Mcl-1 Expression Research Articles

292 (PB072) - Development of selective MCL-1 heterobifunctional degraders

Background: MCL1 is a member of the Bcl-2 family that plays a key role in cellular homeostasis through the regulation of apoptosis as well as other less characterized functions. The growing recognition of MCL-1 role in cancer cell survival and its association with the development of anticancer drug resistance makes it an attractive target for cancer therapy. Several inhibitors have been developed during the past decade and some of them have entered clinical trials, but no drugs have been approved for clinical use so far. Targeted protein degradation using heterobifunctional degraders has emerged as a novel therapeutic modality in drug discovery. This technology has many potential advantages over traditional inhibitors, e.g. reduced side effects and drug resistance, extended duration of action, elevated selectivity, and the opportunity to target “undruggable” proteins. In this study, we report the development of the first-in-class MCL-1 bifunctional degraders with in vivo PD effect. Material and Methods: Compounds’ affinity to CRBN (Cereblon), an E3 ligase, and to MCL-1 was determined by FP and SPR, respectively. AlphaLISA and HTRF were utilized to confirm ternary complex formation. Cell viability was assessed by CellTiter-Glo Assay. Apoptosis induction and changes in mitochondrial membrane potential were assessed by flow cytometry using Annexin/PI and TMRE staining, respectively. Targeted protein degradation in cells treated with compounds in the absence or presence of apoptosis inhibitor was assessed by WB. The mechanism of action of the compounds was confirmed in cancer cells pre-treated with proteasome or NEDD8-Activating Enzyme inhibitors, or CRBN-targeting siRNA, and then treated with the compounds. MCL-1 abundance and expression of apoptosis markers were confirmed by WB. PK studies were conducted in CD1 mice and Sprague Dawley rats, and PD studies in CB-17 SCID mice xenografted with MV4–11 cells. Results: Synthesized compounds bind CRBN and MCL-1 with high affinity, form a ternary complex in vitro and induce the degradation of MCL-1 in cells, which is caspase- and proteasome-dependent and leads to apoptosis induction. These series of compounds showed a nM range cytotoxic activity in haematological cancer cell lines with the best compounds having an IC50 <100 nM. Moreover, MCL-1 degradation in vivo can be achieved by IP administration leading to the initiation of the programmed cell death. Conclusions: Our compounds have a broad cytotoxic activity toward cultured human cancer cells and a promising in vivo pharmacokinetic profile. Our data indicate that targeting MCL-1 by employing bifunctional degraders represents a potentially new and effective strategy for cancer treatment. No conflict of interest.

Jinhua Qinggan granules attenuates acute lung injury by promotion of neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceAcute lung injury (ALI) is one of the fatal complications of respiratory virus infections such as influenza virus and coronavirus, which has high clinical morbidity and mortality. Jinhua Qinggan granules (JHQG) has been approved by China Food and Drug Administration in the treatment of H1N1 influenza and mild or moderate novel coronavirus disease 2019 (COVID-19), which is an herbal formula developed based on Maxingshigan decoction and Yinqiao powder that have been used to respiratory diseases in China for thousands of years. However, the underlying mechanism of JHQG in treating infectious diseases remains unclear. Aim of the studyThis study investigated the effects of JHQG on neutrophil apoptosis and key signaling pathways in lipopolysaccharide (LPS) -induced ALI mice in order to explore its mechanism of anti-inflammation. Materials and methodsThe effect of JHQG on survival rate was observed in septic mouse model by intraperitoneal injection of LPS (20 mg/kg). To better pharmacological evaluation, the mice received an intratracheal injection of 5 mg/kg LPS. Lung histopathological changes, wet-to-dry ratio of the lungs, and MPO activity in the lungs and total protein concentration, total cells number, TNF-α, IL-1β, IL-6, and MIP-2 levels in BALF were assessed. Neutrophil apoptosis rate was detected by Ly6G-APC/Annexin V-FITC staining. Key proteins associated with apoptosis including caspase 3/7 activity, Bcl-xL and Mcl-1 were measured by flow cytometry and confocal microscope, respectively. TLR4 receptor and its downstream signaling were analyzed by Western blot assay and immunofluorescence, respectively. ResultsJHQG treatment at either 6 or 12 g/kg/day resulted in 20% increase of survival in 20 mg/kg LPS-induced mice. In the model of 5 mg/kg LPS-induced mice, JHQG obviously decreased the total protein concentration in BALF, wet-to-dry ratio of the lungs, and lung histological damage. It also attenuated the MPO activity and the proportion of Ly6G staining positive neutrophils in the lungs, as well as the MIP-2 levels in BALF were reduced. JHQG inhibited the expression of Mcl-1 and Bcl-xL and enhanced caspase-3/7 activity, indicating that JHQG partially acted in promoting neutrophil apoptosis via intrinsic mitochondrial apoptotic pathway. The levels of TNF-α, IL-1β, and IL-6 were significantly declined in LPS-induced mice treated with JHQG. Furthermore, JHQG reduced the protein expression of TLR4, MyD88, p-p65 and the proportion of nuclei p65, suggesting that JHQG treatment inhibited TLR4/MyD88/NF-κB pathway. ConclusionJHQG reduced pulmonary inflammation and protected mice from LPS-induced ALI by promoting neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway, suggesting that JHQG may be a promising drug for treatment of ALI.

SHP-1/STAT3-Signaling-Axis-Regulated Coupling between BECN1 and SLC7A11 Contributes to Sorafenib-Induced Ferroptosis in Hepatocellular Carcinoma.

Ferroptosis is a type of iron-dependent cell death pertaining to an excess of lipid peroxidation. It has been suggested that sorafenib—an anti-angiogenic medication for hepatocellular carcinoma (HCC)—induces ferroptosis, but the underlying mechanism for this remains largely unknown. We employed siRNA-mediated gene silencing to investigate the role of Src homology region 2 domain-containing phosphatase-1 (SHP-1), following sorafenib treatment, in cystine/glutamate-antiporter-system-Xc−-regulated cystine uptake. Co-immunoprecipitation was also performed to examine the interactions between MCL1, beclin 1 (BECN1), and solute carrier family 7 member 11 (SLC7A11), which functions as the catalytic subunit of system Xc−. The results of this study showed that sorafenib enhanced the activity of SHP-1, dephosphorylated STAT3, downregulated the expression of MCL1 and, consequently, reduced the association between MCL1 and BECN1. In contrast, increased binding between BECN1 and SLC7A11 was observed following sorafenib treatment. The elevated interaction between BECN1 and SLC7A11 inhibited the activity of system Xc−, whereas BECN1 silencing restored cystine intake and protected cells from ferroptosis. Notably, ectopic expression of MCL1 uncoupled BECN1 from SLC7A11 and rescued cell viability by attenuating lipid peroxidation. The results revealed that ferroptosis could be induced in HCC via SHP-1/STAT3-mediated downregulation of MCL1 and subsequent inhibition of SLC7A11 by increased BECN1 binding.

Adenosine Triphosphate Protects from Elevated Extracellular Calcium-Induced Damage in Human Proximal Kidney Cells: Using Deep Learning to Predict Cytotoxicity.

