Potent AKT Inhibitor Research Articles

The effect of food and acid-reducing agents on the pharmacokinetic profile of capivasertib: Results from a randomised, cross-over study.

This two-part, adaptive study assessed the effect of food and an acid-reducing agent (rabeprazole) on the pharmacokinetics (PK) and safety of capivasertib, a potent AKT inhibitor, in clinical development for cancer treatment. In Part 1, healthy participants (n=24) were randomised to receive single-dose capivasertib after overnight fasting, a high-fat, high-calorie meal and with rabeprazole post-overnight fasting in one of six treatment sequences. Based on Part 1 results, a new group of participants (n=24) were randomised (Part 2) to receive capivasertib after overnight fasting, a low-fat, low-calorie meal and modified fasting (food restricted from 2 hours before dosing to 1 hour post-dose) in one of six treatment sequences. Blood samples were collected for PK analyses. Following a high-fat, high-calorie meal, capivasertib exposure increased versus overnight fasting (geometric mean ratio [GMR 90% CI]: area under the concentration-time curve [AUCinf ] 1.32 [1.22, 1.43]; maximum concentration [Cmax ] 1.23 [1.08, 1.41]), but was comparable to that post-modified fasting (GMR: AUCinf 1.13 [0.99, 1.29]; Cmax 0.85 [0.70, 1.04]). AUCinf was similar and Cmax was lower with/without rabeprazole (GMR: AUCinf 0.94 [0.87, 1.02]); Cmax 0.73 [0.64, 0.84]). Capivasertib exposure was similar after a low-fat, low-calorie meal versus overnight fasting (GMR: AUCinf 1.14 [1.05, 1.25]; Cmax 1.21 [0.99, 1.48]) or modified fasting (GMR: AUCinf 0.96 [0.88, 1.05]; Cmax 0.86 [0.70, 1.06]). Safety was consistent with that in larger trials. This study demonstrates that administering capivasertib with food or acid-reducing agents does not lead to clinically relevant PK or safety profile changes.

AT7867 Inhibits the Growth of Colorectal Cancer Stem-Like Cells and Stemness by Regulating the Stem Cell Maintenance Factor Ascl2 and Akt Signaling.

Cancer stem cells (CSCs) are the core factors leading to recurrence, insensitivity to radiotherapy and chemotherapy, and immunotherapy resistance in patients with colorectal cancer. AT7867, a potent oral AKT inhibitor, was found to have antitumor activity in colorectal cancer; however, the effect on colorectal cancer stem cells is still unclear. This study was conducted to clarify the molecular mechanism underlying the CSC growth inhibitory effects of AT7867. We cultured colorectal cancer cells (CRCs) in a serum-free medium and enriched colorectal cancer stem cells. Subsequently, the effects of AT7867 on CSCs were analyzed by CCK-8, colony formation, flow cytometry, and immunofluorescence assays. The results indicated that AT7867 induces G2/M phase arrest and cell apoptosis in cancer stem cells. Subsequently, we identified Ascl2 as the main gene affecting the stemness of colorectal cancer in AT7867 by RNA sequencing. The current study showed that Ascl2 is involved in the metastasis, invasion, and proliferation of CRCs. The next experiments demonstrated that overexpression of Ascl2 did affect the therapeutic effect of AT7867 on CRC stemness. Furthermore, compared with other Akt inhibitors, AT7867 could promote the differentiation of colorectal cancer stem cells. Thus, AT7867 might be a potential antitumor drug candidate to treat CRC by targeting CSCs.

198 (PB078) - Activity and safety of ipatasertib (ipat) for AKT activating mutation and/ or PTEN loss/loss of function solid tumors from MyTACTIC

Background: MyTACTIC (NCT04632992) is a multiarm basket study evaluating the safety/efficacy of targeted therapies as single agents or combinations in advanced unresectable/metastatic solid tumors with specific genomic alterations. We analyzed the potent oral AKT inhibitor ipat in a pan-tumor population with AKT activating mutation and/or PTEN loss/loss of function from MyTACTIC.

