Akt Inhibitor Research Articles

Lactate promotes HMGB1 phosphorylation and release via Akt signaling pathway in gastric cancer cells HGC-27

Objective: To investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. Methods: Gastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. Results: The release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml (P<0.001 and P=0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P=0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P=0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P=0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P=0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml (P=0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. Conclusion: Lactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway.

Metformin Suppresses Stemness of Non-Small-Cell Lung Cancer Induced by Paclitaxel through FOXO3a

Cancer stem cells (CSCs) play a pivotal role in drug resistance and metastasis. Among the key players, Forkhead box O3a (FOXO3a) acts as a tumor suppressor. This study aimed to unravel the role of FOXO3a in mediating the inhibitory effect of metformin on cancer stemness derived from paclitaxel (PTX)-resistant non-small-cell lung cancer (NSCLC) cells. We showed that CSC-like features were acquired by the chronic induction of resistance to PTX, concurrently with inactivation of FOXO3a. In line with this, knockdown of FOXO3a in PTX-sensitive cells led to changes toward stemness, while overexpression of FOXO3a in PTX-resistant cells mitigated stemness in vitro and remarkably curbed the tumorigenesis of NSCLC/PTX cells in vivo. Furthermore, metformin suppressed the self-renewal ability of PTX-resistant cells, reduced the expression of stemness-related markers (c-MYC, Oct4, Nanog and Notch), and upregulated FOXO3a, events concomitant with the activation of AMP-activated protein kinase (AMPK). All these changes were recapitulated by silencing FOXO3a in PTX-sensitive cells. Intriguingly, the introduction of the AMPK dominant negative mutant offset the inhibitory effect of metformin on the stemness of PTX-resistant cells. In addition, FOXO3a levels were elevated by the treatment of PTX-resistant cells with MK2206 (an Akt inhibitor) and U0126 (a MEK inhibitor). Collectively, our findings indicate that metformin exerts its effect on FOXO3a through the activation of AMPK and the inhibition of protein kinase B (Akt) and MAPK/extracellular signal-regulated kinase (MEK), culminating in the suppression of stemness in paclitaxel-resistant NSCLC cells.

CXADR promote epithelial–mesenchymal transition in endometriosis by modulating AKT/GSK-3β signaling

ABSTRACT Endometriosis is a benign high prevalent disease exhibiting malignant features. However, the underlying pathogenesis and key molecules of endometriosis remain unclear. By integrating and analysis of existing expression profile datasets, we identified coxsackie and adenovirus receptor (CXADR), as a novel key gene in endometriosis. Based on the results of immunohistochemistry (IHC), we confirmed significant down-regulation of CXADR in ectopic endometrial tissues obtained from women with endometriosis compared with healthy controls. Further in vitro investigation indicated that CXADR regulated the stability and function of the phosphatases and AKT inhibitors PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2) and PTEN (phosphatase and tensin homolog). Loss of CXADR led to phosphorylation of AKT and glycogen synthase kinase-3β (GSK-3β), which resulted in stabilization of an epithelial–mesenchymal transition (EMT) factor, SNAIL1 (snail family transcriptional repressor 1). Therefore, EMT processs was induced, and the proliferation, migration and invasion of Ishikawa cells were enhanced. Over-expression of CXADR showed opposite effects. These findings suggest a previously undefined role of AKT/GSK-3β signaling axis in regulating EMT and reveal the involvement of a CXADR-induced EMT, in pathogenic progression of endometriosis.

Synthesis of Novel Anticancer Coumarin-Triazole-Chalcone Hybrids as Potential AKT Inhibitor

Synthesis of Novel Anticancer Coumarin-Triazole-Chalcone Hybrids as Potential AKT Inhibitor

Identification of Dual-Target Inhibitors for Epidermal Growth Factor Receptor and AKT: Virtual Screening Based on Structure and Molecular Dynamics Study.

Epidermal growth factor EGFR is an important target for non-small cell lung (NSCL) cancer, and inhibitors of the AKT protein have been used in many cancer treatments, including those for NSCL cancer. Therefore, searching small molecular inhibitors which can target both EGFR and AKT may help cancer treatment. In this study, we applied a ligand-based pharmacophore model, molecular docking, and MD simulation methods to search for potential inhibitors of EGFR and then studied dual-target inhibitors of EGFR and AKT by screening the immune-oncology Chinese medicine (TCMIO) database and the human endogenous database (HMDB). It was found that TCMIO89212, TCMIO90156, and TCMIO98874 had large binding free energies with EGFR and AKT, and HMDB0012243 also has the ability to bind to EGFR and AKT. These results may provide valuable information for further experimental study.

