Identification of Genes Transcriptionally Responsive to the Loss of MLL Fusions in MLL-Rearranged Acute Lymphoblastic Leukemia

Introduction

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.

Methods

For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.

Results

Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.

Conclusion

We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.     

Flt3 Does Not Play a Critical Role in Murine Myeloid Leukemias Induced by MLL Fusion Genes

Leukemias harboring MLL translocations are frequent in children and adults, and respond poorly to therapies. The receptor tyrosine kinase FLT3 is highly expressed in these leukemias. In vitro studies have shown that pediatric MLL-rearranged ALL cells are sensitive to FLT3 inhibitors and clinical trials are ongoing to measure their therapeutic efficacy. We sought to determine the contribution of Flt3 in the pathogenesis of MLL-rearranged leukemias using a myeloid leukemia mouse model. Bone marrow from Flt3 null mice transduced with MLL-ENL or MLL-CBP was transplanted into host mice and Flt3 ^−/− leukemias were compared to their Flt3 wild type counterparts. Flt3 deficiency did not delay disease onset and had minimal impact on leukemia characteristics. To determine the anti-leukemic effect of FLT3 inhibition we studied the sensitivity of MLL-ENL leukemia cells to the FLT3 inhibitor PKC412 ex vivo. As previously reported for human MLL-rearranged leukemias, murine MLL-ENL leukemia cells with higher Flt3 levels were more sensitive to the cytotoxicity of PKC412. Interestingly, Flt3 deficient leukemia samples also displayed some sensitivity to PKC412. Our findings demonstrate that myeloid leukemias induced by MLL-rearranged genes are not dependent upon Flt3 signaling. They also highlight the discrepancy between the sensitivity of cells to Flt3 inhibition in vitro and the lack of contribution of Flt3 to the pathogenesis of MLL-rearranged leukemias in vivo.     

LncRNA CD27-AS1 promotes acute myeloid leukemia progression through the miR-224-5p/PBX3 signaling circuit

Acute myeloid leukemia (AML) is a hematological malignancy with a low cure rate, especially in the elderly. Previous studies have shown that long non-coding RNA (lncRNA) may be an important factor in the pathogenesis of hematological malignancies, including acute myeloid leukemia (AML). However, the biological roles and clinical significances of most lncRNAs in AML are not fully understood. LncRNA CD27 Antisense RNA 1 (CD27-AS1), as a member of lncRNA family, has rare reports on its function. In present study, we found that the expression of CD27-AS1 examined by quantitative real-time PCR was markedly increased in the AML patients (N = 40) compared with healthy volunteers (N = 40). The overall survival time was significantly shorter in patients with higher CD27-AS1 expression than that in patients with lower CD27-AS1 (P < 0.01). Furthermore, downregulation of CD27-AS1 in AML cells suppressed proliferative ability, arrested cell cycle in G0/G1 phase, and induced apoptosis. However, CD27-AS1 overexpression further enhanced the malignant phenotype of AML cells. Additionally, CD27-AS1 was proved to increase PBX3 expression through sponging miR-224-5p. CD27-AS1 knockdown blocked the MAPK signaling through PBX3 silencing and further inhibited the cell growth of AML cells. Taken together, we demonstrate that CD27-AS1 may be a potential prognostic biomarker of AML, and our finding also provides a new insight for non-coding RNA-based therapeutic intervention of AML.Subject terms: Growth factor signalling, Oncogenesis     

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.

Methodology/Principal Findings

We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.

Conclusions/Significance

Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.     

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.

     

The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability

The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCF^Skp2 and APC^Cdc20. Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the Mll^N and Mll^C subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia.     

A Comprehensive Comparative Analysis of the Histomorphological Features of ALK-Rearranged Lung Adenocarcinoma Based on Driver Oncogene Mutations: Frequent Expression of Epithelial-Mesenchymal Transition Markers than Other Genotype

Molecular classification of lung cancer correlates well with histomorphological features. However, specific histomorphological features that differentiate anaplastic lymphoma kinase (ALK)-rearranged tumors from ALK-negative tumors have not been fully evaluated. Eighty ALK-rearranged and 213 ALK-negative (91 epidermal growth factor receptor-mutated; 29 K-ras-mutated; 93 triple-negative) resected lung adenocarcinomas were analyzed for several histomorphological parameters and histological subtype. ALK-rearranged tumors were associated with younger age at presentation, frequent nodal metastasis, and higher stage of disease at diagnosis. ALK-rearranged tumors were more likely to show a solid predominant pattern than ALK-negative tumors (43.8%; 35/80; p<0.001). Unlike ALK-negative tumors, a lepidic predominant pattern was not observed in ALK-rearranged tumors (p<0.001). In multivariate analysis, the most significant morphological features that distinguished ALK-rearranged tumors from ALK-negative tumors were cribriform formation (odds ratio [OR], 3.253; p = 0.028), presence of mucin-containing cells (OR, 4.899; p = 0.008), close relationship to adjacent bronchioles (OR, 5.361; p = 0.001), presence of psammoma bodies (OR, 4.026; p = 0.002), and a solid predominant pattern (OR, 13.685; p = 0.023). ALK-rearranged tumors exhibited invasive histomorphological features, aggressive behavior and frequent expression of epithelial-mesenchymal transition markers (loss of E-cadherin and expression of vimentin) compared with other genotype (p = 0.015). Spatial proximity between bronchus and ALK-rearranged tumors and frequent solid histologic subtype with p63 expression may cause diagnostic difficulties to differentiate squamous cell carcinoma in the small biopsy, whereas p40 was rarely expressed in ALK-rearranged adenocarcinoma. Knowledge of these features may improve the diagnostic accuracy and lead to a better understanding of the characteristic behavior of ALK-rearranged tumors.     

DOT1L Inhibition Sensitizes MLL-Rearranged AML to Chemotherapy

DOT1L, the only known histone H3-lysine 79 (H3K79) methyltransferase, has been shown to be essential for the survival and proliferation of mixed-linkage leukemia (MLL) gene rearranged leukemia cells, which are often resistant to conventional chemotherapeutic agents. To study the functions of DOT1L in MLL-rearranged leukemia, SYC-522, a potent inhibitor of DOT1L developed in our laboratory, was used to treat MLL-rearranged leukemia cell lines and patient samples. SYC-522 significantly inhibited methylation at H3K79, but not H3K4 or H3K27, and decreased the expression of two important leukemia-relevant genes, HOXA9 and MEIS1, by more than 50%. It also significantly reduced the expression of CCND1 and BCL2L1, which are important regulators of cell cycle and anti-apoptotic signaling pathways. Exposure of MLL-rearranged leukemia cells to this compound caused cell cycle arrest and promoted differentiation of those cells, both morphologically and by increased CD14 expression. SYC-522 did not induce apoptosis, even at 10 µM for as long as 6 days. However, treatment with this DOT1L inhibitor decreased the colony formation ability of primary MLL-rearranged AML cells by up to 50%, and promoted monocytic differentiation. Notably, SYC-522 treatment significantly increased the sensitivity of MLL-rearranged leukemia cells to chemotherapeutics, such as mitoxantrone, etoposide and cytarabine. A similar sensitization was seen with primary MLL-rearranged AML cells. SYC-522 did not affect chemotherapy-induced apoptosis in leukemia cells without MLL-rearrangement. Suppression of DOT1L activity inhibited the mitoxantrone-induced increase in the DNA damage response marker, γH2AX, and increased the level of cPARP, an intracellular marker of apoptosis. These results demonstrated that SYC-522 selectively inhibited DOT1L, and thereby altered gene expression, promoted differentiation, and increased chemosensitivity by preventing DNA damage response. Therefore, inhibition of DOT1L, in combination with DNA damaging chemotherapy, represents a promising approach to improving outcomes for MLL-rearranged leukemia.     

