Disease-stabilizing treatment based on all-trans retinoic acid and valproic acid in acute myeloid leukemia - identification of responders by gene expression profiling of pretreatment leukemic cells

Håkon Reikvam, Randi Hovland, Rakel Brendsdal Forthun, Sigrid Erdal, Bjørn Tore Gjertsen, Hanne Fredly, Øystein Bruserud, Håkon Reikvam, Randi Hovland, Rakel Brendsdal Forthun, Sigrid Erdal, Bjørn Tore Gjertsen, Hanne Fredly, Øystein Bruserud

Abstract

Background: Acute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy.

Methods: Primary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment.

Results: Patients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment.

Conclusions: Responders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment.

Trial registrations: ClinicalTrials.gov no. NCT00175812 ; EudraCT no. 2004-001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332 ; EudraCT no. 2007-2007-001995-36, registered October 14, 2009.

Keywords: Acute myeloid leukemia; All-trans retinoic acid; Gene expression profiling; Valproic acid.

Conflict of interest statement

Ethics approval and consent to participate

All studies were approved by the local Ethics Committee (Region III, University of Bergen, Norway) and samples collected after written informed consent.

Study registrations: Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Mutational profiling of responders and non-responders to AML-stabilizing treatment based on ATRA plus valproic acid. Primary AML cells derived from 41 patients (Additional file 1: Table S2, patients 1–2 and 15–43) were analyzed for AML-associated mutations (see Additional file 1: Table S4). The 41 patients included 12 responders and 29 non-responders to the treatment. The patient numbers at the top of the figure refer to the numbers given in Additional file 1: Table S2, and the figure presents the results only for those mutations that were detected for at least one of these patients. The classification of the mutations can be seen in the left part of the figure. The karyotype classification is given at the bottom of the figure, whereas more detailed information about the cytogenetic abnormalities are included in Additional file 1: Table S2
Fig. 2
Fig. 2
Comparison of the global gene expression profiles for responders and non-responders to the AML-stabilizing treatment based on ATRA and valproic acid – an analysis of the differentially expressed genes based on the function of their encoded proteins. Differentially expressed genes were identified by SAM, and the functional analysis of the encoded proteins was based on the Panther database. Only genes encoding annotated proteins were included in this analysis. The figure thus presents the representative distribution of the genes with known functions that showed differential expression according to the Panther protein class (PS) category. The name of each of the identified classes is given in the figure along with number of genes in each category. Only classes containing ≥ 5 genes are named in the figure. The genes included in each of the five major classes nucleic acid binding, transcription factor, enzyme modulator, hydrolase and receptor are listed in Table 2, and important biological functions of individual genes are described in Additional file 1: Table S6
Fig. 3
Fig. 3
Comparison of the global gene expression profiles – a comparison of primary AML cells sampled before treatment (day 1), after treatment with ATRA alone (day 3) and after triple therapy with ATRA, valproic acid and theophyllamine (day 8). Each part of the figure shows the results for one analysis. The upper part presents the comparison of pretreatment samples and cells collected after 2 days of ATRA therapy (day 3 versus pretreatment samples), the middle figure show the effect of adding valproic acid plus theophyllamine to the ATRA therapy (day 8 versus day 3 samples) and the lower figure shows the effect of the triple combination (pretreatment samples versus AML cells sampled on day 8). The same strategies were used for all three analyses. Differentially expressed genes were first identified by SAM, and the functional analyses of the encoded proteins were based on the Panther database. Only genes encoding annotated proteins were included in these analyses. The figures thus present the representative distribution of the genes with known functions that showed differential expression according to the Panther protein class (PS) category..The name of each of the identified classes is given in the figure along with number of genes in each category. Only classes containing ≥ 2 genes are named. The genes included in each of the five major classes nucleic acid binding, transcription factor, enzyme modulator, hydrolase and receptor are listed in Table 2, and important biological functions of individual genes are described in Additional file 1: Table S6

References

    1. Döhner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. New Eng J Med. 2015;373(12):1136–1152. doi: 10.1056/NEJMra1406184.
    1. Latagliata R, Bongarzoni V, Carmosino I, Mengarelli A, Breccia M, Borza PA, D'Andrea M, D'Elia GM, Mecarocci S, Morano SG, et al. Acute myelogenous leukemia in elderly patients not eligible for intensive chemotherapy: the dark side of the moon. Ann Oncol. 2006;17(2):281–285. doi: 10.1093/annonc/mdj112.
    1. Pollyea DA, Kohrt HE, Medeiros BC. Acute myeloid leukaemia in the elderly: a review. Br J Haematol. 2011;152(5):524–542. doi: 10.1111/j.1365-2141.2010.08470.x.
    1. Ossenkoppele G, Lowenberg B. How I treat the older patient with acute myeloid leukemia. Blood. 2015;125(5):767–774. doi: 10.1182/blood-2014-08-551499.
    1. Thol F, Schlenk RF, Heuser M, Ganser A. How I treat refractory and early relapsed acute myeloid leukemia. Blood. 2015;126(3):319–327. doi: 10.1182/blood-2014-10-551911.
    1. Skavland J, Jorgensen KM, Hadziavdic K, Hovland R, Jonassen I, Bruserud O, Gjertsen BT. Specific cellular signal-transduction responses to in vivo combination therapy with ATRA, valproic acid and theophylline in acute myeloid leukemia. Blood Cancer J. 2011;1 doi: 10.1038/bcj.2011.2.
    1. Fredly H, Reikvam H, Gjertsen BT, Bruserud O. Disease-stabilizing treatment with all-trans retinoic acid and valproic acid in acute myeloid leukemia: serum hsp70 and hsp90 levels and serum cytokine profiles are determined by the disease, patient age, and anti-leukemic treatment. Am J Hematol. 2012;87(4):368–376. doi: 10.1002/ajh.23116.
    1. Fredly H, Stapnes Bjornsen C, Gjertsen BT, Bruserud O. Combination of the histone deacetylase inhibitor valproic acid with oral hydroxyurea or 6-mercaptopurin can be safe and effective in patients with advanced acute myeloid leukaemia--a report of five cases. Hematology. 2010;15(5):338–343. doi: 10.1179/102453310X12647083620967.
    1. Ryningen A, Stapnes C, Lassalle P, Corbascio M, Gjertsen BT, Bruserud O. A subset of patients with high-risk acute myelogenous leukemia shows improved peripheral blood cell counts when treated with the combination of valproic acid, theophylline and all-trans retinoic acid. Leuk Res. 2009;33(6):779–787. doi: 10.1016/j.leukres.2008.10.005.
    1. Lubbert M, Kuendgen A. Combining DNA methyltransferase and histone deacetylase inhibition to treat acute myeloid leukemia/myelodysplastic syndrome: achievements and challenges. Cancer. 2015;121(4):498–501. doi: 10.1002/cncr.29083.
    1. Corsetti MT, Salvi F, Perticone S, Baraldi A, De Paoli L, Gatto S, Pietrasanta D, Pini M, Primon V, Zallio F, et al. Hematologic improvement and response in elderly AML/RAEB patients treated with valproic acid and low-dose Ara-C. Leuk Res. 2011;35(8):991–997. doi: 10.1016/j.leukres.2011.02.021.
    1. Stapnes C, Gjertsen BT, Reikvam H, Bruserud O. Targeted therapy in acute myeloid leukaemia: current status and future directions. Expert Opin Investig Drugs. 2009;18(4):433–455. doi: 10.1517/14728220902787628.
    1. Fredly H, Ersvaer E, Kittang AO, Tsykunova G, Gjertsen BT, Bruserud O. The combination of valproic acid, all-trans retinoic acid and low-dose cytarabine as disease-stabilizing treatment in acute myeloid leukemia. Clin Epigenet. 2013;5(1):13. doi: 10.1186/1868-7083-5-13.
    1. Fredly H, Gjertsen BT, Bruserud O. Histone deacetylase inhibition in the treatment of acute myeloid leukemia: the effects of valproic acid on leukemic cells, and the clinical and experimental evidence for combining valproic acid with other antileukemic agents. Clin Epigenet. 2013;5(1):12. doi: 10.1186/1868-7083-5-12.
    1. Siddikuzzaman, Guruvayoorappan C, Berlin Grace VM. All trans retinoic acid and cancer. Immunopharmacol Immunotoxicol. 2011;33(2):241–249. doi: 10.3109/08923973.2010.521507.
    1. Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood. 2008;111(5):2505–2515. doi: 10.1182/blood-2007-07-102798.
