Functional role of ZAP70 in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common type of leukaemia. It is common in older people and rare in people under 40. CLL is characterised by the accumulation of monoclonal CD5+CD23+ B lymphocytes in the blood, bone marrow and secondary lymphatic tissues. There are 2 main subtypes of CLL based on the IgHV status: Un-mutated CLL (UM-CLL) and Mutated CLL (M-CLL). M-CLL leads to good prognosis whereas UM-CLL is the more aggressive form of the disease that could lead to Richter syndrome. Prognostics markers for CLL are the IgHV mutational status, chromosomal aberrations, CD38 expression and ZAP70 expression. Of these 4 prognostic markers, some are very labour intensive and not easy to perform on a routine basis, however ZAP70 expression has been a reliable predictor for the prognosis of CLL. In this study, I investigated the role of ZAP70 in CLL. This was done using P505-15, a dual selective SYK and ZAP70 inhibitor, to reduce kinase activity of ZAP70. This was followed by siRNA knockdown experiments to disentangle effects of SYK and ZAP70 and reduce off target effects from other kinases. In addition, to better characterise protein interactors of ZAP70, B cell lines expressing both BirA ligase as well as ectopic expression of ZAP70 were generated. Then, immunoprecipitation of endogenous ZAP70 in patient CLL cells was carried out followed by mass spectrometry analysis. BCR signalling is known to play a fundamental role in CLL cell survival and proliferation. Here, I have shown that P505-15 reduces calcium mobilization, cell survival and proliferation in ZAP70 positive CLL cells. Knockdown of ZAP70 using siRNA in CLL patient cells was achieved, however efficiency varied between patients. Knockdown of ZAP70 using siRNA showed no significant change in calcium mobilization compared to non specific siRNA. Using the ZAP70 knockdown cells, I further investigated changes downstream of the BCR signalling pathway. Comparing siZAP70 to siNSC, siZAP70 showed an increase in pAKT levels in CLL cells post anti IgM activation, however no changes in NF-κB activity were observed. Finally, mass spectrometry using BJAB cells and CLL cells positive for ZAP70, confirmed the role of ZAP70 in BCR signalling. In addition, it unexpectedly unveiled ZAP70’s role in CLL cell migration and it’s potential role in the DNA damage pathway in CLL cells.CRU

Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Prognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.This project was funded by the Wellcome Trust. NB is a fellow of the European Hematology Association and was supported by the Academy of Medical Sciences. EP is a European Hematology Association Advanced Research Fellow. GV is a Wellcome Trust Senior Fellow in Clinical Science. IV is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression

Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and disease subgroups

International audienceIn multiple myeloma, next generation sequencing (NGS) has expanded our knowledge of genomic lesions, and highlighted a dynamic and heterogeneous composition of the tumor. Here, we used NGS to characterize the genomic landscape of 418 multiple myeloma cases at diagnosis and correlate this with prognosis and classification. Translocations and copy number changes (CNAs) had a preponderant contribution over gene mutations in defining the genotype and prognosis of each case. Known and novel independent prognostic markers were identified in our cohort of proteasome inhibitor and IMiD-treated patients with long follow-up, including events with context-specific prognostic value, such as deletions of the PRDM1 gene. Taking advantage of the comprehensive genomic annotation of each case, we used innovative statistical approaches to identify potential novel myeloma subgroups. We observed clusters of patients stratified based on the overall number of mutations and number/type of CNAs, with distinct effects on survival, suggesting that extended genotype of multiple myeloma at diagnosis may lead to improved disease classification and prognostication