Qiliqiangxin: A multifaceted holistic treatment for heart failure or a pharmacological probe for the identification of cardioprotective mechanisms?

The active ingredients in many traditional Chinese medicines are isoprene oligomers with a diterpenoid or triterpenoid structure, which exert cardiovascular effects by signalling through nutrient surplus and nutrient deprivation pathways. Qiliqiangxin (QLQX) is a commercial formulation of 11 different plant ingredients, whose active compounds include astragaloside IV, tanshione IIA, ginsenosides (Rb1, Rg1 and Re) and periplocymarin. In the QUEST trial, QLQX reduced the combined risk of cardiovascular death or heart failure hospitalization (hazard ratio 0.78, 95% confidence interval 0.68-0.90), based on 859 events in 3119 patients over a median of 18.2 months; the benefits were seen in patients taking foundational drugs except for sodium-glucose cotransporter 2 (SGLT2) inhibitors. Numerous experimental studies of QLQX in diverse cardiac injuries have yielded highly consistent findings. In marked abrupt cardiac injury, QLQX mitigated cardiac injury by upregulating nutrient surplus signalling through the PI3K/Akt/mTOR/HIF-1α/NRF2 pathway; the benefits of QLQX were abrogated by suppression of PI3K, Akt, mTOR, HIF-1α or NRF2. In contrast, in prolonged measured cardiac stress (as in chronic heart failure), QLQX ameliorated oxidative stress, maladaptive hypertrophy, cardiomyocyte apoptosis, and proinflammatory and profibrotic pathways, while enhancing mitochondrial health and promoting glucose and fatty acid oxidation and ATP production. These effects are achieved by an action of QLQX to upregulate nutrient deprivation signalling through SIRT1/AMPK/PGC-1α and enhanced autophagic flux. In particular, QLQX appears to enhance the interaction of PGC-1α with PPARα, possibly by direct binding to RXRα; silencing of SIRT1, PGC-1α and RXRα abrogated the favourable effects of QLQX in the heart. Since PGC-1α/RXRα is also a downstream effector of Akt/mTOR signalling, the actions of QLQX on PGC-1α/RXRα may explain its favourable effects in both acute and chronic stress. Intriguingly, the individual ingredients in QLQX-astragaloside IV, ginsenosides, and tanshione IIA-share QLQX's effects on PGC-1α/RXRα/PPARα signalling. QXQL also contains periplocymarin, a cardiac glycoside that inhibits Na+ -K+ -ATPase. Taken collectively, these observations support a conceptual framework for understanding the mechanism of action for QLQX in heart failure. The high likelihood of overlap in the mechanism of action of QLQX and SGLT2 inhibitors requires additional experimental studies and clinical trials.

Enzyme-Responsive Branched Glycopolymer-Based Nanoassembly for Co-Delivery of Paclitaxel and Akt Inhibitor toward Synergistic Therapy of Gastric Cancer.

Combined chemotherapy and targeted therapy holds immense potential in the management of advanced gastric cancer (GC). GC tissues exhibit an elevated expression level of protein kinase B (AKT), which contributes to disease progression and poor chemotherapeutic responsiveness. Inhibition of AKT expression through an AKT inhibitor, capivasertib (CAP), to enhance cytotoxicity of paclitaxel (PTX) toward GC cells is demonstrated in this study. A cathepsin B-responsive polymeric nanoparticle prodrug system is employed for co-delivery of PTX and CAP, resulting in a polymeric nano-drug BPGP@CAP. The release of PTX and CAP is triggered in an environment with overexpressed cathepsin B upon lysosomal uptake of BPGP@CAP. A synergistic therapeutic effect of PTX and CAP on killing GC cells is confirmed by in vitro and in vivo experiments. Mechanistic investigations suggested that CAP may inhibit AKT expression, leading to suppression of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Encouragingly, CAP can synergize with PTX to exert potent antitumor effects against GC after they are co-delivered via a polymeric drug delivery system, and this delivery system helped reduce their toxic side effects, which provides an effective therapeutic strategy for treating GC.

