Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

Stopfer, Lauren; Mesfin, Joshua M.; Joughin, Brian A.; Lauffenburger, Douglas A.; White, Forest M.

doi:10.1101/2020.03.03.968750

Cited by 4 publications

(4 citation statements)

References 58 publications

(59 reference statements)

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“…Taken together, we propose a mechanism whereby disruption of the CDK4/CDK6/CCND1 complex leads to upregulation and subsequent degradation of cell cycle proteins ( Figure 5). Similar changes in the immunopeptidome have also been reported, while this manuscript was in preparation, in melanoma cell lines after CDK4/6i treatment (27) . Nonetheless, our study is the first to demonstrate pathway-dependent degradation and presentation.…”

Section: Discussionsupporting

confidence: 83%

Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

Charles

Bourne

Mun

et al. 2020

Preprint

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Purpose: Cyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also demonstrate antineoplastic activity through triggering T cell-mediated immunity. One of the potential mechanisms responsible for this immunological effect might be qualitative and quantitative changes in human leukocyte antigen (HLA) ligands on the cell surface after treatment with CDK4/6i. These changes may increase the immunogenicity of breast cancer cells offering potential synergies for combinations with cancer immunotherapies. Experimental Design:We investigated the ability of two CDK4/6 inhibitors (CDK4/6i), Abemaciclib and Palbociclib, to alter the immunopeptidome at subclinical, non-toxic, levels in different breast cancer cell lines. Biochemical isolation of HLA ligands, identification by mass spectrometry and subsequent network analysis after drug treatment were used to characterize the changes in the immunopeptidome. The mechanisms for altered CDK4/6 presentation were explored.Results: Low-dose treatment with 100nM of Abemaciclib and Palbociclib led to upregulation of cell surface HLA levels and induced hundreds of HLA ligands in breast cancer cell lines. These new ligands were significantly and most strongly enriched for peptides derived from proteins involved in the "G1/S phase transition of cell cycle" pathway and included among others, HLA ligands from CDK4, CDK6, Cyclin D1 and Cyclin E1. An increase in transcript, protein, and subsequent ubiquitination for Cyclin D1, which could lead to enhanced degradation of the target protein, was identified as a potential mechanism for the altered presentation of peptides.Conclusions: CDK4/6i treatment gave rise to drug-induced antigens through cell cycle disruption and increased antigen presentation. Interestingly, these induced HLA ligands are often sourced from the proteins of the CDK4/CDK6/CCND1 complex or more downstream interaction partners, providing evidence that inhibition of a distinct cellular pathway leads to increased presentation of the proteins involved. These findings suggested CDK4/6i provided a tool for highly selective induction of HLA ligands that may be targeted by T cell-based immunotherapeutics. Translational RelevanceThese data demonstrated that low-dose treatment of breast cancer cells with CDK4/6 inhibitors, Abemaciclib and Palbociclib, induced marked changes in presentation of HLA ligands, especially from proteins involved in the G1/S phase transition, the phase in which these drugs arrest the cells. Enhanced ubiquitination and degradation was identified as a mechanism for the altered presentation for one of the relevant proteins. The induced HLA ligands may provide ideal specific targets for combination immunotherapies. The data show for the first time that selective inhibition of a distinct pathway can lead to specific presentation of HLA ligands in breast cancer cells. This work supports the rationale for testing the combination of low-dose CDK4/6i with immunotherapeutic agents, such as immune checkpoint blockade antibodies or T-cell-based...

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Section: Discussionsupporting

confidence: 83%

Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

Charles

Bourne

Mun

et al. 2020

Preprint

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“…Methylthio (C) and TMT (K, N-terminus) were set as static modifications. Standard FDR corrected p value cutoff at the peptide level was set to <0.05 for native proteins and to <0.01 for phosphopeptides (Billiard et al, 2018;Bouchal et al, 2015;Liu et al, 2016;Mournetas et al, 2021;Stopfer et al, 2020). Percent coisolation excluding peptides from quantitation was set at 50.…”

Section: Discussionmentioning

confidence: 99%

Deep Proteome Profiling of Human Mammary Epithelia at Lineage and Age Resolution

et al. 2021

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“…However, the allotypic peptides might be a better reference as demonstrated in this study. The search for a reliable internal standard is an important prerequisite to ensure the comparability and robustness of immunopeptidomic analysis and is especially relevant for promising applications such as quantitative immunopeptidomics 47,48 . So far, protocols exist for the validation of LC-MS/MS based immunopeptidomics pipelines according to pharmaceutical good manufacturing practices (GMP) for peptide identification 31 .…”

Section: Discussionmentioning

confidence: 99%

An innovative approach for HLA typing, molecular tumor testing and the validation of tumor exclusive antigens

Ghosh

Bichmann

Scheid

et al. 2020

Preprint

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The immunopeptidome, representing the point of contact between somatic and T cells, is key for adaptive immunity. Each presented peptide holds an abundance of information not yet well understood. Up to now, the scientific focus has been the definition of pathogenic or tumor derived epitopes and the deconvolution of HLA peptide motifs of the entire immunopeptidome. Here we go one step further and assess the properties of individual peptides to identify defined HLA allotype-specific and frequently presented peptides. Such allotypic peptides represent a versatile tool to determine HLA allotypes or serve as internal standard for characterization of cancer antigens and differentially processed antigens. Finally, individual tissue-and dignity-specific antigens were defined, and the latter were successfully implemented for molecular tumor testing.Using mass spectrometry based immunopeptidomics a database was generated consisting of ~900 HLA-typed samples. The identified allotypic peptides enabled a HLA class I allotype determination, which was 95% correct in our in-house dataset and 98% in an external dataset.These abundant peptides were implemented as internal standard for a semi-quantitative investigation of established tumor antigens and antigens processed differentially in malignant and benign tissue. Defined dignity-specific antigens allowed a 87% correct tumor detection across numerous tumor types.In summary, we describe a machine learning approach for mining immunopeptidomic data in order to develop a classification method, allowing to differentiate HLA class I-allotypes of a sample or distinguish between healthy and malignant state of tissues. Furthermore, based on this method, we developed a procedure for the validation of tumor exclusive antigens. Our results support classification of immunopeptidomic data sets using machine learning and highlight their potential utility for biomarker development.

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Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

Cited by 4 publications

References 58 publications

Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

Deep Proteome Profiling of Human Mammary Epithelia at Lineage and Age Resolution

An innovative approach for HLA typing, molecular tumor testing and the validation of tumor exclusive antigens

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