Abstract 4646: A novel truncated variant of the hematopoietic Hβ-1 tubulin isotype with implications for stem cell biology

The αβ-tubulin dimer is the building block of the microtubule cytoskeleton, which is important for many cellular functions. Several α and β tubulin isotypes have been identified so far; Hβ-1 tubulin (human β tubulin Class VI) is a hematopoietic-specific tubulin isotype whose expression is restricted to megakaryocytes and platelets. TCGA analysis showed that β-1 is highly and specifically expressed in acute myeloid leukemia (AML). β-1 may therefore be an attractive target for the development of novel therapeutics for AML. We developed a highly-specific polyclonal antibody against the last 8 C-terminal amino acids and analyzed a panel of AML cell lines and primary samples by western blot. We identified the presence of a novel truncated 35kD band, distinct from the canonical 50kD tubulin. This 35kD band, which we have named as Acute Leukemia Isoform β1 (ALIBI), was present in 10 of the 19 AML cell lines tested as well as in a large number of AML clinical samples, as evidenced by immunoblotting, and corroborated by RNA-sequencing. We determined that ALIBI was a splice variant of Hb1 tubulin, lacking exons 1 and 2 with partial retention of exon 3 and intron 3. Mass array methylation analysis revealed differential promoter and intragenic methylation pattern in canonical b1 tubulin versus ALIBI, suggesting that there is an epigenetic switch that determines the expression of each tubulin isoform. Immunofluorescence analysis revealed that ALIBI, unlike all tubulin isotypes, does not incorporate into microtubules but instead accumulates in the nucleus and in distinct puncta in the cytoplasm. Exogenous expression of ALIBI, also resulted in a similar cellular distribution. Using a murine leukemia mouse model we found significant enrichment of ALIBI in the leukemic stem cell fraction, while in normal mouse development ALIBI was present in fetal liver and hematopoietic progenitor cells. Based on this observation, as well as the relationship between leukemic stem cells and embryonic stem cells, we probed for the presence of ALIBI within murine embryonic stem cells (mESCs) and found ALIBI to be present in cultured mESCs, and down-regulated upon differentiation when full length b1 tubulin was expressed. Taken together, these data suggest that ALIBI has an important, heretofore unrecognized, role in stem cell biology. However, much remains unknown regarding the function of ALIBI within the nucleus, the factors that regulate its expression over the canonical Hβ1 isoform, and