Activation Status Of Signaling Pathways Research Articles

Abstract 6631: Association between poliovirus receptor (PVR) expression in tumor and exclusion of immune cells expressing T cell immunoreceptor with Ig and ITIM domain (TIGIT) from tumor area

Abstract Context: TIGIT has grown to be an increasingly important target for immunotherapy of cancer. However, it is unclear if there is an association between abundance of TIGIT expressing immune cells in tumor microenvironment and effectiveness of TIGIT-based intervention. This study utilizes an exploratory novel duplex immunohistochemical (IHC) assay to assess if the distribution patterns and/or the abundance of TIGIT-positive immune cells are related to the expression levels of TIGIT’s receptor PVR in tumor. Methods: Individual formalin-fixed, paraffin-embedded surgical resections and tissue microarray cores of 75 cervical, 84 esophageal, 79 non-small cell lung , and 81 small cell lung carcinoma specimens were stained using a duplex chromogenic IHC assay that detects PVR and TIGIT sequentially on a single slide. The slides were examined for (1) overall intensity and percentage of tumor staining positive for PVR, (2) percentage of tumor area occupied by TIGIT-expressing immune cells, and (3) distribution pattern of TIGIT-positive cells. For each specimen, overall PVR intensity was multiplied by percentage of tumor positive for PVR to generate the PVR Expression Score. Results: PVR and TIGIT staining at any intensity was widely observed in all four tumor types ranging from 91% to 100% of specimens positive for PVR and 77% to 100% of specimens positive for TIGIT. Little to no differences in the average percentages of PVR tumor positivity or TIGIT tumor area positivity were found among the four tumor types. However, individual tumor specimens differed widely in these two parameters. Among all 319 tumor specimens evaluated, the percentages of tumor expressing PVR at any intensity ranged from 0 to 100% with approximately 1/3 of the specimens having less than 50% and 3% showing no PVR expression. Percentages of tumor area occupied by TIGIT-positive cells ranged from 0% to 30% with approximately 40% of the specimens having less than 1%. Distribution patterns of TIGIT-expressing cells in each specimen was classified as peritumoral, intratumoral, or mixed. Specimens classified as peritumoral exhibited higher PVR Expression Scores than those classified as intratumoral. Specimens classified as having a mixed TIGIT-positive cell distribution pattern had an average PVR Expression Score that was intermediate between specimens having intratumoral and peritumoral TIGIT distribution patterns. Interestingly, no correlation was found between tumor PVR Expression Scores and abundance of TIGIT expressing cells in tumor area. Conclusion: The correlation between high tumor PVR expression levels and peritumoral distribution patterns of TIGIT-positive cells suggests PVR/TIGIT signaling pathway activation status could be important in infiltration of T cells into tumor parenchyma. Citation Format: Sara A. Moore, Julie Cheung, Aram B. Cholanians, June Clements, Jessica L. Baumann, Tsu-Shuen Tsao. Association between poliovirus receptor (PVR) expression in tumor and exclusion of immune cells expressing T cell immunoreceptor with Ig and ITIM domain (TIGIT) from tumor area [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6631.

Chemical-Biology-derived in vivo Sensors: Past, Present, and Future.

To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology.

Abstract A22: Cervical cancer immunomodulation through stat3 and p65 NFκb: Effects beyond the tumor microenvironment

