Author:

David Draper, PhD, Scientist, In Vitro Operations

Date:

May 31, 2017

Pre-clinical research on the development of small molecule inhibitors that block the activity of disease-associated kinases continues to be a major focus of cancer therapy. Therefore, robust platforms that can quantify the phosphorylation state of these kinases are valuable to drug development efforts. Using the MV-4-11 acute myeloid leukemia (AML) model, this update describes new in vitro and ex vivo phospho-flow services offered at MI Bioresearch that measure phospho-kinase signaling with consistency and reproducibility. This platform uses fluorescence-labeled antibodies that recognize proteins only when phosphorylated on specific amino acid residues that regulate their function. Furthermore, we demonstrate how phospho-flow can integrate high throughput immunophenotyping with phospho-protein detection, which provides an advantage over ELISA-based and other conventional techniques.

The switch between homeostasis and the onset of cancer is controlled by a complex network of intracellular signaling systems that regulate gene expression. These molecular pathways are composed of a cascade of signaling proteins that drive a diverse range of biological functions. These include, but are not limited to, cell cycle regulation, apoptosis, cell survival, angiogenesis, cell migration, and the immune response. The dysregulation of any of these pathways has the potential to promote cancer pathogenesis by blocking the ability of the host to control cell proliferation and tumor growth and/or subsequent metastatic disease.1,2,3

The schematic in Figure 1 illustrates how constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway can promote tumor growth. The activation of this and other pathways is largely controlled by protein kinases, which are enzymes that turn on downstream proteins by adding a phosphate group to serine, threonine, or tyrosine residues. The phosphorylation state of these proteins is often used as a readout for signaling activation. Conversely cell signaling can be shut down by protein phosphatases that remove these phosphate groups.

Fig. 1: Constitutive activation of AKT drives tumor growth. Dysregulation of the PI3K/AKT mTOR pathway is common to a range of malignancies in humans and is especially prevalent in breast cancer. Mutations in several genes that regulate this pathway, which include PIK3CA, PIK3R1, PTEN and AKT1, have been reported in breast cancer patients. AKT signaling has also become the target of leukemia research. Constitutive activation of AKT in 72% of AML patients by phosphorylation at Ser473 has been reported.(4) Furthermore, pre-clinical studies have demonstrated that inhibition of AKT with the pan-AKT inhibitor MK-2206 dephosphorylates AKT and its downstream target S6 and halts tumor growth.( 5)
Fig. 1: Constitutive activation of AKT drives tumor growth. Dysregulation of the PI3K/AKT mTOR pathway is common to a range of malignancies in humans and is especially prevalent in breast cancer. Mutations in several genes that regulate this pathway, which include PIK3CA, PIK3R1, PTEN and AKT1, have been reported in breast cancer patients. AKT signaling has also become the target of leukemia research. Constitutive activation of AKT in 72% of AML patients by phosphorylation at Ser473 has been reported.4 Furthermore, pre-clinical studies have demonstrated that inhibition of AKT with the pan-AKT inhibitor MK-2206 dephosphorylates AKT and its downstream target S6 and halts tumor growth. 5

To demonstrate how phospho-flow can be used to measure changes in protein phosphorylation, MV-4-11 cells were treated in vitro with the pan-AKT inhibitor developed by Merck & Co., MK-2206 (Selleckchem; Houston, TX). Figure 2 shows that MK-2206 induces dephosphorylation of AKT and S6 in MV-4-11 cells following two and 24 hours of treatment. Treatment with MK-2206 also induces apoptosis, which was measured by caspase-3 activation and annexin V binding (Figure 3).

MK-2206 Treatment Reduces Phosphorylation of AKT
and S6 in MV-4-11 Cell Cultures

Fig. 2: MK-2206 treatment decreases phosphorylation levels of AKT and S6 in MV-4-11 cells. MV-4-11 cells were seeded in culture plates and treated with 10 µM MK-2206 or left untreated. p-AKT and p-S6 levels were measured by phospho-flow. Median fluorescence intensities (MFI) are presented in both histogram overlays and bar graphs. Red and Blue histograms represent MK-2206 treated and untreated cultures respectively. Each experimental condition was performed in duplicate. Data shown is representative of 3 experiments.
Fig. 2: MK-2206 treatment decreases phosphorylation levels of AKT and S6 in MV-4-11 cells. MV-4-11 cells were seeded in culture plates and treated with 10 µM MK-2206 or left untreated. p-AKT and p-S6 levels were measured by phospho-flow. Median fluorescence intensities (MFI) are presented in both histogram overlays and bar graphs. Red and Blue histograms represent MK-2206 treated and untreated cultures respectively. Each experimental condition was performed in duplicate. Data shown is representative of three experiments.

