Reverse Phase Protein Analysis Research Articles

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma–carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.

Phosphorylation of GSK3α/β correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and β isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML. MethodsWe analyzed GSK3α/β phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival. ResultsHigh levels of p-GSK3α/β correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/β levels were lower. Phosphorylated GSK3α/β expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/β as an indicator of AKT activation, RPPA revealed that p-GSK3α/β positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with β-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/β expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells. ConclusionsThese findings suggest that AKT-mediated phosphorylation of GSK3α/β may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/β may serve as an important adverse prognostic factor for a subset of AML patients.

Tumor Suppressor Role for B55 alpha PP2A Isoform in AML

Tumor Suppressor Role for B55 alpha PP2A Isoform in AML

Cathepsin G Is Broadly Expressed By AML and Is an Effective Immunotherapeutic Target Against AML in Vivo

Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted.Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL).We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P<0.05; Figure 1A) after 2 weeks compared with mice treated with HIV-CTL (27% residual AML) or that were left untreated (34% residual AML). A similar reduction in AML was seen in the spleens of mice treated with CG1-CTL. Since normal myeloid cells also express CG, to demonstrate the specificity of CG1-CTL for AML, we engrafted NSGS mice with human HLA-A2+ cord blood (CB). After confirming engraftment by detecting human (h) CD45+/HLA-A2+/mouse (m)CD45- cells in peripheral blood, we treated mice with CG1-CTL or HIV-CTL (0.25 x 106). A similar percentage of hCD45+/HLA-A2+/mCD45- cells was detected in the BM of CB-engrafted mice in both CG1-CTL (24% CB) and HIV-CTL (27% CB) after 6 weeks, suggesting the safety of targeting CG1.After demonstrating the efficacy of targeting CG1 in vivo, we then studied CG1 presentation and CG expression in AML patients. To determine CG1 presentation, primary AML blasts, U937-A2 and HLA-A2-trasnduced HL60 (HL60-A2) cell lines were lysed then immunoprecipitated with anti-HLA-A2. Peptides were eluted using standard acid elution methodology and then analyzed by mass spectrometry. CG1 peptide was detected in 6 of 9 primary AML samples and in HL60-A2 and U937-A2 cells. Reverse phase protein analysis (RPPA) was used to study CG protein expression in a large cohort of AML patients and normal donors (ND) to include the following: 511 newly diagnosed patients, 21 ND peripheral blood leukocyte controls, 21 ND CD34+ cell controls and 10 ND BM controls. CG expression in the 511 AML patients samples demonstrated a Gaussian distribution and was higher than CG expression by ND CD34+ cells in 230 samples, equal to normal CD34+ cells in 234 samples and less than normal CD34+ cells in 47 samples. As expected, G-CSF primed ND CD34+ cells had high expression of CG. There were no significant differences in CG expression within the different AML FAB subtypes, although M4 eos and M5b subtypes showed the lowest expression levels. In multivariate Cox model analysis, higher CG expression significantly predicted shorter overall survival (OS) in all patients (P=0.04) (Figure 1B). The association of CG with shorter OS was most significant in patients who had intermediate cytogenetics and FLT3 mutation (P=0.02). Moreover, CG levels were determined in paired samples taken at diagnosis and relapse, and the levels were compared using paired t test and Pearson product-moment correlation analysis. Among 47 cases with paired diagnosis and relapse samples, there was significantly higher CG protein expression in relapse samples (P=0.0001).In summary, we found CG to be an important antigen in AML. CG is associated with worse outcomes in AML patients, however in vivo mouse data show that targeting CG eliminates AML while sparing normal hematopoiesis. Together these data indicate that CG has a promising role as a novel immunotherapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reverse Phase Protein Analysis of Mesenchymal Stem Cells from AML Patients and Healthy Donors Reveals Distinct Patterns of Protein Expression Reflecting Differences in Senescence, Differentiation and Survival Signaling

