Proteomic Landscape Research Articles

Predictive molecular markers of chemoradiation resistance in cervical cancer.

e17505 Background: Cisplatin based concurrent chemoradiation therapy (CCRT) has been recognized as the standard treatment for locally advanced cervical cancer. Although patients treated with CCRT have better chances of survival and prognosis, over 30–40% of patients do not achieve a complete response and may develop locoregional recurrence. Hence predictive biomarkers-based treatment selection of right cohort could be a strong approach for personalized treatment. Therapy resistance occur due to multifactorial events and targeting key molecules involved in treatment resistance could combat treatment resistance. Herein, we profiled both the global proteomic and phosphoproteomic landscape of the concurrent chemotherapy resistant cervical cancer to identify potential predictive biomarkers as well as therapeutic targets. Methods: We employed mass spectrometry based global proteomic and phosphoproteomic strategies to identify proteomic signature and dysregulated kinase involved in CCRT resistance. The biopsy tissue samples were collected before the start of the therapy and the protein was extracted, digested from each sample and labelled with 10 plex TMT (Tandem Mass Tags). The pooled peptides were fractionated by basic pH liquid chromatography analyzed on Orbitrap-Fusion-Tribrid mass spectrometer (ThermoFisher Scientific). For the phosphoproteomic experiment, the peptides were enriched using IMAC based phosphopeptide enrichment method. Liquid chromatography-mass spectrometry/ experiments together with thorough bioinformatics analysis were carried out to characterize the global proteome and phosphoproteome. Results: Analysis of protein expression and phosphorylation led to the identification of dysregulated protein expression and phosphorylation between treatment sensitive and resistant patient cohorts. Further analysis and validation experiment revealed the potential of differentially expressed protein syntaxin 3 (STX3) as a predictive biomarker for treatment resistance. Phosphoproteomic analysis revealed the enrichment of DNA repair pathway in treatment-resistant cohort and the phosphorylation of the proteins involved in DNA repair such as SMC1A, HMGN1, MGMT, DDB2, MSH6 are dysregulated between the cohort suggesting role of DNA repair pathway in the treatment resistance. Kinase-substrate enrichment analysis revealed the activation of kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM in non-responders. Conclusions: We have identified an accurate and reliable biomarker STX3 to predict CCRT resistance in cervical cancer. Further, the phosphoproteomic profiling revealed dysregulation of DNA repair pathways which may be involved in treatment resistance. Targeting the activated kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM could combat treatment resistance in cervical cancer patients.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

Proteomic and Clinicopathologic Assessment of Penile Amyloidosis: A Single Institutional Review of 12 Cases.

There is a paucity of data on penile amyloidosis. We aimed to assess the frequency of different amyloid types in surgical specimens from the penis involved by amyloidosis and correlate relevant clinicopathologic parameters with proteomic findings. Since 2008, our reference laboratory has performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) for amyloid typing. The institutional pathology archive and reference laboratory database were queried to retrospectively identify all penile surgical pathology specimens with LC-MS/MS results between January 1, 2008, and November 23, 2022. Archived H&E-stained and Congo red-stained sections were re-reviewed. Twelve cases of penile amyloidosis were identified, which represented 0.35% (n = 3,456) of penile surgical specimens. AL-type amyloid was most frequent (n = 7), followed by keratin-type amyloid (n = 3) and ATTR (transthyretin)-type amyloid (n = 2). AL-type amyloid cases often showed diffuse dermal/lamina propria deposition, whereas all keratin-type amyloid cases were localized to the superficial dermis. Two cases with keratin-type amyloid had concomitant cutaneous findings (penile intraepithelial neoplasia and condyloma). This series, the largest to date, demonstrates that penile amyloidosis has a heterogeneous proteomic landscape. To the best of our knowledge, this is the first study describing ATTR (transthyretin)-type penile amyloid.

Transcriptomic and Proteomic Landscape of Sugarcane Response to Biotic and Abiotic Stressors.

Sugarcane, a C4 plant, provides most of the world's sugar, and a substantial amount of renewable bioenergy, due to its unique sugar-accumulating and feedstock properties. Brazil, India, China, and Thailand are the four largest sugarcane producers worldwide, and the crop has the potential to be grown in arid and semi-arid regions if its stress tolerance can be improved. Modern sugarcane cultivars which exhibit a greater extent of polyploidy and agronomically important traits, such as high sugar concentration, biomass production, and stress tolerance, are regulated by complex mechanisms. Molecular techniques have revolutionized our understanding of the interactions between genes, proteins, and metabolites, and have aided in the identification of the key regulators of diverse traits. This review discusses various molecular techniques for dissecting the mechanisms underlying the sugarcane response to biotic and abiotic stresses. The comprehensive characterization of sugarcane's response to various stresses will provide targets and resources for sugarcane crop improvement.

Integrated analysis of circulating and tissue proteomes reveals that fibronectin 1 is a potential biomarker in papillary thyroid cancer

Papillary thyroid cancer (PTC) is the most frequent subtype of thyroid cancer, but 20% of cases are indeterminate (i.e., cannot be accurately diagnosed) based on preoperative cytology, which might lead to surgical removal of a normal thyroid gland. To address this concern, we performed an in-depth analysis of the serum proteomes of 26 PTC patients and 23 healthy controls using antibody microarrays and data-independent acquisition mass spectrometry (DIA-MS). We identified a total of 1091 serum proteins spanning 10–12 orders of magnitude. 166 differentially expressed proteins were identified that participate in complement activation, coagulation cascades, and platelet degranulation pathways. Furthermore, the analysis of serum proteomes before and after surgery indicated that the expression of proteins such as lactate dehydrogenase A and olfactory receptor family 52 subfamily B member 4, which participate in fibrin clot formation and extracellular matrix-receptor interaction pathways, were changed. Further analysis of the proteomes of PTC and neighboring tissues revealed integrin-mediated pathways with possible crosstalk between the tissue and circulating compartments. Among these cross-talk proteins, circulating fibronectin 1 (FN1), gelsolin (GSN) and UDP-glucose 4-epimerase (GALE) were indicated as promising biomarkers for PTC identification and validated in an independent cohort. In differentiating between patients with benign nodules or PTC, FN1 produced the best ELISA result (sensitivity = 96.89%, specificity = 91.67%). Overall, our results present proteomic landscapes of PTC before and after surgery as well as the crosstalk between tissue and the circulatory system, which is valuable to understand PTC pathology and improve PTC diagnostics in the future.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.

Abstract A07: Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns

Abstract Acute myeloid leukemia (AML) represents a heterogeneous hematopoietic disorder characterized by accumulation of immature hematopoietic precursors with differentiation block. FLT3 internal tandem duplications (FLT3-ITD) are commonly occurring genetic alterations in AML and are associated with poor prognosis. Preclinical studies showed combining FLT3 inhibitor with MDM2 inhibitor, which is a negative regulator of p53, was synergistic in FLT3-ITD/TP53 wild type (WT) AML. We performed single-cell proteomic evaluation of leukemia landscape in FLT3-ITD patients treated with FLT3i and MDM2i to assess proteomic profiles associated with response and resistance. We performed CyTOF analysis of leukemia cells in serially collected samples from six FLT3-ITD and TP53 WT AML patients following treatment with FLT3i+MDM2i treatment. Using 51 features assessed in CyTOF, we first performed UMAP dimension reduction and clustering to identify distinct cells in leukemia compartment. Notably, the frequencies of blasts identified through CyTOF data analysis were compatible with clinical lab reports. In line with previous reports, we also detected that NPM mutant AML cells did not express CD34. Of note, CD34+ and CD34- leukemia cells were clustered together in NPM1 mut patients, indicating that they have overlapping proteomic profiles. Interestingly, the CD34+ leukemia cells were eliminated at the early time points in CR patients while the CD34- leukemia cells were still detectable after two months. On the other hand, CD34+ leukemia cells in nonresponders(NR) persisted despite therapy. These findings indicate CD34+ leukemia cells were more sensitive to the treatment compared to NPM1 mutant CD34- leukemia cells. Next, we interrogated leukemia proteomic landscape in serial samples, evaluated the therapy-induced alterations in proteomic profiles and sought to identify potential adaptive mechanisms. To this end, we performed differential expression analysis and observed that signaling pathways (p-4EBP1, p-GSK3, p-MEK1/2, p-S6) and differentiation markers(CD33, CD68,CXCR4 HLADR) were more enriched on day 8 post treatment in NR patients, revealing that compensatory signaling activity, phenotypic profiles and differentiation status could be associated with therapy response. Moreover, we found that leukemia cells in NR patients had distinct phenotypic profiles(CD11b,CD68,CXCR4), higher levels of anti-apoptotic molecules (BCL2,MCL1) and enriched survival pathways (p-GSK3, YTHDF2) compared to baseline. In contrast, we did not observe rebound increases in CR patients post-therapy. These findings demonstrate high levels of BCL2 and MCL1 and preferential survival of more differentiated cells may be associated with therapy resistance and treatment failure in NR patient treated with MDM2i and FLT3i. In conclusion, multiplexed single-cell proteomic analysis permitted longitudinal monitoring of leukemia landscape and identified proteomic alterations associated with therapy resistance. Citation Format: Li Li, Muharrem Muftuoglu, Mahesh Basyal, Naval Daver, Michael Andreeff. Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A07.

EEF2 improves dense connective tissue repair and healing outcome by regulating cellular death, autophagy, apoptosis, proliferation and migration

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human Achilles tendon rupture and its association with long-term patient-reported outcomes. Moreover, the potential regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A mass-spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.Trial registration: NCT02318472 registered 17 December 2014 and NCT01317160 registered 17 March 2011, with URL http://clinicaltrials.gov/ct2/show/NCT02318472 and http://clinicaltrials.gov/ct2/show/study/NCT01317160.

OGFOD1 modulates the transcriptional and proteomic landscapes to alter isoproterenol-induced hypertrophy susceptibility

Cardiac hypertrophy is associated with increased translation. However, little is known of the mechanisms that regulate translation in hypertrophy. Members of the 2-oxoglutarate-dependent dioxygenase family regulate several aspects of gene expression, including translation. An important member of this family is OGFOD1. Here, we show OGFOD1 accumulates in failing human hearts. Upon OGFOD1 deletion, murine hearts showed transcriptomic and proteomic changes, with only 21 proteins and mRNAs (0.6%) changing in the same direction. Additionally, OGFOD1-KO mice were protected from induced hypertrophy, supporting a role for OGFOD1 in the cardiac response to chronic stress.

Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas

Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets.

Comparison of proteomic landscape of extracellular vesicles in pleural effusions isolated by three strategies.

Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nano-flow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that, compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in "extracellular exosome." Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.

An ERK1/2‐driven RNA‐binding switch in nucleolin drives ribosome biogenesis and pancreatic tumorigenesis downstream of RAS oncogene

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post‐transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post‐transcriptionally modulates gene expression to promote malignancy remains largely unclear. Using quantitative RNA interactome capture analysis, we here reveal that oncogenic RAS signaling reshapes the RNA‐bound proteomic landscape of pancreatic cancer cells, with a network of nuclear proteins centered around nucleolin displaying enhanced RNA‐binding activity. We show that nucleolin is phosphorylated downstream of RAS, which increases its binding to pre‐ribosomal RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This nucleolin‐dependent enhancement of ribosome biogenesis is crucial for RAS‐induced pancreatic cancer cell proliferation and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA‐binding activity of nucleolin and highlight a crucial role for this mechanism in RAS‐mediated tumorigenesis.

