Changes In Phosphoproteins Research Articles

Phosphoproteomic changes in response to anoxia are tissue-specific in the anoxia-tolerant crucian carp (Carassius carassius).

Crucian carp (Carassius carassius), a freshwater fish, can survive chronic anoxia for several months at low temperatures. Consequently, anoxia-related physiological and biochemical adaptations in this species have been studied for more than half a century. Still, despite for the well-known role of protein phosphorylation in regulating cellular processes, no studies have comprehensively characterized the phosphoproteome in crucian carp. In this study, we report the global phosphoproteome in crucian carp brain and liver during anoxia and reoxygenation. By applying a bottom-up proteomic approach on enriched phosphopeptides we found that the brain phosphoproteome shows surprisingly few changes during anoxia-reoxygenation exposure with only 109 out of 4200 phosphopeptides being differentially changed compared to normoxic controls. By contrast, in the liver 395 out of 1287 phosphopeptides changed. Although most changes occurred in the liver phosphoproteome, the pattern of changes indicated metabolic depression and decreased translation in both brain and liver. We also found changes in phosphoproteins involved in apoptotic regulation and reactive oxygen species handling in both tissues. In the brain, some of the most changed phosphopeptides belonged to proteins involved in central nervous system development and neuronal activity at the synaptic cleft. Changed phosphoproteins specific for liver tissue were related to glucose metabolism, such as glycolytic flux and glycogenolysis. In conclusion, protein phosphorylation in response to anoxia and reoxygenation showed both common and tissue-specific changes related to the functional differences between brain and liver.

Analysis of kidney proteomes to identify biological pathways associated with vancomycin-induced nephrotoxicity in mice by tandem mass tag-labeled quantitative and parallel reaction monitoring phosphoproteomics.

Vancomycin (VCM)-induced nephrotoxicity impedes its treatment applications. Thus, it is important to clarify the relevant mechanism. This study investigated phosphoprotein changes attributable to the VCM nephrotoxicity mechanisms. Biochemical, pathological and phosphoproteomic analyses based on C57BL/6 mice were performed to explore the mechanisms.VCM-treated mice showed increased levels of blood urea nitrogen and creatinine, and signs of acute tubular necrotic lesions. Phosphoproteomic profiling identified 3025 differentially phosphorylated phosphopeptides between the model and control group. Gene Ontology enrichment analysis demonstrated that Molecular Function "oxidoreductase activity" and Cellular Component "peroxisome" were markedly enriched. KEGG pathway analysis identified an enrichment in peroxisome pathway and PPAR (peroxisome proliferator-activated receptor) signaling pathways. Parallel reaction monitoring analysis revealed a significant downregulation of CAT, SOD-1, AGPS, DHRS4, and EHHADH at phosphorylation level by VCM. Notably, the phosphorylation of ACO, AMACR, and SCPX was downregulated by VCM, which are the fatty acid β-oxidation-related proteins involved in PPAR signaling pathways. The phosphorylated PEX5 involved in peroxisome biogenesis was upregulated by VCM. Collectively, these findings indicated that VCM-induced nephrotoxicity is closely associated with peroxisome pathway and PPAR signaling pathways. The current study provides important insight into the mechanisms of VCM nephrotoxicity and will aid in the development of preventive and therapeutic strategies against this nephropathy.

Abstract 3461: Preclinical and clinical evaluation of the RAF/MEK clamp avutometinib (VS-6766) in combination with the mTOR inhibitor everolimus for the treatment of KRAS mutated non-small cell lung cancer

Abstract Background: KRAS mutations (mt) are found in ~30% of non-small cell lung cancers (NSCLC). Despite the approval and development of KRAS G12C and KRAS G12D inhibitors respectively, mechanisms of resistance to MAPK pathway inhibitors are emerging and combination strategies are needed for patients with KRAS mt NSCLC . Avutometinib (VS-6766) is a unique RAF/MEK clamp that inhibits MEK kinase activity and blocks RAF-mediated phosphorylation of MEK. We studied the preclinical and clinical activity of the combination of avutometinib with the mTOR inhibitor everolimus in KRAS mt NSCLC. Methods: A panel of KRAS mt NSCLC cell lines were treated for 1 hour with clinically relevant concentrations of avutometinib and everolimus and changes in phosphoproteins were measured using an antibody array. We then tested for synergy of avutometinib and everolimus in 3D proliferation assays and in the H441 NSCLC xenograft model. A clinical trial is ongoing (NCT02407509) testing the combination of 3.2 mg avutometinib with 5 mg of everolimus administered twice weekly 3 weeks on/1 week off in 28-day cycles (previously defined as the recommended phase 2 dose) in a cohort of patients with KRAS mt NSCLC. Results: Avutometinib inhibited the MAPK pathway (p-MEK, p-ERK, p-90RSK) with an increase in p-PRAS40, suggesting activation of the PI3K pathway as an adaptive resistance mechanism. Everolimus inhibited the PI3K pathway (p-p70S6K and p-RPS6). Among a panel of KRAS mt NSCLC cell lines, avutometinib + everolimus showed synergistic anti-proliferative activity across KRAS G12C, G12V and G12D variants (mean synergy score of ~18). In the H441 KRAS G12V NSCLC xenograft model, there was a significant reduction in tumor volume and increase in survival with the combination of avutometinib and everolimus (87% TGI; 66 days vs 36.5 days in control group) compared to control. In the clinical trial expansion, 16 patients with KRAS mt NSCLC have been treated so far with avutometinib and everolimus (5 G12V, 3 Q61H, 2 G12C, 2 G12A, 2 G12D, 1 G13A, 1 G13D; median prior lines = 2). The current objective response rate (ORR) among the 14 patients who have at least one post assessment scan is 3/14 (21%; 1 G12V, 1 G12A, 1 G13A) with 11/14 showing a reduction in tumor size as best response. The current progression free survival (PFS) is 5.3 months (95% CI 2.8-7.4 months) with 4 patients still on study. Updated data on the planned cohort size of 20 patients will be presented. Conclusion: The combination of avutometinib and everolimus overcomes the activation of the PI3K/AKT/mTOR pathway which is an adaptive resistance mechanism to MAPK pathway inhibition. We have shown that avutometinib and everolimus induce synergistic anti-tumor effects preclinically, and preliminary data suggest clinically meaningful ORR and PFS in patients with KRAS mt NSCLC including non-G12C variants. Citation Format: Simon Rodney, Adam R. Stewart, Victoria Sanchez Perez, Cienne Morton, Lisa A. Pickard, Taleen Shakouri, Toby Prout, Mona Parmar, Alison J. Turner, Silvia Coma, Jonathan Pachter, Laura Finneran, Emma Hall, James Spicer, Anna Minchom, Udai Banerji. Preclinical and clinical evaluation of the RAF/MEK clamp avutometinib (VS-6766) in combination with the mTOR inhibitor everolimus for the treatment of KRAS mutated non-small cell lung cancer [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 3461.

Afatinib Reverses EMT via Inhibiting CD44-Stat3 Axis to Promote Radiosensitivity in Nasopharyngeal Carcinoma

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC. Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo. In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis. Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.

Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF).

Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 ± 0.0 vs. 1.0 ± 0.0; P < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham (0.144 ± 0.01 vs. 0.127 ± 0.01; P < 0.05). In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.

Abstract 1002: Targeting adaptation to cancer treatment by drug combinations

Abstract Adaptation of tumors to therapeutic interventions contributes to dismal long-term patient outcomes. Adaptation to therapy involves the co-action of functionally related proteins that together activate cell survival programs and compensate for the therapeutic impact. Oncogenic dependencies to such adaptive events, however, can generate new therapeutic vulnerabilities that can be targeted with drug combinations. The precision medicine approaches in which targeted drugs are matched to pre-existing genomic aberrations fail to address the adaptive responses and resulting vulnerabilities. Here, we provide the mathematical formulation, implementation, and validation of the TargetScore method. The TargetScore identifies collective adaptive responses to targeted interventions as concurrent changes of phospho-proteins that are connected within a signaling network. Based on the adaptive responses, the method predicts drug-induced vulnerabilities. Using TargetScore, we inferred the adaptive responses with short-term (i.e., days) stress and long-term (i.e., months) acquired resistance to inhibitors of antiapoptotic mediators, MCL1 and BCL2. With experiments guided by the predictions, we identified synergistic interactions between inhibitors of PARP, SHP2, and MCL1 in breast cancer cells. TargetScore is readily applicable to existing precision oncology efforts by matching targeted drug combinations to emerging molecular signatures under therapeutic stress. Citation Format: Heping Wang, Augustin Luna, Gonghong Yan, Xubin Li, Ozgun Babur, Chris Sander, Gordon Mills. Targeting adaptation to cancer treatment by drug combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1002.

