoma cell proofed by the induction of cell cycle arrest and apoptosis through down regulation of cyclin E-CDK2 complex.

Theoretical Studies on the Selectivity Mechanisms of Glycogen Synthase Kinase 3β (GSK3β) with Pyrazine ATP-competitive Inhibitors by 3DQSAR, Molecular Docking, Molecular Dynamics Simulation and Free Energy Calculations.

Background Glycogen synthase kinase-3 (GSK3) is associated with various key biological processes and has been considered as an important therapeutic target for the treatment of many diseases. Great efforts have been made on the development of GSK3 inhibitors, especially ATP-competitive GSK3β inhibitor, but it is still a great challenge to develop selective GSK3β inhibitors because of the high sequence homology with other kinases.Objective In order to reveal the selectivity mechanisms of GSK3β inhibition at the molecular level, a series of ATP-competitive GSK3β inhibitor was analyzed by a systematic computational method, combining 3D-QSAR, molecular docking, molecular dynamic simulations and free energy calculations.Methods Firstly, 3D-QSAR with CoMFA was built to explore the general structure activity relationships. Secondly, CDOCKER and Flexible docking were employed to predicted the reasonable docking poses of all studied inhibitors. And then, both GSK3β and CDK2 complexes were selected to conduct molecular dynamics simulations. Finally, the free energy calculations were employed to find the key selective-residues.Results CoMFA model suggested the steric, hydrophobic fields play key roles in the bioactivities of inhibitors, and the binding mechanisms were well analyzed through molecular docking. The binding free energies predicted are in good agreement with the experimental bioactivities and the free energy calculations showed that the binding of GSK3β/inhibitors was mainly contributed from hydrogen bonding and hydrophobic interaction.Conclusion Some key residues for selective binding were highlighted, which may afford important guidance for the rational design of novel ATP-competitive GSK3β inhibitors.

Antitumor activity of cyclin-dependent kinase inhibitor alsterpaullone in Epstein-Barr virus-associated lymphoproliferative disorders.

Epstein‐Barr virus (EBV) is a well‐established tumor virus that has been implicated in a wide range of immunodeficiency‐associated lymphoproliferative disorders (LPDs). Although rituximab, a CD20 mAb, has proven effective against EBV‐associated LPDs, prolonged use of this drug could lead to resistance due to the selective expansion of CD20− cells. We have previously shown that cyclin‐dependent kinase (CDK) inhibitors are able to specifically suppress the expression of viral late genes, particularly those encoding structural proteins; however, the therapeutic effect of CDK inhibitors against EBV‐associated LPDs is not clear. In this study, we examined whether CDK inhibitors confer a therapeutic effect against LPDs in vivo. Treatment with alsterpaullone, an inhibitor of the CDK2 complex, resulted in a survival benefit and suppressed tumor invasion in a mouse model of LPDs. Inhibition of CDK efficiently induced G1 cell cycle arrest and apoptosis in EBV‐positive B cells. These results suggest that alsterpaullone suppresses cell cycle progression, resulting in the antitumor effect observed in vivo.

SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition.

The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

Cyclin-CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa.

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional maturation process to acquire the competence to fertilize the female egg. During this process, called capacitation, the actin cytoskeleton dramatically changes its organization. First, actin fibers polymerize, forming a network over the anterior part of the sperm cells head, and then it rapidly depolymerizes and disappears during the exocytosis of the acrosome content (the acrosome reaction (AR)). Here, we developed a computational model representing the actin dynamics (AD) process on mature spermatozoa. In particular, we represented all the molecular events known to be involved in AD as a network of nodes linked by edges (the interactions). After the network enrichment, using an online resource (STRING), we carried out the statistical analysis on its topology, identifying the controllers of the system and validating them in an experiment of targeted versus random attack to the network. Interestingly, among them, we found that cyclin-dependent kinase (cyclin–CDK) complexes are acting as stronger controllers. This finding is of great interest since it suggests the key role that cyclin–CDK complexes could play in controlling AD during sperm capacitation, leading us to propose a new and interesting non-genomic role for these molecules.

