The Non-Canonical, R-Loop Regulatory Function of PIWIL4 Maintains Genomic Integrity and Leukemic Potential of AML Cells
Authors: Bamezai Shiva , Vegi Naidu , Pulikkottil Jose Alex , Müller Julia , Grummt Ingrid , Song Chenlin , Döhner Konstanze , Döhner Hartmut , Feuring-Buske Michaela , Rawat Vijay Pal Singh , Buske Christian 
Published: October 2019

Abstract
During DNA replication and transcription, RNA:DNA hybrids are formed as part of three stranded nucleic acid structures known as R-loops. R-loops occur frequently in the genome at highly transcribed regions, ribosomal genes, mitochondria and intergenic regions, and are predominantly resolved by Ribonuclease (RNase) H family of enzymes. However, unscheduled and unresolved R-loops represent a potent source of DNA damage, especially in rapidly dividing cells such as cancer cells. It is imperative for cancer cells to prevent accumulation of unresolved R-loops in order to limit DNA damage. So far, the mechanism how leukemic cells prevent accumulation of R-loops is not well understood. In this study, we show that an RNase H-like protein, PIWIL4, is aberrantly and highly expressed in AML patients, prevents R-loop accumulation via its RNase H activity and thereby acts as important regulator of leukemic growth. In our initial analysis, we observed that the recombinant human PIWIL4 protein digested radiolabeled-RNA-containing R-loops in vitro, exhibiting an RNase H-like activity with increasing efficiency, in incremental concentrations and time durations. Moreover, immunoprecipitation of PIWIL4 followed by liquid chromatography mass spectrometry (LC/MS) in HEK cells showed that PIWIL4 was bound with multiple nuclear and nucleolar RNA processing factors that are associated with formation of R-loops. Published RNA-seq and microarray datasets revealed that, among all cancers, PIWIL4 was significantly highest expressed in myeloid leukemia. Quantitative real time PCR (qRT-PCR) of acute myeloid leukemia (AML) patients revealed that PIWIL4 showed an average of 21.6 ± 5.0-fold higher expression in AML patients (n=68; p<0.0001), compared to healthy CD34+ bone marrow (BM) and BM mononuclear cells (n=3). Western blot of AML patient samples and intracellular (IC) staining confirmed higher PIWIL4 protein expression levels in AML cells compared to cord blood CD34+ HSPCs. Piwil4 expression increased by 6-8 fold in murine BM healthy HPSCs within 48h after transduction with MLL-AF9, AML1-ETO9A and CDX2 oncogenes compared to empty vector (n=3, p<0.0001). Stable knockdown of PIWIL4 in AML cell lines and primary AML samples using shRNA, followed by IC staining and confocal microscopy using an antibody against R-loops (S9.6) revealed a marked increase in accumulation of R-loops within 72h post-transduction in PIWIL4 depleted cells, in contrast to healthy cord blood HSPCs which remained unaffected (n=3). PIWIL4 depleted AML cells exhibited an accumulation of DNA damage associated gH2AX foci, replication stress associated BrdU foci, higher levels of phosphorylated ATR (p-ATR), a marked increase in apoptosis and block in the G2M phase of the cell cycle. Depletion of PIWIL4 significantly impaired clonogenic potential of AML patient samples in vitro (avg. 4.9 ± 0.9-fold reduction, p<0.0001, n=3). In vivo, PIWIL4 depletion in cell lines delayed onset of leukemia (n=8, p<0.001) and in AML patient cells reduced leukemic engraftment in xenografts 12 weeks post-transplantation (avg. scr - 50.6±21% vs avg. shRNA-14.6±10, n=6). Of note, PIWIL4 depletion in cord blood CD34+ HSPCs had no impact on colony formation or differentiation in vitro. RNA-seq of PIWIL4 depleted THP-1 cell line followed by GSEA revealed a significant reduction in expression of ribosomal genes and increased expression of G2M checkpoint repair pathway (n=2, p<0.05, FDR<0.05). qRT-PCR of pre-rRNA (45S rRNA) showed a significant reduction in rRNA transcription in shRNA transduced cell lines (avg. 2.5 ± 0.3-fold reduction, n=3, p<0.01). Overexpression of PIWIL4 or RNase H1 in PIWIL4 depleted AML cell lines rescued R-loop and gH2AX signals, induced a decrease in p-ATR and gH2AX protein levels, and rescued the impact on apoptosis and growth phenotype in colony assays. RNA polymerase I inhibitor CX-5461, known to stabilize R-loop associated secondary structures, acted synergistically with PIWIL4 depletion and induced complete cell death of PIWIL4 depleted AML cells compared to scrambled control at IC50 concentrations. Thus, collectively, we could show for the first time that PIWIL4 is a functional RNase H like enzyme in AML cells, suppresses formation of R-loops, thereby preventing DNA damage and apoptosis of AML cells. Our data also suggest that impairing resolution of R-loops is a powerful therapeutic tool in AML.

Disclosures
Buske: Roche: Honoraria, Research Funding; Bayer: Research Funding; Janssen: Honoraria, Research Funding.