Quantitative proteomics profiling of B-cell cancers

Abstract

Approximately 800,000 leukaemia and lymphoma cases are diagnosed worldwide each year; with incidence spanning the extremes of both age and economic development. Burkitt’s lymphoma (BL) is an aggressive B-cell lymphoid tumour, prevalent in pre-industrialised countries, with the primary sufferers aged between 4 and 7. Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western World; typically presenting with a leukaemic population of CD5+ B cells in patients over 60 years of age. CLL is a heterogeneous disease with a variable clinical course which presents challenges in prognosis and treatment. A more comprehensive understanding of CLL and B-cell cancer biology and the identification of disease biomarkers and therapeutic targets has the potential to improve clinical outcomes. Murine models offer a controlled and accessible means of studying B-cell cancers. Transgenic mice have been developed which use the immunoglobulin heavy chain gene enhancer (Eμ) to impose B-cell specific proto-oncogene expression. Eμ-myc and Eμ-TCL1 mice model aspects of BL and CLL, respectively. To achieve non-biased, quantitative proteomics profiling of B-cell cancers, this investigation has utilised isobaric labelling, two-dimensional liquid chromatography and mass spectrometry. The proteomes of whole cell lysates of human and mouse B-cell cancers were quantitated against non-cancer B-cell controls to determine global cancer-specific protein expression. A parallel analysis of pre-terminal and terminal mouse plasma was conducted using sub-proteome enrichment and size exclusion chromatography to identify potential disease biomarkers. A method was developed which more effectively utilises quantitative isobariclabelled data to conclude differences in protein expression. Proteomic characterisation quantitated 7391 proteins (FDR <1%) for Eμ-myc and EμTCL1 B-cell tumours relative to pre-tumour and WT controls, identifying over 2000 differentially expressed proteins, amongst which were anticipated findings such as myc and TCL1. Common and tumour-specific regulation of pathways, potential targets of inhibition and cell surface proteins were characterised; most notably of which was the interleukin 5 receptor in Eμ-TCL1 tumours. Treatment with interleukin 5 induced proliferation and survival of Eμ-TCL1 tumour cells, validating this novel finding. This constitutes one of the most comprehensive characterisations of B-cell cancers to date. 2095 proteins were profiled in Eμ-myc and Eμ-TCL1 plasma identifying tumour lysis products as the major signature in terminal tumours. Additionally signatures of protein secretion, shedding and immune response were present in a tumour-specific manner. An early, pre-terminal signature of tumour development was also identified in the Eμ-TCL1 model. Profiling of CLL samples quantified 5956 proteins across 14 samples with findings agreeing with expected differential expression in CLL, relative to healthy B cells; the most comprehensive proteomics characterisation of CLL to date. The results suggested novel targets of immunotherapy and inhibitors, especially in the context of B-cell receptor signalling. Novel biology such as global spliceosome upregulation was also uncovered. CLL subtype-specific differences were identified, however, the strongest signature was that of a subtype-independent pattern of protein expression. This investigation has provided unparalleled characterisations of B-cell neoplasms suggesting novel biological mechanisms and therapeutic targets, particularly in CLL. This supports the further characterisation of B-cell cancers, and other cancers, by the presented methodologies.

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ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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