Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

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Association of A313g Glutathione S-Transferase P1 (GSTP1) Inborn Polymorphism with Susceptibility to De Novo MDS

Blood ◽

10.1182/blood.v120.21.1445.1445 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1445-1445

Author(s):

Sophia Zachaki ◽

Chryssa Stavropoulou ◽

Aggeliki Daraki ◽

Marina Kalomoiraki ◽

Panagoula Kollia ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Type A ◽

Common Mechanism ◽

Genotype Distribution ◽

Distribution Analysis ◽

Wild Type ◽

Increased Risk ◽

Variant Genotype

Abstract Abstract 1445 Models for the pathogenesis of myelodysplastic syndromes (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in detoxification of a variety of electrophilic compounds, such as benzo [a]-pyrene and other polycyclic aromatic hydrocarbons (PAHs), chemotherapy drugs and products of oxidative stress. GSTP1 acts through a common mechanism of conjugating reactive oxygen species (ROS) with glutathione, enabling their detoxification and elimination and thus defending tissues against DNA damage. The corresponding gene is subject to a single-nucleotide polymorphism (A313G) leading to abolished enzyme activity. Thus, individuals homozygous for the variant G allele (G/G) have a lower conjugating activity than individuals homozygous for the wild type A allele (A/A), while heterozygotes (A/G) display intermediate activity. The aim of the present study was to evaluate whether the GSTP1 polymorphism influences susceptibility to MDS and/or promote specific chromosomal aberrations. We conducted a case-control study in 310 de novo MDS patients and 370 unrelated healthy controls using both a conventional PCR-RFLP assay and a novel Real-Time PCR genotyping method using hybridization probe technology. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. Comparison of the genotype distribution between controls and MDS cases revealed a significantly higher frequency of the variant genotypes (heterozygotes A/A and homozygotes G/G) among MDS patients, as compared to controls (p<0.0001, χ2=31.167, df=2). The most marked statistical difference between MDS patients and controls was observed between the wild-type (A/A) and the homozygous variant genotype (G/G), since subjects carrying the G/G variant genotype showed a 4.1-fold increased risk of MDS prevalence than subjects carrying the wild-type A/A genotype (p=0.000, χ2=30.5, d.f.=1, OR=4.098, 95%CI=[2.433–6.897]). Allele frequencies distribution analysis between patients and controls, showed that MDS patients exhibited a 1.9-fold increased risk of carrying at least one variant G allele, as compared to the controls (p<0.0001, d.f.=1, OR =1.9, 95%CI=[1.48–2.34]). There was no association between the GSTP1 polymorphism and gender or any specific cytogenetic subgroup, while stratification of patients according to age showed a differential GSTP1 genotype distribution (p=0.007). Our results, derived from the larger series of primary MDS cases tested for the GSTP1 genetic background, reveal an increased incidence of the GSTP1 variant genotypes among MDS patients, providing evidence for a potential pathogenetic role of the GSTP1 polymorphism on de novo MDS risk. Disclosures: No relevant conflicts of interest to declare.

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Role of CD133 As a Marker of Previous Myelodysplasia in De Novo Elderly Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.1063.1063 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1063-1063

Author(s):

Massimo Breccia ◽

Maria Stefania De Propris ◽

Mariella D'Angiò ◽

Caterina Stefanizzi ◽

Sara Raponi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Remission Rate ◽

Response To Treatment ◽

Prognostic Role ◽

Count Response ◽

Acute Myeloid

Abstract Conflicting results have been reported regarding the correlation between CD133, a surface marker of immature progenitors, and outcome in acute myeloid leukemia (AML). The expression of this antigen has also been reported in myelodysplastic syndromes (MDS), in particular in high-risk diseases, but always in small cohorts of patients and without a focus on the prognostic role of this antigen. Aim of our study was to establish a clinico-biologic correlation between CD133 expression and disease features at baseline in a large series of AML patients of different ages, with particular regard to the older age.Seven hundred AML patients consecutively diagnosed at a single Institution were retrospectively analyzed and enrolled in this study. There were 395 males and 305 females, with a median age of 54 years (range 1.1-90.4). A previous MDS phase was recognized in 124 patients. Several clinical and biologic features were recorded at baseline and retrospectively collected, such as age, gender, FAB and WHO morphologic classification, cytogenetic analysis, molecular alterations, hematologic parameters (Hb, platelet and WBC count), response to treatment and outcome. Overall, 157 patients expressed CD133. This first analysis was carried out on the older patient population (≥65 years) on the basis of the CD117 positivity. Seventy-three older patients expressed CD133 at baseline, whereas 36 patients were CD117+CD133-. Comparison between the two groups showed a significant prevalence of a previously recognized MDS phase in CD133+ patients (27% vs 13%, p=0.01), higher incidence of a complex karyotype or typical MDS cytogenetic aberrations (trisomy 8, del20q, del5q) (30% vs 8%, p=0.001) and of dysplastic morphologic features detectable in patients without a previous dysplastic identification (63% vs 27%, p=0.002). Forty-three patients in the CD133+ group and 21 patients in the CD133- group received intensive chemotherapy: the remission rate was 52% and 64%, respectively (p=0.06). The relapse rate was 25.5% in the CD133+ and 19% in the CD133- group, respectively (p=0.08). No differences were observed with regard to the hematologic parameters at baseline or in overall survival between the two groups. We then assessed the characteristics of cases negative for CD117, but CD133+ (13 patients) that were compared to the entire cohort of cases that were CD117+CD133+ (144 patients): again we found that, independently from the positivity for CD117, CD133 identified patients with a previously reported MDS phase (61% of patients CD117-CD133+), with a higher median age (69 years) and with dysplastic morphologic changes (100% CD117-CD133+). Taken together, our findings strongly suggest that CD133 can identify at diagnosis a previous MDS phase. In particular, the presence of this antigen in the setting of older de novo AML patients should be used to recognize early a subset of patients who, for the associated biologic features, could benefit from the use of hypomethylating agents as first line treatment. Further analyses, aimed at identifying the prognostic role of this antigen in a large cohort of patients treated with azacitidine, are warranted. Disclosures No relevant conflicts of interest to declare.

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TAMI-37. STEAROYL-COA DESATURASE 1 IS ESSENTIAL FOR THE GROWTH OF IDH MUTANT GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.821 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi206-vi206

Author(s):

Tomohiro Yamasaki ◽

Lumin Zhang ◽

Tyrone Dowdy ◽

Adrian Lita ◽

Mark Gilbert ◽

...

Keyword(s):

Glioma Cell ◽

De Novo ◽

Glioma Cells ◽

Model Systems ◽

De Novo Lipogenesis ◽

Wild Type ◽

Knock Out ◽

Stearoyl Coa Desaturase ◽

Scd1 Expression

Abstract BACKGROUND Increased de novo lipogenesis is a hallmark of cancer metabolism. In this study, we interrogated the role of de novo lipogenesis in IDH1 mutated glioma’s growth and identified the key enzyme, Stearoyl-CoA desaturase 1 (SCD1) that provides this growth advantage. MATERIALS ANDMETHODS We prepared genetically engineered glioma cell lines (U251 wild-type: U251WT and U251 IDHR132H mutant: U251RH) and normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH). Lipid metabolic analysis was conducted by using LC-MS and Raman imaging microscopy. SCD1 expression was investigated by The Cancer Genome Atlas (TCGA) data analysis and Western-blotting method. Knock-out of SCD1 was conducted by using CRISPR/Cas9 and shRNA. RESULTS Previously, we showed that IDH1 mut glioma cells have increased monounsaturated fatty acids (MUFAs). TCGA data revealed IDH mut glioma shows significantly higher SCD1 mRNA expression than wild-type glioma. Our model systems of IDH1 mut (U251RH, NHARH) showed increased expression of this enzyme compared with their wild-type counterpart. Moreover, addition of D-2HG to U251WT increased SCD1 expression. Herein, we showed that inhibition of SCD1 with CAY10566 decreased relative cell number and sphere forming capacity in a dose-dependent manner. Furthermore, addition of MUFAs were able to rescue the SCD1 inhibitor induced-cell death and sphere forming capacity. Knock out of SCD1 revealed decreased cell proliferation and sphere forming ability. Decreasing lipid content from the media did not alter the growth of these cells, suggesting that glioma cells rely on de novo lipid synthesis rather than scavenging them from the microenvironment. CONCLUSION Overexpression of IDH mutant gene altered lipid composition in U251 cells to enrich MUFA levels and we confirmed that D-2HG caused SCD1 upregulation in U251WT. We demonstrated the glioma cell growth requires SCD1 expression and the results of the present study may provide novel insights into the role of SCD1 in IDH mut gliomas growth.

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Detection of the JAK2V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System

Archives of Pathology & Laboratory Medicine ◽

10.5858/2006-130-997-dotjmi ◽

2006 ◽

Vol 130 (7) ◽

pp. 997-1003

Author(s):

Randall J. Olsen ◽

Zhouwen Tang ◽

Daniel H. Farkas ◽

David W. Bernard ◽

Youli Zu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Clinical Laboratory ◽

Melting Curve ◽

Myeloproliferative Disorder ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Wild Type ◽

Acute Myeloid

Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.

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Role of Granulocyte-Macrophage Colony-Stimulating Factor in Acute Myeloid Leukemia/Myelodysplastic Syndromes

Journal of Global Oncology ◽

10.1200/jgo.2017.009332 ◽

2018 ◽

pp. 1-6

Author(s):

Neemat M. Kassem ◽

Alya M. Ayad ◽

Noha M. El Husseiny ◽

Doaa M. El-Demerdash ◽

Hebatallah A. Kassem ◽

...

Keyword(s):

Gene Expression ◽

Myeloid Leukemia ◽

Serum Levels ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor ◽

Acute Myeloid

Purpose Granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine stimulates growth, differentiation, and function of myeloid progenitors. We aimed to study the role of GM-CSF gene expression, its protein, and antibodies in patients with acute myeloid leukemia/myelodysplastic syndromes (AML/MDS) and their correlation to disease behavior and treatment outcome. The study included 50 Egyptian patients with AML/MDS in addition to 20 healthy volunteers as control subjects. Patients and Methods Assessment of GM-CSF gene expression was performed by quantitative real-time polymerase chain reaction. GM-CSF proteins and antibodies were assessed by enzyme-linked immunosorbent assay. Results There was significant decrease in GM-CSF gene expression ( P = .008), increase in serum level of GM-CSF protein ( P = .0001), and increase in anti–GM-CSF antibodies ( P = .001) in patients with AML/MDS compared with healthy control subjects. In addition, there was a significant negative correlation between serum levels of GM-CSF protein and initial peripheral blood blasts, percentage as well as response to therapy. Conclusion Any alteration in GM-CSF gene expression could have implications in leukemogenesis. In addition, GM-CSF protein serum levels could be used to predict outcome of therapy. GM-CSF antibodies may also play a role in the pathogenesis of AML/MDS. The use of these GM-CSF parameters for disease monitoring and as markers of disease activity needs further research.

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Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽

10.3390/pathogens9090774 ◽

2020 ◽

Vol 9 (9) ◽

pp. 774

Author(s):

Virginio Cepas ◽

Victoria Ballén ◽

Yaiza Gabasa ◽

Miriam Ramírez ◽

Yuly López ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

De Novo ◽

Wild Type ◽

Planktonic Cells ◽

E Coli ◽

Qrt Pcr ◽

Transposon Insertion ◽

De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

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G-CSF and GM-CSF Have Opposite Effects on the Growth and Survival of Cells Containing Abnormal RUNX1.

