Molecular targets for therapy

Targeted inhibition of cooperative mutation- and therapy-induced AKT activation in AML effectively enhances response to chemotherapy

Abstract

Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.

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Fig. 1: PI3K/AKT signaling is constitutively activated in inv(16)/KITD816Y AML.
Fig. 2: Gene set enrichment analyses (GSEA) reveal common functional transcriptional programs in inv(16)/KITD816Y murine AML and human inv(16) AML patients harboring KIT, FLT3, and RAS co-mutations.
Fig. 3: PI3K and DNA-PK inhibitors reduce doxorubicin-induced activation of AKT signaling leading to increased apoptosis of inv(16)/KITD816Y AML cells.
Fig. 4: PI3K and DNA-PK inhibitors markedly enhance doxorubicin-induced comet-tail formation/DNA damage.
Fig. 5: Combinations of doxorubicin and PI3K or DNA-PK inhibitors synergistically inhibit growth and survival of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic BM cells.
Fig. 6: Combinations of standard chemotherapy (cytarabine/doxorubicin) and PI3K or DNA-PK inhibitor significantly prolong survival of mice with inv(16)/KITD816Y AML.
Fig. 7: Model for therapeutic inhibition of mutation- and anthracycline-dependent AKT activation to enhance chemotherapy response in AML.

References

  1. 1.

    Dohner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424–47.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  2. 2.

    Theilgaard-Monch K, Boultwood J, Ferrari S, Giannopoulos K, Hernandez-Rivas JM, Kohlmann A, et al. Gene expression profiling in MDS and AML: potential and future avenues. Leukemia. 2011;25:909–20.

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med. 2016;374:2209–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Allen C, Hills RK, Lamb K, Evans C, Tinsley S, Sellar R, et al. The importance of relative mutant level for evaluating impact on outcome of KIT, FLT3 and CBL mutations in core-binding factor acute myeloid leukemia. Leukemia. 2013;27:1891–901.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Sallmyr A, Fan J, Datta K, Kim KT, Grosu D, Shapiro P, et al. Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML. Blood. 2008;111:3173–82.

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Pearsall EA, Lincz LF, Skelding KA. The role of DNA repair pathways in AML chemosensitivity. Curr Drug Targets. 2018;19:1205–19.

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Arafeh R, Samuels Y. PIK3CA in cancer: the past 30 years. Semin Cancer Biol. 2019;59:36–49.

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Alzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: at the bench and bedside. Semin Cancer Biol. 2019;59:125–32.

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Lu HY, Qin J, Han N, Lei L, Xie F, Li C. EGFR, KRAS, BRAF, PTEN, and PIK3CA mutation in plasma of small cell lung cancer patients. Onco Targets Ther. 2018;11:2217–26.

    PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Sun S, Zhang Y, Zheng J, Duan B, Cui J, Chen Y, et al. HDAC6 inhibitor TST strengthens the antiproliferative effects of PI3K/mTOR inhibitor BEZ235 in breast cancer cells via suppressing RTK activation. Cell Death Dis. 2018;9:929.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. 11.

    Patra S, Young V, Llewellyn L, Senapati JN, Mathew JBRAF. KRAS and PIK3CA mutation and sensitivity to trastuzumab in breast cancer cell line model. Asian Pac J Cancer Prev. 2017;18:2209–13.

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Myers MB, Banda M, McKim KL, Wang Y, Powell MJ, Parsons BL. Breast cancer heterogeneity examined by high-sensitivity quantification of PIK3CA, KRAS, HRAS, and BRAF mutations in normal breast and ductal carcinomas. Neoplasia. 2016;18:253–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Nakanishi Y, Walter K, Spoerke JM, O’Brien C, Huw LY, Hampton GM, et al. Activating mutations in PIK3CB confer resistance to PI3K inhibition and define a novel oncogenic role for p110beta. Cancer Res. 2016;76:1193–203.

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Zhang Z, Liu J, Wang Y, Tan X, Zhao W, Xing X, et al. Phosphatidylinositol 3-kinase beta and delta isoforms play key roles in metastasis of prostate cancer DU145 cells. FASEB J. 2018;32:5967–75.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Ngeow J, Sesock K, Eng C. Breast cancer risk and clinical implications for germline PTEN mutation carriers. Breast Cancer Res Treat. 2017;165:1–8.

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Smith IN, Briggs JM. Structural mutation analysis of PTEN and its genotype-phenotype correlations in endometriosis and cancer. Proteins. 2016;84:1625–43.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Fedele CG, Ooms LM, Ho M, Vieusseux J, O’Toole SA, Millar EK, et al. Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers. Proc Natl Acad Sci USA. 2010;107:22231–6.

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Janku F, Yap TA, Meric-Bernstam F. Targeting the PI3K pathway in cancer: are we making headway?. Nat Rev Clin Oncol. 2018;15:273–91.

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Liu N, Li X, Huang H, Zhao C, Liao S, Yang C, et al. Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth. Oncotarget. 2014;5:5453–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Bozulic L, Surucu B, Hynx D, Hemmings BA. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell. 2008;30:203–13.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Li X, Lu Y, Liang K, Liu B, Fan Z. Differential responses to doxorubicin-induced phosphorylation and activation of Akt in human breast cancer cells. Breast Cancer Res. 2005;7:R589–97.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Feng J, Park J, Cron P, Hess D, Hemmings BA. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem. 2004;279:41189–96.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Dragoi AM, Fu X, Ivanov S, Zhang P, Sheng L, Wu D, et al. DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA. EMBO J. 2005;24:779–89.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Xu N, Lao Y, Zhang Y, Gillespie DA. Akt: a double-edged sword in cell proliferation and genome stability. J Oncol. 2012;2012:951724.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. 25.

    Huang WC, Hung MC. Induction of Akt activity by chemotherapy confers acquired resistance. J Formos Med Assoc. 2009;108:180–94.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther. 2002;1:707–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Toulany M, Maier J, Iida M, Rebholz S, Holler M, Grottke A, et al. Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells. Cell Death Discov. 2017;3:17072.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001;61:3986–97.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Chen X, Guo Y, Ouyang T, Li J, Wang T, Fan Z, et al. Co-mutation of TP53 and PIK3CA in residual disease after neoadjuvant chemotherapy is associated with poor survival in breast cancer. J Cancer Res Clin Oncol. 2019;145:1235–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    de la Rochefordiere A, Kamal M, Floquet A, Thomas L, Petrow P, Petit T, et al. PIK3CA pathway mutations predictive of poor response following standard radiochemotherapy +/- cetuximab in cervical cancer patients. Clin Cancer Res Off J Am Assoc Cancer Res. 2015;21:2530–7.

    Article  CAS  Google Scholar 

  31. 31.

    Wang L, Lin N, Li Y. The PI3K/AKT signaling pathway regulates ABCG2 expression and confers resistance to chemotherapy in human multiple myeloma. Oncol Rep. 2019;41:1678–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Grimwade D, Ivey A, Huntly BJ. Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood. 2016;127:29–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Chen W, Xie H, Wang H, Chen L, Sun Y, Chen Z, et al. Prognostic significance of KIT mutations in core-binding factor acute myeloid leukemia: a systematic review and meta-analysis. PLoS ONE. 2016;11:e0146614.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 34.

    Rapin N, Bagger FO, Jendholm J, Mora-Jensen H, Krogh A, Kohlmann A, et al. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients. Blood. 2014;123:894–904.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles RA, et al. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol. 2006;24:3904–11.

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Yui S, Kurosawa S, Yamaguchi H, Kanamori H, Ueki T, Uoshima N, et al. D816 mutation of the KIT gene in core binding factor acute myeloid leukemia is associated with poorer prognosis than other KIT gene mutations. Ann Hematol. 2017;96:1641–52.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Schlenk RF, Kayser S, Bullinger L, Kobbe G, Casper J, Ringhoffer M, et al. Differential impact of allelic ratio and insertion site in FLT3-ITD-positive AML with respect to allogeneic transplantation. Blood. 2014;124:3441–9.

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Illmer T, Schaich M, Ehninger G, Thiede C, DAs Group. Tyrosine kinase mutations of JAK2 are rare events in AML but influence prognosis of patients with CBF-leukemias. Haematologica. 2007;92:137–8.

    PubMed  Article  Google Scholar 

  39. 39.

    Schnittger S, Bacher U, Kern W, Haferlach C, Haferlach T. JAK2 seems to be a typical cooperating mutation in therapy-related t(8;21)/ AML1-ETO-positive AML. Leukemia. 2007;21:183–4.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Zhang Q, Wu X, Cao J, Gao F, Huang K. Association between increased mutation rates in DNMT3A and FLT3-ITD and poor prognosis of patients with acute myeloid leukemia. Exp Ther Med. 2019;18:3117–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Spiekermann K, et al. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood. 2006;107:1791–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Park SH, Chi HS, Cho YU, Jang S, Park CJ. Effects of c-KIT mutations on expression of the RUNX1/RUNX1T1 fusion transcript in t(8;21)-positive acute myeloid leukemia patients. Leuk Res. 2013;37:784–9.

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Zhao L, Melenhorst JJ, Alemu L, Kirby M, Anderson S, Kench M, et al. KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice. Blood. 2012;119:1511–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Martelli AM, Evangelisti C, Chiarini F, McCubrey JA. The phosphatidylinositol 3-kinase/Akt/mTOR signaling network as a therapeutic target in acute myelogenous leukemia patients. Oncotarget. 2010;1:89–103.

    PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Brenner AK, Andersson Tvedt TH, Bruserud O. The complexity of targeting PI3K-Akt-mTOR signalling in human acute myeloid leukaemia: the importance of leukemic cell heterogeneity, neighbouring mesenchymal stem cells and immunocompetent cells. Molecules. 2016;21:1512.

  46. 46.

    Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood. 2003;102:972–80.

    CAS  Article  Google Scholar 

  47. 47.

    Stein MK, Morris LK, Martin MG. Next-generation sequencing identifies novel RTK VUSs in breast cancer with an emphasis on ROS1, ERBB4, ALK and NTRK3. Pathol Oncol Res. 2018;26:593–5.

  48. 48.

    Mora-Jensen H, Jendholm J, Rapin N, Andersen MK, Roug AS, Bagger FO, et al. Cellular origin of prognostic chromosomal aberrations in AML patients. Leukemia. 2015;29:1785–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Knudsen KJ, Rehn M, Hasemann MS, Rapin N, Bagger FO, Ohlsson E, et al. ERG promotes the maintenance of hematopoietic stem cells by restricting their differentiation. Genes Dev. 2015;29:1915–29.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Pronk CJ, Rossi DJ, Mansson R, Attema JL, Norddahl GL, Chan CK, et al. Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell. 2007;1:428–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Theilgaard-Monch K, Raaschou-Jensen K, Schjodt K, Heilmann C, Vindelov L, Jacobsen N, et al. Pluripotent and myeloid-committed CD34+ subsets in hematopoietic stem cell allografts. Bone Marrow Transplant. 2003;32:1125–33.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Ianevski A, He L, Aittokallio T, Tang J. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinformatics. 2017;33:2413–5.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  53. 53.

    Brachmann SM, Kleylein-Sohn J, Gaulis S, Kauffmann A, Blommers MJ, Kazic-Legueux M, et al. Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations. Mol Cancer Ther. 2012;11:1747–57.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    Speranza MC, Nowicki MO, Behera P, Cho CF, Chiocca EA, Lawler SE. BKM-120 (Buparlisib): a phosphatidyl-inositol-3 kinase inhibitor with anti-invasive properties in glioblastoma. Sci Rep. 2016;6:20189.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010;24:679–86.

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Pardee TS, Zuber J, Lowe SW. Flt3-ITD alters chemotherapy response in vitro and in vivo in a p53-dependent manner. Exp Hematol. 2011;39:473–85.e4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Dos Santos C, McDonald T, Ho YW, Liu H, Lin A, Forman SJ, et al. The Src and c-Kit kinase inhibitor dasatinib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents. Blood. 2013;12:1900–13.

    Article  CAS  Google Scholar 

  58. 58.

    Xiao Y, Deng T, Su C, Shang Z. MicroRNA 217 inhibits cell proliferation and enhances chemosensitivity to doxorubicin in acute myeloid leukemia by targeting KRAS. Oncol Lett. 2017;13:4986–94.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Ueno Y, Mori M, Kamiyama Y, Saito R, Kaneko N, Isshiki E, et al. Evaluation of gilteritinib in combination with chemotherapy in preclinical models of FLT3-ITD(+) acute myeloid leukemia. Oncotarget. 2019;10:2530–45.

    PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

    Murthy RK, Loi S, Okines A, Paplomata E, Hamilton E, Hurvitz SA, et al. Tucatinib, trastuzumab, and capecitabine for HER2-positive metastatic breast cancer. N Engl J Med. 2020;382:597–609.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  61. 61.

    Stone RM, Larson RA, Dohner H. Midostaurin in FLT3-mutated acute myeloid leukemia. N Engl J Med. 2017;377:1903.

    PubMed  Article  PubMed Central  Google Scholar 

  62. 62.

    Altman JK, Foran JM, Pratz KW, Trone D, Cortes JE, Tallman MS. Phase 1 study of quizartinib in combination with induction and consolidation chemotherapy in patients with newly diagnosed acute myeloid leukemia. Am J Hematol. 2018;93:213–21.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  63. 63.

    Zelenetz AD, Barrientos JC, Brown JR, Coiffier B, Delgado J, Egyed M, et al. Idelalisib or placebo in combination with bendamustine and rituximab in patients with relapsed or refractory chronic lymphocytic leukaemia: interim results from a phase 3, randomised, double-blind, placebo-controlled trial. Lancet Oncol. 2017;18:297–311.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

The authors thank all bone marrow donors and AML patients, Bente Langelung Kristensen for professional animal care, and colleagues at the FACS Facility at BRIC. This work was supported by a grant from the Danish Cancer Society (ME, R167-A10932-17-S2), KT-M is supported by a clinical research fellowship and a center grant from the Novo Nordisk Foundation (Grant no. 100191, Novo Nordisk Foundation Center for Stem Cell Biology, DanStem; Grant no. NNF17CC0027852, KT-M, BTP). This work was further supported by grants from the Danish Council for Strategic Research (Grant no. 133100153, KT-M), the Danish Cancer Society (Grant no. R72-A4572-13-S2, KT-M), Børnecancerfonden (2016-0255), and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Foundation (KT-M), and Tømrermester Jørgen Holm og Hustru Elisa, Brødrene Hartmans Fond, F. Hansen’s Mindelegat (KT-M), and the Intramural Research Program, National Human Genome Research Institute, NIH (LZ and PL).

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ME and KT-M conceived and designed the study. ME, KR, CV, AC, MA, and SE collected and assembled the data. ME, KR, MA, and KT-M analyzed and interpreted the data. ME, KR, and KT-M drafted and wrote the manuscript. MA, LZ, KJW, PL, and BTP interpreted the data and contributed to the writing of the manuscript.

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Correspondence to Kim Theilgaard-Mönch.

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Estruch, M., Reckzeh, K., Vittori, C. et al. Targeted inhibition of cooperative mutation- and therapy-induced AKT activation in AML effectively enhances response to chemotherapy. Leukemia (2020). https://doi.org/10.1038/s41375-020-01094-0

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