Pathogenesis of human B cell lymphomas.

The mechanisms that drive normal B cell differentiation and activation are frequently subverted by B cell lymphomas for their unlimited growth and survival. B cells are particularly prone to malignant transformation because the machinery used for antibody diversification can cause chromosomal translocations and oncogenic mutations. The advent of functional and structural genomics has greatly accelerated our understanding of oncogenic mechanisms in lymphomagenesis. The signaling pathways that normal B cells utilize to sense antigens are frequently derailed in B cell malignancies, leading to constitutive activation of prosurvival pathways. These malignancies co-opt transcriptional regulatory systems that characterize their normal B cell counterparts and frequently alter epigenetic regulators of chromatin structure and gene expression. These mechanistic insights are ushering in an era of targeted therapies for these cancers based on the principles of pathogenesis.

Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is.

Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.

RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6.

The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.

Safeguarding intestine cells against enteropathogenic Escherichia coli by intracellular protein reaction, a preventive antibacterial mechanism.

A critical problem in the fight against bacterial infection is the rising rates of resistance and the lack of new antibiotics. The discovery of new targets or new antibacterial mechanisms is a potential solution but is becoming more difficult. Here we report an antibacterial mechanism that safeguards intestine cells from enteropathogenic Escherichia coli (EPEC) by shutting down an infection-responsive signal of the host intestine cell. A key step in EPEC infection of intestinal cells involves Tir-induced actin reorganization. Nck mediates this event by binding with Tir through its SH2 domain (Nck-SH2) and with WIP through its second SH3 domain (Nck-SH3.2). Here we report the design of a synthetic peptide that reacts precisely with a unique cysteine of the Nck-SH3.2 domain, blocks the binding site of the Nck protein, and prevents EPEC infection of Caco-2 cells. Oral update of this nontoxic peptide before EPEC administration safeguards mice from EPEC infection and diarrhea. This study demonstrates domain-specific blockage of an SH3 domain of a multidomain adaptor protein inside cells and the inhibition of Tir-induced rearrangement of the host actin cytoskeleton as a previously unknown antibacterial mechanism.

Utility of the Ba/F3 cell system for exploring on-target mechanisms of resistance to targeted therapies for lung cancer.

Molecular targeted therapies are the standard of care for front-line treatment of metastatic non-small-cell lung cancers (NSCLCs) harboring driver gene mutations. However, despite the initial dramatic responses, the emergence of acquired resistance is inevitable. Acquisition of secondary mutations in the target gene (on-target resistance) is one of the major mechanisms of resistance. The mouse pro-B cell line Ba/F3 is dependent on interleukin-3 for survival and proliferation. Upon transduction of a driver gene, Ba/F3 cells become independent of interleukin-3 but dependent on the transduced driver gene. Therefore, the Ba/F3 cell line has been a popular system to generate models with oncogene dependence and vulnerability to specific targeted therapies. These models have been used to estimate oncogenicity of driver mutations or efficacies of molecularly targeted drugs. In addition, Ba/F3 models, together with N-ethyl-N-nitrosourea mutagenesis, have been used to derive acquired resistant cells to investigate on-target resistance mechanisms. Here, we reviewed studies that used Ba/F3 models with EGFR mutations, ALK/ROS1/NTRK/RET fusions, MET exon 14 skipping mutations, or KRAS G12C mutations to investigate secondary/tertiary drug resistant mutations. We determined that 68% of resistance mutations reproducibly detected in clinical cases were also found in Ba/F3 models. In addition, sensitivity data generated with Ba/F3 models correlated well with clinical responses to each drug. Ba/F3 models are useful to comprehensively identify potential mutations that induce resistance to molecularly targeted drugs and to explore drugs to overcome the resistance.

Heterotrimeric Gq proteins as therapeutic targets?

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein-coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family-specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

TTK inhibitor promotes radiosensitivity of liver cancer cells through p21.

The monopolar spindle 1 ((hMps1/TTK) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To explore the possible relationship between TTK inhibition and radiosensitivity, we examined whether TTK inhibition influences cellular susceptibility of radiation. And we further revealed its mechanisms. We found that the expression of TTK was obviously higher in liver cancer tissues compared to the normal liver tissues. Kaplan-Meier Plotter demonstrated that patients with low TTK expression levels had a longer overall survival than patients with high TTK expression levels. TTK inhibitor AZ3146 could simulated liver cancer cells to accumulate in the G2/M phase, which ultimately enhances DNA damage with more γ-H2AX foci and more apoptosis and necrosis induced by radiation, which prompted that TTK inhibition sensitized liver cancer cells to radiation. In addition, TTK inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced mitotic catastrophe (MC) induced by radiation in a p21-mediated manner. In this study, we present evidences that the TTK inhibitor promotes the radiosensitivity of liver cancer cells through regulating cell cycle in p21-mediated manner in vitro, indicating that TTK inhibitor may be an attractive radiosensitizer for the patients with liver cancer.

A study on MAPK/ERK and CDK2-Cyclin-E signal switch "on and off" in cell proliferation by bis urea derivatives of 1, 4-Diisocyanatobenzene.