MK-2206 Induces Apoptosis in MV-4-11 Cells

Fig. 3: MK-2206 treatment induces apoptosis in MV-4-11 cells. MV-4-11 cells were treated for 24 hours with 0, 1, or 10 µM concentrations of MK-2206. Apoptosis was measured using a fluorescent antibody that binds the active form of caspase-3 (top panel) and annexin V/7-AAD binding (bottom panel).
Fig. 3: MK-2206 treatment induces apoptosis in MV-4-11 cells. MV-4-11 cells were treated for 24 hours with 0, 1, or 10 µM concentrations of MK-2206. Apoptosis was measured using a fluorescent antibody that binds the active form of caspase-3 (top panel) and annexin V/7-AAD binding (bottom panel).

To demonstrate that phospho-flow can produce similar results to conventional techniques, a western blot analysis was performed that revealed a similar pattern of AKT and S6 dephosphorylation following MK-2206 treatment (Figure 4).

Fig. 4: Western block analysis generated results that were consistent with phospho-flow analysis. MV-4-11 cells were treated with 10µM MK-2206 or left untreated for 24 hours. Total protein extracts were transferred to PVDF membranes and probed with anti-p-AKT (Ser473) and anti-p-S6 (Ser240/244) antibodies to measure phosphorylation levels. Densitometry revealed MK-2206 treatment reduced p-AKT and p-S6 signals by 13.2% and 34.1% respectively. α-tubulin was probed for as a loading control.
Fig. 4: Western blot analysis generated results that were consistent with phospho-flow analysis. MV-4-11 cells were treated with 10µM MK-2206 or left untreated for 24 hours. Total protein extracts were transferred to PVDF membranes and probed with anti-p-AKT (Ser473) and anti-p-S6 (Ser240/244) antibodies to measure phosphorylation levels. Densitometry revealed MK-2206 treatment reduced p-AKT and p-S6 signals by 13.2% and 34.1% respectively. α-tubulin was probed for as a loading control.

To demonstrate how phospho-flow can be used in combination with immunophenotyping, mice bearing disseminated MV-4-11 tumor cells were dosed with MK-2206 or vehicle control and AKT and S6 phosphorylation were measured ex vivo by phospho-flow. Figure 5 shows that in vivo MK-2206 treatment induces dephosphorylation of both AKT and S6 in MV-4-11 tumor cells in the bone marrow but only AKT in the mouse lymphocytes.

The Effect of In Vivo MK-2206 Treatment on AKT and
S6 Phosphorylation is Different in Tumor and Host Cells

Fig. 5: In vivo treatment with MK-2206 reduces p-AKT and p-S6 levels in MV-4-11 tumor cells detected in bone marrow by flow cytometry. Mice harboring disseminated MV-4-11 tumors were dosed twice with MK-2206 (180 mg/kg) or vehicle (N=5 mice/group) on days 17 and 18 after implantation. On day 19, bone marrow was flushed from femurs and tibias for analysis. Immunophenotyping was used to distinguish MV-4-11 tumor cells (top panel) vs. mouse lymphocytes (bottom panel) by staining samples with anti-human CD45 and anti-mouse CD45 antibodies respectively. Data are presented as mean ± SD. Statistical analysis was performed using student’s t-test.
Fig. 5: In vivo treatment with MK-2206 reduces p-AKT and p-S6 levels in MV-4-11 tumor cells detected in bone marrow by flow cytometry. Mice harboring disseminated MV-4-11 tumors were dosed twice with MK-2206 (180 mg/kg) or vehicle (N=5 mice/group) on days 17 and 18 after implantation. On day 19, bone marrow was flushed from femurs and tibias for analysis. Immunophenotyping was used to distinguish MV-4-11 tumor cells (top panel) from mouse lymphocytes (bottom panel) by staining samples with anti-human CD45 and anti-mouse CD45 antibodies respectively. Data are presented as mean ± SD. Statistical analysis was performed using student’s t-test.

The results above demonstrate that phospho-flow has multi-purpose utility. It is valuable for screening new drugs for their effects on the activation of cell signaling pathways in vitro. Phospho-flow can also measure drug effects in vivo by multiplexing the technique with immunophenotyping cell surface markers to distinguish analysis in different cell subsets.

Click here, if you missed our latest webinar regarding phospho-flow cytometry and would like to download the full presentation and discussion now.

1Marmiroli, Sandra, et al. “Phosphorylation, signaling, and cancer: targets and targeting.” BioMed Research International 2015 (2015).

2Bianco, Roberto, et al. “Key cancer cell signal transduction pathways as therapeutic targets.” European Journal of Cancer 42.3 (2006): 290-294.

3Nitulescu, George Mihai, et al. “Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review).” International Journal of Oncology 48.3 (2016): 869-885.

4Min, Y. H., et al. “Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable.” Leukemia 17.5 (2003): 995-995.

5Lu, Jeng-Wei, et al. “MK-2206 induces apoptosis of AML cells and enhances the cytotoxicity of cytarabine.” Medical Oncology 32.7 (2015): 1-9.