SIRT2 inhibition by AGK2 enhances mycobacteria-specific stem cell memory responses by modulating beta-catenin and glycolysis

Bacille Calmette-Guerin (BCG) generates limited long-lasting adaptive memory responses leading to short-lived protection against adult pulmonary tuberculosis (TB). Here, we show that host sirtuin 2 (SIRT2) inhibition by AGK2 significantly enhances the BCG vaccine efficacy during primary infection and TB recurrence through enhanced stem cell memory (TSCM) responses. SIRT2 inhibition modulated the proteome landscape of CD4+ Tcells affecting pathways involved in cellular metabolism and T-cell differentiation. Precisely, AGK2 treatment enriched the IFNγ-producing TSCM cells by activating β-catenin and glycolysis. Furthermore, SIRT2 specifically targeted histone H3 and NF-κB p65 to induce proinflammatory responses. Finally, inhibition of the Wnt/β-catenin pathway abolished the protective effects of AGK2 treatment during BCG vaccination. Taken together, this study provides a direct link between BCG vaccination, epigenetics, and memory immune responses. We identify SIRT2 as a key regulator of memory Tcells during BCG vaccination and project SIRT2 inhibitors as potential immunoprophylaxis against TB.

Abstract 5498: SHP2 inhibition enhances antitumor effect of mirdametinib in a pediatric brain tumor model bearing CDC42SE2BRAF fusion by rewiring the proteome and phosphoproteome landscape

Abstract Pediatric gliomas are the most common type of pediatric brain tumors representing wide range of molecularly and clinically heterogenous subtypes. The hyperactivity of mitogen-activated protein kinases (MAPK) pathway has been identified in the majority of pediatric glioma suggesting its therapeutic potential. However, pharmacologic targeting single MAPK pathway’s component is limited due to the development of drug resistance and differential response associated with tumor molecular landscape. Therefore, effective combination strategy in the framework of precision medicine is needed. Here we report combination benefit and molecular underpinning therapeutic response of brain penetrant MEK inhibitor (mirdametinib) and SHP2 inhibitor (SHP099) in a pediatric patient derived xenograft (PDX) and xenoline developed at our institution. Our model was derived from a pediatric patient who was diagnosed with rare high-grade subtype of glioma, anaplastic pleomorphic xanthoastrocytoma, and did not respond to MEK inhibitor, trametinib. Integrative multi-omics revealed molecular fidelity between our model and its patient tumor counterpart including the presence of 7q35 fusion, CDC52SE2-BRAF, CDKN2A/B loss, and MAPK pathway hyperactivation. In vitro studies using our xenoline IU-X128 demonstrated synergy between SHP099 and mirdametinib to curtail cell proliferation (p<0.05). Moreover, this combination was well tolerated in our PDX, IU-RHT128, and potentiated anti-tumor effect of the single agent within clinically achievable doses. Reverse Phase Proteome Array (RPPA) identified MAPK reactivation via Mushasi RNA binding protein-PI3K-AKT crosstalk as a potential innate resistance mechanism to single agent MEK inhibitor in the PDX tumor. Further, tandem mass tags (TMT)-LC-MS/MS profiling on tumor treated with single agent SHP099 or mirdametinib and their combination revealed that combination therapy does not only revert certain proteome and phosphoproteome reprogramming from single agent treatment but also created a novel landscape which can be associated with anti-tumor effect. In this case, kinase network reprograming leading to MAPK reactivation was identified in mirdametinib treated tumor which was attenuated in the combination treatment. In summary, our results demonstrated that combination SHP099 and mirdametinib is superior to single agent alone in the pediatric A-PXA brain tumor model with proteome and phosphoproteome reprogramming of multiple networks as potential molecular mechanisms underlying therapeutic benefit of combination therapy. Ultimately, clinical translation of this finding will potentially benefit patient of this malignant rare pediatric glioma subset which currently does not have standard therapy. Citation Format: Nur P. Damayanti, Anthony Alfonso, Josue D. Ordaz, Erika Dobrota, M. Reza Saadatzadeh, Pankita Pandya, Barbara J. Bailey, Khadijeh Bijangi-Vishehsaraei, Harlan E. Shannon, Kathy Coy, Melissa Trowbridge, Anthony L. Sinn, Rosa Gallager, Julia Wulfkuhle, Emanuel Petricoin, Amber Mosley, Mark S. Marshall, Alex Lion, Michael J. Fergusson, Karl Balsara, Karen E. Pollok. SHP2 inhibition enhances antitumor effect of mirdametinib in a pediatric brain tumor model bearing CDC42SE2BRAF fusion by rewiring the proteome and phosphoproteome landscape. [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 5498.

Abstract 5312: Next generation quantitative proteomic analysis of urothelial lesions reveals novel diagnostic biomarkers to distinguish pathologic pitfalls and protein-protein interactions

Abstract There are several unmet needs remained to be solved in bladder urothelial lesions. Especially, the molecular biology of inverted urothelial papilloma (IUP) as a precursor disease of urothelial carcinoma is poorly understood. Also, muscle-invasive urothelial carcinoma (MIUC) of the bladder, showing a highly aggressive tumor behavior, has prompted the quest for robust biomarkers associated with invasion. A total of 265 tissue specimens from urinary bladder were enrolled for proteomic analysis and validation. Candidate biomarkers were selected through bioinformatic analysis, followed by validation. The latter comprised 2D and 3D invasion and migration assays, also a selection of external public datasets to evaluate mRNA expression and an in-house patient-derived tissue microarray (TMA) cohort to evaluate protein expression with immunohistochemistry (IHC). From the overall proteomic landscape, we found divergent ‘NU-like’ (low-risk) and ‘PUC-like’ (high-risk) signatures in IUP. The latter were characterized by altered metabolism, biosynthesis, and cell-cell interaction functions, indicating oncologic significance. Further machine learning-based analysis revealed SERPINH1, PKP2, and PYGB as potential diagnostic biomarkers discriminating IUP from PUC. The immunohistochemical validation confirmed PYGB as a specific biomarker to distinguish between IUP and PUC with inverted growth. For biomarkers predicting invasion, our multilayered platform-based analysis identified tubulin beta 6 class V (TUBB6) as a promising prognostic biomarker predicting MIUC of the bladder. The in vitro 2D and 3D migration and invasion assays consistently showed that inhibition of TUBB6 mRNA significantly reduced cell migration and invasion ability in two BUC cell lines with aggressive phenotype (TUBB6 migration, P = .0509 and P < .0001; invasion, P = .0002 and P = .0044; TGFBI migration, P = .0214 and P = .0026; invasion, P < .0001 and P = .0001; T24 and J82, respectively). Validation through multiple public datasets, including The Cancer Genome Atlas (TCGA) and selected GSE (Genomic Spatial Event) databases, confirmed TUBB6 as a potential biomarker predicting MIUC. Further protein-based validation with our TMA cohort revealed concordant results, highlighting the clinical implication of TUBB6 expression in BUC patients (overall survival: P < .001). We propose PYGB and TUBB6 as a novel IHC biomarker to predict IUP diagnosis or invasion and poor prognosis in routine practice, also select the optimal treatment in BUC patients. Citation Format: Han Suk Ryu, Soo Young Park, Da Sol Kim, Cheng Hyun Lee. Next generation quantitative proteomic analysis of urothelial lesions reveals novel diagnostic biomarkers to distinguish pathologic pitfalls and protein-protein interactions. [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 5312.