Abstract 4413: CDK6 overexpression promotes resistance to CDK4/6 inhibitors in breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) are highly active therapies in estrogen receptor-positive (ER+) breast cancer. Unfortunately, tumor relapse on these agents is frequently encountered in clinical practice with limited data on the biologic mechanisms that can mediate resistance. We utilized cell line models, patient derived xenografts and patient samples to interrogate mechanisms of resistance to CDK4/6 inhibitors. A survey of genomic alterations associated with limited benefit of CDK4/6iidentified several alterations involving the Hippo pathway which could potentially impact CDK6 kinase expression. Immunohistochemistry from these patients revealed increased YAP nuclear translocation and high levels of CDK6 protein specifically in the group of patients with short progression-free survival (PFS) on CDK4/6i. A PDX model of acquired resistance to CDK4/6i also showed that prolonged treatment with CDK4/6i led to a subset of tumors that developed resistance, all of which showed Hippo pathway suppression and CDK6 upregulation. Finally, cell lines models exposed to CDK4/6i showed multiple clones that featured upregulation of CDK6 through suppression of the Hippo pathway or amplification of the CDK6 gene. Identifying CDK6 overexpression as a recurrent feature of CDK4/6i resistant tumors, we next investigated the function(s) of CDK6 in our models. We performed knockdown of CDK6 by shRNA and found its overexpression to be necessary for drug resistance in these models while reinforced CDK6 could restore resistance or confer resistance when overexpressed in naïve cells. Given the close homology of CDK4 and CDK6, we asked whether any differences might exist between CDK4 and CDK6 complexes, performing immunoprecipitation and mass spectrometry (IP-MS) and found CDK6 to bind a distinct partition of proteins from CDK4 including INK4 proteins. In vitro kinase assays revealed that INK4 proteins impaired the ability of CDK4/6i to block CDK6 activity. INK4 proteins were found to cooperate with CDK6 in mediating drug resistance as their knockdown restored drug sensitivity to resistant cells, while INK4 overexpression promoted resistance in CDK6-high cells. Taken together, the data revealed that high levels of a CDK6-INK4 complex to be a recurrent mechanism of resistance to CDK4/6i and suggest novel CDK6 inhibitors as a strategy to overcome resistance. Citation Format: Qing Li, Pedram Razavi, Zhiqiang Li, Arnaud F. Da Cruz Paula, Edi Brogi, Maurizio Scaltriti, Jorge S. Reis-Filho, Sarat Chandarlapaty. CDK6 overexpression promotes resistance to CDK4/6 inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4413.

Triptolide enhances TRAIL sensitivity of pancreatic cancer cells by activating autophagy via downregulation of PUM1

BackgroundTriptolide (TPL) can enhance the sensitivity of pancreatic cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but available research is limited to whether TPL can affect the relevant downstream signaling pathways of TRAIL. Current knowledge is far from adequate to fully understand the mechanisms by which TPL increases TRAIL sensitivity of pancreatic cancer. PurposeWe aimed to find TPL-regulated upstream components of the signaling pathways of TRAIL to further understand the regulatory mechanism by which TPL increases the sensitivity to TRAIL. MethodsMicroarray analysis and the adherent cell cytometry system Celigo were used to identify the TRAIL-related genes. Western blot analysis, cell proliferation assays, tumorigenicity assays in nude mice, flow cytometry, and transmission electron microscopy were performed to analyze the function of Pumilio RNA-binding family member 1 (PUM1) in TPL-mediated enhancement of sensitivity to TRAIL. The effect of PUM1 silencing on the p27–CDK2 complex was examined by immunoprecipitation. ResultsPUM1 expression was decreased by TPL and TPL + TRAIL but was not decreased by TRAIL alone. PUM1 silencing enhanced low-concentration-TRAIL-induced suppression of proliferation and promotion of apoptosis and increased p27 expression and the amount of the p27–CDK2 complex in pancreatic cancer cells. PUM1 overexpression attenuated the effects of TPL treatment (TRAIL-induced cell proliferation suppression and apoptosis promotion), while PUM1 silencing and TPL enhanced low-concentration-TRAIL-induced autophagy activation in pancreatic cancer cells. Moreover, PUM1 overexpression attenuated the effect of TPL treatment on TRAIL-induced autophagy activation in pancreatic cancer cells. ConclusionPUM1 silencing increased the sensitivity of pancreatic cancer cells to TRAIL in vivo and in vitro, indicating that PUM1 may be a new target for increasing the sensitivity of cancer cells to TRAIL. In addition, our results indicate that TPL enhances TRAIL sensitivity of pancreatic cancer cells by activating autophagy via downregulation of PUM1. This novel concept may have significant implications for the development of new strategies to enhance TRAIL sensitivity of tumors.