Atpase Family AAA Domain-Containing Protein 2 (ATAD2) As a Novel Target in Multiple Myeloma

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P<0.0005 for both EFS and OS). We have begun to validate these genes for impact on genomic instability and on MM cell growth and survival. Here, we present the functional validation of AAA domain containing protein 2 (ATAD2), which is one of the top-most genes in our 30 gene signature in MM and has also been part of a chromosomal instability signature representing six different cancer types (Nature Genetics, 38: 9, 2006) and a mitotic chromosomal instability signature identified in breast cancer [Sci Transl Med, 2013]. Using The Cancer Genome Atlas data we have also correlated elevated ATAD2 expression with poor OS in pulmonary and pancreatic cancers (P<0.02). ATAD2 is a member of the AAA ATPase family of proteins containing two conserved ATPase domains and a bromodomain. Bromodomain containing proteins are involved in the regulation of gene expression and are frequently dysregulated in MM as well as other cancers. ATAD2 bromodomain selectively recognizes acetylated histone 4 (acetylated at K5 and K12) and has been shown to regulate many cellular processes including cell proliferation. ATAD2 has been shown to interact with and stimulate transcriptional activity of MYC and has also been shown to contribute to invasion and migration in several cancers. We have confirmed elevated ATAD2 expression relative to normal PBMC by Western blotting in all twelve myeloma cell lines tested. To further delineate its role in MM, we evaluated the impact of its knockdown on various parameters of growth and genome maintenance using shRNAs. Relative to control shRNA, knockdown with multiple different shRNAs resulted in near complete cell death in 3 MM cell lines (JJN3, H929 and RPMI) in five days. Reduced cell viability was accompanied by increased apoptosis as seen by annexin V/PI staining. Relative to control, ATAD2 knockdown in RPMI cells led to increase in the apoptotic cell fraction by 56% and in H929 cells by 42%. To investigate genomic impact of elevated ATAD2 expression, the live cell fraction from control and ATAD2-knockdown cells was evaluated, right after selection, for impact on the expression of γ-H2AX (a DNA break marker), pRPA32 (a marker of DNA end resection, a distinct step in the initiation of homologous recombination; HR) and recombinase RAD51 (a key player in HR). ATAD2-knockdown in H929 cells inhibited spontaneous DNA breaks, DNA end resection as well as RAD51 expression, suggesting that elevated ATAD2 contributes to increased spontaneous DNA damage and HR activity observed in MM cells. To further confirm its impact on genome stability, the live cell fraction from control and knockdown cells was evaluated for micronuclei (a marker of genomic instability). Relative to control shRNA-transduced cells, knockdown of ATAD2 cells reduced micronuclei by 58% in H929 and 53% in JJN3 cells.

In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma.