Identification of therapeutic targets of the hijacked super-enhancer complex in EVI1-rearranged leukemia

AbstractDeregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1–G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.

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Effect of Down-Regulation of miR-23b-3p on the Differentiation of Acute Myeloid Leukemia via Wilms Cancer Gene 1

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2696 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1377-1382

Author(s):

Lixia Cao ◽

Jing Zhang ◽

Huijuan Ren ◽

Yanqiu Han

Keyword(s):

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Morphological Changes ◽

Hot Spot ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Down Regulation ◽

Blank Group ◽

Acute Myeloid

miRNA has always been a hot spot research. We assessed the effect of down-regulation of miR-23b-3p on the differentiation of acute myeloid leukemia (AML). Human AML cell line U937 was divided into blank group, NC group and miR-23b-3p low expression group (transfected with miR-23b-3p inhibitor) and miR-23b-3p followed by analysis of WT1 level and relationship between miR-23b-3p and WT1 by dual luciferase reporter assay. All-trans retinoic acid is used to induce differentiation, and then the morphological changes of cells and CD11b level were detected. When miR-23b-3p level was reduced, WT1 mRNA and protein level was also decreased. Dual luciferase assay showed that miR-23b-3p bound to WT1 3’-UTR. Inhibition of miR-23b-3p significantly decreased cell proliferation. Swiss Giemsa staining showed that most of cells were in the differentiation stage with low miR-23b-3p expression. The differentiation marker CD11b was significantly higher than other groups, indicating that low miR-23b-3p expression can promote cell differentiation and reduce cell proliferation to a certain extent. Under low miR-23b-3p expression, the positive rate of CD11b was significantly increased. Down-regulating miR-23b-3p can inhibit WT1 to a certain extent and promote the differentiation of AML, which provides a guidance for the gene-level treatment of AML.

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Long-Term Expansion and Self Renewal Is Contained in the CD34+, but Not the CD34- Subfraction of Nucleophosmin Mutated Acute Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v112.11.1349.1349 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1349-1349

Author(s):

Carolien Woolthuis ◽

Lina Han ◽

Djoke van Gosliga ◽

Philip Kluin ◽

Edo Vellenga ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hox Genes ◽

Mutant Protein ◽

Self Renewal ◽

Cd34 Cells ◽

Stromal Layer ◽

Acute Myeloid

Abstract Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.

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Evidence for CD34+ Hematopoietic Progenitor Cell Involvement in Acute Myeloid Leukemia with NPM1 Gene Mutation: Implications for the Cell of Origin

Blood ◽

10.1182/blood.v112.11.307.307 ◽

2008 ◽

Vol 112 (11) ◽

pp. 307-307 ◽

Cited By ~ 1

Author(s):

Maria Paola Martelli ◽

Valentina Pettirossi ◽

Elisabetta Bonifacio ◽

Federica Mezzasoma ◽

Nicla Manes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Myeloid Leukemia ◽

Hox Genes ◽

Leukemic Cells ◽

Mutant Protein ◽

Cell Of Origin ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity >90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

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Distinct Regulation of β- and γ-Catenin throughout Hematopoietic Development Contrasts with Their Cooperative Roles In Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v116.21.1573.1573 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1573-1573

Author(s):

Rhys Gareth Morgan ◽

Lorna Pearn ◽

Kate Liddiard ◽

Robert Hills ◽

Alan Burnett ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Wnt Signaling ◽