Methylenetetrahydrofolate reductase gene polymorphisms contribute to acute myeloid leukemia and chronic myeloid leukemia susceptibilities: Evidence from meta-analyses

PurposeThe expression of methylenetetrahydrofolate reductase (MTHFR) is associated with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Most studies have linked the common functional C677T and A1298C polymorphisms of the MTHFR gene and susceptibility to AML and CML, but the results were not consistent. The aim of the present study was to derive a more precise estimation of the relationship.MethodsMeta-analyses assessing the association of MTHFR C677T and A1298C variations with AML and CML were conducted. Eligible articles were identified from the PubMed and EMBASE databases. All statistical analyses were conducted using Review Manager Software.Results10 and 10 studies were included in the meta-analysis about the role of C677T polymorphism on the AML and CML risks, respectively; 6 and 4 studies were included about the role of A1298C polymorphism on the AML and CML risks, respectively. Overall, both the C677T and A1298C polymorphisms were significantly associated with CML risk under the recessive model (P = 0.04, OR = 1.35, 95% CI = 1.02–1.79 for C677T and P = 0.003, OR = 2.17, 95% CI = 1.29–3.63 for A1298C). In addition, the risk of CML was higher in 1298CC genotype carriers than in 1298AA genotype carriers (P = 0.004, OR = 2.17, 95% = 1.28–3.69). Conversely, the overall data failed to indicate a significant association of C677T or A1298C polymorphisms with AML risk under any model.ConclusionsThe findings provide evidence that C677T and A1298C polymorphisms are risk factors for CML risk.     

Toll-like receptor 8 and 9 polymorphisms in Crimean-Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is an acute viral hemorrhagic fever. The clinical course and outcome of the CCHF infection are different in humans. Toll-like receptors (TLRs) are a family of pathogen recognition receptors. TLR8 and TLR9 contribute to the recognition of viruses. We investigated frequency of TLR8 Met1Val, TLR8 -129C/G, TLR9 -1486T/C and TLR9 2458G/A polymorphisms in CCHF patients and healthy controls. Our study was conducted between June 1 and August 31, 2007 in Cumhuriyet University Hospital, Turkey. TLR genotypes were detected using the PCR-RFLP assay in 85 CCHF patients and 171 healthy controls. We found that heterozygous plus homozygous mutant genotypes frequency for TLR8 Met1Val and for TLR9 -1486T/C were significantly higher in CCHF patients than controls (p = 0.038 and p = 0.009, respectively). The frequency of TLR8 -129G/G genotype in the fatal CCHF patients was significantly higher than that of the non-fatal patients (p = 0.026). The frequency of TLR9 -1486C/C genotype was significantly higher in fatal CCHF patients than in healthy controls (p = 0.009) and in patients with severe disease compared to non-severe disease (p = 0.044). Our findings suggest that TLR8 Met1Val, TLR8 -129C/G, and TLR9 -1486T/C polymorphisms are important on clinical course of CCHF disease.     

The impact of HLA-C matching depends on the C1/C2 KIR ligand status in unrelated hematopoietic stem cell transplantation

It was previously shown that chronic myeloid leukemia (CML) patients transplanted with peripheral blood progenitor cells (PBPC) from HLA-C allele-matched donors had better clinical outcome when lacking the HLA-C-encoded KIR epitope C2. We investigated whether this holds true in other diseases and in HLA-C allele-mismatched patients. Twenty-four myelodysplastic syndrome (MDS), 39 acute myeloid leukemia (AML)/CML, and 34 acute lymphoblastic leukemia/non-Hodgkin lymphoma patients received unrelated unmanipulated PBPC. HLA matching was analyzed retrospectively (including DNA-based direct sequencing of HLA-C). Only in AML/CML, the C2 ligand was associated with impaired overall survival (OS, p?<?0.05). We next calculated the impact of donor/recipient HLA-C allele matching within the C1 and C2 groups. Surprisingly, AML/CML and MDS patients with C2 ligands profited from HLA-C allele mismatching (OS, p?<?0.01), whereas in the C1 group, allele matching was beneficial (p?<?0.05). HLA-C allele mismatching in the C2 KIR ligand group was associated with lower TRM (OR 0.48, p?<?0.009) and lower relapse rate (OR 2.7 p?<?0.1) when compared to allele-matched C2 patients. Thus, patients could be assigned to a low- and a high-risk group according to their C1/C2 ligand status and the HLA-C allele matching degree. These data suggest that four-digit allele matching of HLA-C has differential effects dependent on the presence of C1 and C2 KIR epitopes in the patient.     

Interferon gamma gene +874A/T polymorphism and intracellular interferon gamma expression in pulmonary tuberculosis

We investigated whether IFN-γ gene +874(A/T) polymorphism influences intracellular interferon gamma expression in T-cell subsets of normal healthy subjects (NHS) and pulmonary tuberculosis patients (PTB). Peripheral blood mononuclear cells were stimulated with live Mycobacterium tuberculosis (MTB) and the intracellular IFN-γ expression was studied using flow cytometry. Genotyping of IFN-γ gene +874(A/T) was done using allele specific polymerase chain reaction. Significantly increased IFN-γ expressing CD3+CD4+ and CD3+CD8+ T cells were observed in NHS with AA genotype compared to TT genotype in unstimulated (p = 0.0308 and p = 0.0157) and MTB stimulated (p = 0.0494 and p = 0.0287) cultures and this difference was not observed in PTB patients. The present study suggests that the variant genotypes of IFN-γ (+874) may be associated with altered expression of IFN-γ at the intracellular level and play an immunoregulatory role at the site of M. tuberculosis infection.     

Birth weight and other perinatal characteristics and childhood leukemia in California

Aims. We conducted a large registry-based study in California to investigate the association of perinatal factors and childhood leukemia with analysis of two major subtypes, acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). Methods. We linked California cancer and birth registries to obtain information on 5788 cases and 5788 controls matched on age and sex (1:1). We examined the association of birth weight, gestational age, birth and pregnancy order, parental ages, and specific conditions during pregnancy and risk of total leukemia, ALL and AML using conditional logistic regression, with adjustment for potential confounders. Results. The odds ratio (OR) per 1000 g increase in birth weight was 1.11 for both total leukemia and ALL. The OR were highest for babies weighing ≥4500 g with reference <2500 g: 1.59 (95% CI: 1.05–2.40) and 1.70 (95% CI: 1.08–2.68) for total leukemia and ALL, respectively. For AML, increase in risk was also observed but the estimate was imprecise due to small numbers. Compared to average-for-gestational age (AGA), large-for-gestational age (LGA) babies were at slightly increased risk of total childhood leukemia (OR = 1.10) and both ALL and AML (OR = 1.07 and OR = 1.13, respectively) but estimates were imprecise. Being small-for-gestational age (SGA) was associated with reduced risk of childhood leukemia (OR = 0.81, 95% CI: 0.67–0.97) and ALL (OR = 0.77, 95% CI: 0.63–0.94), but not AML. Being first-born was associated with decreased risk of AML only (OR = 0.70; 95% CI: 0.53–0.93). Compared to children with paternal age <25 years, children with paternal age between 35 and 45 years were at increased risk of total childhood leukemia (OR = 1.12; 95% CI: 1.04–1.40) and ALL (OR = 1.23; 95% CI: 1.04–1.47). None of conditions during pregnancy examined or maternal age were associated with increased risk of childhood leukemia or its subtypes. Conclusions. Our results suggest that high birth weight and LGA were associated with increased risk and SGA with decreased risk of total childhood leukemia and ALL, being first-born was associated with decreased risk of AML, and advanced paternal age was associated with increased risk of ALL. These findings suggest that associations of childhood leukemia and perinatal factors depend highly on subtype of leukemia.     

High FLT3 expression and IL10 (G1082A) polymorphism in poor overall survival in calla acute lymphoblastic leukemia

Patients with acute lymphoblastic leukemia presenting the immunophenotypic marker CD10+ (calla), usually has treatment profile good. The FLT3 molecular marker is listed as a prognostic factor, an important leukaemogenic marker in acute leukemias, also the polymorphism (G1082A) of the IL10 interleukin can to present pleiotropic effects in many diseases and could is associated to development of ALL. However, the FLT3 expression is variability among patients with calla-ALL. The aim of this study was to determine the FLT3 expression, to associate with the genotypes and allelic of G1082A (IL10) in 50 patients with calla-ALL and assess the overall survival at 98 months follow-up. The expression was assessed by quantitative real time PCR (RT-PCR), the G1082A polymorphism was identified by allele-specific PCR and for immunophenotypic classification was used specific markers of B lineage-calla. We observed that patients who died showed higher FLT3 expression (p = 0.005), worse survival (p = 0.0137) and the IL10G allele may favor the survival, because the IL10 GG and IL10 GA genotypes showed a low FLT3 expression (p < 0.05).