Abstract 244: Effects of upregulation of Sept6 v4 splice variant in AML

Evidence is now emerging that the evolutionarily conserved septin family of genes are contributors to diverse cellular processes including cytokinesis, apoptosis, actin and tubulin dynamics, endo and exocytosis, membrane dynamics and polarity. However their exact roles remain uncertain and much remains to be discovered about their role in physiological and pathological situations. Considerable data indicate that septins are implicated in human neoplasia and the SEPT6 gene can be a fusion partner of MLL in acute leukaemia. We investigated the genomic architecture, transcript profile, mRNA and protein expression of SEPT6. The 12 exons of SEPT6 undergo complex splicing events giving rise to 7 distinct transcripts (SEPT6_v1, v2, v3, v4, v4*, v4** and v4***) that encode 4 polypeptides SEPT6_i1-4). Of note are 4 discrete transcripts encoding the same 427 amino acid polypeptide (SEPT6_i4): a phenomenon also seen in other septins. We used a quantitative RT-PCR strategy to define the expression of these transcripts in cell lines and in fetal and adult tissues and show tissue specific patterns of expression. SEPT6 had previously been shown to be a fusion partner of MLL in AML thus we tested the hypothesis that SEPT6 expression would be altered in AML and influenced by the genetic events underpinning the disease. We analysed samples from 310 patients classified by their karyotype [t(8;21), t(15;17), inv(16), 11q23, complex rearrangements, normal karyotype and other] and find that patients with t(15;17) had low expression of SEPT6_v4** compared to patients with inv(16) where high expression of SEPT6_v4** was seen. Furthermore, a principal component analysis segregates the t(15;17) and inv(16) patients by SEPT6 expression. We therefore wondered what the effect of perturbed expression of isoform 4 might be on cells. We show that SEPT6_i3 and i4 localise to both the cytoplasm and nucleus and their overexpression results in abnormal cell size and shape and also multinucleation. Over-expression of SEPT6_i3 and i4 alters stability of microtubules and the rates of their assembly and disassembly in HeLa cells. The microtubule network in these cells resists disruption in response to depolymerisation on ice compared to vector only-expressing cells. When allowed to repolymerise, the SEPT6_i3 and i4 over-expressing cells repolymerise tubulin more slowly compared to control cells. Using RNA interference, we also show increased number of cells with abnormal nuclei (size, shape, and