Clinical laboratory characteristics and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR.

OBJECTIVE: To evaluate the incidence, clinical laboratory characteristics, and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR. METHODS: We collected a total of 359 Ph-negative MPN patients with classical mutations in driver genes JAK2, MPL, or CALR, and divided them into two groups based on whether they had additional atypical variants of driver genes JAK2, MPL, or CALR: 304 patients without atypical variants of driver genes and 55 patients with atypical variants of driver genes. We analyzed the relevant characteristics of these patients. RESULTS: This study included 359 patients with Ph-negative MPNs with JAK2, MPL, or CALR classical mutations and found that 55 (15%) patients had atypical variants of JAK2, MPL, or CALR. Among them, 28 cases (51%) were male, and 27 (49%) were female, with a median age of 64 years (range, 21-83). The age of ET patients with atypical variants was higher than that of ET patients without atypical variants [70 (28-80) vs. 61 (19-82), p = 0.03]. The incidence of classical MPL mutations in ET patients with atypical variants was higher than in ET patients without atypical variants [13.3% (2/15) vs. 0% (0/95), p = 0.02]. The number of gene mutations in patients with atypical variants of driver genes PV, ET, and Overt-PMF is more than in patients without atypical variants of PV, ET, and Overt-PMF [PV: 3 (2-6) vs. 2 (1-7), p < 0.001; ET: 4 (2-8) vs. 2 (1-7), p < 0.05; Overt-PMF: 5 (2-9) vs. 3 (1-8), p < 0.001]. The incidence of SH2B3 and ASXL1 mutations were higher in MPN patients with atypical variants than in those without atypical variants (SH2B3: 16% vs. 6%, p < 0.01; ASXL1: 24% vs. 13%, p < 0.05). CONCLUSION: These data indicate that classical mutations of JAK2, MPL, and CALR may not be completely mutually exclusive with atypical variants of JAK2, MPL, and CALR. In this study, 30 different atypical variants of JAK2, MPL, and CALR were identified, JAK2 G127D being the most common (42%, 23/55). Interestingly, JAK2 G127D only co-occurred with JAK2V617F mutation. The incidence of atypical variants of JAK2 in Ph-negative MPNs was much higher than that of the atypical variants of MPL and CALR. The significance of these atypical variants will be further studied in the future.

Mitochondrial tRNA pseudouridylation governs erythropoiesis.

Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. In this study, we investigated the role of tRNA pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By utilizing patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic PUS1 mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA iPSCs and anemia in the MLASA mouse model. Both MLASA iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels due to pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mTOR inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment effectively ameliorated anemia phenotypes in the MLASA patient. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for anemia patients facing challenges related to protein translation.

Loss of RNA-binding protein CELF2 promotes acute leukemia development via FAT10-mTORC1.

RNA-binding proteins (RBPs) are critical regulators for RNA transcription and translation. As a key member of RBPs, ELAV-like family protein 2 (CELF2) has been shown to regulate RNA splicing and embryonic hematopoietic development and was frequently seen dysregulated in acute myeloid leukemia (AML). However, the functional role(s) of CELF2 in hematopoiesis and leukemogenesis has not been fully elucidated. In the current study, we showed that Celf2 deficiency in hematopoietic system led to enhanced HSCs self-renewal and differentiation toward myeloid cells in mice. Loss of Celf2 accelerated myeloid cell transformation and AML development in MLL-AF9-induced AML murine models. Gene expression profiling integrated with RNA immunoprecipitation sequencing (RIP-Seq), together with biochemical experiments revealed that CELF2 deficiency stabilizes FAT10 mRNA, promotes FAT10 translation, thereby increases AKT phosphorylation and mTORC1 signaling pathway activation. Notably, combination therapy with a mTORC1 inhibitor (Rapamycin) and a MA9/DOTL1 inhibitor (EPZ-5676) reduced the leukemia burden in MLL-AF9 mice lacking Celf2 in vivo. Our study elucidated a novel mechanism by which the CELF2/FAT10-AKT/mTORC1 axis regulates the proliferation of normal blood cells and the development of AML, thus providing potential therapeutic targets for myeloid leukemia suppression.

Pseudouridine synthase 1 regulates erythropoiesis via transfer RNAs pseudouridylation and cytoplasmic translation.

Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA), characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However, the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1, mimicking the common mutation in human MLASA. Pus1-mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically, mutant erythroblasts showed defective pseudouridylation of targeted tRNAs, altered tRNA profiles, decreased translation efficiency of ribosomal protein genes, and reduced globin synthesis, culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis, cytoplasmic translation, and erythropoiesis.

Disruption of mitochondrial energy metabolism is a putative pathogenesis of Diamond-Blackfan anemia.

Energy metabolism in the context of erythropoiesis and related diseases remains largely unexplored. Here, we developed a primary cell model by differentiating hematopoietic stem progenitor cells toward the erythroid lineage and suppressing the mitochondrial oxidative phosphorylation (OXPHOS) pathway. OXPHOS suppression led to differentiation failure of erythroid progenitors and defects in ribosome biogenesis. Ran GTPase-activating protein 1 (RanGAP1) was identified as a target of mitochondrial OXPHOS for ribosomal defects during erythropoiesis. Overexpression of RanGAP1 largely alleviated erythroid defects resulting from OXPHOS suppression. Coenzyme Q10, an activator of OXPHOS, largely rescued erythroid defects and increased RanGAP1 expression. Patients with Diamond-Blackfan anemia (DBA) exhibited OXPHOS suppression and a concomitant suppression of ribosome biogenesis. RNA-seq analysis implied that the substantial mutation (approximately 10%) in OXPHOS genes accounts for OXPHOS suppression in these patients. Conclusively, OXPHOS disruption and the associated disruptive mitochondrial energy metabolism are linked to the pathogenesis of DBA.

Establishing a novel Fanconi anemia signaling pathway-associated prognostic model and tumor clustering for pediatric acute myeloid leukemia patients.

Considering the connection between the Fanconi anemia (FA) signaling pathway and tumor development, we aim to investigate the links between the FA gene expression and the survival prognosis of acute myeloid leukemia (AML) patients. Our study begins by identifying two distinct clusters of pediatric AML patients. Following the batch matching of the TARGET-AML, TCGA-LAML GSE71014, GSE12417, and GSE37642 cohorts, the samples were divided into a training set and an internal validation set. A Lasso regression modeling analysis was performed to identify five signatures: BRIP1, FANCC, FANCL, MAD2L2, and RFWD3. The AML samples were stratified into high- and low-risk groups by evaluating the risk scores. The AML high-risk patients showed a poorer overall survival prognosis. To predict the survival rates, we developed an FA Nomogram incorporating risk score, gender, age, and French-American-British classification. We further utilized the BEAT-AML cohort for the external validation of FA-associated prognostic models and observed good clinical validity. Additionally, we found a correlation between DNA repair, cell cycle, and peroxide-related metabolic events and FA-related high/low risk or cluster 1/2. In summary, our novel FA-associated prognostic models promise to enhance the prediction of pediatric AML prognosis.

Mutation spectrum, expression profiling, and prognosis evaluation of Fanconi anemia signaling pathway genes for 4259 patients with myelodysplastic syndromes or acute myeloid leukemia.

BACKGROUND: Individuals diagnosed with Fanconi anemia (FA), an uncommon disorder characterized by chromosomal instability affecting the FA signaling pathway, exhibit heightened vulnerability to the onset of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). METHODS: Herein, we employed diverse bioinformatics and statistical analyses to investigate the potential associations between the expression/mutation patterns of FA pathway genes and MDS/AML. RESULTS: The study included 4295 samples, comprising 3235 AML and 1024 MDS from our and nine other online cohorts. We investigated the distinct proportion of race, age, French-American-British, and gender factors. Compared to the FA wild-type group, we observed a decrease in the expression of FNACD2, FANCI, and RAD51C in the FA mutation group. The FA mutation group exhibited a more favorable clinical overall survival prognosis. We developed a random forest classifier and a decision tree based on FA gene expression for cytogenetic risk assessment. Furthermore, we created an FA-related Nomogram to predict survival rates in AML patients. CONCLUSIONS: This investigation facilitates a deeper understanding of the functional links between FA and MDS/AML.

PHF6 maintains acute myeloid leukemia via regulating NF-κB signaling pathway.

