“There are currently limited therapeutic options for elderly patients with myeloid malignancies, who are ineligible for stem cell transplantation,” commented Dr. Sayantani Sinha, a postdoctoral research fellow in Dr. Stanley Lee’s lab at Fred Hutchinson Cancer Center. “The aggressive nature of these leukemias motivates us to further identify other novel sensitizers” for targeted, combination therapies. Hematologic cancers – lymphomas, myelomas, and leukemias – have recurrent mutations in RNA splicing factors. RNA splicing factors are proteins that help with proper processing of RNA to produce the template for making proteins. In cells with mutations in splicing factors, there is abnormal accumulation of R-loop structures which are RNA:DNA hybrids formed during transcription. While a causal link to cancer has yet to be confirmed, the accumulation of these RNA:DNA hybrids can increase genomic instability and lead to cell death. Others have shown that inhibiting pathways that promote cell survival in the presence of R-loop accumulation and genomic instability effectively tips the scale to cause cell death. The Lee lab and their collaborators sought to identify compensatory pathways for dealing with R-loop accumulation in splicing factor mutant forms of leukemia. Their findings identified a new target, poly ADP-ribose polymerase 1/2 (PARP1/2), that promotes survival in certain splicing factor mutant leukemias with accumulated R-loops. Importantly, they demonstrate that PARP inhibitors can induce cell death by limiting the PARP1-dependent compensatory survival pathway that overcomes the stress of R-loop accumulation, independent of PARP roles in DNA repair and homologous recombination. These findings were published recently in Cancer Research.
The researchers used a mouse model of leukemia that represents a genotype found in ~10% of human leukemias – MLL-AF9 oncogenic fusion and splicing factor Srsf2P95H/+. Cells harvested from the bone marrow of these mice were used to screen for sensitivity to drugs that either inhibit DNA damage response, DNA replication or metabolic processes. The screen revealed sensitivity to previously reported survival factors (Type-I PRMT, CDK4/6, and ATR) as well as PARP1/2 proteins that had not been described. The researchers also tested sensitivity to PARP1/2 inhibitors using splicing factor SRSF2 mutant human leukemia cells and for other splicing factor mutants in both mouse and human cells and primary leukemia patient biopsies obtained from the Fred Hutch-UW Hematopoietic Diseases Repositories (FH-UW HDR). “We made an unexpected finding that a genetically specific set of leukemia patients, in this case those harbouring somatic mutations in splicing factors SRSF2 and U2AF1, depend on PARP1 to resolve excess R-loops and transcription-replication conflicts,” summarized Dr. Sinha.
“During the course of this study, we also developed a novel proximity ligation-based assay to detect PARP1 activity at R-loops that we plan to further develop into a potential biomarker for PARP inhibitor sensitivity in leukemia and other cancers,” shared Dr. Sinha. This assay was developed and performed because the researchers wanted to determine if the sensitivity of these cancer cells to PARP1 inhibition was due to PARP1 presence at R-loops. Others had reported PARP1 at R-loops and predicted that PARP1 may aid in R-loop formation and/or resolution. Indeed, using this proximity ligation assay for PARP1 and R-loop co-localization, the researchers found that splicing factor mutant leukemia cells that were sensitive to PARP1 inhibition also had more PARP1 activity at R-loops than leukemia cells without the splicing factor mutations.
Altogether, “our study provides pre-clinical evidence that PARP inhibitors may be considered as a suitable therapeutic option for leukemia patients,” stated Dr. Sinha. She continued, “The first-generation PARP inhibitors that are approved for solid tumors target both PARP1 and PARP2. Given the role of PARP2 in regulating erythropoiesis, these first generation of PARP inhibitors exhibit toxicity. Our study shows that splicing mutant leukemias are sensitive to a novel PARP1-specific inhibitor (AZD5305), which has a much improved toxicity profile compared to first-generation PARP inhibitors.”
The future directions of the Lee lab are to evaluate newly developed PARP1-specific inhibitors to survey for drugs with reduced toxicity, improved efficacy, and to provide alternatives for combination therapies. “We will also be delving deeper into how R-loop regulation is perturbed in hematologic malignancies, which will help identify other potential therapeutic targets,” commented Dr. Sinha. This work was supported by the Cancer Consortium “which provided critical funding that allowed us to test several ideas at the beginning of this study,” concluded Dr. Sinha.
The spotlighted research was funded by the National Institutes of Health, Kuni Foundation, Edward P. Evans Foundation, AIRC, Leukemia and Lymphoma Society, AACR, Mugee Research Award for Pediatric Cancer, Randy Shaver Cancer Research and Community Fund Award, Masonic Cancer Center, University of Minnesota Foundation donors, American Society of Hematology, Vera and Joseph Dresner Foundation, Mark Foundation For Cancer Research, and Merck.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Elizabeth Swisher, Derek Stirewalt, Sergei Doulatov, and Stanley Lee contributed to this work.
Liu ZS, Sinha S, Bannister M, Song A, Arriaga-Gomez E, McKeeken AJ, Bonner EA, Hanson BK, Sarchi M, Takashima K, Zong D, Corral VM, Nguyen E, Yoo J, Chiraphapphaiboon W, Leibson C, McMahon MC, Rai S, Swisher EM, Sachs Z, Chatla S, Stirewalt DL, Deeg HJ, Skorski T, Papapetrou EP, Walter MJ, Graubert TA, Doulatov S, Lee SC, Nguyen HD. 2023. R-loop accumulation in spliceosome mutant leukemias confers sensitivity to PARP1 inhibition by triggering transcription-replication conflicts. Cancer Res. Online ahead of print.