FTO (IC50 = 2.6 μM)

FTO, a mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO’s m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro.[1]
Directly attaching to FTO, FB23-2 inhibits its m6A demethylase activity in a specific manner. Human acute myeloid leukemia (AML) cell line cells and primary blast AML cells are significantly suppressed in proliferation and are encouraged to differentiate/apoptose in vitro, simulating FTO depletion. [1]

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The structure of the FTO/FB23 complex suggests that the optimization on the carboxylic acid of FB23 would not disturb the affinity and specificity for FTO. To improve the permeability of FB23, we synthesized derivatives of the benzyl carboxylic acid on the basis of the bioisosterism principle. The benzohydroxamic acid, termed as FB23-2 (Figures 1H and S1B), displays significantly improved anti-proliferative activity on NB4 and MONOMAC6 cells with an IC50 of 0.8 – 1.5 μM (Figure 1I), and maintains inhibitory activity on FTO demethylation in vitro (Figure 1J). To establish the absolute configuration, we determined the X-ray crystal structure of FB23-2, which unambiguously shows an intramolecular hydrogen bond between the amino hydrogen and the carbonyl of hydroxamic acid (Figure 1H, right panel). In addition, we analyzed the relative configuration of FB23-2 in solution using the Nuclear Overhauser Effect (NOE), which is a transfer of nuclear spin polarization through space, rather than chemical bonds. The strong NOE correlation between H-1 and H-10 in the NOESY spectrum also supports the intramolecular hydrogen bonding (Figure S1E). With this evidence in hand, the docking of FB23-2 to FTO resulted in an excellent fit of FB23-2 in a position of perfect overlap to the crystallographically determined binding mode of inhibitor FB23 bound to FTO (Figure S1F). Next, we detected the cellular uptake of FB23-2 by LC-MS/MS quantitation (Figure 1K). Of note, FB23-2 was detected around 0.05–0.2 nmol/million cells in MONOMAC6 and NB4 cells, which is several folds higher than the cellular uptake of FB23 (see Figure 1G). Meanwhile, FB23 was also detected in tiny amounts in the FB23-2 treated AML cells, which is likely a hydrolysis product of FB23-2. The increased intracellular concentration of FB23-2 likely contributes to its improved anti-proliferation effect in AML cells.

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FB23-2 increases RNA methylation in a panel of AML cells [1]
Researchers checked the changes of m6A on RNA when AML cells were treated by the FTO inhibitor. The treatment of NB4 and MONOMAC6 cells with FB23-2 resulted in a substantial increase of m6A abundance in transcriptomes as detected by m6A dot blot assay (Figure 2A). LC-MS/MS quantitation further confirmed the increase of cellular m6A in mRNA of AML cells after exposure to FB23-2 (Figure 2B). The m6Am is another substrate of FTO, and we observed similar increases of m6Am abundance in AML cells exposed to FB23-2 (Figure 2B). The overall level of m6Am is much lower than that of m6A, however. Since recent reports independently showed that FTO minimally affected the expression levels of mRNA starting with m6Am (Akichika et al., 2019; Sun et al., 2019; Wei et al., 2018), it is likely that m6A, rather than m6Am, is the main substrate of FTO in AML cells (Su et al., 2018).

FB23-2 minimally altered proliferation of human normal bone marrow (BM) cells isolated from a healthy donor (Figure 2C). Consistent with the effect of FTO KD on the proliferation of MLL-AF9 (MA9) and FLT3ITD/NPM1 AML murine cells (Li et al., 2017b), FB23-2 significantly suppressed the proliferation of BM cells from these two models in a dose-dependent manner (Figure 2D), accompanied with increased m6A abundance (Figure 2E). Moreover, we determined the anti-leukemia effects of FB23-2 in a panel of additional AML cell lines with different genetic backgrounds, including MA9.3ITD (with MLL-AF9 and FLT3ITD mutation), MA9.3RAS (with MLL-AF9 and NRAS mutations), U937 (with t(10;11) translocation), ML-2 (with t(6;11) translocation), and MV4-11 (with t(4;11) translocation). As expected, FB23-2 efficiently inhibited the proliferation of these AML cell lines with IC50 ranging from 1.9 μM to 5.2 μM, and increased m6A abundance in these cell lines as well (Figures 2E and 2F). Together, these results demonstrate that FB23-2 exhibits FTO inhibition and anti-leukemia effects broadly.

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FB23-2 displays a high selectivity toward FTO [1]
Researchers performed the selectivity profile of FTO inhibitors. Similar to MA, neither FB23 nor FB23-2 inhibits ALKBH5 demethylation in vitro (Figure S2A). As shown by Drug Affinity Responsive Target Stability (DARTS) assay (Lomenick et al., 2009), FB23-2 could not directly bind to ALKBH5 in AML cell lysates (Figure S2B), and showed only marginal effects on the transcription level and protein stability of ALKBH5 (Figures S2C and S2D). In addition, we checked the inhibitory effects of FTO inhibitors on epigenetic targets involved in AML and/or other cancers, including the Histone deacetylases (HDAC), Disruptor of Telomere Silencing 1-like (DOTL1), Bromodomain-containing “reader” proteins (BRD), Lysine-specific demethylase 1 (LSD1), and Jumonji domain-containing histone demethylases (Shortt et al., 2017). FB23 and FB23-2 slightly attenuated the activities of these targets in vitro, while the positive control inhibitors display significant activities (Table S2). Similarly, 20 μM FB23-2 minimally inhibited TET1 protein in vitro (Figure S2E) and did not alter the abundance of 5mC or 5hmC in AML cells (Figure S2F). The major histone methylations in NB4 cells were unaltered by FB23-2 (Figure S2G). In addition, we performed a much broader enzymatic specificity test of FB23-2. The inhibitory effect of 10 μM FB23-2 on activities of 405 human kinases was mapped onto the kinome phylogenetic tree (Figure S2H). Kinases with more than 40% of inhibition were further evaluated. Six kinases were inhibited by FB23-2 with IC50 around 3.0 – 13.4 μM. The inhibitory efficiency of FB23-2 on these kinases is much lower than that of well-established kinase inhibitors. FB23-2 also barely inhibited the oncogenic proteases (Table S3). Although MA primary inhibits both COX-1 and COX-2 to different extents (Vane et al., 1998), neither FB23 nor FB23-2 was observed to significantly inhibit cyclooxygenases even at 50 μM (Figure S2I). Taken together, these results indicate that our inhibitors display a high enzymatic selectivity for FTO.

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FB23-2 exhibits FTO-dependent anti-proliferation activity and promotes myeloid differentiation and apoptosis [1]
In line with the negative regulation of FTO on ASB2 and RARA expression in AML cells (Li et al., 2017b), FB23 and FB23-2 treatment significantly increased their abundance at the mRNA and protein levels in NB4 and MONOMAC6 cells (Figures 3A and 3B). FTO positively regulates MYC and CEBPA in an m6A modification-dependent manner (Su et al., 2018). Similar with shRNA-induced FTO KD, FB23 or FB23-2 indeed inhibited MYC and CEBPA expression in both NB4 and MONOMAC6 cells (Figure 3C).

In order to further determine whether the inhibitory effect of FB23-2 on AML cell proliferation relies on FTO, we generated stable FTO KO NB4 AML cells using CRISPR-Cas9. FB23-2 dramatically suppressed proliferation of AML cells but exhibited a much milder effect on AML cells with stable FTO KO (Figure 3D), suggesting that the inhibitory effect of FB23-2 on the proliferation of AML cells depends on the suppression of an activated FTO signaling. Consistently, we found that the FTO KD and 60 μM FB23 or 3 μM FB23-2 had comparable effects in NB4 cells (Figure 3E). To verify the direct interaction between FB23-2 and FTO, we performed a DARTS assay. As expected, the FTO protein becomes protease-resistant in the presence of FB23-2 (Figure 3F), indicating that FB23-2 indeed binds to FTO in cell lysates.

Researchers further characterized the effects of FB23-2 on AML cells. In line with the inhibitory effects of FTO on myeloid differentiation and apoptosis in AML cells (Li et al., 2017b), FB23-2 substantially accelerated all-trans retinoic acid (ATRA)-induced myeloid differentiation in NB4 and MONOMAC6 cells in a dose-dependent manner (Figures 4A and 4B). Furthermore, FB23-2 induced apoptosis (Figures 4C and 4D) and cell cycle arrest at G1 stage in AML cells (Figures 4E and 4F). Collectively, these results suggest that FB23-2 exhibits FTO-dependent activity in AML cells.

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FB23 and FB23-2 target similar signaling pathways to FTO KD in AML cells [1]
To investigate which genes and signaling pathways are responsible for the anti-leukemic function of FTO inhibitors, Researchers performed transcriptome-wide RNA-sequencing (RNA-seq) analysis of FTO KD, FB23 treated, or FB23-2 treated NB4 AML cells as well as control cells. Via independent analysis of three different comparisons, we found that FTO KD, FB23 treatment, and FB23-2 treatment all caused the significant suppression of MYC targets, E2F targets, and G2M checkpoint signal cascades, which may contribute to the inhibitory effects of FTO inhibitors and FTO KD on cell cycle and proliferation (Figures 5A and S3A–S3D). In addition, all three treatments consistently activated apoptosis and p53 pathways (Figure 5A). Global gene set enrichment analysis (GSEA) indicated that FTO KD and FB23 or FB23-2 treatment display similar effects on regulating a set of functionally important signaling pathways (Figures S3E–S3G). Notably, the vast majority of pathways (41 out of 43, 95.3%) increased by FTO KD could also be enriched by FB23-2 (Figures 5B and S3H); similarly, the majority of signaling pathways suppressed by FB23-2 are also inhibited by FTO KD (Figures 5C and S3H). These results strongly suggest that FTO inhibitors, especially FB23-2, had the same effect on critical signalling pathways that control cell cycle, cell proliferation, and cell survival in AML cells as FTO KD.

In xeno-transplanted mice, FB23-2 significantly slows the development of human AML cell lines and primary cells.[1]
FB23-2 suppresses leukemia progression and improves the survival of leukemic mice.[1]
FB23-2 exhibits therapeutic efficacy in treating a patient-derived xeno-transplantation (PDX) AML mouse model[1]
FB23-2 is safe in mice and displays a favorable pharmacokinetic profile.[1]

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FB23-2 suppresses leukemia progression and improves the survival of leukemic mice [1]
We next assessed the therapeutic effects of FB23-2 in vivo with a xeno-transplantation leukemic model. NOD/LtSz-scid IL2RG-SGM3 (NSGS) mice (Wunderlich et al., 2010) were xeno-transplanted with MONOMAC6 AML cells, and 10 days post xeno-transplantation, FB23-2 (2 mg/kg) or vehicle control was intraperitoneally injected into the individual mice daily for 10 days. Notably, FB23-2 injection substantially delayed the onset of full-blown leukemic symptoms and significantly prolonged survival by almost doubling the median survival (Figure 7C). Compared with the vehicle, FTO inhibitor treatment suppressed leukemia malignancy, including reduced splenomegaly and hepatomegaly (Figure 7D). FACS analysis confirmed that FB23-2 injection suppressed the abundance of human AML cells in the recipient mice (Figures 7E and S5C). To further interpret the effect of FB23-2 on differentiation of AML cells in vivo, we collected peripheral blood (PB), BM, and spleen samples of FB23-2- and vehicle control- treated xenograft mice and stained them with anti-human CD15 and anti-human CD11b. As determined by FACS, FB23-2 treatment promoted AML cell differentiation in vivo (Figures 7F and 7G). Wright-Giemsa staining of PB smears revealed that leukemic blasts from FTO inhibitor-treated AML mice were inhibited and partially differentiated; consistently, H&E staining of spleen and liver also showed less AML cell dissemination in FB23-2-treated mice (Figure 7H). Taken together, our data suggests that pharmacological inhibition of FTO by FB23-2 substantially suppresses leukemia progression and prolongs survival.

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FB23-2 exhibits therapeutic efficacy in treating a patient-derived xeno-transplantation (PDX) AML mouse model [1]
We assessed the therapeutic potential of FB23-2 in treating human primary AML cells. Four AML patients with diverse cytogenetics were tested (Table S8). FB23-2 suppressed proliferation of all four sets of primary AML cells, with IC50 values ranging from 1.6 μM to 16 μM (Figure 8A). FB23-2 also induced cell apoptosis (Figure S6A), decreased colony-forming unit (CFU) capacity (Figure 8B), and accelerated ATRA-mediated myeloid differentiation (Figure S6B) of these primary AML cells. Furthermore, FB23-2 treatment also upregulated the expression of both ASB2 and RARA (Figure 8C), two direct targets of FTO, and elevated global mRNA m6A abundance (Figure 8D), thus supporting our conclusion that FB23-2 displays therapeutic effects via directly targeting FTO signaling in patient-derived primary AML cells.

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Lastly, we tested the in vivo therapeutic efficacy of FB23-2in a PDX AML mouse model. Primary AML cells were xeno-transplanted into sublethally irradiated NSGS mice. We monitored the engraftment of AML leukemia cells in vivo by FACS analysis of the percentage of donor AML cells in PB in recipient mice. When recipient mice had 3–5% donor-derived AML cells, the recipient mice were treated with FB23-2 or DMSO for 17 days. The disease latency of FB23-2-treated mice (median survival time of 58 days) was significantly prolonged compared with that of control mice (median survival time of 48 days) (Figure 8E). Furthermore, FACS analysis of engrafted AML cells in recipient mice revealed a significantly reduced proportion of AML blast cells in PB (Figure 8F) and BM (Figure 8G) upon FB23-2 treatment. Consistent with our findings that FB23-2 induced differentiation of AML cell lines in vitro, we found that more differentiated myeloid cells with an increased ratio of cytoplasm/nucleus were present in FB23-2-treated mice (Figure 8H). The leukemia cells from FB23-2-treated PDX mice gave rise to significantly fewer CFUs with markedly reduced sizes of colonies than the leukemia cells from the DMSO-treated PDX mice did (Figures 8I and 8J), thus suggesting that the leukemia malignancy of FB23-2 treated AML cells was significantly impaired. Notably, not only were the bulk AML cells affected, but also leukemia stem cells (LSCs, defined by CD34+CD38−) were significantly eliminated by FB23-2 in vivo in the treated mice (Figure 8K).

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To further evaluate the number of functional LSCs in primary PDX mice, we performed a secondary transplantation. The secondary recipients of AML cells from primary DMSO-treated PDX mice (control) had markedly higher engraftment compared to the secondary recipient mice with AML cells from primary FB23-2 treated PDX mice (Figure 8L). All of the control, secondary PDX mice died within 66 days while 50% of the secondary PDX mice with FB23-2-treated AML cells still survived after 100 days (Figure 8M), thus suggesting that the number of functional LSCs that are able to regenerate leukemia in vivo in secondary recipients was significantly reduced after FB23-2 treatment in the primary recipient mice. Taken together, our data indicate that FB23-2 induced differentiation of AML cells significantly reduced the number of functional primary AML LSCs in vivo.

Kinases and proteases profiling[1]
The kinases and proteases profiling were conducted by Eurofins Pharma Discovery Services. Inhibitory kinases profiling was conducted against a panel of 405 kinases in the presence of FB23-2at 1 μM and 10 μM, respectively. Taken the Met (h) kinase as an example of kinases profiling, Met (h) was incubated with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 250 μM KKKGQEEEYVFIE, 1 mM sodium orthovanadate, 5 mM sodium-6-glycerophosphate, 10 mM Mg(OAc)2, and [γ−33P]-ATP (specific activity and concentration as required). The reaction was initiated by the addition of the Mg(OAc)2/ATP mix. After incubation for 40 min at room temperature, the reaction was terminated by the addition of phosphoric acid to a final concentration of 0.5%. 10 μl of the reaction was then spotted onto a P30 filtermat and washed four times for 4 min in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. The results were calculated with an equation, Inhibition (%) = (Max-Signal)/(Max-Min) × 100%. The reactions without enzyme but all other components served as Min, and the reactions with DMSO served as Max. The percentage inhibitions of 405 kinases by 10 μM were mapped on to the kinome phylogenetic tree. Each group had two repeats. For IC50 determination, the inhibitory percentage of FB23-2 at varying concentrations was obtained, and the IC50 value for each tested kinase was calculated with nonlinear regression analysis using equation in GraphPad Prism 5.[1]
Taken the Caspase2 as an example of inhibitory proteases profiling, 1 μM or 10 μM FB23-2 was pre-incubated with human recombinant Caspase2 in a reaction buffer of 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% Glycerol, 10 mM DTT at 37 °C for 15 min, followed by adding the 25 μM substrate Z-VDVAD-AFC. After incubation for 1 hr, signals of AFC were quantified with spectrofluorimetric method and the DMSO group was treated as 100%. Each group had two repeats.

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Effect of FTO inhibitors on COX-1 and COX-2 enzymes[1]
The inhibitions of COX1 and COX2 enzymes by FTO inhibitors FB23 and FB23-2 were evaluated using the COX Fluorescent Inhibitor Screening Assay Kit following the manufacturer’s protocols. Briefly, COX-1 and COX-2 were incubated with test compounds at room temperature for 5 min, respectively, then 10 μl ADHP (10-acetyl-3,7-dihydroxyphenoxazine) was added to the sample and background wells (without COX enzymes). Reactions were initiated by quickly adding 10 μl of Arachidonic Acid and incubated for 2 min at room temperature. An excitation wavelength of 535 nm and an emission wavelength of 595 nm were used to obtain the signals.

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HPLC-based assay of the inhibition of m6A demethylation in RNA[1]
In vitro ssRNA demethylations were performed with some modifications on the reported assay (Huang et al., 2015). The reactions, containing 0.25 μM FTOΔN31 or 3 μM ALKBH5ΔN66, 5 μM 15-mer ssRNA (5′-AUUGUCA(m6A)CAGCAGC-3′), 300 μΜ 2OG, 280 μΜ (NH4)2Fe(SO4)2, 2 mM L-ascorbic acid, and inhibitors at required concentrations in 50 mM Tris-HCl (pH 7.5 – 8.0), were incubated at 25 °C for 30 min. The reactions were terminated by heating for 5 min at 90 °C, and then the mixtures were subjected to digestion by nuclease P1 and alkaline phosphatase. The IC50 values were quantitated based on the inhibitory percentages of m6A demethylation in the presence of inhibitors at indicated concentrations, using nonlinear regression, dose-response fit on GraphPad Prism 5.0™. All reactions were performed in triplicate.

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Crystallization and structure determination of FTO/FB23 complex [1]
Crystallizations were conducted with hanging-drop vapor-diffusion method at 18 °C. 8 mg/ml of FTOΔN31 protein was incubated with 5-folds FB23 and mixed with a reservoir solution containing 100 mM sodium citrate (pH 5.4), 11.5% (w/v) polyethylene glycol 3350, and 8% isopropanol. The crystals were cryo-protected using extra 20% (v/v) glycerol. Diffraction data were collected on the BL18U1 and BL17U1 beamline at the Shanghai Synchrotron Research Facility (SSRF). All X-ray data were processed using HKL2000 programs (Otwinowski and Minor, 1997), and converted to structure factors within the CCP4 program (Collaborative Computational Project, 1994). The structure was solved by molecular replacement in Phaser using the structure of FTO/MA complex (PDB code 4QKN) as the searching model. The model of structural complex FTO/FB23 was computationally refined with the program REFMAC5.

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Nuclear Magnetic Resonance (NMR) titration[1]
Phosphate buffer (20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 5% DMSO) was used for NMR data acquisition on a Bruker Avance III-600 MHz spectrometer equipped with a cryogenically cooled probe at 25 °C. Experimental samples contained 200 μM FB23 and FTO protein at 0 μM, 1 μM, 2 μM, and 3 μM, respectively.

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Cellular thermal shift assay (CETSA)[1]
CETSA was conducted according to the protocol as previously described (Martinez Molina et al., 2013). NB4 and MONOMAC6 cells were collected and lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM DTT. 50 μM FB23 or DMSO was added to the supernatant and incubated at 25 °C for 25 min. After denaturing at various temperatures for 5 min, samples were centrifuged, and the supernatants were analyzed by western blot. All experiments were performed in triplicate.

While MA9 and FLT3/NPM1 primary cells isolated from AML mice, five human AML cells (MA9.3ITD, MA9.3RAS, U937, ML2, and MV4-11), and human primary AML cells are treated with DMSO or 5 μM FB23-2 for 72 h for dot blot assay, NB4 and MONOMAC6 cells are treated with DMSO or FB23-2 at varying concentrations.[1]

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Cell proliferation assays 5,000 cells/well NB4, FTO KO NB4, and MONOMAC6 AML cells were seeded and treated with DMSO or FTO inhibitors for 72 hr. The cell proliferations were determined with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay according to the manufacturer’s instructions. 10,000 cells/well human AML cells (MA9.3ITD, MA9.3RAS, U937, ML2, and MV4-11) and four primary cells from AML patients were seeded and subjected to FTO inhibitor treatment for 96 hr as indicated. 10,000 cells/well MA9 and FLT3/NPM1 primary cells isolated from AML mice and 5,000 cells/well shNS and shFTO NB4 cells were seeded and treated with FTO inhibitors for 24 hr, 48 hr, 72 hr, and 96 hr for proliferation determination.[1]

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Quantitation of FB23 and FB23-2 in AML cells NB4 and MONOMAC6 cells were treated with 10 μM FB23 or FB23-2 for 24 hr, respectively. Viable cells were distinguished with 0.1% trypan blue, counted and then harvested with PBS by several washings. Cells were diluted into 100 μl with 50% (v/v) water/methanol and followed by several shock freeze-thaw cycles. The supernatants were collected for analysis. The Ultimate 3000 system coupled with a TSQ Quantiva mass spectrometer was applied to determine the cellular concentration of compound FB23 and FB23-2. Analytes were separated on a XSELECT™ HSS T3 column (100 mm × 3.0 mm, 2.5 μm; Waters, USA). The mobile phases used for elution were (A) 0.1% (v/v) formic acid/water and (B) 0.1% (v/v) formic acid/acetonitrile. The mass spectrometer was operated in the negative MRM mode. Parent-to-product transitions were m/z 375.1→339.1, 375.1→298.1 for FB23, and m/z 390.3→318.0, 390.3→289.9 for FB23-2, respectively.[1]