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the accumulation of immature and abnormally differentiated myeloid cells in bone marrow. Here with in vivo and in vitro models, we demonstrate that the Plant homeodomain finger gene 6 (PHF6) plays an important role in apoptosis and proliferation in myeloid leukemia. Phf6 deficiency could delay the progression of RUNX1-ETO9a and MLL-AF9-induced AML in mice. PHF6 depletion inhibited the NF-κB signaling pathways by disrupting the PHF6-p50 complex and partially inhibiting the nuclear translocation of p50 to suppress the expression of BCL2. Treating PHF6 over-expressed myeloid leukemia cells with NF-κB inhibitor (BAY11-7082) significantly increased their apoptosis and decreased their proliferation. Taken together, in contrast to PHF6 as a tumor suppressor in T-ALL as reported, we found that PHF6 also plays a pro-oncogenic role in myeloid leukemia, and thus potentially to be a therapeutic target for treating myeloid leukemia patients.

High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating blood cancer with high heterogeneity and ill-fated outcome. Despite numerous advances in AML treatment, the prognosis remains poor for a significant proportion of patients. Consequently, it is necessary to accurately and comprehensively identify biomarkers as soon as possible to enhance the efficacy of diagnosis, prognosis and treatment of AML. In this study, we aimed to identify prognostic markers of AML by analyzing the cohorts from TCGA-LAML database and GEO microarray datasets. Interestingly, the transcriptional level of microtubule-associated protein TBCB in AML patients was noticeably increased when compared with normal individuals, and this was verified in two independent cohorts (GSE9476 and GSE13159) and with our AML patients. Furthermore, univariate and multivariate regression analysis revealed that high TBCB expression was an independent poor prognostic factor for AML. GO and GSEA enrichment analysis hinted that immune-related signaling pathways were enriched in up-regulated DEGs between two populations separated by the median expression level of TBCB. By constructing a protein-protein interaction network, we obtained six hub genes, all of which are immune-related molecules, and their expression levels were positively linked to that of TBCB. In addition, the high expression of three hub genes was significantly associated with a poor prognosis in AML. Moreover, we found that the tumor microenvironment in AML with high TBCB expression tended to be infiltrated by NK cells, especially CD56bright NK cells. The transcriptional levels of NK cell inhibitory receptors and their ligands were positively related to that of TBCB, and their high expression levels also predicted poor prognosis in AML. Notably, we found that the down-regulation of TBCB suppressed cell proliferation in AML cell lines by enhancing the apoptosis and cell cycle arrest. Finally, drug sensitivity prediction illustrated that cells with high TBCB expression were more responsive to ATRA and midostaurin but resistant to cytarabine, dasatinib, and imatinib. In conclusion, our findings shed light on the feasibility of TBCB as a potential predictor of poor outcome and to be an alternative target of treatment in AML.

R274X-mutated Phf6 increased the self-renewal and skewed T cell differentiation of hematopoietic stem cells.

The PHD finger protein 6 (PHF6) mutations frequently occurred in hematopoietic malignancies. Although the R274X mutation in PHF6 (PHF6R274X) is one of the most common mutations identified in T cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) patients, the specific role of PHF6R274X in hematopoiesis remains unexplored. Here, we engineered a knock-in mouse line with conditional expression of Phf6R274X-mutated protein in the hematopoietic system (Phf6R274X mouse). The Phf6R274X mice displayed an enlargement of hematopoietic stem cells (HSCs) compartment and increased proportion of T cells in bone marrow. More Phf6R274X T cells were in activated status than control. Moreover, Phf6R274X mutation led to enhanced self-renewal and biased T cells differentiation of HSCs as assessed by competitive transplantation assays. RNA-sequencing analysis confirmed that Phf6R274X mutation altered the expression of key genes involved in HSC self-renewal and T cell activation. Our study demonstrated that Phf6R274X plays a critical role in fine-tuning T cells and HSC homeostasis.

High-expression of the innate-immune related gene UNC93B1 predicts inferior outcomes in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with dismal prognosis. Identification of better biomarkers remained a priority to improve established stratification and guide therapeutic decisions. Therefore, we extracted the RNA sequence data and clinical characteristics of AML from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression database (GTEx) to identify the key factors for prognosis. We found UNC93B1 was highly expressed in AML patients and significantly linked to poor clinical features (p < 0.05). We further validated the high expression of UNC93B1 in another independent AML cohort from GEO datasets (p < 0.001) and performed quantitative PCR of patient samples to confirm the overexpression of UNC93B1 in AML (p < 0.005). Moreover, we discovered high level of UNC93B1 was an independent prognostic factor for poorer outcome both in univariate analysis and multivariate regression (p < 0.001). Then we built a nomogram model based on UNC93B1 expression, age, FAB subtype and cytogenetic risk, the concordance index of which for predicting overall survival was 0.729 (p < 0.001). Time-dependent ROC analysis for predicting survival outcome at different time points by UNC93B1 showed the cumulative 2-year survival rate was 43.7%, and 5-year survival rate was 21.9%. The differentially expressed genes (DEGs) between two groups divided by UNC93B1 expression level were enriched in innate immune signaling and metabolic process pathway. Protein-protein interaction (PPI) network indicated four hub genes (S100A9, CCR1, MRC1 and CD1C) interacted with UNC93B1, three of which were also significantly linked to inferior outcome. Furthermore, we discovered high UNC93B1 tended to be infiltrated by innate immune cells, including Macrophages, Dendritic cells, Neutrophils, Eosinophils, and NK CD56dim cells. We also found UNC93B1 had a significantly positive correlation with CD14, CD68 and almost all Toll-like receptors. Finally, we revealed negatively correlated expression of UNC93B1 and BCL2 in AML and conjectured that high-UNC93B1 monocytic AML is more resistant to venetoclax. And we found high MCL-1 expression compensated for BCL-2 loss, thus, we proposed MCL-1 inhibitor might overcome the resistance of venetoclax in AML. Altogether, our findings demonstrated the utility of UNC93B1 as a powerful poor prognostic predictor and alternative therapeutic target.

Incidence and risk factors for second malignancies among patients with myeloproliferative neoplasms.

OBJECTIVE: The clinical characteristics and survival of patients with myeloproliferative neoplasms (MPNs) with secondary cancer were analyzed to explore the possible risk factors for secondary cancer in MPN patients. METHODS: The clinical characteristics of 1060 Chinese patients with MPN were retrospectively analyzed. The Kaplan-Meier method was used to analyze the survival. The Cox multivariate regression model was used to analyze the risk factors for developing secondary cancer in patients with MPNs. RESULTS: The 1060 patients with MPN had a median follow-up of 10 years (range 1-50) and a median age of 55 years (range 21-86), and 497 (45.2%) were male. The proportion of PV, ET, and PMF was 52.2%, 33.5%, and 14.3%, respectively. About 28.1% (298/1060) of 1060 MPN patients died. The median survival times of the PV, ET, and PMF groups were 20, 24, and 12 years, respectively (p < 0.0001). In age- and sex-matched healthy Chinese patients, the standardized incidence ratio (SIR) value of developing secondary cancer in MPN patients was 6.41 (95% CI: 4.90-9.48). The median survival time was 14 years in the MPN with secondary cancer group. The Cox multivariate analysis showed that age ≥ 65 years (p < 0.0001, HR = 5.027, 95% CI [2.823, 8.952]), MF-1 (p = 0.001, HR = 2.887, 95% CI [1.503, 5.545]) were risk factors for developing secondary cancer. CONCLUSIONS: The survival of MPN patients with secondary cancer was significantly worse than that of patients without secondary cancer. Compared with normal subjects, MPN patients had a 6.41-fold increased risk of developing secondary cancer, and age ≥ 65 years and MF-1 were risk factors for developing secondary cancer in MPN patients.

Mass cytometry analysis identifies T cell immune signature of aplastic anemia and predicts the response to cyclosporine.

Aplastic anemia (AA) is an auto-activated T cell-mediated bone marrow failure. Cyclosporine is often used to treat non-severe AA, which demonstrates a more heterogeneous condition than severe AA. The response rate to cyclosporine is only around 50% in non-severe AA. To better predict response to cyclosporine and pinpoint who is the appropriate candidate for cyclosporine, we performed phenotypic and functional T cell immune signature at single cell level by mass cytometry from 30 patients with non-severe AA. Unexpectedly, non-significant differences of T cell subsets were observed between AA and healthy control or cyclosporine-responder and non-responders. Interestingly, when screening the expression of co-inhibitory molecules, T cell trafficking mediators, and cytokines, we found an increase of cytotoxic T lymphocyte antigen 4 (CTLA-4) on T cells in response to cyclosporine and a lower level of CTLA-4 on CD8+ T cells was correlated to hematologic response. Moreover, a decreased expression of sphingosine-1-phosphate receptor 1 (S1P1) on naive T cells and a lower level of interleukin-9 (IL-9) on T helpers also predicted a better response to cyclosporine, respectively. Therefore, the T cell immune signature, especially in CTAL-4, S1P1, and IL-9, has a predictive value for response to cyclosporine. Collectively, our study implies that immune signature analysis of T cell by mass cytometry is a useful tool to make a strategic decision on cyclosporine treatment of AA.

Phenotypic and genotypic correlation evaluation of 148 pediatric patients with Fanconi anemia in a Chinese rare disease cohort.

BACKGROUND: Fanconi anemia (FA) is a rare autosomal recessive, X-linked or autosomal dominant disease. Few large-scale FA investigations of rare disease cohorts have been conducted in China. METHODS: We enrolled 148 patients diagnosed with FA according to evidence from the clinical phenotype, family history, and a set of laboratory tests. Next, the clinical manifestations and correlation between the genotype and phenotype of FA pediatric cases were investigated. RESULTS: The most common FA subtype in our cohort was FA-A (51.4 %), followed by FA-D2 and FA-P. Finger (26 %) and skin (25 %) deformities were the most common malformations. Based on family history, blood system diseases (51 %) had the highest incidence rate, followed by digestive system tumours. A set of new or prognosis-related mutation sites was identified. For example, c.2941 T > G was a new most common missense mutation site for FANCA. FANCP gene mutation sites were mainly concentrated in exons 12/14/15. The mutations of FANCI/FANCD2 were mainly located at the α helix and ß corners of the protein complex. FA-A/D1 patients with splicing or deletion mutations showed more severe disease than those with missense mutations. Chromosome 1/3/7/8 abnormalities were closely linked to the progression of FA to leukemia. CONCLUSION: Our study investigated the clinical features and genotype/phenotype correlation of 148 Chinese pediatric FA patients, providing new insight into FA.

[Screening of proliferation related lncRNAs in leukemia cell lines by lentivirus shRNA library combined with second-generation sequencing].

Long non-coding RNA (lncRNA) has become an important regulator of many cellular processes, including cell proliferation. Although studies have shown that a variety of lncRNAs play an important role in the occurrence and development of hematopoietic malignancies, a more comprehensive and unbiased method to study the function of lncRNAs in leukemia cell lines is lacking. Here, we used short hairpin RNA (shRNA) library combined with high-throughput sequencing to screen lncRNAs that may affect the proliferation of leukemia cell lines, and identified lncRNA C20orf204-203 among 74 candidate lncRNAs in this study. Further experiments showed that C20orf204-203 was localized in the cytoplasm in both K562 and THP-1 cell lines. C20orf204-203 knockdown decreased the proliferation of K562 and THP-1 cell lines accompanied with the increased proportion of early apoptotic cells. We observed the increased mRNA level of BAD gene while decreased protein level of TP53 and BCL2. The expression of Caspase 3 decreased and Caspase 3-cleaved protein increased in THP-1 cell line. However, their changes were inconsistent in the two cell lines. Our experimental results showed that knockdown of lncRNA C20orf204-203 in leukemia cell lines affected cell proliferation although the mechanism of action in different cell lines may differ. Importantly, our research demonstrated the feasibility of using shRNA library combined with high-throughput sequencing to study the role of lncRNA in leukemia cell lines on a large scale.

Polyclonal evolution of Fanconi anemia to MDS and AML revealed at single cell resolution.

BACKGROUND: Fanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive. METHODS: Herein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage. RESULTS: Our finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage. CONCLUSIONS: We thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.

