Combination Of AKT Inhibitors Research Articles

Mucin 4 Confers Gemcitabine Resistance and an Unfavorable Prognosis in Patients with Cholangiocarcinoma via AKT Activation.

Cholangiocarcinoma (CCA) exhibits aggressive biological behavior and a poor prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line chemotherapy for advanced CCA but has a response rate of only 20-30%. Therefore, investigating treatments to overcome GEM resistance in advanced CCA is crucial. Among mucin (MUC) family members, MUC4 showed the greatest increase in the resistant versus parental sublines. MUC4 was upregulated in whole-cell lysates and conditioned media from gemcitabine-resistant (GR) CCA sublines. MUC4 mediated GEM resistance by activating AKT signaling in GR CCA cells. The MUC4-AKT axis induced BAX S184 phosphorylation to inhibit apoptosis and downregulated GEM transporter human equilibrative nucleoside transporter 1 (hENT1) expression. The combination of AKT inhibitors and GEM or afatinib overcame GEM resistance in CCA. In vivo, capivasertib (an AKT inhibitor) increased GEM sensitivity in GR cells. MUC4 promoted EGFR and HER2 activation to mediate GEM resistance. Finally, MUC4 expression in patient plasma correlated with MUC4 expression. Paraffin-embedded specimens from non-responders expressed significantly more MUC4 than did those from responders, and this upregulation was associated with poor progression-free survival and overall survival. In GR CCA, high MUC4 expression promotes sustained EGFR/HER2 signaling and AKT activation. The combination of AKT inhibitors with GEM or afatinib might overcome GEM resistance.

An effective AKT inhibitor-PARP inhibitor combination therapy for recurrent ovarian cancer

BackgroundAlthough the use of PARP inhibitor has received considerable amount of attention in ovarian cancer, PARP inhibitor resistance still emerges with disease progression. PI3K/AKT pathway inhibitors have been proposed to synergize with PARP inhibition to slow tumor growth, but the exact molecular mechanisms are still elusive.MethodsUtilizing tumor samples from recurrent EOC patients with platinum resistance and prior PARP inhibitor use, Mini PDX and PDX models were established to study the anti-tumor effect of AKT inhibitor (LAE003) and LAE003/PARP inhibitor (Olaparib) in combination. Five ovarian cancer cell lines were treated with Olaparib or LAE003 or in combination in vitro. Cell viability and apoptosis rate were measured after the treatments. Combination index by the Chou–Talalay was used to evaluate in vitro combination effect of Olaparib and LAE003. The protein expression level of PARP1 and PAR was measured by Western blot in cell lines and by immunohistochemistry in PDX tumor tissues.ResultsTumor cells from two out of five platinum-resistant ovarian cancer patients previously treated with PARP inhibitor were sensitive to AKT inhibition in Mini-PDX study. Inhibition of AKT further increased the response of tumor cells to Olaparib in a PDX model derived from a recurrent platinum-resistant ovarian cancer patient. Additive anti-proliferation effect of LAE003 and Olaparib was also observed in three ovarian cancer cell lines with high PARP1 protein level. Interestingly, mechanism study revealed that AKT inhibition decreased PARP enzyme activity as measured by PAR level and/or reduced PARP1 protein level in the tumor cell lines and PDX tumor tissues, which may explain the observed combined anti-tumor effect of LAE003 and Olaparib.ConclusionCollectively, our results suggest that the combination of AKT inhibitor and PARP inhibitor could be a viable approach for clinical testing in recurrent ovarian cancer patients.

