Proteomic Tumor Research Articles

Decoding the MMP14 Integrin Link: Key Player in the Secretome Landscape.

Rapid progress has been made in the exciting field of secretome research in health and disease. The tumor secretome, which is a significant proportion of the tumor proteome, is secreted into the extracellular space to promote intercellular communication and thus tumor progression. Among the many molecules of the secretome, integrins and matrix metalloproteinase 14 (MMP14) stand out as the interplay of adhesion and proteolysis drives invasion. Integrins serve as mechanosensors that mediate the contact of cells with the scaffold of the extracellular matrix and are significantly involved in the precise positioning and activity control of the membrane-bound collagenase MMP14. As a secretome proteinase, MMP14 influences and modifies the secretome itself. While integrins and MT-MMPs are membrane bound, but can be released and are therefore border crossers between the cell surface and the secretome, the extracellular matrix is not constitutively cell-bound, but its binding to integrins and other cell receptors is a stringently regulated process. To understand the mutual interactions in detail, we first summarize the structure and function of MMP14 and how it is regulated at the enzymatic and cellular level. In particular, the mutual interactions between integrins and MMP14 include the proteolytic cleavage of integrins themselves by MMP14. We then review the biochemical, cell biological and physiological effects of MMP14 on the composition and associated functions in the tumor secretome when either bound to the cell membrane, or located on extracellular microvesicles, or as a proteolytically shed non-membrane-bound ectodomain. Novel methods of proteomics, including the analysis of extravesicular vesicles, and new methods for the quantification of MMP14 will provide new research and diagnostic tools. The proteolytic modification of the tumor secretome, especially by MMP14, may bring an additional aspect to tumor secretome studies and will have an impact on the diagnosis and most likely also on the therapy of cancer patients.

CT-Scan-Assessed Body Composition and Its Association with Tumor Protein Expression in Endometrial Cancer: The Role of Muscle and Adiposity Quantities.

Background: Endometrial cancer is strongly associated with obesity, and tumors often harbor mutations in major cancer signaling pathways. To inform the integration of body composition into targeted therapy paradigms, this hypothesis-generating study explores the association between muscle mass, body fat, and tumor proteomics. Methods: We analyzed data from 113 patients in The Cancer Genome Atlas (TCGA) and Cancer Proteomic Tumor Analysis Consortium (CPTAC) cohorts and their corresponding abdominal CT scans. Among these patients, tumor proteomics data were available for 45 patients, and 133 proteins were analyzed. Adiposity and muscle components were assessed at the L3 vertebral level on the CT scans. Patients were stratified into tertiles of muscle and fat mass and categorized into three groups: high muscle/low adiposity, high muscle/high adiposity, and low muscle/all adiposities. Linear and Cox regression models were adjusted for study cohort, stage, histology type, age, race, and ethnicity. Results: Compared with the high-muscle/low-adiposity group, both the high-muscle/high-adiposity (HR = 4.3, 95% CI = 1.0-29.0) and low-muscle (HR = 4.4, 95% CI = 1.3-14.9) groups experienced higher mortality. Low muscle was associated with higher expression of phospho-4EBP1(T37 and S65), phospho-GYS(S641) and phospho-MAPK(T202/Y204) but lower expression of ARID1A, CHK2, SYK, LCK, EEF2, CYCLIN B1, and FOXO3A. High muscle/high adiposity was associated with higher expression of phospho-4EBP1 (T37), phospho-GYS (S641), CHK1, PEA15, SMAD3, BAX, DJ1, GYS, PKM2, COMPLEX II Subunit 30, and phospho-P70S6K (T389) but with lower expression of CHK2, CRAF, MSH6, TUBERIN, PR, ERK2, beta-CATENIN, AKT, and S6. Conclusions: These findings demonstrate an association between body composition and proteins involved in key cancer signaling pathways, notably the PI3K/AKT/MTOR, MAPK/ERK, cell cycle regulation, DNA damage response, and mismatch repair pathways. These findings warrant further validation and assessment in relation to prognosis and outcomes in these patients.

Leveraging Multi-omics to Disentangle the Complexity of Ovarian Cancer.

To better understand ovarian cancer lethality and treatment resistance, sophisticated computational approaches are required that address the complexity of the tumor microenvironment, genomic heterogeneity, and tumor evolution. The ovarian cancer tumor ecosystem consists of multiple tumors and cell types that support disease growth and progression. Over the last two decades, there has been a revolution in -omic methodologies to broadly define components and essential processes within the tumor microenvironment, including transcriptomics, metabolomics, proteomics, genome sequencing, and single-cell analyses. While most of these technologies comprehensively characterize a single biological process, there is a need to understand the biological and clinical impact of integrating multiple -omics platforms. Overall, multi-omics is an intriguing analytic framework that can better approximate biological complexity; however, data aggregation and integration pipelines are not yet sufficient to reliably glean insights that affect clinical outcomes.