Quantitative phosphoproteomics reveals the role of wild soybean GsSnRK1 as a metabolic regulator under drought and alkali stresses

Drought and alkali stresses cause detrimental effects on plant growth and development. SnRK1 protein kinases act as key energy and stress sensors by phosphorylation-mediated signaling in the regulation of plant defense reactions against adverse environments. To understand SnRK1-dependent phosphorylation events in signaling pathways triggered by abiotic factors, we employed quantitative phosphoproteomics to compare the global changes in phosphopeptides and phosphoproteins in 2kinm mutant Arabidopsis (SnRK1.1 T-DNA knockout and SnRK1.2 knockdown by β-estradiol-induced RNAi) complemented with wild soybean GsSnRK1(wt) or dominant negative mutant GsSnRK1(K49M) in response to drought and alkali stresses. Among 4014 phosphopeptides (representing 2380 phosphoproteins) identified in this study, we finalized 74 phosphopeptides (representing 61 phosphoproteins), and 75 phosphopeptides (representing 57 phosphoproteins) showing significant changes in phosphorylation levels under drought and alkali treatments respectively. Function enrichment and protein-protein interaction analyses indicated that the differentially-expressed phosphoproteins (DPs) under drought and alkali stresses were mainly involved in signaling transduction, stress response, carbohydrate and energy metabolism, transport and membrane trafficking, RNA splicing and processing, DNA binding and gene expression, and protein synthesis/folding/degradation. These results provide assistance to identify bona fide and novel SnRK1 phosphorylation substrates and shed new light on the biological functions of SnRK1 kinase in responses to abiotic stresses. SignificanceThese results provide assistance to identify novel SnRK1 phosphorylation substrates and regulatory proteins, and shed new light on investigating the potential roles of reversible phosphorylation in plant responses to abiotic stresses.

Quantitative phosphoproteomic analysis reveals chemoresistance-related proteins and signaling pathways induced by rhIL-6 in human osteosarcoma cells

BackgroundIL-6 plays a pivotal role in resistance to chemotherapeutics, including lobaplatin. However, the underlying mechanisms are still unclear. This study was to investigate the changes in phosphoproteins and their related signaling pathways in the process of IL-6-induced chemoresistance to lobaplain in osteosarcoma cells.MethodsWe performed a quantitative phosphoproteomic analysis of the response of SaOS-2 osteosarcoma cells to recombinant human IL-6 (rhIL-6) intervention prior to lobaplatin treatment. The cells were divided into the control group (Con), the lobaplatin group (Lob), and the rhIL-6-and-lobaplatin group (IL-6). Three biological replicates of each group were included. The differentially expressed phosphoproteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Netphos 3.1 was used for the prediction of kinases, and STRING was used for the visualization of protein–protein interactions. The conserved motifs surrounding the phosphorylated residues were analyzed using the motif-x algorithm. Western blot analysis was performed to verify the differential expression of p-FLNC, its predicted kinase and the related signaling pathway. The results of the bioinformatic analysis were validated by immunohistochemical staining of clinical specimens.ResultsIn total, 3373 proteins and 12,183 peptides, including 3232 phosphorylated proteins and 11,358 phosphorylated peptides, were identified and quantified. Twenty-three significantly differentially expressed phosphoproteins were identified in the comparison between the IL-6 and Lob groups, and p-FLNC ranked second among these phosphoproteins. GO and KEGG analyses revealed the pivotal role of mitogen-activated protein kinase signaling in drug resistance induced by rhIL-6. Four motifs, namely, -SPxxK-, -RxxSP-, -SP-, and -SPK-, demonstrated higher expression in the IL-6 group than in the Lob group. The western blot analysis results verified the higher expression of p-FLNC, AKT1, and p-ERK and the lower expression of p-JNK in the IL-6 group than in the Con and Lob groups. The immunohistochemical staining results showed that p-FLNC, AKT1 and p-ERK1/2 were highly expressed in platinum-resistant clinical specimens but weakly expressed in platinum-sensitive specimens, and platinum-resistant osteosarcoma specimens demonstrated weak expression of p-JNK.ConclusionsThis phosphoproteomic study is the first to reveal the signature associated with rhIL-6 intervention before lobaplatin treatment in human osteosarcoma cells. p-FLNC, AKT1, and MAPK signaling contributes to resistance to lobaplatin in osteosarcoma SaOS-2 cells and may represent molecular targets to overcome osteosarcoma chemoresistance.

