Citations

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient’s tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusions: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Fabrice Lucien, Patricio C. Gargollo, Mohamed E. Ahmed, Yohan Kim, Emily Baer, Doris Phelps, Cristine M. Charlesworth, Benjamin J. Madden, Liguo Wang, Peter J. Hougthon, John Cheville, Haidong Dong, Candace F. Granberg. Surfaceome profiling of rhabdomyosarcoma reveals B7-H3 as a mediator of immune evasion [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A036.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

Background Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown. Methods We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'. Results MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN. Conclusions FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.

Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient's tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusion: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Yohan Kim, Mohamed E. Ahmed, Doris Phelps, Emily Baer, Cristine Charlesworth, Benjamin Madden, Liguo Wang, Haidong Dong, Peter Houghton, John Cheville, Patricio Gargollo, Candace Granberg, Fabrice Lucien. Surfaceome profiling identifies the immune checkpoint molecule B7-H3 as a key player of immune-evasion in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3048.

Bipolar disorder is treated with mood stabilizers. mRNA data show that these drugs regulate synaptic protein expression. To determine protein levels and post‐translational processing of proteins and to determine if paliperidone (PALI), risperidone active metabolite, regulates synaptic proteins similar to mood stabilizers, we determined protein expression profiles in the PFC with chronic drug treatments. LiCl, valproate (VAL), or PALI was given daily by i.p. injections to 4 male Sprague‐Dawley rats/group for 28 d. Controls = 0.9% saline and 0.3% tartaric acid. Brains were dissected 24 h after final injections. Synaptoneurosomal proteins were extracted and separated by DIGE. Simultaneously, phosphorylation changes were determined (ProQ diamond staining). Reproducibly present data in all biological replicates were analyzed and compared with their respective controls using Interpreter (Ludesi). Proteins with differential expression and phosphorylation were identified by nano‐LC MS. PALI, LiCl, and VAL treatment resulted in similar protein expression profiles and phosphorylation patterns at the synaptoneurosomal level, suggesting that the mode of action for this drug is similar to that of mood stabilizers and supporting its use for this purpose. Interestingly, the expression profile for PALI was more similar to that of LiCl than that of VAL. Supported by a grant from Janssen Pharmaceutica, Inc.

Significance Statement Membranous nephropathy (MN) results from antibodies targeting an antigen in the glomerular basement membrane (GBM). The target antigens identified so far include PLA2R, THSD7A, NELL1, SEMA3B, and EXT1/EXT2. Using laser microdissection and mass spectrometry analysis, the authors identified a novel protein, protocadherin 7 (PCDH7), that is present in the GBM of a subset of patients with MN who are negative for all of the known antigens associated with MN. PCDH7 shows granular GBM staining and colocalizes with Ig in the GBM. Furthermore, antibodies to PCDH7 were detected in both the serum and kidney biopsy tissue from individuals with PCDH7-associated MN but not from controls. These findings suggest that PCDH7-associated MN defines a distinct type of MN. Background Membranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%–90% of target antigens in MN. Methods We performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue. Results MS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7. Conclusions MN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.

Significance Statement Approximately 20% of patients with lupus nephritis, the most common renal manifestation of systemic lupus erythematosus, show membranous lupus nephritis on kidney biopsy, and nearly 10% eventually develop ESKD. Recently, two proteins, Exostosin 1 and Exostosin 2 (EXT1/EXT2), were shown to be present in a subset of membranous lupus nephritis kidney biopsy specimens. In an examination of 374 membranous lupus nephritis kidney biopsy specimens, the authors found 32.6% to be EXT1/EXT2-positive. Kidney biopsy specimens from patients with EXT1/EXT2-positive membranous lupus nephritis showed less chronicity features (glomerulosclerosis, interstitial fibrosis, and tubular atrophy) compared with those from EXT1/EXT2-negative patients. EXT1/EXT2-negative patients were also more likely to reach ESKD than EXT1/EXT2-positive patients. These findings suggest that the presence of EXT1/EXT2 is favorable, and that EXT1/EXT2-positive patients have better renal outcomes compared with EXT1/EXT2-negative patients. Background In patients with secondary (autoimmune) membranous nephropathy, two novel proteins, Exostosin 1 and Exostosin 2 (EXT1/EXT2), are potential disease antigens, biomarkers, or both. In this study, we validate the EXT1/EXT2 findings in a large cohort of membranous lupus nephritis. Methods We conducted a retrospective cohort study of patients with membranous lupus nephritis, and performed immunohistochemistry studies on the kidney biopsy specimens against EXT1 and EXT2. Clinicopathologic features and outcomes of EXT1/EXT2-positive versus EXT1/EXT2-negative patients were compared. Results Our study cohort included 374 biopsy-proven membranous lupus nephritis cases, of which 122 (32.6%) were EXT1/EXT2-positive and 252 (67.4%) were EXT1/EXT2-negative. EXT1/EXT2-positive patients were significantly younger ( P =0.01), had significantly lower serum creatinine levels ( P =0.02), were significantly more likely to present with proteinuria ≥3.5 g/24 h ( P =0.009), and had significantly less chronicity features (glomerulosclerosis, P =0.001 or interstitial fibrosis and tubular atrophy, P <0.001) on kidney biopsy. Clinical follow-up data were available for 160 patients, of which 64 (40%) biopsy results were EXT1/EXT2-positive and 96 (60%) were EXT1/EXT2-negative. The proportion of patients with class 3/4 lupus nephritis coexisting with membranous lupus nephritis was not different between the EXT1/EXT2-positive and EXT1/EXT2-negative groups (25.0% versus 32.3%; P= 0.32). The patients who were EXT1/EXT2-negative evolved to ESKD faster and more frequently compared with EXT1/EXT2-positive patients (18.8% versus 3.1%; P =0.003). Conclusions The prevalence of EXT1/EXT2 positivity was 32.6% in our cohort of membranous lupus nephritis. Compared with EXT1/EXT2-negative membranous lupus nephritis, EXT1/EXT2-positive disease appears to represent a subgroup with favorable kidney biopsy findings with respect to chronicity indices. Cases of membranous lupus nephritis that are EXT1/EXT2-negative are more likely to progress to ESKD compared with those that are EXT1/EXT2-positive.

Abstract Background: The objective of the study was to explore disease pathways activated in the inflammatory foci of skin lesions in cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). Methods: Skin biopsies acquired from active CLE and DM lesions, patient (PC) and also healthy controls (HC) were investigated. Biopsy sections were examined by a pathologist and inflammatory foci were laser micro-dissected, captured and proteins within captured tissue were detected in a hypothesis free manner by mass-spectrometry. Protein pathway analysis was performed by string-db.org platform. Findings of interest were confirmed by immunohistochemistry (IHC).Results: Proteome investigation identified interleukin (IL)-16 to be the only detectable and abundant cytokine differentially expressed in CLE compared to DM. Caspase-3, enzyme that cleaves IL-16 into its active form, was detected in low levels. Significantly higher proportion of IL-16 and Caspase-3 positive cells were identified in CLE lesions in comparison to DM, PC and HC. Interferon-regulated proteins (IRP) were abundant in both CLE and DM. Proteomic results indicate more abundant complement deposition in CLE skin lesions. Conclusions: Using hypothesis free mass-spectrometry investigation of CLE inflammatory infiltrates, we identified that IL-16 is the only detectable and highly abundant cytokine, while IRP was a common feature of both CLE and DM. IHC confirmed high expression of IL-16 and caspase-3 in CLE. Our novel molecular findings indicate that IL-16 detection could be useful in differential diagnostics between the two conditions that can display similar histopathological appearance. Potentially, IL-16 could be of interest as a future therapeutic target for CLE.