The bone marrow microenvironment (BME) critically supports hematopoietic stem cells and protects leukemia cells from chemotherapy, immune surveillance, and related stresses. A critical component of the BME is the mesenchymal stem cell (MSC). Dan Link’s group demonstrated that MSC are essential for human hematopoiesis, particularly as a source of SDF-1, which regulates homing, proliferation, and differentiation of HSC. Moreover, studies from our group and others have demonstrated that MSC protect leukemia cells from chemotherapy. At present, very little is known about MSC derived from AML patients, and an understanding of the proteomic makeup of these cells in the leukemia microenvironment could help to elucidate mechanisms involved in supporting their pro-tumor function. We used reverse phase protein array analysis (RPPA) to compare the expression of 151 proteins in MSC derived from AML BMs (N = 106) with those from healthy donors (N = 71). The expression of 45 of these proteins was deemed significantly different (p < 0.01) between the two sets. AML MSC expressed higher levels of p53 and p21 (CDKN1A), and the expression of the latter was correlated with other proteins within each MSC set. Using beta-galactosidase staining, AML MSC were found to undergo senescence more frequently than normal MSC. Elevated p21 in AML MSC is consistent with this finding. While 15 proteins were positively, and 20 proteins negatively, correlated with p21 expression in normal MSC, there were only three proteins positively, and nine negatively, correlated in AML-derived MSC. In normal MSC, SMAD1 (a key component in MSC growth and differentiation involving multiple receptors like TGF beta and BMP) expression and AKT signaling were low when p21 is expressed. However, in AML MSC this association was not seen, albeit a negative correlation with ITGAL was observed. SMAD1 expression was higher in normal MSC. In normal MSC, the expression of SMAD1 was negatively correlated with PPARG and NPM1, and was positively correlated with the expression of phosphorylated ELK. The opposite relationship was seen in AML MSC (i.e., PPARG and NPM1 exhibited positive correlation with SMAD1 and phosphorylated ELK was negatively correlated with the protein). While the significance of these relationships remains to be determined it is interesting to note that PPARG is a key regulator of adipocyte differentiation in MSC, so perhaps this alteration of SMAD/PPARG in AML MSC could impede their differentiation potential. In an accompanying abstract from our group, we report that AML MSC are primed toward osteoblastic differentiation and do not differentiate into adipocytes (Battula VL et al, ASH 2014). The RPPA data on PPARG is consistent with this finding. SMAD1 also positively regulates miR-21. Since p21 is a miR-21 target, it seems possible that the differences in expression could be attributed to SMAD1 and miR-21 signaling. We analyzed miR-21 expression in normal and AML-derived MSC (N = 10, each) using qRT-PCR and found a statistically significant (p =0.014) increase in its expression in normal MSC relative to their disease counterparts. When anti-miR-21 was transduced into healthy donor MSC, which caused a 3-fold increase in p21 (but no difference in cyclin D1 expression, another miR-21 target whose expression was also increased in AML MSC). AML MSC also exhibited higher protein expression of the B55 alpha subunit (PPP2R2A) of protein phosphatase 2A (PP2A). This expression contrasted interestingly with that of leukemia cells, since we have previously reported low PPP2R2A levels in AML blasts associated with shorter remission durations (Ruvolo et al Leukemia 2011). Furthermore, AKT phosphorylation was negatively correlated with PPP2R2A expression in AML blasts, and normal MSC, but there was no correlation between PPP2R2A and phosphorylated AKT in AML MSC. Also, expression of PPP2R2A was positively correlated with the expression of the survival protein NOL3 (ARC) which may provide new clues to possible survival mechanisms in AML MSC. In summary, these findings represent insights into the proteomic profiling of normal and AML MSC. Results suggest that senescence (via p21), differentiation potential (involving SMAD/PPARG pathway), and survival signaling (including PP2A/AKT) are altered in AML MSC. Studies are underway to determine how these variations in MSC properties impact the AML microenvironment. DisclosuresCarter:Tetralogic Pharmaceuticals: Research Funding.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.

