Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

Abstract

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.

Fig. 1.

Gene expression analysis identifies IL1RAP as up-regulated in CML CD34⁺ cells and following retroviral P210 BCR/ABL1 expression in CB CD34⁺ cells. Global gene-expression analyses were performed on CD34⁺ cells obtained at diagnosis from chronic-phase CML patients and on CB CD34⁺ cells, 2 d after retroviral P210 BCR/ABL1 transduction. Heatmaps showing up-regulated genes (red) and down-regulated genes (green) matching to the GO category “integral to plasma membrane” are displayed for primary CD34⁺ cells obtained from normal bone marrows (NBM) (n = 6) and CML (Ph⁺) patients (n = 10) (A) and for retroviral P210 BCR/ABL1-expressing cells in comparison with empty vector MIG control-expressing cells (B). IL1RAP, marked in the figure, is the only highly up-regulated gene in common between the two gene lists. The up-regulation of the IL1RAP transcript was confirmed by real-time PCR using 18S as endogenous control (C). IL1RAP expression is presented as fold change in relation to NBM-C.

Fig. 2.

The kinase activity of P210 BCR/ABL1 induces up-regulation of IL1RAP on the cell surface. Flow cytometric analysis confirmed that IL1RAP expression is induced upon retroviral P210 BCR/ABL1 expression of cord blood CD34⁺ cells, 3 d after transduction (A). CD34⁺GFP⁺ cells were gated according to the gates in the contour plots. The histogram shows the expression of IL1RAP for negative-control staining (white), MIG control (green), MIG-P210 (blue), and MIG-P210 kinase inactive (KI) (light red). KU812 cells were also treated with 5 μM imatinib and IL1RAP expression was assessed 2 d later in live cells (7-AAD⁻) (B). The histogram shows the expression of IL1RAP for imatinib-treated cells (yellow) and nonimatinib-treated cells (gray). One representative experiment out of three is shown. MIG; MSCV-IRES-GFP, murine stem cell virus-internal ribosome entry site-green fluorescent protein.

Fig. 3.

IL1RAP is up-regulated on the cell surface of CML CD34⁺CD38⁻ cells. FACS analysis of CD34⁺ cells from five CML patients in chronic-phase (CML1-5) and from two NBM samples (NBM1, -2). (A) FACS dot-plot showing gating for CD34⁺CD38⁺ or CD34⁺CD38⁻ cells in a representative CML patient. (B) Histogram showing IL1RAP expression within CD34⁺CD38⁻ cells. Light red represents control-stained samples and blue represents IL1RAP-stained samples. The sorting gates for CD34⁺CD38⁻IL1RAP⁻ and CD34⁺CD38⁻IL1RAP⁺ cells are outlined in the histograms. IL1RAP is up-regulated in CML CD34⁺CD38⁻ cells compared with their normal counterparts.

Fig. 4.

IL1RAP expression distinguishes Ph⁺ from Ph⁻ CML cells within the CD34⁺CD38⁻ cell compartment. Flow-drop-FISH on CML CD34⁺CD38⁻IL1RAP⁻ and CD34⁺CD38⁻IL1RAP⁺ cells from CML1-5 revealed an almost complete separation between Ph⁻ and Ph⁺ cells, respectively. (Left) Black bars represent BCR/ABL1⁻ cells and red bars represent BCR/ABL1⁺ cells. Outlined at the top of each bar is the number of Ph⁺ cells of the total nuclei scored within individual samples. The IL1RAP status of sorted cells is shown beneath each bar. (Right) Pictures show BCR/ABL1⁺ (one red, one green, and two fused signals) versus BCR/ABL1⁻ (two green and two red signals) cells. White arrows points to fused signals.

Fig. 5.

IL1RAP expression distinguishes Ph⁺ from Ph⁻ CML LTC-IC. (A) Number of LTC-CFCs derived from CD34⁺CD38⁻IL1RAP⁻ and CD34⁺CD38⁻IL1RAP⁺ cells. Black bars represent IL1RAP⁻ cells and yellow bars represent IL1RAP⁺ cells. (B) Interphase FISH on LTC-CFC. Black bars represent BCR/ABL1⁻ cells and red bars represent BCR/ABL1⁺ cells. Outlined at the top of each bar is the number of Ph⁺ cells of the total nuclei scored within individual samples. The IL1RAP status of sorted LTC-IC is shown beneath each bar. A near-complete discrimination of BCR/ABL1⁻ and BCR/ABL1⁺ cells was found among LTC colonies derived from CD34⁺CD38⁻IL1RAP⁻ or CD34⁺CD38⁻IL1RAP⁺ cells, respectively.

Fig. 6.

Killing of CML CD34⁺CD38⁻ cells by antibody targeting of IL1RAP. (A) Histogram showing IL1RAP expression in the Ph⁻ KG-1 and Ph⁺ KU812 cell lines. Light red shows control-stained samples and blue shows KMT-1–stained samples. The leukemic cell line KG-1 is devoid of IL1RAP expression, whereas KU812 cells express IL1RAP. As a consequence, only a low level of antibody-induced cell death was observed in KG-1, but a dose-dependent ADCC effect was observed using KMT-1 on KU812 cells (B). In line with the level of IL1RAP expression, no obvious ADCC effect was seen using NBM CD34⁺ (C) and CD34⁺CD38⁻ (D) cells, whereas KMT-1 induced a strong dose-dependent ADCC effect in both CML CD34⁺ (C) and CML CD34⁺CD38⁻ cells (D). As a control for nonspecific ADCC effects, a rabbit IgG antibody was also used in the experiments. Each graph shows the average and SD of antibody-induced cell death from a minimum of three independent experiments.