Abstract 1420: GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines

Abstract Androgen deprivation therapy (ADT) with or without antiandrogens such as enzalutamide is the mainstay of treatment in advanced prostate cancer (PC). However, new therapeutic strategies are needed for patients who relapse on antiandrogen therapy. The glucocorticoid receptor (GR) has been implicated as a potential antiandrogen bypass mechanism. We previously showed that ORIC-101, a potent and selective, orally bioavailable small molecule GR antagonist, reverses GR-mediated resistance to enzalutamide in preclinical PC models. In the clinic, we are evaluating ORIC-101 in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Activation of the PI3K/AKT pathway through PTEN loss is a poor prognostic factor and another potential resistance mechanism to anti-hormone directed therapies. Reciprocal crosstalk between androgen signaling and PI3K/AKT pathway activation forms the basis for the clinical development of AKT inhibitors (AKTi) in combination with ADT or antiandrogens (NCT03072238, NCT04087174). In this study, we tested preclinically whether activated GR confers resistance to the combination of AKT inhibitors with enzalutamide and whether co-treatment with ORIC-101 reverses GR-mediated resistance to an enzalutamide/AKTi doublet. We first evaluated the effects of enzalutamide plus AKTi treatment on GR levels, GR target genes and activity in three prostate cancer cell lines: PTEN-null line LNCaP and PTEN-wildtype lines VCaP and CWR22PC. We tested three AKT inhibitors: the ATP-competitive inhibitors ipatasertib (GDC-0068) and capivasertib (AZD5363), in addition to the allosteric inhibitor MK2206, in CSS media and 10% FBS, with or without supplementation of the synthetic glucocorticoid, dexamethasone. An increase in the protein and mRNA levels of GR and GR target genes was observed after 7 days of enzalutamide treatment. In the AKTi plus enzalutamide setting, AKT inhibition did not block the enzalutamide-mediated GR upregulation in either PTEN-null or PTEN-wildtype cells. We next evaluated whether increased GR activity mediates resistance to the enzalutamide/AKTi doublet with ipatasertib or capivasertib, and whether this resistance can be reversed with ORIC-101. GR activation with dexamethasone promoted tumor cell growth and androgen-regulated gene expression in the presence of enzalutamide and ipatasertib or capivasertib, using a 21-day proliferation assay in CWR22PC cells. ORIC-101 was able to reverse these dexamethasone-induced effects, confirmed by reduced secreted PSA levels and cell number measured at end of study. These findings indicate that GR upregulation and activation, an established resistance mechanism for antiandrogens, may drive resistance when combined with an AKT inhibitor, and the GR antagonist ORIC-101 is able to overcome this resistance and restore antitumor activity. Citation Format: Shravani Barkund, Haiying Zhou, Lori S. Friedman, Anneleen Daemen. GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1420.

LncRNA H19 downregulation confers erlotinib resistance through upregulation of PKM2 and phosphorylation of AKT in EGFR-mutant lung cancers.

First-generation EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib have significant activity in NSCLC patients with activating EGFR mutations. However, EGFR-TKI resistance inevitably occurs after approximately 12 months of treatment. Acquired mechanisms of resistance, other than secondary mutations in EGFR (T790M) which account for 50-60%, are less well understood. Here, we identified lncRNA H19 as a significantly downregulated lncRNA in vitro models and clinical specimens with acquired EGFR-TKI resistance, H19 knockdown or overexpression conferred resistance or sensitivity, respectively, both in vitro and in vivo models. H19 downregulation contributed to erlotinib resistance through interaction and upregulation of PKM2, which enhanced the phosphorylation of AKT. AKT inhibitors restored the sensitivity of erlotinib-resistant cells to erlotinib. In EGFR-mutant patients treated with EGFR-TKIs, low H19 levels were associated with a shorter progression-free survival (PFS) (P=0.021). These findings revealed a novel mechanism of low-level H19 in the regulation of erlotinib resistance in EGFR-mutant lung cancers. Combination of AKT inhibitors and EGFR-TKIs could be a rational therapeutic approach for some subgroups of EGFR-mutant lung cancer patients.