Exercise and tumor proteome: insights from a neuroblastoma model.

The impact of exercise on pediatric tumor biology is essentially unknown. We explored the effects of regular exercise on tumor proteome profile (as assessed with liquid chromatography with tandem mass spectrometry) in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma (HR-NB). Tumor samples of 14 male mice (aged 6-8 wk) that were randomly allocated into an exercise (5-wk combined aerobic and resistance training) or nonexercise control group (6 and 8 mice/group, respectively) were analyzed. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used to generate a protein-protein interaction (PPI) network and enrichment analyses. The Systems Biology Triangle (SBT) algorithm was applied for analyses at the functional category level. Tumors of exercised mice showed a higher and lower abundance of 101 and 150 proteins, respectively, than controls [false discovery rate (FDR) < 0.05]. These proteins were enriched in metabolic pathways, amino acid metabolism, regulation of hormone levels, and peroxisome proliferator-activated receptor signaling (FDR < 0.05). The SBT algorithm indicated that 184 and 126 categories showed a lower and higher abundance, respectively, in the tumors of exercised mice (FDR < 0.01). Categories with lower abundance were involved in energy production, whereas those with higher abundance were related to transcription/translation, apoptosis, and tumor suppression. Regular exercise altered the abundance of hundreds of intratumoral proteins and molecular pathways, particularly those involved in energy metabolism, apoptosis, and tumor suppression. These findings provide preliminary evidence of the molecular mechanisms underlying the potential effects of exercise in HR-NB.NEW & NOTEWORTHY We used liquid chromatography with tandem mass spectrometry to explore the impact of a 5-wk exercise intervention on the tumor proteome profile in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma. Exercise altered the abundance of hundreds of proteins and pathways, particularly those involved in energy metabolism and tumor suppression. These molecular changes could mediate, at least partly, the potential antitumorigenic effects of exercise.

Abstract B090: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths, with a 5-year survival rate of just 13%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non- Hispanic Black (defined here as African and/or African American ancestry) patients compared to other races. To identify molecular features that may contribute to racial health disparities in PDAC, we used quantitative mass spectrometry to determine the proteomic signatures unique to tumor racial origin in a cohort of 30 Caucasian and 12 African American patients. This analysis of the bulk tumor proteome revealed 183 proteins were differentially expressed between these groups. The protein with the highest expression in tumors from Black patients relative to Whites was Ring Finger Protein 2 (RNF2) that acts as an epigenetic transcriptional repressor in developmental processes pertaining to cellular differentiation. To determine the molecular pathways in PDAC regulated by RNF2, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 and a primary PDAC cell line derived from a Black patient to identify binding sites for RNF2 in PDAC cells. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6, which is an important determinant in PDAC subtype delineation with higher GATA6 expression associated with the less aggressive and more chemosensitive Classical subtype. Modulating RNF2 levels/activity by exogenous expression, short hairpin RNA (shRNA) knockdown, and small molecule inhibition confirms that GATA6 is a target of RNF2 repression in PDAC. Multi-omic (transcriptomic, proteomic, and phosphoproteomic) characterization was performed on MIA-PaCa2 cells with CRISPR-Cas9 knockout (KO) of RNF2. Differential gene expression (DE) analysis revealed changes in platinum drug resistance, EMT, and innate immune response, which are characteristic of the aggressive Inflammatory PDAC subtype we previously reported using proteomics. Total and phospho-proteomics corroborated these results identifying changes in DNA repair, inflammation response, TNFα mediated signaling, and apoptotic signaling. These results reflect the enrichment of Inflammatory subtype markers in tumors originating in Black patients. In agreement with these results, RNF2 KO shows a significant reduction in migration, as well as an increase in sensitivity to oxaliplatin. Our work identifies differences in the underlying proteome of PDAC tumors associated with tumor racial origin and describes how elevated expression of RNF2 in PDACs from Black patients contribute to inflammation and subtype delineation. This study delineates the associations between tumor racial origin, subtype specification, and response to therapy that could mitigate PDAC health disparities and be broadly applied across all PDAC patients. Citation Format: Anthony E Johansen-Sallee, Alexa M Barber, Henry C.-H. Law, Vignesh Vudatha, Jose Trevino, Nicholas T Woods. Evaluating the molecular determinants of PDAC racial health disparities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B090.

