The Fox(o) and the HDAC

In this issue of Blood, Long et al¹  describe a FOXO1/3-HDAC8-p53–mediated pathway leading to resistance and persistence of FLT3-ITD⁺ acute myeloid leukemia (AML) stem cells after FLT3-inhibition.

A fox, known for his cunning and wiliness, frequently features in many fables by the Greek slave Aesop and others. In the fable “The Fox and the Hare,” the hare stares at the fox expressing his wonder about the fox’s insidiousness. The fox replies by inviting the hare for dinner that evening, so they may discuss the subject. The hare accepts the invitation, but upon arrival at the fox’s house realizes that only plates and bowls were laid on the dining table, but no food. The hare cries out what a fool he has been to accept the invitation, as he was meant to serve as dinner, and runs away.

FOXO(e)s, transcription factors, which normally act as tumor suppressors,²  slyly may also promote leukemogenesis.³  In fact, the stem cell in AML features several cunning fox(o)es, which lead to stem cell resistance to standard chemotherapeutic regimen, as well as their persistence. Modulations of signaling pathways or autophagy, (over)expression of certain proteins or enzymes such as drug efflux pumps, topoisomerase and others, gene alterations in genes, such as Fms-like tyrosine kinase 3 (FLT3), etc, microRNAs influencing cell damage, cell cycle, or apoptosis, and cell death,⁴  immune evasion mechanisms or interactions with the tumor microenvironment,⁵  may lead to resistance to chemotherapy. However, various mechanisms of resistance also occur in AML treated with targeted therapies, such as tyrosine kinase inhibitors (TKIs).

AML associated with internal tandem duplications in FLT3 (FLT3-ITD⁺ AML) affects approximately one-third of AML patients, whereby FLT3-ITD portends a poor prognosis, leading to increased risk of relapse and inferior overall survival.⁶  The advent of well-tolerated FLT3-targeting TKI, however, has improved the outcome of patients with FLT3-ITD⁺ AML, when used in combination therapies, although they do not eliminate leukemia stem cells. In 2017, midostaurin, a multikinase inhibitor, was approved for frontline treatment of AML patients with mutations of FLT3 in combination with standard chemotherapy. Later, the FLT3 inhibitor gilteritinib was approved, and quizartinib is in clinical trial. However, resistance to FLT3 inhibitors, especially when used as monotherapy, seems to be the cause for the unsatisfactory clinical response. Predominant mechanisms of resistance are mutations in the activation loop or the ”gatekeeping domain” in the tyrosine kinase domain of FLT3,⁷  activation of prosurvival signaling pathways, but mutations in other genes, such TET2 or IDH1, have also been observed, particularly in patients treated with the FLT3 inhibitor crenolanib.⁸  In addition, the bone marrow microenvironment and its production of FLT3 ligand or fibroblast growth factor 2 has been implicated in the resistance of FLT3-ITD⁺ AML cells to FLT3 inhibitors.⁹ 

In this article by Long et al, the authors unravel a novel pathway of resistance of FLT3-ITD⁺ AML cells to FLT3 inhibitors via forkhead box protein (FOXO1/3)-mediated upregulation of histone deacetylase 8 (HDAC8). Upregulation of HDAC8 is shown to inactivate the tumor suppressor protein p53, thereby promoting the persistence of FLT3-ITD⁺ AML cells in the presence of an FLT3 inhibitor (see figure).

Using 2 different cell lines with mutated FLT3 and primary human AML cells, the authors demonstrate that treatment with quizartinib leads to upregulation of HDAC8, whose perturbed expression has previously been associated with poor prognosis in various cancers.¹⁰  HDACs, in general, regulate genome stability and gene expression by the modification of histones and chromatin remodeling. Targeting of HDAC8 in combination with FLT3 inhibitor therapy increased the death of FLT3-ITD⁺ AML cells and prolonged survival in immunosuppressed nonobese diabetic/severe combined immunodeficiency mice transplanted with FLT3-mutated cell lines. Gene expression analysis of FLT3-mutated cells treated with an HDAC inhibitor revealed significant upregulation of the p53 pathway. Conversely, depletion of p53 prevented the apoptosis of FLT3-ITD⁺ AML cells treated with the HDAC and/or FLT3 inhibitor. Hypothesizing that increased expression of HDAC8 was due to enhanced transcription, the authors elegantly identified the role of the transcription factors FOXO1/3 for the upregulation of HDAC8 and the promotion of resistance to FLT3 inhibitors. Targeting HDAC8 also activated p53. In xenotransplantation studies with human FLT3-ITD⁺ AML cells, the authors confirmed that pharmacological targeting of HDAC8 and FLT3 led to a superior outcome compared with treatment with the FLT3 inhibitor alone and reduced leukemia-initiating capacity, as measured in secondary transplantation assays. Last, the authors demonstrate that inhibition of HDAC8 and the respective tyrosine kinase may also be beneficial in cells harboring other activating tyrosine kinase mutations.

In summary, this paper suggests a novel pathway of resistance to TKIs via upregulation of HDAC8. Combination of FLT3 inhibitors with HDAC8 inhibitors may be a feasible strategy for elimination of leukemic stem cells in AML. Although it has been shown in different cancers that HDAC inhibition can overcome therapy resistance by different mechanisms, it remains to be seen whether the other HDACs may also contribute to therapy resistance in a similar FOXO1/3-p53–dependent manner. Certain HDAC inhibitors have entered clinical trials or clinical medicine in several hematological cancers. However, the future will show whether the development of inhibitors of other cunning fox(o)es, which promote leukemic progression, can make the hare, that is, HDAC and other leukemic stem cell targets, finally, run away.