Abstract 6310: Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level

Abstract BACKGROUND: Protein phosphorylation is an important cellular regulatory process and a key indicator of signaling activation or deactivation. However, the fast switch-on/off of the process makes difficult the timely and accurate detection of the phosphorylation status. It is even more challenging to monitor the protein phosphorylation during cancer immune response in vivo, whereas immune responses vary among different cell types and can sometimes only be detected in certain rare populations like Treg (T regulatory) and TCM (T central memory) cells. The ability of Phosphoflow cytometry to accurately and rapidly detect many phospho-protein targets simultaneously at single cell level provides significant advantages over conventional methods. Ex vivo phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in monitoring of disease and therapy progress. RESULTS: We established an ex vivo phosphoflow system to evaluate fast changes of pStat5 at single cell level in mouse models. Method validation was carried out step by step to ensure the reproducibility of this assay. The method development included optimizing gating strategy for non-phosphorylated proteins, selecting the reagents and optimizing the procedures of Lyse/Fix, perm, setting up positive controls under in vitro and ex vivo conditions, and optimizing phosphorylated protein staining and detection. Our data showed that the test articles induced differential pStat5 expression in different cell types, and in a dose-dependent manner. SUMMARY: We have established reliable ex vivo phosphoflow method to detect the fast changes of phospho-proteins at single cell level in animal models. This powerful method will enable us to examine protein post-translational modification in complicated host systems. Citation Format: Qiyao Zhang, Jingjing Wang, Yun Ge, Yajie Xiong, Beibei Qin, Qingyang Gu, Qunsheng Ji. Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6310.

Metastatic Melanoma Patient-Derived Xenografts Respond to MDM2 Inhibition as a Single Agent or in Combination with BRAF/MEK Inhibition.

Over 60% of patients with melanoma respond to immune checkpoint inhibitor (ICI) therapy, but many subsequently progress on these therapies. Second-line targeted therapy is based on BRAF mutation status, but no available agents are available for NRAS, NF1, CDKN2A, PTEN, and TP53 mutations. Over 70% of melanoma tumors have activation of the MAPK pathway due to BRAF or NRAS mutations, while loss or mutation of CDKN2A occurs in approximately 40% of melanomas, resulting in unregulated MDM2-mediated ubiquitination and degradation of p53. Here, we investigated the therapeutic efficacy of over-riding MDM2-mediated degradation of p53 in melanoma with an MDM2 inhibitor that interrupts MDM2 ubiquitination of p53, treating tumor-bearing mice with the MDM2 inhibitor alone or combined with MAPK-targeted therapy. To characterize the ability of the MDM2 antagonist, KRT-232, to inhibit tumor growth, we established patient-derived xenografts (PDX) from 15 patients with melanoma. Mice were treated with KRT-232 or a combination with BRAF and/or MEK inhibitors. Tumor growth, gene mutation status, as well as protein and protein-phosphoprotein changes, were analyzed. One-hundred percent of the 15 PDX tumors exhibited significant growth inhibition either in response to KRT-232 alone or in combination with BRAF and/or MEK inhibitors. Only BRAFV600WT tumors responded to KRT-232 treatment alone while BRAFV600E/M PDXs exhibited a synergistic response to the combination of KRT-232 and BRAF/MEK inhibitors. KRT-232 is an effective therapy for the treatment of either BRAFWT or PAN WT (BRAFWT, NRASWT) TP53WT melanomas. In combination with BRAF and/or MEK inhibitors, KRT-232 may be an effective treatment strategy for BRAFV600-mutant tumors.

A Brain Signaling Framework for Stress-Induced Depression and Ketamine Treatment Elucidated by Phosphoproteomics.

Depression is a common affective disorder characterized by significant and persistent low mood. Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, is reported to have a rapid and durable antidepressant effect, but the mechanisms are unclear. Protein phosphorylation is a post-translational modification that plays a crucial role in cell signaling. Thus, we present a phosphoproteomics approach to investigate the mechanisms underlying stress-induced depression and the rapid antidepressant effect of ketamine in mice. We analyzed the phosphoprotein changes induced by chronic unpredictable mild stress (CUMS) and ketamine treatment in two known mood control centers, the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). We initially obtained >8,000 phosphorylation sites. Quantitation revealed 3,988 sites from the mPFC and 3,196 sites from the NAc. Further analysis revealed that changes in synaptic transmission-related signaling are a common feature. Notably, CUMS-induced changes were reversed by ketamine treatment, as shown by the analysis of commonly altered sites. Ketamine also induced specific changes, such as alterations in synapse organization, synaptic transmission, and enzyme binding. Collectively, our findings establish a signaling framework for stress-induced depression and the rapid antidepressant effect of ketamine.

Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis

Soybean (Glycine max (L.) Merrill) is an important component of the human diet and animal feed, but soybean production is limited by abiotic stresses especially salinity. We recently found that rhizobia inoculation enhances soybean tolerance to salt stress, but the underlying mechanisms are unaddressed. Here, we used quantitative phosphoproteomic and metabonomic approaches to identify changes in phosphoproteins and metabolites in soybean roots treated with rhizobia inoculation and salt. Results revealed differential regulation of 800 phosphopeptides, at least 32 of these phosphoproteins or their homologous were reported be involved in flavonoid synthesis or trafficking, and 27 out of 32 are transcription factors. We surveyed the functional impacts of all these 27 transcription factors by expressing their phospho-mimetic/ablative mutants in the roots of composite soybean plants and found that phosphorylation of GmMYB183 could affect the salt tolerance of the transgenic roots. Using data mining, ChIP and EMSA, we found that GmMYB183 binds to the promoter of the soybean GmCYP81E11 gene encoding for a Cytochrome P450 monooxygenase which contributes to the accumulation of ononin, a monohydroxy B-ring flavonoid that negatively regulates soybean tolerance to salinity. Phosphorylation of GmMYB183 was inhibited by rhizobia inoculation; overexpression of GmMYB183 enhanced the expression of GmCYP81E11 and rendered salt sensitivity to the transgenic roots; plants deficient in GmMYB183 function are more tolerant to salt stress as compared with wild-type soybean plants, these results correlate with the transcriptional induction of GmCYP81E11 by GmMYB183 and the subsequent accumulation of ononin. Our findings provide molecular insights into how rhizobia enhance salt tolerance of soybean plants.

Altered Levels of Proteins and Phosphoproteins, in the Absence of Early Causative Transcriptional Changes, Shape the Molecular Pathogenesis in the Brain of Young Presymptomatic Ki91 SCA3/MJD Mouse

Spinocerebellar ataxia type 3 (SCA3/MJD) is a polyQ neurodegenerative disease where the presymptomatic phase of pathogenesis is unknown. Therefore, we investigated the molecular network of transcriptomic and proteomic triggers in young presymptomatic SCA3/MJD brain from Ki91 knock-in mouse. We found that transcriptional dysregulations resulting from mutant ataxin-3 are not occurring in young Ki91 mice, while old Ki91 mice and also postmitotic patient SCA3 neurons demonstrate the late transcriptomic changes. Unlike the lack of early mRNA changes, we have identified numerous early changes of total proteins and phosphoproteins in 2-month-old Ki91 mouse cortex and cerebellum. We discovered the network of processes in presymptomatic SCA3 with three main groups of disturbed processes comprising altered proteins: (I) modulation of protein levels and DNA damage (Pabpc1, Ddb1, Nedd8), (II) formation of neuronal cellular structures (Tubb3, Nefh, p-Tau), and (III) neuronal function affected by processes following perturbed cytoskeletal formation (Mt-Co3, Stx1b, p-Syn1). Phosphoproteins downregulate in the young Ki91 mouse brain and their phosphosites are associated with kinases that interact with ATXN3 such as casein kinase, Camk2, and kinases controlled by another Atxn3 interactor p21 such as Gsk3, Pka, and Cdk kinases. We conclude that the onset of SCA3 pathology occurs without altered transcript level and is characterized by changed levels of proteins responsible for termination of translation, DNA damage, spliceosome, and protein phosphorylation. This disturbs global cellular processes such as cytoskeleton and transport of vesicles and mitochondria along axons causing energy deficit and neurodegeneration also manifesting in an altered level of transcripts at later ages.

Exploratory analysis of the effect of taselisib on downstream pathway modulation and correlation with tumor response in ER-positive/HER2-negative early-stage breast cancer from the LORELEI trial.

1050 Background: Taselisib (T) is an oral, potent, selective inhibitor of Class I PI3-kinase with enhanced activity against PIK3CA mutant cancer cells. Results from the LORELEI trial have demonstrated a significant improvement in ORR (objective response rate) by centrally assessed magnetic resonance imaging in all randomized patients as well as in the PIK3CA mutant (MT) cohort treated with neoadjuvant T plus letrozole (L) compared to placebo (P) plus L. Here we present the results of exploratory analyses of selected pathway-related phosphoproteins. Methods: Baseline (BL) and week3 (W3) tumor biopsies were obtained from 334 patients enrolled in the trial. Phosphoproteins (pAKT, pPRAS40 and pS6) were analyzed by IHC. BL levels as well as changes from BL to W3 were correlated with response assessed either by ORR or cell cycle arrest (Ki67 at W3 &lt; 2.7%). Results: In the overall population, BL phosphoproteins levels were similar between the T and P arms. Higher pAKT (p &lt; 0.001) and pPRAS40 (p = 0.004) levels were observed in MT vs wild-type (WT), whereas the opposite result was found for pS6 (p = 0.03). Treatment-induced absolute changes of phosphoproteins adjusted for BL levels were not significantly different between the T and P arms in the overall population, except for pPRAS40 with higher decrease in the T arm (p = 0.014). After stratification for PIK3CA genotype, a significantly greater decrease in expression levels was observed for pPRAS40 (p &lt; 0.001) and pS6 (p = 0.020) in MT tumors treated with T. The treatment effects were not significantly different in the WT population. A trend for an association between decrease in pS6 levels at W3 and improved ORR was observed in the MT (p = 0.08) and T (p = 0.09) subgroups. The magnitude of pS6 suppression at W3 was higher in tumors achieving a cell cycle arrest in the MT/T subgroup (biserial correlation = -0.473). Conclusions: Exploratory analyses of phosphoproteins showed bioactivity of taselisib as indicated by downstream pathway suppression. Translational research aiming to integrate these results with additional exploratory biomarkers data is currently ongoing. Clinical trial information: NCT02273973.