In Silico Studies of Piperidine Derivatives as Protein Kinase B Inhibitors through 3D-QSAR, Molecular Docking and Molecular Dynamics Simulation

Background: Protein kinase B (Akt) is a serine/threonine-protein kinase that drives the diverse physiological process. Akt is a promising therapeutic target, which involves cancer cell growth, survival, proliferation and metabolism. Objective: The study aims to design highly active Akt inhibitors, and to elucidate the structural requirements for their biological activity, we analyzed the key binding features and summarized the structural determinants for their bioactivities. Methods: A series of piperidine derivatives have been investigated employing three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking and molecular dynamics simulation. Results: The statistics of the comparative molecular field analysis (CoMFA) model (Q2=0.631, R2=0.951) and the comparative molecular similarity index analysis (CoMSIA) model (Q2=0.663, R2=0.966) indicated that our 3D-QSAR model was accurate and reliable. Besides, the stability of receptor-ligand interactions under physiological conditions was then evaluated by molecular dynamics simulation, in agreement with the molecular docking results. Conclusion: Our study provided valuable insights for the discovery of potent Akt inhibitors.

Abstract P5-16-11: Ipatasertib (ipat) in combination with palbociclib (palbo) and fulvestrant (fulv) in patients (pts) with hormone receptor-positive (HR+) HER2-negative advanced breast cancer (aBC)

Abstract Background: Ipat is a potent oral AKT inhibitor that has been studied in multiple clinical trials, primarily in breast and prostate cancers. Combining fulv and AKT inhibition demonstrated efficacy in pts with HR+ aBC regardless of PI3K/AKT pathway alterations [Jones, Lancet Oncol 2020]. IPATunity150 (NCT04060862) was designed as a phase III trial with an open-label phase Ib portion adding ipat to palbo plus fulv in biomarker-unselected HR+ HER2-negative aBC. The biological rationale was to prevent or delay resistance to CDK4/6 inhibition plus endocrine therapy (ET). AKT1 alterations and PTEN loss have been implicated in resistance to CDK4/6 inhibitors [Wander, Cancer Discov 2020; Costa, Cancer Discov 2020]. We report results from the open-label phase Ib portion. Patients and Methods: The primary objective was to assess safety and pharmacokinetics (PK) of ipat in combination with palbo and fulv; several efficacy parameters were also analyzed. Pts with measurable disease who had not previously received a CDK4/6 inhibitor and had experienced relapse during adjuvant ET were treated with ipat at a dose of 300 mg/d, d1-21 q28d, plus standard-of-care doses of palbo (125 mg/d, d1-21 q28d) plus fulv (500 mg q28d with a loading dose in cycle 1). The selected ipat dose was lower than the 400 mg typically used in other studies because of the anticipated drug-drug interaction (DDI) when combining ipat (a sensitive CYP3A4 substrate and mild-to-moderate CYP3A inhibitor) with palbo (a weak time-dependent CYP3A4 inhibitor and CYP3A substrate). Results: Of the 20 pts treated, 20% were Asian, 65% had primary endocrine resistance (relapse ≤2 years after starting adjuvant ET), 80% had received prior (neo)adjuvant chemotherapy, and 60% had liver and/or lung metastases. At the data cutoff (19 Mar 2021; median follow-up 6.1 months), median treatment duration was 5.1, 5.9, and 5.3 months for ipat, palbo, and fulv, respectively. Treatment was ongoing in 13 pts. Grade 3/4 adverse events (AEs) occurred in 80% of pts (no grade 5). The most common AEs were diarrhea (80% any grade, 10% grade 3, no grade 4) and neutropenia (75% any grade, 45% grade 3, 20% grade 4). Other notable grade ≥3 AEs were grade 3 liver function test elevations in 10%. There were no cases of febrile neutropenia and only 1 case of pneumonitis (grade 1). AEs led to at least one dose reduction of ipat in 6 pts (30%; diarrhea n=3 [with vomiting in 1 pt], neutropenia n=3 [with fatigue in 1 pt]) and of palbo in 9 pts (45%; all for neutropenia). One pt (5%) discontinued ipat and palbo permanently due to ongoing neutropenia after protocol-defined dose reductions. As expected, a DDI led to increased ipat exposure (AUC0-24,ss ~60% and Cmax ~40%) when ipat and palbo were combined. Based on population PK analysis, palbo AUC0-24,ss was ~30% higher than reported from the PALOMA 1 and 2 trials, which was expected and consistent with previously reported physiologically based PK modeling of palbo exposure when administered with moderate CYP3A inhibitors [Yu, J Clin Pharmacol 2017]. All 20 pts had at least one post-baseline tumor assessment. Best overall response rate was 45% (95% CI: 23-68%), including confirmed responses in 7 pts (35%; 5% complete response, 30% partial response) at the clinical cutoff date. Median duration of response was 9.6 months (95% CI: 7.1-not estimable). An additional 10 pts (50%) had stable disease. Progression-free survival results were immature (events in 7 pts). There was no obvious association between efficacy and mutations in PIK3CA/AKT1 as tested in ctDNA. Conclusion: The triplet combination of ipat, fulv, and palbo had an acceptable safety profile generally consistent with that of the individual study drugs; ipat exposure was increased through a predicted DDI. Updated results will be presented. Citation Format: Mafalda Oliveira, Aditya Bardia, Sung-Bae Kim, Naoki Niikura, Cristina Hernando, Gustavo Werutsky, Yoland Antill, Pedro Liedke, Catherine Oakman, Eriko Tokunaga, Seth Wander, Vanessa Krause, Toshinari Yamashita, Frauke Schimmoller, Jacob Rotmensch, Heidi Savage, Rucha Sane, Nicholas Turner. Ipatasertib (ipat) in combination with palbociclib (palbo) and fulvestrant (fulv) in patients (pts) with hormone receptor-positive (HR+) HER2-negative advanced breast cancer (aBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-11.

CRISPR/Cas9-mediated Bag-1 knockout increased mesenchymal characteristics of MCF-7 cells via Akt hyperactivation-mediated actin cytoskeleton remodeling.

Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation.

TRIB3 promotes proliferation, migration, and invasion of retinoblastoma cells by activating the AKT/mTOR signaling pathway.

Tribbles pseudokinase 3 (TRIB3) is a member of the tribbles-related family, which has been determined in various cancers, including renal cell carcinoma, acute promyelocytic leukemia, colorectal cancer, endometrial cancer, and glioma. However, its role in retinoblastoma (RB) has not yet been explored. The expression level of TRIB3 was detected in RB tissues and cell lines using qRT-PCR. The effects of TRIB3 on cell proliferation and invasion capacities were analyzed with MTT, crystal violet, and transwell assays. Western blot and rescue assays were conducted to explore the underlying mechanism. This study found that TRIB3 was upregulated in human RB tissues compared to adjacent normal tissues both at the mRNA and protein levels. Overexpression of TRIB3 significantly promoted cell proliferation and invasion of RB cells, while TRIB3 knockdown inhibited these processes. Moreover, the mechanism deciphering experiments showed that TRIB3 overexpression can increase AKT and mTOR phosphorylation. Conversely, TRIB3 knockdown decreased the phosphorylation of AKT and mTOR. Additionally, MK2206, a potent AKT inhibitor, blocked the promotive effects of TRIB3 in RB cells. This study demonstrated that TRIB3 acts as an oncogene and plays a crucial role in the proliferation and invasion of RB cells via regulating the AKT/mTOR signaling pathway. Therefore, TRIB3 may serve as a potential target in the diagnosis and/or treatment of RB.

NET1 promotes HCC growth and metastasis in vitro and in vivo via activating the Akt signaling pathway.

Neuroepithelial cell transforming gene 1 (NET1), a member of the guanine nucleotide exchange factor family, is involved in various cancers, including gastric cancer, breast cancer and glioma. However, the role of NET1 in hepatocellular carcinoma (HCC) remains largely uncovered. In this study, we found that NET1 expression was upregulated in HCC, and that upregulated NET1 expression was closely associated with poor prognosis and some clinical characteristics in HCC patients. Whilst forced expression of NET1 in HCC cells was observed to significantly promote cell growth and metastasis in vitro and in vivo; downregulation of NET1 was shown to exhibit an opposite inhibitory effect. RNA-seq analysis and gene set enrichment analysis demonstrated that knockdown of NET1 significantly suppressed the level of Akt phosphorylation level and the expression of Akt downstream genes in HCC cells. Moreover, MK2206, a potent Akt inhibitor was shown to block the NET1-induced effects in HCC. Taken together, this study demonstrated that, through the Akt signaling pathway, NET1 plays an oncogenic role in HCC progression and metastasis. Hence, NET1 may potentially be used as a potential therapeutic target and prognostic marker of HCC.

Terpenoids from Azadirachta indica are potent inhibitors of Akt: Validation of the anticancer potentials in hepatocellular carcinoma in male Wistar rats.

Hepatocellular carcinoma (HCC) is the commonest primary malignancy with poor patient prognosis and a high mortality rate. In this study, phytochemicals characterized from Azadirachta indica were screened against the catalytic site of Akt, and the anticancer potentials of the extracted leads (terpenoids) were determined in hepatocellular carcinoma in male Wistar rats. The lead compounds are terpenoids; hence, the extraction of terpenoids from A. indica. Gas chromatography-mass spectrometry (GCMS) was employed for the characterization of the extract. Diethylnitrosamine (DEN)-induced hepatocellular carcinoma in male Wistar rats were treated with the terpenoids extract. The hit, lupeol demonstrates inhibition of Akt and is a potential drug candidate. The terpenoids extract downregulate Akt mRNA and demonstrated anti-Akt downstream signaling effects; anti-inflammatory, anti-angiogenesis, pro-apoptotic, and cell cycle arrest, it also demonstrated cellular regeneration, hepatoprotection, antioxidant potentials, and cellular repairs in hepatocellular carcinoma in male Wistar rats. PRACTICAL APPLICATIONS: Hepatocellular Carcinoma (HCC) is the most common primary malignancy with poor patient prognosis and a high mortality rate. Akt, a serine/threonine kinase is at the crossroad of cell survival, the progression of the cell cycle, cell signaling, cell growth, cell division, and inactivation of pro-apoptotic factors. The inhibition of Akt is an effective therapeutic strategy against HCC. In this study, terpenoids from Azadirachta indica are potent inhibitors of Akt and hitherto demonstrate anticancer potentials. A. indica leaves are readily available globally and more also it is readily cultivated in African and Asia, continents with the highest prevalence of HCC. A. indica terpenoids extract demonstrate anti-HCC potentials and hence should be exploited in this regard.

Design, Synthesis, In vitro and In silico Evaluation of New Hydrazonebased Antitumor Agents as Potent Akt Inhibitors