Moluodan promotes DSS-induced intestinal inflammation involving the reprogram of macrophage function and polarization

Ethnopharmacological relevanceMoluodan (MLD) is a traditional Chinese medicine that is composed of 18 herbal medicines based on traditional Chinese medicine theory and practice. It has long been used in treating chronic gastritis and its components were traditionally used in dealing with intestinal inflammation. However, its specific pharmacological mechanism is still unclear. Aim of the studyThe upper and lower digestive tract diseases are correlated. In clinical practice, some chronic gastritis patients are also accompanied by intestinal inflammation. Due to the unclear pharmacological mechanism of MLD and its effect on intestinal inflammation, there is doubt whether MLD is still suitable for this type of patient. Therefore, this study aims to elucidate the pharmacological mechanism of MLD and identify its effect in the mouse model of intestinal inflammation. Materials and methodsMice intestinal inflammation model was induced by 2.5% dextran sulfate sodium (DSS). The mice were given different concentrations of MLD via oral gavage (0.25, 0.5 g/kg b.w.). Pharmacodynamic indicators were assessed including body weight, colon length, disease activity index (DAI), bloody stool score, inflammatory factors, histological change, etc. RAW264.7 macrophage cells were used for in vitro experiments that illuminated the role of MLD in reprogramming macrophage function and polarization. RT-qPCR and western blots were performed to measure the mRNA and protein levels of macrophage polarization marker and effector molecules. The functions of polarized macrophages were tested using ROS detection probes, Edu assay and wound healing assay. ResultsThe administration of MLD exhibited obvious hemostatic effects, while unexpectedly accentuating various aspects of the DSS-induced intestinal inflammation in mice, including increased body weight loss and colon shortening, elevated disease activity index, and intensified colonic tissue damage. Additionally, MLD treatment induced more severe inflammatory cell infiltration and higher proinflammatory cytokines expression in colon tissue. Further results showed that MLD promoted M1 macrophage polarization and stimulated its proinflammatory cytokines expression, while only slightly affecting the function of M2 macrophage. Western blot analysis revealed that MLD induced the phosphorylation of AKT and NF-κB. The polarization of M1 macrophages induced by MLD was inhibited by either an Akt inhibitor or a NF-κB inhibitor. ConclusionsAlthough MLD has an obvious hemostatic effect, it generally promoted the severity of DSS-induced colitis in mice by facilitating macrophage polarization toward the M1 phenotype through the AKT/NF-κB pathway. Our study suggested that MLD may not be suitable for colitis, especially during the acute inflammation stage.

DDDR-22. PREDICTING AND TARGETING CLONALLY EXPANDING, TMZ-RESISTANT TUMOR CELLS IN GLIOBLASTOMA

Abstract Emerging evidence supports the notion that phenotypic plasticity contributes to disease progression and drug resistance in malignant glioma. We have recently described a rare population of ALDH1A1+ tumor cells in newly diagnosed, treatment-naive glioblastoma that can adapt to the exposure of the standard chemotherapeutic temozolomide (TMZ). Their subclonal growth leads to AKT-driven, TMZ-resistant cellular hierarchies. Accumulation of ALDH1A1+/pAKT+ cells can therefore be noted subsequent to TMZ in patient relapse tissue and in PDX models of disease. We have also shown that this series of events requires a sequential targeting approach where these subclones are allowed to enrich under TMZ and, only in a second step, are treated with AKT inhibitors. This “enrich and kill” strategy doubles the TMZ-based survival benefit in preclinical PDX models (Kebir et al., Clin Cancer Res 2023). Here, we took advantage of short-term expanded patient-derived cell cultures, which allow the study of early stages of this type of adaptive plasticity in controlled conditions. Specifically, we characterized the human lysine-specific demethylase 5B (KDM5B) as a prospective indicator for subclonal expansion in rare cells under first-time exposure to TMZ. We used genetic reporters, pharmacological interference, CHIP-seq analysis and cellular barcoding to investigate the dynamics of KDM5B expression and the related intracellular cross-signaling. We monitored patient cell samples over prolonged periods of time, and we found that KDM5Bhigh treatment-naive glioblastoma cells preferentially contribute to the dynamics of ALDH1A1 subclones and drug resistance under the influence of TMZ. These findings may lay ground for the development of biomarker-assisted clinical trials. This work is supported by DFG/GRC-CRU337/2 (proj#405344257).

Tanshinone IIA promotes the proliferation and differentiation ability of primary muscle stem cells via MAPK and Akt signaling

Salvia miltiorrhiza Bunge is a widely-used traditional Chinese medicine to treat a variety of diseases including muscle disorders. The underlying pharmacological mechanisms of which active component and how it functions are still unknown. Tanshinone IIA (Tan IIA) is the main active lipophilic compound in Salvia miltiorrhiza Bunge. Muscle stem cells (MuSCs) play a crucial role in maintaining healthy physiological function of skeletal muscle. For the purpose of this study, we investigated the effects of Tan IIA on primary MuSCs as well as mechanism. The EdU staining, cell counts assay and RT-qPCR results of proliferative genes revealed increased proliferation ability of MuSCs after Tan IIA treatment. Immunofluorescent staining of MyHC and RT-qPCR results of myogenic genes found Tan IIA contributed to promoting differentiation of MuSCs. In addition, enrichment analysis of RNA-seq data and Western blot assay results demonstrated activated MAPK and Akt signaling after treatment of Tan IIA during proliferation and differentiation. The above proliferative and differentiative phonotypes could be suppressed by the combination of MAPK inhibitor U0126 and Akt inhibitor Akti 1/2, respectively. Furthermore, HE staining found significantly improved myofiber regeneration of injured muscle after Tan IIA treatment, which also contributed to muscle force and running performance recovery. Thus, Tan IIA could promote proliferation and differentiation ability of MuSCs through activating MAPK and Akt signaling, respectively. These beneficial effects also significantly contributed to muscle regeneration and muscle function recovery after muscle injury.