Tumori testisa u pasa

The aim of this research paper was to review the literature on testicular tumors in dogs, as the future of diagnostics and choice of therapy for oncological patients lies in tumor markers and proteomics research. Older dogs are prone to developing testicular tumors, which are usually benign, but in some cases can be malignant. The suspicion of a testicular tumor often arises incidentally during the clinical examination of a patient for another problem or during a systematic examination. It occurs most frequently in non-castrated individuals over the age of 10 years, and only very rarely do patients with testicular tumors show any symptoms. Histopathological examination is considered an objective diagnostic method for differentiating the types of testicular tumors in dogs. A testicular biopsy is also possible but does not guarantee a therapeutic effect. Histologically, testicular tumors in dogs are divided into germ cell tumors, testicular stromal tumors and mixed testicular tumors. Previous research determined serum and immunohistochemical markers for testicular tumors in dogs, and it has been found that there is a significant difference in the expression of anti-Müllerian hormone, testosterone and 17-beta-estradiol in the serum and tissue of testicular tumors in dogs, which is useful for diagnosis and treatment decisions. Recent research has focused on proteomics, which analyzes the entire protein component, and biomedical research has already begun to use it for diagnostic and prognostic markers of testicular tumors. To date, no proteomic studies have been performed on testicular tumors in dogs, but the expression of various proteins associated with oncogenic effects has been determined in dogs with different tumors compared to healthy dogs.

Peptide set test: a peptide-centric strategy to infer differentially expressed proteins.

The clinical translation of mass spectrometry-based proteomics has been challenging due to limited statistical power caused by large technical variability and inter-patient heterogeneity. Bottom-up proteomics provides an indirect measurement of proteins through digested peptides. This raises the question whether peptide measurements can be used directly to better distinguish differentially expressed proteins. We present a novel method called the peptide set test, which detects coordinated changes in the expression of peptides originating from the same protein and compares them to the rest of the peptidome. Applying our method to data from a published spike-in experiment and simulations demonstrates improved sensitivity without compromising precision, compared to aggregation-based approaches. Additionally, applying the peptide set test to compare the tumor proteomes of tamoxifen-sensitive and tamoxifen-resistant breast cancer patients reveals significant alterations in peptide levels of collagen XII, suggesting an association between collagen XII-mediated matrix reassembly and tamoxifen resistance. Our study establishes the peptide set test as a powerful peptide-centric strategy to infer differential expression in proteomics studies. Peptide set test (PepSetTest) is publicly available at https://github.com/JmWangBio/PepSetTest.

Pan-cancer analyses suggest kindlin-associated global mechanochemical alterations

Kindlins serve as mechanosensitive adapters, transducing extracellular mechanical cues to intracellular biochemical signals and thus, their perturbations potentially lead to cancer progressions. Despite the kindlin involvement in tumor development, understanding their genetic and mechanochemical characteristics across different cancers remains elusive. Here, we thoroughly examined genetic alterations in kindlins across more than 10,000 patients with 33 cancer types. Our findings reveal cancer-specific alterations, particularly prevalent in advanced tumor stage and during metastatic onset. We observed a significant co-alteration between kindlins and mechanochemical proteome in various tumors through the activation of cancer-related pathways and adverse survival outcomes. Leveraging normal mode analysis, we predicted structural consequences of cancer-specific kindlin mutations, highlighting potential impacts on stability and downstream signaling pathways. Our study unraveled alterations in epithelial–mesenchymal transition markers associated with kindlin activity. This comprehensive analysis provides a resource for guiding future mechanistic investigations and therapeutic strategies targeting the roles of kindlins in cancer treatment.

Abstract 24: Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumor in thyroid tissue, with a 6-month medium overall survival. The effectiveness of conventional treatments in extending the survival of patients is limited. Therefore, there is an urgent need to develop novel systemic therapeutic interventions. Adoptive cell therapy has revolutionized cancer immunotherapy by successfully treating B-cell malignancies through chimeric antigen receptor-based approaches. For solid tumors, there is a need to identify a broader spectrum of tumor target antigens, and the field has now shifted towards leveraging T cell receptors (TCRs) which can recognize both intracellular and extracellular antigen-derived epitopes. Our team has established an immunogenic epitope discovery pipeline, followed by a specialized protocol for empiric validation of candidate epitopes. We characterized immunogenic epitopes by coupling tandem mass spectrometry of MHC-eluted peptides with tumor proteome and MHC-peptide binding prediction databases. By integrating multi-omics data (genomics, transcriptomics, and immunopeptidomics) from both in-house and publicly available ATC databases, we find that ATC exhibits significant upregulation of pathways associated with microtubule-based movement, chromosome segregation, and kinetochore complex components. We have identified several HLA-A2- and A24-restricted immunogenic peptides representing these pathway-associated proteins that covered more than 60% of the patient population. These immunogenic peptides were subsequently utilized to isolate antigen-specific T cells from peripheral blood and identify cognate TCR. These peptide-specific T lymphocytes elicit cytotoxicity toward multiple HLA-matched ATC cell lines and tumor samples in vitro and in vivo. In conclusion, our studies contribute to the development of a new treatment paradigm for treating ATC. Citation Format: Shao-Hsi Hung, Ying C. Henderson, Pan Ke, Yulun Chiu, Shailbala Singh, Yunyun Chen, Stephen Y. Lai, Cassian Yee. Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 24.

Abstract 4064: hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice

Abstract Introduction: Tumor-infiltrating cytotoxic CD8+T cells frequently undergo an altered state of differentiation referred to as "exhaustion" and, as a result, they fail to control tumor growth. IL-15 is a cytokine which stimulates the generation, proliferation and cytotoxic function of tumor CD8+T and NK cells. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials. The aim of this study was to overcome the exhaustion of the tumor-infiltrating CD8+T cells and to increase their cytotoxicity, using Fenofibrate (FF), a PPAR-α agonist, in combination with hetIL-15. Experimental Procedures: We have evaluated the therapeutic efficacy of hetIL-15 immunotherapy as a monotherapy and in combination with FF in the murine EO771 orthotopic breast cancer model. The effects of hetIL-15 and/or FF on immune cells and on tumor microenvironment were analyzed by flow cytometry, transcriptomics, metabolomics and proteomics. The metabolic profile of tumor-infiltrating T cells was performed with Seahorse analysis. Results and Conclusions: hetIL-15 monotherapy resulted in tumor eradication in 40% of treated mice and increased survival. Seahorse analysis of tumor-infiltrating CD8+T cells confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity. Upon hetIL-15 treatment, tumor-infiltrating CD8+T cells showed elevated extracellular acidification rate (ECAR), resulting in a pronounced shift in the OCR/ECAR ratio in comparison to control, confirming their increased proliferating status. These tumor-infiltrating CD8+T cells also showed increased mitochondrial potential and/or mass and fatty acid (FA) uptake, as evidenced by increased MitoTracker and Bodipy staining, respectively. Transcriptomic analysis revealed increased expression of genes involved in several metabolic pathways such as oxidative phosphorylation, FA oxidation and glycolysis. Monotherapy with FF had no effect on tumor growth delay, whereas the FF- hetIL-15 combination resulted in complete eradication of the tumors in 85%. Combination therapy reshaped the tumor microenvironment, resulting in higher IFN-γ and in differentially expressed metabolites as shown by tumor proteomics and metabolomics, respectively. The combination treatment increased the mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype. We conclude that hetIL-15 supports a favorable metabolic profile of intratumoral effector lymphocytes, important for their function. Our data suggest that metabolic reprogramming of tumor specific CD8+T cells using FF is a promising strategy to overcome T cell exhaustion and to promote survival in a metabolically hostile tumor microenvironment. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Katherine C. Goldfarbmuren, Breana Myers, George N. Pavlakis, Barbara K. Felber. hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4064.

Abstract 4897: Personalized cell surface target identification from pan-cancer RNA-seq and proteomics profiling of tumors

Abstract In its third phase, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program collected pan-cancer data from 1,069 patients profiling the transcriptome, proteome, and phospho-proteome of tumors and adjacent tissues from ten cancer types. To identify potential immunotherapeutic candidates from this CPTAC cohort, we searched for cell-surface proteins that are highly expressed in tumor subtypes compared to their expression in normal tissues and cell types from ARCHS4 and the Genotype-Tissue Expression (GTEx) resources. For each cancer type, tumor samples were first clustered into subtypes based on RNA-seq data, and then cell surface targets were prioritized for each cluster. These targets were subsequently confirmed for high protein expression in the tumor subtypes. Tumor samples were also processed by their classification into immune subtypes to identify targets specific to hot (CD8+/IFNG+) and cold (CD8-/IFNG-) tumors of each cancer type. For each cancer type, the computational pipeline identified ~30 cell surface proteins that are highly expressed in the tumor and lowly expressed across tissues and cell types in both the ARCHS4 and GTEx backgrounds. Many of these targets are also robustly differentially expressed at the proteome level. Several targets were shared across tumor types, including VTCN1 and TMPRSS4. Altogether, this rational approach to identify potential targets demonstrates a pipeline that can be applied for personalized cancer diagnosis and therapeutic development. Citation Format: Giacomo B. Marino, Eden Z. Deng, Daniel J.B. Clarke, Weiping Ma, Pei Wang, Adam C. Resnick, Avi Ma'ayan. Personalized cell surface target identification from pan-cancer RNA-seq and proteomics profiling of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4897.