A series of novel substituted bisurea 1,4-Diisocyanatobenzene compounds were designed, synthesized and introduced as potent anticancer compounds and screened for their in vitro anti-proliferative activities in human cancer cell lines. The structures of all titled compounds were characterized using Fourier-transform infrared mass spectra, nuclear magnetic resonance spectroscopy, elemental analysis and evaluated their sustainability using biological experiments. A selected group of ten derivatives were apprised for their anti-proliferative activity. The compounds 3d and 3e displayed potent anticancer activity with low IC50 value of 5.40, and 5.89 µM against HeLa cancer cell lines. The observed apoptosis data has demonstrated that compounds 3d and 3e induce the activaties of caspase-9 and caspase-3, the compounds 3d and 3e regulated fungal zone inhibition. Due to promising growth inhibitions, the all synthesized compounds were allowed to campaign includes quantum-polarized-ligand, quantum mechanical and molecular mechanical, docking experiments. The compounds 3d and 3e have exhibited a higher affinity for ERK/MAP kinase and CDK2 proteins. The molecular docking interactions have demonstrated two stage inhibition of cancer cells by binding with ERK/MAP kinase and CDK2 leads to inactivation of cell proliferation,cell cycle progression,cell divisionanddifferentiation, and hypo-phosphorylation of ribosome leading cells to restricts at point boundary of the G1/S phase. The long-range molecular dynamics, 150 ns, simulations were also revealed more consistency by 3d. Our study conclude good binding propensity for active-tunnel of ERK/MAP kinase and CDK2 proteins, by 3d (1,1'-(1,4-phenylene) bis(3-(2-chlorobenzyl)urea)), to suggest that the designed and synthesized 3d is to use as selective novel nuclei in anti-cancer chemotherapeutics.

CCNE1 Is a Putative Therapeutic Target for ARID1A-Mutated Ovarian Clear Cell Carcinoma.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is resistant to platinum chemotherapy and is characterized by poor prognosis. Today, the use of poly (ADP-ribose) polymerase (PARP) inhibitor, which is based on synthetic lethality strategy and characterized by cancer selectivity, is widely used for new types of molecular-targeted treatment of relapsed platinum-sensitive ovarian cancer. However, it is less effective against OCCC. METHODS: We conducted siRNA screening to identify synthetic lethal candidates for the ARID1A mutation; as a result, we identified Cyclin-E1 (CCNE1) as a potential target that affects cell viability. To further clarify the effects of CCNE1, human OCCC cell lines, namely TOV-21G and KOC7c (ARID1A mutant lines), and RMG-I and ES2 (ARID1A wild type lines) were transfected with siRNA targeting CCNE1 or a control vector. RESULTS: Loss of CCNE1 reduced proliferation of the TOV-21G and KOC7c cells but not of the RMG-I and ES2 cells. Furthermore, in vivo interference of CCNE1 effectively inhibited tumor cell proliferation in a xenograft mouse model. CONCLUSION: This study showed for the first time that CCNE1 is a synthetic lethal target gene to ARID1A-mutated OCCC. Targeting this gene may represent a putative, novel, anticancer strategy in OCCC treatment.

Protein kinase C: perfectly balanced.

Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For conventional and novel isozymes, this is effected by binding the lipid second messenger, diacylglycerol, but for atypical PKC isozymes, this is effected by binding protein scaffolds. PKC shot into the limelight following the discovery in the 1980s that the diacylglycerol-sensitive isozymes are "receptors" for the potent tumor-promoting phorbol esters. This set in place a concept that PKC isozymes are oncoproteins. Yet three decades of cancer clinical trials targeting PKC with inhibitors failed and, in some cases, worsened patient outcome. Emerging evidence from cancer-associated mutations and protein expression levels provide a reason: PKC isozymes generally function as tumor suppressors and their activity should be restored, not inhibited, in cancer therapies. And whereas not enough activity is associated with cancer, variants with enhanced activity are associated with degenerative diseases such as Alzheimer's disease. This review describes the tightly controlled mechanisms that ensure PKC activity is perfectly balanced and what happens when these controls are deregulated. PKC isozymes serve as a paradigm for the wisdom of Confucius: "to go beyond is as wrong as to fall short."

Concepts and advances in cancer therapeutic vulnerabilities in RAS membrane targeting.

For decades oncogenic RAS proteins were considered undruggable due to a lack of accessible binding pockets on the protein surfaces. Seminal early research in RAS biology uncovered the basic paradigm of post-translational isoprenylation of RAS polypeptides, typically with covalent attachment of a farnesyl group, leading to isoprenyl-mediated RAS anchorage at the plasma membrane and signal initiation at those sites. However, the failure of farnesyltransferase inhibitors to translate to the clinic stymied anti-RAS therapy development. Over the past ten years, a more complete picture has emerged of RAS protein maturation, intracellular trafficking, and location, positioning and retention in subdomains at the plasma membrane, with a corresponding expansion in our understanding of how these properties of RAS contribute to signal outputs. Each of these aspects of RAS regulation presents a potential vulnerability in RAS function that may be exploited for therapeutic targeting, and inhibitors have been identified or developed that interfere with RAS for nearly all of them. This review will summarize current understanding of RAS membrane targeting with a focus on highlighting development and outcomes of inhibitors at each step.

DEK-targeting aptamer DTA-64 attenuates bronchial EMT-mediated airway remodelling by suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathway in asthma.

This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-ß1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-ß1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-ß1 expression, respectively, on the contrary, TGF-ß1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-ß1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-ß1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients.

Cordycepin Inhibits Cancer Cell Proliferation and Angiogenesis through a DEK Interaction via ERK Signaling in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement. SIGNIFICANCE STATEMENT: Cordycepin provides multiple strategies in antitumors, but its mechanisms are not fully elucidated, especially on cholangiocarcinoma (CCA). We reported that cordycepin inhibited the viability of CCA cells, induced apoptosis via extracellular signal-regulated kinase 1/2 deactivation and DEK inhibition, and reduced the angiogenetic capabilities of CCA both in vivo and in vitro.

Identification of LEM-14 inhibitor of the oncoprotein NSD2.

The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC50 of 132 µM and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC50 of 418 µM (NSD1), IC50 of 111 µM (NSD2) and IC50 of 60 µM (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors.

MicroRNA-107 induces cell cycle arrests by directly targeting cyclin E1 in ovarian cancer.

Deregulated expression of microRNAs plays oncogenic or anti-oncogenic roles in various cancers. However, expression of miR-107 was not consistent among several types of cancer, and the effect of miR-107 in ovarian cancer remains unclear. In this study, we found that expression miR-107 was significantly decreased in ovarian cancer patients and in cell lines. Ectopic expression of miR-107 suppressed cell proliferation and G1 phase to S transition of cell cycle, and was associated with downregulation of cyclin E1 (CCNE1) expression. Mechanistically, CCNE1 was confirmed to be a direct target of miR-107 through the dual-luciferase reporter assay. Knockdown of CCNE1 dramatically impeded cell cycle in G1/S phase transition similarly as miR-107 overexpression did. In addition, overexpression of CCNE1 reversed the inhibition of cell proliferation induced by miR-107 overexpression. Finally, miR-107 had anti-cancer potential by suppressing tumor initiation and progression in vivo. Our finding indicates that miR-107 serves as a tumor suppressor by decreasing CCNE1 expression levels, which may provide potential therapeutic strategies in ovarian cancer treatment.

Inhibition of cyclin E1 overcomes temozolomide resistance in glioblastoma by Mcl-1 degradation.

Glioblastoma (GBM) is one of the major causes of brain cancer-related mortality worldwide. Temozolomide (TMZ) is an important agent against GBM. Acquired TMZ-resistance severely limits the chemotherapeutic effect and leads to poor GBM patient survival. To study the underlying mechanism of drug resistance, two TMZ resistant GBM cell lines, A172 and U87, were generated. In this study, the TMZ resistant cells have less apoptosis and cell-cycle change in response to the TMZ treatment. Western blot results revealed that cyclin E1 was upregulation in TMZ resistant cells. Inhibition or depletion of cyclin E1 re-sensitized the resistant cells to the TMZ treatment, which indicated the induction of cyclin E1 is the cause of TMZ resistance in GBM cells. Furthermore, we also found the expression of cyclin E1 stabilized the expression of Mcl-1, which contributes to the TMZ resistance in GBM cells. Finally, our in vivo xenograft data showed that the combination of flavopiridol, a cyclin E1/CDK2 inhibitor, overcomes the TMZ resistant by inducing higher apoptosis. Overall, our data provided a rationale to overcome the TMZ resistant in GBM treatment by inhibiting the cyclin E1 activity.

Inhibition of cyclin E1 sensitizes hepatocellular carcinoma cells to regorafenib by mcl-1 suppression.

BACKGROUND: To clarify the effects of cylcin E1 expression on HCC tumor progression, we studied the expression of cyclin E1 and inhibitory efficacy of regorafenib and sorafenib in HCC cells, and investigated a potential therapy that combines regorafenib treatment with cyclin E1 inhibition. METHODS: Western blotting for caspase-3 and Hoechst 33225 staining was used to measure the expression level of apoptosis-related proteins under drug treatment. RESULTS: Our results showed that enhanced expression of cyclin E1 after transfection compromised apoptosis in HCC cells induced by regorafenib or sorafenib. Conversely, down-regulation of cyclin E1 gene expression or inhibition of cyclin E1 by the cyclin-dependent kinase (CDK) inhibitors dinaciclib (DIN) or flavopiridol sensitized HCC cells to regorafenib and sorafenib by inducing apoptosis. The expression of Mcl-1, which is modulated by STAT3, plays a key role in regulating the therapeutic effects of CDK inhibitors. Xenograft experiments conducted to test the efficacy of regorafenib combined with DIN showed dramatic tumor inhibitory effects due to induction of apoptosis. Our results suggested that the level of cyclin E1 expression in HCCs may be used as a pharmacodynamic biomarker to assess the antitumor effects of regorafenib or sorafenib. CONCLUSIONS: Combining regorafenib and CDK inhibitors may enhance the clinical efficiency of the treatment of HCCs.