Modulation of activated oncogene <Emphasis Type="Italic">c-kit</Emphasis> expression with RNA-interference

Overexpression of oncogene c-kit is detected in 80% patients with acute myeloid leukemia (AML). A transgenic model cell line expressing oncogene c-kit was obtained by transduction with a recombinant retrovirus. We have designed small interfering RNAs (siRNAs) that efficiently suppress the expression of activated oncogene c-kit. Further, small hairpin RNAs (shRNAs) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells, as well as Kasumi-1 cells from a patient with AML.     

Clinical and Prognostic Implications of Roundabout 4 (Robo4) in Adult Patients with Acute Myeloid Leukemia

Background

Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.

Methods

We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.

Results

Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.

Conclusions

BM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.     

Prognostic role of Wnt and Fzd gene families in acute myeloid leukaemia

Wnt-Fzd signalling pathway plays a critical role in acute myeloid leukaemia (AML) progression and oncogenicity. There is no study to investigate the prognostic value of Wnt and Fzd gene families in AML. Our study screened 84 AML patients receiving chemotherapy only and 71 also undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT) from the Cancer Genome Atlas (TCGA) database. We found that some Wnt and Fzd genes had significant positive correlations. The expression levels of Fzd gene family were independent of survival in AML patients. In the chemotherapy group, AML patients with high Wnt2B or Wnt11 expression had significantly shorter event-free survival (EFS) and overall survival (OS); high Wnt10A expressers had significantly longer OS than the low expressers (all P < .05), whereas, in the allo-HSCT group, the expression levels of Wnt gene family were independent of survival. We further found that high expression of Wnt10A and Wnt11 had independent prognostic value, and the patients with high Wnt10A and low Wnt11 expression had the longest EFS and OS in the chemotherapy group. Pathway enrichment analysis showed that genes related to Wnt10A, Wnt11 and Wnt 2B were mainly enriched in ‘cell morphogenesis involved in differentiation’, ‘haematopoietic cell lineage’, ‘platelet activation, signalling and aggregation’ and ‘mitochondrial RNA metabolic process’ signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo-HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.     

c-kitpos GATA-4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling

Background

Resident c-kit positive (c-kit^pos) cardiac stem cells (CSCs) could be considered the most appropriate cell type for myocardial regeneration therapies. However, much is still unknown regarding their biological properties and potential.

Methodology/Principal Findings

We produced clones of high and low expressing GATA-4 CSCs from long-term bulk-cultured c-kit^pos CSCs isolated from adult rat hearts. When c-kit^pos GATA-4 high expressing clonal CSCs (cCSCs) were co-cultured with adult rat ventricular cardiomyocytes, we observed increased survival and contractility of the cardiomyocytes, compared to cardiomyocytes cultured alone, co-cultured with fibroblasts or c-kit^pos GATA-4 low expressing cCSCs. When analysed by ELISA, the concentration of IGF-1 was significantly increased in the c-kit^pos GATA-4 high cCSC/cardiomyocyte co-cultures and there was a significant correlation between IGF-1 concentration and cardiomyocyte survival. We showed the activation of the IGF-1 receptor and its downstream molecular targets in cardiomyocytes co-cultured with c-kit^pos GATA-4 high cCSCs but not in cardiomyocytes that were cultured alone, co-cultured with fibroblasts or c-kit^pos GATA-4 low cCSCs. Addition of a blocking antibody specific to the IGF-1 receptor inhibited the survival of cardiomyocytes and prevented the activation of its signalling in cardiomyocytes in the c-kit^pos GATA-4 high cCSC/cardiomyocyte co-culture system. IGF-1 supplementation or IGF-1 high conditioned medium taken from the co-culture of c-kit^pos GATA-4 high cCSCs plus cardiomyocytes did extend the survival and contractility of cardiomyocytes cultured alone and cardiomyocytes co-cultured with c-kit^pos GATA-4 low cCSCs.

Conclusion/Significance

c-kit^pos GATA-4 high cCSCs exert a paracrine survival effect on cardiomyocytes through induction of the IGF-1R and signalling pathway.     

