Discovery of Novel AKT Inhibitors with Enhanced Anti-Tumor Effects in Combination with the MEK Inhibitor

<div><p>Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via <i>PI3K/PTEN</i> mutation or loss) while cell lines with activating mutations in the MAPK pathway (<i>KRAS/BRAF</i>) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.</p></div

Effect of GSK2141795 on AKT signaling and growth inhibition in human cancer cell lines.

<p><i>A</i>, BT474 (left) and LNCaP (right) cell lines were treated with DMSO or GSK2141795 for 1 h. Western analysis was performed to assay levels of phosphorylated and total GSK-3, PRAS40, AKT and ERK, phosphorylated FOXO, Caspase 9, and MEK. Tubulin was used as a loading control. <i>B</i>, Scatter plot of EC₅₀'s for anti-proliferative effect of GSK2141795 against various haematological cancer cell lines. Cell lines are grouped according to their disease classification and sub-divided into cellular origin of B cell (black diamond), pre-B cell (red diamond), T cell (blue diamond) or other (grey diamond). <i>C</i>, Scatter plot of EC₅₀'s for various solid cancer cell lines treated with GSK2141795. Cells were treated as described above. Cell lines are grouped by tissue of origin then further divided by genetic status. <i>KRAS/BRAF^V600E</i> mutation status is represented by color with mutant (red), wild type (black) or other (blue); whereas <i>PIK3CA</i> or <i>PTEN</i> status represented with shape, mutant (diamond) and wild type (circle).</p

Biochemical and Cellular Activity of GSK2110183 and GSK2141795.

#<p>IC₅₀ or K_i of selected kinases inhibited >50% at 0.5 µM from larger kinase panel.</p><p>*Data represents K_i (nM) values.</p>$<p>Data represents mean ± std dev from multiple experiments for cellular assays.</p

Dose responsive PD/PK relationship of GSK2110183 and GSK2141795 in BT474 tumor xenografts.

<p>Mice bearing BT474 tumors were treated with vehicle or GSK2110183 at 10, 30 or 100/kg (A) or GSK2141795 at 3, 10 and 30 mg/kg (B) daily for 7 days (QDx7; n = 3/group). Tumors were harvested and analysed by ELISA for phosphorylated and total PRAS40 levels. The concentration of compound in the tumor (black triangles; ng/g) and blood (grey squares; ng/mL) was quantified by LC/MS-MS. Data represents mean ± s.d. *p<0.01.</p

Anti-tumor activity of GSK2141795 in vivo.

<p>Mice bearing either BT474 (<i>A</i>) or SKOV3 (<i>B</i>) tumors were treated with vehicle (black line) or GSK2141795 at 10 (red line), 20 (blue line) or 30 (gold line) mg/kg, once daily for 21 d (QDx21). Duration of treatment is shown by the horizontal grey line. Tumor volume was measured twice per week. Data represents mean ± SEM. #p<0.05, *p<0.01.</p

Effect of GSK2141795 on cell cycle.

<p>LNCaP, BT474, A3 and I9.2 cells lines were treated with GSK2141795 for 24; bars, SD.</p

Combination anti-tumor effect of AKT and MEK inhibitors in mouse models of pancreatic cancer.

<p>Mice bearing HPAC (<i>A</i>) or CAPAN-2 (<i>B</i>) tumor xenografts were treated with either vehicle (black line), 0.3 mg/kg GSK1120212 (purple line), 30 mg/kg GSK2141795 (green line) or the combination of both (blue line). Data represents mean ± SEM. #p<0.05, *p<0.01. <i>C</i>, Immunohistochemical evaluation of HPAC tumor xenografts treated with vehicle, GSK1120212 at 0.3 mg/kg QDx3 (MEK inh), GSK2141795 at 30 mg/kg QDx3 (AKT inh) or the combination of GSK1120212 plus GSK2141795 at 0.3 and 30 mg/kg, respectively (combo). Tumor tissues were stained with markers of proliferation (Ki67), apoptosis (Cleaved Caspase 3; CC3), MAPK pathway activation (phospho-ERK, dually phospho-ERK and total ERK) and AKT pathway activation (phospho-PRAS40, total PRAS40, phospho-S6 kinase, total S6K, phospho-AKT and total AKT). Quantitative image analysis of various immune-staining is represented as a percentage of the vehicle control (100%). Data represents mean ± s.d.</p

Effect of GSK2110183 on AKT signaling and growth inhibition in human cancer cell lines.

<p><i>A</i>, BT474 (left) and LNCaP (right) cell lines were treated with DMSO or GSK2110183 for 1 h. Western analysis was performed to assay levels of phosphorylated and total GSK-3, PRAS40, AKT and ERK, phosphorylated Foxo, Caspase 9, and MEK. Tubulin was used as a loading control. <i>B</i>, Scatter plot of EC₅₀'s for anti-proliferative effect of GSK2110183 against various haematological cancer cell lines. Cell lines are grouped according to their disease classification and sub-divided into cellular origin of B cell (black diamond), pre-B cell (red diamond), T cell (blue diamond) or other (grey diamond). <i>C</i>, Scatter plot of EC₅₀'s for various solid cancer cell lines treated with GSK2110183. Cells were treated as described above. Cell lines are grouped by tissue of origin then further divided by genetic status. <i>KRAS/BRAF^V600E</i> mutation status is represented by color with mutant (red), wild type (black) or other (blue); whereas <i>PIK3CA</i> or <i>PTEN</i> status represented with shape, mutant (diamond) and wild type (circle).</p