Prognostic value analysis of cholesterol and cholesterol homeostasis related genes in breast cancer by Mendelian randomization and multi-omics machine learning.

The high incidence of breast cancer (BC) prompted us to explore more factors that might affect its occurrence, development, treatment, and also recurrence. Dysregulation of cholesterol metabolism has been widely observed in BC; however, the detailed role of how cholesterol metabolism affects chemo-sensitivity, and immune response, as well as the clinical outcome of BC is unknown. With Mendelian randomization (MR) analysis, the potential causal relationship between genetic variants of cholesterol and BC risk was assessed first. Then we analyzed 73 cholesterol homeostasis-related genes (CHGs) in BC samples and their expression patterns in the TCGA cohort with consensus clustering analysis, aiming to figure out the relationship between cholesterol homeostasis and BC prognosis. Based on the CHG analysis, we established a CAG_score used for predicting therapeutic response and overall survival (OS) of BC patients. Furthermore, a machine learning method was adopted to accurately predict the prognosis of BC patients by comparing multi-omics differences of different risk groups. We observed that the alterations in plasma cholesterol appear to be correlative with the venture of BC (MR Egger, OR: 0.54, 95% CI: 0.35-0.84, p<0.006). The expression patterns of CHGs were classified into two distinct groups(C1 and C2). Notably, the C1 group exhibited a favorable prognosis characterized by a suppressed immune response and enhanced cholesterol metabolism in comparison to the C2 group. In addition, high CHG score were accompanied by high performance of tumor angiogenesis genes. Interestingly, the expression of vascular genes (CDH5, CLDN5, TIE1, JAM2, TEK) is lower in patients with high expression of CHGs, which means that these patients have poorer vascular stability. The CAG_score exhibits robust predictive capability for the immune microenvironment characteristics and prognosis of patients(AUC=0.79). It can also optimize the administration of various first-line drugs, including AKT inhibitors VIII Imatinib, Crizotinib, Saracatinib, Erlotinib, Dasatinib, Rapamycin, Roscovitine and Shikonin in BC patients. Finally, we employed machine learning techniques to construct a multi-omics prediction model(Risklight),with an area under the feature curve (AUC) of up to 0.89. With the help of CAG_score and Risklight, we reveal the signature of cholesterol homeostasis-related genes for angiogenesis, immune responses, and the therapeutic response in breast cancer, which contributes to precision medicine and improved prognosis of BC.

Abstract 15796: Akt1 Y350f Mutation: A Protective Mechanism Against Pulmonary Hypertension, Suppressing Endothelial-to-Mesenchymal Transition in vivo and in vitro

Background: Pulmonary hypertension (PH) is a life-threatening condition characterized by elevated blood pressure in the lungs due to narrowed arteries. Endothelial-to-mesenchymal transition (EndMT) contributes to enhanced proliferation and metabolic changes in the pulmonary arteries of PH patients. Our previous reports have highlighted the role of Akt nitration at tyrosine 350 (Y350) residue in promoting proliferation and metabolic switch in PH models. To investigate the protective potential of inhibiting Akt nitration, we developed a mouse model with a mutation of Akt Y350 to phenylalanine (Y350F), preventing nitration and activation of Akt. Hypothesis: We hypothesized that Akt1 Y350F mutant mice would be protected in preclinical PH models due to inhibited EndMT. Methods: Both mutant Y350F and wild-type Akt1 mice were treated with Sugen5416 (20mg/kg/week i.p.)/hypoxia (10% O2) (Su/Hx) for four weeks to explore pathological mechanisms. Endothelial cells were isolated for in vitro evaluation under EndMT inducers, including Sin-1 chloride and TGF-β1. Results: The wild-type Su/Hx model exhibited vascular remodeling, increased right ventricular systolic pressure (RVSP) (p&lt;0.001), and right ventricular hypertrophy. In contrast, the Akt Y350F mutant model displayed significant protection, with reduced vascular remodeling, pulmonary pressure, and right heart hypertrophy. Akt nitration-induced EndMT markers, TGF-β1 (p&lt;0.05) and TWIST1 (p&lt;0.01), were elevated in wild-type Su/Hx lung tissue. However, the Akt Y350F mutation prevented Akt activation and diminished EndMT markers in the Akt1 Y350F Su/Hx lungs. In isolated cells and lung tissue, induction of EndMT resulted in a progressive PH phenotype, while Akt inhibition exhibited a protective role against PH progression in the Akt Y350F mutant model. Conclusion: Inhibition of Akt nitration through the Y350F mutation restricts vascular remodeling and halts EndMT both in vivo and in vitro. Our findings suggest that strategies targeting Akt nitration could be effective in controlling PH.

