Capillary Isoelectric Focusing Immunoassay Research Articles

Specific Cerebrospinal Fluid SerpinA1 Isoform Pattern in Alzheimer's Disease.

SerpinA1 (α1-antitrypsin) is a soluble glycoprotein, the cerebrospinal fluid (CSF) isoforms of which showed disease-specific changes in neurodegenerative disorders that are still unexplored in Alz-heimer’s disease (AD). By means of capillary isoelectric focusing immunoassay, we investigated six serpinA1 isoforms in CSF samples of controls (n = 29), AD-MCI (n = 29), AD-dem (n = 26) and Lewy body disease (LBD, n = 59) patients and correlated the findings with CSF AD core biomarkers (Aβ42/40 ratio, p-tau, t-tau). Four CSF serpinA1 isoforms were differently expressed in AD patients compared to controls and LBD patients, especially isoforms 2 and 4. AD-specific changes were found since the MCI stage and significantly correlated with decreased Aβ42/40 (p < 0.05) and in-creased p-tau and t-tau levels in CSF (p < 0.001). Analysis of serpinA1 isoform provided good di-agnostic accuracy in discriminating AD patients versus controls (AUC = 0.80) and versus LBD patients (AUC = 0.92), with best results in patients in the dementia stage (AUC = 0.97). SerpinA1 isoform expression is altered in AD patients, suggesting a common, albeit disease-specific, in-volvement of serpinA1 in most neurodegenerative disorders.

Development and Technical Validation of an Immunoassay for the Detection of APP669-711 (Aβ-3-40) in Biological Samples.

The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.

CSF SerpinA1 in Creutzfeldt-Jakob disease and frontotemporal lobar degeneration.

ObjectiveSerpinA1 (alpha‐1 antitrypsin) is an acute inflammatory protein, which seems to play a role in neurodegeneration and neuroinflammation. In Alzheimer’s disease and synucleinopathies, SerpinA1 is overexpressed in the brain and the cerebrospinal fluid (CSF) showing abnormal patterns of its charge isoforms. To date, no comprehensive studies explored SerpinA1 CSF isoforms in Creutzfeldt–Jakob disease (CJD) and frontotemporal lobar degeneration (FTLD).MethodsUsing a capillary isoelectric focusing immunoassay, we analyzed CSF SerpinA1 isoforms in control cases (n = 31) and patients with a definite or probable diagnosis of CJD (n=77) or FTLD (n = 30), belonging to several disease subtypes.ResultsThe overall SerpinA1 signal was significantly higher than in controls in CJD subtypes linked to abnormal prion protein (PrPSc) type 1, such as sporadic CJD (sCJD) MM(V)1, and in FTLD‐TDP. Moreover, CJD linked to PrPSc type 1 and FTLD‐TAU groups showed a significant relative increase of acidic and basic isoforms in comparison with controls, thereby forming two distinct SerpinA1 isoform profiles.InterpretationCJD linked to PrPSc type 1 and FTLD show a differential upregulation and post‐translational modifications of CSF SerpinA1. Further studies are needed to clarify whether these findings may reflect a common, albeit disease‐specific, pathogenetic mechanism related to neurodegeneration.

Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro

Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. Commercial hCG preparations for ART or research purposes were compared in vitro. The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and β-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate. Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and β-arrestin 2 recruitment (EC50 0.03-2.0 μg/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P ≥ 0.05; n = 3). N/A. The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG,quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity. hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women. This study was supported by a grant of the Italian Ministry of Education, University and Research (PRIN 2015XCR88M). The authors have no conflict of interest.

Detection of intrathecal immunoglobulin G synthesis by capillary isoelectric focusing immunoassay in oligoclonal band negative multiple sclerosis

Oligoclonal immunoglobulin G bands (OCBs) restricted to the cerebrospinal fluid indicate intrathecal inflammation. Using isoelectric focusing and immunoblotting, they are detected in about 95% of patients with clinically definite multiple sclerosis (MS). To elucidate whether in the remaining 5% OCBs are truly absent or alternatively missed due to insufficient sensitivity of the routine measurement, we employed a new, highly sensitive nanoscale method for OCB detection. Capillary isoelectric focusing followed by immunological detection served to analyze OCBs in 33 well-characterized OCB-negative and 10 OCB-positive MS patients as well as in 100 OCB-negative control patients with non-inflammatory neurological diseases and 30 OCB-positive control patients with inflammatory neurological diseases. We detected intrathecal immunoglobulin G production in 10 out of 33 MS patients (30%), initially diagnosed as being OCB-negative, and in all 10 OCB-positive MS patients, but in only 3 out of 100 non-inflammatory neurological controls (3%) and in 29 of 30 inflammatory neurological controls (97%). At least about one-third of MS patients without intrathecal immunoglobulin G synthesis according to standard methods are OCB-positive. Advanced methods for OCB detection may increase the analytical sensitivity for detecting OCB in patients with MS who are OCB-negative according to current routine methods.