Abstract A49: Acquired resistance of non-small cell lung cancer to EGFR-TKI: Role of AKT3

Abstract The discovery of EGFR-TKIs has had a dramatic impact on the treatment of NSCLC with EGFR activation mutation. Although they improve the overall survival, the main obstacle is the emergence of acquired resistance. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance), generated and showing “off-target resistance,” were employed to investigate the molecular and cellular characteristics of the acquired resistance to EGFR-TKIs. AKT activation (p-AKT) in both parental cells was more sensitive to EGFR-TKIs than that in resistant cells. We found that mRNA and protein of AKT3, but not AKT1 and AKT2, were upregulated in both HCC827/IR and H1975/AR cells. Immunoprecipitation of AKT3 demonstrated its contribution to AKT activation. Knockdown of AKT3 in both resistant cells decreased basal AKT activation (p-AKT). However, the insensitive inhibition of gefitinib on AKT activation (p-AKT) in HCC827/IR cells and AZD9291in H1975/AR cells remained even if AKT3 was knocked down. AKT3 protein, but not AKT1 and AKT2, was found to be overexpressed in resistant cells treated with gefitinib or AZD9291 as well as AKT3 KD, indicating the crucial role of AKT3 in acquired resistance to EGFR-TKIs. Combination of AKT inhibitors with AZD9291 showed synergistic inhibition on cell viability of H1975/AR cells. Therefore, AKT3 upregulation might be one of the mechanisms for acquired resistance to EGFR-TKIs, and it might serve as a predictive biomarker for EGFR-TKIs therapy. Citation Format: Shih-Hsiang Huang, Jin-Yuan Shih, Ching-Chow Chen. Acquired resistance of non-small cell lung cancer to EGFR-TKI: Role of AKT3 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A49.

S-15 in combination of Akt inhibitor promotes the expansion of CD45RA\u2212CCR7+ tumor infiltrating lymphocytes with high cytotoxic potential and downregulating PD-1+Tim-3+ cells as well as regulatory T cells

BackgroundAutologous tumor-infiltrating lymphocytes (Tils) immunotherapy is a promising treatment in patients with advanced hepatocellular cancer. Although Tils treatment has shown great promise, their persistence and the efficacy after adoptive-transfer are insufficient and remain a challenge. Studies have demonstrated that IL-15 and Akt inhibitor can regulate T cell differentiation and memory. Here, we constructed S-15 (Super human IL-15), a fusion protein consisting of human IL-15, the sushi domain of the IL-15 receptor α chain and human IgG-Fc. Herein we compared the effects of S-15 with IL-2 or in combination with Akti on the expansion and activation of Tils.MethodsHepatocellular cancer tissues were obtained from 6 patients, Tils were expanded using IL-2, IL-2/S-15, IL-2/Akti or in combination IL-2/S-15/Akti. At day 10, anti-CD3 antibody was added to the culture media and expanded to day 25. The composition, exhaustion and T-cell differentiation markers (CD45RA/CCR7) were analyzed by flow cytometry.ResultsWe found that IL-2/S-15/Akti expanded Tils and showed the highest percentage of central memory CD45RA−CCR7+ phenotype prior to anti-CD3 antibody activation and after anti-CD3 antibody activation. T cells cultured with IL-2/S-15/Akti exhibited a mixture of CD4+, CD8+, and CD3+CD4−CD8− T cells; S-15 in combination with Akt inhibitor downregulated the expression of PD-1+Tim-3+ on Tils and decreased the Tregs in Tils. Additionally, the Tils expanded in the presence of the Akt inhibitor and S-15 showed enhanced antitumor activity as indicated by the increase in IFN-γ producing tumor infiltrating CD8+ T cells and without comprising the Tils expansion.ConclusionOur study elucidates that IL-2/S-15/Akti expanded Tils and represent a viable source for the cellular therapy for patients with hepatocellular cancer.

The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells.

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. Glucose transporters are potential targets for the development of anticancer drugs. In search of anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, a potent allosteric Akt inhibitor, when combined with WZB117, showed a synergistic effect on growth inhibition and apoptosis induction in 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. Mechanism studies revealed that MK-2206 and WZB117 exert a synergistic cytotoxic effect in both MCF-7 and MDA-MB-231 breast cancer cells by inhibiting Akt phosphorylation and inducing DNA damage. The combination may also compromise DNA damage repair and ultimately lead to apoptosis. Our findings suggest that the combination of Akt inhibitors and GLUT1 inhibitors could be a novel strategy to combat breast cancer.