Blood ◽

10.1182/blood.v106.11.2760.2760 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2760-2760

Author(s):

Fariborz Mortazavi ◽

Arun Sharma ◽

Marie Baraoidan ◽

Vinzon Ibanez ◽

SiJun Yang ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Cell Line ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Leukemia Cells ◽

Growth And Survival ◽

Gm Csf ◽

Molecular Abnormalities ◽

Empty Vector ◽

Human Cd34

Abstract Primary leukemia cells can respond very differently to cytokines such as G-CSF and GM-CSF, demonstrating marked increases in colony-forming ability to one but not the other (Park et al, Blood1989; 74: 56–65). To examine if such differences might correlate with underlying molecular abnormalities, we examined the growth and survival response of hematopoietic cells containing abnormal RUNX1 variants (a frequent occurrence in leukemia and myelodysplastic syndrome) to treatment with G-CSF and GM-CSF. In the RUNX1-ETO containing cell-line Kasumi-1, GM-CSF decreased colony forming ability whereas G-CSF preserved or increased it. The 32Dcl3 murine myelomonocytic cell-line terminally differentiates in response to G-CSF or mGM-CSF. We transduced 32Dcl3 with RUNX1-ETO or Empty Vector: G-CSF promoted the growth and survival of 32D RUNX1-ETO but caused 32D Empty Vector to terminally differentiate and die. In contrast, 32D RUNX1-ETO cultured in mGM-CSF demonstrated accelerated terminal differentiation and death compared to 32D Empty Vector. Many of the RUNX1 variants seen in leukemia and myelodysplastic syndrome, including RUNX1-ETO, lack the RUNX1 C-terminal transcription regulating domain (TRD). In human CD34+ hematopoietic cells transduced with RUNX1 with TRD deleted (CD34 RUNX1noTRD), colony forming ability was retained even after >40 days of culture in IMDM 10% FBS, SCF 10ng/ml, IL-3 30ng/ml, G-CSF 100ng/ml (CD34 Empty Vector lost colony forming ability after 14 days). However, culture in media supplemented with 100ng/ml GM-CSF instead of G-CSF significantly impaired colony forming ability of CD34 RUNX1noTRD compared to CD34 Empty Vector. In conclusion, RUNX1 variants seen in acute myeloid leukemia and myelodysplastic syndrome promote growth and survival in G-CSF but accelerate differentiation and death in response to GM-CSF. The molecular basis for this effect appears to relate to the presence or absence of the RUNX1 C-terminal transcription regulating domain. These findings may have clinical relevance.

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Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.1168.1168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1168-1168

Author(s):

Jerry C. Cheng ◽

Deepa Shankar ◽

Stanley F. Nelson ◽

Kathleen M. Sakamoto

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Western Blot ◽

Blot Analysis ◽

Hematopoietic Cells ◽

Wild Type ◽

Qrt Pcr ◽

T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

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

Blood ◽

10.1182/blood.v108.11.2213.2213 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2213-2213

Author(s):

J. Pulikkan ◽

A. Peer Zada ◽

M. Geletu ◽

V. Dengler ◽

Daniel G. Tenen ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Dominant Negative ◽

Pcr Analysis ◽

Wild Type ◽

Rt Pcr ◽

Promoter Assay ◽

Type C ◽

Acute Myeloid

Abstract 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.

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BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

Blood ◽

10.1182/blood.v110.11.3365.3365 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3365-3365

Author(s):

Jean-Yves Metais ◽

Ashley E. Dunfee ◽

Rodrigo T. Calado ◽

Cynthia E. Dunbar

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Gene Product ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Marker Genes ◽

Empty Vector ◽

Myeloid Progenitor ◽

The Impact

Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

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