A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation.

The serum half-life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high-throughput method for semi-quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry.

N-truncated Aβ2-X starting with position two in sporadic Alzheimer's disease cases and two Alzheimer mouse models.

According to the modified amyloid hypothesis, the key event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid β-peptides (Aβs) in plaques and cerebral blood vessels. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid-β protein precursor (AβPP). Vast evidence suggests that Aβx-42 isoforms play an important role in triggering neurodegeneration due to their high abundance, amyloidogenic propensity and toxicity. Although N-truncated Aβ peptides and Aβx-42 species appear to be the crucial players in AD etiology, the Aβ2-X isoforms did not receive much attention yet. The present study is the first to show immunohistochemical evidence of Aβ2-X in cases of AD and its distribution in AβPP/PS1KI and 5XFAD transgenic mouse models using a novel antibody pAB77 that has been developed using Aβ2-14 as antigen. Positive plaques and congophilic amyloid angiopathy (CAA) were observed in AD cases and in both mouse models. While in AD cases, abundant CAA and less pronounced plaque pathology was evident, the two mouse models showed predominantly extracellular Aβ deposits and minor CAA staining. Western blotting and a capillary isoelectric focusing immunoassay demonstrated the high specificity of the antibody pAb77 against Aβ-variants starting with the N-terminal Alanine-2.

Detection and Differentiation of Threonine- and Tyrosine-Monophosphorylated Forms of ERK1/2 by Capillary Isoelectric Focusing-Immunoassay.

The extracellular signal regulated kinases ERK1/2 play important roles in the regulation of diverse cellular functions and have been implicated in several human diseases. In addition to the fully activated, diphosphorylated ERK1/2 protein, monophosphorylated forms of ERK1/2 have been observed, which may have distinct biological functions. We report here on the highly sensitive detection and differentiation of unphosphorylated, threonine-phosphorylated (pT), tyrosine-phosphorylated (pY) and diphosphorylated ERK1 and ERK2 by capillary isoelectric focusing followed by immunological detection (CIEF-immunoassay). Eight different phosphorylated and unphosphorylated forms of ERK1/2 were resolved according to charge. The unequivocal identification and differentiation of ERK1 and ERK2 forms monophosphorylated at either threonine or tyrosine was achieved by competitive blocking with specific phospho-peptides and different phosphorylation-sensitive antibodies. The suitability of the additional pT-ERK1/2 and pY-ERK1/2 differentiation for the time-resolved in-depth study of phospho-form distribution in response to specific stimuli is demonstrated in human neuroblastoma SH-SY5Y and monocytic THP-1 cell lines, and in human peripheral blood mononuclear cells.

Capillary isoelectric focusing immunoassay as a new nanoscale approach for the detection of oligoclonal bands.

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput-approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB-CIEF-immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.

Abstract 33: Interplay of DNA damage and cell cycle signaling on Replication Protein A in human cells

Abstract Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-stranded break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phase of the cell cycle and all three subunits of RPA contain phosphorylation sites. The exact set of HR-relevant phosphorylation sites of RPA is unknown. In this study, the phosphorylation sites of chromatin-bound RPA in S and G2 phase were identified. After DNA damage, phosphorylation included pSer4/8, pSer12, pThr21, pSer23 and pSer33 of RPA2. Phosphorylation of these sites increased with time after DNA damage. Using a general ATM/ATR phosphorylation antibody, only RPA2 had substantial pSer/pThr ATM/ATR signals. Additionally, pTyr was detected on RPA1 and RPA2 and was removed in response to DSBs, indicating the regulatory action of a phosphatase. A capillary isoelectric focusing immunoassay used under native conditions revealed in high resolution isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Strikingly, RPA is a phosphoprotein in control G2 cell lysates with up to 5 putative phospho-isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33 and RPA1 pTyr. DNA damaged lysates contained 9 isoforms, containing up to 14 phosphates. DNA damage isoforms contained RPA2 pSer4/8, pSer12, pThr21, pSer23, pSer33 and other not yet identified phosphorylation sites. Citation Format: Kerry Brader, Adam Mosel, Shengqin Liu, Elizabeth Kremmer, Kaitlin Goettsch, Heinz-Peter Nasheuer, Greg Oakley, Gloria Borgstahl. Interplay of DNA damage and cell cycle signaling on Replication Protein A in human cells. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 33. doi:10.1158/1538-7445.CANSUSC14-33

Interplay of DNA damage and cell cycle signaling at the level of human replication protein A.

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites.

Molecular systems pharmacology: isoelectric focusing signature of protein kinase Cδ provides an integrated measure of its modulation in response to ligands.

Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure–activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.

Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples

We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson’s disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.

Analysis of Amino-Terminal Variants of Amyloid-β Peptides by Capillary Isoelectric Focusing Immunoassay

Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβ(x-10), Aβ(x-16), and Aβ(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.

Abstract 5521: Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies.