Background: Akt is overexpressed or activated in a variety of human cancers, including gliomas, lung, breast, ovarian, gastric and pancreatic carcinomas. Akt inhibition leads to the induction of apoptosis and inhibition of tumor growth and therefore extensive efforts have been devoted to the discovery of potent antitumor drugs targeting Akt. Objectives: The objective of this work was to identify potent anticancer agents targeting Akt. Methods: New hydrazone derivatives were synthesized and investigated for their cytotoxic effects on 5RP7 H-ras oncogene transformed rat embryonic fibroblast and L929 mouse embryonic fibroblast cell lines. Besides, the apoptotic effects of the most active compounds on 5RP7 cell line were evaluated using flow cytometry. Their Akt inhibitory effects were also investigated using a colorimetric assay. In silico docking and Absorption, Distribution, Metabolism and Excretion (ADME) studies were also performed using Schrödinger’s Maestro molecular modeling package. Results and Discussion: Compounds 3a, 3d, 3g and 3j were found to be effective on 5RP7 cells (with IC50 values of <0.97, <0.97, 1.13±0.06 and <0.97 μg/mL, respectively) when compared with cisplatin (IC50= 1.87±0.15 μg/mL). It was determined that these four compounds significantly induced apoptosis in 5RP7 cell line. Among them, N'-benzylidene-2-[(4-(4-methoxyphenyl)pyrimidin- 2-yl)thio]acetohydrazide (3g) significantly inhibited Akt (IC50= 0.5±0.08 μg/mL) when compared with GSK690693 (IC50= 0.6±0.05 μg/mL). Docking studies suggested that compound 3g showed good affinity to the active site of Akt (PDB code: 2JDO). According to in silico ADME studies, the compound also complies with Lipinski's rule of five and Jorgensen's rule of three. Conclusion: Compound 3g stands out as a potential orally bioavailable cytotoxic agent and apoptosis inducer targeting Akt.

Ischemic Postconditioning Alleviates Intestinal Ischemia-Reperfusion Injury by Enhancing Autophagy and Suppressing Oxidative Stress through the Akt/GSK-3β/Nrf2 Pathway in Mice.

Aims Ischemic postconditioning (IPO) has a strong protective effect against intestinal ischemia-reperfusion (IIR) injury that is partly related to autophagy. However, the precise mechanisms involved are unknown. Methods C57BL/6J mice were subjected to unilateral IIR with or without IPO. After 45 min ischemia and 120 min reperfusion, intestinal tissues and blood were collected for examination. HE staining and Chiu's score were used to evaluate pathologic injury. We test markers of intestinal barrier function and oxidative stress. Finally, we used WB to detect the expression of key proteins of autophagy and the Akt/GSK-3β/Nrf2 pathway. Results IPO significantly attenuated IIR injury. Expression levels of LC3 II/I, Beclin-1, and p62 were altered during IIR, indicating that IPO enhanced autophagy. IPO also activated Akt, inhibited GSK-3β/Nrf2 pathway. Conclusion Our study indicates that IPO can ameliorate IIR injury by evoking autophagy, activating Akt, inactivating GSK-3β, and activating Nrf2. These findings may provide novel insights for the alleviation of IIR injury.β/Nrf2 pathway.

Circumventing AKT-Associated Radioresistance in Oral Cancer by Novel Nanoparticle-Encapsulated Capivasertib.

Background: Development of radioresistance in oral squamous cell carcinoma (OSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in recent decades. Effective strategies to overcome radioresistance are necessary to improve the therapeutic outcomes of radiotherapy in OSCC patients. Methods: Cells and xenograft tumors were irradiated using the Small Animal Radiation Research Platform. AKT inhibitor capivasertib (AZD5363) was encapsulated into cathepsin B-responsible nanoparticles (NPs) for tumor-specific delivery. Cell viability was measured by alamarBlue, cell growth was determined by colony formation and 3D culture, and apoptosis was assessed by flow cytometry with the staining of Fluorescein isothiocyanate (FITC) Annexin V and PI. An orthotopic tongue tumor model was used to evaluate the in vivo therapeutic effects. The molecular changes induced by the treatments were assessed by Western blotting and immunohistochemistry. Results: We show that upregulation of AKT signaling is the critical mechanism for radioresistance in OSCC cells, and AKT inactivation by a selective and potent AKT inhibitor capivasertib results in radiosensitivity. Moreover, relative to irradiation (IR) alone, IR combined with the delivery of capivasertib in association with tumor-seeking NPs greatly enhanced tumor cell repression in 3D cell cultures and OSCC tumor shrinkage in an orthotopic mouse model. Conclusions: These data indicate that capivasertib is a potent agent that sensitizes radioresistant OSCC cells to IR and is a promising strategy to overcome failure of radiotherapy in OSCC patients.

Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small-molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 h, even after compound washout. Our findings suggest that AKT degradation may confer prolonged pharmacological effects compared with inhibition, and highlight the potential advantages of AKT-targeted degradation.

Abstract C076: In vitro and in vivo combination of ARQ 751 with PARP inhibitors, CDK4/6 inhibitors, Fulvestrant and Paclitaxel

Abstract Background: Dysregulation of the PI3K/AKT signaling pathway is associated with various types of cancers. AKT can be activated through activated receptor tyrosine kinases, gain-of-function mutations of PIK3CA, PTEN deficiency, or AKT amplification or activating mutations such as AKT1-E17K. It has been shown that the PI3K/AKT pathway has contributed to the resistance of conventional chemotherapy, radiation and other targeted therapies. AKT inhibitors have been extensively studied in the clinic and enhance the effects of targeted therapy, chemotherapy and radiation therapy. ARQ 751 is a second-generation AKT inhibitor, which has distinct physico-chemical properties as compared to ArQule’s first-generation inhibitor, miransertib (ARQ 092). ARQ 751 is currently in a phase 1 clinical study in molecularly defined cancer patients. In this study, we assessed the combined effect of ARQ 751 with PARP inhibitors, CDK4/6 inhibitors, an estrogen receptor antagonist and a chemotherapeutic agent both in vitro and in vivo. Methods: In vitro anti-proliferative studies were performed using MTS or Celltiter-Glo as either single agents or in combination with other therapeutic agents. The combination index was calculated based on Cho-Talalay method. In vivo efficacy was tested in patient-derived breast cancer tumors bearing AKT1-E17K mutations and breast cancer cells with PIK3CA mutations. Reverse phase protein arrays (RPPA) were performed on xenograft tumor tissues. Results: ARQ 751 in combination with the PARP inhibitors exhibited enhanced anti-proliferative activity in MDA-MB-468 breast cancer cells. The combination of ARQ 751 and olaparib also suppressed anchorage-independent growth in MDA-MB-231 and HCC1143 cells. In combination with the CDK4/6 inhibitor, ribociclib, ARQ 751 demonstrated superior cell growth inhibition in comparison to the single agents. A synergistic effect was observed at a majority of combination concentration points. In the in vivo efficacy study in a xenograft model with HCC-1954 breast cancer cells, combined treatment of ARQ 751 at 25 mg/kg and paclitaxel at 15 mg/kg showed enhanced antitumor activity with TGI of (90%) in comparison to ARQ 751 alone (46%) or paclitaxel alone (44%) after 3-weeks of treatment. Furthermore, an estrogen receptor positive patient-derived tumor xenograft model harboring the AKT1-E17K mutation was used to assess the effect of ARQ 751 in combination with either fulvestrant or palbociclib or both agents. Combination of ARQ 751 at 25 mg/kg with either fulvestrant at 2.5 mg/kg or palbociclib at 50 mg/kg exerted tumor growth inhibition of 91% or 93%, respectively, as compared to 69% for ARQ 751, 68% for fulvestrant and 38% for palbociclib. When the three agents were combined, tumor regression (TGI >100%) was observed. In order to understand the molecular mechanism involved in the superiority of the combined effect, a RPPA study from xenograft tumor tissues is being performed to assess any alterations in several key pathways. Conclusions: ARQ 751, a highly potent and selective next-generation AKT inhibitor, is combinable with various therapeutic agents including PARP inhibitors, an ER antagonist, CDK4/6 inhibitors, and a chemotherapeutic agent, in vitro and in vivo. The data support the rationale for testing these combinations in the clinic. Citation Format: Yi Yu, Alden Harring, Eva Volckova, Ronald E Savage, Brian Schwartz. In vitro and in vivo combination of ARQ 751 with PARP inhibitors, CDK4/6 inhibitors, Fulvestrant and Paclitaxel [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C076. doi:10.1158/1535-7163.TARG-19-C076

