REPERTOIRE OF PROTEIN KINASES ENCODED IN THE GENOME OF ZEBRAFISH SHOWS REMARKABLY LARGE POPULATION OF PIM KINASES

In recent times, zebrafish has garnered lot of popularity as model organism to study human cancers. Despite high evolutionary divergence from humans, zebrafish develops almost all types of human tumors when induced. However, mechanistic details of tumor formation have remained largely unknown. Present study is aimed at analysis of repertoire of kinases in zebrafish proteome to provide insights into various cellular components. Annotation using highly sensitive remote homology detection methods revealed ``substantial expansion'' of Ser/Thr/Tyr kinase family in zebrafish compared to humans, constituting over 3% of proteome. Subsequent classification of kinases into subfamilies revealed presence of large number of CAMK group of kinases, with massive representation of PIM kinases, important for cell cycle regulation and growth. Extensive sequence comparison between human and zebrafish PIM kinases revealed high conservation of functionally important residues with a few organism specific variations. There are about 300 PIM kinases in zebrafish kinome, while human genome codes for only about 500 kinases altogether. PIM kinases have been implicated in various human cancers and are currently being targeted to explore their therapeutic potentials. Hence, in depth analysis of PIM kinases in zebrafish has opened up new avenues of research to verify the model organism status of zebrafish

The role of histone kinases in controlling transcription in B cell lymphoma and leukaemia

Protein kinases are central mediators of signal transduction pathways and transcriptional regulation. Lymphoid malignancies are characterised by aberrant activation of key signal transduction pathways and specific gene expression programmes. Consequently, targeting kinases involved in these signal transduction pathways is a promising therapeutic strategy. Because gene expression is regulated at the level of chromatin, the aim of this study was to assess the effects of chromatin-modifying kinases on histone phosphorylation and transcriptional regulation in B cell lymphoma and the consequences of kinase inhibition for tumour cell viability and the chromatin structure of target genes. The kinase PIM1, whose mRNA is highly expressed in the aggressive activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), but not germinal centre B cell-like DLBCL (GCB-DLBCL), has been shown to associate with the transcription factor MYC and to regulate the expression of MYC target genes by phosphorylating histone H3S10. Therefore, effects of PIM1 on viability and gene expression were evaluated in ABC-DLBCL and in the MYC-dependent Burkitt lymphoma (BL). However, pan-PIM kinase inhibition or knockdown of PIM1 did not effectively reduce viability of ABC-DLBCL or Burkitt lymphoma cell lines. Further, the expression of the MYC- and PIM1-bound GNL3 gene was largely unaffected by alterations in PIM kinase levels or activity. In conclusion, PIM kinases do not seem to be bona fide therapeutical targets in DLBCL and BL. The second part of this project aimed to understand the effects of Ibrutinib on chromatin structure in chronic lymphocytic leukaemia (CLL) cells. Ibrutinib inhibits Bruton’s tyrosine kinase, and thus B cell receptor (BCR) signalling, and is currently being tested in clinical trials for the treatment of CLL. In vitro, Ibrutinib inhibited BCR-induced gene expression and histone H3T6 and T11 phosphorylation. A possible kinase targeting H3T6 and H3T11 downstream of the BCR might be zipper-interacting protein kinase (ZIPK), a ZIPK inhibitor blocked H3T6p and H3T11p and gene expression. Short-term Ibrutinib treatment appeared to inhibit histone turnover but did not reduce H3K4me3, H3K9ac, H2A.Z or POL II recruitment at target genes, indicating that it inhibits only some aspects of transcription. In contrast, long-term Ibrutinib treatment decreased H3K4me3 and H3K9ac in promoter regions, possibly by an indirect, gene silencing-dependent mechanism. In summary, the results suggest that Ibrutinib blocks progression of CLL by inhibiting only some branches of BCR signalling and interestingly, many transcription-associated changes to the chromatin remain unaltered, while transcription is effectively inhibited