R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

Plasmodium falciparum DHFR and DHPS Mutations Are Associated With HIV-1 Co-Infection and a Novel DHPS Mutation I504T Is Identified in Western Kenya.

Antifolate resistance is significant in Kenya and presumed to result from extensive use and cross-resistance between antifolate antimalarials and antibiotics, including cotrimoxazole/Bactrim used for HIV-1 chemotherapy. However, little is known about antifolate-resistant malaria in the context of newly diagnosed HIV-1 co-infection prior to administration of HIV-1 chemotherapy. Blood samples from a cross-sectional study of asymptomatic adult Kenyans enrolled during voluntary HIV testing were analyzed by PCR for Plasmodium spp. More than 95% of volunteers with identifiable parasite species (132 HIV-1 co-infected) were infected with Plasmodium falciparum alone or P. falciparum with Plasmodium ovale and/or Plasmodium malariae. Deep sequencing was used to screen for mutations in P. falciparum dihydrofolate reductase (dhfr) (N51I, C59R, S108N, I164L) and dihydropteroate synthase (dhps) (S436H, A437G, K540E, A581G) from 1133 volunteers. Individual mutations in DHPS but not DHFR correlated with HIV-1 status. DHFR haplotype diversity was significantly different among volunteers by gender and HIV-1 status. DHPS haplotype diversity by HIV-1 status was significantly different between volunteers paired by age and gender, indicating that patterns of resistance were independent of these variables. Molecular simulations for a novel DHPS mutation (I504T) suggested that the mutated protein has increased affinity for the endogenous ligand DHPPP and decreased affinity for drug binding. A sub-group of monoclonal infections revealed that age and parasitemia were not correlated and enabled identification of a rare septuple-mutant haplotype (IRNL-HGEA). In our study, adult Kenyans newly diagnosed with HIV-1 infection were predominantly infected with moderately resistant P. falciparum, with patterns of infecting parasite genotypes significantly associated with HIV-1 status. Together with the discovery of DHPS I504T, these data indicate that antifolate resistance continues to evolve in Kenya. Further, they highlight the need to understand the effects of associated mutations on both fitness and resistance of P. falciparum in the context of HIV-1 co-infection to better inform treatment for asymptomatic malaria.