Leukemia (2001) 15, 1537–1543  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu The inhibitor selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic MV Blagosklonny1, T Fojo1, KN Bhalla3, J-S Kim1, JB Trepel1, WD Figg1, Y Rivera2 and LM Neckers2

Departments of 1Developmental Therapeutics and 2Cell and Cancer Biology, Medicine Branch, National Cancer Institute, NIH, Bethesda and Rockville, MD, USA; and 3Moffitt Cancer Center, Tampa, FL, USA

The Bcr-Abl fusion protein drives leukemogenesis and can ren- Materials and methods der leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90- associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, Cell lines and reagents but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most HL60 and Jurkat, human leukemia cell lines, were obtained cytotoxic drugs, but were found to be sensitive to GA. Further- from American Type Culture Collection (Manassas, VA, USA). more, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, HL60-Bcr-Abl, a Bcr-Abl stable transfected HL-60 cells, were 8,16 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and described previously. Paclitaxel (Taxol), was Bristol-Myers cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an (Princeton, NJ, USA) product. Adriamycin (doxorubicin) was inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells obtained from Sigma (St Louis, MO, USA) and dissolved in to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic DMSO as a 2 mg/ml stock solution. STI 571 was kindly pro- effects of DOX in parental HL60 cells. These results indicate vided by Novartis Pharma (East Hanover, NJ, USA). The pro- that sensitization of Bcr-Abl-expressing cells, but not desensi- tization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, teasome inhibitor PS-341 was a kind gift from Dr Peter Elliott, GA differentially affects leukemia cells depending on their Bcr- Proscript, Inc. (Cambridge, MA, USA). Abl expression and selectively increases in Bcr-Abl- expressing cells. Leukemia (2001) 15, 1537–1543. Keywords: oncogenes; Bcr-Abl; chemotherapy; cytoprotection; gendanamycin; HSP90 Immunoblot analysis

Cells were lysed and soluble proteins were harvested in TNES buffer (50 mm Tris HCl pH 7.5, 100 mm NaCl, 2 mm EDTA, Introduction 1mm sodium orthovanadate, 1% (v/v) NP40) containing pro- tease inhibitors (20 ␮g/ml aprotinin, 20 ␮g/ml leupeptin, 1 mm Chronic myelogenous leukemia (CML) is characterized by a PMSF). Proteins were resolved with SDS-PAGE as previously reciprocal t(9;22) chromosomal translocation, known as the described.15,17 Immunoblotting was performed using rabbit , that fuses the truncated Bcr gene polyclonal anti-human PARP (Upstate Biotechnology, Lake to the truncated c-Abl.1,2 Bcr-Abl is also often found in adult Placid, NY, USA), for Bcr using monoclonal anti-human Bcr acute lymphocytic leukemia (ALL).3 The Bcr-Abl gene enco- antibodies N 2 (Oncogene Science, Calbiochem, San Diego, des for character chimeric p210 and p185 proteins.4 These CA, USA). are constitutively active tyrosine kinases which may induce resistance to apoptotic cell death.5–12 A number of protein kinases, including Raf-1, Src, and ErbB, depend on association with the protein, heat-shock MTT assay protein 90 (Hsp90), for proper function and stability. The benzoquinone ansamycin geldanamycin (GA) binds to Hsp90 Fifteen thousand cells were plated in 0.1 ml in 96-well flat and specifically inhibits this chaperone’s function, resulting in bottom plates and then exposed to tested agents (final volume ␮ degradation of HSP90-associated proteins.13,14 The chimeric 0.2 ml per well). At the indicated time, 20 l of 5 mg/ml MTT BCR-ABL exists in a complex with Hsp90, and GA causes solution in PBS was added to each well for 4 h. After removal ␮ degradation of BCR-ABL after 3–5 h of treatment.15 Since Bcr- of the medium, 170 l of DMSO was added to each well to Abl expression is associated with resistance to cytotoxic dissolve the formazan crystals. The absorbance at 540 nm was agents, we hypothesized that treatment of Bcr-Abl-expressing determined using a Biokinetics plate reader (Bio-Tek Instru- cells with GA would restore their sensitivity to chemothera- ments, Inc, Winooski, VT, USA). Triplicate wells were assayed peutic agents. Indeed our results confirm this prediction. for each condition and standard deviations were determined. Importantly, while sensitizing Bcr-Abl-expressing cells, GA protected parental cells from cytotoxic effects of doxo- rubicin (DOX). Number of dead and live cells

Cells were plated in 24-well plates in 1 ml of medium, or in 96-well plates in 0.2 ml, and were treated with drugs. After the indicated time, cells were counted in triplicate on a Coul- ter Z1 cell counter (Hialeah, FL, USA). In addition, cells were Correspondence: MV Blagosklonny, Medicine Branch, Bldg 10, R incubated with trypan blue and the numbers of blue (dead) 12N226, NIH, Bethesda, MD 20892, USA cells and transparent (live) cells were counted in a hemocyto- Received 30 January 2001; accepted 26 June 2001 meter. Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1538

Figure 2 Reversal of resistance/sensitivity ratios in HL60 and K562 cells by GA. Effects of increasing concentrations of GA on the cytotox- icity caused by 500 nm DOX in either K562 (closed triangles) or HL60 (open triangles) cells. MTT assay was performed after 3 days as described in Materials and methods. At GA = 0 nM axis X, the values represent the cytotoxicity of 500 ng/ml alone. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d. Figure 1 Bcr-Abl downregulation and cytotoxic effects of GA in K562 cells. (a) K562 cells were incubated with indicated concen- trations of GA and then immunoblot for Bcr-Abl was performed as Results described in Materials and methods. Multiple lower molecular bands may represent products of BCR-ABL degradation in GA-treated cells. (b) K562 cells and HL60 cells (shown for comparison) were incubated Pharmacological depletion of Bcr-Abl and cytotoxicity with GA. MTT assay was performed after 3 days, as described in Materials and methods. Results were calculated as the percent of In agreement with previous report,15 treatment of K562 cells values obtained with untreated cells and represent mean ± s.d. with 30–810 nm GA completely depleted Bcr-Abl protein (Figure 1a). Similarly, 30 nm of GA caused a near-maximal growth inhibition of K562 cells (Figure 1b). Previous studies have shown that K562 leukemia cells expressing Bcr-Abl are Cell cycle analysis resistant to numerous therapeutic agents including anti-CD95 mAb, high-dose AraC, , paclitaxel, actinomycin D, 5,8,9,16,18,19 Cells were washed with PBS and resuspended in 75% ethanol staurosporine, and inhibitors. In con- in PBS and kept at 4°C for at least 30 min. Prior to analysis, trast, GA was more cytotoxic in K562 cells than in HL60 cells cells were washed again with PBS and resuspended and incu- (Figure 1b). bated for 30 min in propidium iodide staining solution con- taining 0.05 mg/ml propidium iodide (Sigma), 1 mm EDTA, 0.1% Triton-X-100 and 1 mg/ml RNAse A in PBS. The suspen- GA sensitized K562 cells but protected HL60 cells sion was then passed through a nylon mesh filter and ana- lyzed on a Becton Dickinson FACScan (Franklin Lakes, NJ, K562 cells were much more resistant to doxorubicin (DOX) USA). than were HL60 cells. (60% and 4% survival, respectively; see Figure 2, X-axis: GA = 0). Addition of 10–100 nm GA decreased survival of DOX-treated K562 cells. If sensitization by GA was due to depletion of Bcr-Abl, this effect should not Nuclear fragmentation assay be observed in HL60 cells. In fact, GA did not sensitize HL60 cells (Figure 2). Unexpectedly, GA rendered these cells more Cells were incubated with drugs for the indicated time. Cells resistant to DOX. Sensitization of K562 coupled with desensit- were washed with PBS, pelleted on to glass slides in a cyto- ization of HL60 resulted in the reversal of their relative sensi- centrifuge, fixed with 90% ethanol with 10% glacial acid and tivities to DOX in the presence of GA (Figure 2). stained with DAPI as described previously.18 Nuclei were When added alone as well as with either PTX or DOX, GA visualized under UV microscopy. depleted p210BCR-ABL in K562 cells (Figure 3a). Also, PTX and

Leukemia Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1539

Figure 3 Combinations of drugs with GA. K562 cells (a) and HL60 cells (b) were treated with 90 nm GA, 500 ng/ml DOX, 100 nm PTX or their combinations, as indicated. After 16 h, cells were lysed and immunoblots for PARP and BCR were performed as described in Materials and methods. K562 cells (c) and HL60 cells (d) were treated as described in (a) and (b). MTT assay was performed after 48 h as described in Materials and methods. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d.

