JAK2V617F Mutation Selectively Exerts the STAT3 Pathway for Enhancing a Neutrophil Activation Marker.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1901-1901
Author(s):  
Seido Oku ◽  
Katsuto Takenaka ◽  
Kotaro Shide ◽  
Takashi Kumano ◽  
Kikushige Yoshikane ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Conflicts Of Interest ◽  
Neutrophil Activation ◽  
Myelogenous Leukemia ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Stat3 Pathway ◽  
Nb4 Cells ◽  
Pi3k Inhibitor Ly294002 ◽  
Activation Marker

Abstract Abstract 1901 Poster Board I-924 Leukocyte alkaline phosphatase (LAP) is considered as a neutrophil activation marker. The level of LAP is quantitated as the LAP score. It is well known that patients with chronic myelogenous leukemia (CML) usually have low LAP scores, whereas those with BCR-ABL negative chronic myeloproliferative disorders (MPD) have elevated LAP scores. In CML patients, the premature release of granulocytes from the bone marrow into the peripheral blood is considered as the cause of low LAP scores. However, the reason for elevated LAP scores in BCR-ABL negative MPD patients has been unclear. An acquired JAK2V617F mutation is observed in most patients with BCR-ABL negative MPD. It has been shown that the JAK2V617F mutation induces constitutive activation of its downstream signaling pathways such as STAT3/STAT5, Ras/MAPK and PI3K pathways. We speculated that an elevated LAP score might be due to the activation of Jak2 downstream pathways through the JAK2V617F mutation. We analyzed LAP expression in BCR-ABL negative MPD patients. JAK2V617F homozygous patients had higher LAP expression than JAK2V617F heterozygous or negative patients. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of JAK2V617F positive patients. The myeloid cell line NB4 was transfected with the JAK2V617F mutation and a wild-type Jak2 using lentivirus vectors. It was observed that the JAK2V617F mutation, but not wild-type Jak2, enhanced cell proliferation. Then the LAP expression in NB4 cells was evaluated after these cells were differentiated by all-trans retinoic acid and granulocyte colony-stimulating factor. It was observed that the JAK2V617F mutation, but not wild-type Jak2, increased LAP expression. Next, we examined which of the Jak2 downstream pathways played a major role in increasing LAP expression and prompting cell proliferation. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, STAT3 siRNA and STAT5 siRNA, we demonstrated that the JAK2V617F mutation primarily used the STAT3 pathway to increase LAP expression. On the other hand, the JAK2V617F mutation used the STAT5, the Ras/MAPK and the PI3K pathways, but not the STAT3 pathway, to prompt proliferation of NB4 cells. In conclusion, we obtained direct evidence that the JAK2V617F mutation induced elevation of LAP scores via the STAT3 pathway, and prompted proliferation of NB4 cells via the STAT5, the Ras/MAPK and the PI3K pathways. Our findings showed the possibility that the JAK2V617F mutation might use specific downstream pathways depending on various phenotypic manifestations of BCR-ABL negative MPD. Disclosures: No relevant conflicts of interest to declare.

Loss of Stress Sensor GADD45a and GADD45b Accelerates BCR-ABL-Driven Leukemogenesis Via Distinct Signaling and Cellular Pathways.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1217-1217
Author(s):  
Xiaojin Sha ◽  
Barbara Hoffman ◽  
Dan Liebermann
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Signaling Pathways ◽  
Conflicts Of Interest ◽  
Multiple Stressors ◽  
Myelogenous Leukemia ◽  
Ras Signaling ◽  
Wild Type ◽  
Cellular Pathways ◽  
Myeloid Progenitors

Abstract Abstract 1217 The bcr/abl oncogene causes chronic myelogenous leukemia (CML) in humans. BCR/ABL is known to localize to the cytoskeleton and to display a constitutively active tyrosine kinase activity that leads to the recruitment of downstream effectors of cell proliferation and survival. This is accomplished via several adapter proteins and signaling pathways, including Ras, PI3K-AKT, PkD2-NFkB and JAK-STAT5, all of which are believed to participate in the pathogenesis of CML. The complex nature of these signaling pathways and how they contribute to the initiation and progression of CML is only partially understood. The Gadd45 family of genes (Gadd45a, Gadd45b & Gadd45g) encode for small (18 kd) nuclear proteins that are rapidly induced by multiple stressors, including genotoxic and oncogenic stress. They are involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli through MAPK and JNK/SAPK pathways. Furthermore Gadd45a has been identified as a mediator of oncogenic Ras signaling. GADD45 proteins are upregulated during myeloid lineage terminal differentiation. To investigate if and how GADD45A and GADD45B play a role in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with wild type, gadd45a or gadd45b null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. It was observed that loss of gadd45a or gadd45b accelerates the development of BCR/ABL driven leukemia in wild type recipients. BCR/ABL transformed gadd45a or gadd45b deficient progenitor recipients exhibited significantly accelerated kinetics of increase in the number of WBC and percentage of myeloid blasts in blood compared to mice reconstituted with the same number of wild type bone marrow cells transduced with BCR/ABL. There was also increase in the rate of accumulation of CD11b+Gr1+ cells in the bone marrow and spleen. Using in vitro and in vivo BrdU assays, enhanced proliferation capacity was observed for BCR/ABL transduced gadd45a, but not gadd45b, deficient myeloid progenitors. However, impaired apoptosis was observed both in BCR/ABL transduced gadd45a and gadd45b deficient myeloid progenitors. These results indicate that both gadd45a and gadd45b function as suppressors of the development of BCR/ABL driven CML, where gadd45a appears to suppress CML via a mechanism involving both inhibition of cell proliferation and enhancement of apoptosis, whereas gadd45b appears to effect only apoptosis. Enhanced JNK signaling was observed in both gadd45a and gadd45b deficient progenitors, whereas enhanced p38 and AKT signaling was observed only in gadd45a deficient myeloid progenitors. Taken together, these data indicate that loss of either gadd45a or gadd45b accelerates BCR-ABL driven CML via distinct signaling and cellular pathways. Further elucidating the role Gadd45 stress sensors play in suppressing the development of leukemia should increase understanding of the molecular/cellular pathology BCR/ABL mediated leukemogenesis, and has the potential to lead to the development of new/improved modalities for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

Homoharringtonine Affects the Expression of PI3K and p-AKT of NB4, SHI-1 Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4381-4381
Author(s):  
Mingzhen Yang ◽  
Xiaoyu Zhang ◽  
Zhenqi Huang ◽  
Qinhua Liu ◽  
Lin Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Acute Myelogenous Leukemia ◽  
Conflicts Of Interest ◽  
Mammalian Target Of Rapamycin ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Growth And Survival ◽  
Acute Myelogenous

Abstract Abstract 4381 Background: Homoharringtonine (HHT) was efficient in therapying patients with acute myeloid leukemia (AML) in China, but little is known about the mechanism of its action. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions and many human cancers, including acute myelogenous leukemia (AML). We try to explore the effect of HHT on PI3K/Akt pathway in AML cells, thus supplying theoretical basis for wider use of HHT. Method: The NB4 and SHI-1 cells were cultured in 20% FCS RPMI-1640 with different concentration of HHT, cell proliferation was detected with MTT, apoptosis was measured by FCM, the protein of PI3K and p-Akt were determined by Western blot. Result: 5ug/L HHT suppressed NB4 and SHI-1 cells proliferation and induced apoptosis after culture 24hr, 100ug/L HHT suppressed 71.29% NB4 and 64.83% SHI-1 cells proliferation respectively. Apoptosis increased obviously with the increasing HHT concentration and the culture time, the leukemia cell apoptosis was significant at 500ug/L HHT, about 41.84% NB4 cells and 46.88% SHI-1 cells were apoptosis when the HHT concentration was 100ug/L. The protein expression of PI3K, and p-Akt gradually declined with HHT concentration increasing, when 500ug/L HHT co-cultured with leukemia cells for 24 hours, The protein expression of PI3K and p-Akt were lowest. The p-Akt of NB4 and SHI-1 cells decreased 28.4% and 34.5% respectively at 5ug/L HHT for 48hr, the PI3K of NB4 and SHI-1 cells decreased 31.56% and 37.38% respectively at 10ug/L HHT for 48hr. Conclusion: HHT could inhibit NB4, SHI-1 cells proliferation and induce leukemia cells apoptosis, and could down-regulate the expression of PI3K and p-Akt significantly, this might be the one of mechanisms that HHT induce NB4 and SHI-1 cells apoptosis, we presume that HHT inhibit proliferation of acute myelogenous leukemia cells through effect of PI3K/Akt signaling pathways. Disclosures: No relevant conflicts of interest to declare.

Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 809-809
Author(s):  
Hajime Akada ◽  
Saeko Akada ◽  
Dongqing Yan ◽  
Robert Hutchison ◽  
Golam Mohi
Keyword(s):  
Polycythemia Vera ◽  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Myeloid Cell ◽  
Negative Regulator ◽  
Jak2v617f Mutation ◽  
Common Mutation ◽  
Wild Type

Abstract Abstract 809 The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F. Disclosures: No relevant conflicts of interest to declare.

C-Myc Is Required for Acute but Not for Chronic Hematopoietic Malignances in Pten-Null Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1632-1632
Author(s):  
Yinshi Guo ◽  
Christopher Seet ◽  
Chao Niu ◽  
Peter Breslin ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Conflicts Of Interest ◽  
Apoptotic Cell ◽  
Akt Signaling ◽  
Apoptotic Cell Death ◽  
Lymphocytic Leukemia ◽  
Mutant Mice ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem

Abstract Abstract 1632 Poster Board I-658 c-Myc, the product of an oncogene, is a common target of most leukemic oncoproteins. Deregulation of c-Myc is commonly found in human leukemic blasts. Transgenic over-expression of c-Myc induces myeloid, erythroid, and lymphocytic leukemia in mice, however whether c-Myc is absolutely required for leukemogenesis has not yet been addressed. Pten, a tumor-suppressing phosphatase, inhibits cell proliferation and promotes apoptotic cell death through repression of PI3K-Akt signaling. Inactivating mutations of Pten and deregulation of PI3K/Akt signaling are both involved in the development of both chronic and acute hematopoietic malignances. Mice with Pten deletions in hematopoietic stem cells (HSCs) develop myeloproliferative disorders (MPD) followed by acute T lymphocytic or myeloid leukemia, reminiscent of disease progression in human chronic myelogenous leukemia. Mice with Pten deletions in lymphocytes develop lymphadenopathy (due to a chronic polyclonal lymphoproliferative disorder) as well as CD4+ T lymphocytic lymphoma. The appearance of these diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether c-Myc is required for the development of these hematopoietic disorders in Pten-mutant mice, we generated inducible Pten and c-Myc double-knockout mice (Pten-/-c-Myc-/-). By comparing the hematopoietic phenotypes of the Pten-/-c-Myc-/- mice with those of Pten-mutant (Pten-/-) mice, we found that both sets of mice developed MPD and lymphadenopathy, however none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPD which developed in Pten-/- mice, which is dominated by granulocytes, megakaryocytes predominate in the MPD of Pten-/-c-Myc-/- mice. We have concluded that c-Myc is required for the development of both T lymphocytic lymphoma and the acute leukemic transformation of Pten-/- MPD, but is not essential for the development of chronic myeloid or lymphoid proliferative disorders. Our study suggests that deregulation of PI3K/Akt signaling in Pten-mutant hematopoietic cells protects these cells from apoptotic cell death, which results in chronic proliferative disorders, while the deregulation of c-Myc resulting from additional mutations promotes hematopoietic cell proliferation and blockage of maturation, and is absolutely required for the development of acute hematopoietic malignances. Disclosures No relevant conflicts of interest to declare.

Loss of Stress Sensor GADD45a Accelerates BCR-ABL-Driven Leukemogenesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1668-1668
Author(s):  
Kaushiki Mukherjee ◽  
Xiaojin Sha ◽  
Ravi Bhatia ◽  
Barbara Hoffman ◽  
Dan Liebermann
Keyword(s):  
Conflicts Of Interest ◽  
Multiple Stressors ◽  
Chronic Phase ◽  
Human Growth ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Wild Type ◽  
M Cell ◽  
Cycle Arrest ◽  
The Status

Abstract Abstract 1668 The bcr-abl fusion oncogene causes chronic myelogenous leukemia (CML) in human. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly upregulated in chronic phase CML samples. However in accelerated and blast phase samples, expression was significantly downregulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype. Disclosures: No relevant conflicts of interest to declare.

Loss of Growth Arrest DNA Damage 45a,b (GADD45a,b) Enhances Oncogenicity in BCR/ABL-Driven Chronic Myelogenous Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3264-3264
Author(s):  
Xiaojin Sha ◽  
Dan A. Liebermann ◽  
Barbara Hoffman
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Chronic Myelogenous Leukemia ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Growth Arrest ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Myeloid Lineage ◽  
Myeloid Progenitors

Abstract 3264 Poster Board III-1 The bcr/abl oncogene causes chronic myelogenous leukemia (CML) in human. BCR/ABL induces the transformation of myeloid lineage through MAPK, JNK/SAPK, PI3K signaling pathways. Growth arrest DNA damage 45A (GADD45A) and GADD45B are upregulated during myeloid lineage terminal differentiation. They are involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli through MAPK and JNK/SAPK pathways. To investigate the effect of GADD45A and GADD45B in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with wild type, gadd45a or gadd45b null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. We found that loss of gadd45a or gadd45b accelerated the development of CML-like disease in wild type recipients. BCR/ABL transformed gadd45a or gadd45b deficient progenitor recipients exhibited a significantly accelerated kinetics of increase in the number of WBC and percentage of myeloid blasts in blood compared to mice reconstituted with the same number of wild type bone marrow cells transduced with BCR/ABL. There was also increase in the rate of accumulation of CD11b+Gr1+ cells in the bone marrow and spleen. Using in vitro and in vivo BrdU assays, enhanced proliferation capacity was observed for both BCR/ABL transduced gadd45a and gadd45b deficient myeloid progenitors. BCR/ABL transduced gadd45a and gadd45b deficient primary myeloid progenitors formed more and bigger colonies compared to BCR/ABL transformed wild type progenitors. Impaired apoptosis was showed in BCR/ABL transduced gadd45a deficient myeloid progenitors. These results indicate that both gadd45a and gadd45b function as suppressors of the development of BCR/ABL driven CML, where gadd45a appears to suppress CML via mechanism involving inhibition of cell proliferation enhancement of apoptosis, whereas gadd45b appears to only inhibit cellular proliferation. Dissecting the molecular nature of signaling paths involved in the suppressive function of gadd45a and gadd45b in BCR/ABL driven CML, as well as analysis of Gadd45 in CML patients, is underway. Disclosures: No relevant conflicts of interest to declare.

The TLE1 Tumor Suppressor Regulates Myc Induced Leukemogenesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2473-2473
Author(s):  
David Sweetser ◽  
Selvi Ramasamy ◽  
Jessica S Blackburn ◽  
David M. Langenau
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Gene Family ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Cell Fate Decisions

Abstract Abstract 2473 The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies. Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability. We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of HMGA2 Cooperates with Jak2V617F in the Development of Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 797-797
Author(s):  
Avik Dutta ◽  
Robert E Hutchison ◽  
Golam Mohi
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Jak2v617f Mutation ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Control Vector ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem

Abstract High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown. MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice. Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of GLI1 and CTNNB1 Knockdown on Notch Signaling and Proliferation of AML and T-ALL Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1042-1042
Author(s):  
Yuki Okuhashi ◽  
Yusuke Takahashi ◽  
Mika Ohtaka ◽  
Erika Shiratori ◽  
Shijyun O ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Leukemia Cell ◽  
Mtor Signaling ◽  
Leukemia Cells ◽  
Nb4 Cells

Abstract Objective: Although Hedgehog (Hh) and Wnt signaling pathways are known to play a role in cancer cell growth, their specific effects on leukemia cells are not fully understood. Previously we reported that the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppressed the growth of leukemia cells. However, a specific inhibitory role of these chemical compounds on Hh and Wnt signaling could not be confirmed since these compounds also have off-target effects. Therefore the effects of specific inhibition of Hh and Wnt signaling pathways must be investigated in detail. In our previous study, we reported that small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 suppressed growth of T-ALL cell lines. In this context, the relationship between Hh, Wnt, Notch and mTOR signaling; known to regulate stemness; has not been fully elucidated. To address these questions, the effects of siRNA-mediated knockdown of GLI1 and CTNNB1 (Catenin beta 1), which respectively mediate Hh and Wnt signaling, on cell proliferation as well as Notch and mTOR signaling were studied in AML and T-ALL cell lines. Methods: Two AML cell lines (NB4 and THP-1) and 2 T-ALL cell lines (DND41 and Jurkat) were used in this study. siRNAs targeting GLI1 (siGL), CTNNB1 (siCT), NOTCH1 (siN1) or control siRNA were transfected into cells by using the pipette tip chamber-based electroporation system. The effects of siGL and siCT transfection on cell proliferation were examined using a colorimetric WST-8 assay and by observing cytospin preparations of the harvested cells. The effects on mRNA and protein expression were examined by quantitative RT-PCR and immunoblotting, respectively. Results: Transfection with siGL and siCT selectively suppressed the expression of GLI1 and CTNNB1, respectively. siRNA treatment did not lead to any significant effects on cell proliferation or morphology in all tested cell lines. In NB4 cells, GLI1 as well as CTNNB1 knockdown increased the level of NOTCH1, the cleaved NOTCH1 fragment (active form of NOTCH1), HES1, and phosphorylated mTOR protein. In the other cell lines, the expression of these proteins was not significantly altered by siGL and siCT. Discussion: siRNA-mediated knockdown experiments suggested that Hh and Wnt signaling had insignificant effect on the proliferation of the leukemia cell lines used in this study. We report a novel interaction, namely, the activation of Notch and mTOR signaling by the suppression of Hh and Wnt signaling in NB4 cells. The molecular mechanism and significance of this phenomenon as well as its translation to other cell lines needs further examination. We propose that this finding must be taken into account when Hh- and Wnt-targeted therapy against leukemia are developed. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

TGF-β1 Supports Leukemia Cell Survival Via Negative Regulation of FLI-1 Transcription Factor, ERK Inactivation and MMP-1 Secretion

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3543-3543 ◽  
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Yasuhito Hatanaka ◽  
Takashi Miida ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Survival ◽  
Conflicts Of Interest ◽  
Human Fibroblasts ◽  
Leukemia Cell ◽  
Neutralizing Antibody ◽  
Mapk Signaling ◽  
Serum Starvation ◽  
Nb4 Cells ◽  
Tgf Β1

Abstract Abstract 3543 Transforming growth factor β (TGF-β) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al., Br J Haematol.2008) and the anti-leukemic effects of action of TGF-β neutralizing antibody under TGF-β abundant and hypoxic bone marrow (BM) microenvironment (ASH, 2012). Friend leukemia virus integration 1 (FLI-1), a member of Ets transcriptional factors, plays a pivotal role in the regulation of extracellular matrix (ECM) genes, and is known to be negatively regulated through TGF-β1-dependent acetylation. We have recently reported that abnormal (high or low) expression of FLI-1 protein analyzed by reverse phase protein arrays is associated with inferior remission duration and reduced survival (Kornblau et al., Blood, 2011). Notably, among 195 proteins tested, FLI-1 expression correlated most with SMAD4, the common mediator in a family of SMAD proteins involved in TGFβ signaling. In this study, we investigated the molecular interactions between TGF-β1 and FLI-1 in AML cells and its functional role in TGF-β-mediated survival. In four AML cell lines, MV4;11, U937, NB4, and OCI-AML3, recombinant TGF-β1 (2ng/mL) induced the TGF-β downstream signaling targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and/or Smad2 phosphorylation, which was reversed by anti-TGF-β neutralizing antibody 1D11 (Genzyme). No consistent change of Smad4 expression was observed in TGF-β1 treated cells. Treatment with rhTGF-β1 inhibited serum starvation-induced apoptosis in MV4;11, U937 and NB4, but not in OCI-AML3 cells. The anti-apoptotic effect of TGF-β1 was associated with G0/G1 cell cycle arrest, which was effectively reversed by anti-TGF-β antibody 1D11. In MV4;11, U937 and NB4 cells, in which rhTGF-β1 promoted cell survival, rhTGF-β1 downregulated expression levels of FLI-1 mRNA and/or protein. However, FLI-1 was upregulated by rhTGF-β1 in OCI-AML3 cells. Since FLI-1 activation is known to cause cell proliferation associated with Ras pathway activation, we investigated MAPK signaling downstream of Ras. Changes in ERK phosphorylation levels after rhTGF-β1 treatment were fully concordant with FLI-1, whereby phospho-ERK was downregulated in MV4;11, U937, and NB4 cells, and upregulated in OCI-AML3 cells. These effects were reversed by anti-TGF-β antibody 1D11. In turn, rhTGF-β1 induced Matrix metalloproteinase-1 (MMP-1) mRNA which inversely correlated with FLI-1 expression. (U937; 6.9 fold increase, OCI-AML3; 3.1 fold decrease). It has been reported that ERK signaling upregulates MMP-1 expression, and that FLI-1 downregulates MMP-1 promoter activity in human fibroblasts. MMP-1, being responsible for degradation of collagenous proteins of ECM, correlates with poor prognosis in leukemia. We also observed that rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in MV4;11, U937, and NB4 cells, but not in OCI-AML3 cells. In summary, TGF-β-induced FLI-1 downregulation and ERK inactivation may be implicated in pathological matrix remodeling via oncogenic MMP-1 transcription in TGF-β abundant BM microenvironment. These findings suggest that FLI-1 and MMP-1 contribute to chemoresistance and poor outcomes in AML and represent potentially targetable molecular aberrations in AML. Disclosures: No relevant conflicts of interest to declare.

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