456P - First-in-human phase I study of TAS-117, an allosteric AKT inhibitor, in patients with advanced solid tumours

456P - First-in-human phase I study of TAS-117, an allosteric AKT inhibitor, in patients with advanced solid tumours

Abstract 4922: The combination of MK-2206 and WZB117 exerts a synergistic cytotoxic effect against breast cancer cells

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. The PI3K/Akt/mTOR signaling pathway plays a crucial role in cancer cell growth, survival, and metabolism. It has been demonstrated that MK-2206, a potent allosteric Akt inhibitor, is cytotoxic against many cancer cell lines and is currently in clinical development. Cancer cells consume more glucose and rely on glycolysis for energy production even in the presence of abundant oxygen, a phenomenon called the Warburg effect. Therefore, glucose transporters responsible for delivering glucose into cells have become targets for the development of anticancer drugs. In search for anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, when combined with WZB117, showed a synergistic effect on growth inhibition of breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Western blot analysis of proteins involved in the Akt/mTOR pathway revealed that Akt phosphorylation was markedly suppressed in both cell lines by MK-2206 and WZB117 treatment. Phosphorylation of mTOR and its downstream effectors p70S6K and 4E-BP-1 was also dramatically downregulated in MCF-7 but the effect was not as significant in MDA-MB-231 cells, suggesting that other mechanisms might also contribute to cytotoxicity induced by MK-2206 and WZB117. Further investigation revealed that the combination of MK-2206 and WZB117 induced γ-H2AX, a DNA damage marker, in both cell lines. Results from the comet assay confirmed that MK-2206 and WZB117 induced DNA damage. Protein levels of Rad51 and Ku80, participating in homologous recombination and non-homologous end joining repair respectively, were also decreased in MCF-7 cells, suggesting that MK-2206 and WZB117 may not only cause DNA damage, but also impair DNA repair. Furthermore, MK-2206 or WZB117 alone significantly induced ROS, and the combination of both further increased the ROS level within a short time period. In conclusion, this new finding indicates that MK-2206 and WZB117 may exert a synergistic cytotoxic effect in both ER(+) MCF-7 and triple-negative MDA-MB-231 breast cancer cells via ROS induction, which may in turn cause DNA damage. The combination may also compromise DNA damage repair, and ultimately lead to cell death. This finding may have clinical implications. Citation Format: Yu-Liang Li, Hao-Cheng Weng, Lih-Ching Hsu. The combination of MK-2206 and WZB117 exerts a synergistic cytotoxic effect against breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4922.

Abstract 5788: Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT

Abstract Objective: AKT is a component of the PI3K/AKT/mTOR (PAM) pathway, which plays an important part in cell proliferation, growth and survival, activated in many cancers and has been validated as a therapeutic target in the clinic. Inhibition of AKT activity can block the PAM pathway and proliferation of various types of tumor cells with loss of tumor suppressor PTEN, mutational activation of p110a catalytic subunit of PI3K and amplification of the gene encoding either AKT or PI3K. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of HZB0071, a small molecule AKT kinase inhibitor, in preclinical models of solid cancer with AKT overexpression. Method: Kinase inhibiting activity of HZB0071 was determined with AKT1, 2, 3 kinase assays. Cellular anti-proliferative activity was evaluated with AKT overexpressed LNcap cell line and DU145 cell line without AKT overexpression. The antitumor activity of HZB0071 was evaluate in vivo and the synergistic effect combined with Paclitaxel in AKT highly expressed patient-derived mouse xenograft (PDX) models of Gastric Cancer ST-02-0013 and cell-derived mouse xenograft (CDX) models of Breast Cancer BT474. Result: HZB0071 displayed potent kinase inhibiting activity for AKT1, 2, 3 with IC50 1.3±0.41 nM, 50.3±13.4 nM and 90.4±5.84 nM, respectively. HZB0071 inhibited cell proliferation in AKT overexpressed LNcap cells with IC50 67±7 nM. In contrast, HZB0071 showed much weaker anti-proliferative activity in DU145 cells with IC50 >10,000 nM. HZB0071 showed antitumor efficacy in AKT highly expressed gastric cancer PDX model ST-02-0013 (TGI = 62% @30 mpk, QD as single agent; TGI = 61% @15 mpk + Paclitaxel @10 mpk, QD, in combination with Paclitaxel), as well as in the breast cancer BT474 CDX model (TGI = 88% @30 mpk + Paclitaxel @15 mpk, QD). Conclusion: We have identified a novel potent AKT inhibitor HZB0071. Preclinical studies shows antitumor efficacy of HZB0071 in AKT overexpressed solid cancer models. HZB0071 represents a promising clinical candidate for treating solid cancers with high AKT expression. Citation Format: Gang Li, Lihong Hu, Weifeng Mao, Dongfang Li, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5788.

