Targeting the splice factor kinases SRPK1 and CLK1 in leukaemia

This study was aimed at investigating the effect of inhibiting SRPK1 in leukaemic cells. It was also aimed at exploring the potential utility of combining conventional leukaemia chemotherapy (such as imatinib) with compounds that inhibit SRPK1.SRPK1 is best known for its role in the phosphorylation of serine/argenine rich proteins (SR-proteins) which are responsible for constitutive and alternative mRNA splicing. Studies have associated elevated levels of SRPK1 with tumour growth, proliferation and invasiveness with inhibition resulting in decreased tumour growth and altering the choice of alternative splice site. Imatinib mesylate and azacytidine remain the drugs of choice for the management of chronic myeloid leukaemia (CML) and acute myelogenous leukaemia (AML) respectively. Studies have shown that both imatinib and azacytidine are able to reduce the growth of proliferating Bcr/Abl+ and AML cells principally through the induction of apoptotic cell death.SRPK1 was inhibited using the small molecule inhibitor SPHINX. SPHINX was combined with either imatinib in a CML cell line (K562) or azacytidine in an AML cell line (Kasumi-1) for up to 72hrs. Results suggest that the SPHINX compound affects the ability of SRPK1 to phosphorylate its substrates in all three cell lines (TK6, K562 and Kasumi-1). Inhibition of SRPK1 was found to reduce cell viability in Kasumi-1 cells and at higher concentration, affect K562 cell viability consistent with the work of Sanidas et al.,(2010). There was also an indication that SRPK1 could be regulating its own expression through a feedback loop in a cell line-dependent manner.Studies with imatinib mesylate and azacytidine showed that both imatinib mesylate and azacytidine are able to reduce cell growth and viability in a dose and time-dependent manner. On combining them with SPHINX, a combination of azacytidine and SPHINX had an additive effective on Kasumi-1 cells but not with imatinib mesylate in K562 cells. Results also showed that imatinib affected the alternative splicing of caspase 9 favouring a pro-apoptotic isoform, caspase 9a. Imatinib mesylate alone also caused an apparent reduction in the expression of SRPK1, CLK1 and SRSF1, suggesting that pathways imatinib affects cell signalling pathways that regulate the expression of these oncogenic splice factor kinases and splice factors. In summary, this thesis presents evidence that targeting SRPK1 could potentially provide therapeutic benefit in the treatment of a range of leukaemias; further research is now needed to explore this novel approach

SPHINX-based combination therapy as a potential novel treatment strategy for acute myeloid leukaemia

Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo. As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines; Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5 μg/ml, Kasumi-1 cells) and imatinib (up to 20 μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth