Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry

Abstract Abstract 1587 Poster Board I-613 In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced. Disclosures Hallek: Roche: Consultancy, Honoraria, Research Funding.

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Mer Receptor Tyrosine Kinase Is A Potential Therapeutic Target in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.1317.1317 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1317-1317

Author(s):

Alisa B. Lee-Sherick ◽

Kelly Menachof ◽

Kristen M. Eisenman ◽

Amy McGranahan ◽

Colleen McGary ◽

...

Keyword(s):

Flow Cytometry ◽

Tyrosine Kinase ◽

Cell Lines ◽

Propidium Iodide ◽

Receptor Tyrosine Kinase ◽

Kinase Inhibitor ◽

Control Cell ◽

Flow Cytometric Analysis ◽

Flow Cytometric ◽

And Control

Abstract Abstract 1317 Acute myelogenous leukemia (AML) is difficult to treat successfully in both adult and pediatric patients using conventional chemotherapy. Mutated or aberrantly expressed proteins on the cell surface of myeloblasts provide a focus for targeted therapy which could potentially augment therapeutic outcome, decrease toxicity to normal tissues, and/or provide a therapy option for those who are not able to tolerate conventional therapy. We report here that the Mer receptor tyrosine kinase is upregulated in approximately 80% of AML cell lines and patient samples, and explore the therapeutic potential of Mer inhibition. We assessed the prevalence of Mer expression in AML. Western blot and flow cytometric analysis demonstrated expression of Mer in greater than 85% (12/14) of AML cell lines. Mer expression was also assessed at the time of diagnosis and relapse in both pediatric and adult patient samples using flow cytometry. We found that Mer was expressed on leukemic blasts in 80% of 36 pediatric and 100% of 10 adult patients at the time of diagnosis with AML. Additionally, 100% of 11 patients expressed Mer at the time of relapse. Furthermore, when analyzing patient samples at relapse compared to the same patient's diagnostic sample, there was a trend toward increased Mer expression. This is in contrast to normal bone marrow myeloid progenitors from healthy donors, which express little or no Mer. Using two independent shRNA constructs directed against Mer, we analyzed the effects of Mer inhibition in two Mer expressing AML cell lines. Mer knock-down and control cell lines were assessed for apoptosis by flow cytometry after serum starvation and staining with Yo-Pro-1 iodide and propidium iodide. Compared to AML cell lines transduced with a non-silencing control shRNA (shControl), cell lines expressing reduced levels of Mer protein demonstrated significantly more apoptosis (p<0.05). Additionally, when these cell lines were plated in equal number in methylcellulose, cell lines with reduced Mer expression demonstrated decreased colony forming potential compared to shControl cells (p<0.01). Mer knock-down and control cell lines were injected into NOD-SCID-gamma mice after sublethal irradiation and the mice were monitored for development of leukemia. Mice injected with myeloblasts expressing decreased levels of Mer demonstrated significantly prolonged symptom-free survival compared to mice transplanted with shControl AML cells (p<0.001). To further explore the effects of Mer inhibition in AML, we used a novel small molecule tyrosine kinase inhibitor (UNC1666), which has high specificity to Mer. Three Mer expressing AML cell lines were treated with UNC1666 in vitro; treatment reduced phosphorylation of Mer and the downstream signaling molecules ERK1/2 and STAT6. Additionally, treatment with UNC1666 resulted in significant induction of apoptosis (p<0.05) by flow cytometric analysis after staining with Yo-Pro-1 iodide and propidium iodide, and dose-dependent inhibition of colony formation in soft agar, when compared to vehicle treated cells In summary, the upregulation of Mer expression in patient samples and the functional effects on survival with Mer shRNA knockdown help validate Mer as a new and attractive AML therapeutic target. Furthermore, a novel Mer tyrosine kinase inhibitor decreased myeloblast cell survival, providing evidence that Mer is a druggable target in AML. Disclosures: Kireev: WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Wang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

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