Insights from Pre-Clinical Studies for New Combination Treatment

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Insights from pre-clinical studies for new combination treatment regimens with the Bcr-Abl kinase inhibitor imatinib mesylate (Gleevec/Glivec) in chronic myelogenous leukemia: a translational perspective P La Rose´e1,2, ME O’Dwyer1 and BJ Druker1

1Oregon Health and Science University, Division of Hematology and Medical Oncology, Portland, OR, USA; and 2III Medizinische Universita¨tsklink, Fakulta¨tfu¨r Klinische Medizin Mannheim der Universita¨t Heidelberg, Mannheim, Germany

Clinical phase I/II studies with the Abl kinase inhibitor imatinib Work from several groups have revealed various mech- mesylate (Gleevec/Glivec, formerly STI571) for the treatment for anisms of resistance both in vitro11 and in vivo.12–15 The chronic myelogenous leukemia (CML) demonstrated the safety and the remarkable efficacy of this molecularly targeted agent. emergence of resistance to imatinib is not a surprise, parti- However, a significant proportion of patients treated in the cularly in advanced phase CML where molecular abnormali- chronic phase of the disease after having failed interferon ties in addition to Bcr-Abl are undoubtedly present. Resistance alpha (IFN) remain predominantly Philadelphia chromosome to antineoplastic agents has been studied extensively and this + positive (Ph ), suggesting a risk of later relapses. Furthermore, research has revealed a complex scenario of mechanisms results in blast crisis patients revealed a high frequency of similar to those reported with imatinib.16 The occurrence of relapses or resistance to imatinib. To circumvent resistance, improve response rates, or prolong survival, pre-clinical evalu- resistance led to the use of multiple antineoplastic agents with ations of combinations of imatinib with other agents have been differingmechanisms of action. Based on these consider- pursued. Some of these have already been translated into clini- ations, several groups investigated in vitro combinations of cal studies. Here, we first summarize evidence from pre-clinical imatinib with various anti-leukemic agents. studies on new combination regimens with imatinib in the treatment of CML. Second, we analyze preliminary clinical data of ongoing combination studies. Finally, we provide a summary of approaches that use novel antileukemic agents with Insights from in vitro cytotoxicity studies – conventional molecularly characterized modes of action. chemotherapeutic agents Leukemia (2002) 16, 1213–1219. doi:10.1038/sj.leu.240555 Keywords: imatinib mesylate; ST1571; combination therapy; CML In vitro studies demonstrate that Bcr-Abl has anti-apoptotic activity and renders leukemic cells resistant to chemotherapy.17,18 Inhibition of Bcr-Abl expression by antisense oli- Introduction gonucleotides reverses the suppression of apoptosis and makes CML cells susceptible to apoptois by antileukemic Chronic myelogenous leukemia is an hematopoietic stem cell drugs.19 McGahon et al19 showed that the anti-apoptotic effect disorder that accounts for 15–20% of all leukemias in adults.1 of Bcr-Abl is dependent on Bcr-Abl kinase activity. Therefore, The hallmark of the disease is the Philadelphia chromosome,2 it was hypothesized that kinase inhibition by imatinib would which is a result of the (9;22)(q34;q11) translocation.3 Mul- also act as a chemo-sensitizer. Indeed, Fang et al20 showed tiple experimental models established the product of the trans- that imatinib significantly increased cytosine arabinoside (ara- location, the deregulated Bcr-Abl tyrosine kinase, as the C) or doxorubicin-induced apoptosis of Bcr-Abl positive cell causative leukemogenic agent (for review, see Refs 4 and 5). lines. The central role of Bcr-Abl in the pathogenesis of CML have Several groups have published in vitro combination studies made it a particularly attractive target for drug therapy.6 The usingimatinib and conventional chemotherapeutic agents. 21–23 Abl kinase inhibitor imatinib mesylate proved to be safe and Table 1 lists the results for data obtained with comparable effective in phase I clinical studies for CML and Ph+ acute parameters (identical cell line (K562), same drugor drug lymphoblastic leukemia (ALL).7,8 These results were confirmed class). All results were generated using tetrazolium-based cell SPOTLIGHT in phase II international multicenter studies which have been line proliferation assays (MTT assay).24 These groups deter- published recently. mined the inhibition of proliferation after continuous exposure Taken together, these data confirm the remarkable efficacy to the combination drugs for 2 days (Topaly et al22), 3 days of imatinib as a single agent for all stages of CML. However, (Thiesing et al21) and up to 7 days (Kano et al23). The data two major concerns arise from the clinical studies: 40% of the were analyzed by the median effect method based on the iso- patients treated for chronic phase CML after interferon failure bologram equation25 (Topaly), the isobologram by hand remain predominantly Ph+. A recent analysis suggests these method (Thiesing) and the Steel and Peckham isobologram26 patients have a higher risk of relapse.9 Primary and secondary (Kano). The methodical differences of data analysis may resistance to imatinib causes frequent relapses in blast crisis explain some of the variability in the reported efficacy. patients.10,89,90 The studies listed in Table 1 also used multiple Bcr-Abl positive (p210Bcr-Abl, p185Bcr-Abl) and negative cell lines. For example, Thiesing et al tested a Bcr-Abl negative cell line, MO7e, and a derivative with engineered p210Bcr-Abl Correspondence: B Druker, Orgeon Health and Science University, expression (MO7p210). Colony-formingassays usingprimary Division of Hematology and Medical Oncology; 3181 Sam Jackson Park Rd, mail code L592, Portland, Oregon 97201, USA; Fax: 1 503 patient cells (chronic phase CML) were also performed. The 494 3688 findings in colony-forming assays confirmed the data gener- Received 25 February 2002; accepted 14 March 2002 ated in cell line proliferation assays with the exception that Pre-clinical investigation of imatinib in conjunction with anti-leukemic agents P La Roseé et al 1214 Table 1 Comparison of three independent studies investigating the Table 2 Summary of conventional chemotherapeutic agents that in vitro cytotoxicity of imatinib with conventional chemothera- have been tested in cytotoxicity studies in combination with imatinib peutic agents Combined drug Synergy Additivity Antagonism Combined drug Study Effect Ara-C

