Hepatitis B virus X protein (HBx) plays critical roles in the development of hepatocellular carcinogenesis (HCC). Yes-associated protein (YAP), a downstream effector of the Hipposignaling pathway, is an important human oncogene. In the present article, we report that YAP is involved in the hepatocarcinogenesis mediated by HBx. We demonstrated that the expression of YAP was dramatically elevated in clinical HCC samples, hepatitis B virus (HBV)-infected hepatoma HepG2.2.15 cell line, and liver cancer tissues of HBx-transgenic mice. Meanwhile, we found that overexpression of HBx resulted in the up-regulation of YAP in stably HBx-transfected HepG2/H7402 hepatoma cell lines, whereas HBx RNA interference reduced YAP expression in a dose-dependent manner in the above-mentioned cell lines, suggesting that HBx up-regulates YAP. Then, we investigated the mechanism underlying the up-regulation of YAP by HBx. Luciferase reporter gene assays revealed that the promoter region of YAP regulated by HBx was located at nt 2232/1115 containing cyclic adenosine monophosphate response element-binding protein (CREB) element. Chromatin immunoprecipitation (ChIP) demonstrated that HBx was able to bind to the promoter of YAP, whereas it failed to work when CREB was silenced. Moreover, we confirmed that HBx activated the YAP promoter through CREB by electrophoretic mobility shift assay and luciferase reporter gene assays. Surprisingly, we found that YAP short interfering RNA was able to remarkably block the HBx-enhanced growth of hepatoma cells in vivo and in vitro. Conclusion: YAP is a key driver gene in HBx-induced hepatocarcinogenesis in a CREB-dependent manner. YAP may serve as a novel target in HBV-associated HCC therapy. (HEPATOLOGY 2012;56:2051-2059 H epatocellular carcinoma (HCC) is the fifthmost common cancer and the third leading cause of cancer death worldwide. 1 Hepatitis B virus (HBV) infection is one of the major causes of HCC. 2 Among the four proteins encoded by HBV, the HBV X protein (HBx) is a multifunctional regulatory protein and plays a crucial role in hepatocellular carcinogenesis. 3 Although it does not bind directly to DNA, HBx modulates transcriptional activation by interacting with nuclear transcription factors, such as activating protein 1 (AP-1), nuclear factor kappa lightchain enhancer of activated B cells (NF-jB), specificity protein 1 (Sp-1), and cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), and affects the cytoplasmic modulation of signal transduction pathways. 4,5

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

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The infection of hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma (HCC), in which HBV X protein (HBx) plays crucial roles. MicroRNAs are involved in diverse biologic functions and in carcinogenesis by regulating gene expression. In the present study, we aim to investigate the underlying mechanism by which HBx enhances hepatocarcinogenesis. We found that miR-205 was downregulated in 33 clinical HCC tissues in comparison with adjacent noncancerous hepatic tissues. The expression levels of miR-205 were inversely correlated with those of HBx in abovementioned tissues. Then, we demonstrated that HBx was able to suppress miR-205 expression in hepatoma and liver cells. We validated that miR-205 directly targeted HBx mRNA. Ectopic expression of miR-205 downregulated HBx, whereas depletion of endogenous miR-205 upregulated HBx in hepatoma cells. Notably, our data revealed that HBx downregulated miR-205 through inducing hypermethylation of miR-205 promoter in the cells. In terms of function, the forced miR-205 expression remarkably inhibited the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo, suggesting that miR-205 is a potential tumor-suppressive gene in HCC. HBx-transgenic mice showed that miR-205 was downregulated in the liver. Importantly, HBx was able to abrogate the effect of miR-205 on tumor suppression in carcinogenesis. Therefore, we conclude that HBx is able to inhibit tumor suppressor miR-205 to enhance hepatocarcinogenesis through inducing hypermethylation of miR-205 promoter during their interaction. Therapeutically, miR-205 may be useful in the treatment of HCC.

NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and a MYC transgene in germinal center B cells (VQ mice). VQ mice developed a highly malignant MM characterized by high proliferation index, hyperactivation of ERK and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved PD1 and TIGIT immune checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced stage MM. Our study provides a strong rationale to develop MEK inhibition-based therapies for treating advanced/relapsed MM.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ~40% in chronic myelomonocytic leukemia (CMML). ASXL1 mutations associate with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients with shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+; Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global level of H3K27ac, and Flt3 upregulation. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands, PD-L1/L2, CD155, and CD80/86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or shRNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wildtype T cells overexpressed PD-1 and TIGIT receptors, leading to a predominant exhausted T cell phenotype. Combined inhibition of MEK and BET led to downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8+ T cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Abstract We and others have previously shown that KrasG12D is a much more potent oncogene than oncogenic Nras in hematological malignancies. We attributed the strong leukemogenic activity of KrasG12D at least partially to its unique capability to hyperactivate wild-type (WT) Nras and Hras. Here, we report that Sos1, a guanine nucleotide exchange factor, is required to mediate this process. Sos1 is overexpressed in KrasG12D/+ cells, but not in NrasQ61R/+ and NrasG12D/+ cells. KrasG12D proteins form a complex with Sos1 in vivo. Sos1 deficiency attenuates hyperactivation of WT Nras, Hras, and the downstream ERK signaling in KrasG12D/+ cells. Thus, Sos1 deletion ameliorates oncogenic Kras-induced myeloproliferative neoplasm (MPN) phenotypes and prolongs the survival of KrasG12D/+ mice. In contrast, Sos1 is dispensable for hyperactivated granulocyte-macrophage colony-stimulating factor signaling in NrasQ61R/+ cells, and Sos1−/− does not affect MPN phenotypes in NrasQ61R/+ mice. Moreover, the survival of KrasG12D/+; Sos1−/− recipients is comparable to that of KrasG12D/+ recipients treated with combined MEK and JAK inhibitors. Our study suggests that targeting Sos1-oncogenic Kras interaction may improve the survival of cancer patients with KRAS mutations.

Idiopathic pulmonary fibrosis (IPF), a lethal respiratory disease with few treatment options, occurs due to repetitive microinjuries to alveolar epithelial cells (AECs) and progresses with an overwhelming deposition of extracellular matrix (ECM), ultimately resulting in fibrotic scars and destroyed the alveolar architecture. Here, an inhaled ribosomal protein‐based mRNA nanoformulation is reported for clearing the intrapulmonary ECM and re‐epithelializing the disrupted alveolar epithelium, thereby reversing established fibrotic foci in IPF. The nanoformulation is sequentially assembled by a ribosomal protein‐condensed mRNA core, a bifunctional peptide‐modified corona and keratinocyte growth factor (KGF) with a PEGylated shielding shell. When inhaled via a nebulizer, the nanoformulations carried by microdrops are deposited in the alveoli, and penetrate into fibrotic foci, where the outer KGFs are detached after matrix metalloproteinase 2 (MMP2) triggering. The RGD motif‐grafted cores then expose and specifically target the integrin‐elevated cells for the intracellular delivery of mRNA. Notably, repeated inhalation of the nanoformulations accelerates the clearance of locoregional collagen by boosting the intralesional expression of MMP13 and alveolar re‐epithelialization mediated by KGFs, which synergistically ameliorates the lung function of a bleomycin‐induced murine model. Therefore, this work provides an alternative mRNA‐inhalation delivery strategy, which shows great potential for the treatment of IPF.