Abstract 5096: ALIBI: a novel, truncated tubulin isotype in AML and stem cells

Abstract

Tubulin isotype expression is tissue restricted, with β-1 tubulin (TUBB1) restricted to hematopoietic tissues. Using the TCGA database, we found that β-1 is highly expressed in acute myeloid leukemia (AML) among all hematologic malignancies. β-1 may therefore be an attractive target for the development of novel therapeutics for AML. Analysis of a panel of AML cell lines using a polyclonal antibody against the C-terminus of β-1 revealed the presence of a band at 35kD, distinct from the canonical tubulin molecular weight of 50kD. This 35kD band, which we call Acute Leukemia Isoform β1 (ALIBI), was present in 10 of the 19 AML cell lines tested of various molecular and morphologic subtypes. In addition, we analyzed 12 AML primary AML patient blast samples and found ALIBI expression in six patient samples, suggesting that ALIBI is clinically relevant and prevalent Immunofluorescent analysis of ALIBI's subcellular localization revealed that ALIBI is present in distinct cytoplasmic aggregates not co-localizing with native microtubules as well as in the nucleus. Interestingly, ALIBI was also enriched in AML patient CD34+/CD38- cells containing leukemia stem cells. Using a murine β-1 antibody in a mouse model of acute leukemia (HOXA9-NUP98/BCR-ABL) we found that ALIBI expression was significantly higher in the leukemic marrow as compared to normal mouse marrow. As leukemia stem cells share features with embryonic stem cells (ESCs) we next investigated ALIBI expression in mouse ESCs after differentiation along megakaryocytic and erythroid lineages. ALIBI, but not canonical β-1, was expressed in ESCs, while hematopoietic differentiation led to loss of ALIBI and expression of only canonical β-1 tubulin, suggesting that expression of canonical β-1 and ALIBI are differentially regulated and that ALIBI expression is associated with stemness. RNASeq and 5′RACE of cell lines and patient samples revealed that ALIBI mRNA is missing exons 1 and 2 of canonical β-1 RNA, while it retains part of exon 3, exon 4 and the intervening intron. These results suggest that ALIBI mRNA is alternatively spliced, leading to the use of a cryptic translational start site in exon 4 flanked by a strong Kozak sequence. Exogenous expression of the putative coding region of ALIBI in ALIBI-negative cell lines produced a protein product with similar molecular weight and subcellular localization as endogenous ALIBI. Studies of ALIBI function using genome editing are currently underway. In conclusion, ALIBI is a novel truncated isoform of tubulin that has unique biologic features never previously described for tubulins. While the exact function of ALIBI within leukemic cells is still being uncovered, it nevertheless is a highly attractive target of novel therapeutic development based on its high expression in AML and its unique biologic properties.

Citation Format: Paul Basciano, Xi Li, Jason Matakas, Susanna Liu, Silvana Di Giandomenico, Siddhartha Sen, Todd Evans, Joseph Scandura, Monica Guzman, Paraskevi Giannakakou. ALIBI: a novel, truncated tubulin isotype in AML and stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5096. doi:10.1158/1538-7445.AM2015-5096