scholarly journals Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2969-2976 ◽  
Author(s):  
Kazushi Inoue ◽  
Hiroya Tamaki ◽  
Hiroyasu Ogawa ◽  
Yoshihiro Oka ◽  
Toshihiro Soma ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Wilms Tumor ◽  
Suppressor Gene ◽  
Hematopoietic Progenitor Cells ◽  
Wt1 Gene ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Oncogenic Function

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

The Wilms’ Tumor Suppressor Wt1 Activates Transcription of the Erythropoietin-Receptor in Hematopoietic Progenitor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1188-1188
Author(s):  
Christof Dame ◽  
Karin M. Kirschner ◽  
Patricia Hagen ◽  
Christiane S. Hussels ◽  
Holger Scholz
Keyword(s):  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Wilms Tumor ◽  
Regulation Of Gene Expression ◽  
Erythropoietin Receptor ◽  
Hematopoietic Progenitor Cells ◽  
Luciferase Reporter ◽  
Recombinant Erythropoietin ◽  
Wild Type ◽  
Hematopoietic Progenitor

Abstract The Wilms’ tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.

The Wilms' tumor suppressor Wt1 activates transcription of the erythropoietin receptor in hematopoietic progenitor cells

2008 ◽  
Vol 22 (8) ◽  
pp. 2690-2701 ◽  
Author(s):  
Karin M. Kirschner ◽  
Patricia Hagen ◽  
Christiane S. Hussels ◽  
Matthias Ballmaier ◽  
Holger Scholz ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Wilms Tumor ◽  
Erythropoietin Receptor ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

scholarly journals Aberrant Overexpression of the Wilms Tumor Gene (WT1) in Human Leukemia

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1405-1412 ◽  
Author(s):  
Kazushi Inoue ◽  
Hiroyasu Ogawa ◽  
Yoshiaki Sonoda ◽  
Takafumi Kimura ◽  
Hideaki Sakabe ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Wilms Tumor ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Cell Populations ◽  
Human Leukemia ◽  
Wt1 Gene ◽  
Hematopoietic Progenitor ◽  
Expression Levels ◽  
Hla Dr

Abstract To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (<10−2) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 × 10−1. Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Safak Yalcin ◽  
Sathish Kumar Mungamuri ◽  
Dragan Marinkovic ◽  
Xin Zhang ◽  
Wei Tong ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

scholarly journals JARID2 Functions as a Tumor Suppressor in Myeloid Neoplasms by Repressing Self-Renewal in Hematopoietic Progenitor Cells

Cancer Cell ◽  
2018 ◽  
Vol 34 (5) ◽  
pp. 741-756.e8 ◽  
Author(s):  
Hamza Celik ◽  
Won Kyun Koh ◽  
Ashley C. Kramer ◽  
Elizabeth L. Ostrander ◽  
Cates Mallaney ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Myeloid Neoplasms

scholarly journals Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1591-1591
Author(s):  
Juliana M. Xavier ◽  
Lauremilia Ricon ◽  
Karla Priscila Vieira ◽  
Longhini Ana Leda ◽  
Carolina Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 796-796 ◽  
Author(s):  
Windy D. Berkofsk-Fessler ◽  
Jonathan D. Licht ◽  
Melanie-Jane McConnell ◽  
Donna S. Neuberg ◽  
Timothy S. Bowler ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Tumor Suppressor Genes ◽  
Hematopoietic Progenitor Cells ◽  
Multiple Testing Correction ◽  
Hematopoietic Progenitor ◽  
Suppressor Genes ◽  
Putative Tumor Suppressor ◽  
Cd34 Cells

Abstract Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1269-1269
Author(s):  
Colleen E. Annesley ◽  
Rachel E. Rau ◽  
Daniel Magoon ◽  
David Loeb ◽  
Patrick Brown
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Wilms Tumor ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Exon 9

Abstract Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

Btg1-Deficiency Promotes ETV6-RUNX1-Mediated Leukemic Transformation By Upregulation of BCL6

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5193-5193
Author(s):  
Esther Tijchon ◽  
Liesbeth van Emst ◽  
Dorette van Ingen Schenau ◽  
Laurens T van der Meer ◽  
Simone de Rijk ◽  
...  
Keyword(s):  
B Cell ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Housekeeping Gene ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Capacity ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Targeted Deletion ◽  
Effector Pathways

Abstract Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The ETV6-RUNX1 translocation arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are commonly observed in BCP-ALL (9%), but are significantly enriched in ETV6-RUNX1-positive leukemia (25%). The BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors. Here, we show that ETV6-RUNX1 interacts both with BTG1 and PRMT1, and this interaction is lost in c-Kit+Ter-119-Btg1-/- fetal liver (FL) derived hematopoietic progenitors (HPCs). Moreover, targeted deletion of Btg1 enhanced the proliferative capacity of ETV6-RUNX1 in FL-HPCs as measured by enhanced colony-forming and serial replating capacity (Figure 1). The combined loss of Btg1 function and ETV6-RUNX1 expression correlated with strong upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53 (Figure 2). Similarly, ectopic expression of BCL6 promoted both proliferation and replating capacity of FL-derived progenitor cells in the presence of SCF, FLT3L and IL-7. This phenotype correlated with a fivefold suppression of p19Arf and a twofold suppression of Tp53 expression. Inhibition of BCL6 in a variety of human BCP-ALL cell lines by the peptide inhibitor RI-BPI resulted in decreased proliferation and induction of apoptosis as measured by Annexin-V staining. These included the ETV6-RUNX1-positive cell lines UOC-B6, AT-2 and REH, the BCR-ABL1-positive cell line SD1, as well as Nalm6. Together our results point to a novel role for BCL6 in promoting cell proliferation of primitive progenitor B cells and suggest that targeted inhibition of BCL6 may be effective in the treatment of various BCP-ALL subtypes. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (>30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P< 0.05, **, P< 0.01. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (>30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P< 0.05, **, P< 0.01. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P< 0.05, **, P< 0.01, ***, P< 0.001. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P< 0.05, **, P< 0.01, ***, P< 0.001. Disclosures No relevant conflicts of interest to declare.

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