The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

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.

Runx1 exon 6-related alternative splicing isoforms differentially regulate hematopoiesis in mice.

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(-) (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6(+)). They also facilitated the transactivation of Runx1bEx6(+). To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6(-) and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1(+)c-Kit(+) cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis.

Enhanced Photovoltaic Performance of Inverted Perovskite Solar Cells through Surface Modification of a NiOx-Based Hole-Transporting Layer with Quaternary Ammonium Halide-Containing Cellulose Derivatives.

In this study, we positioned three quaternary ammonium halide-containing cellulose derivatives (PQF, PQCl, PQBr) as interfacial modification layers between the nickel oxide (NiOx) and methylammonium lead iodide (MAPbI3) layers of inverted perovskite solar cells (PVSCs). Inserting PQCl between the NiOx and MAPbI3 layers improved the interfacial contact, promoted the crystal growth, and passivated the interface and crystal defects, thereby resulting in MAPbI3 layers having larger crystal grains, better crystal quality, and lower surface roughness. Accordingly, the photovoltaic (PV) properties of PVSCs fabricated with PQCl-modified NiOx layers were improved when compared with those of the pristine sample. Furthermore, the PV properties of the PQCl-based PVSCs were much better than those of their PQF- and PQBr-based counterparts. A PVSC fabricated with PQCl-modified NiOx (fluorine-doped tin oxide/NiOx/PQCl-0.05/MAPbI3/PC61BM/bathocuproine/Ag) exhibited the best PV performance, with a photoconversion efficiency (PCE) of 14.40%, an open-circuit voltage of 1.06 V, a short-circuit current density of 18.35 mA/cm3, and a fill factor of 74.0%. Moreover, the PV parameters of the PVSC incorporating the PQCl-modified NiOx were further enhanced when blending MAPbI3 with PQCl. We obtained a PCE of 16.53% for this MAPbI3:PQCl-based PVSC. This PQCl-based PVSC retained 80% of its initial PCE after 900 h of storage under ambient conditions (30 °C; 60% relative humidity).

Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis.

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.

RUNX1 deficiency cooperates with SRSF2 mutation to induce multilineage hematopoietic defects characteristic of MDS.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematologic malignancies with a propensity to progress to acute myeloid leukemia. Causal mutations in multiple classes of genes have been identified in patients with MDS with some patients harboring more than 1 mutation. Interestingly, double mutations tend to occur in different classes rather than the same class of genes, as exemplified by frequent cooccurring mutations in the transcription factor RUNX1 and the splicing factor SRSF2. This prototypic double mutant provides an opportunity to understand how their divergent functions in transcription and posttranscriptional regulation may be altered to jointly promote MDS. Here, we report a mouse model in which Runx1 knockout was combined with the Srsf2 P95H mutation to cause multilineage hematopoietic defects. Besides their additive and synergistic effects, we also unexpectedly noted a degree of antagonizing activity of single mutations in specific hematopoietic progenitors. To uncover the mechanism, we further developed a cellular model using human K562 cells and performed parallel gene expression and splicing analyses in both human and murine contexts. Strikingly, although RUNX1 deficiency was responsible for altered transcription in both single and double mutants, it also induced dramatic changes in global splicing, as seen with mutant SRSF2, and only their combination induced missplicing of genes selectively enriched in the DNA damage response and cell cycle checkpoint pathways. Collectively, these data reveal the convergent impact of a prototypic MDS-associated double mutant on RNA processing and suggest that aberrant DNA damage repair and cell cycle regulation critically contribute to MDS development.

Combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment.

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.

SRSF2 Is Essential for Hematopoiesis, and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing.

Myelodysplastic 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.