Spliceosome Mutations Induce R Loop-Associated Sensitivity to ATR Inhibition in Myelodysplastic Syndromes.

Heterozygous somatic mutations in spliceosome genes (U2AF1, SF3B1, ZRSR2, or SRSF2) occur in >50% of patients with myelodysplastic syndrome (MDS). These mutations occur early in disease development, suggesting that they contribute to MDS pathogenesis and may represent a unique genetic vulnerability for targeted therapy. Here, we show that RNA splicing perturbation by expression of the U2AF1(S34F) mutant causes accumulation of R loops, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA, and elicits an ATR response. ATR inhibitors (ATRi) induced DNA damage and cell death in U2AF1(S34F)-expressing cells, and these effects of ATRi were enhanced by splicing modulating compounds. Moreover, ATRi-induced DNA damage was suppressed by overexpression of RNaseH1, an enzyme that specifically removes the RNA in RNA:DNA hybrids, suggesting that the ATRi sensitivity of U2AF1(S34F)-expressing cells arises from R loops. Taken together, our results demonstrate that ATR may represent a novel therapeutic target in patients with MDS carrying the U2AF1(S34F) mutation and potentially other malignancies harboring spliceosome mutations.Significance: This study provides preclinical evidence that patients with MDS or other myeloid malignancies driven by spliceosome mutations may benefit from ATR inhibition to exploit the R loop-associated vulnerability induced by perturbations in splicing. Cancer Res; 78(18); 5363-74. ©2018 AACR.

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFß, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFß markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.

Tissue-specific interferon alpha subtype response to SIV infection in brain, spleen, and lung.

Interferon alpha (IFNalpha) is a type I interferon that plays a major role in host defense. There are 13 different IFNalpha genes in humans, but much of the work concerning their role in viral defense has been limited to studying either subtype 2 or pan IFNalpha due to the inability to distinguish between highly similar genetic and amino acid sequences. Because of recent advances in molecular and biochemical techniques, it is possible to study the regulation of individual subtypes. It has been reported that HIV/SIV infection results in impaired IFNalpha responses in certain tissues. Using a pigtailed macaque SIV model, we examined the subtype response during acute infection in 3 tissues that are known to be infected with HIV/SIV, but whose IFNalpha subtype response has not been extensively studied: the brain, spleen, and lung. We found that the expression and regulation of specific subtypes occur in a tissue-specific manner. There was more limited IFNalpha subtype expression in the lung and brain, where predominantly macrophages are infected compared to the spleen, which contains both infected CD4+ lymphocytes and macrophages. Understanding the IFNalpha subtype response in tissues known to be infected with HIV/SIV can help tailor adjunctive treatment regimens to highly active antiretroviral therapy.

RCP is a human breast cancer-promoting gene with Ras-activating function.

Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.

LOMA: a fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens.

BACKGROUND: Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals. RESULTS: In this paper, we study how the efficiency of random PCR amplification affects hybridization signals. We describe a model that predicts the amplification efficiency of a given random primer on a target viral genome. The prediction allows us to filter false-negative probes of the genome that lie in regions of poor random PCR amplification and improves the accuracy of pathogen detection. Subsequently, we propose LOMA, an algorithm to generate random primers that have good amplification efficiency. Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly. CONCLUSION: The blind use of a random primer with attached universal tag (random-tagged primer) in a PCR reaction on a pathogen sample may not lead to a successful amplification. Thus, the design of random-tagged primers is an important consideration when performing PCR.