Post-treatment de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma

BackgroundDasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed.ResultsHere, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.ConclusionsLoss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies.

Abstract 450: Global Phosphopeptide Analyses Identifies a Profile that Distinguishes Advanced Heart Failure Patients Capable of Cardiac Recovery Following LVAD Unloading

The classical view of the human heart characterizes it as an organ with limited ability to recover and restore itself. Consequently, left ventricular assist devices (LVAD), which provide mechanical circulatory support and profound ventricular unloading, have been used as both a destination therapy as well as a bridge to transplantation in heart failure patients. However, evidence from many prospective studies indicates that a subset of patients with LVAD implants can significantly improve the structure and function of their heart (i.e. “responders”) and undergo LVAD explantation. This intriguing phenomenon provides great promise for heart failure patients, although the underlying mechanisms driving this recovery are largely unknown. To identify global changes in signaling pathways operative in the normal and failing human heart and to distinguish patients who respond to LVAD therapy from non-responders we performed phosphopeptide profiling and label-free quantitation of 38 patients. Our analysis utilized cardiac tissue from 10 donor controls, 6 recovered heart failure patients (responder), and 22 patients that did not respond to LVAD therapy (non-responders). Phosphopeptide enrichment and mass spectrometry-based analyses identified 15,816 unique phosphopeptides, 2,017 of which were shared between all samples. Label-free quantitation and bioinformatic analysis further classified 288 peptides [≥ 2 fold change (p&lt;0.01)] within this dataset that distinguish control tissue from those in heart failure. Most intriguing however, was our assessment of the phosphopeptide profile of heart failure patients at the time of LVAD implantation which, using a signature of 67 peptides [≥ 2 fold change (p&lt;0.01)], allowed complete separation of responder and non-responder samples via statistical analysis. Thus, this panel of 67 phosphopeptides enabled us to determine which patients would experience cardiac recovery following ventricular unloading prior to LVAD implantation. Overall this study characterizes unique changes in phosphoproteins and signaling pathways between the normal and failing human heart and specifies those which define hearts capable of cardiac recovery which may guide strategies to improving current heart failure therapies.

Abstract 996: A translational phosphoproteomic approach to study differences in KRAS signaling in pancreatic, colorectal and lung cancers

Abstract Aims To understand any context-dependent differences in signaling pathways between pancreatic (PAN), colorectal (CR) and lung (LU) cancers with KRAS mutations using a targeted phosphoproteomic approach in cell lines and patient-derived cancer cells exposed to targeted anticancer drugs ex-vivo. Materials and Methods We studied a panel of 30 KRAS mutant cell lines: 10 PAN, 10 CR and 10 LU cell lines. Cancer cells were also immuno-magnetically isolated from pleural effusions and ascites of patients with KRAS mutant CR and LU cancer and exposed to a DMSO control and clinically relevant concentrations of PI3K (pictilisib), AKT (AZD5363), mTOR (everolimus), EGFR (gefitinib), BRAF (vemurafenib), MEK (trametinib) and HSP90 (luminespib) inhibitors for 1 hr. Dynamic changes in a panel of 52 relevant phosphoproteins were studied using the Luminex 200 platform. Hierarchical clustering and logistic regression were used to find differences in dynamic changes in phosphoproteins between KRAS mutant, PAN, CR and LU cancer cells. Results Supervised clustering studying exposure to different drugs revealed that when exposed to the PI3K inhibitor, pictilisib, KRAS mutant LU cancers did not significantly cluster together; p=0.008, p=0.104 following Benjamini-Hochberg correction. Independently, logistic regression showed significant differences in signaling of KRAS mutant cells when exposed to the PI3K inhibitor, pictilisib. PAN and CR cancers showed an increase in p-MEK while LU cancer cells did not; p=0.0195. LU cancer cell lines showed significantly more reduction of p-AKT compared to PAN and CR cell lines when exposed to the PI3K inhibitor; p=0.0423. As expected, exposure to vemurafenib increased p-MEK levels across the majority of the KRAS mutant cell lines, however compensatory reductions in p-mTOR levels were seen significantly more in PAN and CR cell lines and not in LU cell lines; p=0.0084. The dynamic phosphoprotein changes caused by pictilisib were validated in cancer cells isolated from serous effusions of 3 KRAS mutant LU and 4 KRAS mutant CR cancer patients. Validation of these findings using multiple other inhibitors and time-points is ongoing. Interpretation/conclusions We hypothesise that the significantly greater reduction in p-AKT and less increase of compensatory p-MEK caused by PI3K inhibition in KRAS mutant LU cells compared to KRAS mutant PAN and CR cell lines represents preferential signaling of these cells through the PI3K pathway. Increase in p-MEK driven by BRAF inhibitors caused a reduction in p-mTOR in PAN and CR but not in LU cell lines also indicating preferential dependence of signaling in KRAS mutant lung cancer cells through the PI3K pathway. These findings are important while designing clinical trials of KRAS mutant cancers and more broadly to precision medicine where mutation status independent of tissue context is often used. Citation Format: Adam Stewart, Elizabeth A. Coker, Anna Minchom, Sebastian Pölsterl, Alexandros Georgiou, Paul Huang, Bissan Al-Lazikani, Udai Banerji. A translational phosphoproteomic approach to study differences in KRAS signaling in pancreatic, colorectal and lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2017-996

Abstract 412: Global Phosphopeptide Analyses Identifies a Profile that Distinguishes Advanced Heart Failure Patients Capable of Cardiac Recovery Following Left Ventricular Assist Device Unloading

The classical view of the human heart characterizes it as an organ with limited ability to recover and restore itself. Consequently, left ventricular assist devices (LVAD), which provide mechanical circulatory support and profound ventricular unloading, have been used as both a destination therapy as well as a bridge to transplantation in heart failure patients. However, evidence from many prospective studies indicates that a subset of patients with LVAD implants can significantly improve the structure and function of their heart (i.e. “responders”) and undergo LVAD explantation. This intriguing phenomenon provides great promise for heart failure patients, although the underlying mechanisms driving this recovery are largely unknown. To identify global changes in signaling pathways operative in the normal and failing human heart and to distinguish patients who respond to LVAD therapy from non-responders we performed phosphopeptide profiling and label-free quantitation of 38 patients. Our analysis utilized cardiac tissue from 10 donor controls, 6 recovered heart failure patients (responder), and 22 patients that did not respond to LVAD therapy (non-responders). Phosphopeptide enrichment and mass spectrometry-based analyses identified 15,816 unique phosphopeptides, 2,017 of which were shared between all samples. Label-free quantitation and bioinformatic analysis further classified 288 peptides [≥ 2 fold change (p&lt;0.01)] within this dataset that distinguish control tissue from those in heart failure. Most intriguing however, was our assessment of the phosphopeptide profile of heart failure patients at the time of LVAD implantation which, using a signature of 67 peptides [≥ 2 fold change (p&lt;0.01)], allowed complete separation of responder and non-responder samples via statistical analysis. Thus, this panel of 67 phosphopeptides enabled us to determine which patients would experience cardiac recovery following ventricular unloading prior to LVAD implantation. Overall this study characterizes unique changes in phosphoproteins and signaling pathways between the normal and failing human heart and specifies those which define hearts capable of cardiac recovery which may guide strategies to improving current heart failure therapies.

Abstract P5-13-01: Transcript analysis of PI3K and immune-related genes and gene signatures in the pre- and post-treatment samples from the window of opportunity study of anastrozole and anastrozole with pictilisib (GDC-0941) in patients with HR-positive early breast cancer (OPPORTUNE study)

