CD34 expression in native human acute myelogenous leukemia blasts: Differences in CD34 membrane molecule expression are associated with different gene expression profiles

Abstract Acute Myelogenous Leukemia (AML) is a disease that clinically evolves over time as many patients who are responsive to therapy upfront acquire resistance to the same agents when applied in the relapse setting. The stem cell model for AML has been invoked to explain primary resistance to standard therapy; the leukemia stem cell (LSC) population representing a therapy-refractory reservoir for relapse. There have been no prospective efforts to formally assess the evolution of the LSC population during patients’ clinical course. We performed a prospective characterization of specimens from a well-defined cohort of patients with AML at diagnosis and relapse to assess the frequency and phenotype of functionally defined LSCs. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Pre-therapy and post-relapse LSC frequencies were assessed using xenotransplantation limiting dilution analyses (LDA). We assessed the frequencies of CD45RA, CD32, TIM-3, CD96, CD47, and CD97 expressing populations that have been previously published to possess LSC activity. Functionally validated pre-therapy and post-relapse LSC populations were identified using fluorescent labeled cell sorting and NSG xenotransplantation. LSC activity was confirmed for each population using secondary xenotransplantation. Gene expression analysis of highly enriched LSC populations from pre-therapy and post-relapse samples was performed using ABI TILDA qPCR analyses following pre-amplification. Results We demonstrated by LDA an 8 to 42-fold increase in LSC frequency between diagnosis and relapse in paired primary patient samples. The increase in LSC activity was not associated with an increase in frequency for phenotypically-defined populations previously reported to possess LSC activity. Rather, we found that LSC activity expanded at relapse to immunophenotypic populations of leukemic cells that did not possess LSC activity prior to treatment. Moreover, in all patients, the number of phenotypically distinct LSC populations (as defined by CD34 and CD38 or CD32 and CD38) detectable at relapse was dramatically expanded. Further, while the majority of the LSC populations’ gene expression profile remained stable between diagnosis and relapse, a subset of genes were enriched in defined LSC populations at relapse including IL3-receptor alpha and IL1-RAP, both previously demonstrated to play a role in LSC biology. Conclusions This study is the first to characterize the natural evolution of LSCs in vivo following treatment and relapse. We demonstrate an increase in LSC activity and greatly increased phenotypic diversity of the LSC population, suggesting a loss of hierarchical organization following relapse. These findings demonstrate that treatment of AML patients with conventional chemotherapy regimens can promote quantitative and qualitative expansion of the LSC compartment. Further, the data indicate that surface antigen immune-phenotype is not predictive of function in relapse and suggest a major limitation to efforts targeting specific surface antigens in the relapse setting. Understanding the mechanisms by which LSC expansion occurs and how to target it will likely improve our currently poor treatment options for patients who relapse. Disclosures: Becker: Millenium: Research Funding.

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MmTRA1b/phospholipid scramblase 1 gene expression is a new prognostic factor for acute myelogenous leukemia

Leukemia Research ◽

10.1016/s0145-2126(03)00189-9 ◽

2004 ◽

Vol 28 (2) ◽

pp. 149-157 ◽

Cited By ~ 18

Author(s):

Akihiro Yokoyama ◽

Takuya Yamashita ◽

Eisuke Shiozawa ◽

Atsuko Nagasawa ◽

Junko Okabe-Kado ◽

...

Keyword(s):

Gene Expression ◽

Prognostic Factor ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Phospholipid Scramblase ◽

Acute Myelogenous ◽

Phospholipid Scramblase 1

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Evaluation of Bone Marrow Specimens With Acute Myelogenous Leukemia for CD34, CD15, CD117, and Myeloperoxidase

Archives of Pathology & Laboratory Medicine ◽

10.5858/2001-125-1063-eobmsw ◽

2001 ◽

Vol 125 (8) ◽

pp. 1063-1069 ◽

Cited By ~ 2

Author(s):

Cherie H. Dunphy ◽

Jacek M. Polski ◽

H. Lance Evans ◽

Laura J. Gardner

Keyword(s):

Bone Marrow ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Flow Cytometric ◽

Acute Myelogenous ◽

Cd34 Expression ◽

French American British ◽

Higher Sensitivity

Abstract Context.—Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. Objective.—To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. Materials and Methods.—Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). Results.—Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. Conclusions.—CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

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A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

Blood ◽

10.1182/blood.v126.23.4266.4266 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4266-4266 ◽

Cited By ~ 1

Author(s):

Tzu-Chieh Ho ◽

Craig T Jordan ◽

Mark W. LaMere ◽

John M. Ashton ◽

Kristen O'Dwyer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cell Lines ◽

Small Molecule ◽

Acute Myelogenous Leukemia ◽

Research Funding ◽

Myelogenous Leukemia ◽

Molecule Inhibitor ◽

Acute Myelogenous ◽

Pre Treatment

Abstract Background Acute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach. Results While the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect. Conclusions We identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. Disclosures Wang: Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

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Abnormal Gene Expression as an Early Indicator of Relapse in Acute Myelogenous Leukemia (AML)

Experimental Hematology Today—1988 ◽

10.1007/978-1-4613-8862-3_15 ◽

1989 ◽

pp. 99-101

Author(s):

M. J. Evinger-Hodges ◽

J. A. Spinolo ◽

I. Cox ◽

K. A. Dicke

Keyword(s):

Gene Expression ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Early Indicator ◽

Acute Myelogenous

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Faculty Opinions recommendation of Dysregulated gene expression networks in human acute myelogenous leukemia stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1160298.620617 ◽

2009 ◽

Author(s):

Paul Liu ◽

R Katherine Hyde

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells ◽

Acute Myelogenous

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Novel Synthetic Histone Deacetylase Inhibitor, SK-7041, Has Potent Anti-Proliferative Activity in Acute Myeloid Leukemia Cell Lines.

Blood ◽

10.1182/blood.v110.11.2854.2854 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2854-2854

Author(s):

Sung-Soo Yoon ◽

Eunkyung Bae ◽

Juwon Park ◽

Yongjun Cha ◽

Young Y. Lee ◽

...

Keyword(s):

Gene Expression ◽

Histone Deacetylase ◽

Cell Lines ◽

Histone Deacetylase Inhibitors ◽

Expression Profiles ◽

Leukemia Cell ◽

Gene Expression Profiles ◽

Myelogenous Leukemia ◽

G1 Arrest ◽

Cancer Cell Growth

Abstract Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, which regulates gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. Thus, histone deacetylase inhibitors (HDACIs) are emerging as a new class of anti-cancer agents for leukemia. In this study, we examined anti-proliferative effects of novel hybrid synthetic HDACI, SK-7041, in various acute myelogenous leukemia (AML) cell lines (KG-1, HL-60, HEL, U937, ML-1). SK-7041 induced the time-dependent hyperacetylation of histones H3 and H4 in AML cell lines. It preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041. All the cell lines tested showed similar patterns of growth inhibition by SK-7041. Their IC50 values were approximately 0.5 mM at 72 hours incubation. SK-7041 effectively induced the apoptosis via activation of caspase-3, -7, -9, and PARP, but not caspase-8. SK-7041 enhanced G1 arrest via decreasing Cyclin D1 expression and increasing p21 expression. Changes in gene expression profiles after treating KG-1 cells with various concentrations of SK-7041 were assessed using a cDNA microarray consisting of distinct cDNAs of cancer-related genes. 7 genes, namely RGL1, FYN, CARD9, ABCA7, TNFRSF6B, CASP9, and ENPP2 were induced substantially (Global M >2.0) and 8 genes, namely PTPN7, CD34, INSIG1, IL16, LHX6, TRIB3, BID, and PDCD4 were reduced substantially (Global M < 2.0). In conclusion, this study showed SK-7041 inhibited cancer cell growth and caused characteristic gene expression profile changes in AML cells. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in AML cells.

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