Phosphorylation Of GSK3β Is Associated With Inferior Survival In Acute Myeloid Leukemia and Is An Indicator Of AKT Activation In AML Blasts and Bone Marrow Mesenchymal Stem Cells

▪Glycogen Synthase Kinase 3β (GSK3β) is a key regulator of cell metabolism, proliferation, survival, and differentiation. The kinase has abundant substrates including many proteins in the canonical WNT pathway. Considering that GSK3β phosphorylation of many pro-survival proteins result in their degradation (e.g MYC, MCL-1), it is not surprising that GSK3β activation by stress challenge leads to cell cycle arrest and/or apoptosis. GSK3β is negatively regulated by serine 9 phosphorylation mediated by Protein Kinase B (AKT). Since AKT activation supports survival of AML cells and inactivation of GSK3β could suppress stress signaling events, we hypothesize that serine 9 phosphorylation of GSK3β (p-GSK3β ) will be detrimental for AML patients. In the current study, we analyzed GSK3β expression by Reverse Phase Protein Analysis (RPPA) in a cohort of 511 AML patients. GSK3β expression was correlated with patient survival data and disease characteristics such as French-American-British (FAB) classification, cytogenetics, and mutational status. High levels of p-GSK3β were found to correlate with adverse outcome for survival and complete remission duration (CR) in patients with intermediate cytogenetics but not in those with unfavorable cytogenetics. CR was only 45 weeks in the third of patients with highest p-GSK3β levels compared to 98 weeks for patients with low levels (p = 0.008; N = 121). Even intermediate cytogenetic patients with FLT3 mutation fared better when p-GSK3β levels were lower (50 versus 24 weeks; p = 0.009; N = 35). Expression of GSK3β and its phosphorylated form was compared with expression of 229 other proteins using RPPA in the AML patient cohort. Consistent with p-GSK3β as an indicator of AKT activation, RPPA revealed that p-GSK3β is positively correlated with phosphorylation of AKT (S473), BAD (S136), and P70S6K. In addition, p-GSK3β negatively correlated with FOXO3A (which is degraded after phosphorylation by AKT). Bone marrow mesenchymal stem cells (BM MSCs) are a critical component of the leukemic microenvironment but how these cells modulate the survival of leukemia cells is not clear. RPPA analysis was performed on BM MSC from healthy donor (N = 71) and BM MSCs from AML patients (N = 106). Interestingly, both total and phosphorylated GSK3 were found to be elevated in the AML samples suggesting that AKT is also activated in the leukemic MSCs. In vitro models of the BM microenvironment suggest that the AKT pathway is activated in both the leukemic and supporting stromal cells so this finding is consistent with these models (Konopleva and Andreeff, Curr Drug Targets. 2007; 8: 685). Unlike in AML blast cells, FOXO3A was not correlated with GSK3β phosphorylation in the MSCs. Examination of miRs in normal versus AML BM MSCs using microarray analysis and validated by qRT-PCR indicate that miR-21 is elevated in the MSC of the normal individuals. As miR-21 is suppressed by FOXO3A, this finding supports the notion that AKT is active in the AML BM MSCs but FOXO3A may not be functional. This possibility is plausible as induction of FOXO3A results in apoptosis in MSC (Djouad Cloning Stem Cells. 2009; 11:407).ConclusionThese findings suggest that AKT mediated phosphorylation of GSK3β may be detrimental to AML patients and p-GSK3β may serve as an important prognostic factor for at least a subset of AML patients. The results also suggest that activation of AKT can occur in both the malignant cells and MSC cells in the leukemic microenvironment. Disclosures:No relevant conflicts of interest to declare.

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

ObjectiveLeptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Leprdb/lb) to determine the role of leptin in skeletal muscle.Methods and FindingsSkeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.ConclusionThese data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.