Abstract Background: The OPPORTUNE Study randomized postmenopausal patients (pts) to receive 2-week preoperative treatment with anastrozole (ANA) plus pictilisib ("ANA+PIC" arm) or ANA alone. Patients had newly diagnosed, operable, ER+, HER2- invasive breast cancer of ≥1 cm size. The primary outcome at interim analysis (n=70) revealed that the addition of PIC significantly increased the anti-proliferative response to ANA as measured by reduction in Ki67 immunohistochemistry (IHC). Multivariate analyses suggested benefit of PIC for patients with luminal B disease (Schmid et al. SABCS 2014). Methods: RNA expression analysis of ∼800 breast cancer-related genes was performed on patients analyzed at the interim analysis, including 14 (ANA) and 20 (ANA+PIC) patients with matched pre- and post- treatment paired tumour samples using the nCounter platform (NanoString). Differential expression of individual genes by arm was assessed using paired and moderated t-tests and statistical significance assessed through false discovery rate (FDR). Ingenuity Pathway Analysis (IPA) of differentially expressed transcripts identified pathways of relevance. Protein expression was analyzed by reverse protein array ( RPPA) in pre- and post-treatment samples. Results: In an unsupervised analysis, down-regulation of genes associated with ER signaling was observed in patients who received single-agent ANA and ANA+PIC, which included genes that regulate the cell cycle, cell death, survival, growth and proliferation and known ER target genes (e.g., PGR, GREB1). In addition, transcripts related to growth factor signaling pathway appeared to be specifically modulated in the ANA+PIC arm, possibly via the upregulation of the expression of RTK ligands. There were no clear changes in PI3K-related phosphoproteins (e.g., AKT, S6, 4E-BP1) in the post-treatment samples by RPPA. However, known PI3K-regulated genes, IRS2 and PIK3IP1, were upregulated in the post-treatment samples and a composite PI3K gene expression signature score (O'Brien et al. 2010) was reduced in both study arms following treatment. This PI3K signature was associated with pre-treatment luminal B status (n=27) and, consistent with this finding, the baseline PI3K gene signature score in the ANA arm, but not the ANA+PIC arm, was inversely associated with the decrease in post treatment Ki67. The tumor immune microenvironment was analyzed though the use of composite gene sets. In our initial observations, analysis of pre- and post-treatment samples showed that 2-week treatment with ANA resulted in a modest increase in transcripts associated with multiple immune signatures, which was further enhanced by the addition of PIC. Conclusions: Gene expression analysis of pre- and post-treatment samples in the OPPORTUNE study demonstrates on-target inhibition of ER and PI3K signaling networks. The analysis of additional paired samples is in progress to further assess if 2-weeks of treatment with a regimen containing an AI in patients with early breast cancer impacts the tumor immune microenvironment. Citation Format: Schmid P, Pinder SE, Bundred N, Wheatley D, Macaskill J, Zammit C, Hu J, Price R, Shia A, Lim L, Parker P, Molinero L, Yu J, O'Brien C, Wilson T, Savage H, Derynck M, Lackner MR, Amler L, Purushotham A, Thompson A, Gendreau S. Transcript analysis of PI3K and immune-related genes and gene signatures in the pre- and post-treatment samples from the window of opportunity study of anastrozole and anastrozole with pictilisib (GDC-0941) in patients with HR-positive early breast cancer (OPPORTUNE study). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-01.

Genome-wide perturbations by miRNAs map onto functional cellular pathways, identifying regulators of chromatin modifiers.

Background:Regulation of gene expression by microRNAs (miRNAs) is critical for determining cellular fate and function. Dysregulation of miRNA expression contributes to the development and progression of multiple diseases. miRNA can target multiple mRNAs, making deconvolution of the effects of miRNA challenging and the complexity of regulation of cellular pathways by miRNAs at the functional protein level remains to be elucidated. Therefore, we sought to determine the effects of expression of miRNAs in breast and ovarian cancer cells on cellular pathways by measuring systems-wide miRNA perturbations to protein and phosphoproteins.Methods:We measure protein level changes by reverse-phase protein array (RPPA) in MDA-MB-231, SKOV3.ip1 and HEYA8 cancer cell lines transfected by a library of 879 human miRNA mimics.Results:The effects of multiple miRNAs–protein networks converged in five broad functional clusters of miRNA, suggesting a broad overlap of miRNA action on cellular pathways. Detailed analysis of miRNA clusters revealed novel miRNA/cell cycle protein networks, which we functionally validated. De novo phosphoprotein network estimation using Gaussian graphical modeling, using no priors, revealed known and novel protein interplay, which we also observed in patient ovarian tumor proteomic data. We identified several miRNAs that have pluripotent activities across multiple cellular pathways. In particular we studied miR-365a whose expression is associated with poor survival across several cancer types and demonstrated that anti-miR-365 significantly reduced tumor formation in animal models.Conclusions:Mapping of miRNA-induced protein and phosphoprotein changes onto pathways revealed new miRNA-cellular pathway connectivity, paving the way for targeting of dysregulated pathways with potential miRNA-based therapeutics.