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/lp/ou_press/targeting-bcr-abl-independent-tki-resistance-in-chronic-myeloid-nh02OgOkQZ
- Publisher
- Oxford University Press
- Copyright
- Copyright © 2022 Oxford University Press
- ISSN
- 0027-8874
- eISSN
- 1460-2105
- DOI
- 10.1093/jnci/djx236
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- See Article on Publisher Site
Abstract
Background: Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL- dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR- ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods: Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib- resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n¼ 4) and a human xenograft mouse model (n¼ 4–6 mice per group). All statistical tests were two-sided. Results: We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alterna- tive activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alterna- tive therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235þHCQ: 45.0[17.9]% vs 24.0[8.4]%, P ¼ .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235þHCQ-treated mice: 38.5 days vs 47.0 days, P ¼ .04). Conclusion: Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance. Received: May 23, 2017; Revised: August 7, 2017; Accepted: October 10, 2017 © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 ARTICLE 468 | JNCI J Natl Cancer Inst, 2018, Vol. 110, No. 5 Chronic myeloid leukemia (CML) is caused by a reciprocal translo- Ethics Statements cation giving rise to the Philadelphia (Ph) chromosome within a CML and normal samples (n ¼ 4 and n ¼ 5, respectively) hemopoietic stem cell (1). This leads to transcription/translation required informed consent in accordance with the Declaration of BCR-ABL, a constitutively active tyrosine kinase (2). CML usually of Helsinki and approval of the National Health Service (NHS) presents in a chronic phase (CP), before progressing to accelerated Greater Glasgow Institutional Review Board. Ethical approval phase (AP) and terminal blast crisis (BC) if left untreated. Imatinib has been given to the research tissue bank (REC 15/WS/0077) has statistically significantly improved life expectancy by inducing and for using surplus human tissue in research (REC 10/S0704/ cytogenetic and molecular responses in the majority of patients in 60). Animal work was carried out with ethical approval from the CP (3). However, the pathway to “cure” has been tempered by drug University of Glasgow under the Animal (Scientific Procedures) intolerance, insensitivity of CML stem cells to TKIs (4–7), and drug Act 1986. Animal experiments were performed in accordance resistance (8,9). with Home Office regulations under an approved project license The mechanisms of drug resistance have been extensively (PPL No: 60/4492). investigated and can be classified as BCR-ABL dependent or inde- pendent. It is known that approximately 50% of patients who relapseonimatinib havemutations within theABL kinase domain, Gene Ontology Term Enrichment Analysis affecting imatinib binding within the kinase pocket (10). Dasatinib, Gene ontology (GO) term enrichment analysis was carried out nilotinib, and/or bosutinib have activity against the majority of using the GO.db (v3.2.2) and GOstats (v2.36.0) Bioconductor imatinib-resistant mutants, except T315I (11). Although the devel- libraries in R; P values were generated using a hypergeometric opment of a TKI active against the T315I mutant has proven chal- test and adjusted for multiple testing (20). lenging, ponatinib (AP24534), a third-generation TKI, has activity against T315I in vitro (12) and in patients (13,14). Ponatinib was tested in the PACE clinical trial in patients with the T315I mutation Statistical Analysis or who are resistant/intolerant to either dasatinib or nilotinib. Findings from PACE show that major molecular response (MMR) is Error bars represent SD. Statistical analyses were performed achieved in 56% of CP patients with the T315I mutation (14), using the two-tailed Student’s t test or Gehan-Breslow although a proportion of patients will ultimately develop or be pro- Wilcoxon test. A P value of .05 or less was taken to be statisti- ven to have ponatinib-resistant disease. cally significant. Patients whose disease fails multiple TKI treatments with- Detailed information on all other methods can be found in out having ABL kinase domain mutations predominantly repre- the Supplementary Material (available online). sent a population with BCR-ABL-independent mechanisms of resistance. For this group of patients, the treatment options are Results very limited, and only 27% of “resistant/intolerant” patients achieved MMR in the PACE trial (14). Although much less is Cellular Modeling of BCR-ABL-Independent Mechanisms known about BCR-ABL-independent resistance, a recent genetic of Resistance study has shown that it can vary between individuals, often suggesting re-activation of signaling pathways involved in CML Imatinib-resistant cell lines are often sensitive to more pathogenesis (15). Additionally, studies have shown that potent second-generation TKIs and/or ponatinib and are increased FGF2 in the BM (16) or activation of LYN (17,18) may therefore not an ideal model to investigate acquired resist- be responsible