Mutational Landscape of Del(9q) Acute Myeloid Leukemia (AML)
Authors: Dolnik Anna , Chakkarappan Sundaram R. , Blätte Tamara J. , Cocciardi Sibylle , Krönke Jan , Andrea Corbacioglu , Holzmann Karlheinz , Paschka Peter , Rücker Frank G. , Kapp-Schwoerer Silke , Gaidzik Verena I. , Schlenk Richard F. , Döhner Hartmut , Döhner Konstanze , Bullinger Lars 
Published: October 2019

Abstract
Background: Deletion of the long arm of chromosome 9, del(9q), is a recurrent genomic abnormality, which occurs at a frequency of ~2% in AML. Interestingly, deletions of 9q are mainly found in t(8;21)-positive AML, as well as in AML with NPM1 (NPM1mut) or CEBPA (CEBPAmut) gene mutation, thereby suggesting that del(9q) can act as cooperating event in these prognostically favorable AML subgroups.
Aims: In order to dissect the biology of AML with del(9q), we comprehensively characterized a large cohort of 9q21 deleted cases (n=45) at the molecular level.
Methods: We performed SNP 6.0 microarray analysis to delineate the minimally deleted region on 9q, and we analyzed gene expression in selected cases to determine whether 9q21 deletions are displaying a characteristic expression pattern. Potential candidate genes were further studied by shRNA based knock-down experiments in cell line models. Finally, we performed whole exome sequencing (WES) of paired diagnostic and remission samples from n=20 del(9q) patients with NPM1mut (n=7), NPM1wt/CEBPAmut (n=7), and t(8;21) (n=6) to identify additional aberrations cooperating with 9q loss in leukemogenesis.
Results: By SNP microarray analysis, we could confirm a minimally deleted region (MDR) on 9q21 encompassing seven genes (GKAP1, KIF27, C9orf64, HNRNPK, RMI1, SLC28A3, NTRK2). By targeted resequencing in n=50 non-9q deleted cases, we detected a mutation in HNRNPK, which was recently confirmed to be recurrently mutated by The Cancer Genome Atlas (TCGA) project. These findings point to HNRNPK as the most important candidate gene of the MDR. HNRNPK encodes for a ubiquitously expressed heterogeneous nuclear ribonucleoprotein (hnRNP), which influences pre-mRNA processing and other aspects of mRNA metabolism, and it is thought to play a role during cell cycle progression. To further evaluate the biology underlying 9q deleted/HNRNPK haploinsufficient cases, gene expression data were generated by microarray technology comparing NPM1mut cases with and without del(9q) (n=11 vs n=119, respectively). These analyses showed deregulated expression of genes involved in splicing and mRNA processing, and there was an overlap with gene expression changes following shRNA-mediated HNRNPK knock-down in AML cell lines, which also suggested a growth advantage for haploinsufficient cells. While these data further support that HNRNPK might play a cooperating role in AML, we were eager to see whether there are additional mutations commonly linked to del(9q). By WES, we detected on average 7.8 somatic protein altering point mutations per sample (missense and nonsense SNVs) and 2.5 frameshift insertions or deletions affecting genes known to play a role in AML as well as genes not yet linked to AML. In accordance with the general mutational spectrum of t(8;21), NPM1 or CEBPA mutant AML, we identified mutations in known epigenetic regulators such as ASXL1, ASXL2, TET2 or DNMT3A, but we also could find novel somatic mutations in additional genes involved in the regulation of the chromatin structure such as BRD3 or BRWD3. Furthermore, we identified mutations in genes associated with mRNA processing and RNA splicing,as well as mutations affecting the RAS- signaling pathway and DNA repair mechanisms.
Conclusions: While ongoing analyses are likely to identify additional gene mutations in del(9q) AML, first results suggest HNRNPK haploinsufficiency as a potential "driver" mutation playing a role in the pathomechanism of 9q deleted AML. A better understanding of the HNRNPK function in normal hematopoietic cells as well as leukemia cells without del(9q), and studying the impact of HNRNPK mutations in AML might enable novel therapeutic approaches for del(9q)/HNRNPKmut AML.
These authors contributed equally to the work: AD and SRC as well as KD and LB.
Supported by: FP7 NGS-PTL project, and DFG SFB 1074 B3 project.

Disclosures
No relevant conflicts of interest to declare.