Abstract 1374: Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancers and has a poor prognosis due to a high proliferation rate and early metastasis. The current standard treatment, a combination of a platinum-based chemotherapy and etoposide, does not consider the underlying molecular profiles and, therefore, displays limited success. Tailoring treatment to the specific attributes of the 4 SCLC subtypes characterized by the predominant expression of either ASCL1, NeuroD1, POU2F3 or YAP1 (SCLC-A/N/P/Y) might improve patient outcomes. Here, we investigated the proteomic landscape of SCLC subsets with the aim to identify novel subtype-specific characteristics of diagnostic and therapeutic relevance. MS-based proteomics on 26 human SCLC cell lines was performed to decipher pathway-level differences between the subtypes. Mitochondria and lipid droplets were stained with Mitotracker Red CMXROS and Bodipy 493/503, respectively, and quantified by flow cytometry and confocal imaging. Differential responses to inhibitors of complex 1 (metformin, IACS-010759), the CPT1 inhibitor perhexiline, the glutaminolysis inhibitor BPTES and the aerobic glycolysis inhibitor UK5099 were tested using MTT-based cell viability assays. Pathway analyses based on proteomic data identified several metabolic pathways including “oxidative phosphorylation” (OXPHOS) to be significantly upregulated in SCLC-A compared to the other subtypes. Furthermore, multiple proteins essential for the electron transport chain (COX4I1, COX5B, NDUFA5) were highly expressed. Analysis of mitochondria showed a significantly higher number of mitochondria in cells (n=3) from the SCLC-A subtype, which also exhibited a more elongated shape. Lipid droplets were more abundant in the other, low OXPHOS subtypes (n=3). Perhexiline treatment confirmed higher OXPHOS activity solely in the SCLC-A subtype. In contrast, low OXPHOS cells were more sensitive to glutaminolysis inhibition, while no difference was seen when glycolysis was inhibited. Suggesting a differential response to clinically used OXPHOS inhibitors, SCLC cell lines with high and low oxidative activity were treated with metformin and IACS-010759 and response to treatment correlated positively with oxidative state. In this study, we demonstrate distinct differences in metabolic processes in the SCLC-A subtype compared to the other subtypes. Specifically, our data show upregulated oxidative phosphorylation as a potential therapeutic target. Understanding the variances in the metabolic phenotype between the molecular subtypes is integral to achieving precision medicine and advancing SCLC patient treatment. Citation Format: Karin Schelch, Anna Schwendenwein, Kristiina Boettiger, Dominik Kirchhofer, Christian Lang, Zsolt Megyesfalvi, Beata Szeitz, Konrad Hoetzenecker, Lukas Unger, Melinda Rezeli, Balazs Dome. Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype [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 1374.

The proteomic landscape of genome-wide genetic perturbations

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

SARS-CoV-2 Variants Show Different Host Cell Proteome Profiles With Delayed Immune Response Activation in Omicron-Infected Cells

The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remains elusive. Leveraging whole cell proteomics, we determined host signalling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signalling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signalling pathways causative for the differential pathogenicity of SARS-CoV-2 variants.

Proteomic Landscape of Human Sperm in Patients with Different Spermatogenic Impairments

Although the proteome of sperm has been characterized, there is still a lack of high-throughput studies on dysregulated proteins in sperm from subfertile men, with only a few studies on the sperm proteome in asthenozoospermic and oligoasthenozoospermic men. Using liquid chromatography-mass spectrometry (LC-MS/MS) along with bioinformatics analyses, we investigated the proteomic landscape of sperm collected from subfertile men (n = 22), i.e., asthenozoospermic men (n = 13), oligoasthenozoospermic men (n = 9) and normozoospermic controls (n = 31). We identified 4412 proteins in human sperm. Out of these, 1336 differentially abundant proteins were identified in 70% of the samples. In subfertile men, 32 proteins showed a lower abundance level and 34 showed a higher abundance level when compared with normozoospermic men. Compared to normozoospermic controls, 95 and 8 proteins showed a lower abundance level, and 86 and 1 proteins showed a higher abundance level in asthenozoospermic and oligoasthenozoospermic men, respectively. Sperm motility and count were negatively correlated with 13 and 35 and positively correlated with 37 and 20 differentially abundant proteins in asthenozoospermic and oligoasthenozoospermic men, respectively. The combination of the proteins APCS, APOE, and FLOT1 discriminates subfertile males from normozoospermic controls with an AUC value of 0.95. Combined APOE and FN1 proteins discriminate asthenozoospermic men form controls with an AUC of 1, and combined RUVBL1 and TFKC oligoasthenozoospermic men with an AUC of 0.93. Using a proteomic approach, we revealed the proteomic landscape of sperm collected from asthenozoospermic or oligoasthenozoospermic men. Identified abundance changes of several specific proteins are likely to impact sperm function leading to subfertility. The data also provide evidence for the usefulness of specific proteins or protein combinations to support future diagnosis of male subfertility.

Quantitative proteomic landscape of unstable atherosclerosis identifies molecular signatures and therapeutic targets for plaque stabilization

Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.