Abstract 5451: Testing a combination of AKT inhibitor (AZD5363) with PARP inhibitor olaparib plus carboplatin in TNBC

Abstract Background: AKT, a hallmark of cancer in a wide range of solid tumors, is a serine/threonine protein kinase. Inhibition of AKT is known to cause the perturbation of cell proliferation signals and induction of apoptosis in several preclinical in vitro and in vivo models. AKT inhibitor AZD5363 shows promise in different solid tumors in patients with activating mutations of AKT (E17K) (Hyman D. M. et al., JCO 2017), the efficacy is not substantial in patients with PIK3CA mutations (Banerji. U, CCR 2017). PI3K-PTEN-AKT pathway alterations are very common in TNBC. Alterations of AKT1/2/3 (15%), PTEN (29%) and PIK3CA (25%) mediated upregulation of the PI3K pathway are common in TNBC (De et al., 2016; cBioPortal). In our Avera cohort (January 2014-June 2017) of TNBC patients (n=93), we have recorded a total of 16.5 % alteration in AKT1/2/3. We have also identified an integral role of the PI3K-AKT pathway in the DDR-mediated antitumor action of PARP inhibitor in TNBC (De et al., 2014). Aim: Here we tested the role of genetic background in determining the effectiveness of a combination of AZD5363 with PARP inhibitor Olaparib (O, Lynparza) plus carboplatin (C) in TNBC. Methods: Using cell line based models of TNBC with defined genetic backgrounds, we studied proliferation (Incucyte) and cell cycle by PI (Accuri). Mechanistic induction of apoptosis was evaluated by live-cell triple immuno-fluorescence (IF) (MitoView Blue+NucView488 Casp3 substrate + CF 594 AnnexinV) with a simultaneous quantification of Annexin (Accuri). Long-term clonogenic response to triple combination was tested using 3D On-Top Matrigel assay. Result: AZD5363 treatment in PTEN-null SUM149 cells with BRCA1 mutation [2288delT (fs>735X)] responded synergistically to O and C with a dose-dependent increase apoptosis but with limited G1 increase. AZD5363 treatment in PTEN-null MDA-MB468 cells with wt BRCA1 led to an additive effect in slowing proliferation when combined with O and C and a very strong G1 increase but limited induction of apoptosis. AZD5363 as a single agent slowed proliferation and increased G1 in PIK3CA (P539R/H1074R) mutated BT20 cells with a concomitant increase in apoptosis. Mechanistically AZD5363 inhibited pophoyrlation of AKT substrates PRAS40 and GSKβ in TNBC cell lines. As expected KRAS/BRAF mutated (G13D/G464V), BRCA1 wt MDA-MB231 cells showed no effect on either proliferation or apoptosis. Best effect on the inhibition of clonogenic growth (reducing 3D colony formation and size as compared to control over single or double treatment) was recorded in SUM149 compared to MDA468 > BT20, > HCC1937. Summary: Here we present the effect of the combination of AZD5363 and O plus C in several TNBC cell line based models. Our data demonstrate that maximum effect of the triple combination is seen in PTEN-null cell line harboring BRCA1 mutations followed by PTEN-null cell BRCA1wt. Mechanistic work utlizing AKT1-E17K transfected cells is ongoing. Citation Format: Jennifer H. Carlson, Mark J. O'Connor, Casey Williams, Pradip De, Nandini Dey, Brian Leyland-Jones. Testing a combination of AKT inhibitor (AZD5363) with PARP inhibitor olaparib plus carboplatin in TNBC [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 5451.

Luteoloside induces G0/G1 arrest and pro-death autophagy through the ROS-mediated AKT/mTOR/p70S6K signalling pathway in human non-small cell lung cancer cell lines

Autophagy has attracted a great deal of interest in tumour therapy research in recent years. However, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, on autophagy remains poorly understood in human lung cells. In the present study, we have investigated the anticancer effects of luteoloside on non-small cell lung cancer (NSCLC) cells and demonstrated that luteoloside effectively inhibited cancer cell proliferation, inducing G0/G1 phase arrest associated with reduced expression of CyclinE, CyclinD1 and CDK4; we further found that treatment with luteoloside did not strongly result in apoptotic cell death in NSCLC (A549 and H292) cells. Interestingly, luteoloside induced autophagy in lung cancer cells, which was correlated with the formation of autophagic vacuoles, breakdown of p62, and the overexpression of Beclin-1 and LC3-II, but not in a human bronchial epithelial cell line (BEAS-2B). Notably, pretreatment of cancer cells with 3-MA, an autophagy inhibitor, protected against autophagy and promoted cell viability but not apoptosis. To further clarify whether luteoloside-induced autophagy depended on the PI3K/AKT/mTOR/p70S6K signalling pathway, a major autophagy-suppressive cascade, cells were treated with a combination of AKT inhibitor (LY294002) and mTOR inhibitor (Rap). These results demonstrated that luteoloside induced autophagy in lung cancer cell lines by inhibiting the pathway at p-Akt (Ser473), p-mTOR and p-p70S6K (Thr389). Moreover, we observed that luteoloside-induced cell autophagy was correlated with production of reactive oxygen species (ROS). NAC-mediated protection against ROS clearly implicated ROS in the activation of autophagy and cell death. In addition, the results showed that ROS served as an upstream effector of the PI3K/AKT/mTOR/p70S6K pathway. Taken together, the present study provides new insights into the molecular mechanisms underlying luteoloside-mediated cell death in NSCLC cells and supports luteoloside as a potential anti-cancer agent for targeting NSCLC through the induction of autophagy, inhibition of proliferation and PI3K/AKT/mTOR/p70S6K signalling.

Abstract 4731: Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer

Abstract Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2017-4731

Abstract 391: Implications of Akt inhibition for neoadjuvant radiotherapy: improving the rectal cancer treatment

Abstract Resistance to therapy is a major obstacle to a favorable outcome in cancer treatment. For rectal cancer, neoadjuvant chemoradiotherapy (nCRT) can lead to complete tumor regression in a significant proportion of patients (up to 40%) but approximately 60% of patients have only partial or no tumor remission after nCRT. A number of works searching for a differential gene expression signature has failed. As those gene signatures relies on the sample set, there is an increasing agreement that biological understanding is necessary to produce a signature in terms of pathways and biological processes. In this regard we applied pathway enrichment analysis to RNA-Seq data in order to identify biological pathways involved in tumor resistance and discriminate rectal cancer patients that is refractory to neoadjuvant treatment. Mitochondrial oxidative phosphorylation and phosphatidylinositol signaling were identified as the main pathways involved in resistance to nCRT in patients with rectal cancer. Both pathways have been reported as involved in resistance to radiotherapy and chemotherapy in solid tumors but according to the status of gene regulation observed and the literature, there is evidence that Akt pathway could be central in promoting resistance.Therefore, we evaluated the status of Akt activation by immunohistochemistry of pre-treatment biopsies of rectal tumors as well as by immunoblot of colorectal cancer cell lines utilizing antibodies against phosphorylated Akt (pS473). As a result, we observed an increased proportion of Akt phosphorylation in the resistant cell line, SW480, and in patients with TRG 0-2 in comparison to those with TRG 3-4 and in the sensitive cell line, SW48. To evaluate the implication of Akt in promoting nCRT resistance, we performed colony formation assays using Akt inhibitors as radiosensitizers. We observed an additive effect using MK2206 at sub-doses in combination to radiotherapy (2 Gy) in the resistant cell line. In fact, Akt inhibition improved in approximately 40% the tumoricidal effects of radiotherapy. Moreover, at IC50 dose this effect is even higher, reaching 60% the improvement of radiotherapy. Furthermore, the treatment of MK2206 with radiotherapy alone or in combination with 5-FU have similar effectiveness. In this setting, as Akt is involved in a range of cellular functions that promote tumor progression and metastasis, we propose that combination of Akt inhibitor and radiotherapy as neoadjuvant treatment would maximize tumor remission and improve the rectal cancer treatment. Citation Format: Fernanda C. Koyama, Camila Ramos, Angelita Habr-Gama, Venâncio Avancini Ferreira Alves, Rodrigo O. Perez, Anamaria Aranha Camargo. Implications of Akt inhibition for neoadjuvant radiotherapy: improving the rectal cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 391.