Abstract Chronic signaling by molecules generated in the tumor microenvironment has local and systemic effects on the immune system. We have previously shown that cervical cancer cells express cytokines as IL-6 and G-CSF, among other factors. These cytokines have been associated with cancer progression in several types of cancer, either by promoting chronic inflammation and/or tolerance. Trying to better understand how cells from the immune system and tumor integrate signals generated by the tumor microenvironment, we have investigated the activation status of signaling pathways involved in cell proliferation and survival, inflammation and immune responses. We observed a cohort of cervical cancer patients, all of whom had cervical biopsies positive for high oncogenic risk human papillomavirus (HPV). In the cervical cancer biopsies, we found constitutive expression of phosphorylated STAT3, Akt, p65 NFκB and CREB, in both the tumor and leukocyte compartments. In the peripheral blood, however, we found constitutive expression of phosphorylated STAT3 and Akt, but significantly lower expression of phosphorylated p65 NFκB than in controls. Data previously published by our laboratory using a HPV16 experimental model showed high constitutive expression of phospho-STAT3 and low negative expression of phospho-p65 NFκB in splenocytes of tumor-bearing mice compared to controls. Given the data from our patients, we hypothesized that the high STAT3 expression and low NFκB expression could have a correlation, with a biologic effect on tumor-bearing subjects. Again, using an HPV16 experimental model, we neutralized IL-6 and G-CSF expression or pharmacologically inhibited STAT3 with the NSC74859 compound. In both cases, we observed significant reduction in tumor growth, decrease in phospho-STAT3 expression (when using the neutralizing antibodies), and importantly, significant increase in phospho-p65 NFκB expression. We also observed a significant increase in T-cell infiltration in tumors from treated animals and increase in antigen-specific T-cell proliferation. Our data indicate that cytokines that signal chronically through STAT3 can inhibit antitumor immune responses through NFκB inhibition. We also suggest that inhibition of STAT3 may be used as therapeutic tool against cervical cancer and other HPV-associated cancers. Financial support: FAPESP 2014/19326-6; 2008/57889-1; CNPq 573799/2008-3. Citation Format: Renata Ariza Marques Rossetti, Gretel Rodriguez Rodriguez, Ildelfonso Alves Da Silva, Jr. Jesus De Paula Carvalho, Maria Beatriz Sartor De Faria Rosa, Luisa Lina Villa, Ana Paula Lepique. Cervical cancer immunomodulation through stat3 and p65 NFκb: Effects beyond the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A22.

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation, induction of apoptosis via the ERK and JNK-dependent mechanism, and suppression of sIL-6R and VEGF production

BackgroundXanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue. MethodsAnti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays. ResultsXN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells. ConclusionsERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells.General Significance: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.

Abstract 3339: Single-cell analysis reveals molecular mechanisms of NRAS-mediated leukemia stem cell self-renewal in a murine model of AML

Abstract Acute myeloid leukemia (AML) is frequently fatal because patients who initially respond to chemotherapy eventually relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al., Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. NRASG12V is required for self-renewal in a murine AML model (Sachs et al., Blood 2014). To study NRAS-mediated leukemia self-renewal, we use a transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, NRASG12V oncogene (Kim et al., Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We hypothesize that NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. To identify the NRASG12V-mediated self-renewing subpopulation, we performed single-cell RNA sequencing on the LSC-enriched cells from our AML model. Hierarchical clustering of LSC single-cell data identified three discrete profiles. Comparing the profiles of NRASG12V-expressing LSCs (“RAS-on”) to doxycycline-treated LSCs (“RAS-Off”) revealed that two of the three LSC-expression profiles are lost when NRASG12V is withdrawn. These data suggest that these two profiles are NRASG12V-dependent consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). One of these LSC subpopulations preferentially expresses genes associated with leukemia self-renewal. On the basis of this single-cell gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V-responsive LSC-subpopulations. We sorted LSCs based on CD36 and CD69 status and found that CD36−CD69+ LSCs (the group that expresses self-renewal gene expression profiles) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD69- LSCs and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p < 0.00001 for each comparison). Accordingly, in vivo mouse reconstituting experiments show that the CD36-CD69+ LSC is the only LSC subgroup that can reconstitute the leukemia in mice (p < 0.005). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of β-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease. Citation Format: Rebecca S. LaRue, Klara E. Noble-Orcutt, Conner Hansen, Ngoc Ha, David A. Largaespada, Zohar Sachs. Single-cell analysis reveals molecular mechanisms of NRAS-mediated leukemia stem cell self-renewal in a murine model of AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3339.