In kidney, extracellular [Ca2+] can modulate intracellular [Ca2+] to control key cellular processes. Hence, extracellular [Ca2+] is normally maintained within narrow range. We tested effect of extracellular ATP on viability of human proximal (HK-2) cells at high calcium. Modulation of intracellular calcium was assessed by imaging cytosolic [Ca2+], and expression of calcium-binding proteins (CaBPs). We present an artificial intelligence enabled deep learning model for prediction of injury and protection against extracellular [Ca2+] in HK-2 cells. HK-2 cells were cultured in calcium-free DMEM supplemented with CaCl2. Morphological changes were detected using light microscopy. Cell viability was determined using MTT Assay. Intracellular [Ca2+] was detected using fluorescence microscopy. For easy detection of HK-2 cells injury, we performed light microscopy image classification based on Convolutional Neural Network. Expression of CaBPs, p21, and Mcl-1 was measured using real-time PCR. We show decreased viability of HK-2 cells cultured in elevated calcium levels, which was prevented by adenosine triphosphate (ATP). Exposure of cells to elevated extracellular [Ca2+] correlated with increasing fluorescence of intracellular calcium indicator, which was attenuated in presence of ATP. Since features cannot be detected easily by human eyes, we propose a customized deep learning-based CNN model for classification of HK-2 cells injury by extracellular calcium with high accuracy of 98%. Our data demonstrated significant increase in mRNA levels of calmodulin, S100A8, S100A14 and CaBP28k, with elevated extracellular [Ca2+]. Expression of these genes was enhanced with ATP. The results suggest that ATP protects human proximal (HK-2) cells against elevated extracellular calcium levels. We present a CNN model as user friendly tool to study calcium dependent injury in (HK-2) cells. Finally, we show that ATP-mediated protection is correlated with enhanced expression of calcium-binding proteins.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1β, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1β, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1β, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1β, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

Myeloid expression of the anti-apoptotic protein Mcl1 is required in anti-myeloperoxidase vasculitis but myeloperoxidase inhibition is not protective

Antibodies to neutrophil and monocyte myeloperoxidase and proteinase 3 are a feature of anti-neutrophil cytoplasmic antibody vasculitis, a disease with significant morbidity for which new treatments are needed. Mice with a myeloid-specific deletion of the anti-apoptotic protein Mcl1 have reduced numbers of circulating neutrophils. Here, we assessed if myeloid-specific Mcl1 was required in murine anti-myeloperoxidase vasculitis and whether inhibition of myeloperoxidase was protective. In a murine model of anti-neutrophil cytoplasmic antibody vasculitis, induced by anti-myeloperoxidase antibody, mice with a myeloid-specific deletion of Mcl1 were protected from disease. They had fewer crescents, neutrophils, and macrophages in the glomeruli, lower serum creatinine levels and reduced albuminuria compared with controls. At baseline and day six after disease induction they had fewer circulating neutrophils than controls. At day six there were also fewer circulating monocytes. Myeloperoxidase inhibition with AZD5904 had no effect on histological or biochemical parameters of disease, and there was also no reduction in albuminuria at day one, two, five or seven after disease induction. These findings persisted when disease was induced without granulocyte-colony stimulating factor, which increases disease severity. A second myeloperoxidase inhibitor, AZM198, also showed no evidence of an effect, although both AZD5904 and AZM198 inhibited human neutrophil extracellular trap formation invitro. Thus, our results show that while myeloid-specific Mcl1 is required in this model of anti-myeloperoxidase vasculitis, myeloperoxidase inhibition is not protective.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n=1140, CCA n=72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Ligand- and structure-based identification of novel CDK9 inhibitors for the potential treatment of leukemia

Cyclin-dependent kinase 9 (CDK9) plays a vital role in controlling cell transcription and has been an attractive target for cancer treatment. Herein, ten predictive models derived from 1330 unique molecules against CDK9 were constructed based on molecular fingerprints and graphs using two conventional machine learning and four deep learning methods. The evaluation results showed that FP-GNN deep learning architecture performed best for CDK9 inhibitors prediction with the highest BA and F1 values of 0.681 and 0.912 for testing set. We then performed virtual screening to identify new CDK9 inhibitors by incorporating the optimal established predictive model and molecular docking. Five compounds were identified to show broad anticancer activity against various cancer cell lines through bioassays. For example, C9 exhibited antiproliferative activities against HeLa, MOLM-13 and MDA-MB-231 with IC50 values of 2.53, 3.92 and 11.65 μM. Kinase inhibition assay results demonstrated that these compounds displayed submicromolar (214 ∼ 504 nM) inhibitory activities against CDK9. Further cellular mechanism evaluation revealed that C9 suppressed the activity of CDK9 and interfered with the expression of Mcl-1 and cleaved PARP in MOLM-13 cells, resulting in the induction of cellular apoptosis. In addition, C9 displayed a good stability in rat liver microsomes, artificial gastrointestinal fluid and plasm. An online platform (called DEEPCDK9Pred) was developed based on the FP-GNN models to predict or design new CDK9 inhibitors. Collectively, our findings demonstrated that FP-GNN algorithm can achieve accurate prediction of CDK9 inhibitors and the subsequent discovery of C9 as a new potential CDK9 inhibitor deserves further structural modification for the treatment of leukemia.

All-trans retinoic acid enhanced the antileukemic efficacy of ABT-199 in acute myeloid leukemia by downregulating the expression of S100A8

Acute myeloid leukemia (AML) is prone to relapse. Targeted therapy with a specific inhibitor of the anti-apoptotic protein Bcl-2 ABT-199 is an effective method for relapsed and refractory patients, but drug resistance is likely, which is primarily related to high Mcl-1 and S100A8 expression. All-trans retinoic acid (ATRA) can inhibit Bcl-2 and Mcl-1 expression. The study purpose was to determine whether ATRA can enhance the antileukemia effect of ABT-199 on AML cells. Our data showed that ATRA combined with ABT-199 exerts a synergistic antileukemic effect by inducing apoptosis and cell cycle arrest in AML. In vivo, combination therapy prolonged the survival of AML xenograft mice. The possible mechanism involves promoting apoptosis through downregulation of S100A8 expression by inhibiting the PI3K/AKT signaling pathway. This study provides a potential treatment strategy and theoretical support for overcoming the clinical ABT-199 resistance problem in AML patients.

Interferon-β promotes the survival and function of induced regulatory T cells

Type I interferons (IFNs) are pleiotropic cytokines and impact various immune cells, including regulatory T cells (Treg cells). The effect of type-I IFNs on the development and function of Treg cells is quite controversial. Here we induced Treg cells (iTreg cells) from naïve CD4+ T cells in vitro in the presence or absence of IFN-β to elucidate its direct effect on the induction of iTreg cells. We found that IFN-β suppressed the proliferation of iTreg cells but enhanced their expression of anti-apoptotic genes Bcl-2 and Mcl-1 during the development of iTreg cells. We also found that IFN-β promoted suppression of conventional T cell proliferation by iTreg cells. These results suggest that IFN-β promotes the survival and immunomodulatory function of iTreg cells.

Protein Kinase CK2 represents a new target to boost Ibrutinib and Venetoclax induced cytotoxicity in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin lymphoma, characterized by frequent relapses. In the last decade, the pro-survival pathways related to BCR signaling and Bcl-2 have been considered rational therapeutic targets in B cell derived lymphomas. The BTK inhibitor Ibrutinib and the Bcl-2 inhibitor Venetoclax are emerging as effective drugs for MCL. However, primary and acquired resistance also to these agents may occur. Protein Kinase CK2 is a S/T kinase overexpressed in many solid and blood-derived tumours. CK2 promotes cancer cell growth and clonal expansion, sustaining pivotal survival signaling cascades, such as the ones dependent on AKT, NF-κB, STAT3 and others, counteracting apoptosis through a “non-oncogene” addiction mechanism. We previously showed that CK2 is overexpressed in MCL and regulates the levels of activating phosphorylation on S529 of the NF-κB family member p65/RelA. In the present study, we investigated the effects of CK2 inactivation on MCL cell proliferation, survival and apoptosis and this kinase’s involvement in the BCR and Bcl-2 related signaling. By employing CK2 loss of function MCL cell models, we demonstrated that CK2 sustains BCR signaling (such as BTK, NF-κB and AKT) and the Bcl-2-related Mcl-1 expression. CK2 inactivation enhanced Ibrutinib and Venetoclax-induced cytotoxicity. The demonstration of a CK2-dependent upregulation of pathways that may antagonize the effect of these drugs may offer a novel strategy to overcome primary and secondary resistance.