Abstract Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC) and genomic analysis has been commonly used to identify patients who may benefit from targeted therapies. Nevertheless, the accuracy of predicting responses to targeted therapies is only 50-80% based on sequencing or FISH. Developing proteomic biomarkers is valuable for evaluating therapeutic effects and better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, low throughput or/and lack of assay reproducibility, accuracy and sensitivity. In this study, a novel capillary isoelectricfocusing (IEF) immunoassay system (ProteinSimple, Santa Clara, CA) was used to study the dynamic phosphorylation status of signaling molecules in tyrosine kinase or/and MEK inhibitor treated NSCLC cells. NanoPro showed the same dynamic Erk phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation enables multiple protein phosphorylation isoforms to be resolved and detected using pan-reactive antibodies. In PD325901 MEK inhibitor treated cells, a specific MEK response pattern to drug treatment was revealed by NanoPro. This pattern demonstrated on-target effect of the drug, which was not detectable by Western blotting. NanoPro also identified a MEK2 response signature that may be associated with NSCLC cell sensitivity to the tyrosine kinase inhibitor, erlotinib, and distinguished erlotinib-sensitive from erlotinib-resistant cells. This MEK2 signature was further confirmed in cells with acquired resistance to erlotinib when compared to respective parental cells. In an HCC827 xenograft study, Western blotting was not able to differentiate human cancer cell signals from mouse stromal cell interference. In contrast, NanoPro detected and distinguished human Erk1 isoforms from the mouse isoforms based on their pI differences and demonstrated that erlotinib effectively inhibited Erk phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. We further used NanoPro to monitor responses of protein phosphorylations to erlotinib and AZD6244 (a MEK1/2 inhibitor) in a NSCLC patient by profiling 18 signaling molecules with as little as 8 μg tumor material. Signals from baseline and inhibitor treated samples were precisely quantified and compared. In conclusion, NanoPro can assess protein phosphorylation both qualitatively and quantitatively to provide more information about the activation status of proteins, which is not readily assessed by traditional Western blotting. Its higher sensitivity, better reproducibility and reduced tissue requirement are appealing for drug development and evaluation of drug effects in targeted therapies. Citation Format: Jinqiu Chen, Jih-Hsiang Lee, Michelle Herrmann, Kang-Seo Park, Madeleine Heldman, Paul K. Goldsmith, Yisong Wang, Giuseppe Giaccone. Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies. [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 5521. doi:10.1158/1538-7445.AM2013-5521

641 Tumour metabolism: back to the future

We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson’s disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.

Abstract LB-74: A high-throughput RNAi sensitization screen of rapamycin identifies targets for rational drug combination strategies

Abstract RNAi screening affords an unbiased and rapid method to identify genes involved in particular cellular processes and is thus a powerful tool for drug target identification and validation. Rapamycin is a well defined inhibitor of mTOR, a protein being actively pursued as an anti-tumor target. In breast cancer patients, however, treatment with mTOR inhibitors has shown only modest activity suggesting that a combined approach may be required to maximize the clinical application of mTOR inhibitors in these patients. In this study, we performed a high-throughput synthetic siRNA-based RNAi screen of the human kinome plus 350 additional genes (4 siRNAs per gene) in combination with rapamycin, to identify candidate genes whose silencing potentiate the inhibitory effects of rapamycin. Screens were conducted in the ER+ breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-468. For the RNAi screen rapamycin (10 nM) or vehicle only (0.1% DMSO) was added 48 hours post siRNA transfection and cell viability was measured after a further 24 hours of incubation. By comparing the normalized effect on cell viability for each siRNA (Z-score) in the control and rapamycin screens we identified 36 candidate genes whose silencing sensitized MCF-7 cells to rapamycin and 56 that sensitized MDA-MB-468 cells. The silencing of six candidate genes sensitized both cell lines to rapamycin, AKT1, ASPA, CDKN2A, MAP3K7IP1, MAPK12 and PCTK3. The sensitization of rapamycin by silencing of AKT1 was confirmed. Rapamycin treatment increased AKT phosphorylation in the cell lines. HDAC inhibitors have been shown to facilitate the dephosphorylation of AKT; therefore, we tested the combination of rapamycin with the HDAC inhibitor MS-275. MS-275 completely blocks rapamycin-induced AKT phosphorylation, and combining it with rapamycin synergistically inhibited the growth of the MCF-7, MDA-MB-468, MDA-MB-435 and MDA-MB-231 breast cancer cell lines. We investigated if other pathways are affected by combining rapamycin with MS-275. De-regulation of the RAF-MEK-ERK pathway is frequently detected in human cancers but the high level of ERK phosphorylation is significantly reduced in cells treated with the rapamycin/MS-275 combination. To profile the unphosphorylated, mono and dual-phosphorylated isoforms of ERK1 and ERK2 we used a capillary isoelectric focusing immunoassay to show that the di-phosphorylated isoform of ERK2 was decreased. Our results highlight the potential of high-throughput siRNA screens to identify rational drug combination strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-74.