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There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

Expression levels of placental alkaline phosphatase (ALPP) and ALPP-like 2 (ALPPL2) are relatively low in most cancer types, limiting potential benefits from ALPP or ALPPL2 targeting therapies. Enhancing their expression would be an attractive approach for targeted therapy. We have undertaken analysis of ALPP and ALPPL2 protein expression in whole cell extracts (WCE) and the surfaceome of 158 cancer cell lines and found ALPP, and to a less extent ALPPL2, to be expressed on the surface at relatively low levels across multiple cancer types. We explored various means to enhance ALPP expression in lung adenocarcinoma (LUAD) and found that induction of cancer cell quiescence via nutrient deprivation, or treatment with EGFR inhibitors, greatly enhanced ALPP surface expression. Mechanistic studies revealed that enhancement of surface ALPP expression in LUAD cells following gefitinib treatment was mediated through repression of MEK/ERK signaling and activation of the transcription factor FoxO3a, which was identified as an upstream transcriptional regulator of ALPP. Using xenograft models of LUAD, we further demonstrated that gefitinib treatment upregulates surface expression of ALPP in LUAD cells but not in normal tissues. Combination therapy with gefitinib and an ALPP antibody conjugated with Monomethylauristatin F resulted in enhanced tumor suppression compared with gefitinib alone. Our findings support a novel combination treatment modality that boosts the efficacy of ALPP-ADC directed therapy. Citation Format: Yihui Chen, Monica Hong, Hanwen Xu, Jody Vykoukal, Soyoung Park, Yining Cai, Ricardo A. León-Letelier, Fu Chung Hsiao, jennifer B. Dennison, Edwin J. Ostrin, Johannes F. Fahrmann, Hiroyuki Katayama, Samir M. Hanash. EGFR inhibition in lung adenocarcinoma upregulates cell surface expression of the placental antigen ALPP and enhances efficacy of ALPP-ADC therapy [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 558.

Extracellular vesicles (EVs) are known effectors of cell signaling and exchange implicated in cancer development and progression, as well as remodeling and immunomodulation of the tumor microenvironment. EVs are detected in various biofluids and are considered to convey signature features of the cells from which they originate. Clinical assessment of plasma-derived EV molecular contents therefore offers promise of “liquid biopsies” for detection or subtyping of cancers as well as dynamic reporting of tumor status during and after treatment. To date, there have been no comprehensive analyses aimed at interrogating the diverse repertoire of EV protein cargoes in small cell lung carcinoma (SCLC). Using plasmas from a cohort of 18 SCLC cases, 16 lung adenocarcinoma (LUAD) cases and 34 controls matched based on smoking status, age, and sex, EVs were enriched via affinity capture using magnetic beads functionalized with phosphatidyl serine binding protein TIM4 in addition to single-step density flotation ultracentrifugation to deplete abundant protein background. In-depth proteomic profiling was performed on enriched EVs via nano liquid chromatography / high-resolution ion-mobility mass spectrometry. Profiles were intersected with proteomic profiles generated from intratumor EVs dissociated from 12 SCLC patient-derived xenograft (PDX) mouse models as well as EVs isolated from cell-conditioned medias of a panel of 17 SCLC cell lines. A total of 496 proteins were quantified in TIM4pos EVs. Unsupervised hierarchical clustering and principal component analyses showed that TIM4pos EV protein cargoes differentiate SCLC from LUAD and matched controls. Differential analyses identified 117 proteins that were elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to controls. Of these 117 features, 101 (86%) were also elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to TIM4pos LUAD EVs. Intersection of proteins elevated in TIM4pos SCLC EVs with proteomic profiles of intratumor EVs from SCLC PDX models and conditioned media-derived EVs from SCLC cell lines yielded 67 and 92 overlapping proteins, respectively. Overlapping proteins were characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition and included a prevalence of neuronal tubulin and actin family members, YWHA family members, and annexins. We developed and applied an optimized EV isolation and proteomics workflow to profile EV protein cargoes and establish circulating EV-associated protein signatures of SCLC with biomarker potential. Our integrated analyses identified novel EV-associated proteins for detection of SCLC that are associated with oncogenic drivers. These studies demonstrate that EVs harbor cancer cell disseminated signatures and that continued efforts to query the EV proteome towards discovery of novel SCLC biomarkers are warranted. Citation Format: Jody Vykoukal, Taketo Kato, Hiroyuki Katayama, Allison Stewart, Ehsan Irajizad, Yining Cai, Fu-Chung Hsiao, Jennifer B. Dennison, Edwin J. Ostrin, Hai T. Tran, Carl M. Gay, Lauren A. Byers, Johannes F. Fahrmann, Samir M. Hanash. Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P078.

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response.MethodsProtein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls.ResultsProteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (pConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

Harnessing the immune response to tumor antigens in the form of autoantibodies, which occurs early during tumor development, has relevance to the detection of cancer at early stages. We conducted an initial screen of antigens associated with an autoantibody response in serous ovarian cancer using recombinant protein arrays. The top 25 recombinants that exhibited increased reactivity with cases compared to controls revealed TP53 and MYC, which are ovarian cancer driver genes, as major network nodes. A mass spectrometry based independent analysis of circulating immunoglobulin (Ig)-bound proteins in ovarian cancer and of ovarian cancer cell surface MHC-II bound peptides also revealed a TP53&ndash, MYC related network of antigens. Our findings support the occurrence of a humoral immune response to antigens linked to ovarian cancer driver genes that may have utility for early detection applications.