DOX slightly downregulated Bcr-Abl. This downregulation of gated combinations of GA with either PS-341, an inhibitor of Bcr-Abl occurred before apoptosis, since PARP was still intact the proteasome, or DOX (Figure 6). As expected, HL60-Bcr- 16 h after the treatment (Figure 3a). In HL60 cells, Bcr levels Abl were more resistant to PS-341 than were the parental cells were not affected by GA (Figure 3b). Consistent with previous (Figure 6a). It is noteworthy that PS-341 and GA antagonized reports,20,21 both DOX and PTX caused apoptosis in HL60 one another in HL60-Bcr-Abl cells but not in parental cells, cells, as evidenced by PARP cleavage (Figure 3b). Remark- consistent with the notion that GA targets Bcr-Abl for protea- ably, GA partially abrogated DOX- and PTX-induced PARP somal degradation. Finally, GA sensitized HL60-Bcr-Abl cells cleavage (Figure 3b). Furthermore, this inhibition of PARP to DOX, but protected the parental cells against DOX-induced cleavage was accompanied by higher survival of HL60 cells cytotoxicity (Figure 6b). In the absence of GA, HL60 cells (Figure 3d). In contrast, GA increased toxicity of PTX and DOX were sensitive to DOX, whereas HL60-Bcr-Abl were resistant in K562 cells (Figure 3c). Since the activity of PTX is cell to DOX (Figure 6b, axis X: GA = 0). In the presence of GA, cycle-dependent, we next investigated by flow cytometry the HL60-Bcr-Abl cells were more sensitive to DOX than were effects of combinations of GA and PTX (Figure 4). Treatment parental cells (Figure 6b). This result was confirmed by exam- with GA caused G1 and G2 arrest. As expected, PTX caused ining nuclear fragmentation, a morphological marker of G2/M arrest. Combination of PTX with GA decreased G2/M apoptosis. DOX induced nuclear fragmentation in HL60, but due to an increase in sub-G1-peaks, consistent with apoptosis. not in HL60-Bcr-Abl cells (Figure 7). GA prevented nuclear fragmentation caused by DOX in HL60 cells, but caused nuclear fragmentation in DOX-treated HL60-Bcr-Abl cells Comparison of HL60 and HL60-Bcr/ABL (Figure 7). We conclude that opposite effects of GA in Bcr- Abl-expressing and non-expressing HL60 cells depend solely To rule out the possibility that differences in cellular back- on Bcr-Abl. ground other than the expression of Bcr-Abl might determine opposite responses of HL60 and K562 cells, we compared parental HL60 cells with HL60 cells stably transfected with The kinase inhibitor, STI 571, sensitizes Bcr-Abl cells Bcr-Abl (HL60-Bcr-Abl cells). HL60-Bcr-Abl cells are much but does not protect parental HL60 cells more resistant to chemotherapy than parental cells.8,16 In con- trast, HL60-Bcr-Abl cells were more sensitive to GA (10– Finally, we compared GA with STI 571, an inhibitor of kinases 100 nm) than parental HL60 cells (Figure 5). We next investi- including Bcr-Abl and Abl.22 Unlike GA, STI 571 did not

Leukemia Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1540

Figure 5 Effects of GA on parental and BCR-ABL-expressing HL60 cells. HL60 and Bcr-Abl-transfected HL60 (HL60-Bcr-Abl) were incu- bated with GA, as indicated. MTT assay was performed after 48 h as described in Materials and methods. Results were calculated as the per- cent of values obtained with untreated cells and represent mean ± s.d.

gate either DOX-induced PARP cleavage (Figure 8b) or DOX- induced cell death (Figure 8d).

Discussion

Apoptosis in response to chemotherapy is often evaded by cancer cells.23 The expression of the BCR-ABL chimeric pro- tein prevents apoptosis caused by a variety of anticancer Figure 4 Cell cycle analysis of GA-treated K562 cells. K562 cells 22 were treated with either 90 nm GA, 100 nm PTX, or their combination agents. Geldanamycin (GA), is an inhibitor of the chaperone (PTX + GA), or left untreated (control). After 24 h, flow cytometry was Hsp90, and it promotes depletion of Hsp90-associated pro- performed as described in Materials and methods. teins, including Bcr-Abl.15 Here we show that GA downregul- ated Bcr-Abl in Ph1-positive K562 cells and in HL60 cells stably transfected with Bcr-Abl. In both cell lines, a depletion deplete the Bcr-Abl protein (Figure 8a). Both STI 571 and GA of the Bcr-Abl protein and a near-maximal toxicity were achi- caused comparable PARP cleavage in HL60-Bcr-Abl cells. eved at concentrations of GA as low as 30 nm. Furthermore, DOX did not induce caspase-3 activation (data not shown) or GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) PARP cleavage in Bcr-Abl cells (Figure 8a). Both GA and STI and, albeit to a lesser degree, to paclitaxel (PTX). 571 significantly increased DOX-induced death of HL60-Bcr- Inhibitors of the Abl kinase, including STI 571, induce Abl cells (Figure 8c). Therefore, degradation of the Bcr-Abl apoptosis in Bcr-Abl-positive cells.6,10,11,22,24–27 In agreement protein by GA and inhibition of the Bcr-Abl kinase by STI 571 with previous results,28–30 we show that STI 571 induced resulted in similar effects in Bcr-Abl-expressing cells: sensitiz- apoptosis in Bcr-Abl-expressing cells and sensitized Bcr-Abl ation of these cells to DOX. The role of Bcr-Abl was confirmed expressing cells to chemotherapeutic agents. Given that GA by using parental HL60 cells (Figure 8b). Since HL60 cells do and STI 571 inhibit Bcr-Abl activities by different mechanisms, not express Bcr-Abl, we measured Raf-1 as a surrogate end these data confirm that inhibition of Bcr-Abl is responsible for point of GA effects. As expected, GA (alone and in sensitization caused by either GA or STI 571. combination) depleted cells of Raf-1, whereas STI 571 did not STI 571 has been remarkably effective in preclinical and (Figure 8b). DOX induced PARP cleavage, whereas GA and clinical trials.31 However, development of resistance to STI STI 571 did not (Figure 8b). Unlike GA, STI 571 did not abro- 571 has been reported,32–34 thus validating GA-induced Bcr-

Leukemia Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1541

Figure 6 Effects of combinations of GA with PS-341 or DOX in parental and BCR-ABL-expressing HL60 cells. (a) Cells were incubated with 300 nm PS-341 alone (GA = 0nm) or with increasing concentrations of GA, as indicated. MTT assay was performed after 48 h as described in Materials and methods. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d. (b) Cells were incubated with 500 ng/ml DOX alone (GA = 0nm) or with increasing concentrations of GA, as indicated. MTT assay was performed after 48 h as described in Materials and methods. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d.

Figure 7 Effects of GA and DOX on nuclear fragmentation of parental and Bcr-Abl-expressing HL60 cells. HL60 and HL60-Bcr-Abl cells were incubated with 500 ng/ml DOX, 90 nm GA, or their combination (DOX + GA), or were left untreated. After 20 h, DAPI staining for nuclear fragmentation was performed as described in Materials and methods.

Abl depletion as an alternative therapeutic approach. Unex- decrease drug-related side-effects, while increasing a thera- pectedly, and unlike STI-571, GA protected parental HL60 peutic index. Further experiments using primary BCR-ABL- cells against DOX-induced apoptosis. Although seemingly negative cells will be necessary to determine the generality of paradoxical, this phenomenon is likely a result of GA’s this observation. reported ability to induce a compensatory elevation in the synthesis of several heat shock proteins including Hsp70.35 It References is well known that high levels of Hsp70 can inhibit apoptosis.36 1 Shtivelman EB, Lifshitz B, Gale RP, Canaani E. Fused transcript of Using GA in this setting recalls the similar strategy of selec- abl and bcr genes in chronic myelogenous leukemia. Nature 1985; tively protecting wt -expressing cells against paclitaxel- 315: 550–554. induced apoptosis by pretreatment with low concentrations of 2 Deininger MWN, Goldman JM, Melo JV. The molecular biology 37 of chronic myeloid leukemia. Blood 2000; 96: 3343–3356. DOX. Thus, use of GA to simultaneously increase the drug 3 Huettner CS, Zhang P, Van Etten RA, Tenen DG. Reversibility of tolerance of normal cells, while improving the sensitivity of acute B-cell leukaemia induced by BCR-ABL1. Nature Genet Bcr-Abl-expressing tumor cells to chemotherapy, might 2000; 24: 57–60.

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Figure 8 Effects of GA and STI 571 on drug-induced caspase activation and cytotoxicity. HL60-Bcr-Abl cells (a) or HL60 cells (b) were treated with 500 ng/ml DOX, 90 nm GA, 1 ␮m STI 571 alone and in combinations, as indicated. After 16 h, cells were lysed and immunoblotted for Bcr-Abl and PARP (a), and Raf-1 and PARP, as described in Materials and methods. (c–d) MTT assay was performed after 40 h, as described in Materials and methods. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d.

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