Design, Synthesis, and Evaluation of a New Series of Thiazole-Based Anticancer Agents as Potent Akt Inhibitors.

In an attempt to develop potent anticancer agents targeting Akt, new thiazole derivatives (1–10) were synthesized and investigated for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma, and NIH/3T3 (healthy) mouse embryonic fibroblast cell lines. The most potent compounds were also investigated for their effects on apoptosis and Akt pathway. The most promising anticancer agent was found to be 2-[2-((4-(4-cyanophenoxy)phenyl)methylene)hydrazinyl]-4-(4-cyanophenyl)thiazole (6), due to its selective inhibitory effects on A549 and C6 cells with IC50 values of 12.0 ± 1.73 µg/mL and 3.83 ± 0.76 µg/mL, respectively. Furthermore, compound 6 increased early and late apoptotic cell population (32.8%) in C6 cell line more than cisplatin (28.8%) and significantly inhibited the Akt enzyme. The molecular docking study was performed to predict the possible binding modes of compounds A, 6, and 8 inside the active site of Akt (PDB code: 4EJN). Molecular docking simulations were found to be in accordance with in vitro studies and, hence, supported the biological activity. A computational study for the prediction of absorption, distribution, metabolism and excretion (ADME) properties of all compounds was also performed. On the basis of Lipinski’s rule of five, the compounds were expected to be potential orally bioavailable agents.

Discovery of chiral dihydropyridopyrimidinones as potent, selective and orally bioavailable inhibitors of AKT

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK2. A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model.

Herbacetin suppresses cutaneous squamous cell carcinoma and melanoma cell growth by targeting AKT and ODC.

Herbacetin is a flavonol compound that is found in plants such as flaxseed and ramose scouring rush herb, it possesses a strong antioxidant capacity, and exerts anticancer effects on colon and breast cancer. However, the effect of herbacetin on skin cancer has not been investigated. Herein, we identified herbacetin as a dual V-akt murine thymoma viral oncogene homolog (AKT) and ornithine decarboxylase (ODC) inhibitor, and illustrated its anticancer effects in vitro and in vivo against cutaneous squamous cell carcinoma (SCC) and melanoma cell growth. To identify the direct target(s) of herbacetin, we screened several skin cancer-related protein kinases, and results indicated that herbacetin strongly suppresses both AKT and ODC activity. Results of cell-based assays showed that herbacetin binds to both AKT and ODC, inhibits TPA-induced neoplastic transformation of JB6 mouse epidermal cells, and suppresses anchorage-independent growth of cutaneous SCC and melanoma cells. The inhibitory activity of herbacetin was associated with markedly reduced NF-κB and AP1 reporter activity. Interestingly, herbacetin effectively attenuated TPA-induced skin cancer development and also exhibited therapeutic effects against solar-UV-induced skin cancer and melanoma growth in vivo. Our findings indicate that herbacetin is a potent AKT and ODC inhibitor that should be useful for preventing skin cancers.