A novel ferroptosis-related gene signature associated with cuproptosis for predicting overall survival in breast cancer patients.

Purpose Ferroptosis and cuproptosis are both metal-dependent regulated cell death that play an important role in cancer. However, the expression patterns and the prognostic values of ferroptosis-related genes (FRGs) associated with cuproptosis in breast cancer (BC) are largely unknown. This study aims to explore the prognostic value of cuproptosis-related FRGs and their relationship with tumor microenvironments in BC. Methods The clinical and RNA sequencing data of BC patients from TCGA, METABRIC and GEO databases were analyzed. The least absolute shrinkage and selection operator regression analysis was used to establish prognostic signatures based on cuproptosis-related FRGs. The overall survival between risk subgroups was assessed by Kaplan-Meier analysis. The changes in risk score during neoadjuvant chemotherapy, and differences in immune cells, immune checkpoints, and drug sensitivity between risk subgroups were also analyzed in this study. Results A successful development of a prognostic signature based on cuproptosis-related FRGs in the TCGA cohort was achieved and it was validated in the METABRIC cohort. Gene set enrichment analysis results revealed the enrichment of steroid biosynthesis and ABC transporters in the high-risk group. Moreover, the signature was also found to be associated with immune cells and immune checkpoints. Lower risk score in patients after neoadjuvant chemotherapy and higher sensitivity of the high-risk group to AKT inhibitor VIII and cisplatin was also observed. Conclusion Cuproptosis-related FRGs can be used as a novel prognostic signature for predicting the overall survival of BC patients. This can provide meaningful insights into the selection of immunotherapy and antitumor drugs for BC.

GPR120/FFAR4 stimulation attenuates airway remodeling and suppresses IL-4- and IL-13-induced airway epithelial injury via inhibition of STAT6 and Akt

BackgroundAirway remodeling is associated with severity and treatment insensitivity in asthma. This study aimed to investigate the effects of G protein-coupled receptor 120 (GPR120) stimulation on alleviating allergic inflammation and remodeling of airway epithelium. Research design and methodsOvalbumin (OVA)-challenged BALB/c mice and type-2-cytokine (IL-4 and IL-13)-exposed 16HBE human bronchial epithelial cells were treated with GSK137647A, a selective GPR120 agonist. Markers of allergic inflammation and airway remodeling were determined. ResultsGSK137647A attenuated inflammation and mucus secretion in airway epithelium of OVA-challenged mice. Stimulation of GPR120 in 16HBE suppressed expression of asthma-associated cytokines and cytokine-induced expression of pathogenic mucin-MUC5AC. These effects were abolished by co-treatment with AH7614, a GPR120 antagonist. Moreover, GPR120 stimulation in 16HBE cells reduced expression of fibrotic markers including fibronectin protein and ACTA2 mRNA and inhibited epithelial barrier leakage induced by type-2 inflammation via rescuing expression of zonula occludens-1 protein. Furthermore, GPR120 stimulation prevented the cytokine-induced airway epithelial remodeling via suppression of STAT6 and Akt phosphorylation. ConclusionsOur findings suggest that GPR120 activation alleviates allergic inflammation and remodeling of airway epithelium partly through inhibition of STAT6 and Akt. GPR120 may represent a novel therapeutic target for diseases associated with remodeling of airway epithelium, including asthma.

Threshold Attenuation of Perk Signaling Is Sufficient to Overcome RAS-Mediated FLT3 Inhibitor Resistance

Introduction Acute myeloid leukemia (AML) with activation of FMS-related receptor tyrosine kinase 3 ( FLT3) confers a poor prognosis, principally due to disease relapse. The FLT3 inhibitor (FLT3i) gilteritinib is standard of care in relapsed/refractory FLT3-mutant AML, however its efficacy is limited by drug resistance, primarily due to RAS mutations. This suggests that RAS signaling is the critical mediator of downstream FLT3 signaling, but there is no standardized treatment for patients who relapse on FLT3i due to RAS pathway activation. Efforts to overcome resistance have been limited both by understanding of the optimal RAS effectors to target as well as the potential toxicity of simultaneously targeting FLT3 and RAS. Here, we demonstrate that while RAS-driven resistance to gilteritinib is mediated through MAPK, not PI3K, attenuation of pERK signaling is sufficient to overcome this resistance both in vitro and in vivo. Methods/ Results FLT3-ITD+ cells (Molm14) with transduced NRAS mutations are resensitized to gilteritinib by MEK inhibitor treatment, proving that MAPK signaling is necessary for RAS-driven resistance. Further, expression of a constitutively active MEK construct (MEKDD) in Molm14 confers resistance to gilteritinib, showing MAPK signaling is sufficient for RAS-driven resistance. Conversely, we show that activated PI3K signaling is neither necessary nor sufficient to drive resistance, as AKT inhibition cannot resensitize NRAS-mutants to gilteritinib, and an activated AKT mutant does not confer gilteritinib resistance. We used a doxycycline-inducible system to increase expression of MEKDD, to titrate levels of ERK signaling activation in Molm14 cells. Surprisingly, we found that the relationship between ERK phosphorylation (pERK) and FLT3i resistance was non-linear and appeared to increase at a threshold of pERK (62%). In keeping with this, suppression of pERK levels in Molm14 cells with concurrent RAS mutations to levels below 70% of baseline using the MEK inhibitor (MEKi) trametinib, was sufficient to resensitize to gilteritinib in proliferation assays. A similar phenomenon was seen in apoptosis assays. Molm14 NRAS Q61K mutant cells treated with 500nM gilteritinib plus increasing doses of trametinib showed apoptosis plateaued at the relatively low dose of 5nM trametinib. Western Blot analysis showed residual pERK activation at this dose. Thus, attenuation of MAPK signaling below a threshold, rather than its ablation, can overcome FLT3i resistance. In a patient-derived xenograft (PDX) model of AML with concurrent FLT3-ITD and NRAS Q61K, the combination of low dose MEK inhibition (PD-901 1.5mg/kg) combined with gilteritinib was equivalent to standard dose MEK inhibition (PD-901 5mg/kg) combined with gilteritinib in suppressing outgrowth of human CD45+ cells in spleen, bone marrow, and blood of NSGS mice in comparison to vehicle-treated control. Standard dose MEK inhibition in combination with gilteritinib was also noted to be intolerable in the majority of mice with only 2 of 6 mice surviving 28 days. Given the similar activity of low and high dose MAPK inhibition in combination with gilteritinib, we hypothesized that suppression of ERK activation below a critical level is sufficient to activate a shared transcriptional program to trigger cell cycle arrest and apoptosis. To test this hypothesis, we performed RNAseq analysis of Molm14 NRAS Q61K cells treated with gilteritinib, standard dosing of trametinib, low dose trametinib, or the combination. There is a gene expression signature of MAPK expression unique to the combination of gilteritinib and trametinib that is dose independent of trametinib, which includes MAPK regulators including ETV4 and ETV5. Conclusions MAPK is the critical effector pathway of RAS-driven gilteritinib resistance, but partial attenuation of pERK signaling is sufficient to inhibit proliferation and induce apoptosis, restoring sensitivity to FLT3i. Applied clinically, this suggests feasibility of low dose MAPK inhibition in combination with gilteritinib to treat resistant FLT3-activated AML. This strategy that would improve tolerability, a major concern in clinical use of MAPK pathway inhibitors.

Development of a Phase 1 Study Evaluating the Activity of Modular CAR T for Multiple Myeloma (MCARTY) Targeting BCMA and CD19 for Improved Persistence