Suppression and Activation of the Malignant Phenotype by Extracellular Matrix in Xenograft Models of Bladder Cancer: A Model for Tumor Cell “Dormancy”

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases.

Abstract 4120: Low expression of PP2A/B55α is associated with increased MiR-191 expression and relapse in acute myeloid leukemia.

Abstract Acute myeloid leukemia (AML) is highly fatal so a better understanding of chemoresistance mechanisms is essential. Protein Phosphatase 2A (PP2A) B55α subunit is under-expressed in AML blast cells compared to normal CD34+ cells and patients with lower levels of the B subunit exhibit shorter remission duration (Ruvolo et al Leukemia, 2011). As remission duration is linked to relapse in AML, we have now examined B55α expression by Reverse Phase Protein Analysis (RPPA) in 47 paired samples taken from AML patients at first diagnosis and at relapse. We find B55α levels are reduced in AML blast cells during relapse. These data suggest suppressing the B subunit may activate survival pathways in the malignant cells that would shorten remission duration and promote relapse. To determine the role of B55α in AML cell survival, expression of the B subunit was blocked in OCI-AML3 cells using shRNA. Cells with B55α shRNA were more resistant to killing by the conventional chemotherapy agent AraC and the BH3 mimetic ABT-737 compared to OCI-AML3 cells containing a non-specific control shRNA. Consistent with RPPA data from AML patients, phosphorylation of AKT and PKCα was increased as were MYC levels in B55α shRNA cells. Though the role for PP2A as a regulator of signal transduction is well established, there is little if any information on how the enzyme regulates survival processes that are mediated by microRNAs (miRs). MiRs are emerging as important regulators of leukemic cell survival and proliferation. We postulated a role for B55α in miR regulation because the B subunit induces MYC, which in turn regulates many miRs including those in the mir17∼92 polycistron. MiR expression profiles were obtained by mircroarray analysis using OCI-AML3 B55α shRNA cells and control shRNA cells. We found that reduction of the B subunit resulted in at least a 2 fold reduction in 30 miRs. MiR-1260 showed the greatest change with a 23 fold decrease in expression. Only three miRs were increased by 2 fold or more in the B55α shRNA cells (i.e. miR-4290; miR-4508; miR-191-5p). The identification of miR-191-5p is especially intriguing as we previously found that AML patients with increased levels of this miR had worse overall survival (Garzon et al Blood 2008). Increased miR-191 expression has been observed in solid tumors, therefore it is possible that this miR supports tumor survival in many cancers. Interestingly, none of the members of the 17∼92 polycistron were affected by loss of the B subunit suggesting that the B subunit regulates miR expression by mechanisms independent of MYC. Since the B subunit has pro-death effects in the AML cells and MYC supports survival it is not surprising that MYC function may be suppressed. While the mechanism how B55α suppresses miR-191-5p and what proteins the miR targets in AML are not clear, a possible AML therapeutic strategy could be to target miR-191 either by activating the B subunit or targeting the miR itself. Citation Format: Peter P. Ruvolo, Vivian Ruvolo, Rodrigo Jacamo, Yihua Qiu, Kevin Coombes, Nianxiang Zhang, Ramiro Garzon, Steven M. Kornblau, Carlo Croce, Michael Andreeff. Low expression of PP2A/B55α is associated with increased MiR-191 expression and relapse in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4120. doi:10.1158/1538-7445.AM2013-4120

Abstract P4-08-01: PI3K/mTOR inhibition overcomes in vitro and in vivo trastuzumab resistance independent of feedback activation of pAKT