    1. Fredly H, Ersvaer E, Stapnes C, Gjertsen BT, Bruserud Ø. The Combination of Conventional Chemotherapy with New Targeted Therapy in Hematologic Malignancies: The Safety and Efficiency of Low-Dose Cytarabine Supports its Combination with New Therapeutic Agents in Early Clinical Trials. Curr Cancer Ther Rev. 2009;5:243–255. doi: 10.2174/157339409789712645.
    1. Dimberg A, Bahram F, Karlberg I, Larsson LG, Nilsson K, Oberg F. Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27(Kip1) Blood. 2002;99(6):2199–2206. doi: 10.1182/blood.V99.6.2199.
    1. Johnson DE, Redner RL. An ATRActive future for differentiation therapy in AML. Blood Rev. 2015;29(4):263–268. doi: 10.1016/j.blre.2015.01.002.
    1. Stapnes C, Ryningen A, Hatfield K, Oyan AM, Eide GE, Corbascio M, Kalland KH, Gjertsen BT, Bruserud O. Functional characteristics and gene expression profiles of primary acute myeloid leukaemia cells identify patient subgroups that differ in susceptibility to histone deacetylase inhibitors. Int J Oncol. 2007;31(6):1529–1538.
    1. Trus MR, Yang L, Suarez Saiz F, Bordeleau L, Jurisica I, Minden MD. The histone deacetylase inhibitor valproic acid alters sensitivity towards all trans retinoic acid in acute myeloblastic leukemia cells. Leukemia. 2005;19(7):1161–1168. doi: 10.1038/sj.leu.2403773.
    1. Bug G, Ritter M, Wassmann B, Schoch C, Heinzel T, Schwarz K, Romanski A, Kramer OH, Kampfmann M, Hoelzer D, et al. Clinical trial of valproic acid and all-trans retinoic acid in patients with poor-risk acute myeloid leukemia. Cancer. 2005;104(12):2717–2725. doi: 10.1002/cncr.21589.
    1. Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH, Schiffer CA, Doehner H, Tallman MS, Lister TA, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol. 2003;21(24):4642–4649. doi: 10.1200/JCO.2003.04.036.
    1. Cheson BD, Bennett JM, Kantarjian H, Pinto A, Schiffer CA, Nimer SD, Lowenberg B, Beran M, de Witte TM, Stone RM, et al. Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood. 2000;96(12):3671–3674.
    1. Cheson BD, Greenberg PL, Bennett JM, Lowenberg B, Wijermans PW, Nimer SD, Pinto A, Beran M, de Witte TM, Stone RM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood. 2006;108(2):419–425. doi: 10.1182/blood-2005-10-4149.
    1. Bruserud O, Hovland R, Wergeland L, Huang TS, Gjertsen BT. Flt 3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt 3-ligand effects in AML cell populations with and without genetic Flt 3 abnormalities. Haematologica. 2003;88(4):416–428.
    1. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–26. doi: 10.1038/nbt.1754.
    1. Staffas A, Kanduri M, Hovland R, Rosenquist R, Ommen HB, Abrahamsson J, Forestier E, Jahnukainen K, Jonsson OG, Zeller B, et al. Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia. Blood. 2011;118(22):5905–5913. doi: 10.1182/blood-2011-05-353185.
    1. Stavrum AK, Petersen K, Jonassen I, Dysvik B. Analysis of gene-expression data using J-Express. Curr Protoc Bioinforma. 2008, Chapter 7:Unit 7.3; doi:10.1002/0471250953.bi0703s21.
    1. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98(9):5116–5121. doi: 10.1073/pnas.091062498.
    1. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–15550. doi: 10.1073/pnas.0506580102.
    1. Mi H, Muruganujan A, Casagrande JT, Thomas PD. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 2013;8(8):1551–1566. doi: 10.1038/nprot.2013.092.
    1. Network CGAR. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. New Eng J Med. 2013;368(22):2059–2074. doi: 10.1056/NEJMoa1301689.
    1. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, Potter NE, Heuser M, Thol F, Bolli N, et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. New Eng J Med. 2016;374(23):2209–2221. doi: 10.1056/NEJMoa1516192.
    1. Kantarjian H, Ravandi F, O'Brien S, Cortes J, Faderl S, Garcia-Manero G, Jabbour E, Wierda W, Kadia T, Pierce S, et al. Intensive chemotherapy does not benefit most older patients (age 70 years or older) with acute myeloid leukemia. Blood. 2010;116(22):4422–4429. doi: 10.1182/blood-2010-03-276485.