Hydroxyurea Thiesing et al Antagonistic (IC50) Carboplatin Topaly et al Antagonistic (IC10–99) Corticosteroids Kano et al Additive (IC80) Etoposide Daunorubicin Thiesing et al Additive (IC75) Idarubicin Doxorubicin Kano et al Additive (IC80) IFN Ara-C Thiesing et al Synergistic (уIC50) Mafosfamide Kano et al Additive (IC80) Mitoxantrone у Etoposide Topaly et al Synergistic ( IC50) Treosulfan Kano et al Additive (IC80) Vincristine у Mafosfamide Topaly et al Synergistic ( IC75) Nimustine hydrochloride 4-OH- Kano et al Additive/Synergistic (IC80) Busulfan cyclophosphamide 4-OH-cyclophosphamide Daunorubicin Only data generated using the same cell line (K562) were con- Doxorubicin sidered for comparison. All three investigators determined joint Fludarabine

SPOTLIGHT action using MTT proliferation assays. Gemcitabine (IC ): inhibitory concentrations of drugs, at which combined activity Taxotere x was defined. Thiotepa Cladribine Hydroxyurea hydroxyurea (HU) was antagonistic when combined with Methotrexate Topotecan imatinib in proliferation assays but the combination had enhanced antiproliferative activity in colony-formingassays. Included are all published results in the studies of Topaly et al, IFN, a standard treatment option for CML patients, is not Fru¨hauf et al, Thiesing et al and Kano et al. Joint action studies included in Table 1, since various cell lines were tested using were performed using myeloid or lymphoid CML blast crisis cell IFN in combination with imatinib. Thiesing et al reported lines or using Bcr-Abl negative cell lines with engineered Bcr-Abl additive activity at the IC60 in K562 cells, whereas Kano et al expression. found synergism testing KU812 and TCC-Y cells at the IC80. Kano et al also tested the activity of imatinib in combination with vincristine and methotrexate. They found synergistic showed no potentiation of toxicity usingBcr-Abl negativecell activity for vincristine in 3/4 of tested cell lines, but antagon- lines or bone marrow mononuclear cells derived from ism in 3/4 cell lines usingmethotrexate. Additional data on healthy donors.21,22 the pre-clinical evaluation of a variety of conventional chemo- Nevertheless, c-kit has an essential role in the maintenance therapeutic agents were reported by Fru¨hauf et al.27 All of the of normal hematopoiesis (for review see Ref. 35). Furthermore, data for the various combinations of imatinib with conven- c-Abl has been linked to lymphoid development.36 Thus, tional chemotherapy are presented in Table 2. inhibition of c-kit by imatinib may impair hematopoiesis and A consistent observation from our studies and that of others inhibition of c-Abl carries the theoretical risk of impairing is that increased synergy is observed at higher levels of inhi- lymphocyte development. Thus, combination with anti-leu- bition.22,28 Our interpretation of this is that maximal inhibition kemic drugs could overall lead to an aggravation of hematol- of Bcr-Abl kinase activity is required to reverse the drug- ogic side-effects. Recently, Ruchatz et al37 presented data resistant phenotype induced by Bcr-Abl expression. The impli- showingthat