Upregulation of microRNA-375 is associated with poor prognosis in pediatric acute myeloid leukemia

A genome-wide serum miRNA expression analysis previously showed the upregulation of microRNA-375 (miR-375) in acute myeloid leukemia (AML) patients compared with healthy controls. The aim of this study was to investigate the expression patterns and the prognostic relevance of miR-375 in pediatric AML. Expression levels of miR-375 in bone marrow mononuclear cells were detected by real-time quantitative PCR in a cohort of 106 patients with newly diagnosed pediatric AML. Expression levels of miR-375 in the bone marrow of pediatric AML patients were significantly higher than those in normal controls (P < 0.001). Then, miR-375 upregulation occurred more frequently in French–American–British classification subtype M7 than in other subtypes (P < 0.001). Regarding to cytogenetic risk, the expression levels of miR-375 in pediatric AML patients with unfavorable karyotypes were dramatically higher than those in intermediate and favorable groups (P = 0.002). Moreover, high miR-375 expression was significantly associated with shorter relapse-free survival (P < 0.001) and overall survival (P < 0.001) in pediatric AML patients. Multivariate analysis further identified miR-375 expression and cytogenetics risk as independent prognostic factors for both relapse-free survival and overall survival. In particular, the prognostic relevance of miR-375 expression was more obvious in the subgroup of patients with intermediate-risk cytogenetics. Our findings suggest for the first time that the upregulation of miR-375 may be one of the molecular mechanisms involved in the development and progression of pediatric AML. Since its correlation with poor relapse-free survival and overall survival, miR-375 may be a novel biomarker to improve the management of pediatric AML patients.     

Expression of PD‐L1 on regulatory B cells in patients with acute myeloid leukaemia and its effect on prognosis

Programmed death‐ligand 1 (PD‐L1) is involved in immunosuppression in variety of tumours. Regulatory B cells (Bregs) are critical immune regulatory cells, and it has been demonstrated that the number of regulatory B cells in patients with acute myeloid leukaemia (AML) is much higher than that in healthy donors (HDs), which is linked to a poor prognosis. This study aimed to determine whether increased expression of PD‐L1, including in Bregs, is associated with a worse prognosis in individuals with AML. The proportion of Bregs, PD‐L1 expression in Bregs and PD‐1 expression in T cells were determined using flow cytometry using patient samples from 21 newly diagnosed AML patients at different stages of treatment and 25 HDs. We confirmed PD‐L1 expression in Bregs, and PD‐1 expression in CD3⁺CD4⁺T cells in bone marrow and peripheral blood samples from AML patients was higher than that in samples from HDs. The complete remission (CR) and progression‐free survival (PFS) of Bregs with high PD‐L1 expression were significantly decreased following induction chemotherapy. PD‐L1 expression is indeed increased in Bregs from individuals with AML, and high PD‐L1 expression is related to a poor prognosis.     

RUNX1 mutation and elevated FLT3 gene expression cooperates to induce inferior prognosis in cytogenetically normal acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a bone marrow malignancy having multiple molecular pathways driving its progress. In recent years, the main causes of AML considered all over the world are genetic variations in cancerous cells. The RUNX1 and FLT3 genes are necessary for the normal hematopoiesis and differentiation process of hematopoietic stem cells into mature blood cells, therefore they are the most common targets for point mutations resulting in AML.MethodsWe screened 32 CN-AML patients for FLT3-ITD (by Allele-specific PCR) and RUNX1 mutations (by Sanger sequencing). The FLT3 mRNA expression was assessed in all AML patients and its subgroups.ResultsEight patients (25%) carried RUNX1 mutation (K83E) while three patients (9.37%) were found to have internal tandem duplications in FLT3 gene. The RUNX1 mutation data were correlated with clinical parameters and FLT3 gene expression profile. The RUNX1 mutations were observed to be significantly prevalent in older males. Moreover, RUNX1 and FLT3-mutated patients had lower complete remission rate, event-free survival rate, and lower overall survival rate than patients with wild-type RUNX1 and FLT3 gene. The RUNX1 and FLT3 mutant patients with up-regulated FLT3 gene expression showed even worse prognosis. Bradford Assay showed that protein concentration was down-regulated in RUNX1 and FLT3 mutants in comparison to RUNX1 and FLT3 wild-type groups.ConclusionThis study constitutes the first report from Pakistan reporting significant molecular mutation analysis of RUNX1 and FLT3 genes including FLT3 expression evaluation with follow-up. This provides an insight that aforementioned mutations are markers of poor prognosis but the study with a large AML cohort will be useful to further investigate their role in disease biology of AML.     