Single-Cell Analysis Reveals Molecular Mechanisms of NRAS-Mediated Leukemia Stem Cell Self-Renewal in a Murine Model of AML

Acute myeloid leukemia (AML) is a lethal malignancy because patients who initially respond to chemotherapy eventually relapse with treatment-resistant disease. Leukemia stem cells (LSCs) reestablish the disease by self-renewal: the ability of a stem cell to reproduce itself and give rise to progeny. LSC self-renewal is therefore critical to relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, the mechanisms that direct hematopoietic stem cell (HSC) proliferation are distinct from the mechanisms that allow HSCs to self-renew (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. To study leukemia self-renewal, we use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We demonstrated that expression of NRASG12V is required for self-renewal in this AML model and that NRASG12V -mediated signaling is distinct among leukemic subsets (Sachs et al. Blood 2014). We hypothesize that NRAS-activated pathways required for LSC self-renewal are limited to a subpopulation of cells with the LSC immunophenotype. Defining the mechanisms of self-renewal has been a challenge because cancer cells are highly heterogeneous and because disengaging proliferation from self-renewal can be difficult experimentally. To overcome these obstacles, we use single-cell technologies (single-cell, whole transcriptome, RNA sequencing and mass cytometry, CyTOF) to define the signaling and transcriptional profiles of individual cells. We performed single-cell RNA sequencing on unsorted leukemia cells and on a sorted, LSC-enriched population. The single-cell transcriptional profile of LSCs was distinct from the bulk population (Fig. 1A). The 100 most differentially expressed genes between these groups are involved in hematopoietic cell fate and differentiation, confirming the biological validity of this technique. Next, we sought to identify an NRASG12V -mediated self-renewing subpopulation among the LSCs. Unsupervised, two-dimensional, hierarchical clustering of LSC single-cell data identified three discrete subpopulations among the LSCs, each expressing a unique gene expression profile (Fig. 1B). Comparing the single-cell transcriptional profiles of NRASG12V -expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of the three LSC-expression profiles seen in RAS-on cells (Groups 1 and 3, Fig. 1B) are lost when NRASG12V is withdrawn (Fig. 1C). These data suggest that these two profiles (Groups 1 and 3) are NRASG12V -dependent, consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). Gene set enrichment analysis of these profiles, modified for single-cell data, revealed that Group 1 preferentially expresses genes associated with leukemia self-renewal. On the basis of this gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V -responsive LSC-subpopulations (Groups 1 and 3). We sorted LSCs based on CD36 and CD69 status and found that CD36- CD69+ LSCs (consistent with Group 1 gene expression) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD36+CD69- LSCs (Group 3) and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p < 0.00001 for each comparison). We have previously shown that colony-forming capacity is an accurate surrogate for in vivo leukemia reconstituting ability and self-renewal in our model (Sachs, et al. Blood 2014). These experiments characterize the NRASG12V -mediated self-renewal transcriptional signature and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of b-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease. [Display omitted] DisclosuresLargaespada:NeoClone Biotechnology, Inc.: Consultancy, Other: stockholder; Genentech Inc: Honoraria, Research Funding; Discovery Genomics Inc.: Consultancy, Other: stockholder; B-MoGen Biotechnologies Inc.: Consultancy, Other: stockholder; Orbimed Inc: Consultancy.

Using activation status of signaling pathways as mechanism-based biomarkers to predict drug sensitivity.

Many complex traits, as drug response, are associated with changes in biological pathways rather than being caused by single gene alterations. Here, a predictive framework is presented in which gene expression data are recoded into activity statuses of signal transduction circuits (sub-pathways within signaling pathways that connect receptor proteins to final effector proteins that trigger cell actions). Such activity values are used as features by a prediction algorithm which can efficiently predict a continuous variable such as the IC50 value. The main advantage of this prediction method is that the features selected by the predictor, the signaling circuits, are themselves rich-informative, mechanism-based biomarkers which provide insight into or drug molecular mechanisms of action (MoA).

Anamorsin Overexpression Leads to Dysregulation of Lipopolysaccharide-Stimulated B Cell Proliferation through Ras Signaling

Introduction: Anamorsin (AM, also called CIAPIN1) was originally isolated as a molecule that conferred resistance to apoptosis caused by growth factor deprivation. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and small compared to wild type (WT) embryos. It suggests that AM is indispensable for embryo growth and hematopoiesis. To determine which signaling pathways AM utilizes for these functions, we analyzed murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. P38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggested that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells. However, functions of AM still remain not fully understood. In order to elucidate functions of AM, we generated AM transgenic (Tg) mice under control of CAG promoter. Since our previous study showed AM overexpression in approximately 30% of B cell type malignant lymphoma (DLBCL and FL) cases, we focused on AM overexpressed B cells from AM Tg mice in this study.Methods and Results: At first, we compared the number of B cells in the peripheral blood and spleen size between AM Tg mice and WT mice and found no difference. Next, we analyzed the lipopolysaccharide (LPS) stimulated B cells. B cells were selected from spleen by using anti-B220 antibody. The purified B cells were exposed to LPS for three days and measured. Unexpectedly, LPS-stimulated proliferation of B cells from AM Tg mice was decreased compared to WT mice. Since we initially confirmed that AM overexpression did not affect TLR4 expression on B cells, we examined TLR4 signaling pathway activation status by detecting phosphorylation of the signal transduction molecules using Western-blotting, and found that phosphorylation of Erk1/2 and IKBα were decreased in LPS-stimulated B cells from AM Tg mice. Next, we performed cDNA microarray analysis to reveal the mechanisms of inhibition of the LPS signaling pathways by comparing the differential gene expression profiles in B cells of AM Tg and WT mice with or without LPS stimulation. We extracted 2375 genes from the data sets that met the following criteria: genes with > 2.0 fold change between LPS- vs LPS+ in AM Tg or WT mice, and secondly with >1.5 fold difference in the rate of gene expression change with LPS stimulation between AM Tg and WT mice, and performed upstream regulator analysis to predict the upstream regulators on the cascade of LPS signaling pathway with Ingenuity Pathway Analysis software. The data suggested that Ras activation was decreased in LPS-stimulated AM Tg B cells compared to WT B cells. Then, we confirmed the impairment of LPS induced Ras activation in AM Tg B cells using Ras activation assay kit. Furthermore, we found that AM mRNA level was increased (1.8-4.0 fold) in WT B cells at 4 hours after LPS stimulation. From these data, it was shown that AM overexpression in AM Tg B cells inhibited Ras signaling pathways and retarded the cell proliferation of LPS-stimulated B cells.Conclusion and Discussion: In our previous study, we showed that AM negatively regulates novel PKCs and p38MAPK by using MEFcells generated from AM KO mice, while in this study it was shown that overexpressed AM negatively regulates Ras signaling pathways by using AM Tg B cells. Although there are no reports other than ours that show the relationships between the signaling molecules and AM, our present data showed the novel role of AM that regulates the signaling molecules in the different manner. LPS increased AM expression via Ras activation in B cells, and AM overexpression inhibited Ras, which suggested AM might induce a negative feedback loop that attenuates Ras activation in LPS stimulated B cells. Our previous data showed that low international prognostic index (IPI) DLBCL patients with AM overexpression had a poor prognosis, especially in the patients who received chemotherapy without rituximab. In those patients, AM overexpression might contribute at least in part to chemotherapy resistance possibly through negative regulation of Ras. In summary, we showed that overexpressed AM is one of negative regulators of LPS induced signaling pathways in B cells. DisclosuresNo relevant conflicts of interest to declare.