CONCLUSION BCMA directed CAR T cell therapy for multiple myeloma (MM) shows promise, but very long-term survival may be limited. Notably, BCMA CAR T persistence in MM is shorter than that seen in with CD19 CAR T cells in B cell malignancies where long-term CD19 CAR T persistence is associated with durable responses. Shorter BCMA CAR T persistence may be explained by the lower surface density of BCMA on tumour compared with that of CD19. Alternatively, CD19 CAR T persistence may be driven by recognition of continuous emergence of newly differentiated B cells. We sought to improve BCMA CAR T persistence by (a) developing a BCMA CAR with high sensitivity to BCMA, and (b) co-expression with a CD19 CAR. Notably, while CD19 is absent or expressed at low level on myeloma cells, CD19 may be expressed on a subpopulation of myeloma stem cells, providing an additional rationale for co-targeting CD19 in MM. We performed functional screening of several mid to high affinity BCMA binders in CAR format. We additionally explored whether a more stable CAR format would improve sensitivity. We explored co-expression of the new BCMA CAR with the CD19 CAR from obecabatagene autoleucel (also known as CAT19), which is known to drive long-term persistence. We also designed and started a clinical study (MCARTY, NCT04795882) testing the BCMA CAR alone or co-expressed with a CD19 CAR in patients with refractory Myeloma. The binding affinity for BCMA was determined for 13 antibody clones from a rat library, before being assessed functionally in single chain variable fragment (scFv) and fragment antigen-binding (Fab) formats in a second generation (41bbζ) CAR backbone. Noting superior target binding, functional efficacy and minimal basal activation of effector cells expressing the D8 binder in Fab format we then assessed this construct against standard BCMA targeting CARs based on the bb2121 and LCAR-B38M binders used in Ide-Cel and Cilta-Cel respectively. Compared to these competitor CARs, D8 demonstrated improved sensitivity to BCMA with improved activation, target kill, and cytokine release in a series of cocultures with plate bound antigen and cell targets expressing low level BCMA. Successful manufacture in an academic setting of dual targeting CAR T cells was achieved by double transduction of autologous CD4/CD8 selected T cells with separate lentiviral vectors titrated for equal transduction. Manufacture by CliniMACS Prodigy included AKT inhibitor VIII to reduce T cell differentiation and LentiBoost, a polaxamer-based transduction enhancer, in a protocol lasting 6-8 days. This resulted in 3 populations of CAR T cells (D8+CAT19-, D8-CAT19+, D8+CAT19+) which can be individually tracked in treated patients. The MCARTY trial is a single centre, Phase 1 dose escalation trial uniquely designed with two cohorts to evaluate the safety of D8 CAR T cells (Cohort 1) and D8/CAT19 CAR T cells (Cohort 2) in patients with relapsed/refractory MM at a dose of 50 or 150x10^6 CARs. Inclusion criteria are: progression or stable disease to last treatment, 3 or more previous lines of therapy, exposure to proteasome inhibitors (PI), immunomodulatory drugs (IMID) and CD38 antibodies, ECOG 0/1. There was no selection based on previous exposure to BCMA targeting agents or tumour BCMA expression. As of 11 July, 8 patients have been treated, 6 patients with D8 CAR alone (n=3 at each dose level) and 2 patients with the lower dose of dual targeting D8/CAT19 CAR T cells. Median age was 57 (range 33-66), high risk cytogenetics in 4 of 7 patients (57%), previous lines of therapy 4 (range 3-8). Doses were well tolerated with no cases of ICANS or CRS≥ grade 3. At a median follow up of 4.5 months (range 1-12), clinical responses ≥PR were seen in all patients (ORR: 100%). Of 6 patients, 5 (83%) CR/sCR, 1 (17%) PR at 3 months. We observed robust expansion of D8 CAR in Cohort 1, and of dual and single targeting CARs in Cohort 2. We report a unique approach to prolong CAR persistence in MM by developing a CAR T cell co-expressing a sensitive BCMA CAR construct and CD19 CAR with proven durability. We report feasibility of double transduction, the first clinical use of a FabCAR and a unique trial design allowing comparison of treatment with single vs dual targeting CAR constructs.

Oncogene SCARNA12 as a potential diagnostic biomarker for colorectal cancer

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, and represents a severe threat to the life and health of individuals. Increasing evidence supports the role of small nucleolar RNAs (snoRNAs) as critical regulatory gene in cancer development. Small Cajal body-specific RNAs (scaRNAs), a subtype of snoRNAs, are named for their subcellular localization within Cajal bodies. SCARNA12, which located at the intronic region of PHB2 in chromosome 12p13.31 with 270 nucleotides (nt) in length. It has been reported function as a diagnostic marker for cervical cancer. However, its biological functions and molecular mechanisms in CRC have yet to be elucidated. In this study, bioinformatics analysis revealed that SCARNA12 was highly expressed in CRC and positively correlated with poor prognosis in CRC patients. Additionally, SCARNA12 showed upregulated expression in CRC cell lines and clinical CRC tissue samples. Moreover, SCARNA12 overexpression in SW620 cells accelerated cell proliferation, suppressed the apoptosis rate, and enhanced tumorigenesis in vivo. The knockdown of SCARNA12 expression in HCT116 and HT29 cells resulted in contrasting effects. The functioning of SCARNA12 is mechanically independent of its host gene PHB2. Notably, the overexpression of SCARNA12 activated PI3K/AKT pathway in SW620 cells, and the malignancy degree of CRC cells was attenuated after treatment with MK2206 (a specific AKT inhibitor). Our findings demonstrated that SCARNA12 plays an oncogenic role in CRC progression and can be used as a potential diagnostic biomarker for CRC.

