SUMMARY Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase—the positive transcription elongation factor complex (P-TEFb)—from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

dMyelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2 flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage ؊ Sca ؉ c-Kit ؉ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes. Multiple classes of genetic aberrations have been suggested as the cause of myelodysplastic syndromes (MDS) (1, 2), including mutations in signal transduction, transcription factors, and epigenetic modifiers (3-5). Interestingly, recent genomewide sequencing studies revealed that mutations in genes encoding splicing factors are commonly associated with MDS and other hematological malignancies (6-15). One of these newly identified genes codes for the SRSF2 splicing factor (also known as SC35), and its mutations have been linked to poor survival among MDS patients (16,17). Most of the SRSF2 mutations occurred at proline 95, and the majority of these mutations changed this proline to histidine (P95H); less-frequent changes to leucine (P95L) and arginine (P95R) and in-frame deletion of 8 amino acids (aa) from P95 to R102 (⌬8aa) have also been reported previously (6,16,(18)(19)(20). However, the causal effect of these mutations on MDS development remains to be established. SRSF2 is one of the founding members of the serine/argininerich (SR) protein family of splicing factors (21). It is involved in both constitutive and regulated splicing. Homozygous germ line Srsf2 knockout (KO) mice are embryonically lethal (22), and conditional knockout (cKO) mice display various tissue-specific phenotypes (22-24). Importantly, Srsf2 downregulation in mouse embryonic fibroblasts results in G 2 /M cell cycle arrest and genomic instability (23). To date, systematic analysis of SRSF2 function in the blood system has not been reported except for its requirement in T cell development (24). Given the tight link of Srsf2 mutations to MDS, we ai...

• Human RUNX1a orthologs are only found in primates.• Alternative splicing of Runx1 involving exon 6 affects the pool size of hematopoietic stem cells.RUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1a was only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6 2 (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the fulllength Runx1b (Runx1bEx6

Immunotherapies have shown promising results in certain cancer patients. For hepatocellular carcinoma (HCC), the multiplicity of an immunotolerant microenvironment within both the tumor, and the liver per se may limit the efficacy of cancer immunotherapies. Since radiation induces immunogenic cell death and inflammatory reactions within the tumor microenvironment, we hypothesized that a combination therapy of radiation and lasting local immunostimulating agents, achieved by intratumoral injection of an adenoviral vector encoding interleukin 12, may reverse the immunotolerant microenvironment within a well-established orthotopic HCC toward a state favorable for inducing antitumor immunities. Our data showed that radiation and IL-12 combination therapy (RT/IL-12) led to dramatic tumor regression in animals bearing large subcutaneous or orthotopic HCC, induced systemic effect against distant tumor, and significantly prolonged survival. Radiation monotherapy induced tumor regression at early times but afterwards most tumors regained exponential growth, while IL-12 monotherapy only delayed tumor growth. Mechanistic studies revealed that RT/IL-12 increased expression of MHC class II and co-stimulatory molecules CD40 and CD86 on tumor-infiltrating dendritic cells, suggesting an improvement of their antigen presentation activity. RT/IL-12 also significantly reduced accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and impaired their suppressive functions by reducing production of reactive oxygen species. Accordingly, tumor-infiltrating CD8+ T cells and NK cells were significantly activated toward the antitumor phenotype, as revealed by increased expression of CD107a and TNF-α. Together, our data showed that RT/IL-12 treatment could reset the intratumoral immunotolerant state and stimulate activation of antitumor cellular immunity that is capable of eliminating large established HCC tumors.

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.