for the survival of cells following BCR-ABL inhibi- ance to all available TKIs (Supplementary Figure 1, A–C, tion. However, ponatinib, which has activity against FGF recep- available online). Hence, we aimed to develop a ponatinib- tor and LYN kinase (12), has been shown to overcome FGF2- resistant cell line with acquired BCR-ABL-independent resist- mediated resistance in CML patients without kinase domain ance. KCL22 cells (human myeloid BC CML cell line) were grown mutations (16) and to be effective against many imatinib- in increasing concentrations of ponatinib for a prolonged period. Pon-Res resistant CML cell lines (19), highlighting the importance of Ponatinib-resistant (KCL22 ) clones continued to proliferate using ponatinib as the TKI of choice for investigation of when exposed to 100 nM ponatinib (Figure 1A). Sequencing of the acquired BCR-ABL-independent resistance in CML. BCR-ABLkinasedomainshowed no kinase domain mutations The goals of the current study were to examine what drives (data not shown). Measuring tyrosine 207 phosphorylation of BCR-ABL-independent resistance and identify clinically relevant CRKL, a direct BCR-ABL substrate, revealed that BCR-ABL activity T315I oncology compounds with activity against ponatinib-resistant cells. was inhibited to similar levels as in KCL22 and parental KCL22 (Figure 1B). Methods Transcriptional Response and mTORC1 Activity Pon-Res Transplantation Experiments Following BCR-ABL Inhibition in KCL22 Cells Pon-Res Human KCL22 cells, labeled with lentiviral firefly lucifer- To investigate the potential mechanism(s) of resistance in Pon-Res Pon-Res ase, were transplanted via tail vein injection into eight- to KCL22 cells, parental KCL22 and KCL22 cells were 12-week-old female NSG mice (four to six mice were assigned treated with ponatinib to fully switch off BCR-ABL signaling per drug arm per experiment). For in vivo treatment, after one (Supplementary Figure 2A, available online), and RNA was week, the mice were treated with vehicle control, HCQ, NVP- harvested for transcriptomic analysis. Of the 5736 gene tran- BEZ235, or the combination of NVP-BEZ235/HCQ for four to five scripts that met the necessary expression thresholds in the weeks. RNA sequencing (RNA-seq) experiment, only 250 were Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 R. Mitchell et al. |469 BCR-ABL-dependent mechanism BCR-ABL-independent mechanism T315I Pon-Res KCL22 KCL22 KCL22 6 6 Input 24h 48h 72h 2 2 0 0 0 Untr 1 10 100 Das Untr 1 10 100 Das Untr 1 10 100 Das Ponatinib, nM Ponatinib, nM Ponatinib, nM 100 100 100 75 75 75 IC50: 11.0nM IC50: 2.5nM 50 50 IC50: 303.6nM 25 25 0 0 Ponatinib, nM Ponatinib, nM Ponatinib, nM Ponanib, nM Ponanib, nM Ponanib, nM Tyr207 Tyr2 0 7 p-CRKL p-CRKL Y207 p-CRKL β-tubulin β-tubul i n β-tubul i n T315I Figure 1. Proliferation of ponatinib-resistant chronic myeloid leukemia cells in the absence of BCR-ABL kinase activity. KCL22 (wild-type BCR-ABL), KCL22 , and Pon-Res KCL22 cells were cultured with or without (Untr) increasing concentrations of ponatinib and 150 nM dasatinib. Proliferation was measured by cell counting using a glass hemocytometer following 24, 48, and 72 hours of drug treatment, and IC50 values were calculated using GraphPad Prism software (A). To assess for BCR-ABL activity, the levels of phosphorylation of CRKL were measured by immunoblot following four hours of drug treatment at varying concentrations of ponatinib and 150 nM dasatinib (B). Error bars ¼ SD. Two independent experiments were performed in triplicate. Untr ¼ untreated. Pon-Res differently expressed between the two cell lines Drug Repurposing Screen in KCL22 Cells (Supplementary Figure 2B, available online). Pathway enrich- We next aimed to identify approved anticancer drug(s) with effi- ment analysis highlighted 42 potentially deregulated path- cacy against TKI-resistant cells. We performed a screen using a ways (Supplementary Figure 2C, available online). More validated oncogene drug library of 119 approved oncology drugs strikingly, while 1661 were differentially expressed following Pon-Res (Supplementary Table 1, available online). KCL22 cells ponatinib-mediated BCR-ABL inhibition in the parental KCL22 were cultured alone or in combination with 100 nM ponatinib. cells, the same treatment had virtually no effect on the tran- Pon-Res The effect of additional drug exposure on cell survival was scriptome of KCL22 cells (Figure 2, A and B). There was measured (omacetaxine mepesuccinate, a US Food and Drug no correlation (r ¼ 0.60) between the two cell lines in the Administration (FDA)–approved but nonselective and toxic transcriptional response of the 5736 genes to ponatinib inhibitor of total protein biosynthesis [22] was used as a control (Figure 2C). This suggested that signaling pathways down- drug). This approach identified 36 drugs that were more effec- stream of BCR-ABL (and normally inhibited by TKIs) remained Pon-Res Pon-Res tive in inhibiting proliferation of KCL22 cells when com- active following BCR-ABL inhibition in KCL22 cells. pared with all BCR-ABL-targeting TKIs tested (Supplementary To investigate further the mechanism(s) of resistance of Pon-Res T315I Pon-Res Figure 3A, available online). Identified drugs included various KCL22 