Abstract Background: Aberrant activity of the PI3K/mTOR pathway has been implicated in resistance to trastuzumab and anti-hormonal therapy for HER2-amplified and ER-positive breast cancer, respectively. Previous studies in our laboratory and others have shown that increased PI3K/mTOR signaling, either through PTEN loss or activating PIK3CA mutations, can confer resistance to trastuzumab therapy. In this study, we assessed the potential of targeting the PI3K/mTOR pathway in overcoming both de novo and acquired trastuzumab resistance. Materials and Methods: The in vitro activity of the pan-PI3K inhibitor BKM120, the mTORC1 inhibitor RAD001 and the dual PI3K/mTORC1/2 inhibitor BEZ235 were evaluated in a panel of 49 human breast cancer and immortalized cell lines. The in vivo activity of these molecules was assessed in six cell line xenografts models representing, ER+/HER2− (KPL-1, ZR75-1), ER+/HER2+ (UACC812, MDA361), ER−/HER2+ (SUM190), PIK3CA mutant (SUM190, MDA361), PTEN-null (ZR75-1) and trastuzumab resistant (BT-TR) breast cancer. Finally, the effect of PI3K/mTOR inhibition on feedback activation of PI3K signaling and compensatory pathways was measured by Western blot, immunohistochemistry (IHC) and reverse phase protein analysis (RPPA) of control and treated cell lysates/tumors. Results: Using a sensitivity cut-off of an IC50 of &amp;lt; 1 µmol/L, 16 of the 18 HER2-amplified breast cancer cell lines and 8 of 10 cell lines with activating mutations in PIK3CA were sensitive to the pan-PI3K inhibitor BKM120. BEZ235 showed the most potent efficacy across the panel with IC50s &amp;lt; 100 nmol/L for each of the 49 cell lines tested. The HER2-amplified/PIK3CA mutant cell lines were also unexpectedly sensitive to the mTORC1 inhibitor RAD001, this was despite the silencing of mTORC1 signaling being followed by a feedback increase in phospho-AKT signaling. Furthermore, each of these molecules showed remarkable in vivo activity across the panel of xenografts models. BKM120, RAD001 and BEZ235 induced both tumor stabilization and regression independent of PTEN, PI3K, ER and HER2 status. Pharmacodynamic analysis of tumor tissue revealed that BEZ235 and RAD001 both inhibited mTORC1 signaling as indicated by a reduction in the levels of phosphorylated ribosomal protein S6 (pS6). However, in contrast to BKM120 and BEZ235, RAD001 did not induce a reduction in the levels of pAKT (S473 or TH308) yet showed comparable in vivo efficacy to each of these molecules. Finally, combined targeting of HER2 and PI3K/mTOR in vitro increased the anti-proliferative activity of the molecules and led to an increased induction of apoptosis. The efficacy of these molecules (alone or in combination with trastuzumab) was assessed in a model of in vivo trastuzumab resistance generated through long-term treatment of the trastuzumab sensitive BT474 cells (BT-TR). All PI3Ki induced complete inhibition of tumor proliferation in monotherapy, while the combination of trastuzumab and each of these molecules induced tumor regression in the trastuzumab resistant tumors. Discussion: These pre-clinical data indicate that targeting the PI3K/mTOR pathway either alone or in combination with trastuzumab is effective strategy for overcoming trastuzumab resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-01.

Concomitant inhibition of DNA methyltransferase and BCL-2 protein function synergistically induce mitochondrial apoptosis in acute myelogenous leukemia cells

DNA methylation and BLC-2 are potential therapeutic targets in acute myeloid leukemia (AML). We investigated pharmacologic interaction between the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the BCL-2 inhibitor ABT-737. Increased BCL-2 expression determined by reverse phase protein analysis was associated with poor survival in AML patients with unfavorable cytogenetics (n = 195). We found that 5-AZA, which itself has modest apoptotic activity, acts synergistically with ABT-737 to induce apoptosis. The 5-AZA/ABT-737 combination enhanced mitochondrial outer membrane permeabilization, as evidenced by effective conformational activation of BAX and ∆ψ(m) loss. Although absence of p53 limited apoptotic activities of 5-AZA and ABT-737 as single agents, the combination synergistically induced apoptosis independent of p53 expression. 5-AZA down-regulated MCL-1, known to mediate resistance to ABT-737, in a p53-independent manner. The 5-AZA/ABT-737 combination synergistically induced apoptosis in AML cells in seven of eight patients. 5-AZA significantly reduced MCL-1 levels in two of three samples examined. Our data provide a molecular rationale for this combination strategy in AML therapy.

The PP2A Subunit B55α Suppresses Survival Signaling in Acute Myeloid Leukemia Cells

Abstract 396Acute myeloid leukemia (AML) is still highly fatal so a better understanding of the mechanisms controlling chemoresistance is critical for the development of more effective therapies. The Protein Kinase B (AKT) regulates diverse cellular functions including protein translation, transcription, cell proliferation and apoptosis and is emerging as an important target for AML therapy. We have reported that activation of AKT predicts poor clinical outcome for patients with AML. Mutations of upstream activators of AKT such as Phosphatidylinositol 3 Kinase (PI3K) and RAS are well characterized and have been implicated in many cancers including AML. However, signal transduction is a dynamic process and the possibility arises that loss of protein phosphatase function can promote kinase activation in the absence of constitutive activation of the kinase by upstream activators. AKT has been shown to be regulated by Protein Phosphatase 2A (PP2A). PP2A is a family of hetero-trimeric serine/threonine phosphatases. While family members share a fairly common catalytic core (i.e. A and C subunits), the functional component of each PP2A isoform lies in its regulatory subunit (i.e. B subunit). Recent data suggests that suppression of PP2A can promote activation of survival signaling including the activation of AKT in AML. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with phosphorylation of AKT at threonine 308 and with AKT activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression had shorter complete remission duration. In the current study, we now found by Reverse Phase Protein Analysis (RPPA) that there is a negative correlation between B55α expression and expression and phosphorylation of a number of survival kinases including Protein Kinase C a (PKCα) and SRC in AML patients. PP2A has been shown to negatively regulate the PKC family of kinases but this is the first report to implicate B55α in that process. While levels of phosphorylated PKCα negatively correlated with B55α, total levels of the kinase also were negatively correlated with the B subunit suggesting that phosphatase likely does not dephosphorylates PKCα but rather suppresses expression of the kinase. While SRC has not been linked to B55α, the kinase has been shown to negatively regulate the PP2A catalytic subunit (PP2A/C). Since PP2A is an obligate hetero-trimer, inactivation of PP2A/C via SRC could result in proteolytic cleavage of the B subunit. Consistent with this notion, treatment of OCI-AML3 cells with the tyrosine kinase inhibitor Dasatinib resulted in reduction of PP2A/C levels. Consistent with a mechanism whereby SRC regulated B55α rather than vice-versa, OCI-AML3 cells expressing B55α shRNA did not display changes in SRC phosphorylation status though there was a >67% loss of the B subunit in these cells. To determine the role of the AKT Phosphatase in AML cell survival, the OCI-AML3 cells expressing B55α shRNA were used to determine if reduction of levels of the phosphatase would promote AKT activation and promote chemoresistance. Cells with B55α shRNA exhibited higher levels of AKT S473 phosphorylation suggesting activation of the kinase. Furthermore, OCI-AML3 cells expressing B55α shRNA were more resistant to killing by the conventional chemotherapy agent AraC. One surprising finding was that OCI-AML3 B55α shRNA transductant cells were more resistant to okadaic acid, a PP2A inhibitor that is highly toxic to AML cells. Despite effects on AKT activation and survival, the reduction of the B subunit had no effect on total PP2A activity as determined by molybdate dye assay. Reduction of B55α did promote expression of at least one PP2A B regulatory subunit (i.e. B56α levels increased nearly 2 fold). Interestingly, B56α is positively regulated by AKT suggesting a possible feedback mechanism may exist between AKT and the various PP2A isoforms. An intriguing possibility arises that while PP2A is generally considered a tumor suppressor, inhibition of the AKT Phosphatase may activate an okadaic acid resistant PP2A isoform with survival function. Taken together, these results suggest that B55α may suppress survival signaling by kinases such as AKT and loss of function of the B subunit may promote resistance to chemotherapy in AML. Disclosures:No relevant conflicts of interest to declare.

