Phosphatidylinositol 3-kinase Inhibitor ZSTK474 Research Articles

Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3β (GSK3β), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3β pathways, and mediates the G1/S transition to control cell proliferation.

The Antileukemia Activity of ZSTK474 on U937 Cells

To investigate the antileukemia activity of phosphatidylinositol-3 kinase (PI3K) inhibitor ZSTK474 on human leukemia cell line U937. MTT, soft agar assay, flow cytometric analysis and western blot were used to detect the effect of ZSTK474 on U937 cell proliferation, tumorigenicity, cell cycle, cell apoptosis and phosphorylation levels of the key factor of PI3K/AKT pathway. Chou-Talalay method was used to evaluate the combination of ZSTK474 with Cytarabine or Homoharringtonine. PI3K inhibitor ZSTK474 could inhibit the proliferation and tumorigenicity of U937 cell, induce G1 cell cycle arrest and promote cell apoptosis, and enhance intracellular ROS production and decrease MMP, downregulate Cyclin D1, p-Rb, BCL-2 and upregulate p27, caspase-9, caspase-3, PARP and BAX. Furthermore, the phosphorylation of PDK1, GSK-3β, AKT and mTOR could be downregulated by ZSTK474. The combination of ZSTK474 with Homoharringtonine was synergistic. ZSTK474 can inhibit the pathway of PI3K/AKT, ZSTK474 alone or in combination with Homoharringtonine shows potential antileukemia activity on U937 cells.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.

Abstract LB-A20: Novel Dual Oncogenic Target Inhibitor against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K)

Abstract The Ras/MEK/ERK and PI3K/Akt/mTor pathways play a central role in the regulation of normal cell growth, division and differentiation. Dysregulation of these signaling pathways driven by oncogenic mutations/activation leading to elevated kinase activity has been demonstrated in many human cancers. Strong evidence suggests the existence of a feedback loop with crosstalk between these two signaling cascades leading to redundancy in survival pathways. Consequently, monotherapy targeting a single cascade may be insufficient to induce tumor cell death due to drug resistance mechanisms. Initial biological results are presented from a series of novel small molecule kinase inhibitors specifically designed to simultaneously target both MEK1 and PI3K. Structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive class of MEK inhibitors PD0325901, respectively, were covalently combined to provide single compound dual inhibitors. Inhibitors showed potent MEK1 inhibition (0.015 < IC50 (nM) < 56.7) and PI3K inhibition (54 < IC50 (nM) < 341) in tumor cell culture inhibition assays. Cellular efficacy studies were accomplished and revealed several of the designed inhibitor analogs demonstrated near complete inhibition of MEK1 and PI3K kinase activity by western blot analysis in A549 lung adenocarcinoma and D54 glioma cell lines at concentrations as low as 5 μM. Inhibitors also produced dose-dependent decreases in cell viability in these cell lines similar to that displayed with the combined administration of equivalent doses of ZSTK474 and PD0325901. In vivo evaluation of selected compounds was undertaken to evaluate oral bioavailability and activity. In preliminary studies conducted in D54 glioma tumor bearing mice, a 95% and 67% inhibition of tumor ERK1/2 and Akt phosphorylation, respectively, was found at 2 h post-administration as determined by western blot analysis. This study reveals that dual targeted small molecules designed to inhibit MEK1/PI3K pathways were effective in target modulation in intact mouse tumor models. Overall, this study details a highly novel approach for combined MEK1/PI3K inhibition with first in vivo results. Citation Format: Brian D. Ross, Hao Hong, Hanxiao Wang, Charles A, Nino, Marcian E. Van Dort. Novel Dual Oncogenic Target Inhibitor against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A20.

Development of novel PET probes targeting phosphatidylinositol 3-kinase (PI3K) in tumors

Phosphatidylinositol 3-kinase (PI3K) activity and protein expression levels are often increased in tumor regions. Since PI3K plays a crucial role in regulating cell growth and proliferation, inhibiting PI3K-dependent pathways could be a promising approach for cancer treatment. In clinical practice, however, evaluation of PI3K expression levels is limited to immunohistochemistry of patient samples, which requires invasive biopsies.Here we report the synthesis of three candidate compounds, FMTA-1, 2 and 3, and evaluate their capacity to detect PI3K expression levels with positron emission tomography (PET). Among the three candidates, FMTA-2 showed a lower IC50 value for PI3K. 18F Radiolabeling of FMTA-2 to produce [18F]FMTA-2 was accomplished and its capacity for detecting PI3K expression levels was evaluated in vitro and in vivo. Cell uptake of [18F]FMTA-2 correlated well with cellular PI3K expression levels, and was suppressed by the ATP-competitive PI3K inhibitor ZSTK474. In an in vivo experiment using tumor-transplanted model mice, a higher signal-to-noise ratio (S/N) was seen with [18F]FMTA-2 in animals transplanted with DMS114 cells (expressing high PI3K levels) relative to DU145 cells (expressing low PI3K levels). However, in vivo pharmacokinetics of [18F]FMTA-2 was undesirable and the absolute amount of this compound that accumulated at the tumor region was low. To the best of our knowledge, this study represents the first trial of a PET tracer for detecting PI3K. Although further improvement of the probe is required prior to clinical application, these results should encourage future work.

Abstract A43: Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a PI3K inhibitor ZSTK474

Abstract Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors remain unclear. We previously identified that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long-term exposure to PI3K inhibitors. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug-naïve cancer cells to PI3K inhibitors and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R exhibited resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine-phosphorylated IRS-1 in high levels, which was dependent on basal IGF1R expression. In these cells, combination with the IGF1R-TKI, OSI-906, improved the efficacy of ZSTK474 in vitro and in vivo. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3K inhibitors with IGF1R inhibitors for treating IGF1R-positive human cancers. Citation Format: Sho Isoyama, Gensei Kajiwara, Naomi Tamaki, Okamura Mutsumi, Hisashi Yoshimi, Naoki Nakamura, Yumiko Nishimura, Takao Yamori, Shingo Dan. Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a PI3K inhibitor ZSTK474. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A43.

Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor

The MAP kinase (Ras/MEK/ERK) and PI3K/Akt/mTOR oncogenic signaling pathways are central regulators of KRAS-mediated transformation. Molecular reciprocity between the Ras/MEK/ERK and PI3K/Akt/mTOR pathways provides cancer cells with the ability to evade treatment when targeting only one pathway with monotherapy. Multi-kinase targeting was explored through the development of a single bivalent chemical entity by covalent linking of high-affinity MEK and PI3K inhibitors. A prototype dual-acting agent (compound 8) designed using the PI3K inhibitor ZSTK474 and the Raf/MEK inhibitor RO5126766 as scaffolds displayed high in vitro inhibition of both PI3K (IC50=172nM) and MEK1 (IC50=473nM). Additionally, compound 8 demonstrated significant modulation of MEK and PI3K signaling pathway activity in human A549 human lung adenocarcinoma cells and pancreatic cancer cells (PANC-1) and also decreased cellular viability in these two cell lines.

Abstract C96: Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors.

Abstract Background: Tumor cells can vary markedly in their response to anti-cancer drugs. Therefore predicting sensitivity or resistance of tumor cells to the drug and developing therapeutic strategies for overcoming the resistance is required to realize personalized medicine for cancer treatment. Although multiple drugs targeting phosphatidylinositol 3-kinase (PI3K) including ZSTK474 are undergoing clinical trials, little is known about the resistance. We previously reported that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to acquired resistance to PI3K inhibitors (PI3Kis). In this study, we examined whether IGF1R contributes to intrinsic resistance to PI3Kis and whether combination of IGF1R tyrosine kinase inhibitor (IGF1R-TKI) and PI3Ki is an effective strategy for cancer cells exhibiting the intrinsic resistance to PI3Kis. Materials and Methods: Correlation analyses between the expression level of IGF1R with the efficacy of PI3Ki across an in vitro panel of 39 human cancer cell lines (termed JFCR39) and a panel of 24 human tumor xenografts implanted in nude mice (termed JFCR24 xenografts) were performed. Specific siRNAs against IGF1R were used to examine the functional involvement of IGF1R in intrinsic resistance. Combination effect of ZSTK474 and an IGF1R-TKI OSI-906 was examined in vitro using isobologram analysis and in vivo using human tumor xenografts implanted in nude mice. Results: Correlation analysis revealed that PI3Ki-naïve cancer cells expressing high levels of IGF1R exhibited resistance to ZSTK474 in vitro and in vivo. Targeted knockdown of IGF1R expression using specific siRNAs enhanced the efficacy of ZSTK474 in intrinsic resistant cell lines overexpressing IGF1R. In these cell lines, higher concentration of ZSTK474 was needed to dephosphorylate Akt compared to IGF1R-negative cell lines, and specific knockdown of IGF1R made it easier to dephosphorylate Akt after ZSTK474 treatment. Finally, we found a synergistic therapeutic effect of ZSTK474 in combination with OSI-906 in vivo, as well as in vitro. Conclusion: PI3Ki-naïve cancer cells overexpressing IGF1R exhibited resistance to ZSTK474, but combination of ZSTK474 with an IGF1R-TKI exerted a remarkable therapeutic response in such cancer cells. We hope that our findings would contribute to personalized medicine using PI3Kis in future clinical application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C96. Citation Format: Sho Isoyama, Gensei Kajiwara, Naomi Tamaki, Hisashi Yoshimi, Naoki Nakamura, Mutsumi Okamura, Yumiko Nishimura, Takao Yamori, Shingo Dan. Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C96.

Development of an Immunohistochemical Protein Quantification System in Conjunction with Tissue Microarray Technology for Identifying Predictive Biomarkers for Phosphatidylinositol 3-Kinase Inhibitors

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials.

Discovery of Phosphatidylinositol 3-Kinase Inhibitory Compounds from the Screening Committee of Anticancer Drugs (SCADS) Library

Identification of new uses for existing drugs is known to be an efficient approach in drug discovery. The identification of a novel phosphatidylinositol 3-kinase (PI3K) inhibitor is important in terms of cancer chemotherapy because PI3K is implicated in many types of cancer. In an effort to discover new PI3K inhibitory compounds, we recently carried out a screening of Screening Committee of Anticancer Drugs (SCADS) library, a compound library mainly composed of antitumor drugs and kinase inhibitors. As a result, six new PI3K inhibitory compounds were identified each of which displayed over 60% inhibition of PI3Kalpha at 10 microM. Baicalein, the most potent of these inhibitors, exhibited 73% inhibition at 1 microM. Further characterization of Baicalein and Akt inhibitor VIII showed that both compounds displayed comparable inhibition against PI3Kbeta and delta, but relatively weak activity against PI3Kgamma. Growth inhibition effects of Akt inhibitor VIII and Baicalein on human cancer cell line panel JFCR39 were also investigated, and the mean logarithm of the concentration required for 50% growth inhibition of cells (Log GI50) was determined to be -5.59 and -4.70, respectively. In addition, COMPARE analysis of the two compounds together with known PI3K inhibitors was carried out by using PI3K inhibitor ZSTK474 as a seed. Our results show that Akt inhibitor VIII displays a similar fingerprint to that of ZSTK474 (r=0.633), while Baicalein does not (r=0.126). These findings suggest the inhibition profile of Baicalein in cells is different from that of a typical PI3K inhibitor.