Signaling pathway differences between EGFR TKI-sensitive and -resistant non-small cell lung cancer cells.

e13510 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have showed dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC), but the biggest problem is resistance. How to overcome resistance is still a serious problem to clinical practice. The aims of this studies was to analyse the signaling pathway differences between the EGFR TKI-sensitive and resistant NSCLC cell lines and to seek potential molecular targets for tumor treatment after resistance. Methods: We selected four types of NSCLC cell lines: PC-9(exon19 del), PC9GR (gefitinib-acquired resistant cell), H1650(exon19 del), H1975 (L858R+T790M), signaling pathway differences between PI3K/Akt/mTOR,MAPK and STAT3 were analysed through western blot, immunoprecipitation and confocal microscope. Results: Activation status of EGFR super signaling pathways: PI3K-Akt, Erk and STAT3 between EGFR TKI- sensitive and resistant NSCLC cells was different under basic state. The phosphorylation levels of Akt ser473, FOXO1 and Erk in EGFR TKI-resistant cells, especially in H1650 cell, were more higher than that in PC9 cell except for p-STAT3. p-STAT3 expression in PC9 was higher than that in resistant NSCLC cells. The further analysis showed that cellular localizations of p-STAT3 between sensitive and resistant cells were different through confocal microscope, p-STAT3 in PC9 and H1975 cells was almost in nucleus, while in PC9GR and H1650 cells was almost in cytoplasm. The assay of mTORC2 kinase activities in vitro showed that mTORC2 kinase activities in the resistant cells were obviously higher than that in sensitive cells. Conclusions: we found that EGFR TKI-sensitive and resistant NSCLC cells had different activation status of signaling pathways especially mTOR associated signaling pathways and different mTORC2 kinase activities. These results indicated that targeting mTOR with specific mTOR inhibitors would be a good strategy for NSCLC treatment after EGFR TKI resistance.

Abstract C153: Quantitative analysis of IGF-1R expression in FFPE human rhabdomyosarcoma tumor tissue by mass spectrometry.

Abstract The IGF-1R pathway is activated in many cancer types which has led to clinical development of many IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R-specific SRM assay was preclinically validated on cell lines expressing a range of IGF1R protein. The expression levels of IGF1R in seven formalin fixed rhabdomyosarcoma (RMS) cell lines were measured by SRM mass spectrometry. We detected a range of IGF1R expression from 68 to 333 amol/ug for these cell lines, values that showed extremely high correlation with antibody based techniques. Analysis of FFPE human rhabdomyosarcoma xenograft explants demonstrates that variable levels of IGF1R expression can be measured accurately. In xenografts where treatment with a therapeutic IGF1R antibody caused tumor regression there was a concomitant loss of IGF1R expression. We have extended these studies by characterizing the expression of IGF1R in a clinical cohort of 25 RMS tumors. IGF1R was detected in 13/14 embryonal RMS tumors and all 11 syncytial RMS tumors. The range of expression in these tissues was 50–250 amol/ug of protein tissue. However, there was one tumor which expressed ∼4500 amol/ug of IGF1R and may represent a stable amplification, or mutation resulting in this extremely high level of IGF1R expression. Additional studies are underway to continue characterization of this tumor. These novel techniques for quantifying IGF1R protein expression in FFPE RMS tumor tissue may enhance our ability to select patients for IGF1R targeted therapy. We are extending this work in two ways. First we are initiating retrospective analysis of IGF1R levels in a sarcoma trial of an IGFR targeted therapy, and second, we are also preparing to use this assay for prospective analysis of FFPE sarcoma tissue in a future IGF1R targeted therapy trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C153.

Quantitative analysis of IGF-1R expression in FFPE human rhabdomyosarcoma tumor tissue by mass spectrometry.

10069 Background: The IGF-1R pathway is activated in many cancer types which has led to clinical development of many IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing’s sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify patients most likely to benefit from anti-IGF1R therapies. Methods: We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue-SRM technology platform which enables r...

