Down-regulation of homeobox genes MEIS1 and HOXA in MLL-rearranged acute leukemia impairs engraftment and reduces proliferation

Rearrangements of the MLL (ALL1) gene are very common in acute infant and therapy-associated leukemias. The rearrangements underlie the generation of MLL fusion proteins acting as potent oncogenes. Several most consistently up-regulated targets of MLL fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally related and have been implicated in other types of leukemias. Each of the four genes was knocked down separately in the human precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mutant and control cells were compared for engraftment in NOD/SCID mice. Engraftment of all mutants into the bone marrow (BM) was impaired. Although homing was similar, colonization by the knockdown cells was slowed. Initially, both types of cells were confined to the trabecular area; this was followed by a rapid spread of the WT cells to the compact bone area, contrasted with a significantly slower process for the mutants. In vitro and in vivo BrdU incorporation experiments indicated reduced proliferation of the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, as evidenced by reduced migration toward an SDF-1 gradient and loss of SDF-1-augmented proliferation in culture. The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.     

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211–8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.     

Increased expression of HOXA9 gene in Hirschsprung disease

OBJECTIVE: Hirschsprung disease is characterized by segmental aganglionosis of the terminal bowel. Neurons of the enteric nervous system arise from neural crest, migrate and colonize intestinal muscle coat where they proliferate and differentiate. The first pathophysiologic hypothesis suggests an absence of neural cell migration. The most recent hypothesis involves disorders of their homing and/or their differentiation due to an altered intestinal microenvironment. PATIENTS AND METHODS: We analyzed the expression of muscle markers and laminin isoforms by immunocytochemistry and of homeotic genes involved in the regionalization of the intestinal mesenchyme during development (HOXA4, HOXA9, HOXD9) by RT-PCR, in colon specimens from two children with Hirschsprung disease (pathological and transition regions) and from healthy adult controls. RESULTS: We showed an increase in HOXA9 gene expression in Hirschsprung disease specimens while HOXA4 and HOXD9 mRNA expressions were unchanged. No significant differences in the muscle markers nor in the laminin isoforms were noted. CONCLUSION: These data suggest that intrinsic dysregulation of the intestinal wall microenvironment could account for the pathophysiology of Hirschsprung disease.     

HOXA genes are included in genetic and biologic networks defining human acute T-cell leukemia (T-ALL)

Using a combination of molecular cytogenetic and large-scale expression analysis in human T-cell acute lymphoblastic leukemias (T-ALLs), we identified and characterized a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA and the TCRB locus. Real-time quantitative polymerase chain reaction (RQ-PCR) analysis showed that the expression of the whole HOXA gene cluster was dramatically dysregulated in the HOXA-rearranged cases, and also in MLL and CALM-AF10-related T-ALL cases, strongly suggesting that HOXA genes are oncogenic in these leukemias. Inclusion of HOXA-translocated cases in a general molecular portrait of 92 T-ALLs based on large-scale expression analysis shows that this rearrangement defines a new homogeneous subgroup, which shares common biologic networks with the TLX1- and TLX3-related cases. Because T-ALLs derive from T-cell progenitors, expression profiles of the distinct T-ALL subgroups were analyzed with respect to those of normal human thymic subpopulations. Inappropriate use or perturbation of specific molecular networks involved in thymic differentiation was detected. Moreover, we found a significant association between T-ALL oncogenic subgroups and ectopic expression of a limited set of genes, including several developmental genes, namely HOXA, TLX1, TLX3, NKX3-1, SIX6, and TFAP2C. These data strongly support the view that the abnormal expression of developmental genes, including the prototypical homeobox genes HOXA, is critical in T-ALL oncogenesis.     

Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia

Identification of the targets of mixed lineage leukemia (MLL) fusion genes will assist in understanding the biology of MLL fusion gene leukemias and in development of better therapies. Numerous studies have implicated HOXA9 as one of the possible targets of MLL fusion proteins. To determine if HOXA9 was required for leukemia development by MLL fusion genes, we compared the effects of the Mll-AF9 knock-in mutation in mice in the presence or absence of Hoxa9. Both groups of mice showed myeloid expansion at 8 weeks and then developed myeloid leukemia with a similar incidence and time course. The leukemia in the mice lacking Hoxa9 generally displayed a more immature myeloid phenotype than that in the mice that were wild-type for Hoxa9. Gene expression profiling revealed that expression of Mll-AF9 led to overexpression of Hoxa5, Hoxa6, Hoxa7, Hoxa9, and Hoxa10. Thus, genes of the Hox-a cluster are important in defining the phenotype but not the incidence of Mll-AF9 leukemia. These results demonstrate that the Mll-AF9 fusion gene disrupts the expression of several Hox genes, none of which as a single gene is likely to be necessary for development of leukemia. Instead, we propose that the "Hox code" minimally defined by the Hoxa5-a9 cluster is central to MLL leukemogenesis.     

Concomitant inhibition of DNA methyltransferase and BCL-2 protein function synergistically induce mitochondrial apoptosis in acute myelogenous leukemia cells

DNA methylation and BLC-2 are potential therapeutic targets in acute myeloid leukemia (AML). We investigated pharmacologic interaction between the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the BCL-2 inhibitor ABT-737. Increased BCL-2 expression determined by reverse phase protein analysis was associated with poor survival in AML patients with unfavorable cytogenetics (n?=?195). We found that 5-AZA, which itself has modest apoptotic activity, acts synergistically with ABT-737 to induce apoptosis. The 5-AZA/ABT-737 combination enhanced mitochondrial outer membrane permeabilization, as evidenced by effective conformational activation of BAX and ?ψ_m loss. Although absence of p53 limited apoptotic activities of 5-AZA and ABT-737 as single agents, the combination synergistically induced apoptosis independent of p53 expression. 5-AZA down-regulated MCL-1, known to mediate resistance to ABT-737, in a p53-independent manner. The 5-AZA/ABT-737 combination synergistically induced apoptosis in AML cells in seven of eight patients. 5-AZA significantly reduced MCL-1 levels in two of three samples examined. Our data provide a molecular rationale for this combination strategy in AML therapy.     

Combinations of recombinant human interferons and retinoic acid synergistically induce differentiation of the human promyelocytic leukemia cell line HL-60

The human acute promyelocytic leukemia cell line HL-60 is induced to differentiate into morphologically and functionally mature monocytelike cells by incubation with a combination of 10 nmol/L retinoic acid (RA) and various concentrations of recombinant immune interferon (rIFN- gamma). These induced cells show marked increases in antibody-dependent cellular cytotoxicity (ADCC), antibody-coated erythrocyte (EA) rosettes, nonspecific esterase, and 5'-nucleotidase activity. rIFN- gamma alone at concentrations of 10 to 1,000 U/mL has essentially no effect on morphological maturation, nitroblue tetrazolium reduction, and immunophagocytosis. However, rIFN-gamma at these concentrations increases EA rosetting in a concentration-dependent manner that is not affected by 10 nmol/L RA. At a concentration of 1,000 U/ml, rIFN-gamma induces moderate increases in nonspecific esterase, 5'-nucleotidase, and ADCC. These parameters are markedly increased by the addition of 10 nM RA, a concentration which alone has no effect on these markers. Based on units of antiviral activity, rIFN-gamma is tenfold more active than rIFN-alpha D in inducing EA rosettes and 40-fold more active in inducing nitroblue tetrazolium reduction and immunophagocytosis. These results, indicating that combinations of rIFN-gamma or rIFN-alpha and RA synergistically induce differentiation of HL-60, suggest that this combination may have clinical utility in the treatment of patients with certain leukemias.