Abstract 1603: Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis

Abstract Background: The presence of minimal intra-pancreatic fat deposition (IPFD) in the healthy human pancreas has been demonstrated in numerous studies. But excess IPFD, or fatty pancreas disease, leads to a wide array of diseases, including but not limited to diabetes, pancreatitis, and pancreatic ductal adenocarcinoma (PDAC). Remarkably, PDAC patients with excessive intra-pancreatic fat accumulation are commonly associated with a poor prognosis compared to those with minimal deposition. However, the interaction between tumor and intra-pancreatic adipose tissue remains elusive. Here, we evaluated the pancreatic fat infiltration in normal people and patients with PDAC respectively, and explored the mechanism of how intra-pancreatic fat affects the progression of PDAC. Methods: Magnetic resonance imaging (MRI) and clinical biopsy specimens were used to assess the degree of pancreatic fat infiltration in PDAC patients and healthy control individuals. The C57BL/6J mice injected orthotopically with KPC cells and adipocytes, high-fat diet (HFD)-induced obesity KC (Pdx1-Cre; KrasLSL-G12D/WT) mice, and KC; ob/ob mice were established to dynamically observe how adipocytes affect tumor progression in vivo. The snRNA-seq of tumor samples and proteomics of conditional medium of PDAC cell lines were performed to identify the mechanism of interaction between cancer cells and adipocytes. Results: We found that patients with PDAC presented excess intra-pancreatic fat deposition more frequently than healthy individuals, and the degree of fat infiltration was positively correlated with the tumor burden of PDAC patients. HFD-induced obesity KC mice and KC; ob/ob mice showed a higher degree of IPFD, which contributed to tumor growth and low survival rates of mice. Additionally, the expression of uncoupling protein 1 was upregulated in PDAC patients and KPC (Pdx1-Cre; KrasLSL-G12D/WT; p53LSL-R172H/WT) mice compared to normal controls. Constantly, the snRNA-seq of PDAC samples demonstrated the increase in thermogenic signature. In vitro, C3H10T1/2 cells treated with conditional medium from pancreatic cancer cell lines or co-cultured with pancreatic cancer cell lines showed increased expression of browning markers and elevated oxygen consumption rate. Specifically, combined analysis of the proteomic of T3M4 and CFPAC-1 conditional medium and the snRNA-seq revealed that pancreatic cancer cells secreted integrin β4 (ITGB4) to improve the thermogenesis of intra-pancreatic adipocytes (IPA), which in turn promoted cancer cell progression. Conclusion: Parts of PDAC patients present excess intra-pancreatic fat accumulation, a process associated with tumor growth and poor prognosis. Mechanistically, pancreatic cancer cells activate the thermogenesis of IPA by secreting ITGB4, and then the thermogenetic adipocytes fuel the progression of the tumor in response. Citation Format: Xinpeng Yin, Yuan Chen, Ruiyuan Xu, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1603.

Abstract 633: Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination

Abstract T cell lymphomas (TCL) are a heterogeneous group of lymphoproliferative disorders with poor prognosis. Aberrant activation of JAK/STAT pathway is associated with lymphoma dissemination in TCL patients. Although the FDA approved the use of inhibitors to target this pathway (Ruxolitinib), significant toxic effects have been reported and their use is limited. Thus making an excellent opportunity to study biological factors that regulate the activation of these pathways. In this sense, we recently showed that thyroid hormones (THs) acting through integrin αVβ3 induced TCL proliferation, which is reduced in vitro and in vivo in the presence of integrin αVβ3 pharmacological inhibitor. Our aim is to evaluate the impact of THs on JAK/STAT pathway activation and their implications on anti-lymphoma therapy. For the in vitro assays we used human and murine TCL cell lines. We used EL4 cells and c57bl/6 syngeneic mice for in vivo analysis. Proteomic profiles were evaluated by LC-MS-MS analysis. The GSE58445 database was used for in silico analysis. We found that THs induced STAT1, 3 and 5 phosphorylation in TCL cells and that the integrin αVβ3 inhibitor, Cilengitide (Cile) blunted these effects (p&amp;lt;0.05). Interestingly, we found that high mRNA expression levels of integrin β3 significantly correlate with worse overall survival in a cohort of TCL patients (p&amp;lt;0.05). Additionally, Ruxolitinib (Ruxo) diminished THs-induced STATs phosphorylation (p&amp;lt;0.05). As Ruxo reported toxic effects we aim to study alternative therapies such as Bexarotene (Bex), a synthetic retinoid used for cutaneous LCT treatment. We found that Bex significantly reduced cell viability in vitro of all TCL subtypes analyzed and combination with Cile resulted in improved anti-lymphoma activity (p&amp;lt;0.01). Also, Ruxo significantly decreased TCL cell viability but to a lesser extent than Bex+Cile (p&amp;lt;0.05). We previously found that BexT4+Cile (T4+, thyroxine supplementation) significantly decreases the in vivo growth of TCL tumors. To understand the mechanisms under this effects we analyzed tumor proteomics profiles and metalloprotease activity after 12 days of treatment. We found that BexT4+Cile regulates proteomic profiles related not only to lymphoma progression (JAK/STAT and PI3K-Akt signaling) but also pathways that impact the tumor microenvironment (TNF and angiogenic factors secretion and Th1/Th2 T cell differentiation). Regarding metalloprotease activity, we found that BexT+Cile inhibited MMP2 and MMP9 activity (p&amp;lt;0.05). Finally, we evaluated the dissemination of TCL in an in vivo model (by vain tail) and found that BexT4+Cile reduced EL4 cell implantation into the liver and kidneys (p&amp;lt;0.05). These results described the mechanisms by which THs induce the activation of JAK/STAT oncogenic pathway and showed how the inhibition of integrin αvβ3 in combination with bexarotene has therapeutic potential for TCL. Citation Format: María M. Debernardi, María V. Revuelta, Helena Sterle, Gonzalo Gonzalez, Ingrid L M Souza, Alejandro Correa, Leandro Cerchietti, Graciela Cremaschi, Florencia Cayrol. Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 633.

Exploring SLC16A1 as an Oncogenic Regulator and Therapeutic Target in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a primary malignant tumor of the liver, typically diagnosed in advanced stages. Surgical resection remains the principal treatment method in clinical practice. Regrettably, the majority of patients receive their diagnosis at an advanced stage, making surgical intervention unfeasible. While chemotherapy serves as the main palliative treatment for advanced CCA, its effectiveness is significantly limited due to the rapid development of chemoresistance. Studying the pathogenesis of CCA and new resistance targets is crucial for improving clinical outcomes. In our current study, we first identified the expression of SLC16A1 in the transcriptome and proteome of human tumors and found abnormal expression of SLC16A1 in various human cancers. Subsequently, we focused our attention on the role of SLC16A1 in CCA. Utilizing bioinformatics analysis, we pioneered the identification of the clinical significance of SLC16A1 in this type of cancer. Specifically, higher expression levels of SLC16A1 were observed in CCA patients with venous invasion and higher T and M stages. Additionally, patients with higher SLC16A1 expression had poorer prognoses. These results suggest the oncogenic role of SLC16A1 in CCA. Further immune infiltration analysis revealed a significant correlation between SLC16A1 and the infiltration levels of cells like neutrophils and macrophages in the tumor microenvironment, indicating SLC16A1's potential involvement in regulating the tumor immune microenvironment of CCA. Moreover, results from functional and pathway enrichment analyses revealed that SLC16A1 might affect clinical outcomes in CCA patients by participating in drug metabolism processes. Finally, through further in vitro and in vivo experiments, we confirmed that SLC16A1, as an oncogene in CCA, promotes the growth of CCA cells and chemoresistance. Knocking down SLC16A1 inhibited the growth of CCA cells and enhanced their sensitivity to 5-Fluorouracil (5-FU). Overall, this study reveals the key role of SLC16A1 in the development of CCA and highlights its significance as a potential target for improving treatment efficacy and chemotherapy sensitivity.

Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.

CTIM-10. A SURGICAL WINDOW-OF-OPPORTUNITY CLINICAL TRIAL EVALUATING THE PCSK9 INHIBITOR EVOLOCUMAB IN PATIENTS WITH GLIOMA

Abstract Gliomas evade the immune system by downregulating cell surface expression of major histocompatibility class (MHC)-I. Antagonism of proprotein convertase subtilisin/kexin type 9 (PCSK9) increases MHC-I expression and can enhance checkpoint inhibition in vivo. Evolocumab is an FDA approved monoclonal antibody (mAb) for hyperlipidemia that inhibits PCSK9. However, large constructs such as mAbs typically have limited penetrance across the blood-brain-barrier. We report the preliminary results of a surgical window-of-opportunity study evaluating if Evolocumab administered subcutaneously pre-preoperatively is detectable in tumor tissue from patients with newly diagnosed or recurrent glioma (PESKe; NCT04937413). Twenty contemporaneous control samples are prospectively collected from patients undergoing craniotomy without receiving Evolocumab. Although non-randomized, these specimens provide valuable information for exploratory comparison of the treated and untreated specimens in terms of drug penetrance and MHC-I expression. To date, 24 patients have been enrolled (M: 14, F: 10) with a median age of 46.5. Of these, 5 received pre-procedure subcutaneous Evolocumab (420mg), while the remaining 19 were analyzed as controls. In treated patients, Evolocumab was detectable via mass spectroscopy (LC-MS/MS) in all analyzed cases, including in tumor tissue from both contrast-enhancing and non-contrast-enhancing regions. Ratios of drug in tumor relative to intra-operative blood were markedly higher in samples from contrast-enhancing regions compared to non-contrast enhancing regions (median ratio of tumor to blood of 0.1503 vs 0.0248 respectively). The tumor proteome was also analyzed using LC-MS/MS to quantitate MHC-I and MHC-II proteins. Initial analysis found increased MHC-I (HLA-A, HLA-B, HLA-C) quantitation via mass spectroscopic analysis of the tumor proteome in treated samples compared to contemporaneous untreated controls. No significant adverse events have been reported in those who received pre-procedure Evolocumab. Ongoing analyses will seek to determine whether increased MHC-I quantities in tumor correspond with increased cell surface expression via immunofluorescence and immunohistochemistry.

BIOM-57. UNRAVELING THE DYSREGULATION OF MIR-17/92 CLUSTER IN AGGRESSIVE PEDIATRIC BRAIN TUMORS: IMPLICATIONS FOR NOVEL THERAPEUTIC STRATEGIES

Abstract The miR-17/92 cluster, commonly referred to as “OncoMir-1,” is extensively studied in cancer due to its oncogenic role, but it has yet to be fully characterized in pediatric brain tumors. In this study, we conduct a comprehensive investigation involving miRNA profiling of 262 pediatric brain tumors, along with sample-matched data from the Pediatric Brain Tumor Atlas, including WGS, RNA-sequencing, proteomics, and phospho-proteomics data. Through miRNA-based unsupervised clustering, we identified four distinct miRNA signatures that transcend histological boundaries. An aggressive cluster (consisting of Medulloblastoma, ATRT, High-Grade Astrocytic Tumors, and some Ependymomas) exhibited the shortest survival and displayed an enriched network of co-expressed miR-17/92 cluster family members and paralogues, specifically miR-106b/25 and miR-106a/363 (p&amp;lt; 0.001). These tumors exhibited significant upregulation of the MIR17HG gene, which encodes the miR-17/92 cluster of miRNAs (p&amp;lt; 0.001). Further exploration of the upstream regulatory mechanisms of MIR17HG identified n-MYC and E2F1/2/3 significantly upregulated (p&amp;lt; 0.01). Additionally, utilizing sample-matched phospho-proteomics data, we found hyperphosphorylation of RB1, the upstream regulator of E2F, at three sites, providing additional evidence of RB1-E2F regulation upstream of MIR17HG. RB1 hyperphosphorylation and subsequent complete inactivation are mediated by the CDK2/Cyclin E complex, which is inhibited by the tumor suppressor p21. Interestingly, p21 (CDKN1A) gene expression was entirely depleted in the aggressive brain tumors group. In summary, our findings reveal the aberrant upregulation of the oncogenic miR-17/92 cluster in a group of aggressive brain tumors, potentially resulting from inactivating hyperphosphorylation of RB1 caused by p21 loss. This event frees E2F transcription factors and enables transcription of MIR17HG. Additionally, our study highlights E2F1/2/3 and CDK2 as potential therapeutic targets not only for aggressive brain tumors but also for other cancers overexpressing the miR-17/92 cluster. Future research will focus on molecular validation and targeting of the miR-17/92 regulatory pathway using available brain tumor cell line models.

Proteogenomic Features Define Novel Subtypes of Mantle Cell Lymphoma

Introductions Recent advancements in mass spectrometry-based proteomics have unveiled novel translational and post-translational aspects of tumor biology. The joint characterization of tumor proteomics with genomics and transcriptomics enables proteogenomic analysis, improving our understanding of the molecular mechanisms, identifying new proteome-specific markers associated with clinical outcomes, and discovering novel treatment approaches. However, proteogenomic features remain largely uncharted due to a scarcity of information on MCL proteome. Methods We conducted a proteogenomic profiling study with 4 normal and 27 MCL B cells derived from healthy donors and MCL patients, integrating whole-exon sequencing (WES), transcriptome sequencing and mass spectrometry-based proteomics with data-independent acquisition (DIA) mode. We assessed the relationship among genetic lesions, RNA and protein expression, and further classified MCL based on omics information using k-means clustering. Results To this end, we first identified 1107 differentially expressed proteins between MCL and normal B cells (FDR&amp;lt;0.05) (329 upregulated and 778 downregulated in MCLs). The up-regulated proteins are highly enriched for RNA splicing and DNA repair pathway while the down-regulated proteins are primarily involved in cytoskeleton and endosomal transport. When integrated with 33 recurrent genetic lesions we previously reported, we found that mutations in CCND1 and NOTCH1 had significant impact on protein expression. Key genes including ATM and BCL10 were differentially expressed between samples with and without NOTCH1 mutation. Next, we computed Pearson correlation coefficients between RNA and protein levels for each gene to define the relationships between them. RB1 (0.88), PML (0.81) and CDK2 (0.81) showed high correlation efficiency, however, the overall correlation between RNA and protein abundance was low (0.26), suggesting that proteome abundance profiles may provide unseen information not previously discovered from WES and transcriptome data. Last, we evaluated protein expression level and their associations with progress free survival and overall survival to identify proteins potentially influencing clinical outcomes. Our comprehensive analysis led us to identify 40 proteins that could play a crucial role in disease progression. To assess the prognostic value of the omics data, we performed multi-omics clustering by incorporating genomics, transcriptomics and proteomics. This enabled us to identify four distinct clusters, each characterized by key driver genetic lesions as well as RNA and protein signatures. Notably, patients in these four clusters exhibited significantly different median overall survival (C1 to C4: not reach, 28.9, 15.8 and 7.9 months; log-rank test, P&amp;lt;0.001). Importantly, the protein signature remained significant even when considering the MIPI stage as a covariate (P&amp;lt;0.001), suggesting its potential as an independent prognostic factor. We then conducted an in-depth protein feature analysis and found that cluster 1 exhibited a significant enrichment of genes involved in ubiquitin-mediated proteolysis pathway. Cluster 2 showed a remarkable upregulation in DNA replication and cell cycle pathways. C3 displayed a significant upregulation of genes associated with BCR pathway and oxidative phosphorylation. Cluster 4 had an upregulation of RNA polymerase, pyrimidine metabolism, and RNA splicing, along with a notable downregulation in the BCR pathway. Our cluster analysis not only stratified MCL patients but also uncovered critical cellular pathways that could serve as potential therapeutic targets. Conclusions In this study, we investigated proteome changes and identified protein signatures associated with overall survival in MCL. Our integrative analysis unveiled four clusters, each characterized with unique genetic features, distinct gene and protein signatures, and different clinical outcomes. This study emphasizes the significance of protein abundance data as a nonredundant layer of information in MCL biology. Moreover, we have provided a protein expression reference map for MCL, offering valuable insights for future research and clinical applications.

Mapping the Spatial Proteome of Head and Neck Tumors: Key Immune Mediators and Metabolic Determinants in the Tumor Microenvironment

Mapping the Spatial Proteome of Head and Neck Tumors: Key Immune Mediators and Metabolic Determinants in the Tumor Microenvironment

Abstract PO-061: Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is considered to be among the most aggressive solid tumors in thyroid tissue, with a 6-month medium overall survival. The effectiveness of conventional treatments, such as surgery, chemotherapy, and radiation therapy, in extending the survival of patients is limited. Therefore, there is an urgent need to develop novel systemic therapeutic interventions that can induce long-lasting responses. Adoptive cell therapy has revolutionized cancer immunotherapy by successfully treating B-cell malignancies through chimeric antigen receptor-based approaches. For solid tumors, there is a need to identify a broader spectrum of tumor target antigens, and the field has now shifted towards leveraging T cell receptors (TCRs) which can recognize both intracellular and extracellular antigen-derived epitopes displayed by major histocompatibility complex (MHC) molecules. Our team has established an immunogenic epitope discovery pipeline, followed by a specialized protocol for empiric validation of candidate epitopes. We characterized immunogenic epitopes by coupling tandem mass spectrometry of MHC-eluted peptides with tumor proteome and MHC-peptide binding prediction databases. By integrating multi-omics data (genomics, transcriptomics, and immunopeptidomics) from both in-house and publicly available ATC databases, we find that ATC exhibits significant upregulation of pathways associated with microtubule-based movement, chromosome segregation, kinetochore complex components, and mitotic cytokinesis. We have identified several HLA-A2- and A24-restricted immunogenic peptides representing these pathway-associated proteins that covered more than 60% of the patient population. NDC80 kinetochore complex components (NDC80) and PDZ binding kinase (PBK) have been selected as top candidates. Peptides derived from NDC80 and PBK were subsequently utilized to isolate antigen-specific T cells from peripheral blood and identify cognate TCR. These peptide-specific T lymphocytes elicit cytotoxicity toward multiple HLA-matched ATC cell lines and tumor samples in vitro and in vivo. Furthermore, understanding the role of these genes in ATC tumorigenesis may identify novel combination strategies with adoptive cell therapy to yield synergistic effects. In conclusion, our studies contribute to the development of a new treatment paradigm for treating ATC, with the prospect of improving survival and outcome in this rare cancer patient population. Citation Format: Shao-Hsi Hung, Ying Henderson, Ke Pan, Yulun Chiu, Shailbala Singh, Yunyun Chen, Stephen Lai, Cassian Yee. Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-061.