Breaking the loop in AML

In this issue of Blood, Bamezai et al¹ reveal an unexpected, moonlighting (ie, working a second job, typically secretly in addition to one’s regular employment) function of the germ cell–associated RNA binding protein (RBP) PIWIL4 in acute myeloid leukemia (AML). The authors show a tumor-specific requirement for PIWIL4 in R-loop homeostasis. Loss of PIWIL4 in AML cells led to uncontrolled R-loop formation, transcriptional stalling, DNA damage, and enhanced sensitivity to ATR inhibition, findings that may inform future therapeutic aprproaches.

R-loops form when a nascent RNA transcript hybridizes to its single-stranded DNA template behind the progressing RNA polymerase complex.^2,3 This creates an RNA-DNA hybrid structure, displacing the nontemplate strand as a single-stranded DNA loop. These transient structures are common, occupying as much as 5% to 10% of the genome, and may contribute to physiological processes such as transcriptional regulation and DNA repair. However, they also contribute to processes detrimental to the cell, including replication stress, DNA damage, and genomic instability. This may represent a particular vulnerability for cancer cells where high rates of both transcription and replication increase the risk of collision between R-loops and replication forks.⁴ As such, cells employ multiple strategies to ensure efficient resolution of R-loops. These include RNA processing and export pathways that direct RNA away from the DNA template, RNA-modifying enzymes, topoisomerases, helicases, and nucleases with RNase H activity, which degrade RNA within an RNA-DNA hybrid.

PIWIL4 is an RBP belonging to the Argonaute family.⁵ Along with the other PIWI family members, it is predominantly expressed in germ cells in the testis. The canonical function of PIWI proteins is the PIWI-interacting RNA (piRNA)-directed silencing of transposable elements, which would otherwise threaten the genomic integrity of germ cells.⁶ However, evidence is beginning to emerge that suggests PIWI proteins may play roles outside of the germ cell and in some cases in a piRNA-independent manner.⁷ The study reported in this issue of Blood begins with the surprising observation that PIWIL4 is expressed at strikingly high levels in AML cells and leukemic stem cells (LSCs). Indeed, AML oncogenes were able to drive increased PIWIL4 expression in hematopoietic stem and progenitor cells (HSPCs). Moreover, the authors show how AML and LSCs depend upon PIWIL4 for survival, engraftment, and propagation. In contrast, nonmalignant HSPCs showed normal function after PIWIL4 depletion. This dispensability for normal hematopoiesis is consistent with a previous mouse model,⁸ confirming a cancer-specific dependency upon PIWIL4. These intriguing findings prompt the question, why is PIWIL4, a germ cell-associated RBP, so important to malignant myeloid cells? Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments showed minimal association with piRNA, leading the authors to conclude that PIWIL4 must be acting independently from its canonical role in the PIWI pathway. Instead, through a series of carefully designed experiments, they elucidate how AML and LSCs coopt the RNase H catalytic activity of PIWIL4 to resolve R-loops. Interestingly, PIWIL4-dependent R-loop resolution was most enriched at the loci of genes essential for AML growth and LSC maintenance. PIWIL4 depletion in AML cells led to accumulation of R-loops, which in turn resulted in stalled transcription of AML and LSC signature genes, replicative stress, DNA damage, and loss of cellular fitness. These detrimental effects were specific to AML cells and were potentiated by pharmacological inhibition of ATR, highlighting a potential approach for AML-specific therapeutic targeting.

The findings of this study are important and provocative and raise new questions. First, the study uncovers a novel moonlighting function for PIWIL4 in the resolution of R-loops and maintenance of genomic integrity. At the same time, the limited rescue with RNase H leaves open the possibility that AML dependence on PIWIL4 may involve other functions beyond R-loop resolution. It also leaves open the question as to whether the R-loop-resolving function of PIWIL4 is unique to AML or might be shared with other cancer types. Second, the study highlights the importance of the complex interplay between RNA and RBPs in the regulation of hematopoiesis and hematopoietic malignancy and an emerging theme that RBPs frequently moonlight beyond a single canonical function.⁹ Finally, the requirement to resolve R-loops reveals a specific vulnerability of AML cells and LSCs that appears not to be shared with normal HSPCs. Precisely why this requirement should be so specific to AML cells is yet to be fully elucidated. However, this question and the ways this cancer-specific vulnerability might be exploited as a therapeutic strategy to target AML without damaging normal myeloid precursors will be the subject of exciting future research.

Conflict-of-interest disclosure: The author declares no competing financial interest.