administration of imatinib to nude mice indeed cation of this is that full doses of imatinib would be required increases the sensitivity to concomitant treatment with idarub- to achive optimal therapeutic responses in combination icin (IDA) as measured by mortality, bone marrow cytopenia regimens. and spleen size. In contrast, Uemura and Griffin38 treated normal mouse bone marrow cells with imatinib ex vivo and analyzed survival usingcolony-formingassays after ionizingradi- Predicting hematologic toxicity of combination regimens ation, a potent pro-apoptotic stimulus. They did not find using pre-clinical data differences in the survival rate comparingimatinib treated with untreated cells, suggesting that imatinib treatment does Pre-clinical studies showed that imatinib is a relatively selec- not affect the survival of normal hematopoietic cells when tive kinase inhibitor. Kinases inhibited at submicromolar con- exposed to concomitant stress factors such as radiation. centrations are Abl (v-Abl, c-Abl, Tel/Abl, Bcr-Abl), PDGF-R alpha and beta, Tel/PDGF-R beta, c-Kit, Argand Tel/Arg. 29–32 Cytotoxicity studies comparingthe anti-proliferative activity of Translation of in vitro studies into clinical trials imatinib against Bcr-Abl expressing cell lines compared to Bcr-Abl negative cell lines confirmed this highly specific target A variety of dose-findingstudies combiningchemotherapeutic profile.29,33 Furthermore, experiments usingprimary bone drugs with imatinib are under way to assess their clinical effi- marrow samples from CML patients and normal donors cacy and toxicity. These combinations were selected for clini- revealed a favorable therapeutic index for imatinib with only cal trials based on pre-clinical data described above, along marginal toxicity toward normal early and lineage committed with existingclinical data that established the respective progenitor cells.29,34 Combination studies analyzingthe in agents as standard treatment options for CML or Ph+ ALL. vitro cytotoxicity of imatinib and chemotherapeutic agents Table 3 lists some of the ongoing clinical studies.

Leukemia Pre-clinical investigation of imatinib in conjunction with anti-leukemic agents P La Rose´e et al 1215 Table 3 Translation of laboratory data into clinical studies

Combined drugs CML stage Ref.

IFN CP 41 PEG-IFN (PEG-Intron) CP Italian study group PEG-IFN (PEG-Intron) CP 42 PEG-IFN (Pegasys) early CP http://www.kompetenznetz-leukaemie.de Ara-C CP http://www.cancernet.nci.nih.gov Ara-C CP 44 Ara-C My-BC http://www.cancernet.nci.nih.gov IFN + ara-C CP http://www.mdanderson.org Idarubicin + ara-C My-BC http://www.mdanderson.org Daunorubicin, vincristine, Ly-BC http://www.cancernet.nci.nih.gov prednisone Cyclophosphamide, vincristine, Ly-BC 47 doxorubicine, dexamethasone Etoposide/mitoxantrone, ara-C My-BC http://www.kompetenznetz-leukaemie.de

Listed is a selection of ongoing clinical studies. Where no reports of preliminary results are available, www-access to study information is provided. PEG-IFN-␣, poly-ethylene-glycosylated-IFN-␣; CP, chronic phase; My-BC, myeloid blast crisis; Ly-BC, lymphoid blast crisis.

