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Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.

Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent haemopoietic stem cell, the hallmark of which is the constitutively activated Bcr-Abl protein tyrosine kinase. During the initial chronic phase of CML the primitive multipotent leukaemic progenitor cells remain growth factor dependent and are capable of producing terminally differentiated cells. Although the available evidence suggests that Bcr-Abl directly affects signalling pathways involved in controlling the development of primitive haemopoietic progenitors the identification of the specific biological consequences of Bcr-Abl activity in these progenitors has been hampered by the lack of suitable systems modelling CML. By transfecting the multipotent haemopoietic cell line FDCP-Mix with a temperature sensitive mutant of Bcr-Abl we have developed the first working model that mirrors the chronic phase of CML. FDCP-Mix cells expressing Bcr-Abl tyrosine kinase activity remain growth factor dependent and retain their ability to differentiate. Normal neutrophilic cells are formed in response to G-CSF and GM-CSF. In addition, the transfected FDCP-Mix cells grown at the permissive temperature for Bcr-Abl tyrosine kinase activity display enhanced survival and proliferation in low concentrations of growth factor. These findings are consistent with the initial subtle changes seen in CML progenitor cells during the chronic phase and confirm that Bcr-Abl effects are context specific, i.e. they depend on the origin and developmental potential of the transfected cells. This questions the significance of studies in non-haemopoietic and differentiation blocked haemopoietic cells.

Fms interacting protein (FMIP) is a substrate for Fms tyrosine kinase, and a nuclear/cytoplasm shuttling protein with a leucine zipper. As the phosphorylation of FMIP is observed in insulin-stimulated preadipocytes, we examined the role of FMIP in adipocyte differentiation, using the mesenchymal multipotent stem cells, C2C12 cells, that can differentiate into adipocytes, muscle cells and osteoblasts. Ectopic expression of FMIP in C2C12 impairs the adipocyte differentiation induced by treatment with insulin, dexamethasone and 3-isobutyl-1-methylxanthine. These cells exhibit muscle phenotype with multinuclear morphology. Furthermore, knockdown of endogenous FMIP expression by small interfering RNA improves adipocytic lineage commitment of C2C12 cells, while impairing muscle differentiation. Upon stimulation with insulin, CCAAT/enhancer binding protein (C/EBP)beta, but not C/EBPalpha, is upregulated in cells expressing ectopic FMIP, whereas in FMIP knockdown cells, C/EBPalpha is constitutively expressed. Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown partially mimics the effects of FMIP in this system. Northern blot analysis and reverse transcriptase-polymerase chain reaction study reveal that ectopic FMIP-expressing cells do not contain the polyadenylated C/EBPalpha mRNA, but contain the C/EBPalpha pre-mRNA, suggesting that FMIP plays a role in RNA processing and/or export. Indeed, a member of the THO complex that plays a role in mRNA export, THOC1, is co-precipitated with FMIP. The data we have acquired on FMIP suggest that it is a target for tyrosine kinase receptors that potentiate mRNA export.

Macrophage colony-stimulating factor (M-CSF or CSF-1) and its cognate receptor, the tyrosine kinase c-fms, are essential for monocyte and macrophage development. We have recently identified an Fms-interacting protein (FMIP) that binds transiently to the cytoplasmic domain of activated Fms molecules and is phosphorylated on tyrosine by Fms tyrosine kinase. FMIP is a substrate not only for Fms but also for protein kinase C (PKC). Mutagenesis reveals that this occurs on serines 5 and 6. Adjacent to these sites is a nuclear localization signal (NLS). We show that this NLS is essential for the predominantly nuclear localization of FMIP. Generation of phosphomimetic substitutions on serine residues 5 and 6 confirms that PKC-mediated phosphorylation on this site leads to translocation of FMIP to the cytosol. Furthermore, the mutant FMIP (FMIPSS5,6AA) was detected abundantly in the nucleus even in the presence of activated PKCalpha. Wild-type FMIP and FMIPSS5,6AA inhibited M-CSF-mediated survival signaling, while FMIPSS5,6EE-expressing cells survived and differentiated into macrophages more efficiently than wild-type cells in the presence of M-CSF or TPA. We conclude M-CSF-mediated activation of PKCalpha can potentiate FMIP action to initiate survival/differentiation signaling.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.