Ethanolic extract of Coleus aromaticus leaves impedes the proliferation and instigates apoptotic cell death in liver cancer HepG2 cells through repressing JAK/STAT cascade.

Liver cancer or hepatocellular carcinoma (HCC) has become a leading cause for cancer burden across the globe, and incidences have tripled since the last two decades. Poor diagnosis of primary liver cancer and limited treatment strategies aggravate the challenges. Researchers globally have shown a steep inclination toward the exploration of plant-based compounds for their nutraceutical and anticancer potential to fit into the role of novel chemotherapeutics. Coleus aromaticus is a well-known culinary herb that earlier has been reported for several medicinal attributes. The current investigation deals with exploring the anticancer potential of ethanolic leaf extract of C. aromaticus (CoL-EtOH) against hepatocellular carcinoma HepG2 cell line. The observations made it evident that CoL-EtOH extract impeded the viability of HepG2 at 400 μg/ml (p< .01). Additionally, the extract also succeeded in escalating ROS production (p< .01) which aided dissipation of mitochondrial membrane potential and disruption of nuclear morphology. CoL-EtOH further activated caspase-8, -9, and -3 which was reaffirmed by increase in apoptosis at 400 μg/ml (p< .01). Moreover, post treatment with CaLEt-OH extract significantly reduced the expression of JAK-1 & STAT-3 genes (p< .01) along with regulated expression of Mcl1, Bcl-2, cyclinD1, p21, and p27 within HepG2 cells. This evidence portrays the promising anticancer potential of CoL-EtOH projecting it as a novel chemotherapeutic agent against HCC. PRACTICAL APPLICATIONS: The herb Coleus aromaticus belonging to Lamiaceae family and Coleus genus is known by various names in different regions of the world and several language-specific vernacular names. The herb has been used in therapeutic and medicinal applications as well as in culinary preparations. Various attributes of the nutritional strength and functional characteristics of the leaves in terms of carotenoids, minerals, phenols, dietary fiber, and antioxidant activity have been reported by several researchers. Carvacrol and thymol are majorly found in the plant, while chlorogenic acid and rosmarinic acid etc. as the phenolic components. The herb has been used in therapeutic and medicinal implications as well as in culinary preparations.

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.

Ipatasertib, an oral AKT inhibitor, in combination with paclitaxel exhibits anti-proliferative and anti-tumorigenic effects in preclinical studies for endometrioid endometrial cancer: Endometrial cancer molecularly targeted therapy consortium (227)