Interferon-␣ and the pre-clinical data for these drugs when combined with imatinib (Table 2). Preliminary results of a study which Several randomized clinical studies have established IFN as a sequentially combines an established regimen for Ph+ ALL or standard treatment option for CML patients.39,40 Since IFN in lymphoid blast crisis CML with imatinib maintenance have combination with imatinib has in vitro activity, we and others been reported.47 The toxicity profile seems to be equivalent initiated phase I/II clinical studies in chronic phase to chemotherapy alone, consistent with the sequential as patients.41,42 The major conclusion from preliminary data is opposed to simultaneous combination. that the combination treatment is feasible and high rates of Longer follow-up, optimized dosing and alternative treat- hematologic and cytogenetic responses are achievable. How- ment schedules are necessary to determine the therapeutic ever, the combination of imatinib with IFN produces a higher efficacy of these combination regimens. rate of hematologic toxicity. This was seen with using conventional IFN and IFN modified by pegylation (PEG-IFN). The optimal dose schedules for phase III studies have yet to be Imatinib in combination with novel antileukemic agents determined. The list of compounds that show activity against Bcr-Abl expressing cell lines is rapidly growing. Several recent review Ara-C articles summarize information about new anti-leukemic agents that translate insights from basic science into clinical Additive to synergistic activity of ara-C with imatinib in pre- application.48–52 Here we focus on in vitro data for combi- clinical studies and the proven clinical efficacy of ara-C in nations of novel agents with imatinib. In particular, we all stages of CML43 prompted several clinical studies of this emphasize novel combinations which are movingtowards combination. Our institution is conductinga phase I/II combi- phase I/II clinical studies. Table 4 gives an overview of nation study of low dose ara-C and imatinib.44 Preliminary reported in vitro data. data indicate a higher toxicity profile of this combination compared to imatinib alone. Although response rates do not SPOTLIGHT appear to be superior to imatinib monotherapy, longer studies Arsenic trioxide (As2O3) usingmaximally tolerated doses are ongoingtodefine 53,54 response rates. Since ara-C was shown to improve the The success of As2O3 in promyelocytic leukemia and the complete hematologic and cytogenetic response rate when historical experience with As2O3, the active ingredient in 45 combined with IFN in chronic phase patients, clinical inves- Fowler’s solution, for CML, has led to studies of As2O3 in Bcr- tigation is also evaluating the combination of IFN/ara-C + Abl-positive leukemia cells. In vitro studies with clinically imatinib (Table 3). achievable concentrations of As2O3 showed induction of apoptosis and differentiation in Ph+ K562 and HL60/Bcr-Abl cells.55 Furthermore, myelomonocytic U937 cells with Multi-agent chemotherapy enforced expression of Bcr-Abl have increased sensitivity to

As2O3 as compared to parental cells, suggesting that Bcr-Abl Several protocols combiningmulti-agentchemotherapy with expression acts as a sensitizer for the cytotoxic activity of 56 imatinib in advanced phase CML have been initiated (see As2O3. This ﬁndingneeds to be interpreted with caution. Table 3). A multi-center phase I/II study in Germany is enrol- Usingpaired cell lines, we have observed that Bcr-Abl can lingpatients with myeloid blast crisis investigatingthe combi- induce either sensitivity or resistance to As2O3 dependingon 91 nation of mitoxantrone, etoposide, ara-C and imatinib. The the cell type. Since As2O3 toxicity is modulated by intra- 57 rationale for this study is derived from favorable clinical cellular glutathione levels, sensitivity to As2O3 may depend results for refractory AML usingetoposide and mitoxantrone 46 on the cellular environment rather than on a speciﬁc sensitiz-

Leukemia Pre-clinical investigation of imatinib in conjunction with anti-leukemic agents P La Rose´e et al 1216 Table 4 Novel anti-leukemic agents in combination with undergo this modiﬁcation and farnesylation may be required imatinib – summary of pre-clinical studies in Bcr-Abl expressingcells for these proteins to function in proliferative and survival pathways. Although proteins such as ras and rhoB are targets of Novel agents Molecular mechanism(s) Ref. farnesyl transferase inhibitors (FTIs), it is not clear that all of the cellular effects of FTIs can be explained by targeting these 17-AAG hsp90 chaperone function 69 two proteins.64 Regardless, FTIs have anti-proliferative proper- inhibition 65 adaphostin tyrosine kinase inhibition 82 ties and have entered phase I studies for acute leukemias. AG490 tyrosine kinase inhibition 83 The FTI SCH66336 was shown to be active in vitro and in

