Cloning of the Full-Length Gene of a Novel Human Acute Monocytic Leukemia Associated Antigen (MLAA-22) and Characterization of Its Functions
Authors: Zhang Wanggang , Gu Liufang , He Aili , Cao Xingmei , Chen Yinxia , Ma Xiaorong , Liu Jie , Zhang Pengyu 
Published: October 2019

Abstract
Abstract 1399

Introduction:
In the past several decades, rapid progress has been made in the development of combination therapies for acute leukemia involving chemotherapy and hematopoietic stem cell transplantation. However, these protocols are not particularly effective in treating minimal residual disease (MRD), which is the source of relapses. Treating patients in complete remission with appropriate immunotherapy is anticipated to be an excellent method to control MRD and cure leukemia.
Our group used inactivated leukemic cells in combination with IL-2, IL-6, GM-CSF and Freund's incomplete adjuvant as a whole-cell vaccine in the 1990s. To improve whole-cell vaccine, we constructed a cDNA expression library from human U937 cells applied the method of serologic analysis of recombinant cDNA expression library (SEREX) to identify acute monocytic leukemia-associated antigens (MLAAs). Thirty-five distinct novel antigens were identified through the SEREX analysis by reaction with sera from acute monocytic leukemia patients. MLAA-22 (GenBank no: AY288965) is a representative gene in this group.

Methods:
Because the sequence of MLAA-22 was obtained from a cDNA library, we first sought to determine if the sequence is complete. Both 5' and 3' ends of MLAA-22 were determined by RLM -RACE in U937 cell line and the full-length of MLAA-22 was confirmed by RT-PCR. After full-length of MLAA-22 was determined, new sequence was analyzed by bioinformatics at both nucleic acid and amino acid sequence levels. Next, we studied the functions of MLAA-22 gene by RNAi. The shRNA lentiviral vector of MLAA-22 was constructed to infect 293T cells and a high titer of the virus particles was obtained from supernatant of 293T. U937 cells were infected with virus particles to down regulate the expression of MLAA-22 mRNA. MLAA-22 gene expression was determined by fluorescent real-time quantitative PCR. After that, a series of changes in phenotype and functions of U937 cell were detected by MTT, Hoechst staining and FCM.

Results:
Compared to the original sequence of MLAA-22 (GenBank no: AY288965), the new sequence is extended by 75 and 606 bp at the 5' and 3' ends, respectively. Full-length of MLAA-22 is 2718 bp. MLAA-22 is located in 17q11.2 and is highly homologous to the putative human gene KIAA0100. The functions of the KIAA0100 gene and protein have not been thoroughly elucidated. The MLAA-22 sequence has a complete open reading frame (ORF) that encodes a protein containing 701 amino acid residues. Studies on the function of MLAA-22 revealed that down-regulated MLAA-22 mRNA>70% in U937 cells resulted in the inhibition of cell proliferation. Furthermore, the apoptosis rate of U937 was significantly higher than that of the control group. Based on these results we speculate that MLAA-22 may be a new anti-apoptotic gene related to the development of acute monocytic leukemia.

Conclusions:
The full-length of MLAA-22 cDNA was 2718bp and it was located in 17q11.2. MLAA-22 may be a new anti-apoptosis gene related to acute monocytic leukemia. It may play an important role in the pathogenesis of acute monocytic leukemia.

Disclosures:
No relevant conflicts of interest to declare.