cells, parental KCL22, KCL22 , and KCL22 conventional chemotherapeutic drugs and more specific kinase cells were treated with dasatinib or ponatinib, and inhibition of inhibitors. Comparison of drug sensitivity between targets downstream of BCR-ABL was measured. This revealed Pon-Res parental KCL22 and KCL22 cells confirmed resistance of inhibition of CRKL and STAT5 phosphorylation, indicative of Pon-Res KCL22 cells to all FDA-approved BCR-ABL-targeting TKIs complete inhibition of BCR-ABL, but sustained phosphorylation and demonstrated that all other drugs that are effective against of the translation regulator ribosomal protein S6 (RPS6) indi- cated activation of mTOR complex 1 (mTORC1) (21), a common parental KCL22 cells at 1 mM retained their activity against Pon-Res downstream node on which multiple oncogenic signaling path- KCL22 cells (Figure 3A). Comparison of untreated and Pon-Res ways converge (Figure 2D; Supplementary Figure 2A, available KCL22 cells grown in the presence of 100 nM ponatinib online). showed that single-agent treatment was similarly effective as Untr 0.1 10 000 Untr 0.1 Untr 0.1 10 000 Relative cell number Relative cell number Relative cell number Relative cell number Relative cell number Relative cell number ARTICLE Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 ARTICLE 470 | JNCI J Natl Cancer Inst, 2018, Vol. 110, No. 5 A BC Pon-Res Pon-Res KCL22+Pon vs KCL22 +Pon KCL22+Pon KCL22 +Pon KCL22+Pon r=0.60 r =36% Pon-Res KCL22 +Pon KCL22+Pon logFC logFC Downregulation q≤0.01 q≤0.05 Upregulation q≤0.01 q≤0.05 T315I Pon-Res KCL22 KCL22 KCL22 Y2 0 7 p-CRKL Y6 9 4 p-STAT5 S240/244 p-RPS6 GAPDH Da s a nib - + - - +- - +- Pona ni b -- --- + - + + Pon-Res Figure 2. Transcriptional response and mTORC1 activity in ponatinib-resistant cells following BCR-ABL inhibition. KCL22 and KCL22 cells were cultured with or Pon-Res without 100 nM ponatinib for 24 hours and RNA harvested for RNA-seq. A) The transcriptional response of KCL22 and KCL22 cells is represented by Volcano plots (up- and downregulation are indicated by magenta and green, respectively; light and dark colors correspond to q-value thresholds of 0.05 and 0.01, respectively; statis- tically nonsignificant changes are colored gray). B) A proportional Venn diagram represents the overlap in statistically significant response to ponatinib (q 0.05) in both cell lines (4073 refers to the number of genes not changed). C) A direct comparison of the transcriptional response of all genes in both cell lines; identical expres- T315I Pon-Res sion is shown by the red line; the true linear relationship is indicated by the blue line. D) KCL22, KCL22 , and KCL22 cells were cultured 6 150 nM dasatinib or 100 nM ponatinib. Phosphorylation of CRKL, STAT5, and RPS6 was measured after 24 hours of drug treatment. when combined with complete BCR-ABL inhibition (Figure 3B). This is believed to be because they are incomplete, substrate- selective mTORC1 inhibitors (24). However, with the develop- Subsequent target association analysis enriched for and high- ment of catalytic mTOR inhibitors, it is still hoped that mTOR lighted microtubule, proteasome, and allosteric mTORC1 inhibi- represents a druggable target in malignancies driven by activa- tors (Supplementary Figure 3B, available online). However, tion of the mTOR pathway. To confirm deeper mTORC1 inhibi- these microtubule and proteasome inhibitors have known tox- Pon-Res tion with catalytic mTOR inhibitors, KCL22 cells were icities in the clinic. With IC50 levels for everolimus, sirolimus, treated with PI-103 and its derivative NVP-BEZ235 (which inhibit and temsirolimus all below 200 nM (indicating on-target effect), both mTORC1 and mTORC2 and have activity against all PI3K regardless of whether used alone or in combination with pona- isoforms [25,26]) and compared with rapamycin (Figure 4A). tinib (Supplementary Figure 3, A–C, available online), we In line with previous studies, rapamycin had little effect on decided to focus our subsequent work on mTOR as a potential phosphorylation of 4E-BP1, whereas PI-103 and NVP-BEZ235 target for TKI resistance. This decision was also supported by (using IC50 concentrations) led to reduction in 4E-BP1 phos- the data shown in Figure 2B and Supplementary Figure 2 (avail- phorylation, demonstrating more potent mTORC1 inhibition able online), which suggest that sustained mTORC1 activity may (Figure 4B). Seventy-two hours of drug treatment led to modest Pon-Res support survival of KCL22 cells following TKI treatment. induction of apoptosis by rapamycin, with more extensive and statistically significant apoptosis observed following NVP- Pon-Res BEZ235 treatment (44.2[9.6]%, P ¼ .02), whereas TKIs had no Sensitivity of KCL22 and TKI-Resistant Primary effect (Figure 4C). Similar effects were seen in colony forming CML Cells to Catalytic mTOR Inhibitors cell (CFC) assay (data not shown). To test if these findings would To date, sirolimus (rapamycin) and rapamycin analogues (allos- replicate using different BCR-ABL-positive cell lines, we gener- Pon-Res teric mTORC1 inhibitors) (Supplementary Table 2, available ated ponatinib-resistant BaF3 cells (BaF3 ), which were online) have only shown modest efficacy in clinical trials (23). also highly sensitive to NVP-BEZ235, showing that sensitivity of -log (q) Pon-Res KCL22 +Pon logFC Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 R. Mitchell et al. |471 generation TKI treatments. Importantly, while ponatinib was A 100 ineffective, the catalytic mTOR inhibitors NVP-BEZ235 (26), Approved TKIs Omacetaxine Gedatolisib (27,28), Apitolisib (29,30), VS-5584 (31), and AZD8055 P onatinib Bosutinib Dasatinib (32) all induced apoptosis over and above the ponatinib-treated Nilotinib arm (Supplementary Figure 5, available online). Imatinib To further understand the mechanism by which NVP- Sirolimus BEZ235 induced death, we performed RNA-seq on parental 60 Everolimus Pon-Res KCL22 and KCL22 . Strikingly, while ponatinib had no Pon-Res Temsirolimus effect on gene transcription in KCL22 cells (Figure 2, A and 40 B), NVP-BEZ235 was sufficient to induce transcriptional changes on the same scale as ponatinib-treated parental KCL22 cells (Figure 4D, compare with Figure 2A). Further analysis showed that the majority of gene changes following ponatinib and NVP-BEZ235 combination treatment were accounted for in the NVP-BEZ235 single arm (Figure 4E). Additionally, comparison of transcriptional changes in ponatinib-treated KCL22 cells and Pon-Res NVP-BEZ235-treated KCL22 cells showed a high correla- tion (r ¼ 0.78) in transcriptional effect, with the majority (80%) 0 10 20 30 40 50 60 70 80 90 100 of statistically significant (q 0.05) changes following BCR-ABL Pon-Res KCL22 , % inhibition inhibition in the parental cells also occurring in NVP-BEZ235- Pon-Res B treated KCL22 cells (Figure 4, F and G). Substantial overlap in transcriptional changes was also shown when NVP-BEZ235- treated KCL22 cells were included in the analysis, demonstrat- 90 ing that targeting mTOR downstream of BCR-ABL rescues the Omacetaxine impaired transcriptional response following BCR-ABL inhibition in TKI-resistant cells (Supplementary Figure 6A, available online). GO enrichment analysis of the differentially expressed Temsirolimus genes showed that ponatinib treatment in KCL22 and NVP- Pon-Res BEZ235 treatment in KCL22 cells affected genes involved 50 Sirolimus in the execution of apoptosis and DNA repair (Supplementary Everolimus Figure 6Bi, available online). A parallel analysis demonstrated that NVP-BEZ235 treatment was additionally associated with changes in protein synthesis/mRNA translation Dasatinib (Supplementary Figure 6Bii, available online). Bosutinib Nilotinib The Effect of Catalytic mTORC1 Inhibition on Autophagy Imatinib -1 0 Pon-Res in KCL22 Cells P onatinib mTORC1 not only regulates mRNA translation, but is also the -1 0 0 10 20 30 40 50 60 70 80 90 100 master regulator of autophagy (macro-autophagy) (33,34). To Pon-Res KCL22 , % inhibition Pon-Res assess autophagy flow, we generated KCL22 cell lines sta- bly expressing fluorescence-tagged human LC3B (mRFP-GFP- Figure 3. Sensitivity of ponatinib-resistant cells to allosteric mTORC1 LC3B) that enable different stages of autophagy to be visualized inhibition. An approved oncology drug library was screened against KCL22 and Pon-Res by fluorescence microscopy (35). The appearance of red/green KCL22 cells. Following 72 hours of 1 mM drug treatment, metabolic activ- ity/proliferation was assessed using resazurin assay. Relative IC50 was calcu- puncta (yellow when overlapped) indicates autophagosomes, lated for each drug used, and a comparison was made between KCL22 and and as GFP is highly susceptible to the low pH within the lyso- Pon-Res Pon-Res KCL22 cells (A) and between KCL22 cells cultured in the absence or somes, a “red only” signal indicates autolysosomes. This proc- presence of 100 nM ponatinib (B). ess can be inhibited by hydroxychloroquine (HCQ), which inhibits autophagy at a late stage by preventing the fusion of ponatinib-resistant cells to mTOR inhibition is not restricted to autophagosomes and lysosomes, leading to build-up of yellow Pon-Res the KCL22 cell line (Supplementary Figure 4A, available fluorescence. NVP-BEZ235 treatment increased puncta exhibit- Pon- online). We then examined whether ponatinib-mediated BCR- ing a “red only” signal (autophagy flow complete) in KCL22 Res ABL inhibition further enhanced the effect of NVP-BEZ235. In cells (Figure 5A). This indicates that NVP-BEZ235 induces line with Figure 3B and Supplementary Figure 3C (available autophagy flow, which can be effectively inhibited when com- online), no increase was observed following ponatinib and NVP- bined with HCQ treatment. BEZ235 combination over NVP-BEZ235 alone when apoptosis or Next, we investigated if autophagy induced by NVP-BEZ235 Pon-Res CFC was measured (Supplementary Figure 4, B and C, available has a protective role. KCL22 cells were treated in combi- online). nation with chloroquine (CQ)-mediated autophagy inhibition. Encouraged by these results and to translate our findings While CQ treatment alone did not lead to statistically significant closer to the clinic, we compared the effect of ponatinib with reduction in colony formation, it statistically significantly various catalytic mTOR inhibitors on available progenitor cells increased the cell death effect of NVP-BEZ235 (P ¼ .04) (Figure derived from the BM of a patient who had failed to achieve com- 5B). Further combination experiments demonstrated that CQ plete cytogenetic response following first-, second-, and third- and NVP-BEZ235 are synergistic in inhibiting proliferation of Pon-Res KCL22 + ponatinib, % inhibition KCL22, % inhibition ARTICLE Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 ARTICLE 472 | JNCI J Natl Cancer Inst, 2018, Vol. 110, No. 5 A B TKIs BCR-ABL (ponanib) Y207 p-CRKL PI3K S240/244 p-RPS6 Allos teric AKT (rapalogs) T3 7 /4 6 p-4E-BP1 mTORC1 mTORC2 S65 p-4E-BP1 RPS6 4E-BP1 Ca ta ly c β-tubul i n (NVP-BEZ235) Pon-Re s KCL22 P = .02 D D E Pon-Res Pon-Res 1 3 KCL22 + KCL22 + 0 2 NVP-BEZ235 Pon + BEZ235 Pon-Res KCL22 1 1 +Pon 140 1038 141 81 568 72 59 470 69 Pon-Res Pon-Res KCL22 KCL22 logFC +Pon+BEZ235 +NVP-BEZ235 Downregulation q≤0.01 q≤0.05 Upregulation q≤0.01 q≤0.05 F G Pon-Res KCL22+Pon vs KCL22 +NVP-BEZ235 174 702 480 94 368 274 80 334 206 Pon-Res KCL22+Pon KCL22 +NVP-BEZ235 KCL22+Pon logFC Figure 4. Transcriptional changes and levels of apoptosis in ponatinib-resistant cells following treatment with catalytic mTOR inhibitors. A) Schematic diagram dem- Pon-Res onstrating the activity of allosteric (blue-green) and catalytic (red) mTOR inhibitors. B) KCL22 cells were cultured with 150 nM dasatinib, 100 nM ponatinib, 10 nM rapamycin, 500 nM PI-103, or 100 nM NVP-BEZ235 or untreated (Untr). Phosphorylation of CRKL, RPS6, and 4E-BP1 was measured four hours following drug treatment. Pon-Res C) KCL22 cells were cultured 6 2 mM imatinib, 2 mM nilotinib, 150 nM dasatinib, 100 nM ponatinib, 10 nM rapamycin, or 100 nM NVP-BEZ235, and apoptosis was Pon-Res measured following 72 hours of drug treatment. Error bars ¼ SD. Three independent experiments were performed. D–G) KCL22 cells were cultured 6 100 nM Pon-Res NVP-BEZ235, alone and in combination with 100 nM ponatinib for 24 hours, and RNA was harvested for RNA-seq. D) The transcriptional response of KCL22 cells Untr Imatinib Nilotinib Dasatinib Ponatinib Rapamycin NVP-BEZ235 -log (q) % apoptos is Pon-Res KCL22 +NVP-BEZ235 logFC Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 R. Mitchell et al. |473 Pon-Res KCL22 cells when CQ is used at the 5–20 mM concentration significantly enhanced the effect of NVP-BEZ235 (NVP-BEZ235 range (combination index for NVP-BEZ235/CQ concentrations of vs NVP-BEZ235þHCQ: 45.0[17.9]% vs 24.0[8.4]%, P ¼ .002). Colony 50 nM/5 mM, 100 nM/10 mM, 150 nM/15 mM and 200 nM/20 mM polymerase chain reaction and fluorescence in situ hybridiza- were 0.18, 0.60. 0.50, and 0.52, respectively) (Supplementary tion confirmed that the vast majority of cells were Ph positive Figure 7, A and B, available online). CRISPR-Cas9 and RNAi tech- (data not shown). To test the effect of this combination on nor- niques were employed to test if the specific inhibition of mal cells, non-CML cells (derived from patients with Ph- autophagy would enhance NVP-BEZ235-induced death. Initially, negative, nonmyeloid hematological malignancies) were ATG7, an E1-like enzyme and essential autophagy gene required treated with ponatinib, NVP-BEZ235 alone, and in combination for LC3 lipidation, was targeted using CRISPR-Cas9. ATG7 knock- with HCQ, and compared with a cytotoxicity of 10 nM omace- down inhibited LC3B-II formation and autophagy, measured by taxine treatment. This revealed that while omacetaxine sub- a decrease in LC3B-II levels and an increase in the autophagy stantially affected the CFC potential of normal progenitor cells, substrate SQSTM1/p62 (36,37)(Figure 5C), and indeed sensitized the combination of NVP-BEZ235 and HCQ had only a minimal cells to death following NVP-BEZ235-mediated mTOR inhibition effect (Figure 6D). (P ¼ .06) (Figure 5D). Similarly, RNAi-mediated ATG7 knockdown statistically significantly increased the effect of the NVP-BEZ235 Discussion on apoptosis, confirming that autophagy plays a protective role Pon-Res in KCL22 cells following inhibition of mTORC1 (P ¼ .002) Despite the promising results in the PACE trial, where ponatinib (Figure 5E). induced rapid and durable responses in CP-CML patients, it is associated with considerable cardiovascular toxicity that may be dose-dependent. Additionally, a proportion of patients taking The Effect of Pharmacological Inhibition of Autophagy ponatinib already have or will develop BCR-ABL-independent and NVP-BEZ235 Treatment In Vivo and in TKI- mechanisms of resistance, and therefore fail ponatinib treat- Resistant Primary CML Cells ment. Therefore, it is hoped that this patient population that currently experiences rare response to TKI treatment and very We next tested whether mTOR inhibition can interfere with leu- short survival may share an alternative drug target that can be kemia initiation when combined with pharmacological autoph- Pon-Res inhibited with a novel compound. We, therefore, for the first agy inhibition. KCL22 cells were labeled with lentiviral luciferase and treated ex vivo with NVP-BEZ235, HCQ, and the time, generated a ponatinib-resistant cell line, which developed BCR-ABL-independent activation of mTOR. This afforded us a combination. Following drug treatment, cells were injected into NSG mice, which were then monitored weekly by luciferase bio- unique opportunity to search for drugs that are effective against ponatinib-resistant CML cells. Our screen and subsequent test- imaging. At week 4, there was a marked delay in leukemia development in mice engrafted with cells treated with the com- ing of catalytic mTOR inhibitors revealed that NVP-BEZ235 had increased potency in inhibiting mTORC1 in ponatinib-resistant bination (Figure 6A). The combination treatment also statisti- cells. This correlated with potent transcriptional response and cally significantly prolonged overall survival of xenografted NSG mice when compared with NVP-BEZ235 single treatment (P ¼ induction of apoptosis, in agreement with previous studies where catalytic mTOR inhibitors, such as OSI-027 and PP242 (or .01) (Figure 6A). To test the tolerability and efficacy of this drug combination, dual PI3K/mTOR inhibitors, such as PI-103 and NVP-BEZ235), Pon-Res have been shown to prevent expansion of Ph-positive acute we injected luciferase-expressing KCL22 cells into NSG mice. Following evidence of engraftment, the xenografted mice lymphoblastic leukemia cells in vivo (38), to sensitize CML cells were treated for up to five weeks. By week 3, bio-imaging to nilotinib (39,40), and to be effective in targeting CML cells showed leukemia development in untreated and HCQ-treated in vitro (41,42). Critically, we also showed that NVP-BEZ235 was mice (Figure 6B). By five weeks, all untreated and HCQ-treated more effective than ponatinib against available primary cells obtained from heavily pretreated TKI-resistant CML patients. mice had to be killed, together with two out of five mice treated with NVP-BEZ235 alone. Mice showed few signs of toxicity, and Although further investigation will be required to confirm the exact mechanism of resistance in each patient (requires opti- the combination statistically significantly extended the survival of mice when compared with NVP-BEZ235 single treatment mized protocols for rare BM-aspirated cells), this provides a (median survival NVP-BEZ235 vs NVP-BEZ235þHCQ: 38.5 days rationale for testing catalytic mTOR inhibitors in the clinic for vs 47.0 days, P ¼ .04) (Figure 6B). patients who do not respond to BCR-ABL inhibitors. Finally, we compared the effect of NVP-BEZ235 in combina- A phase I dose-finding study of NVP-BEZ235 is ongoing in tion with HCQ with ponatinib on cells derived from the BM of patients with relapsed or refractory acute leukemia four patients who had failed to achieve cytogenetic response (NCT01756118). Based on a phase I trial in patients with advanced solid tumors, the recommended dose for NVP-BEZ235 following first-, second-, or third-generation TKI treatments (Supplementary Table 3, available online). Importantly, NVP- is 300 mg twice daily (BID), which is still expected to inhibit BEZ235 had a greater effect on survival of progenitor cells in mTORC1/2 according to pharmacodynamic data (43). However, these patients (Figure 6C). HCQ treatment statistically recent results from a phase II trial for patients with everolimus- Figure 4. Continued to NVP-BEZ235 alone (left) and in combination with ponatinib (right) is represented by Volcano plots (up- and downregulation are indicated by magenta and green, respectively; light and dark colors correspond to q-value thresholds of 0.05 and 0.01, respectively; statistically nonsignificant changes are colored gray). E) A propor- tional Venn diagram represents the overlap in statistically significant response (q 0.05) to ponatinib alone (dark blue), NVP-BEZ235 alone (green), and the combina- Pon-Res tion (light blue) in the KCL22 cells. F) A proportional Venn diagram represents the overlap in statistically significant response (q 0.05) to ponatinib in KCL22 Pon-Res cells (red) and NVP-BEZ235 in KCL22 cells (green). G) A direct comparison of the transcriptional responses of all 1718 genes to treatment common to both experi- ments; identical expression is shown by the red line, and the true linear relationship is indicated by the blue line. One independent experiment was performed in quadruplicate. Untr ¼ untreated. ARTICLE Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 ARTICLE 474 | JNCI J Natl Cancer Inst, 2018, Vol. 110, No. 5 B Pon-Re s Pon-Res KCL22 KCL22 mRFP-GFP-LC3 Untr NVP-BEZ235 P = .04 5 µm 5 µm _ _ P < .001 HCQ NVP-BEZ235+HCQ P < .001 5 µm 5 µm __ Pon-Res KCL22 CRISPR-ATG7 CRISPR-Ctrl ATG7 CRISPR-Ctrl CRISPR-ATG7 LC3B-I LC3B-I I P = .06 SQSTM1 S240/244 p-RPS6 β-tubul i n -+ -+ -+ - + NVP-BEZ235 HCQ -+ -- + + - + Pon-Re s KCL22 P = .002 shCtrl P < .001 shATG7 P < .001 Untr NVP-BEZ235 ATG7 β-tubulin Pon-Res Figure 5. Autophagic response following mTOR inhibition in ponatinib-resistant cells. A) KCL22 cells expressing mRFP-GFP-LC3 were cultured 6 100 nM NVP- BEZ235 alone (top panel) or in combination with 10 mM hydroxychloroquine (HCQ; bottom panel). Scale bars ¼ 5 mm. Autophagy flow (top panel) and inhibition of Pon-Res autophagy flow (bottom panel) was visualized following 24 hours of drug treatment. B) KCL22 cells were cultured 6 150 nM dasatinib, 100 nM ponatinib, and 100 nM NVP-BEZ235 with and without chloroquine-mediated autophagy inhibition. Colony forming potential was measured following 72 hours of drug treatment. C–E) Pon-Res KCL22 cells were infected with lentivirus-expressing sgRNA (C and D) or shRNA-targeting (E) ATG7 or empty vector/scrambled (Scr) shRNA as control. Following knockdown, cells were treated with 100 nM ponatinib (D), 100 nM NVP-BEZ235 (C–E) alone, or in combination with 10 mM HCQ (C). C and E) Stable ATG7 knockdown, inhibition of autophagy (LC3-II and SQSTM1 levels), and mTORC1 activity were measured in puromycin-selected cells by immunoblot. Colony forming potential (D) or apoptosis (E) was measured following 72 hours of drug treatment. Error bars ¼ SD. Statistical analysis was performed using the two-tailed Student’s t test. CQ ¼ chloro- quine; Untr ¼ untreated. Untr Dasatinib Ponatinib CQ NVP-BEZ235 CQ+BEZ235 Untr Ponatinib NVP-BEZ235 % apoptosis Total number of colonies Relative colony number Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 R. Mitchell et al. |475 Untr HCQ NVP-BEZ235 Pon-Res LUC+ End of experiment P = .01 KCL22 BEZ235+HCQ ↓↓ Drug trea tment in vitro Vehi cl e HCQ NVP-BEZ235 BEZ235+HCQ 10 15 20 25 30 35 40 45 Days Days Untr HCQ NVP-BEZ235 BEZ235+HCQ 04 4 4 4 31 4 34 3 4 35 3 4 36 2 42 4 Pon-Res LUC+ KCL22 Untr HCQ NVP-BEZ235 P = .04 BEZ235+HCQ Drug trea tment in vivo Untr HCQ NVP-BEZ235 BEZ235+HCQ 10 15 20 25 30 35 40 45 50 55 Days Days Untr HCQ NVP-BEZ235 BEZ235+HCQ 06 6 6 6 27 6 28 6 4 35 6 42 3 6 47 5 49 1 52 1 CD BM MNCs Non-CML (TKI-resistant Pts 1-4) P < .001 P = .002 Pt 1 100 P = .002 Pt 2 Pt 3 Pt 4 Figure 6. Sensitivity of xenografted ponatinib-resistant cells and primary chronic phase TKI-resistant chronic myeloid leukemia cells to NVP-BEZ235 and hydroxychlor- Pon-Res oquine (HCQ)-mediated autophagy inhibition. A) KCL22 cells were labeled with firefly luciferase and treated ex vivo with 100 nM NVP-BEZ235, alone and in combi- nation with 10 mM HCQ. Seventy-two hours following drug treatment, cells were transplanted intravenously into sublethally irradiated NSG mice (four mice per group, two independent experiments). Thirty minutes after the transplant, the mice were injected with D-luciferin substrate to ensure the success of the transplantation and the cell viability. Leukemic progression was measured weekly by luciferase bio-imaging (left). Overall survival was monitored by Kaplan-Meier analysis (right). A table Pon-Res showing the number of mice at risk is shown below the graph. B) Firefly luciferase labeled KCL22 cells were transplanted intravenously into NSG mice (five to six mice per group, two independent experiments). Mice were then treated with NVP-BEZ235 (45 mg/kg, oral gavage), HCQ (60 mg/kg, intraperitoneal injection), and the combination for up to five weeks. Leukemia progression and overall survival were measured by luciferase bio-imaging (left) and Kaplan-Meier analysis (right), Untr Ponatinib HCQ NVP-BEZ235 BEZ235+HCQ Untr Ponatinib HCQ NVP-BEZ235 BEZ235+HCQ Omacetaxine 5 weeks 3 weeks Killed 4 weeks Relative colony number Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Percent survival Percent survival Relative colony number ARTICLE Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 ARTICLE 476 | JNCI J Natl Cancer Inst, 2018, Vol. 110, No. 5 resistant pancreatic neuroendocrine tumors (NCT01658436) Centre (C596/A18076) and the BSU facilities at the Cancer show that many patients experience toxicities on 300 mg BID, Research UK Beatson Institute (C596/A17196), Scottish although this may also reflect the fragility of the heavily pre- Universities Life Science Alliance (MSD23_G_Holyoake- treated patients with this aggressive cancer (44). Chan), Scottish National Blood Transfusion Service, Cancer It is clear that the depth of response to TKI is the major Research UK programme funding (C11074/A11008), the driver for sustained remissions, hence the need to rapidly Howat Foundation and Friends of Paul O’Gorman (flow reduce overall leukemic cell burden and ideally to reduce the cytometry support). GVH is a Kay Kendall Leukaemia Fund numbers of BM-located cells (45). We showed that HCQ treat- (KKLF) Intermediate Research Fellow (KKL698)/Leadership ment (a nonspecific autophagy inhibitor that is being tested in Fellow/John Goldman Fellow. LH is a KKLF Intermediate more than 30 active clinical trials [46]) inhibited autophagy flow Research Fellow (KKL1148)/John Goldman Fellow. BC is sup- and enhanced death following NVP-BEZ235, both in vitro and in ported, in part, by National Cancer Institute grant CA95111. a xenograft model of CML. Importantly, we also showed that PS is funded by the Brain Tumour Charity, Cancer Research genetic autophagy inhibition sensitized CML cells to death, indi- UK (CRUK) and Association for International Cancer cating that the main additive effect of HCQ was due to a block in Research (AICR) and is supported by the National Institute the autophagy process, but not off target effect. for Health Research University College London Hospitals A recent phase I trial in patients with advanced solid tumors Biomedical Research Centre. BJD is funded by the Howard and melanoma shows that HCQ is safe and tolerable and has Hughes Medical Institute and National Institutes of Health some antitumor activity when used in combination with temsirolimus-mediated mTOR inhibition (47). Although our grant R37CA065823. results on normal blood cells suggest that HCQ and NVP- BEZ235-mediated mTOR inhibition may be tolerated with Notes regards to myelosuppression, results from phase I trials are awaited for different innovative catalytic mTOR inhibitors such Affiliations of authors: Glasgow Polyomics (GH, PH), Wolfson as Gedatolisib, Apitolisib, VS5584, and AZD8055 (all in phase I; Wohl Cancer Research Centre (RM, PB, GVH), Paul O’Gorman Apitolisib in phase II). The outcome of these studies may deter- Leukaemia Research Centre (LEMH, AM, AH, TLH), Institute of mine the most suitable catalytic mTOR inhibitor (in terms of Cancer Sciences, University of Glasgow, Glasgow, UK; Scottish efficacy and tolerability) to be taken forward for combination National Blood Transfusion Service, Gartnavel General Hospital, studies. Glasgow, UK (EKA); Cancer Research UK, Beatson Institute, Given that the mechanism(s) of resistance to TKIs may vary Garscube Estate, Glasgow, UK (KH, DJ, JO, KMR, ES); Faculty of from patient to patient, potential limitations of this study Health and Medical Sciences, University of Adelaide, Adelaide, should be considered. First, the in vitro studies of primary CML Australia and Imperial College, London, UK (JVM); Strathclyde cells in response to the catalytic mTOR inhibitor(s) presented Institute of Pharmacy and Biomedical Sciences, University of here were confined to a relatively small number of TKI-resistant Strathclyde, Glasgow, UK (EC); He ´ matologie Clinique 1G, Centre CML patients’ samples. Additionally, HCQ is nonspecific Hospitalier Lyon Sud, Pierre Be ´ nite, France (VMS, FEN); Division autophagy inhibitor, and development of more specific and/or of Hematology and Medical Oncology, Oregon Health and more potent autophagy inhibitors might be required to inhibit Science University, Knight Cancer Institute, Portland, OR (BJD); autophagy in BM-located cells in CML patients. Institute of Translational Medicine, Department of Molecular We conclude that catalytic mTOR inhibitors may be effective and Clinical Cancer Medicine, University of Liverpool, UK (REC); for patients with BCR-ABL-independent resistance and that Department of Haematology, Milton Keynes Hospital NHS pharmacological autophagy inhibition will further enhance Foundation Trust, Milton Keynes, UK (SM); Institute of their efficacy. This is particularly important for this heavily pre- Molecular, Cell and Systems Biology, College of Medical, treated population because treatment options for patients who Veterinary and Life Sciences, University of Glasgow, UK (PH); fail all currently available TKIs, including ponatinib, are very Samantha Dickson Brain Cancer Unit, UCL Cancer Institute, limited. Paul O’Gorman Building, London, UK (PS); Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA (BC). Funding The funders had no role in the design of the study; the col- This work was supported by Medical Research Council lection, analysis, or interpretation of the data; the writing of the (G0600782 and G0900882, CHOICES, ISCRTN No. 61568166), manuscript; or the decision to submit the manuscript for the Kay Kendall Leukaemia Fund (KKL404 and KKL501), publication. Leuka, Glasgow Experimental Cancer Medicine Centre, TLH has previously received research support from Bristol- which is funded by Cancer Research UK and the Chief Myers Squibb and Novartis. FEN is a consultant for Novartis, Scientist’s Office (Scotland), Cancer Research UK Glasgow ARIAD, and Pfizer, has received research grants from Novartis, Figure 6. Continued respectively. A table showing the number of mice at risk is shown below the graph. Untr ¼ untreated. C) Bone marrow (BM)–derived mononuclear cells (MNCs) from four TKI-resistant patients (Pts 1–4) were cultured in SFM supplemented with PGF and treated with 100 nM ponatinib, 100 nM NVP-BEZ-235 alone, or in combination with HCQ-mediated autophagy inhibition for 72 hours. Survival of progenitor cells was measured by colony forming cell (CFC) assay. Each dot represents average of two to three technical replicates. D) BM cells were collected on four occasions from patient No. 1 (Pt 1) during the period of 2013–2016. Ph-negative CD34 cells (n¼ 4) were cultured in SFM supplemented with PGF and treated with 100 nM ponatinib, 100 nM NVP-BEZ-235, 10 mM HCQ, 10 nM omacetaxine, and the combination of NVP- BEZ-235 and HCQ. Survival of progenitor cells was measured by CFC assay following 72 hours of drug treatment. Error bars ¼ SD. Statistical analyses were performed using the Gehan-Breslow Wilcoxon test (A and B) or the two-tailed Student’s t test (C and D). CML ¼ chronic myeloid leukemia; HCQ ¼ hydroxychloroquine; Untr ¼ untreated. Downloaded from https://academic.oup.com/jnci/article/110/5/467/4643200 by DeepDyve user on 12 July 2022 R. Mitchell et al. |477 17. Donato NJ, Wu JY, Stapley J, et al. BCR-ABL independence and LYN kinase and has received speakers fees from Novartis, Bristol-Myers overexpression in chronic myelogenous leukemia cells selected for resist- Squibb, ARIAD, and Pfizer. BJD is currently principal investigator ance to STI571. 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Journal
"JNCI: Journal of the National Cancer Institute" – Oxford University Press
Published: May 1, 2018
Keywords: autophagy; protein-tyrosine kinase inhibitor; mtor serine-threonine kinases; mtor inhibitors; genes; transcription, genetic; cell lines; pharmacology; mice; imatinib mesylate