TAS0612, a Novel RSK, AKT, and S6K Inhibitor, Exhibits Antitumor Effects in Preclinical Tumor Models.

The MAPK and PI3K pathways are involved in cancer growth and survival; however, the clinical efficacy of single inhibitors of each pathway is limited or transient owing to resistance mechanisms, such as feedback signaling and/or reexpression of receptor-type tyrosine kinases (RTK). This study identified a potent and novel kinase inhibitor, TAS0612, and characterized its properties. We found that TAS0612 is a potent, orally available compound that can inhibit p90RSK (RSK), AKT, and p70S6K (S6K) as a single agent and showed a strong correlation with the growth inhibition of cancer cells with PTEN loss or mutations, regardless of the presence of KRAS and BRAF mutations. Additional RSK inhibitory activity may differentiate the sensitivity profile of TAS0612 from that of signaling inhibitors that target only the PI3K pathway. Moreover, TAS0612 demonstrated broad-spectrum activity against tumor models wherein inhibition of MAPK or PI3K pathways was insufficient to exert antitumor effects. TAS0612 exhibited a stronger growth-inhibitory activity against the cancer cell lines and tumor models with dysregulated signaling with the genetic abnormalities described above than treatment with inhibitors against AKT, PI3K, MEK, BRAF, and EGFR/HER2. In addition, TAS0612 demonstrated the persistence of blockade of downstream growth and antiapoptotic signals, despite activation of upstream effectors in the signaling pathway and FoxO-dependent reexpression of HER3. In conclusion, TAS0612 with RSK/AKT/S6K inhibitory activity may provide a novel therapeutic strategy for patients with cancer to improve clinical responses and overcome resistance mechanisms.

Ezetimibe inhibits triple-negative breast cancer proliferation and promotes cell cycle arrest by targeting the PDGFR/AKT pathway

Cholesterol levels were strongly associated with tumor progression and metastasis. Targeted cholesterol metabolism has broad prospects in tumor treatment. Ezetimibe, the only FDA-approved inhibitor of cholesterol absorption, has been reported to be able to inhibit angiogenesis in liver cancer. However, the efficacy and specific mechanisms of Ezetimibe in the treatment of Triple-Negative Breast Cancer (TNBC)have not been reported. Our research shows Ezetimibe inhibits TNBC cell proliferation and blocks the cell cycle in the G1 phase. Mechanistically, Ezetimibe inhibits the activation of PDGFRβ/AKT pathway, thereby promoting cell cycle arrest and inhibiting cell proliferation. By overexpressing PDGFRβ in TNBC cells, we found that PDGFRβ significantly reduced the inhibitory effect of Ezetimibe on TNBC cell proliferation and the cell cycle. Similarly, SC79, an AKT agonist, can reduce the proliferation inhibitory and cycle-blocking effects of Ezetimibe on TNBC cells. Furthermore, the AKT inhibitor MK2206 enhanced the inhibitory effect of Ezetimibe on the cell cycle and proliferation ability of TNBC cells overexpressing PDGFRβ. In xenograft tumor models, we also found that Ezetimibe inhibited TNBC growth, an effect that can be blocked by overexpression of PDGFR or activation of AKT. In summary, we have demonstrated that EZ inhibits the PDGFR/AKT pathway, thereby halting TNBC cycle progression and tumor growth.

Safety and Toxicology Study of Hu7691, a Novel AKT Inhibitor, following Oral Administration in Rats.

Hu7691 represents a novel Pan-Akt kinase inhibitor, demonstrating excellent selectivity towards non-AGC kinase families and pronounced inhibitory effects on the proliferation of multiple tumor cell lines. However, there is currently a notable absence of in vivo toxicological research evidence concerning Hu7691. This study represents the first investigation into the 14-day repeated-dose toxicity of Hu7691 in male and female Sprague Dawley (SD) rats. Male rats were administered daily doses of 12.5, 50, 100, and 150 mg/kg/day, while female rats received doses of 12.5, 25, 50, and 75 mg/kg/day for 14 consecutive days. Hematological assessments, organ weights, and histopathological examinations revealed corresponding alterations, suggesting potential target organs for toxicity including the spleen, thymus, and gastrointestinal tract. It is worth noting that the test substance may also impact the liver, kidneys, heart, and ovaries. The No Observed Effect Level (NOAEL) was determined to be no greater than 12.5 mg/kg/day. Based on the observed gender-related toxicity differences in preliminary trials, it is recommended that the high dose reference dose for male animals in formal experiments should not be less than 100 mg/kg/day, while for female animals, it should be less than 50 mg/kg/day.