Identification and characterization of pathogenic ZNF687 variant in acute myeloid leukemia
Analysis of Beat AML sequencing data revealed a recurrent frameshift variant (R939Pfs*Ter36) in Zinc Finger 687 (ZNF687) that was observed in ~1% of patients and has not been previously described. ZNF687 encodes a transcription factor containing DNA binding C2H2 zinc finger and has been studied in select other malignancies, including Paget's disease of bone that is associated with giant cell tumor (PDB/GCT) and hepatocellular carcinoma. It is part of a network with ZMYND8 and two additional zinc finger proteins, ZNF592 and ZNF532, that associate with H3K4 demethylation machinery and regulates gene transcription. The R939Pfs*Ter36 frameshift induced overexpression of ZNF687 compared with wild-type, and the overexpressed protein was mislocalized to the cytoplasm. We further performed RNA sequencing on HEK293 cells expressing the frameshift variant, wild-type, or a previously identified variant (P937R) that has been reported as a germline ZNF687 variant characterized to predispose to PDB/GCT. We observed the R939Pfs*Ter36 frameshift to induce activation of oxidative phosphorylation, inactivation of histone methylation machinery, and significant downregulation of lysine methyltransferase 2D (KMT2D) compared with wild-type and P937R. Overall, these findings suggest that ZNF687 R939Pfs*Ter36 plays an important role in AML pathogenesis, which could aid in diagnostic precision, and may impact therapeutic responses. Overall design: Six-well plates were seeded with 3e5 HEK293 cells overnight. The cells were transfected with ZNF687 WT, frameshift, missense variant containing plasmids and pMIG empty using Fugene6 (Promega) according to the manufacturer's protocol. Cells were cultured and harvested for protein lysate extraction following the observation of GFP fluorescence 72h after transfection. RNA was purified using the QIAGEN RNeasy Mini Kit. Total RNA integrity was evaluated using the Bioanalyzer (Agilent). RNA-seq libraries were prepared from total RNA using the Illumina Stranded mRNA kit (Illumina). Libraries were then profiled on a TapeStation D1000 tape (Agilent) and quantified using an NGS Library Quantification Kit (Roche/Kapa) on a StepOnePlus Real Time PCR Workstation (Thermo/ABI). Libraries were sequenced on a NovaSeq 6000 (Illumina). Fastq files were generated from the resulting base call files using bcl2fastq (Illumina). The paired-end reads were aligned to hg38 (NCBI build 39) and aligned reads were analyzed using the Rsubread Package.