Abstract A31: High-grade serous ovarian cancer subtyping identifies pathways for targeted therapy.

Abstract Introduction: We recently identified three prognostic groups for high-grade serous ovarian cancers (HGS-OvCa) patients treated with platinum and taxane using transcriptomics and clinical data (Chen, Huhtinen, et al. Cancer Res. 2015). In this contribution we first use pathway analysis methods to identify transcript markers and pathways that drive resistance to platinum and experimentally inhibit of the key pathways to restore sensitivity to platinum. Two of the prognostic groups (Poor I and Poor II) were associated with poor survival but have distinct expression profiles. Poor II express high stromal response genes and confirms the C1 subtype characterized by Tothill et al. (2008), whereas Poor I is previously unidentified and characterized by genome-wide hypermethylation. Our objectives here are to: 1) identify key pathways for Poor I and Poor II based on transcriptomics data (data unpublished), 2) inhibit the herein identified pathways to suggest combinatorial therapeutics options for platinum resistance HGS-OvCa patients (data unpublished), 3) test the impact of hypermethylation drug Decitabine to platinum resistance in vitro (data partially unpublished). Methods: Expression levels were qRT-PCR validated using well described prospective patient cohort. The role of hypermethylation and specific pathways on cell viability were studied in vitro using commercial (CAOV4, NIHOVCAR3, OVCAR8, TYKnu) and spheroidal primary HGS-OvCa cell lines. Results: Poor I subtype presents with significantly higher methylation of CpG loci (p < 0.001) as compared with Good or Poor II subtypes. We observed significant up-regulation of three isoforms of DNA methyltransferase 3B (DNMT3B) in Poor I samples. The role of hypermethylation was further validated in vitro using patient derived cell lines with platinum resistance. Inhibition of methylation by Decitabine exhibited strong effect on cell viability specifically on Poor I characteristic HGS-OvCa cell lines. The data suggests that clinical trials testing the effect of Decitabine should be focused to patients with Poor I disease. To evaluate signaling pathways that are associated with platinum-taxane resistance we evaluated common pathways enriched in Poor I and Poor II using the transcriptomics data available at The Cancer Genome Atlas repository. The most prominent pathway is MAPK signaling pathway of which four genes (FAS, MAPK1, AKT and NR4A1) emerged from the analysis. Pathway analysis further suggested ERK5 and AKT as upstream regulators for NR4A1 in platinum-taxane resistance in HGS-OvCa. The impact of ERK5 and AKT pathways on platinum resistance was evaluated in vitro using commercial and primary HGS-OvCa cell lines with wild-type BRCA1/2. The combination of AKT-inhibitor API-2 and Cisplatin resulted in a marked decrease in cell viability over Cisplatin alone whereas AKT-inhibitor alone had only little effect. In contrast, ERK5 inhibition with XMD8-92 combined with Cisplatin did not have an effect on HGS-OvCa cell viability. However, XMD8-92 enhanced the effect of API-2 in subset of the cell lines. Thus, we tested whether combination of the three drugs results in better response. In general, the addition of XMD8-92 did not improve the effect of API-2 and Cisplatin. However, in two highly platinum resistant cell lines, CAOV-4 and M022i primary cell line, the combination of API-2, XMD8-92 and Cisplatin produced the highest reduction in cell viability. In conclusion, we have identified pathways that are likely driving drug resistance in HGS-OvCa and tested the impact of platinum combined with targeted AKT and ERK5 inhibition in vitro. We further show that the Poor I type HGS-OvCa cells are sensitive to Decitabine. Thus, Decitabine may be an effective (combinatorial) treatment option for patients with the Poor I subtype disease. Combination of AKT and ERK5 inhibitors with platinum can also be effective for a subset of platinum resistant ovarian cancer patients. Citation Format: Kaisa Huhtinen, Ping Chen, Katja Kaipio, Piia Mikkonen, Viljami Aittomäki, Rony Lindell, Johanna Hynninen, Annika Auranen, Seija Grenman, Rainer Lehtonen, Sampsa Hautaniemi, Olli Carpén. High-grade serous ovarian cancer subtyping identifies pathways for targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A31.

Additive effect by combination of Akt inhibitor, MK-2206, and PDGFR inhibitor, tyrphostin AG 1296, in suppressing anaplastic thyroid carcinoma cell viability and motility

The phosphatidylinositol-3-kinase/Akt pathway and receptor tyrosine kinases regulate many tumorigenesis related cellular processes including cell metabolism, cell survival, cell motility, and angiogenesis. Anaplastic thyroid carcinoma (ATC) is a rare type of thyroid cancer with no effective systemic therapy. It has been shown that Akt activation is associated with tumor progression in ATC. Here we observed the additive effect between an Akt inhibitor (MK-2206) and a novel platelet-derived growth factor receptor inhibitor (tyrphostin AG 1296) in ATC therapy. We found an additive effect between MK-2206 and tyrphostin AG 1296 in suppressing ATC cell viability. The combination of MK-2206 and tyrphostin AG 1296 induces additive apoptosis, additive suppression of the Akt signaling pathway, as well as additive inhibition of cell migration and invasion of ATC cells. Furthermore, the combination of MK-2206 and tyrphostin AG 1296 induced additive suppression of ATC tumor growth in vivo. In summary, our studies suggest that the combination of Akt and receptor tyrosine kinase inhibitors may be an efficient therapeutic strategy for ATC treatment, which might shed new light on ATC therapy.

Synergy with combination of AKT inhibitor (MK-2206) and paclitaxel in head and neck squamous cell carcinoma.

e13532 Background: Though there have been many advances in the treatment of HNSCC, the 5-year survival rate remains at 50%. This is partly due to molecular alterations such as the activation of the PI3k-AKT pathway in HNSCC, which has been associated with treatment failure. We stained human tissue samples and saw increased expression of p-AKT and pS6 in HNSCC compared to normal and dysplasia. This led us to investigate the role of the AKT inhibitor MK-2206 in combination with an already established chemotherapeutic agent, paclitaxel. Methods: Cell viability and Combination index were used to determine synergy in 4 HNSCC cell lines. PI exclusion and Annexin/PI staining were used to determine cell death and apoptosis, respectively. Autophagy was measured through changes in LC3BII protein levels using western blot analysis and a stably transfected GFP-LC3BII cell line was used to detect changes in protein localization under confocal microscopy. Results: All cell lines were sensitive to paclitaxel while only some were sensitive to MK-2206. However, there was effective synergy to the combination treatment in all cell lines. Apoptotic synergy was observed in some cell lines indicating that the combination stimulates cell death in the appropriate context. In one autophagy competent cell line, MK-2206 induced robust autophagy while paclitaxel blocked a latter step in the pathway leading to an accumulation of autophagosomes prior to induction of apoptosis. Conclusions: Paclitaxel and MK-2206 appears to be an effective combination in HNSCC possibly through their effect on autophagy trafficking, leading to an accumulation of autophagosomes and eventual apoptosis. Clinical trials examining this combination are warranted.

IS1-4 - Synergistic Anti-Tumor Effect of Sorafenib in Combination with AKT Inhibitors in Hepatocellular Carcinoma

ABSTRACT Background Sorafenib is currently the only approved agent for hepatocellular carcinoma (HCC). However, its clinical efficacy in HCC is limited. Previous studies indicate that compensatory activation of AKT may contribute to the resistance to sorafenib. We hypothesized that combination of sorafenib and AKT inhibitors could enhance the antitumor effect of sorafenib in HCC. Methods We analyzed the cellular and molecular effects of sorafenib in combination with inhibitors of AKT in human HCC cell lines, and evaluated their combinational effects against HCC in vitro and in vivo. Results HCC cells, including Huh7, Hep3B, and SKhep1 cells, expressed high levels of p-AKT in a prompt and sustained manner upon treated with sorafenib. Down-regulation of AKT by siRNA or inhibition of phospho-AKT by AKT inhibitors (MK-2206 or perifosine) enhanced the anti-proliferative effect of sorafenib in cultured HCC cells. The combination indexes of sorafenib and AKT inhibitors were Conclusions Inhibition of AKT by AKT inhibitors improved the antitumor effect of sorafenib against HCC in vitro and in vivo. Combination of AKT inhibitors and sorafenib warrants further consideration for clinical development in HCC. (The study was supported by NSC97-2628-B-002-004-MY3 and NSC100-2325-B-002-043-.)

Imatinib mesylate (STI571) enhances amrubicin-induced cytotoxic activity through inhibition of the phosphatidylinositol 3-kinase/Akt pathway in small cell lung cancer cells

Small cell lung cancer (SCLC) is characterized by autocrine mechanisms. Stem cell factor (SCF) and its receptor c-kit can activate Akt and extracellular signal-regulated kinase (Erk) pathways. Imatinib mesylate (STI571) can inhibit c-kit tyrosine kinase activity, but clinical trials have resulted in failure. We investigated the possibility of SCF/c-kit-targeted therapy against SCLC. Using c-kit-positive SCLC cells (H209 and H69 cells) and SCF as a model of the autocrine mechanisms, the effects of SCF, LY294002, PD98059 or STI571 on Akt and Erk were assessed by Western blot analysis. The cell growth inhibitions of cisplatin, etoposide irinotecan and amrubicin (AMR) with or without SCF, LY294002, PD98059 or STI571 were evaluated by MTT assay. Treatment with SCF activated Akt and Erk and the activations were inhibited by STI571 in H209 but not in H69 cells. LY294002 and PD98059 inhibited SCF-induced Akt and Erk activation in H209 cells, respectively. STI571 alone did not exert growth inhibition in the SCF-treated cells. In H209 cells, SCF decreased the cytotoxicity of AMR, but not of other drugs. In H69 cells, SCF did not affect sensitivity to any drugs. LY294002 but not PD98059 restored or enhanced AMR-sensitivity in SCF-treated H209 or untreated H69 cells, respectively. STI571 restored the AMR-sensitivity of SCF-treated H209 cells to the basal level. If the SCF/c-kit contributes to Akt activation in vivo, the combination of STI571 and AMR may be effective against SCLC. Additionally, using a combination of AKT inhibitors and AMR may be a promising treatment in the future.

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-α-induced Interleukin-6 Synthesis in Osteoblasts

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl-CHIRO-inositol 2-( R)-2- O-methyl-3-O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.

Possible involvement of phosphatidylinositol 3-kinase/Akt signal pathway in vasopressin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells

We previously reported that p38 mitogen-activated protein (MAP) kinase takes a part in arginine vasopressin (AVP)-induced heat shock protein 27 (HSP27) phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the phosphorylation of HSP27 in these cells. AVP time-dependently induced the phosphorylation of PI3K and Akt. Akt inhibitor, 1 l-6-hydroxymethyl- chiro-inositol 2- (R)-2- O-methyl-3- O-octadecylcarbonate, partially suppressed the phosphorylation of HSP27. The AVP-induced HSP27 phosphorylation was attenuated by LY294002, a PI3K inhibitor. The combination of Akt inhibitor and SB203580, a p38 MAP kinase inhibitor, completely suppressed the AVP-induced phosphorylation of HSP27. Furthermore, LY294002 or Akt inhibitor did not affect the AVP-induced phosphorylation of p38 MAP kinase and SB203580 did not affect the phosphorylation of PI3K or Akt. These results suggest that PI3K/Akt plays a part in the AVP-induced phosphorylation of HSP27, maybe independently of p38 MAP kinase, in aortic smooth muscle A10 cells.