Block In Differentiation Of Myeloid Cells Research Articles

Isocitrate Dehydrogenase 2 Mutation Drives Bone Marrow Macrophage Dysfunction without a Complete Block in Hematopoietic Differentiation

Myelodysplastic syndromes (MDS) represent a broad category of myeloid malignancies characterized by myeloid skewed hematopoiesis, blood cytopenias and increased risk of transformation to leukemia. While not common, isocitrate dehydrogenase 2 (IDH2) mutations are represented in clonal hematopoiesis and in MDS, are found in patients with higher International Prognosis Scoring System (IPSS) disease scores and confer a higher risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite which disrupts hematopoiesis. However, the mechanisms that induces clonal progression and transformation remains incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutation on mature myeloid cell functions and the bone marrow microenvironment (BMME) remains to be elucidated. Using RAW264.7 cells, a monocyte/macrophage-like murine cell line, we retrovirally infected cells with human wt IDH2 or the R172K IDH2 mutant, a common IDH2 mutation. After confirming a 30-fold increase of 2HG (p<.005) and observing evidence of hypermethylation in the IDH2 mutant RAW cells, we tested several key functions of bone marrow macrophages: ability to engulf apoptotic cells (efferocytosis), erythropoietic support, and cytokine production. After labeled apoptotic neutrophils were fed as efferocytic targets to RAW cells at a 10:1 ratio for 6 hours, we observed a 10% decrease of efferocytosis in IDH2 mutants (p < 0.05). Next, we cocultured RAW cells with primary murine late-stage erythroid progenitors and quantified their capacity to form colonies (CFU-E) in the presence of erythropoietin. Compared to wt, IDH2 mutant RAW cells inhibited CFU-E formation by 25% (p < 0.0075). Cytokine array on cell culture supernatant from mutant RAW cells had elevated proinflammatory chemokines/cytokines (CCL5, CXCL10, TNF-a) and decreased cytokines related to inflammation resolution and efferocytosis (GDF-15, Gas6, Endostatin, CCL2). To test these findings further in vivo we generated a murine bone marrow transplant model with retrovirally infected hematopoietic stem cells (HSC: lineage-, c-Kit+, Sca-1+) containing the human wt or R172K IDH2 mutation with an mCherry reporter and transplanted into CD45.1 congenic mice (10 mutant, 10 wt, 1:1 male:female). Mutant IDH2 HSCs demonstrated better engraftment (85-90%) compared to wt (70-80%) and also had higher levels of mCherry positivity within CD45.2+ cells (80-90% mutant vs 40-60% in wt). Longitudinal blood draws showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes in IDH2 mutants by 16 weeks post-transplant. In contrast to current literature, IDH2 mutant cells did not exhibit a complete block in myeloid cell differentiation, as the mutant clone gave rise to mature monocytes, macrophages, and neutrophils. We also harvested bone marrow at 16 weeks post transplantation. Compared to wt IDH2, the mutant IDH2 bone marrow cells exhibited myeloid skewing hematopoiesis and a 3-fold increase in bone marrow monocytes (CD45+, Ly6G-, Ly6C++, F4/80+, p < 0.00001). In the IDH2 mutant bone marrow cells, myeloid progenitors were also expanded: common myeloid progenitors (CMPs: 4-fold increase p < 0.001, CD45+, lineage-, c-Kit+, Sca-1-, CD34+, CD16/32-), and granulocytic monocytic precursors (GMPs: 3-fold increase p < 0.0001, CD45+, c-Kit+, Sca-1-, CD34+, CD16/32+). We also observed a previously unreported loss of long-term hematopoietic stem cells (LT-HSC: 13.5-fold reduction, p < 0.00001, CD45+, lineage-, c-Kit+, Sca-1+, Flt3-, CD48-, CD150+). When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow derived macrophages fed normal apoptotic neutrophils at a 10:1 ratio for 3 or 24 hours. In summary, we identify defects in efferocytic function and cytokine production in monocytes and macrophages derived from IDH2 mutant hematopoietic stem and progenitor cells. We have previously shown that defects in the efferocytic function of bone marrow macrophages induce increases in inflammatory signals and contribute to skewing of hematopoiesis. Since these macrophage dysfunctions have not previously been attributed to IDH2 single mutation, they represent a potential novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and transformation to leukemia.

USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism

AbstractRas mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic KrasG12D/+. USP22 deficiency in KrasG12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient KrasG12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient KrasG12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient KrasG12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis.

Elevated PIN1 expression by C/EBPα-p30 blocks C/EBPα-induced granulocytic differentiation through c-Jun in AML

The transcription factor CCAAT enhancer-binding protein alpha (C/EBPalpha) has an important role in granulopoiesis. The tumor suppressor function of C/EBPalpha is shown by the findings that loss of expression or function of C/EBPalpha in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPalpha mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPalpha (C/EBPalpha-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPalpha-p30 in AML. In the present study, we show that C/EBPalpha-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPalpha-p30 results in recruitment of E2F1 in the PIN1 promoter. We show that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPalpha mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also show that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination, and c-Jun blocks granulocyte differentiation mediated by C/EBPalpha. Our data suggest that the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPalpha mutation.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn't block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

P53-independent apoptosis associated with c-Myc-mediated block in myeloid cell differentiation.

Previously we have shown that deregulated expression of c-myc in M1 myeloid leukemic cells blocked IL-6-induced differentiation and its associated growth arrest; however, the cells proliferated at a significantly reduced rate compared to untreated cells. The basis for the increased doubling time of IL-6-treated M1myc cells was found to be due to the induction of a p53-independent apoptotic pathway. The apoptotic response was not completely penetrant; in the same population of cells both proliferation and apoptosis were continuously ongoing. Down-regulation of Bcl-2 was insufficient to account for the apoptotic response, since deregulated expression of Bcl-2 delayed, but did not block, the onset of apoptosis. Furthermore, our results indicated that the IL-6-induced partial hypophosphorylation of the retinoblastoma gene product (Rb), observed in M1myc cells, was not responsible for the apoptotic response. Finally, the findings in M1 cells were extended to myeloid cells derived from the bone marrow of wild type and p53-deficient mice, where the deregulated expression of c-myc was also shown to block terminal differentiation and induce apoptosis independent of p53. These findings provide new insights into how myc participates in the neoplastic process, and how additional mutations can promote more aggressive tumors. Oncogene (2000) 19, 2967 - 2977

The promyelocytic leukemia protein PML interacts with the proline-rich homeodomain protein PRH: a RING may link hematopoiesis and growth control.

Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML is disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis.