To the Editor:
The FMS-like tyrosine kinase-3 (FLT3) is mutated in ~30% of acute myeloid leukemia (AML) patients. Common FLT3 mutations are internal tandem duplications (FLT3-ITD) and point mutations in its c-terminal tyrosine kinase domain (FLT3-TKD). The resulting active FLT3 receptors promote cell proliferation and resistance to programmed cell death (apoptosis) . The poor prognosis of patients with FLT3-ITD has spurred an intensive search for FLT3 inhibitors (FLT3i) . These include the nanomolar FLT3i quizartinib, which has produced promising benefits in AML patients with FLT3-ITD . TKD mutations in FLT3-ITD arise during the therapy of patients with quizartinib and confer drug resistance. The broad-range tyrosine kinase inhibitors midostaurin and gilteritinib are used with standard chemotherapy to treat AML. Their pleiotropic actions frequently evoke hematological toxicity [1, 2]. Marbotinib is a hybrid FLT3i containing elements of quizartinib and bis(1H-indol-2-yl)methanone. Marbotinib specifically inhibits FLT3-ITD and FLT3-TKD [4, 5].
Epigenetic modifiers of the histone deacetylase (HDAC) family control the development and survival of blood cells. Compared to normal cells, certain leukemic cell types frequently have aberrant expression levels and activities of epigenetic modifiers that belong to the histone deacetylase (HDAC) family. Accordingly, inhibitors of zinc-dependent HDACs (HDACi) are approved drugs for the treatment of subtypes of leukemia and lymphoma. These agents are vorinostat, romidepsin, belinostat, and chidamide. Further HDACi are tested clinically (www.clinicaltrials.org), such as the benzamide entinostat (MS-275) [6, 7]. HDACi decrease FLT3-ITD through ubiquitin-dependent proteasomal degradation and apoptosis-associated caspase activation. Combinations of HDACi and FLT3i synergistically kill FLT3-ITD-positive cells through accelerated elimination of FLT3-ITD and DNA replication stress/DNA damage induction. HDAC1, HDAC2, and particularly HDAC3 maintain the stability of FLT3-ITD . A cellular self-digestion process termed autophagy also eliminates FLT3-ITD . Nonetheless, autophagy can promote FLT3i resistance [10, 11]. HDACs modulate autophagy in leukemic cells [6, 12], but it is unknown if HDACi modulate FLT3-ITD through autophagy and if this has biological relevance.
Despite initial hope in HDACi, their limited clinical efficacy as monotherapies and toxicological issues make additional research necessary. This includes the development of nanomolar HDACi with a low impact on normal cells and the identification of effective drug combinations [7, 13]. KH16 and KH29 are recently developed HDACi, which we have named yanostat-1/yanostat-2 [12, 14]. We reveal that their inhibitory profiles are superior to those of SAHA and MS-275 in AML cells carrying FLT3-ITD. Low doses of HDACi cause hormesis effects through FLT3-ITD. Specific inhibition of FLT3-ITD with the nanomolar FLT3i marbotinib and quizartinib abrogates undesired hormesis and is synergistically lethal in combination with nanomolar doses of KH16.
KH16 and KH29 carry the hydroxamic acid as zinc binding moiety being present in SAHA and have nanomolar activity against HDACs . We applied KH16 and KH29 to human FLT3-ITD-positive MV4-11 and MOLM-13 AML cells for 24-48 h. Flow cytometry demonstrated that nanomolar concentrations of KH16 and KH29 induced apoptosis (37 nM < IC50 < 85 nM). KH16 and KH29 were at least 6.3-fold more effective apoptosis inducers than the established HDACi MS-275 and SAHA (533 nM < IC50 < 814 nM) (Fig. 1A–D; Table 1; Fig. S1A, B). Thus, KH16 and KH29 are significantly more potent inducers of apoptosis in FLT3-ITD-positive leukemic cells than the clinical grade HDACi vorinostat and entinostat.
Human embryonic kidney and retinal pigment epithelial (RPE1) cells do not undergo apoptosis when they are exposed to KH16 and KH29 [12, 14]. We extended these analyses. We incubated MV4-11 cells, RPE1 cells, and human keratinocytes (HaCaT cells) with rounded IC50 concentrations of KH16, KH29, and SAHA for 24-72 h. Flow cytometry showed that the HDACi induced apoptosis significantly in MV4-11 cells but not in RPE1 and HaCaT cells (Fig. S2). To additionally consider the responses of normal human blood cells to KH16 and KH29, we isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and treated the cells with rounded IC50 concentrations of KH16, KH29, MS-275, and SAHA for 24 h. None of these HDACi compromised the viability of PBMCs (Fig. 1E). We further distinguished the main leukocyte populations by employing antibodies for lineage-specific surface markers. We found that HDACi did not induce apoptosis in B-cells, T-cells, monocytes, dendritic cells, natural killer (NK) cells, and polymorphonuclear neutrophils (PMNs) (Fig. 1F). Thus, despite being potent inducers of apoptosis in leukemic cells with FLT3-ITD, KH16 and KH29 do not kill normal cells.
Immunoblot analyses with lysates from MV4-11 and MOLM-13 cells that were incubated with equimolar concentrations of KH16, KH29, and MS-275 verified that 25 nM of KH16 and KH29 induced an accumulation of hyperacetylated histone H3 (Fig. 1G, H). This effect increased dose-dependently. Up to 100 nM MS-275 did not induce an accumulation of acetylated histone H3.
We confirmed that KH16 and KH29 induced apoptosis by immunoblots assessing activation of caspase-3 and cleavage of its target PARP1. Moreover, we detected a dose-dependent induction of pro-apoptotic BIM and a reduction of anti-apoptotic BCL-XL. KH16 and KH29 caused these molecular alterations more effectively than MS-275. Whereas 25 nM KH16 and KH29 induced the autophagy protein p62, 100 nM KH16 and KH29 decreased p62. 25–100 nM MS-275 stabilized p62 (Fig. 1G, H).
Unexpectedly, 25–50 nM KH16 and KH29, and 25–100 nM MS-275, evoked an accumulation of total and phosphorylated FLT3-ITD (Fig. 1G, H). At 100 nM concentrations of KH16 and KH29, this effect was lost. This held for the phosphorylation of the kinases ERK1/ERK2, the transcription factor STAT5, and the levels of the DNA repair protein RAD51. Doses of 100 nM KH16 and KH29 attenuated these tumor-promoting targets of FLT3-ITD (Fig. 1G, H).
We aimed to define the biological importance of the accumulation of p-FLT3-ITD and FLT3-ITD in response to low doses of KH16 (Fig. 1G, H). We hypothesized that this increase in FLT3-ITD attenuated pro-apoptotic effects of HDACi. We experimentally addressed this with a nanomolar dose of marbotinib. We found that 25 nM KH16 and 2 nM marbotinib combined favorably against MV4-11 cells (Fig. 2A). We also noted this effect using 25 nM KH16 and 2 nM quizartinib. Compared to KH16 plus FLT3i, combinations of 25 nM SAHA and the FLT3i were significantly less effective apoptosis inducers. In such combinations, increasing the dose of SAHA to 650 nM gave similar results as 25 nM KH16 (Fig. 2B). According to these results, combinations of KH16 plus marbotinib or quizartinib are low nanomolar inducers of apoptosis in FLT3-ITD-positive leukemic cell populations.
Immunoblot analyses verified that 50 nM KH16 plus 2 nM marbotinib activated caspase-3, induced the DNA damage marker ɣH2AX, and decreased the anti-apoptotic proteins BCL-XL and MCL1. Histone hyperacetylation was equally evoked by KH16 ± marbotinib (Fig. 2C). These findings suggest that low doses of HDACi cause hormesis through kinase signaling. KH16 plus marbotinib decreased the autophagy regulators p62 and HDAC6 (Fig. 2C), indicating a breakdown of autophagy. Inhibition of autophagy with chloroquine potentiated the ability of 50-100 nM KH16 to attenuate p62 and FLT3-ITD in MV4-11 and MOLM-13 cells. Moreover, this combination treatment caused a reduction and cleavage of the UNC-51-like autophagy-activating kinase-1 (ULK1) (Figs. 2D; S3). Caspase-dependent mechanisms likely conferred this loss and processing of proteins that belong to the autophagy system .
We analyzed how the combination of 25 nM KH16 and 2 nM marbotinib affected PBMCs and subpopulations therein. We noted that these drugs alone and in combination did not significantly induce apoptosis of B-cells, T-cells, monocytes, dendritic cells, NK cells, and PMNs (Fig. 2E). These data suggest that these inhibitors selectively kill leukemic cells carrying mutant FLT3 and spare normal blood cells.
In conclusion, the epigenetic drugs KH16 and KH29 are innovative lead compounds that deserve further consideration as treatment options for leukemia. One needs to consider the threshold concentrations at which HDACi evoke caspase-dependent apoptosis and abrogate cytoprotective autophagy. This finding stresses the need for HDACi that are effective at low nanomolar concentrations. Specific FLT3 kinase inhibitors disable undesired hormesis effects that HDACi cause. This allows such drug combinations to favorably combine against leukemic cells that carry the clinically unfavorable marker FLT3-ITD.
The datasets that we generated during and analyzed during this study are available from the corresponding authors on reasonable request.
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Work done in the group of OHK is funded by the German Research Foundation/Deutsche Forschungsgemeinschaft (DFG) KR2291/9-1, project number 427404172; KR2291/12-1, project number 445785155; DFG KR2291/14-1, project number 469954457; KR2291/15-1, project number 495271833; KR2291/16-1, project number 496927074; KR2291/17-1, project number 502534123; KR2291/18-1, project number 528202295; funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 393547839 – SFB 1361; the DAAD Egypt/Germany; the Brigitte und Dr. Konstanze Wegener-Stiftung (projects 65/110); and the Walter Schulz-Stiftung. Work done in the group of WS is funded by the DFG project number 469954457, SI868/22-1, and the Alexander von Humboldt Foundation Project EGY 1191187 (HSI). We thank Prof. Dr. F.-D. Böhmer, Institute of Molecular Cell Biology, CMB, Jena University Hospital, Jena, Germany, for MV4-11 and MOLM-13 cells (originally from the DSMZ, authenticated by DNA fingerprint profiling using eight different and highly polymorphic short tandems repeats, at the Leibniz-Institute DSMZ, Braunschweig, Germany). We thank the Blood transfusion unit of the University Medical Center Mainz for providing us with buffy coats for PBMC isolation.
Open Access funding enabled and organized by Projekt DEAL.
MBe, SM, and OHK declare the patent “Synthesis, pharmacology and use of new and selective FMS-like tyrosine kinase 3 (FLT3) inhibitors, WO2019/034538”; SM and OHK declare “Novel HDAC6 inhibitors and their uses, WO2016020369A1”; OHK declares “The use of molecular markers for the preclinical and clinical profiling of inhibitors of enzymes having histone deacetylase activity, WO/2004/027418”.
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Zeyn, Y., Hausmann, K., Halilovic, M. et al. Histone deacetylase inhibitors modulate hormesis in leukemic cells with mutant FMS-like tyrosine kinase-3. Leukemia 37, 2319–2323 (2023). https://doi.org/10.1038/s41375-023-02036-2