Loss of Titan (Samd9L), a Candidate -7/7q- Responsible Gene Encoding An Actin Remodeling Regulator, Develops MDS/AML in Cooperation with Evi1 or Fbxl10.

Abstract 2963

Poster Board II-939

From a common microdeletion cluster located in chromosome subband 7q21.3, we identified three candidate responsible genes (Kasumi=Samd9, Titan=Samd9L and Miki=LOC253012) which encode myeloid-tumor suppressors (BBRC 2009). As we presented previously (ASH annual meeting, 2008), Miki encodes a centrosomal protein and is involved in myelodysplasia and chromosomal instability. On the other hand, Kasumi and Titan, that encode 60% identical proteins, are poorly characterized. Recent reports revealed that bi-allelic point mutations in Kasumi gene cause a rare fatal skin disease, Normophosphatemic Familial Tumoral Calcinosis (NFTC). To identify the contributions of these genes to leukemogenesis, we initially generated Titan deficient mice (mouse genome contains only Titan and lacks Kasumi gene). Although heterozygous (titan+/−) and homozygous (titan-/-) mice were born and grown normally without hematological abnormalities, they naturally developed AML at high frequency after they reached 20 months old. This unusually long latency suggests that additional gene alterations are required for leukemia development. Thus we attempted to accelerate the onset of leukemia by retroviral insertional mutagenesis. Virus infection induced various myeloid leukemias after 10 to 12 months in almost all titan+/− and titan-/- mice. Inverse PCR detected two common virus integration sites specific for titan+/− and titan-/- mice, which induced deregulated expression of a zinc finger transcription factor, Evi1, and a histone H3K36 demethylase, Fbxl10. We next performed mouse BMT using titan-/- and +/+ bone marrow cells transduced with Evi1 retrovirally. Mice transplanted with titan(+/+)/Evi1 overexpressing cells developed MDS or AML after 7 months. By contrast, most mice transplanted with titan(-/-)/Evi1 overexpressing cells developed AML within 6 months after BMT, confirming co-operation between loss of Titan and Evi1 overexpression in myeloid leukemogenesis. Both Evi1 and Fbxl10 are reported to downregulate p15^Ink4b tumor suppressor gene. Moreover, in human secondary MDS, DNA methylation in the promoter region of p15^Ink4b is closely associated with 7q deletion. Thus we compared Fbxl10 and p15^Ink4b expressions between AML/MDS samples with or without 7q deletion. We found that 7q deletion was correlated with higher Fbxl10 and with lower p15^Ink4b levels, suggesting that silencing of p15^ink4b through transcriptional and epigenetical mechanisms would be involved in leukemia with 7q deletion. To elucidate the function of Kasumi and Titan, we firstly immunoprecipitated Titan binding proteins from FLAG-Titan expressing cells and identified two specific bands around 150 and 70KDa. Mass spectrometry analysis showed that they correspond to Flightless1 (Fli1) and Scinderin (Scin), respectively. Because both Fli1 and Scin belong to the gelsolin superfamily proteins that bind to and sever actin filaments, we speculated that Titan is involved in cell movement via actin remodeling. To analyze this, we established Titan-knockdown (K/D) mouse fibloblasts by introducing Titan-specific shRNA-expressing vectors and observed their migration under time-lapse microscopy. In wound-healing assay, Titan-K/D cells migrated slower towards wound edge with loss of polarity. Each cell moved restlessly by quickly changing the direction. In these cells, lamellipodial protrusions rapidly formed and retracted. In accordance with this phenotype, activity of Rac1, a Rho GTPase, increased in Titan-K/D cells. This abnormality in cell migration is likely involved in pathogenesis of NFTC, i.e., severe inflammations in skin and mucosae. Moreover, since recent reports revealed the contribution of hyperactivated Rac1 to transformation of hematopoietic stem cells through abnormal actin remodeling, our findings suggest that deletion of Kasumi or Titan is a cue to cause AML/MDS through aberrant Rac1 activation.