A targeting nanoplatform for chemo-photothermal synergistic therapy of small-cell lung cancer.

The precise delivery of drugs to tumor sites and the thermoresistance of tumors remain major challenges in photothermal therapy (PTT). Somatostatin receptor 2 (SSTR2) is proposed as an ideal target for the precise treatment of SCLC. We developed a targeting nano-drug delivery system comprising anti-SSTR2 monoclonal antibody (MAb) surface-modified nanoparticles co-encapsulating Cypate and gambogic acid (GA). The formed SGCPNs demonstrated excellent monodispersity, physiological stability, preferable biocompatibility, and resultant efficient photothermal conversion efficacy. SGCPNs were quickly internalized by SSTR2-overexpressing SCLC cells, triggering the release of GA under acidic and near-infrared (NIR) laser irradiation environments, leading to their escape from lysosomes to the cytosol and then diffusion into the nucleus. SGCPNs can not only decrease the cell survival rate but also inhibit the activity of heat shock protein 90 (HSP90). SGCPNs can be precisely delivered to xenograft tumors of SSTR2-positive SCLC in vivo. Upon NIR laser irradiation, therapy of SGCPNs showed significant tumor regression. In conclusion, SGCPNs provide a new chemo-photothermal synergistic treatment strategy for targeting SCLC.

CD11b + CD43 hi Ly6C lo splenocyte-derived macrophages exacerbate liver fibrosis via spleen-liver axis.

BACKGROUND AND AIMS: Monocyte-derived macrophages (MoMFs), a dominant population of hepatic macrophages under inflammation, play a crucial role in liver fibrosis progression. The spleen serves as an extra monocyte reservoir in inflammatory conditions; however, the precise mechanisms of involvement of the spleen in the pathogenesis of liver fibrosis remain unclear. APPROACH AND RESULTS: By splenectomy and splenocyte transfusion, it was observed that splenic CD11b + cells accumulated intrahepatically as Ly6C lo MoMFs to exacerbate CCl 4 -induced liver fibrosis. The splenocyte migration into the fibrotic liver was further directly visualized by spleen-specific photoconversion with KikGR mice and confirmed by CD45.1 + /CD45.2 + spleen transplantation. Spleen-derived CD11b + cells purified from fibrotic livers were then annotated by single-cell RNA sequencing, and a subtype of CD11b + CD43 hi Ly6C lo splenic monocytes (sM-1s) was identified, which was markedly expanded in both spleens and livers of mice with liver fibrosis. sM-1s exhibited mature feature with high expressions of F4/80, produced much ROS, and manifested preferential migration into livers. Once recruited, sM-1s underwent sequential transformation to sM-2s (highly expressed Mif , Msr1 , Clec4d , and Cstb ) and then to spleen-derived macrophages (sMφs) with macrophage features of higher expressions of CX 3 CR1, F4/80, MHC class II, and CD64 in the fibrotic hepatic milieu. Furthermore, sM-2s and sMφs were demonstrated capable of activating hepatic stellate cells and thus exacerbating liver fibrosis. CONCLUSIONS: CD11b + CD43 hi Ly6C lo splenic monocytes migrate into the liver and shift to macrophages, which account for the exacerbation of liver fibrosis. These findings reveal precise mechanisms of spleen-liver axis in hepatic pathogenesis and shed light on the potential of sM-1 as candidate target for controlling liver diseases.

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.

A novel immunogenic cell death-related classification indicates the immune landscape and predicts clinical outcome and treatment response in acute myeloid leukemia.

BACKGROUND: Immunogenic cell death (ICD) is closely related to anti-tumor therapy and regulates the tumor microenvironment (TME). This study aims to explore the molecular characteristics of ICD in acute myeloid leukemia (AML) and to analyze the value of ICD-related biomarkers in TME indication, prognosis prediction, and treatment response evaluation in AML. METHODS: Single-sample gene set enrichment analysis was used to calculate the ICD score. LASSO regression was used to construct a prognostic risk score model. We also analyzed differences in clinical characteristics, immune landscape, immunotherapy response, and chemotherapy sensitivity between high-risk and low-risk patients. RESULTS: This study identified two ICD-related subtypes and found significant heterogeneity in clinical prognosis, TME, and immune landscape between different ICD subtypes. Subsequently, a novel ICD-related prognostic risk score model was developed, which accurately predicted the prognosis of AML patients and was validated in nine AML cohorts. Moreover, there were significant correlations between risk scores and clinicopathological factors, somatic mutations, TME characteristics, immune cell infiltration, immunotherapy response, and chemosensitivity. We further validated the model gene expression in a clinically real-world cohort. CONCLUSIONS: The novel ICD-related signatures identified and validated by us can serve as promising biomarkers for predicting clinical outcomes, chemotherapy sensitivity, and immunotherapy response in AML patients, guiding the establishment of personalized and accurate treatment strategies for AML.

Nras Q61R/+ and Kras-/- cooperate to downregulate Rasgrp1 and promote lympho-myeloid leukemia in early T-cell precursors.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of T-cell ALL. Although genetic mutations hyperactivating cytokine receptor/Ras signaling are prevalent in ETP-ALL, it remains unknown how activated Ras signaling contributes to ETP-ALL. Here, we find that in addition to the frequent oncogenic RAS mutations, wild-type (WT) KRAS transcript level was significantly downregulated in human ETP-ALL cells. Similarly, loss of WT Kras in NrasQ61R/+ mice promoted hyperactivation of extracellular signal-regulated kinase (ERK) signaling, thymocyte hyperproliferation, and expansion of the ETP compartment. Kras-/-; NrasQ61R/+ mice developed early onset of T-cell malignancy that recapitulates many biological and molecular features of human ETP-ALL. Mechanistically, RNA-sequencing analysis and quantitative proteomics study identified that Rasgrp1, a Ras guanine nucleotide exchange factor, was greatly downregulated in mouse and human ETP-ALL. Unexpectedly, hyperactivated Nras/ERK signaling suppressed Rasgrp1 expression and reduced Rasgrp1 level led to increased ERK signaling, thereby establishing a positive feedback loop to augment Nras/ERK signaling and promote cell proliferation. Corroborating our cell line data, Rasgrp1 haploinsufficiency induced Rasgrp1 downregulation and increased phosphorylated ERK level and ETP expansion in NrasQ61R/+ mice. Our study identifies Rasgrp1 as a negative regulator of Ras/ERK signaling in oncogenic Nras-driven ETP-like leukemia.

Integrative analysis of the prognostic value and immune microenvironment of mitophagy-related signature for multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a fatal malignant tumor in hematology. Mitophagy plays vital roles in the pathogenesis and drug sensitivity of MM. METHODS: We acquired transcriptomic expression data and clinical index of MM patients from NCI public database, and 36 genes involved in mitophagy from the gene set enrichment analysis (GSEA) database. Least absolute shrinkage and selection operator (LASSO) Cox regression analysis was conducted to construct a risk score prognostic model. Kaplan-Meier survival analysis and receiver operation characteristic curves (ROC) were conducted to identify the efficiency of prognosis and diagnosis. ESTIMATE algorithm and immune-related single-sample gene set enrichment analysis (ssGSEA) was performed to uncover the level of immune infiltration. QRT-PCR was performed to verify gene expression in clinical samples of MM patients. The sensitivity to chemotherapy drugs was evaluated upon the database of the genomics of drug sensitivity in cancer (GDSC). RESULTS: Fifty mitophagy-related genes were differently expressed in two independent cohorts. Ten out of these genes were identified to be related to MM overall survival (OS) rate. A prognostic risk signature model was built upon on these genes: VDAC1, PINK1, VPS13C, ATG13, and HUWE1, which predicted the survival of MM accurately and stably both in training and validation cohorts. MM patients suffered more adverse prognosis showed more higher risk core. In addition, the risk score was considered as an independent prognostic element for OS of MM patients by multivariate cox regression analysis. Functional pathway enrichment analysis of differentially expressed genes (DEGs) based on risk score showed terms of cell cycle, immune response, mTOR pathway, and MYC targets were obviously enriched. Furthermore, MM patients with higher risk score were observed lower immune scores and lower immune infiltration levels. The results of qRT-PCR verified VDAC1, PINK1, and HUWE1 were dysregulated in new diagnosed MM patients. Finally, further analysis indicated MM patients showed more susceptive to bortezomib, lenalidomide and rapamycin in high-risk group. CONCLUSION: Our research provided a neoteric prognostic model of MM based on mitophagy genes. The immune infiltration level based on risk score paved a better understanding of the participation of mitophagy in MM.

MiR-3663-3p participates in the anti-hepatocellular carcinoma proliferation activity of baicalein by targeting SH3GL1 and negatively regulating EGFR/ERK/NF-κB signaling.

Baicalein is a purified flavonoid that exhibits anticancer effects in hepatocellular carcinoma (HCC). However, its underlying molecular mechanisms remain largely unclear. In this study, we found that baicalein inhibited HCC cell growth, induced apoptosis, and blocked cell cycle arrest at the S phase in vitro, as well as reduced HCC tumor volume and weight in vivo. Quantitative reverse transcriptase-PCR (qRT-PCR) results suggested that miR-3663-3p was downregulated in HCC tissues. After baicalein treatment, miR-3663-3p expression was upregulated in HCC cells. Transfection of miR-3663-3p suppressed HCC cell proliferation and colony formation, increased the proportion of apoptotic cells in vitro, and reduced the volume and weight of tumors in vivo. The results of dual-luciferase reporter assay showed that miR-3663-3p could directly bind to the 3'-UTR of SH3GL1. SH3GL1 overexpression partly reduced the growth-inhibiting effect of miR-3663-3p. Both baicalein treatment and miR-3663-3p overexpression downregulated the expression of SH3GL1 and inactivated the Erk1/2, p-NF-κB/p65, and EGFR signaling pathways. Overall, our data suggest that baicalein may act as a novel HCC suppressor, and that the miR-3663-3p/SH3GL1/EGFR/ERK/NF-κB pathway plays a vital role in HCC progression. Thus, baicalein treatment or miR-3663-3p induction may be a promising strategy for HCC therapy.

Silencing of long noncoding RNA HOXD-AS1 inhibits proliferation, cell cycle progression, migration and invasion of hepatocellular carcinoma cells through MEK/ERK pathway.

Accumulating findings reveal that long noncoding RNAs (lncRNAs) as crucial regulatory molecules serve vital functions in the progression of hepatocellular carcinoma (HCC). This study aims to investigate the biological roles and mechanisms of lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in HCC cells based on transcriptome analysis. The Cancer Genome Atlas data analysis and experimental validation showed that HOXD-AS1 was increased in HCC tissues/cell lines and positively relevant to histologic grade. The subcellular localization results indicated HOXD-AS1 was dispersed both in the nucleus as well as the cytoplasm of HCC cells. In vitro loss-of-function experiments revealed that silencing of HOXD-AS1 could dramatically suppress the proliferation, migration, and invasion, and induce S or/and G2/M phase cell cycle arrest as well as apoptosis of Bel-7402 and MHCC97H cells accompanying the changes in expression levels of cyclin B1, cyclin D1, BCL-2, BAX, and MMP2. In vivo assay also showed that HOXD-AS1 silencing could markedly reduce xenograft tumor volume and weight of HCC cells. Transcriptome and bioinformatic analysis indicated that a total of 1103 genes were significantly altered by HOXD-AS1 silencing, of which 132 genes exhibited a significant correlation with HOXD-AS1 expression in HCC tissues. Gene Ontology (GO) enrichment analysis revealed differentially expressed genes were remarkably enriched in several cancer-related biological processes (cell proliferation, cell cycle, apoptosis, migration, angiogenesis, and hypoxic response). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that HOXD-AS1 has the potential to affect p53, tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK) pathway, and Western blot results further validated that HOXD-AS1 silencing could inhibit the MEK/ERK pathway in Bel-7402 cells. Collectively, HOXD-AS1, as an oncogenic lncRNA, might exert crucial functions in HCC progression and serve as a potential diagnostic biomarker and therapeutic target for HCC.

Ring1 promotes the transformation of hepatic progenitor cells into cancer stem cells through the Wnt/ß-catenin signaling pathway.

The proliferation of hepatic progenitor cells (HPCs) is observed in reactive conditions of the liver and primary liver cancers. Ring1 as a member of polycomb-group proteins which play vital roles in carcinogenesis and stem cell self-renewal was increased in HCC patients and promoted proliferation and survival of cancer cell by degrading p53. However, the mechanisms of Ring1 driving the progression of hepatocarcinogenesis have not been elucidated. In this study, forced expression Ring1 and Ring1 siRNA lentiviral vectors were utilized to stably overexpression and silence Ring1 in HPC cell line (WB-F344), respectively. Our finding indicated that overexpression of Ring1 in HPCs promoted colony formation, cell multiplication, and invasion in vitro, conversely depletion of Ring1 repressed the biological functions of HPCs relative to controls. The expression of ß-catenin was upregulated in the HPCs with overexpression of Ring1, and the correlation analysis also showed that ß-catenin and Ring1 had a significant correlation in the liver cancer tissues and adjacent tissues. The activation of the Wnt/ß-catenin signaling pathway significantly increased the expression of liver cancer stem cells related (LCSCs)-related molecular markers CD90 and EpCAM, which led to the transformation of HPCs into LCSCs. Most importantly, the injection of HPCs with overexpressed Ring1 into the subcutaneous of nude mice leads to the formation of poorly differentiated HCC neoplasm. Our findings elucidate that overexpression of Ring1 the activated Wnt/ß-catenin signaling pathway and drove the transformation of HPCs into cancer stem cell-like cells, suggesting Ring1 has extraordinary potential in early diagnosis of HCC.

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.

Improvement of human platelet aggregation post-splenectomy with paraesophagogastric devascularization in chronic hepatitis B patients with cirrhotic hypersplenism.

Thrombocytopenia is a common hematological abnormality in patients with cirrhotic hypersplenism. Splenectomy with paraesophagogastric devascularization (SPD) is a conventional surgical therapy which can reverse pancytopenia in these patients. Platelets are traditionally recognized for their central role in hemostasis. However, the status of platelet aggregation in chronic hepatitis B patients with cirrhotic hypersplenism before and after SPD has not been reported yet. A total of 41 cirrhotic patients and 31 healthy controls were included in this study. Platelet aggregation was detected by AggRAM® Advanced Modular System (Helena Laboratories, USA). ELISA was used to detect the cytokines closely related to platelet aggregation. Expressions of platelet membrane glycoproteins (GPs) were evaluated by flow cytometric analysis. Platelet aggregation was found to be decreased distinctly in the cirrhotic patients, and to be restored to normal level after SPD. The cirrhotic patients showed higher plasma levels of the cytokines HMGB1, PEDF, vWF, cAMP and cGMP, which also improved partially after SPD. Moreover, the cirrhotic patients had much lower expression of GPIIb/IIIa, GPIbα and P-selectin than either the healthy controls or SPD patients at basal or activated level. Generally, SPD benefits cirrhotic patients with bleeding tendencies by improving platelet counts and aggregation. GPIIb/IIIa may be the key membrane protein responsible for the change in platelet aggregation before and after SPD.

p53-/- synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia.

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells through activating ß-adrenergic signaling.

Psychological stress promotes tumor progression and has a large impact on the immune system, particularly the spleen. The spleen plays an important role in tumor behavior. However, the role and mechanism of the spleen in hepatocellular carcinoma progression induced by stress is unclear. Here, we showed that the spleen plays a critical role in hepatocellular carcinoma growth induced by restraint stress. Our results demonstrated that restraint stress promoted hepatocellular carcinoma growth, changed the spleen structure, and redistributed splenic myeloid cells to tumor tissues. Interestingly, we found that splenectomy could inhibit hepatocellular carcinoma growth and prevent increases in myeloid cells and macrophages in tumor tissues in stressed mice. Restraint stress significantly elevated the concentration of norepinephrine in the spleen, serum and tumor tissues. Meanwhile, propranolol, an inhibitor of ß-adrenergic signaling, could inhibit hepatocellular carcinoma growth and prevent the redistribution of splenic myeloid cells induced by restraint stress, suggesting that restraint stress promotes hepatocellular carcinoma growth and redistributes splenic myeloid cells through ß-adrenergic signaling. Mechanistic studies revealed that restraint stress upregulated the expressions of CXCL2/CXCL3 in tumor tissues and changed the expression of CXCR2 in myeloid cells. SB225002, an inhibitor of CXCR2, could prevent the recruitment of myeloid cells in tumor tissues and inhibit tumor growth in stressed mice. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells to tumor tissues via activating ß-adrenergic signaling. The CXCR2-CXCL2/CXCL3 axis contributed to the recruitment of myeloid cells in tumor tissues induced by restraint stress.

The Spleen Promotes the Secretion of CCL2 and Supports an M1 Dominant Phenotype in Hepatic Macrophages During Liver Fibrosis.

BACKGROUND/AIMS: Liver fibrosis is a complex process of tissue remodeling in response to injury. Hepatic macrophages have been identified as a key player in this process. As the largest lymphoid organ in the body, the spleen exerts both local and systemic effects on immune cell response. Splenectomy can improve hepatic function during the treatment of liver cirrhosis. However, whether the spleen influences disease progression through the modulation of hepatic macrophages remains unclear. METHODS: We examined ex vivo hepatic macrophage responses from splenectomized or sham operated rats and performed splenocyte adoptive transfer studies, in combination with in vivo CCL2 blockade, in splenectomized or sham operated rats. RESULTS: We found that splenectomy reduced fibrosis severity and monocyte/ macrophage infiltration within the injured liver. Splenectomy also reduced secretion of the monocyte chemokine CCL2 by hepatic macrophages. Ex vivo, splenocytes, especially splenic macrophages, promoted CCL2 secretion via upregulation of SOCS3 signaling in hepatic macrophages. Migration of splenic monocytes in response to conditioned medium from hepatic macrophages was inhibited by the blockade of SOCS3-CCL2-CCR2 signaling. Splenectomy also attenuated the establishment of an M1-dominant hepatic macrophage phenotype whilst the adoptive transfer of splenocytes could partly reverse this effect and exacerbate fibrosis. However, CCL2 blockade following adoptive splenocyte transfer restored the protective effects of splenectomy. CONCLUSION: Our study demonstrates that splenic macrophages can promote hepatic macrophage secretion of CCL2, which in turn facilitates monocyte recruitment and the establishment of an M1-dominant hepatic macrophage phenotype, and thus increase the severity of liver fibrosis.

Repetitive restraint stress changes spleen immune cell subsets through glucocorticoid receptor or ß-adrenergic receptor in a stage dependent manner.

Immune system is sensitive to stress. Spleen is the largest peripheral immune organ innervated with sympathetic nerves and controlled by adrenomedullary system in the body. However, the alterations and mechanism of spleen immune cell subsets caused by repetitive restraint stress (RRS) is poorly understood. In this study, we found that RRS reduced spleen index in mice, and induced an expansion of white pulp and involution of the red pulp. Meanwhile, the percentage of CD3+CD8+ T lymphocytes, CD11b+F4/80+ macrophages, CD11b+Ly-6G-Ly-6Chi monocytic myeloid derived suppressor cells (mMDSCs) and CD11b+Ly-6G+Ly-6Cint granulocytic myeloid derived suppressor cells (gMDSCs) in spleen were significantly changed by RRS. Mechanistically, we found that the expression of norepinephrine (NE) and ß-adrenergic receptor (ß-AR) in spleen were up-regulated after 21 days of RRS, but not 7 days. The expression of corticosterone (CORT) and glucocorticoid receptor (GR) in spleen were up-regulated after 7 days of RRS but were lower after 21 days of RRS, even though they were still higher than that in mice without stress. By treating the stressed mice with RU486 (antagonist of GR) or propranolol (antagonist of ß-AR), we demonstrated that GR was responsible for the changes of spleen induced by 7 days of RRS and ß-AR was for 21 days of RRS. Our data suggest that RRS changes spleen immune cell subsets through GR or ß-AR in a stage dependent manner.

Spleen Regulates Hematopoietic Stem/Progenitor Cell Functions Through Regulation of EGF in Cirrhotic Hypersplenism.

BACKGROUND: Hematopoietic abnormality is a common cause of cirrhotic hypersplenism (CH) complications and death; it causes serious adverse effects and is associated with bleeding, anemia, infection in CH patients. However, the underlying mechanism is unclear. AIMS: We aimed to investigate the effects of the spleen on hematopoiesis and hematopoietic stem/progenitor cells (HSPCs) in CH patients. METHODS: Eleven CH patients were enrolled to assess the effects of the spleen on HSPC functions. Hematopoietic changes were examined by flow cytometry analysis. HSPC functions were detected with colony-forming assays and in vitro cell cultures. Enzyme-linked immunosorbent assay (ELISA) was used to test the concentration of epithelial growth factor (EGF). RESULTS: The number of HSPCs was decreased in CH patients and was rescued after splenectomy. Serum from CH patients dysregulated HSPCs function, and serum from splenectomy patients restored the dysregulated HSPC function in vitro. The concentration of EGF was decreased in CH patients and was restored to normal level after splenectomy. EGF rescued the dysregulated HSPCs function in vitro. CONCLUSIONS: The spleen can regulate the functions of HSPCs in CH patients by regulating EGF signaling. EGF may be a therapeutic target for CH treatment.

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.

Deficiency of ß Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice.

Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous Nras(G12D/+) mutation. In this model, Nras(G12D/+) hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common ß chain of the GM-CSF receptor (ßc) is dispensable for Nras(G12D/+) HSC function; loss of ßc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in Nras(G12D/+) HSCs. Therefore, ßc(-/-) does not abrogate CMML in Nras(G12D/+) mice. However, ßc deficiency indeed significantly reduces Nras(G12D/+)-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease.

Nras(G12D/+) promotes leukemogenesis by aberrantly regulating hematopoietic stem cell functions.

Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras(G12D/+)) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras(G12D/+) in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras(G12D/+) signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras(G12D/+) serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras(G12D/+) HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras(G12D/+) HSCs and prevents the expansion of Nras(G12D/+) HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis.

Notch1 gene mutations target KRAS G12D-expressing CD8+ cells and contribute to their leukemogenic transformation.

Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8(+) T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/ß-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells.