Efficacy of tyrosine kinase inhibitors on a mouse chronic myeloid leukemia model and chronic myeloid leukemia stem cells

•Ponatinib lowers the chimerism of BCR-ABL1-positive cells in PB more than imatinib or dasatinib.•Ponatinib prolongs the survival of CML mice much longer than imatinib or dasatinib.•Ponatinib exhibits stronger efficacy in the inhibition of splenomegaly.•Ponatinib exhibits stronger efficacy in reduction of CML LSC-containing CML LSK cells. Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by constitutively active BCR-ABL1 tyrosine kinase resulting from the t(9;22) Philadelphia translocation. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), is a revolutionary molecular target inhibitor for CML. However, leukemic stem cells (LSCs) eventually become resistant to imatinib and thereby cause relapse. The next-generation BCR-ABL1 TKI dasatinib is also unable to eliminate CML LSCs. On the other hand, the third-generation BCR-ABL1 TKI ponatinib is not well studied in terms of its efficacy on CML LSCs. Here, we evaluate the efficacy of ponatinib against CML LSC-containing lin–Sca-1+c-Kit+ (LSK) cells using a mouse CML-like model. To this end, we compared the efficacy of imatinib, dasatinib, and ponatinib on CML LSK cells and showed that ponatinib is more effective at eliminating CML LSK cells. Our results suggest that ponatinib could be potentially useful for achieving treatment-free remission in CML patients. Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by constitutively active BCR-ABL1 tyrosine kinase resulting from the t(9;22) Philadelphia translocation. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), is a revolutionary molecular target inhibitor for CML. However, leukemic stem cells (LSCs) eventually become resistant to imatinib and thereby cause relapse. The next-generation BCR-ABL1 TKI dasatinib is also unable to eliminate CML LSCs. On the other hand, the third-generation BCR-ABL1 TKI ponatinib is not well studied in terms of its efficacy on CML LSCs. Here, we evaluate the efficacy of ponatinib against CML LSC-containing lin–Sca-1+c-Kit+ (LSK) cells using a mouse CML-like model. To this end, we compared the efficacy of imatinib, dasatinib, and ponatinib on CML LSK cells and showed that ponatinib is more effective at eliminating CML LSK cells. Our results suggest that ponatinib could be potentially useful for achieving treatment-free remission in CML patients. In chronic myeloid leukemia (CML), the malignancy of hematopoietic stem cells (HSCs) is driven by the oncogene BCR-ABL1 [1Rowley JD. 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Therefore, cure of CML with imatinib is rare, and most patients require continued treatment to prevent recurrence. Dasatinib is a second-generation TKI with greater potency for BCR-ABL1 inhibition than imatinib [12Cortes J Rousselot P Kim DW et al.Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis.Blood. 2007; 109: 3207-3213Crossref PubMed Scopus (382) Google Scholar, 13Hochhaus A Kantarjian HM Baccarani M et al.Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy.Blood. 2007; 109: 2303-2309Crossref PubMed Scopus (540) Google Scholar, 14Guilhot F Apperley J Kim DW et al.Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase.Blood. 2007; 109: 4143-4150Crossref PubMed Scopus (331) Google Scholar]. Although dasatinib targets an earlier progenitor population than imatinib in primary CML, it does not eliminate CML LSCs [15Copland M Hamilton A Elrick LJ et al.Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction.Blood. 2006; 107: 4532-4539Crossref PubMed Scopus (548) Google Scholar]. Therefore, the eradication of CML LSCs is one of the main goals in CML studies. CML originates from HSCs [16Fialkow PJ Jacobson RJ Papayannopoulou T Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage.Am J Med. 1977; 63: 125-130Abstract Full Text PDF PubMed Scopus (675) Google Scholar]. CML LSCs share several features with normal HSCs and thus cannot be eradicated by conventional and targeted therapies. Some TKIs have an antiproliferative effect on CML LSCs, but induce only modest levels of apoptosis [17Holtz MS Slovak ML Sawyers CL Forman SJ Bhatia R Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation.Blood. 2002; 99: 3792-3800Crossref PubMed Scopus (224) Google Scholar]. It has been reported that highly quiescent CML LSCs are detected in the bone marrow (BM) and are more active than normal HSCs [18Holyoake T Jiang X Eaves C Eaves A Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.Blood. 1999; 94: 2056-2064Crossref PubMed Google Scholar]. Importantly, highly quiescent CML LSCs are more resistant to TKIs. CML LSCs survive independently of BCR-ABL1 activity, resulting in LSC persistence in the presence of TKIs [19Jiang X Zhao Y Smith C et al.Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies.Leukemia. 2007; 21: 926-935Crossref PubMed Scopus (287) Google Scholar]. Many signaling pathways that support BCR-ABL1–independent CML LSC survival have been reported, and several inhibitors for these pathways have been examined, including PI3K/AKT/FOXO signaling, hedgehog signaling, JAK/STAT signaling, WNT signaling, and BM niche-induced signaling [20Houshmand M Simonetti G Circosta P et al.Chronic myeloid leukemia stem cells.Leukemia. 2019; 33: 1543-1556Crossref PubMed Scopus (80) Google Scholar]. Recently, integrin-linked kinase (ILK) has been found to be a factor responsible for TKI resistance of CML LSCs [21Rothe K Babaian A Nakamichi N et al.Integrin-linked kinase mediates therapeutic resistance of quiescent CML stem cells to tyrosine kinase inhibitors.Cell Stem Cell. 2020; 27 (110–124.e9)Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Ponatinib is a highly potent third-generation TKI and can inhibit critical kinase domain mutations including the T315I mutation [22O'Hare T Shakespeare WC Zhu X et al.AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.Cancer Cell. 2009; 16: 401-412Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar]. In addition to BCR-ABL1, ponatinib also inhibits SRC and receptor tyrosine kinases such as the vascular endothelial growth factor receptors (VEGFRs), fibroblast growth factor receptors (FGFRs), fms-like tyrosine kinase 3 (FLT3), KIT, and platelet-derived growth factor receptors (PDGFRs). Genetic alterations of these tyrosine kinases, which promote fusion proteins or activating mutations, have been implicated in the pathogenesis of multiple hematologic malignancies. Ponatinib is known to have powerful inhibitory activity against many tyrosine kinases, and one study did report that the inhibitory activity of ponatinib against FGFR overcomes FGF2-mediated imatinib resistance in CML [23Traer E Javidi-Sharifi N Agarwal A et al.Ponatinib overcomes FGF2-mediated resistance in CML patients without kinase domain mutations.Blood. 2014; 123: 1516-1524Crossref PubMed Scopus (42) Google Scholar]. Hence, ponatinib is potentially useful for multiple hematologic malignancies. However, because its inhibitory activity for VEGFRs is linked to several vascular events, its clinical use is restricted to patients with the T315I mutation. Probably for this reason, few studies have examined the efficacy of ponatinib against CML LSCs. Therefore, in this study, we examined whether ponatinib can eradiate CML LSCs in a mouse CML-like model. To this end, we compared the killing efficiency of three TKIs: imatinib, dasatinib, and ponatinib. Our data indicate that ponatinib is more efficient in killing CML LSC-containing CML LSK cells than imatinib or dasatinib. Imatinib, dasatinib, and ponatinib were purchased from LC Laboratories (Woburn, MA). The cDNA encoding human BCR-ABL1 (a gift from H. Honda, Tokyo Women's Medical University) was cloned into the EcoRI site of the MSCV-IRES-GFP vector. Retroviral packaging cells (Plat-E) [24Morita S Kojima T Kitamura T Plat-E: an efficient and stable system for transient packaging of retroviruses.Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1328) Google Scholar] were transiently transfected with the MSCV-BCR-ABL1-IRES-GFP plasmid using polyethylenimine (Sigma-Aldrich, St. Louis, MO) and used for transplantation into mice as described later. Day 4, 5-fluorouracil (5FU)-treated BALB/c mice were culled to isolate whole bone marrow (BM) cells. The cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Thermo Fisher Scientific, Waltham, MA) supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, 50 µmol/L ß-mercaptoethanol, 10 ng/mL mouse interleukin (IL)-6, 50 ng/mL human thrombopoietin (TPO), and 50 ng/mL mouse stem cell factor (SCF) overnight. On the next day, the cells were infected with the aforementioned retrovirus carrying MSCV-BCR-ABL-IRES-GFP using RetroNectin (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). Five thousand green fluorescent protein (GFP)-positive cells were transplanted intravenously into lethally irradiated (9.5 Gy) BALB/c mice together with 1 × 105 bone marrow mononuclear cells from nonirradiated BALB/c mice. The following antibodies (BioLegend, San Diego, CA) were used: CD11b (M1/70)-APC and Gr-1(RB6-8C5)-APC for the peripheral blood (PB) analysis and a lineage marker cocktail (CD5(53–7.3)-biotin, TER-119(TER-119)-biotin, CD11b(M1/70)-biotin, Gr-1(RB6-8C5)-biotin and B220(RA3-6B2)-biotin), c-Kit(2B8)-PE-Cy7, and Sca-1(D7)-APC for the lin–Sca-1+c-Kit+ (LSK) cell analysis. Flow cytometric analyses were performed using FACSAria and FACSVerse (BD Biosciences, San Jose, CA). Statistical differences were determined using one-way analysis of variance with Tukey's correction for multiple comparisons and the log-rank (Mantel–Cox) test for survival analyses using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). The experimental design using a mouse CML-like model is illustrated in Supplementary Figure E1A (online only, available at www.exphem.org). Briefly, BM cells from 5FU-treated mice were subjected to the retroviral transduction of BCR-ABL1. The transduced cells were transplanted into lethally irradiated mice to establish a mouse CML-like model. All mice died with CML within 45 days of the transplantation (Supplementary Figure E1B). First, we monitored the chimerism of BCR-ABL1-positive cells based on the GFP expression in the PB of CML mice every 3 days from 7 days post transplant to determine the start time of the therapy (Supplementary Figure E1C). The chimerism gradually increased during the observation period. Fluctuations in the chimerism of eight CML mice were similar from 7 to 10 days post transplant, indicating that this is the appropriate time window for the treatment. Thus, we decided to start the TKI treatment 7 days post transplant. Next we determined the optimal dosages of imatinib, dasatinib, and ponatinib for the model. As the dosages of imatinib, dasatinib, and ponatinib in common use are 0.2, 0.02, and 0.02 mg/g, respectively, we examined the following dosages: imatinib (0.1, 0.2, and 0.4 mg/g), dasatinib (0.01, 0.02, 0.04, 0.08, and 0.16 mg/g), and ponatinib (0.01, 0.02, 0.04, 0.08, and 0.16 mg/g). The TKIs were given on a 5-day on/2-day off schedule from 7 to 35 days post transplant. The chimerism of BCR-ABL1-positive cells in PB and the body weight of CML mice were measured every other week. Imatinib ≥0.2 mg/g, dasatinib ≥0.02 mg/g, and, and ponatinib doses ≥0.02 mg/g exhibited similar efficacy for the chimerism of GFP-positive cells in PB (Supplementary Figure E1D). High dosages of TKIs (≥0.6 mg/g imatinib, ≥0.08 mg/g dasatinib, and ≥0.04 mg/g ponatinib) caused short survival times, probably because of side effects (Supplementary Figure E1E). A high dosage of TKIs (≥0.6 mg/g imatinib, ≥0.04 mg/g dasatinib, and ≥0.04 mg/g ponatinib) also caused a reduction in body weight (Supplementary Figure E1F). Therefore, we chose 0.2 mg/g imatinib, 0.02 mg/g dasatinib, and 0.02 mg/g ponatinib as the appropriate dosages, which are consistent with dosages commonly used for the mouse model. Next, we compared the efficacy of imatinib, dasatinib, and ponatinib in the mouse CML-like model. CML mice were treated with 0.2 mg/g imatinib, 0.02 mg/g dasatinib, and 0.02 mg/g ponatinib. The TKIs were given on a 5-day-on/2-day-off schedule (Figure 1A). The chimerism of BCR-ABL1-positive cells in PB from the CML mice was measured every other week. We repeated the comparison experiments three times. In all three experiments, the chimerism of BCR-ABL1-positive cells in PB from the ponatinib-treated groups was much lower than that in the other groups (Figure 1B). The proportion of BCR-ABL1/Mac1/Gr-1 triple-positive cells in PB from ponatinib-treated CML mice was around 30% at 28 days post transplant, whereas those for imatinib- and dasatinib-treated CML mice were >50% (Supplementary Figure E2, online only, available at www.exphem.org). The chimerism of the CML mice from the dasatinib-treated groups was as low as that of mice from the ponatinib-treated groups initially, but gradually increased later. The chimerism of mice from the imatinib-treated groups was higher than that of the other groups during the entire observation period. Ponatinib significantly prolonged the survival of CML mice compared with imatinib or dasatinib (Figure 1C). Of note, all except one CML mouse from the ponatinib-treated group survived during the observation period. On the other hand, all CML mice treated with imatinib or dasatinib died during the observation period. Unexpectedly, dasatinib was less effective than imatinib in terms of prolonging the survival of CML mice, although dasatinib was more effective at reducing BCR-ABL1–positive cells in PB. Ponatinib is significantly more effective at maintaining the chimerism of BCR-ABL1–positive cells in PB low and prolonging the survival of CML mice compared with imatinib or dasatinib. To clarify the molecular basis for the better therapeutic effects of ponatinib in the mouse CML-like model, we compared the impact of imatinib, dasatinib, and ponatinib on CML LSCs. CML mice treated with TKIs were culled 4 weeks post transplant for the analyses, as illustrated in Figure 2A. Ponatinib reduced spleen weight more effectively than imatinib or dasatinib (Figure 2B). The efficacies of imatinib and dasatinib in spleen weight reduction were equivalent. CML LSCs are known to be in the BCR-ABL1–positive hematopoietic stem/progenitor population, which is defined by the surface marker phenotype lin–Sca-1+c-Kit+ (LSK). The proportion of BCR-ABL1–positive LSK cells in the BM and spleen of CML mice treated with dasatinib or ponatinib was clearly reduced compared with that of mice treated with imatinib (Figure 2C). Furthermore, ponatinib reduced the absolute number of CML LSK cells in the BM and spleen more effectively than imatinib (Figure 2D,E). Ponatinib also exhibited stronger efficacy in reducing the number of CML LSK cells in the spleen than dasatinib, and the efficacy of ponatinib in CML LSK cells in the BM tended to be stronger than that of dasatinib. The strong efficacy of ponatinib in killing CML LSK cells could be due to its multi-inhibition activities for SRC and receptor tyrosine kinases such as VEGFRs, FGFRs, FLT3, KIT, and PDGFRs, in addition to its inhibitory activity on BCR-ABL1 including BCR-ABL1 mutants. Taken together, these data strongly indicate that ponatinib is more effective at killing CML LSC-containing LSK cells than imatinib or dasatinib in a mouse CML-like model, indicating a new application of ponatinib for the eradication of CML LSCs. TK has research support from Otsuka Pharmaceutical Company, Ltd. The other authors declare no competing interests. We acknowledge the IMSUT FACS Core Laboratory and the IMSUT Animal Research Center. This project is supported by a grant from Otsuka Pharmaceutical Company, Ltd. Supplementary Figure E1, Supplementary Figure E2.Supplementary Figure E2(A) Experimental design. Each group; n=6Show full caption(B) Representative FACS plots of white blood cells in PB from CML mice treated with imatinib, dasatinib or ponatinib every other week post-transplantation.Representative spleen images from CML mice treated with imatinib, dasatinib or ponatinib.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (B) Representative FACS plots of white blood cells in PB from CML mice treated with imatinib, dasatinib or ponatinib every other week post-transplantation. Representative spleen images from CML mice treated with imatinib, dasatinib or ponatinib.