Expression of the runt homology domain of RUNX1 disrupts homeostasis of hematopoietic stem cells and induces progression to myelodysplastic syndrome.

Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit(+)Sca-1(+)Lin(-) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit(+)Sca-1(+)Lin(-) cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.

Validating a targeted next-generation sequencing assay and profiling somatic variants in Chinese non-small cell lung cancer patients.

Non-small cell lung cancer (NSCLC) is featured with complex genomic alterations. Molecular profiling of large cohort of NSCLC patients is thus a prerequisite for precision medicine. We first validated the detection performance of a next-generation sequencing (NGS) cancer hotspot panel, OncoAim, on formalin-fixed paraffin-embedded (FFPE) samples. We then utilized OncoAim to delineate the genomic aberrations in Chinese NSCLC patients. Overall detection performance was powerful for mutations with allele frequency (MAF) ≥ 5% at >500 × coverage depth, with >99% sensitivity, high specificity (positive predictive value > 99%), 94% accuracy and 96% repeatability. Profiling 422 NSCLC FFPE samples revealed that patient characteristics, including gender, age, lymphatic spread, histologic grade and histologic subtype were significantly associated with the mutation incidence of EGFR and TP53. Moreover, RTK signaling pathway activation was enriched in adenocarcinoma, while PI(3)K pathway activation, oxidative stress pathway activation, and TP53 pathway inhibition were more prevalent in squamous cell carcinoma. Additionally, novel co-existence (e.g., variants in BRAF and PTEN) and mutual-exclusiveness (e.g., alterations in EGFR and NFE2L2) were found. Finally, we revealed distinct mutation spectrum in TP53, as well as a previously undervalued PTEN aberration. Our findings could aid in improving diagnosis, prognosis and personalized therapeutic decisions of Chinese NSCLC patients.

Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients.

Next-generation sequencing (NGS) has become a promising approach for tumor somatic mutation detection. However, stringent validation is required for its application on clinical specimens, especially for low-quality formalin-fixed paraffin-embedded (FFPE) tissues. Here, we validated the performance of an amplicon-based targeted NGS assay, OncoAim™ DNA panel, on both commercial reference FFPE samples and clinical FFPE samples of Chinese colorectal cancer (CRC) patients. Then we profiled the mutation spectrum of 648 Chinese CRC patients in a multicenter study to explore its clinical utility. This NGS assay achieved 100% test specificity and 95-100% test sensitivity for variants with mutant allele frequency (MAF) ≥ 5% when median read depth ≥ 500×. The orthogonal methods including amplification refractory mutation system (ARMS)-PCR and Sanger sequencing validated that NGS generated three false negatives (FNs) but no false positives (FPs) among 516 clinical samples for KRAS aberration detection. Genomic profiling of Chinese CRC patients with this assay revealed that 63.3% of the tumors harbored clinically actionable alterations. Besides the commonly mutated genes including TP53 (52.82%), KRAS (46.68%), APC (24.09%), PIK3CA (18.94%), SMAD4 (9.47%), BRAF (6.15%), FBXW7 (5.32%), and NRAS (4.15%), other less frequently mutated genes were also identified. Statistically significant association of specific mutated genes with certain clinicopathological features was detected, e.g., both BRAF and PIK3CA were more prevalent in right-side CRC (p < 0.001 and p = 0.002, respectively). We concluded this targeted NGS assay is qualified for clinical practice, and our findings could help the diagnosis and prognosis of Chinese CRC patients.

Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein.

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.

Usp18 deficient mammary epithelial cells create an antitumour environment driven by hypersensitivity to IFN-λ and elevated secretion of Cxcl10.

The theory of cancer immunoediting refers to mechanisms by which the immune system can suppress or promote tumour progression. A major challenge for the development of novel cancer immunotherapies is to find ways to exploit the immune system's antitumour activity while concomitantly reducing its protumour activity. Using the PyVmT model of mammary tumourigenesis, we show that lack of the Usp18 gene significantly inhibits tumour growth by creating a tumour-suppressive microenvironment. Generation of this antitumour environment is driven by elevated secretion of the potent T-cell chemoattractant Cxcl10 by Usp18 deficient mammary epithelial cells (MECs), which leads to recruitment of Th1 subtype CD4(+) T cells. Furthermore, we show that Cxcl10 upregulation in MECs is promoted by interferon-λ and that Usp18 is a novel inhibitor of interferon-λ signalling. Knockdown of the interferon-λ specific receptor subunit IL-28R1 in Usp18 deficient MECs dramatically enhances tumour growth. Taken together, our data suggest that targeting Usp18 may be a viable approach to boost antitumour immunity while suppressing the protumour activity of the immune system.

hNOA1 interacts with complex I and DAP3 and regulates mitochondrial respiration and apoptosis.

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O(2) consumption approximately 20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as gamma-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.

Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics.

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1/SNX13 attenuates Galphas-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1/SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1/SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.