Low expression of PP2A regulatory subunit B55α is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients

The regulation of Protein Kinase B (AKT) is a dynamic process that depends on the balance between phosphorylation by upstream kinases for activation and inactivation by dephosphorylation by protein phosphatases. Phosphorylated AKT is commonly found in acute myeloid leukemia (AML) and confers an unfavorable prognosis. Understanding the relative importance of upstream kinases and AKT phosphatase in the activation of AKT is relevant for the therapeutic targeting of this signaling axis in AML. The B55α subunit of Protein Phosphatase 2A (PP2A) has been implicated in AKT dephosphorylation but its role in regulating AKT in AML is unknown. We examined B55α protein expression in blast cells derived from 511 AML patients using Reverse Phase Protein Analysis (RPPA). B55α protein expression was lower in AML cells compared to normal CD34+ cells. B55α protein levels negatively correlated with T308 phosphorylation levels. Low levels of B55α were associated with shorter complete remission duration demonstrating that decreased expression is an adverse prognostic factor in AML. These findings suggest that decreased B55α expression in AML is at least partially responsible for increased AKT signaling in AML and suggests that therapeutic targeting of PP2A could counteract this.

505 Role for PP2A Regulatory Subunit B55alpha as a Regulator of AKT and Other Signaling Pathways in AML

Abstract Abstract 1668 Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures: No relevant conflicts of interest to declare.

A randomized phase II study of MK-2206 in comparison with everolimus in refractory renal cell carcinoma.(NCI 8727).

TPS192 Background: Up-regulation of the phosphoinositide-3 phosphate kinase (PI3K) pathway is associated with poor prognosis in patients with renal cell carcinoma (RCC). Our data show that metastatic RCC patients treated with anti-vascular endothelial growth factor (VEGF) inhibitory therapy who have up-regulated PI3K pathway have a shorter progression free survival (PFS). A 80 patient Clinical Therapeutics Evaluation Program sponsored study of patients who received sorafenib +/- interferon alpha, AKT S473 was the single strongest predictor for clinical outcome in a panel of clinical and tissue variables. Similarly, reverse phase protein analysis of nephrectomy samples in patients who received presurgical bevacizumab showed that up-regulation of AKT was associated with a shorter PFS. We hypothesize that blockade of AKT in patients who are refractory to anti-VEGF therapy will eliminate one of the key drivers of tumor growth and proangiogenic signaling, and will result in prolongation of PFS. MK-2206 is a selective allosteric inhibitor of AKT. MK-2206 does not bind to the active site of AKT, and consequently does not compete with either ATP or peptide substrate for binding to AKT. This study will randomize patients who progressed on anti-VEGF therapy between MK-2206 and everolimus, a mammalian target of rapamycin inhibitor. Methods: This is an open-label, multi-center, randomized phase II study. To be eligible, patients have to have metastatic RCC, and should have progressed on an anti-VEGF agent. Up to two prior therapies are allowed. Patients will be randomized using a 2:1 ratio to receive either MK2206 or everolimus. The primary endpoint of the study is PFS. The anticipated median PFS is 4.9 months and 8.2 months for the everolimus and MK2206 arms, respectively. The secondary endpoints include safety, overall survival (OS), overall response rate (ORR) and time to failure (TTF). In addition, correlative studies evaluating primary tumor AKT levels, tumor virtual karyotype and pre versus post therapy cytokine and angiogenesis factor levels will be examined and correlate to outcome. As of February 1, 2011, the trial is open for accrual.

Role for PP2A Regulatory Subunit B55α as A Regulator of AKT and Other Signaling Pathways In AML.

AbstractAbstract 1668Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures:No relevant conflicts of interest to declare.