    1. Zhang XZ, Yin AH, Lin DJ, Zhu XY, Ding Q, Wang CH, Chen YX. Analyzing gene expression profile in K562 cells exposed to sodium valproate using microarray combined with the connectivity map database. J Biomed Biotechnol. 2012;2012:654291.
    1. Wang J, Fong CC, Tzang CH, Xiao P, Han R, Yang M. Gene expression analysis of human promyelocytic leukemia HL-60 cell differentiation and cytotoxicity induced by natural and synthetic retinoids. Life Sci. 2009;84(17–18):576–583. doi: 10.1016/j.lfs.2009.02.001.
    1. Schlenk RF, Lubbert M, Benner A, Lamparter A, Krauter J, Herr W, Martin H, Salih HR, Kundgen A, Horst HA, et al. All-trans retinoic acid as adjunct to intensive treatment in younger adult patients with acute myeloid leukemia: results of the randomized AMLSG 07–04 study. Ann Hematol. 2016;95(12):1931–1942. doi: 10.1007/s00277-016-2810-z.
    1. Ryningen A, Stapnes C, Paulsen K, Lassalle P, Gjertsen BT, Bruserud O. In vivo biological effects of ATRA in the treatment of AML. Expert Opin Investig Drugs. 2008;17(11):1623–1633. doi: 10.1517/13543784.17.11.1623.
    1. Balmer JE, Blomhoff R. Gene expression regulation by retinoic acid. J Lipid Res. 2002;43(11):1773–1808. doi: 10.1194/jlr.R100015-JLR200.
    1. Zheng PZ, Wang KK, Zhang QY, Huang QH, Du YZ, Zhang QH, Xiao DK, Shen SH, Imbeaud S, Eveno E, et al. Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia. Proc Natl Acad Sci U S A. 2005;102(21):7653–7658. doi: 10.1073/pnas.0502825102.
    1. Park MH, Cho SA, Yoo KH, Yang MH, Ahn JY, Lee HS, Lee KE, Mun YC, Cho DH, Seong CM, et al. Gene expression profile related to prognosis of acute myeloid leukemia. Oncol Rep. 2007;18(6):1395–1402.
    1. Bullinger L, Schlenk RF, Gotz M, Botzenhardt U, Hofmann S, Russ AC, Babiak A, Zhang L, Schneider V, Dohner K, et al. PRAME-induced inhibition of retinoic acid receptor signaling-mediated differentiation--a possible target for ATRA response in AML without t(15; 17) Clin Cancer Res. 2013;19(9):2562–2571. doi: 10.1158/1078-0432.CCR-11-2524.
    1. Rucker FG, Lang KM, Futterer M, Komarica V, Schmid M, Dohner H, Schlenk RF, Dohner K, Knudsen S, Bullinger L. Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks. Epigenetics. 2016;11(7):517–525. doi: 10.1080/15592294.2016.1187350.
    1. Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, Metzeler KH, Poeppl A, Ling V, Beyene J, et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med. 2011;17(9):1086–1093. doi: 10.1038/nm.2415.
    1. Ng SW, Mitchell A, Kennedy JA, Chen WC, McLeod J, Ibrahimova N, Arruda A, Popescu A, Gupta V, Schimmer AD, et al. A 17-gene stemness score for rapid determination of risk in acute leukaemia. Nature. 2016;540(7633):433–437. doi: 10.1038/nature20598.
    1. Steinmetz B, Hackl H, Slabakova E, Schwarzinger I, Smejova M, Spittler A, Arbesu I, Shehata M, Soucek K, Wieser R. The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid. Cell Cycle. 2014;13(18):2931–2943. doi: 10.4161/15384101.2014.946869.
    1. Rommer A, Steinmetz B, Herbst F, Hackl H, Heffeter P, Heilos D, Filipits M, Steinleitner K, Hemmati S, Herbacek I, et al. EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p 21/WAF in human myeloid cells. PLoS One. 2013;8(2):e56308. doi: 10.1371/journal.pone.0056308.
    1. Wieser R. New functions for ecotropic viral integration site 1 (EVI1), an oncogene causing aggressive malignant disease. Cell Cycle. 2012;11(21):3915. doi: 10.4161/cc.22392.
    1. Verhagen HJMP, Smit MA, Rutten A, Denkers F, Poddighe PJ, Merle PA, Ossenkoppele GJ, Smit L. Primary acute myeloid leukemia cells with overexpression of EVI-1 are sensitive to all-trans retinoic acid. Blood. 2016;127(4):458–463. doi: 10.1182/blood-2015-07-653840.
    1. Kronenwett R, Butterweck U, Steidl U, Kliszewski S, Neumann F, Bork S, Blanco ED, Roes N, Graf T, Brors B, et al. Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia. Oncogene. 2005;24(34):5313–5324. doi: 10.1038/sj.onc.1208596.

Source: PubMed

Sponsorer og samarbeidspartnere

Medisinsk tilstand

Legemiddelintervensjoner

3
Abonnere