Abstract 4399: In vitro and in vivo evaluations of correlation between the activation status of signaling pathways and the efficacy of anticancer compounds in a panel of JFCR39 human cancer cell lines

Abstract The phosphoinositide 3-kinase (PI3K) pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway are frequently activated in cancer. Activation of these pathways contribute to cell survival, cell cycle progression and cell growth in the tumor, and are expected to influence the efficacy of antitumor compounds including conventional antitumor drugs, PI3K pathway inhibitors and Ras/MAPK pathway inhibitors. However, it is not well understood whether the efficacies of anticancer compounds can be predicted based on these aberrations and on the activation status of signaling pathway members. To clarify this issue, we first examined the mutation, expression and phosphorylation status of PI3K and Ras/MAPK pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Second, we evaluated the in vitro efficacy of various anticancer compounds including PI3K and Ras/MAPK pathway inhibitors and conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the drug efficacies in vitro. For example, KRAS/BRAF mutation prominently correlated with the efficacy of a MEK inhibitor and the inefficacy of PI3K inhibitors, respectively, while phosphorylation levels of Akt correlated with the efficacy of PI3K inhibitors. Some of these correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study molecular pharmacology of the PI3K and Ras/MAPK pathways in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4399. doi:10.1158/1538-7445.AM2011-4399

Abstract 2211: Quantitative analysis of IGF-1R expression and signaling pathway activation in FFPE xenograft and human tumor tissues

Abstract The IGF-1R signal pathway is activated in many cancer types which has led to active clinical development of both antibody and small molecule IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF-1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify and select patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R- specific SRM assay was developed and preclinically validated on cell lines expressing a range of IGF1R protein. The final assay has a LOD of &amp;lt;20 attomol/ug and an LOQ of ∼35 attomol/ug, values that are well in line with expected levels of expression in primary tumor sourced cell lines and xenograft explants. The expression levels of IGF1R in seven formalin fixed rhabdomyosarcoma (RMS) cell lines were measured by SRM mass spectrometry. We detected a range of IGF1R expression from 68 to 333 amol/ug for these cell lines, values that showed extremely high correlation with antibody based techniques (Cao et al, Cancer Res. 68:8039). We extended this quantitation study by measuring expression of IGF-1R in human rhabdomyosarcoma xenograft explants and a cohort of ∼40 embryonal and alveolar rhabdomyosarcoma tumors. Interestingly, in xenografts treated with a therapeutic IGF1R antibody, tumor regression was seen, and a loss of IGF1R expression was noted by SRM analysis. Our goal is to define expression levels of IGF1R in these tumors, and to validate this assay for use as a companion diagnostic in follow on clinical trials of IGF1R inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2011-2211

88 In silico and biological evaluation of correlation between efficacy of PI3K inhibitors with the activation status of signaling pathways in a panel of JFCR39 human cancer cell lines

88 In silico and biological evaluation of correlation between efficacy of PI3K inhibitors with the activation status of signaling pathways in a panel of JFCR39 human cancer cell lines

Functional Classification of Peripheral T-Cell Lymphomas as an Approach to Improve Outcome Prediction and Therapy Selection

Many types of peripheral T-cell lymphoma (PTCL) are currently classified by the World Health Organization (WHO) based on their predominant site of organ involvement (eg, intestinal or cutaneous types). However, this approach and traditional staging scores such as the IPI can provide limited prognostic information, especially in those PTCL types where morbidity and mortality are primarily related to immune dysregulation and cytokine syndromes driven by the lymphoma cells. These "immune participatory" PTCLs (including, commonly, angioimmunoblastic T-cell lymphoma and many extranodal types) can therefore have poor outcomes even at low tumor burdens. For these reasons, a classification that includes functional profiling of the lymphoma cells may add valuable prognostic information. Such data, including cytokine expression patterns and T-cell receptor signaling pathway activation status, whether normal or abnormal, need to be considered in future classification systems, especially when incorpating targeted therapy.

Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization.