Targeting miR-155 in FLT3-ITD mutated AML: ready for prime time?

The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor, which is present in about 30% of CN-AML and confers a poor prognosis (1). FLT3 encodes a receptor tyrosine kinase expressed on hematopoietic progenitor cells involved in stem cell differentiation and proliferation (2). FLT3 activating mutations such as internal tandem duplication (ITD) lead to constitutive, ligand-independent activation of this receptor, conferring a growth and survival advantage. The mutation itself has not been shown to independently drive leukemic transformation in vivo (1,3). Rather, FLT3-ITD must collaborate with additional oncogenic mutations to trigger hematopoietic malignancy (3). Despite extensive research throughout the last decades, only Midastaurin has been recently approved by the FDA as a first line treatment in combination with chemotherapy (4) highlighting the difficulties for establishing targeted therapies. Therefore, novel approaches such as targeting downstream effectors of FLT3-ITD signaling are relevant to explore new therapeutic targets.

MicroRNAs (miRNAs) are small noncoding RNAs that control post-transcriptional gene expression in various biological and pathological processes. MiRNA expression has been shown to be highly dysregulated in AML. Specifically, miR-155 is the most significantly overexpressed miRNA in FLT3-ITD mutated AML (5-7). MiR-155 is a known oncogene that accelerates formation of lymphomas and when overexpressed in HSPCs leads to a myeloproliferative disorder as previously shown by O’Connell et al. (8,9). Our group recently linked miR-155 upregulation to MLL-rearranged AML (10), a subgroup with high FLT3 levels (2) and not only, as previously reported, to FLT3-ITD positive AML (5,7,11) or FAB M4/5 AML (12), implying a broader role for this miRNA in AML. This finding is in line with previous reports in B-cell lymphomas, where miR-155 overexpression was detected in all screened subtypes, regardless of cytogenetics (13). Additionally, high miR-155 expression levels were associated with an inferior overall survival in CN-AML (14) and were found to be part of a leukemic stem cell miRNA signature (15). Enforced expression of miR-155 in myeloid cells has been shown to have both oncogenic and tumor suppressor functions in AML. Palma et al. proposed an anti-leukemic role for miR-155 through the induction of apoptosis and myeloid differentiation in the AML cell line OCI-AML3 (11), whereas several groups have reported miR-155 as an oncogene in leukemia (14,16-19) and therefore a potential therapeutic target (16). Based on the published data, it appears that the function of miR-155 is context-dependent. Narayan et al. recently proposed a novel dose-dependent role for miR-155 in the regulation of AML, which might partially explain the observed discrepancy (20). Considering that miR-155 has hundreds of predicted targets, determining its regulated downstream pathways is highly context-dependent and thus challenging. Of note, although miR-155 is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), miR-155^−/− mice did not show impaired myeloid differentiation or any perturbations in the HSPC compartment (21).

Wallace et al. now uncovered the downstream mechanism of miR-155 in FLT3-ITD AML (22). Mice homozygous for FLT3-ITD (FLT3-ITD^+/+) develop a myeloproliferative disease (MPD) defined by a myeloid-specific cell expansion, however, the mutation itself does not drive leukemic transformation. Interestingly, a comparable phenotype was observed through engineered overexpression of miR-155 in HSPCs. Through crossing homozygous FLT3-ITD (FLT3-ITD^+/+) mice with miR-155 knock out (miR-155^−/−) mice generating FLT3-ITD^+/+/miR-155^−/− mice, Wallace et al. showed that deletion of miR-155 in FLT3-ITD homozygous mice weakens the MPD phenotype, suggesting a functional relationship. More importantly, this direct genetic approach resolved previous controversies about the functional relationship of miR-155 and FLT3-ITD in AML cells.

FLT3-ITD^+/+/miR-155^−/− mice had a decreased myeloid progenitor compartment compared to FLT3-ITD^+/+ mice as well as reduction in proliferation of LSK and myeloid progenitor cell populations, indicating a role for miR-155 in promoting myeloid progenitor expansion in the pre-malignant context of FLT3-ITD mediated MPD. Using an RNA sequencing approach of sorted LSK cells, the authors identified the interferon pathway as highly enriched in FLT3-ITD^+/+/miR-155^−/−vs. FLT3 ITD^+/+ LSK cells. This was further confirmed by Western blot analysis of STAT1, a master regulator of interferon responses, which showed highly increased STAT1 protein levels in FLT3-ITD^+/+/miR-155^−/− cells. Based on this finding, the authors concluded that miR-155 promotes proliferation of myeloid progenitor cells by reducing the anti-proliferative effects of interferon signaling in FLT3-ITD^+/+ leukemia. By mining their hypothesis with the TCGA-LAML dataset, the authors confirmed that IFN-α and IFN-γ were significantly downregulated in FLT3-ITD AML compared with FLT3-WT AML. Subsequent genetic depletion through CRISPR/Cas9 of miR-155 in human AML cell lines showed elevated Interferon signaling and STAT1 levels. To identify the relevant targets of miR-155 in the context of FLT3-ITD mutated AML, the authors showed the upregulation of established miR-155 targets including Ship1, Pu.1 and Cebpb in FLT3-ITD^+/+/miR-155^−/− compared to FLT3-ITD^+/+ mice and further confirmed the expression levels of these targets in the human TCGA-LAML dataset. CEBPb is a known interferon regulator whereas Ship1 is an inhibitor of AKT signaling, demonstrating that miR-155 works through several targets to modulate multiple signaling pathways and responses in FLT3-ITD mutated AML. Finally, Wallace et al. translated their findings to primary human FLT3-ITD mutated AML cells, in which they could demonstrate reduced survival and increased apoptosis after treatment with a miR-155 inhibitor in vitro.

The findings of Wallace et al. highlight the relevance of miR-155 in FLT3-ITD driven AMLs and open a new path to the possibility that the depletion or inhibition of miR-155 may provide a therapeutic angle. While much has been learned about miR-155 biology, there are still many unanswered questions that add complexity to its role in AML and it is yet to be determined if inhibition of this miRNA in vivo is ready for prime time and will eventually lead to a better outcome for AML patients.

Acknowledgments

Funding: A Rouhi was supported by the DFG (SFB 1074, project A5) as well as the gender equality program by the DFG (SFB 1074, project Z2), a fellowship from the Canadian Institutes of Health Research and the Baustein Startförderung Program of the Medical Faculty, Ulm University. F Kuchenbauer was supported by grants from Deutsche Krebshilfe grant 109420 (Max-Eder program); fellowship 2010/04 by the European Hematology Association; and by the Deutsche Forschungsgemeinschaft (DFG) (SFB 1074, project A5) and the Wilhelm Sander Stiftung (2015.153.1).

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Pei-Pei Xu (Department of Hematology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2017.09.13). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Papaemmanuil E, Dohner H, Campbell PJ. Genomic Classification in Acute Myeloid Leukemia. N Engl J Med 2016;375:900-1. [Crossref] [PubMed]
2. Kuchenbauer F, Kern W, Schoch C, et al. Detailed analysis of FLT3 expression levels in acute myeloid leukemia. Haematologica 2005;90:1617-25. [PubMed]
3. Frohling S, Scholl C, Levine RL, et al. Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles. Cancer Cell 2007;12:501-13. [Crossref] [PubMed]
4. Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med 2017;377:454-64. [Crossref] [PubMed]
5. Jongen-Lavrencic M, Sun SM, Dijkstra MK, et al. MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia. Blood 2008; [Crossref] [PubMed]
6. Garzon R, Volinia S, Liu CG, et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood 2008;111:3183-9. [Crossref] [PubMed]
7. Garzon R, Garofalo M, Martelli MP, et al. Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci U S A 2008;105:3945-50. [Crossref] [PubMed]
8. Costinean S, Zanesi N, Pekarsky Y, et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A 2006;103:7024-9. [Crossref] [PubMed]
9. O'Connell RM, Rao DS, Chaudhuri AA, et al. Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder. J Exp Med 2008;205:585-94. [Crossref] [PubMed]
10. Schneider E, Staffas A, Rohner L, et al. MicroRNA-155 is upregulated in MLL-rearranged AML but its absence does not affect leukemia development. Exp Hematol 2016;44:1166-71. [Crossref] [PubMed]
11. Palma CA, Al Sheikha D, Lim TK, et al. MicroRNA-155 as an inducer of apoptosis and cell differentiation in Acute Myeloid Leukaemia. Mol Cancer 2014;13:79. [Crossref] [PubMed]
12. Xue H, Hua LM, Guo M, et al. SHIP1 is targeted by miR-155 in acute myeloid leukemia. Oncol Rep 2014;32:2253-9. [Crossref] [PubMed]
13. Eis PS, Tam W, Sun L, et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci U S A 2005;102:3627-32. [Crossref] [PubMed]
14. Marcucci G, Maharry KS, Metzeler KH, et al. Clinical role of microRNAs in cytogenetically normal acute myeloid leukemia: miR-155 upregulation independently identifies high-risk patients. J Clin Oncol 2013;31:2086-93. [Crossref] [PubMed]
15. Lechman ER, Gentner B, Ng SW, et al. miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells. Cancer Cell 2016;29:214-28. [Crossref] [PubMed]
16. Khalife J, Radomska HS, Santhanam R, et al. Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia. Leukemia 2015;29:1981-92. [Crossref] [PubMed]
17. Chuang MK, Chiu YC, Chou WC, et al. A 3-microRNA scoring system for prognostication in de novo acute myeloid leukemia patients. Leukemia 2015;29:1051-9. [Crossref] [PubMed]
18. Gerloff D, Grundler R, Wurm AA, et al. NF-kappaB/STAT5/miR-155 network targets PU.1 in FLT3-ITD-driven acute myeloid leukemia. Leukemia 2015;29:535-47. [Crossref] [PubMed]
19. Lee DW, Futami M, Carroll M, et al. Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155. Oncogene 2012;31:4085-94. [Crossref] [PubMed]
20. Narayan N, Morenos L, Phipson B, et al. Functionally distinct roles foar different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukemia. Leukemia 2017;31:808-20. [Crossref] [PubMed]
21. Thai TH, Calado DP, Casola S, et al. Regulation of the germinal center response by microRNA-155. Science 2007;316:604-8. [Crossref] [PubMed]
22. Wallace JA, Kagele DA, Eiring AM, et al. miR-155 promotes FLT3-ITD-induced myeloproliferative disease through inhibition of the interferon response. Blood 2017;129:3074-86. [Crossref] [PubMed]