ABSTRACT
Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.
ABSTRACT
Many hematologic malignancies are not curable with chemotherapy and require novel therapeutic approaches. Chimeric antigen receptor (CAR) T-cell therapy is 1 such approach that involves the transfer of T cells engineered to express CARs for a specific cell-surface antigen. CD38 is a validated tumor antigen in multiple myeloma (MM) and T-cell acute lymphoblastic leukemia (T-ALL) and is also overexpressed in acute myeloid leukemia (AML). Here, we developed human CD38-redirected T cells (CART-38) as a unified approach to treat 3 different hematologic malignancies that occur across the pediatric-to-adult age spectrum. Importantly, CD38 expression on activated T cells did not impair CART-38 cells expansion or in vitro function. In xenografted mice, CART-38 mediated the rejection of AML, T-ALL, and MM cell lines and primary samples and prolonged survival. In a xenograft model of normal human hematopoiesis, CART-38 resulted in the expected reduction of hematopoietic progenitors, which warrants caution and careful monitoring of this potential toxicity when translating this new immunotherapy into the clinic. Deploying CART-38 against multiple CD38-expressing malignancies is significant because it expands the potential for this novel therapy to affect diverse patient populations.
Subject(s)
Hematologic Neoplasms , Leukemia, Myeloid, Acute , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Adult , Animals , Child , Humans , Mice , Hematologic Neoplasms/therapy , Hematologic Neoplasms/metabolism , Leukemia, Myeloid, Acute/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , T-LymphocytesABSTRACT
With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.
Subject(s)
Proteome , Proteomics , Humans , Mass Spectrometry/methods , Proteome/analysis , Proteomics/methods , Reproducibility of Results , WorkflowABSTRACT
As a consequence of acquired or intrinsic disease resistance, the prognosis for patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Novel, less toxic drugs are clearly needed. One of the most promising emerging therapeutic strategies for cancer treatment is targeted immunotherapy. Immune therapies have improved outcomes for patients with other hematologic malignancies including B-cell ALL; however no immune therapy has been successfully developed for T-ALL. We hypothesize targeting CD38 will be effective against T-ALL. We demonstrate that blasts from patients with T-ALL have robust surface CD38 surface expression and that this expression remains stable after exposure to multiagent chemotherapy. CD38 is expressed at very low levels on normal lymphoid and myeloid cells and on a few tissues of nonhematopoietic origin, suggesting that CD38 may be an ideal target. Daratumumab is a human immunoglobulin G1κ monoclonal antibody that binds CD38, and has been demonstrated to be safe and effective in patients with refractory multiple myeloma. We tested daratumumab in a large panel of T-ALL patient-derived xenografts (PDX) and found striking efficacy in 14 of 15 different PDX. These data suggest that daratumumab is a promising novel therapy for pediatric T-ALL patients.
Subject(s)
ADP-ribosyl Cyclase 1/antagonists & inhibitors , Antibodies, Monoclonal/pharmacology , Membrane Glycoproteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , ADP-ribosyl Cyclase 1/metabolism , Adolescent , Adult , Animals , Antibodies, Monoclonal/adverse effects , Child , Child, Preschool , Female , Gene Expression Regulation, Leukemic/drug effects , Humans , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
The proteins of the cellular plasma membrane (PM) perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the PM proteome has been challenging to isolate and characterize, and is poorly represented in standard LC-MS/MS analyses. In this study, we employ sucrose gradient ultracentrifugation for the enrichment of the PM proteome, without chemical labeling and affinity purification, together with GeLCMS and use subsequent bioinformatics tools to select proteins associated with the PM/cell surface, herein referred to as the surfaceome. Using this methodology, we identify over 1900 cell surface associated proteins in a human acute myeloid leukemia cell line. These surface proteins comprise almost 50% of all detected cellular proteins, a number that substantially exceeds the depth of coverage in previously published studies describing the leukemia surfaceome.