Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells

Abstract

Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations.

Fig. 1.

iCaspase-8 specifically triggers apoptosis in cells expressing BCR-ABL. (A) iCaspase-8 becomes active after phosphorylation of Crk at Tyr-221 by BCR-ABL. (B) iCaspase-8 (MSCV-IRES-GFP-WRE) was expressed in K562 cells. Forty-eight hours after infection, cell death % (with SEM) was analyzed by PI staining (*P < 0.05 by Student t test). (C and D) Normal Ba/F3 cells, or Ba/F3 cells expressing BCR-ABL, FLT3^D835Y, or TEL-PDGFRβ, were infected with a retroviral vector (MSCV-IRES-GFP-WRE) encoding vector alone (GFP), iCaspase-8 (iC8), iCaspase-8^Y/F (iC8^Y/F), or iCaspase-8^C/S (iC8^C/S). Forty-eight hours after infection, the cells were fixed, stained with anti-cleaved caspase 3-antibody (Alexa 647), and analyzed by flow cytometry. Shown are representative flow cytometry plots (C) and average cell death % with SEM (% cells with cleaved-caspase-3 signal) in the GFP-positive population (D; *P < 0.05 GFP vs. iC8 by Student’s t test). (E) Ba/F3 cell lysates were subjected to immunoblotting with anti–pan-Crk and anti–phospho-Tyr-221 antibodies.

Fig. 2.

iCaspase-8 activation is caused by intramolecular dimerization. (A) Schematic diagram of iCaspase-8. Each of the two caspase-8 moieties is composed of the small (S) and large (L) subunits. The linker connecting the two subunits will be autoprocessed upon activation of iCaspase-8, resulting in 10-, 20-, and 70-kDa proteolytic fragments. (B) iCaspase-8 or iCaspase-8^Y/F was expressed in Ba/F3 cells (control or BCR-ABL). Forty-eight hours after infection, cell lysates were prepared and loaded onto a Supedex-200 column. Each chromatographic fraction was collected and subjected to immunoblotting with anti-human Caspase-8 antibody to detect iCaspase-8. Intact and cleaved iCaspase-8 is shown by an arrow and an arrowhead, respectively.

Fig. 3.

iCaspase-8 eliminates BCR-ABL–positive cells while sparing normal cells. The mixture of Ba/F3 cells (normal:BCR-ABL = 9:1) was infected with an empty vector or iCaspase-8 (MSCV-IRES-GFP-WRE), and FACS sorted to select GFP-positive cells. The sorted cells were further cultured for 7 d and analyzed by qPCR for the BCR-ABL message. qPCR results were normalized against actin (Right; *P < 0.05 by Student’s t test). The experiments were repeated four times, and there was no significant difference in cell growth between the two groups (Left; P > 0.05 by Student’s t test). Error bars are SEM.

Fig. 4.

iCaspase-8 selectively kills BCR-ABL–positive mouse HSCs. (A) Mouse KLS cells were cotransduced with BCR-ABL (MSCV-IRES-GFP) and iCapsase-8 (MSCV-IRES-YFP-WRE) or empty vector. BCR-ABL was either wild type (WT) or carried an imatinib-resistant mutation (E255K, T315I, M351T). GFP-YFP double-positive cells were further selected by FACS and plated in methylcellulose medium with or without 2 µM imatinib. The colony numbers were counted 7 d after plating (*P < 0.05 YFP vs. iC8 by Student t test). (B) Mouse KLS cells were transduced with iCapsase-8 (MSCV-IRES-GFP-WRE) or empty vector. GFP positive cells were selected by FACS and plated in methylcellulose medium. The colony numbers were counted 7 d after plating. Averages with SEM are shown.

Fig. 5.

iCaspase-8 selectively kills blast crisis CML cells in mouse. (A) Lin⁻ BCR-ABL⁺ NUP98-HOXA9⁺ cells isolated from mice with blast crisis CML were infected with control vector or iCaspase-8 (MSCV-IRES-GFP-WRE). Forty-eight hours after infection, GFP and YFP (BCR-ABL) coexpressing cells were selected and plated in methylcellulose medium for colony forming assays. The colony numbers were counted 7 d after plating. Average colony numbers with SEM are shown (*P < 0.05 by Student’s t test). (B) Cells infected with BCR-ABL, NUP98-HOXA9, and either control vector or iCaspase-8 were transplanted and survival was monitored (control, n = 23; iCaspase-8, n = 20; *P < 0.05 by Kaplan–Meier analysis with log-rank test). (C and D) Spleens were collected from each mouse and weighed (C). Average weights and SEM are shown (*P < 0.05 by Student’s t test). Representative spleens are shown in D: spleens from control (Left) and iCaspase-8 mice [surviving mice (Center) and leukemic mice (Right)].

Fig. 6.

iCaspase-8 selectively kills patient-derived CML cells in vitro. (A) Schematic diagram of the experimental process. Ficoll-purified white blood cells were isolated from patients with blast crisis stage of CML. The cells were stained with PE-conjugated anti-CD34 antibody and CellTrace Violet. CD34/CellTrace Violet double-positive cells were selected by FACS. CD34⁺/CellTrace Violet⁺ cells were transduced with vector control or iCaspase-8 (pCDH-EF1-IRES-GFP). Forty-eight hours later, the cells were stained with Annexin V (conjugated with Alexa 647). (B) Average cell death % with SEM (% cells with Annexin V signal) in the GFP-positive population are shown (*P < 0.05 by Student’s t test) (C). Representative flow cytometry plots (with % cell population) are shown (Left): Annexin V vs. CellTrace Violet in GFP⁺ or iCaspase-8⁺ populations. Average cell survival % with SEM in CellTrace Violet^high and CellTrace Violet^low populations are shown (Right; *P < 0.05 by Student t test).