As2O3 induction of mitochondrial 28, 58 vivo against a murine model of Bcr-Abl-induced leukemia and damage prolonged survival in p190Bcr-Abl transgenic mice without Bcr-Abl downregulation apparent side-effects.66,67 It also inhibited the colony forma- bestatin, actinonin aminopeptidase inhibition 84 tion of CML cells at nanomolar to low micromolar doses. decitabine DNA hypomethylation 28 homoharringtonine inhibition of protein synthesis 23 Daley’s group recently reported in vitro sensitivity of imatinib- leptomycin nuclear entrapment of Bcr- 75, 85 resistant Bcr-Abl-positive cell lines and cells derived from Abl patients with imatinib resistance to this molecule. Moreover, PD184352 MAPK kinase inhibition 86 they observed that SCH66336 sensitized cells to imatinib, PS341 proteasome inhibition 87 even in imatinib-resistant cell lines.68 This opens the potential SCH66336, L-744,832 farnesyl transferase inhibition 68, 69 to regain imatinib sensitivity after resistance has emerged. TRAIL induction of apoptosis 88 Phase I/II trials of this combination will be initiated shortly. SPOTLIGHT trichostatin A histon deacetylase inhibition 28 Topaly et al69 also reported highly synergistic activity of a different FTI (L-744832) in lymphoid blast crisis EM-3 cells and As2O3, arsenic trioxide; hsp90, heat-shock protein 90; PS341, proteasome inhibitor 341; MAPK, mitogen-activated protein kinase; detected no cross-resistance to imatinib resistant BVI73-R and 17-AAG, allylamino-17-demethoxygeldanamycin; TRAIL, tumor K562-R cells. This conﬁrms the potential of this group of necrosis factor ␣-related apoptosis-inducing ligand. molecules, especially in regards to circumventing imatinib resistance.

ingeffect of Bcr-Abl. Combination studies in K562 or HL60/Bcr-Abl cells showed that the pro-apoptotic activity of Homoharringtonine 58 imatinib is enhanced by As2O3. We also observed additive to synergistic anti-proliferative activity of As2O3 and imatinib The cephalotaxine alkaloid homoharringtonine (HHT) has in cell line assays and the combination showed substantially activity against CML in vitro and in vivo. Its mechanisms of decreased colony formation of lineage-committed bone mar- action and clinical applications have recently been reviewed 28 70 row progenitor cells derived from CML patients. As2O3 by Kantarjian et al. Several groups have investigated the in specifically downregulates Bcr-Abl protein expression before vitro cytotoxicity of HHT in conjunction with imatinib. Kano induction of apoptosis and this appears to be regulated at the et al reported additive anti-proliferative activity in 4/4 Bcr-Abl- 59 71 translational level. This is of interest, particularly in regards positive cell lines at IC50 and IC80 whereas Scappini et al to imatinib resistance, as Bcr-Abl overexpression is associated observed slight synergism at a low dose range in KBM-5 cells. with resistance to imatinib.14 Based on the promisingpre- Experiments with imatinib-resistant cell lines showed mixed clinical data, we are initiatinga multi-center phase I/II study, results with crossresistance in K562-r cells while LAMA-r cells 72 that combines As2O3 with imatinib for CML chronic phase remained sensitive to HHT. These results warrant further patients who do not achieve a major cytogenetic response clinical investigation of HHT in combination with imatinib. with imatinib monotherapy. Since HHT has been shown to improve the cytogenetic response rate followingtherapy with IFN in early chronic phase patients,73 combination studies includingimatinib and Decitabine (5-aza-2-deoxycytidine) HHT Ϯ IFN in early chronic phase may be a promising strategy. This cytidine analogue was initially generated to overcome ara-C resistance and was found to be a potent hypomethylat- ingagent. 60 DNA methylation, an important epigenetic mech- New experimental approaches anism of cell regulation, has significant implications for the pathophysiology of malignancies.61 Decitabine induces re- As listed in Table 4, a variety of other agents have been tested expression of silenced tumor suppressor genes and has direct in conjunction with imatinib in vitro. These include signal cytotoxic effects through insertion into DNA (for review, see transduction inhibitors (the geldanamycin derivative 17-AGG, Ref. 62). We were interested in the cytotoxic activity of decita- the MEK1/2 inhibitor PD 184352 and the tyrphostins AG490 bine in combination with imatinib and found synergistic anti- and adaphostin), trichostatin A, a specific histone deacetylase proliferative activity in a wide dose range.28 Clinical data inhibitor, the proteasome inhibitor PS341, the aminopeptidase usingdecitabine for the treatment of CML blast crisis have inhibitors bestatin and actinonin, the TNF-␣-related apoptosis shown promisingresults. 63 Hence, clinical phase I/II studies inducingligand(TRAIL) and leptomycin B, a nuclear export for the combination of imatinib and decitabine are planned. blocker. Evolvingknowledgeabout mechanisms of resistance to imatinib will enable targeting of specific molecular abnor- malities that cause resistance. For example, point mutations in Farnesyl transferase inhibitors the Abl kinase domain have been observed in some relapsed patients that result in reactivation of the Bcr-Abl kinase.12 Protein farnesylation is an important post-translational modi- Thus, the Bcr-Abl kinase remains a good target in these fication of intracellular proteins. A multitude of proteins patients. Whether alternative inhibitors can be synthesized to

Leukemia Pre-clinical investigation of imatinib in conjunction with anti-leukemic agents P La Roseé et al 1217 overcome resistance caused by Abl kinase mutations has yet 5 Thijsen SFT, Schuurhuis GJ, van Oostveen JW, Ossenkoppele GJ. to be determined (for an overview on Bcr-Abl kinase inhibi- Chronic myeloid leukemia from basics to bedside. Leukemia tors, see Dummond and Holyoake74). Combinations may also 1999; 13: 1646–1674. 6 Druker BJ, Lydon NB. Lessons learned from the development of an be beneficial for ex vivo purging of CML autografts as reported Abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J 75 for imatinib in conjuction with leptomycin B. Clin Invest 2000; 105: 3–7. 7 Druker BJ, Talpaz M, Resta DJ, PengB, BuchdungerE, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. Concluding remarks and future perspective Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–1037. Early in the clinical development of imatinib in vitro studies 8 Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF. Ford JM, of combination strategies were performed and have been Capdeville R. Talpaz M. Activity of a specific inhibitor of the BCR- translated into clinical trials. Translation of in vitro cytotoxi- ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia city studies into clinical trials is particularly important for and acute lymphoblastic leukemia with the Philadelphia chromo- patients with resistance to imatinib. The major pre-clinical some. N Engl J Med 2001; 344: 1038–1042. 9 O’Dwyer ME, Mauro MJ, Blasdel C, Farnsworth M, Kurilik G, Fong studies that led to clinical protocols were based on cytotoxi- D, Hsieh YC, Mori M, Capdeville R, Druker BJ. Lack of cytogenetic city data usingcell lines or primary CML cells. This is indeed response is an adverse prognostic factor of chronic phase CML the first step in definingcombinations with potential thera- patients treated with imatinib mesylate (STI571). Blood 2001; 98 peutic synergy, however, a variety of factors may lead to the (Suppl. 1): 137a (Abstr.). over- or underestimation of the in vivo relevance of the 10 Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller C, data.76,77 Frey78 pointed out that ‘the only certainty involved Ottmann OG, Schiffer CA, Talpaz M, Guilhot F, Deininger MWN, in the evaluation of joint action is that conclusions must be Fischer T, O’Brien S, Stone R, Gambacorti-Passerini C, Russel N, Reiffers J, Shea T, Chapuis B, Coutre S, Tura S, Morra E, Larson drawn in the face of uncertainty’. In addition, anti-leukemic RA, Saven A, Peschel C, Gratwohl A, Mandelli F, Ben-Am M, effects may be due to the cellular cytotoxicity of the agents Gathmann I, Capdeville R, Paquette RL, Druker BJ. 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