GPR56 contributes to the development of acute myeloid leukemia in mice.

The G protein-coupled receptor 56 (GPR56) was identified as part of the molecular signature of functionally validated leukemic stem cells isolated from patients with acute myeloid leukemia (AML). This report now demonstrates particularly high expression of GPR56 in patients with mutant NPM1 and FLT3-length mutation and association of high GPR56 expression with inferior prognosis in a large patient cohort treated in two independent multicenter phase III trials. Functional relevance of GPR56 expression was validated in mice, in which co-expression of Gpr56 significantly accelerated HOXA9-induced leukemogenesis and vice versa knockdown of Gpr56 delayed onset of HOXA9/MEIS1-induced AML. Overexpression of Gpr56 grossly changed the molecular phenotype of Hoxa9-transduced cells affecting pathways involved in G protein-coupled receptors (GPRCs) and associated intracellular signaling. Blockage of surface GPR56 by an anti-GPR56 antibody successfully impaired engraftment of primary human AML cells. In summary, these data demonstrate that high expression of GPR56 is able to contribute to AML development and characterize the GPR56 as a potential novel target for antibody-mediated antileukemic strategies.

The leukemogenicity of Hoxa9 depends on alternative splicing.

Although the transforming potential of Hox genes is known for a long time, it is not precisely understood to which extent splicing is important for the leukemogenicity of this gene family. To test this for Hoxa9, we compared the leukemogenic potential of the wild-type Hoxa9, which undergoes natural splicing, with a full-length Hoxa9 construct, which was engineered to prevent natural splicing (Hoxa9FLim). Inability to undergo splicing significantly reduced in vivo leukemogenicity compared to Hoxa9-wild-typed. Importantly, Hoxa9FLim could compensate for the reduced oncogenicity by collaborating with the natural splice variant Hoxa9T, as co-expression of Hoxa9T and Hoxa9FLim induced acute myeloid leukemia (AML) after a comparable latency time as wild-type Hoxa9. Hoxa9T on its own induced AML after a similar latency as Hoxa9FLim, despite its inability to bind DNA. These data assign splicing a central task in Hox gene mediated leukemogenesis and suggest an important role of homeodomain-less splice variants in hematological neoplasms.

CALM/AF10-positive leukemias show upregulation of genes involved in chromatin assembly and DNA repair processes and of genes adjacent to the breakpoint at 10p12.

The t(10;11)(p12;q14) is a recurring chromosomal translocation that gives rise to the CALM/AF10 fusion gene, which is found in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma. We analyzed the fusion transcripts in 20 new cases of CALM/AF10-positive leukemias, and compared the gene expression profile of 10 of these to 125 patients with other types of leukemia and 10 normal bone marrow samples. Based on gene set enrichment analyses, the CALM/AF10-positive samples showed significant upregulation of genes involved in chromatin assembly and maintenance and DNA repair process, and downregulation of angiogenesis and cell communication genes. Interestingly, we observed a striking upregulation of four genes located immediately centromeric to the break point of the t(10;11)(p12;q14) on 10p12 (COMMD3 (COMM domain containing 3), BMI1 (B lymphoma Mo-MLV insertion region 1 homolog), DNAJC1 (DnaJ (Hsp40) homolog subfamily C member 1) and SPAG6 (sperm associated antigen 6)). We also conducted semiquantitative reverse transcriptase-PCR analysis on leukemic blasts from a murine CALM/AF10 transplantation model that does not have the translocation. Commd3, Bmi1 and Dnajc1, but not Spag6 were upregulated in these samples. These results strongly indicate that the differential regulation of these three genes is not due to the break point effect but as a consequence of the CALM/AF10 fusion gene expression, though the mechanism of regulation is not well understood.

FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia.

The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294-335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6.

Proteomic identification of the MYST domain histone acetyltransferase TIP60 (HTATIP) as a co-activator of the myeloid transcription factor C/EBPalpha.

The transcription factor C/EBPalpha (CEBPA) is a key player in granulopoiesis and leukemogenesis. We have previously reported the interaction of C/EBPalpha with other proteins (utilizing mass spectrometry) in transcriptional regulation. In the present study, we characterized the association of the MYST domain histone acetyltransferase Tat-interactive protein (TIP) 60 (HTATIP) with C/EBPalpha. We show in pull-down and co-precipitation experiments that C/EBPalpha and HTATIP interact. A chromatin immunoprecipitation (ChIP) and a confirmatory Re-ChIP assay revealed in vivo occupancy of the C/EBPalpha and GCSF-R promoter by HTATIP. Reporter gene assays showed that HTATIP is a co-activator of C/EBPalpha. The co-activator function of HTATIP is dependent on its intact histone acetyltransferase (HAT) domain and on the C/EBPalpha DNA-binding domain. The resulting balance between histone acetylation and deacetylation at the C/EBPalpha promoter might represent an important mechanism of C/EBPalpha action. We observed a lower expression of HTATIP mRNA in undifferentiated U937 cells compared to retinoic acid-induced differentiated U937 cells, and correlated expression of CEBPA and HTATIP mRNA levels were observed in leukemia samples. These findings point to a functional synergism between C/EBPalpha and HTATIP in myeloid differentiation and suggest that HTATIP might be an important player in leukemogenesis.