SHP2 Inhibitors Show Anti-Myeloma Activity and Synergize With Bortezomib in the Treatment of Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. The protein tyrosine phosphatase SHP2 is a central node regulating RAS/mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) signaling pathway which plays a crucial role in the pathogenesis and proteasome inhibitor (PI) resistance of MM. Several preclinical studies have demonstrated that SHP2 inhibitors exerted antitumor activity in cancer-harboring diverse mutations in the RAS pathway, offering the potential for targeting myeloma. In this study, we showed that pharmacological inhibition of SHP2 activity using SHP099 and RMC-4550 efficiently inhibited the proliferation of MM cells by inducing apoptosis and cell cycle arrest. As per the mechanism, SHP2 inhibitors activated the level of cleaved caspase3, BAK, and P21 and downregulated ERK phosphorylation in MM cells. Moreover, the blockade of SHP2 exhibited anti-myeloma effect in vivo in a mouse xenograft model. In addition, SHP2 inhibitors synergized the antineoplastic effect of bortezomib in bortezomib-sensitive MM cells and showed identical efficacy in targeting bortezomib-resistant MM cells. Overall, our findings suggest that SHP2-specific inhibitors trigger anti-myeloma activity in vitro and in vivo by regulating the ERK pathway and enhancing cytotoxicity of bortezomib, providing therapeutic benefits for both bortezomib naïve and resistant MM.

Rapamycin Promotes the Expansion of Myeloid Cells by Increasing G-CSF Expression in Mesenchymal Stem Cells.

Rapamycin, also known as sirolimus, an inhibitor of mammalian target of rapamycin (mTOR), is a regulatory kinase responsible for multiple signal transduction pathways. Although rapamycin has been widely used in treating various hematologic diseases, the effects of rapamycin are still not fully understood. Here we found that both oral and intraperitoneal administration of rapamycin led to the expansion of myeloid lineage, while intraperitoneal administration of rapamycin impaired granulocyte differentiation in mice. Rapamycin induced bone marrow mesenchymal stem cells to produce more G-CSF in vitro and in vivo, and promoted the myeloid cells expansion. Our results thus demonstrated that intraperitoneal administration of rapamycin might promote the expansion of myeloid lineage while impair myeloid cell differentiation in vivo.

Osteopontin is required for the maintenance of leukemia stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder with a poor prognosis. The clinical significance of Leukemia stem cells (LSCs) plays an important role in the generation of AML and is the main cause of the recurrence after remission. Osteopontin (OPN), an extracellular matrix protein, has been implicated in hematopoietic malignancies. However, the specific role and the underlying mechanism of AML cell autocrined OPN in leukemia maintenance remain unknown. Here, we showed that knockdown of Opn expression significantly prolonged the survival of mice with MLL-AF9 cell-induced AML and markedly reduced the tumor burden. The LSCs from the Opn-knockdown groups exhibited decreased numbers and impaired function as determined by immunophenotype, colony-forming and limiting dilution assays. Further analysis revealed that Opn prevents LSCs from undergoing apoptosis and cell cycle arrest. Repression of OPN in human AML cell lines in vitro mimics the phenotypes observed in the mouse model. Overall, our data indicated that OPN is a potent therapeutic target for eradicating LSCs in AML.

[Effect of Rheb1 in the Development of Mouse Megakaryocyte-Erythroid Progenitor Cells].

OBJECTIVE: To investigate the effect of Rheb1 in the development of mouse megakaryocyte-erythroid progenitor cells and its related mechanism. METHODS: Rheb1 was specifically knocked-out in the hematopoietic system of Vav1-Cre;Rheb1fl/fl mice(Rheb1Δ/Δ mice). Flow cytometry was used to detect the percentage of red blood cells in peripheral blood and erythroid cells in bone marrow in Vav1-Cre;Rheb1fl/fl mice and control mice. The CFC assay was used to detect the differentiation ability of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Real-time fluorescence quantification PCR was used to detect the relative expression of PU.1,GATA-1,GATA-2,CEBPα and CEBPß of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Rapamycin was added to the culture medium, and it was used to detect the changes in cloning ability of megakaryocyte-erythroid progenitor cells from wild-type mice in vitro. RESULTS: After Rheb1 was knocked out, the development and stress response ability of megakaryocyte-erythroid progenitor cells in mice were weaken and the differentiation ability of megakaryocyte-erythroid progenitor cells in vitro was weaken. Moreover, the expression of GATA-1 of megakaryocyte-erythroid progenitor cells was decreased. Further, rapamycin could inhibit the differentiative capacity of megakaryocyte-erythroid progenitor cells in vitro. CONCLUSION: Rheb1 can regulate the development of megakaryocyte-erythroid progenitor cells probably through the mTOR signaling pathway in mice.