Multidrug resistance determinants in acute myeloid leukemia developed in persons exposed to ionizing radiation due to the Chernobyl accident

The results of multidrug resistance determinants expression analysis on leukemic cells of 56 acute myeloid leukemia (AML) patients by immunophenotyping are presented. Of these, there were 21 persons exposed to ionizing radiation due to the Chemobyl accident with radiation-associated AML and 35 patients with spontaneous leukemia. The aim of this study was to determine if transport proteins (P-glycoprotein, LRP, and MDR1), apoptosis-related proteins (Fas, Bcl-2, Bax, p53, and Bcl-X(L)), and topoisomerase IIalpha expression in AML patients with the history of radiation exposure differed from those in spontaneous AML cases. Leukemic cells in patients with radiation-associated diseases compared to spontaneous AML more often overexpressed antiapoptotic oncoprotein Bcl-2 (12/21 vs. 6/35, p < 0.005) and less often demonstrated expression of Fas receptor (12/21 vs. 30/35, p < 0.05). Moreover, leukemic cells were simultaneously Fas negative and Bcl-2 positive in 4 out of 21 patients exposed to ionizing radiation but none of spontaneous cases had similar phenotype (p < 0.05). Leukemic cells in patients with radiation-associated AML compared to spontaneous cases more often were P-glycoprotein positive (12/20 vs 9/31, p < 0.05). P-glycoprotein overexpression significantly correlated with resistant disease in patients with radiation-associated AML (r = 0.47, p < 0.05), but was not a prognostic variable for the treatment outcome in terms of overall survival. Defects in pathways of drug-induced apoptosis and function of pump, that actively effluxes drugs could contribute significantly to developing drug resistance in radiation-associated AML.     

U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.     

Geographic heterogeneity of the AML1-ETO fusion gene in Iranian patients with acute myeloid leukemia

Background:

The human AML1 gene, located on chromosome 21, can be fused to the AML1- eight-twenty-one (ETO) oncoprotein on chromosome eight, resulting in a t(8;21)(q22;q22) translocation. Acute myeloid leukemia (AML) associated with this translocation is considered a distinct AML with a favorable prognosis. Due to the various incidences of the translocation, which is associated with geographic diversities, investigation of molecular epidemiology is important to increase the awareness of physicians and hematologists regarding the frequency this chromosomal aberration.

Methods:

The patients were classified according to the French–American–British classification into eight groups: M0–M7. Determination of the prevalence of the AML1-ETO fusion gene was accomplished by TaqMan real-time PCR. Bone marrow samples from 113 patients with newly-diagnosed, untreated AML -M1, -M2, and -M4, and 20 healthy controls admitted to the Ghaem Hospital in Mashhad, Iran were studied.

Results:

The AML1-ETO fusion gene was detected up 50% of the M2 subgroup and absent in the M1 and M4 subtypes and healthy controls. Comparison of the prevalence of the t(8;21) translocation with results of previous studies showed that it varies between countries. This result may be due to geographic or ethnic differences, or both.

Conclusions:

The relatively high prevalence of the t(8;21) translocation in Iran was similar to that found in other Asian countries. It was closely associated with female gender, relatively young age, and FAB-M2 subtype. Its distribution varied considerably with geographic area. Therefore, further studies are needed to provide epidemiological data important for the establishment of optimal therapeutic strategies applicable to patients of each region. Key Words: Acute myeloid leukemia, AML1-ETO, M2, Prevalence, t(8;21)     

CASP8 SNP D302H (rs1045485) Is Associated with Worse Survival in MYCN-Amplified Neuroblastoma Patients

Background

Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis.

Patients and Methods

We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays.

Results and Conclusion

We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.     

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rg^null mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.     

Microarray-based analysis and clinical validation identify ubiquitin-conjugating enzyme E2E1 (<Emphasis Type="Italic">UBE2E1</Emphasis>) as a prognostic factor in acute myeloid leukemia

Background

Previous research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions.

Methods

We performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis.

Results

Microarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p?<?0.05). Next, we validated our findings in an independent cohort of AML samples collected in our hospital. We found that ubiquitin-conjugating enzyme E2E1 (UBE2E1) expression was adversely correlated with AML survival (p?=?0.04). Multivariable analysis showed that UBE2E1 ^high patients had a significant shorter OS and shorter progression-free survival after adjusting other known prognostic factors (p?=?0.03). At last, we found that UBE2E1 expression was negatively correlated with patients’ response to induction chemotherapy (p?<?0.05).

Conclusions

In summary, we demonstrated that UBE2E1 expression was a novel prognostic factor in adult, non-APL AML patients.