Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated 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 who had 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 levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. 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 short hairpin RNAs 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 wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in 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.

Expression of NrasQ61R and MYC transgene in germinal center B cells induces a highly malignant multiple myeloma in mice.

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 an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain 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.

Unique dependence on Sos1 in Kras G12D -induced leukemogenesis.

We and others have previously shown that Kras G12D 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 Kras G12D/+ cells, but not in Nras Q61R/+ and Nras G12D/+ cells. KrasG12D proteins form a complex with Sos1 in vivo. Sos1 deficiency attenuates hyperactivation of WT Nras, Hras, and the downstream ERK signaling in Kras G12D/+ cells. Thus, Sos1 deletion ameliorates oncogenic Kras-induced myeloproliferative neoplasm (MPN) phenotypes and prolongs the survival of Kras G12D/+ mice. In contrast, Sos1 is dispensable for hyperactivated granulocyte-macrophage colony-stimulating factor signaling in Nras Q61R/+ cells, and Sos1 -/- does not affect MPN phenotypes in Nras Q61R/+ mice. Moreover, the survival of Kras G12D/+ ; Sos1 -/- recipients is comparable to that of Kras G12D/+ 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.

Kras Is Critical for B Cell Lymphopoiesis.

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell-specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre-B cell stage and late B cell maturation, resulting in the reduction of BM pre-, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell-specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre-BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre-B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Kras is Required for Adult Hematopoiesis.

Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony-stimulating factor (GM-CSF)-evoked ERK1/2 activation is only moderately decreased in Kras(-/-) myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9-12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras(-/-) BM cells is greatly compromised, which is attributable to defects in the self-renewal of Kras(-/-) HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859-1871.

A Prospective Randomized Study of Anterior Cruciate Ligament Reconstruction With Autograft, γ-Irradiated Allograft, and Hybrid Graft.

PURPOSE: To compare the results of patients who underwent anterior cruciate ligament (ACL) reconstruction with autograft, γ-irradiated allograft, or hybrid graft in a prospective randomized study with a minimum clinical follow-up period of 5 years. METHODS: In this prospective, randomized, comparative study, 102 patients with ACL tears underwent ACL reconstruction with autograft (gracilis and semitendinosus tendons), γ-irradiated allograft (tibialis anterior tendons), or hybrid graft (γ-irradiated tibialis anterior tendon allograft and semitendinosus tendon autograft). Laboratory testing of the erythrocyte sedimentation rate and C-reaction protein level were performed; clinical results were evaluated with the KT-1000 arthrometer (MEDmetric, San Diego, CA), Lachman test, Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (both objective and subjective). RESULTS: There were 32 patients in the autograft group, 31 in the hybrid graft group, and 32 in the γ-irradiated allograft group at last follow-up. No differences were found among the 3 groups regarding patient demographic data and the duration from injury to operation (P > .05). The C-reaction protein and erythrocyte sedimentation rate values were statistically higher in the γ-irradiated allograft group than in the other 2 groups on the third, seventh, and fourteenth days (P < .05). No significant differences were found between the autograft and hybrid graft groups (P > .05). The KT-1000 examination showed more anterior laxity in the γ-irradiated allograft group than in the other 2 groups (P < .05). No significant differences in the Lachman test and pivot-shift test findings were found among the 3 groups (P > .05). The Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (subjective and objective) showed no differences among the 3 groups (P > .05). CONCLUSIONS: Patients undergoing primary ACL reconstruction with hybrid graft or autograft had satisfactory and similar objective and subjective clinical results. On KT-1000 measurement of anteroposterior knee laxity, both the autograft and hybrid graft groups showed statistically significant differences compared with the γ-irradiated allograft group. LEVEL OF EVIDENCE: Level II, prospective comparative study.

The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF.

Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT.

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3'UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

Hepatitis B virus X protein upregulates oncogene Rab18 to result in the dysregulation of lipogenesis and proliferation of hepatoma cells.

Hepatitis B virus X protein (HBx) contributes to the development of hepatocellular carcinoma (HCC) through inducing dysregulation of lipogenesis. However, the mechanism by which HBx induces the abnormal lipogenesis is not well known. In this study, we report that the oncogene Rab18, a member of Ras family, enhances the HBx-induced hepatocarcinogenesis through inducing dysregulation of lipogenesis and proliferation. Our data showed that the expression levels of Rab18 were positively associated with those of HBx in clinical HCC tissues. HBx was able to upregulate the expression of Rab18 in p21-HBx transgenic mice and hepatoma cell lines. Next, we identified the mechanism by which HBx upregulated Rab18. The results demonstrated that cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) were able to stimulate Rab18 promoter through activating transcription factor activator protein 1 (AP-1) and cyclic adenosine 3',5'-monophosphate response element-binding (CREB). In addition, we identified another pathway that HBx activated Rab18. We found that miR-429 was able to directly target the 3' untranslated region of Rab18, suggesting that Rab18 is one of the target genes of miR-429. Then, we found that HBx was able to downregulate miR-429 in hepatoma cells. The oil red O staining showed that HBx resulted in the dysregulation of lipogenesis through Rab18. Moreover, Rab18 contributed to the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. HBx enhances hepatocarcinogenesis through leading to the dysregulation of lipogenesis and proliferation of hepatoma cells, involving two pathways such as HBx/COX-2/5-LOX/AP-1/CREB/Rab18 and HBx/miR-429/Rab18.

Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18.

Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2'-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.

The oncoprotein HBXIP uses two pathways to up-regulate S100A4 in promotion of growth and migration of breast cancer cells.

We have reported that hepatitis B X-interacting protein (HBXIP) promotes the proliferation and migration of breast cancer cells. However, the underlying mechanism is poorly understood. In this study, we report that HBXIP works in the event through up-regulating S100A4. We observed that HBXIP expression was positively correlated to that of S100A4 in 87 clinical breast cancer tissue samples. Then, we identified that HBXIP was able to up-regulate S100A4 expression in breast cancer cells. Notably, we observed the HBXIP nuclear localization, implying that HBXIP may be associated with the promoter of S100A4. Chromatin immunoprecipitation assay (ChIP) and electrophoretic mobility shift assay (EMSA) showed that HBXIP was able to bind to the nucleotides +200~+239 region of S100A4 promoter, containing two putative recognition motif of transcription factor STAT4 and GRß. It suggests that HBXIP is able to activate S100A4 promoter via interacting with STAT4 in breast cancer cells, leading to the up-regulation of S100A4. In addition, we identified another pathway of S100A4 up-regulation mediated by HBXIP. We found that HBXIP activated the PTEN/PI3K/AKT signaling by inducing DNA methylation of PTEN, which subsequently boosted S100A4 expression. In function, we demonstrated that HBXIP enhanced the growth or migration of breast cancer cells through S100A4 in vivo and in vitro. Collectively, we conclude that HBXIP up-regulates S100A4 through activating S100A4 promoter involving STAT4 and inducing PTEN/PI3K/AKT signaling to promote growth and migration of breast cancer cells. Our finding provides new insight into the mechanism of HBXIP in promotion of the development of breast cancer.

Hepatitis B virus X protein modulates oncogene Yes-associated protein by CREB to promote growth of hepatoma cells.

UNLABELLED: 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 Hippo-signaling 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 -232/+115 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.

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1ß (IL-1ß) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1ß monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1ß/IL-1rn levels under steady-state, and that loss of repression of IL-1ß signaling may underlie pre-leukemic lesion and AML progression.

Role of ASXL1 in hematopoiesis and myeloid diseases.

Next-generation sequencing technology, including whole-exome or whole-genome sequencing and target gene sequencing, has allowed the molecular characterization of somatic mutation spectrums in hematologic diseases. Mutations in Additional sex combs-like 1 (ASXL1), a chromatin regulator, are identified in clonal hematopoiesis of indeterminate potential (CHIP), indicating ASXL1 mutations as early events in leukemogenesis. Not surprisingly, they occur at high frequency in myeloid malignancies and are associated with poor prognosis. Therefore, understanding how mutant ASXL1 drives clonal expansion and leukemogenesis will serve as the basis for the future development of preventative and/or therapeutic strategies for myeloid diseases with ASXL1 mutations. Here, we discuss the biology of ASXL1 and its role in controlling normal and malignant hematopoiesis. In addition, we review the clinical relevance of ASXL1 mutations in CHIP and myeloid diseases.

Gata2 +9.5 enhancer regulates adult hematopoietic stem cell self-renewal and T-cell development.

Mammalian GATA2 gene encodes a dual zinc finger transcription factor, which is essential for hematopoietic stem cell (HSC) generation in the aorta, gonad, mesonephros (AGM) region, HSC self-renewal, and specification of progenitor cell fates. Previously, we demonstrated that Gata2 expression in AGM is controlled by its intronic +9.5 enhancer. Gata2 +9.5 deficiency removes the E-box motif and the GATA site and depletes fetal liver HSCs. However, whether this enhancer has an essential role in regulating adult hematopoiesis has not been established. Here, we evaluate Gata2 +9.5 enhancer function in adult hematopoiesis. +9.5+/- bone marrow cells displayed reduced T cell reconstitution in a competitive transplant assay. Donor-derived analysis demonstrated a previously unrecognized function of the +9.5 enhancer in T cell development at the lymphoid-primed multipotent progenitor stage. Moreover, +9.5+/- adult HSCs displayed increased apoptosis and reduced long-term self-renewal capability in comparison with wild-type (WT) HSCs. These phenotypes were more moderate than those of Gata2+/- HSCs. Consistent with the phenotypic characterization, Gata2 expression in +9.5+/- LSKs was moderately higher than that in Gata2+/- LSKs, but lower than that in WT LSKs. Our data suggest that +9.5 deficiency compromises, without completely abrogating, Gata2 expression in adult HSCs.

Ponatinib sensitizes myeloma cells to MEK inhibition in the high-risk VQ model.

Multiple myeloma (MM) is a malignant plasma cell cancer. Mutations in RAS pathway genes are prevalent in advanced and proteasome inhibitor (PI) refractory MM. As such, we recently developed a VQ MM mouse model recapitulating human advanced/high-risk MM. Using VQ MM cell lines we conducted a repurposing screen of 147 FDA-approved anti-cancer drugs with or without trametinib (Tra), a MEK inhibitor. Consistent with its high-risk molecular feature, VQ MM displayed reduced responses to PIs and de novo resistance to the BCL2 inhibitor, venetoclax. Ponatinib (Pon) is the only tyrosine kinase inhibitor that showed moderate MM killing activity as a single agent and strong synergism with Tra in vitro. Combined Tra and Pon treatment significantly prolonged the survival of VQ MM mice regardless of treatment schemes. However, this survival benefit was moderate compared to that of Tra alone. Further testing of Tra and Pon on cytotoxic CD8+ T cells showed that Pon, but not Tra, blocked T cell function in vitro, suggesting that the negative impact of Pon on T cells may partially counteract its MM-killing synergism with Tra in vivo. Our study provides strong rational to comprehensively evaluate agents on both MM cells and anti-MM immune cells during therapy development.

Inhaled mRNA Nanoformulation with Biogenic Ribosomal Protein Reverses Established Pulmonary Fibrosis in a Bleomycin-Induced Murine Model.

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.

Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack.

Emerging evidence has shown that hepatitis B virus (HBV) X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma. Complement regulatory proteins including CD46, CD55 and CD59 contribute to escape of tumor cells from complement-dependent cytotoxicity (CDC). However, little is known about the potential role of HBx in anti-CDC activity during hepatocarcinogenesis. In the present study, we for the first time report that HBx decreases the sensitivity of hepatoma and hepatic cells to CDC. Coincidentally, we demonstrated that HBx increased the promoter activity of CD59, as well as their messenger RNA and protein levels. Moreover, flow cytometry showed the increased expression level of CD59 protein on the surface of HBx-positive cells. Of interest, we found that HBx up-regulated CD59 by binding with cAMP response element-binding to the promoter region of the CD59 gene using chromatin immunoprecipitation assay. In addition, we showed that HBx up-regulated CD59 by let-7i at post-transcriptional regulation level. Our data showed that the deposition of C5b-9 were decreased on the cell surface in HepG2-X cells relative to HepG2 cells, suggesting that increased CD59 mediated by HBx prevents the formation of functional membrane attack complex. Furthermore, we demonstrated that down-regulation of CD59 was sufficient to abolish the resistance capability of CDC in HBx-positive cells by RNA interference (siRNA) in vitro and in vivo. Thus, we conclude that HBx contributes to cells resistance to CDC through CD59. Therapeutically, CD59 may serve as a target in HBV-associated hepatoma patients.

Embryonic Expression of NrasG 12 D Leads to Embryonic Lethality and Cardiac Defects.

Ras proteins control a complex intracellular signaling network. Gain-of-function mutations in RAS genes lead to RASopathy disorders in humans, including Noonan syndrome (NS). NS is the second most common syndromic cause of congenital heart disease. Although conditional expression of the NrasG12D/ + mutation in adult hematopoietic system is leukemogenic, its effects on embryonic development remain unclear. Here, we report that pan-embryonic expression of endogenous NrasG12D/ + by Mox2-Cre in mice caused embryonic lethality from embryonic day (E) 15.5 and developmental defects predominantly in the heart. At E13.5, NrasG12D/ + ; Mox2Cre/ + embryos displayed a moderate expansion of hematopoietic stem and progenitor cells without a significant impact on erythroid differentiation in the fetal liver. Importantly, the mutant embryos exhibited cardiac malformations resembling human congenital cardiac defects seen in NS patients, including ventricular septal defects, double outlet right ventricle, the hypertrabeculation/thin myocardium, and pulmonary valve stenosis. The mutant heart showed dysregulation of ERK, BMP, and Wnt pathways, crucial signaling pathways for cardiac development. Endothelial/endocardial-specific expression of NrasG12D/ + caused the cardiac morphological defects and embryonic lethality as observed in NrasG12D/ + ; Mox2Cre/ + mutants, but myocardial-specific expression of NrasG12D/ + did not. Thus, oncogenic NrasG12D mutation may not be compatible with embryonic survival.