<b>Objectives:</b> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. The PI3K/ AKT pathway is altered in the vast majority of endometrioid endometrial cancers (ECs). A recent clinical trial demonstrated that IPAT combined with paclitaxel showed a significant prolonged overall survival in breast cancer patients. Thus, we aimed to evaluate the antitumorigenic effects of IPAT in combination with paclitaxel in EC cell lines and an EC mouse model. <b>Methods:</b> The human endometrioid EC cell lines Ishikawa <i>(PTEN</i> mutant), and HEC-1A (<i>PTEN</i> wild type, <i>PIK3CA</i> mutant, <i>KRAS</i> mutant) were exposed to varying concentrations and durations of IPAT (Genentech) and paclitaxel. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and paclitaxel, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Western immunoblotting determined the effects of IPAT and paclitaxel on apoptosis (MCL, BCL-xl), cellular stress (ATF, PERK, Bip, PDI, IER), and DNA damage (RAD50). LKB1^fl/^flp53^fl/fl mice were used to evaluate the effect of IPAT, paclitaxel, and the combination of these two drugs on EC growth. The mice were treated with placebo, IPAT (50mg/kg daily orally for four weeks), paclitaxel (10 mg/kg IP weekly), or IPAT/paclitaxel starting eight weeks after tumor induction via AdCre injection. <b>Results:</b> IPAT and paclitaxel inhibited cellular proliferation in a dose-dependent fashion in both cell lines after 72 hours of treatment. The median IC50 for IPAT was 0.13µM for Ishikawa and 4.9µM for HEC-1A, respectively. The median IC50 for paclitaxel was 5.6nM for Ishikawa and 1.9nM for HEC-1A, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and paclitaxel induced the activity of cleaved caspase-3 by 3.5-fold in Ishikawa and 1.12-fold in HEC-1A, while simultaneously increasing expression of the apoptotic proteins MCL-1 and BCL-xl. IPAT and paclitaxel also increased Bip, PERK, ATF4, PDI, and IER protein expression. Treatment of cells with IPAT and paclitaxel decreased phosphorylated-S6 (p-S6) expression and induced the expression of p-AKT and RAD50. Compared to control mice, treatment of LKB1^fl/flp53^fl/fl mice with IPAT reduced tumor weight by 62.1%, and treatment with paclitaxel decreased tumor weight by 73.9% (p<0.05). The combination of IPAT and paclitaxel reduced tumor growth by 78.3% and showed improved efficacy over IPAT alone (p<0.05), but not over Taxol alone. <b>Conclusions:</b> The combination of IPAT and paclitaxel demonstrates synergy in human endometrioid EC cell lines and improved efficacy over IPAT alone in the LKB^1fl/fl/p53^fl/fl mouse model of EC. These results suggest that IPAT with paclitaxel is worthy of further exploration in clinical trials for EC.

Oleic acid, a monounsaturated fatty acid, exhibits antiproliferative and anti-tumorigenic effects in pre-clinical studies for endometrioid endometrial cancer (261)

<b>Objectives:</b> Oleic acid (OA), a monounsaturated fatty acid commonly found in olive oil, has been studied for its potential protective qualities in inflammatory conditions and cancers. In breast cancer cells, OA has been shown to reduce the expression of the oncogene <i>Her-2/ neu</i> and promote apoptosis. The relationship between the intake of olive oil and endometrial cancer risk and progression has been largely unexplored. Thus, we aimed to evaluate the effect of OA on cell proliferation and tumor growth in endometrial cancer cell lines and a transgenic mouse model of endometrial cancer. <b>Methods:</b> The human endometrioid endometrial cancer cell lines, ECC-1 and KLE, were exposed to varying concentrations of OA. Cell proliferation was assessed by MTT assay. Apoptosis was assessed by cleaved caspase-3, -8, and -9 assays. Cell cycle progression was measured by Cellometer. Cellular stress was measured by DCFH-DA assay. Mitochondrial membrane potential was assessed by TMRE assay. Western immunoblotting was performed to determine the effects of OA on BCL-xl, MCL-1, CDK4, CyclinD1, Calnexin, and Bip. The LKB1^fl/flp53^fl/fl mouse model of endometrioid endometrial cancer was used to determine the antitumorigenic activity of OA. The mice were treated with OA (10 mg/kg, oral gavage, daily) versus control for four weeks. <b>Results:</b> OA inhibited proliferation in a dose-dependent manner in both cell lines, with a 60% reduction at a dose of 500 µM in the ECC-1 cell line and 250 µM in the KLE cell line. OA at a dose of 200 µM significantly increased the activity of cleaved caspase-3, 8, and 9 by 2.2, 1.4, and 1.7-fold in the ECC-1 cells (p<0.05), respectively, and 1.4, 1.3, and 1.4-fold in the KLE cells (p<0.05), respectively. OA simultaneously decreased expression of Bcl-xl and MCL-1 in both cell lines. OA inhibited cell cycle progression by G1 phase arrest in both cell lines in a dose-dependent fashion (p=0.02) and decreased expression of the cell cycle-related proteins, CDK4, and cyclin D1. Treatment with OA significantly increased ROS production at doses of 50 and 200 µM (p<0.05) and induced Calnexin and Bip expression in both cell lines. TMRE assay showed decreased mitochondrial membrane potential with OA at doses of 50 and 200 µM (p<0.05). Treatment of LKB1^fl/flp53^fl/fl mice with OA was well-tolerated and reduced tumor weight by 53% compared with control mice (p<0.05). <b>Conclusions:</b> We found that OA, the main monounsaturated fatty acid of olive oil, had antiproliferative and antitumorigenic effects in endometrioid endometrial cancer. These findings provide a preclinical rationale for OA as a potential dietary intervention and therapeutic adjunct in endometrial cancer patients.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in serous endometrial cancer: Endometrial Cancer Molecularly Targeted Therapy Consortium (238)

<b>Objectives:</b> Ipatasertib (IPAT) is an investigational, orally-administered, ATP-competitive, highly selective inhibitor of protein kinase B (AKT) that has demonstrated antitumor activity in metastatic prostate cancer and longer progression-free survival in triple-negative breast cancer. Hyperactivation of the PI3K/AKT signaling pathway is frequently seen in endometrial cancer (EC), including serous histology tumors. Thus, we evaluated the antiproliferative efficacy of IPAT in combination with standard-of-care carboplatin in human serous EC cell lines. <b>Methods:</b> The human serous EC cell lines, ARK-1 and SPEC-2, were exposed to varying concentrations of IPAT (Genetech) and carboplatin. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and carboplatin, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was measured by DCFDA assay. Western immunoblotting determined the effects of IPAT and carboplatin on apoptotic (MCL-1 and BCL-2) and cellular stress (Bip and PERK) pathways in the ARK-1 and SPEC-2 cell lines. <b>Results:</b> IPAT and carboplatin significantly inhibited cell proliferation in a dose-dependent manner after 72 hours of treatment. The median IC₅₀ for IPAT was 8µM in the ARK-1 and 5µM in SPEC-2. The median IC₅₀ for carboplatin was 50µM in the ARK-1 and 65µM in SPEC-2. Simultaneous exposure of cells to various doses of carboplatin in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and carboplatin induced the activity of cleaved-caspase-3 by 2.3-fold in ARK-1 and 3.8-fold in SPEC-2 (p<0.05) while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. DCFDA assay analysis demonstrated that IPAT and carboplatin induced ROS products by 1.39-fold and 1.17-fold in the ARK-1 and SPEC-2 cell lines (p<0.05), respectively, and increased Bip and PERK protein expression. The effects of IPAT combined with carboplatin on apoptosis and cellular stress were greater when compared to IPAT or carboplatin alone in both cell lines (p<0.05). <b>Conclusions:</b> The combination of IPAT and carboplatin showed synergistic antiproliferative activity in human serous EC cell lines. Thus, IPAT plus carboplatin may be a promising combination in the treatment of serous EC. Studies in transgenic and patient-derived xenograft EC mouse models are underway to assess the in vivo efficacy of IPAT +/- carboplatin.

MiRNA-16-1 Suppresses Mcl-1 and Bcl-2 and Sensitizes Chronic Lymphocytic Leukemia Cells to BH3 Mimetic ABT-199.

ObjectiveChronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199. Materials and Methods In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays. Results MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relative to blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05). ConclusionOur findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients.

Pulmonary Delivery of Extracellular Vesicle-Encapsulated Dinaciclib as an Effective Lung Cancer Therapy.

Simple SummaryThe clinical outcomes of lung cancer remain poor. The targeted delivery of treatment and the implementation of a method to overcome drug resistance are essential for the improvement of cancer therapy. The aim of our study was to assess the treatment effectiveness of engineered extracellular vesicles (EV) carrying both dinaciclib, a potent CDK inhibitor, and the proapoptotic factor TRAIL for a combinatorial lung cancer therapy. We showed that the engineered complexed EV agent, EV-T-Dina, was stable both in vitro and in vivo. Importantly, EV-T-Dina can overcome the drug-resistance of lung cancer cells, and when nebulized and administered by the pulmonary route, it demonstrated high efficacy and satisfactory safety for the treatment of lung cancers. The underlying mechanism for the synergistic killing of cancer cells by dinaciclib and TRAIL was associated with the concomitant downregulation of the anti-apoptotic factors cFLIP, MCL-1, and Survivin. Thus, the aerosolized EV-T-Dina potentially constitutes a novel and effective therapy for lung cancers.The clinical outcomes of lung cancer remain poor, mainly due to the chemoresistance and low bioavailability of systemically delivered drugs. Therefore, novel therapeutic strategies are urgently needed. The TNF-related apoptosis-inducing ligand (TRAIL)-armed extracellular vesicle (EV-T) has proven to be highly synergistic for the killing of cancer cells with the potent cyclin-dependent kinase (CDK) inhibitor Dinaciclib (Dina). However, both optimal drug formulations and delivery strategies are yet to be established to facilitate the clinical application of the combination of EV-T and Dina. We hypothesize that Dina can be encapsulated into EV-T to produce a complexed formulation, designated EV-T-Dina, which can be nebulized for pulmonary delivery to treat lung cancer with potentially improved efficacy and safety. The prepared EV-T-Dina shows good stability both in vitro and in vivo and is very efficient at killing two highly TRAIL-resistant cancer lines. The ability to overcome TRAIL resistance is associated with the concomitant downregulation of the expression of cFLIP, MCL-1, and Survivin by Dina. The EV-T-Dina solution is nebulized for inhalation, showing unique deposition in animal lungs and importantly it demonstrates a significant suppression of the growth of orthotopic A549 tumors without any detectable adverse side events. In conclusion, the aerosolized EV-T-Dina constitutes a novel therapy, which is highly effective and safe for the treatment of lung cancers.

Design, synthesis, and biological evaluation of novel 7-substituted 10,11-methylenedioxy-camptothecin derivatives against drug-resistant small-cell lung cancer in vitro and in vivo

A series of novel 7-substituted 10,11-methylenedioxy-camptothecin (FL118) derivatives were designed, synthesized, and biologically evaluated. All the FL118 analogues showed significant cytotoxic activities in vitro with IC50 values in the nanomolar range and were more potent than topotecan. The most active compound 9c exhibited more significant anti-tumor activity against small-cell lung cancer (NCI–H446, H69, drug-resistant H69AR cells, drug-resistant NCI–H446/Irinotecan cells and drug-resistant NCI–H446/EP cells) in vitro. Additionally, 9c could also induce the expression of apoptosis proteins such as caspase-3, caspase-9, and PARP in small-cell lung cancer. Further studies showed that 9c induced apoptosis by inhibiting the expression of Mcl-1, Bcl-2, XIAP and survivin in small-cell lung cancer. In vivo9c also showed better anti-tumor efficacy, with the tumor growth inhibition rates were 40.4% (0.75 mg/kg), 73.7% (1.5 mg/kg), and 95.5% (3 mg/kg). It is noteworthy that 9c also demonstrated potent inhibition of drug-resistant tumor growth in NCI–H446/Irinotecan and NCI–H446/EP xenograft models, the tumor growth inhibition rates were 93.42% and 84.46%, respectively. Taken together, these findings indicated that compound 9c displays outstanding antitumor activity and drug-resistance in small-cell lung cancer both in vivo and in vitro, which could be worth further research as a novel anti-tumor drug against small-cell lung cancer.