Cell Lines ◽

Nuclear Localization ◽

Myeloid Leukemia ◽

Nuclear Translocation ◽

Differentiated Cells ◽

Cd34 Cells ◽

Normal Human ◽

Acute Myeloid

Abstract Abstract 1573 Wnt proteins are important developmental regulators and are known to play a role in maintenance of hematopoietic stem cells (HSC). Wnt signaling has also been identified as one of the most frequently dysregulated processes associated with acute myeloid leukemia (AML), though the significance of this observation is as yet poorly understood. Here we investigate the role of two Wnt signaling proteins; β-catenin and γ-catenin and their respective roles in both normal human hematopoiesis and in AML. These proteins have dual and overlapping roles as transcriptional activators of Wnt target genes in the nucleus, and as structural components of the cytoskeleton. To determine the potential scope of influence of these proteins, we first examined their expression levels and subcellular location throughout normal human hematopoiesis using multi-parameter flow cytometric analysis and confocal microscopy. As expected β-catenin was strongly expressed in human cord blood derived HSC (212 MFI ±124, n=6) and at lower levels in differentiated subsets; surprisingly however β-catenin expression was maintained in granulocytic (1182 MFI±568) and monocytic cells (284 MFI±107). Nuclear localization was independent of cytoplasmic expression level, being strongly nuclear-localized in early progenitors and predominantly cytoplasmic in differentiated cells (58%±5 nuclear-localized in CD34+ cells vs 27%±1 in granulocytes, P=0.008). The expression pattern of γ-catenin was similar to β-catenin but showed a reciprocal pattern of subcellular localization, with levels of nuclear γ-catenin strongest in differentiated cells (10%±2 in CD34+ cells vs 44%±3 in monocytes P=0.0005). These data imply complementary roles for β and γ-catenin in normal hematopoiesis and show that nuclear localization of these proteins is regulated independently and irrespective of their expression level. In AML patients, β-catenin dysregulation has been previously reported; however, we also observed frequent overexpression of γ-catenin (over 5 fold in 25% of patients). This overexpression was associated with lower remission rates (OR 1.23 per log increase, P=0.03, CI 1.02–1.49) arising from resistant disease (OR 1.57 per log increase, P=0.003, CI 1.16–2.14) in a cohort of 243 AML patients adjusted for baseline diagnostic variables. In contrast to normal hematopoiesis, we found that nuclear localization of γ-catenin correlated with nuclear localization of β-catenin in AML (R=0.5, n=59) suggesting that the capacity to independently regulate the nuclear entry/retention of these catenins is disrupted in AML. To investigate this, we examined the effect of ectopic overexpression of γ-catenin in normal cord blood derived CD34+ cells and AML cell lines. Three-fold overexpression of γ-catenin failed to induce nuclear translocation of γ- or β-catenin in normal progenitors, which exhibited no major developmental defects. In contrast, in 3 of 4 AML cell lines, overexpression of γ-catenin strongly promoted its nuclear localization (9-16 fold) and was associated with a block in agonist-induced differentiation - a phenotype previously associated with β-catenin. In accord with this, we found that as in primary AML, nuclear translocation of γ-catenin in AML cell lines was associated with translocation of β-catenin (2-22 fold). In conclusion, we propose that in normal hematopoiesis, nuclear translocation of β- and γ-catenin is tightly and independently regulated for each catenin. In contrast, most AML cells lack this regulation resulting in correlated nuclear levels of β- and γ-catenin. In addition, we found while overexpression of γ-catenin has little consequence for normal cells; in malignant cells γ-catenin facilitates nuclear translocation of β-catenin. Disclosures: No relevant conflicts of interest to declare.

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Targeting Acute Myeloid Leukemia Stem Cells by MUC1-C Subunit Inhibition

Blood ◽

10.1182/blood.v116.21.848.848 ◽

2010 ◽

Vol 116 (21) ◽

pp. 848-848 ◽

Cited By ~ 1

Author(s):

Dina Stroopinsky ◽

Jacalyn Rosenblatt ◽

Keisuke Ito ◽

Li Yin ◽

Hasan Rajabi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Myeloid Leukemia ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Control Peptide ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 848 Introduction: Acute myeloid leukemia (AML) arises from a malignant stem cell population that is resistant to cytotoxic therapy and represents a critical reservoir of conferring disease recurrence. A major focus of investigation is the identification of unique markers on leukemia stem cells (LSCs) that differentiate them from normal hematopoietic stem cells and thereby serve as potential therapeutic targets. MUC1 is a high molecular weight transmembrane glycoprotein that is aberrantly expressed in many epithelial tumors and confers cell growth and survival. We have developed an inhibitor of the MUC1-C receptor subunit that blocks oligomer formation and nuclear localization. In the present study, we have examined expression of MUC1 on LSCs as compared to normal hematopoietic stem cells and studied the effect of MUC1-C inhibition on the functional properties of LSCs. Methods and Results: Using multichannel flow cytometric analysis, we isolated the LSC compartment as defined by CD34+/CD38-/lineage- cells from bone marrow specimens obtained from patients with active AML. The majority of LSCs strongly expressed MUC1 with a mean percentage of 77% (n=6). These findings were confirmed by immunocytochemical staining of LSCs isolated by flow cytometric sorting. MUC1 expression was not detectable on the CD34- fraction of AML cells, but was present on the granulocyte-macrophage progenitor (GMP) fraction (CD34+/CD38+ cells) (mean=83%; n=6). In contrast, MUC1 expression was not observed on CD34+ progenitors isolated from normal donors (18%, n=6). In concert with these findings, RT-PCR analysis for MUC1 RNA demonstrated expression in CD34+ cells isolated from AML patients, but not normal volunteers. Notably, we also found that MUC1 expression selectively identifies malignant hematopoietic progenitors in a patient with chimerism between normal and leukemia derived stem cells. The presence of MUC1+CD34+ cells was detected in a patient with AML who achieved a morphologic complete remission following sex mismatched allogeneic transplantation. Using Bioview technology, we found that MUC1 is expressed only in the recipient (XX) CD34+ cells, representing residual malignant cells, whereas the donor (XY) derived CD34+ cells, representing the majority of the progenitors, lacked MUC1 expression. We subsequently examined the effects of MUC1-C inhibition on the capacity of leukemic progenitors to proliferate and support colony formation. MUC1-C inhibition with the GO-203 cell-penetrating peptide resulted in downregulation of the β-catenin pathway, an important modulator of cell division and survival, which is known to support the LSC phenotype. No significant change was detected with a control peptide, or with MUC1-C inhibition of progenitors isolated from a normal control. Furthermore, MUC1-C inhibition resulted in apoptosis, as demonstrated by flow cytometric staining for AnnexinV in AML CD34+ cells, but not in CD34+ progenitors isolated from normal volunteers (mean Annexin positive cells 53% and 5%, respectively, n=4). Consistent with these findings, the MUC1-C inhibitor, but not the control, peptide resulted in cell death of CD34+ cells isolated from AML patients, but not normal controls. Most significantly, exposure of CD34+ AML cells to the MUC1-C inhibitor resulted in loss of their capacity for colony formation in vitro with mean colonies of 4 and 40 for those cells exposed to the MUC1 inhibitor and a control peptide (n=2). In contrast, colony formation by normal hematopoietic stem cells was unaffected. Conclusions: MUC1 is selectively expressed by leukemic progenitors and may be used to differentiate malignant from normal hematopoietic stem cell populations. MUC1-C receptor subunit inhibition results in (i) downregulation of b-catenin signaling, (ii) induction of apoptosis and cell death, and (iii) disruption of the capacity to induce leukemia colony formation. Disclosures: Stone: genzyme: Consultancy; celgene: Consultancy; novartis: Research Funding. Kufe:Genus Oncology: Consultancy, Equity Ownership.

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Highly Sensitive Monitoring of Minimal Residual Disease in FLT3-itd-Positive Acute Myeloid Leukemia Patients with a Mutation Specific Quantitative-PCR Method,

Blood ◽

10.1182/blood.v118.21.3530.3530 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3530-3530

Author(s):

Joanna Schiller ◽

Inka Praulich ◽

Michael Hallek ◽

Karl-Anton Kreuzer

Keyword(s):

Myeloid Leukemia ◽

Research Funding ◽

Tandem Duplication ◽

Residual Disease ◽

Patient Specific ◽

Base Pairs ◽

Qrt Pcr ◽

Highly Sensitive ◽

Minimal Residual ◽

Acute Myeloid

Abstract Abstract 3530 Minimal residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) can predict relapse clearly in advance and therefore allows early therapeutic intervention. Moreover, recent studies have highlighted the significance of personalized treatment on the basis of MRD status for improving outcome in AML. The FLT3 internal tandem duplication (FLT3 -ITD) is one of the most frequent mutations in AML patients occurring in 15–35% of all cases and approximately in 20–30% of cytogenetic normal (CN) AML. Clinically, FLT3 -ITDs have been strongly associated with high leukocytosis, high blast counts, normal karyotype and, most importantly, poor clinical outcome. However, due to the high sequence variability of individual FLT3 -ITD commonly a universal PCR approach is applied which has a low sensitivity (approx. 1: 5×102). For this reasons FLT3 -ITD is regarded as not suitable for longitudinal MRD measurements. The aim of this study was to develop a novel cDNA-based, highly sensitive quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay for the detection of the FLT3 -ITD mutation level. Furthermore, the prognostic value of FLT3 -ITD-based MRD detection in AML patients was compared with other genetic markers like NPM1 mutations, MLL -partial tandem duplication (MLL -PTD) and the expression of PML /RARα fusion gene or WT1 over-expression. To design patient-specific qRT-PCR, FLT3 -ITDs were amplified with universal primers, purified corresponding bands from agarose gel and directly sequenced. On the basis of the FLT3 -ITD, individual mutation-specific primers were designed. The expression of FLT3 -ITD was determined using complementary DNA samples at different points in time diagnosis and subsequent treatment. For estimation of the sensitivity and specificity of this approach we used a dilution of FLT3 -ITD cDNA in a pool of FLT3 -unmutated cDNA. From a total of 394 newly diagnosed AML patients 55 (14%) were FLT3 /ITD positive. Retrospectively we analyzed ITD mutation of FLT3 in 39 available cases. The length of ITD ranged from 3 to 144 base pairs (median 46). Nine patients had extra insertions of 2–38 base pairs between two repeats. For the FLT3 -ITD quantification we developed patient-specific qRT-PCR for 29 individuals with mutation-specific forward primers and studied 83 peripheral blood and 61 bone marrow samples. Three cases with WT1, fifteen with NPM1, three with MLL -PTD and five with PML /RARα fusion genes expression levels were compared with FLT3 -ITD expression levels. 26 of 29 assays (90%) were highly specific (1:104 − 1:105) and yielded similar results when compared to other high sensitive assays for molecular markers like NPM1 or PML /RARα. In three cases (10%) a co-amplification of the wild-type could not be avoided resulting in lower sensitivity (1:103). We could show that FLT3 -ITD positivity reliably predicted relapse up to 10 months in advance. 92% patients, who achieved FLT3 -ITD negativity with our assay, did not relapse. Furthermore we compared paired PB and BM samples at diagnosis and after induction therapy in 5 cases. The difference in FLT3 -ITD expression were not statistically significant (p=0.8) which is in line with recent studies. We conclude that highly sensitive detection of individual FLT3 -ITD posses equal prognostic power in AML like established molecular MRD markers. Using this approach MRD guided treatment decisions appear to be justified and should be incorporated in future studies. Disclosures: Hallek: Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kreuzer:Alexion: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Shire: Honoraria, Research Funding.

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Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1+ and/or FLT3+

Blood ◽

10.1182/blood.v118.21.4908.4908 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4908-4908

Author(s):

Pervin Topcuoglu ◽

Klara Dalva ◽

Sinem Civriz Bozdag ◽

Onder Arslan ◽

Muhit Ozcan ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Aberrant Expression ◽

Hla Dr ◽

Cd36 Expression ◽

Cd34 Cells ◽

Cd56 Expression ◽

Acute Myeloid

Abstract Abstract 4908 Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1 and/or FLT-3 Positive Pervin Topçuoglu, Klara Dalva, Sinem Civriz Bozdag, Önder Arslan, Muhit Özcan, Osman Ýlhan, Hamdi Akan, Meral Beksaç, Günhan Gürman Aim: We aimed to evaluate immunophenotypical (IP) features in AML pts with NPM1+ and/or FLT3+ except on acute promyelocytic leukemia. Patients&Method: Between Nov 2009 and Feb 2011, we retrospectively analyzed IP features by flow cytometry (FCM) in 51 pts (46M;17F) with new diagnosed AML. Median age was 46 years (range: 14–71 ys). The mutations of NMP1 and FLT-3 TKD&ITD were determined by the methods of RQ-PCR or RFLP, respectively in the samples of bone marrow (n=31) or periheral blood (n=20) at the diagnosis. Antigenic expression of leukemic cells was analyzed by four-color FCM (FITC, PE, PerCP&APC) based by Nomdedeu et al researh (Leuk res 2011; 35:163) (Table-1). Results: We detected NMP1+ mutation in 16 patients. Of these, three were associated with mutations of FLT3-ITD (n=2) or -TKD (n=1). Twelve patients had FLT3+ (9 ITD or 3 TKD). More than half of the patients without any mutation were CD15+/CD34+/HLA-DR+ and 11.5% for CD34 negative. Similarly, the patients with FLT-3 positive were mostly CD34+ as the pts w/o any mutations. Contrary, most of the pts with NMP1+ were CD34 negative (56.3%) (Table 1). When evaluated the complete IP in leukemic cells, the expression of CD123 was significantly marked in the patients with NPM1+ and/or FLT3+ than those w/o mutations (p=0.008). While the co-expression of CD7 and CD117 was found in 67% of the pts w/o any mutations, 30% of the pts with NMP1 and/or FLT-3 ITD (p=0.01). CD56 expression was detected in more pts with NMP1+ than those with FLT-3+ (40% vs 8%, p=0.04). Besides, CD36 expression was positive in the all pts with FLT3-ITD than TKD+ (p=0.005). More intensive CD33 expression was seen in NMP1+ pts. The expression of CD64 was similar in all three mutations. Conclusion: Though NMP1 mutation was associated more CD34+ cells, more FLT3+ pts had CD34 positivity. The expression of CD123 was especially associated with the mutations. Aberrant expression of CD56 was in more pts with NPM1+, but CD36 for FLT3-ITD. These data might be a step for a study aiming to show a correlation between the type of mutations combined with IP features of leukemic cells and clinical characteristics or disease course of AML pts. Disclosures: No relevant conflicts of interest to declare.

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A Highly Selective Anti-ROR1 Monoclonal Antibody Inhibits Human Acute Myeloid Leukemia CD34+ Cell Survival and Self-Renewal.

Blood ◽

10.1182/blood.v120.21.2560.2560 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2560-2560

Author(s):

Larissa Balaian ◽

Anil Sadarangani ◽

George F. Widhopf ◽

Rui-kun Zhong ◽

Charles Prussak ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Myeloid Leukemia ◽

The Self ◽

Self Renewal ◽

Leukemia Stem Cell ◽

Cd34 Cells ◽

Selective Expression ◽

Dose Dependent ◽

Acute Myeloid

Abstract Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

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