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m6A dot blot assay NB4 and MONOMAC6 cells were treated with DMSO or FB23-2 at varying concentrations for 72 hr, while MA9 and FLT3/NPM1 primary cells isolated from AML mice, five human AML cell (MA9.3ITD, MA9.3RAS, U937, ML2, and MV4-11), and human primary AML cells were treated with DMSO or 5 μM FB23-2 for 72 hr for dot blot assay. Total RNA was separated with miRNeasy Mini Kit, and poly (A)+ RNA was further enriched with PolyATract mRNA isolation System IV in accordance with the manufacturer’s instructions. The RNA samples were diluted in RNA binding buffer, denatured at 65 °C for 5 min. Then one volume of 20 x SSC buffer was added into the RNA samples before dotted onto the Amersham Hybond-N+ membrane with Bio-Dot Apparatus. The RNA samples were cross-linked onto the membrane via UV irradiation. The membrane was stained with 0.02% methylene blue (MB) as loading control. After UV crosslinking and MB staining, the membrane was washed with PBST, blocked with 5% nonfat dry milk for 1 hr at room temperature and incubated with m6A antibody (1 : 2000) at 4 °C overnight. Finally, the membrane was then incubated with the HRP-conjugated goat anti-rabbit IgG and developed with Amersham ECL Prime Western Blotting Detection Reagent.[1]

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LC-MS/MS quantitation of m6A and m6Am in AML cells NB4 and MONOMAC6 cells were cultured with DMSO or 20 μM FB23-2 for 72 hr. mRNA was isolated in line with the dot blot assay, followed by the removal of contaminated rRNA with RiboMinus Transcriptome Isolation Kit. 300 ng mRNA was decapped with 5 units RppH with the thermopol buffer, then the products were digested by nuclease P1 for 1 hr at 42 °C. Subsequently, 1 unit of alkaline phosphatase and NH4HCO3 (100 mM) were added and incubated for another 1 hr at 37 °C. The Ultimate 3000 system coupled with a TSQ Quantiva mass spectrometer was applied to quantitate the cellular levels of A, m6A, and m6Am. Samples were centrifuged and loaded onto a XSELECT™ CSH™ C18 column (100 mm × 3.0 mm, 2.5 μm) and eluted by the gradient methanol. The parent-to-product transitions for A, m6A, and m6Am were 268.1/136.1, 282.1/150.1, and 296.2/150.1, respectively.

Animal model[1]
The NSGS mice were bred and subjected to the xeno-transplantation model. For the AML mouse model, 0.2 × 106 MONOMAC6 cells were directly transplanted into NSGS mice via tail vein. After 10 days, FB23-2 (2 mg/kg/day) and DMSO vehicle were intraperitoneally injected into the mice for a continuous 10 days. The mice were euthanized by CO2 inhalation if they exhibited classical AML symptoms including hunched posture, paralysis, and reduced body weight. Meanwhile, the PB, spleen, and liver samples were collected for further analysis.
PDX models were generated by injecting primary BM cells from AML patient Pt 2017_63 (2 X 106 per mouse) into the tail veins of 6- to 8-week-old sublethally irradiated (2.5 Gy) NSGS mice (NOD.Cg-PrkdcscidIl2rgtm1WjlTg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ), which were purchased from The Jackson Laboratory. When recipient mice had 3–5% donor-derived AML cells in PB, 6 mg/kg/day FB23-2 was delivered by i.p. for 17 days, vehicle DMSO was administrated as control. Mice were weighed daily during treatment and doses were recalculated to make sure the mice received a consistent dose of 6 mg/kg/day. One day after the 17-day full treatment, mice were randomly picked up, and then PB cells were collected and analyzed for the engraftment of leukemia cells by FACS using anti-human-CD45 and anti-mouse-CD45. When the mice became moribund, BM cells were collected and analyzed for the engraftment of leukemia cells by FACS using anti-human-CD45 and anti-mouse-CD45. In addition, the LSCs population was determined as the human CD34+CD38−population. For second transplantation, the patient AML cells, collected from the spleen of primary NSGS mice which were transplanted with BM cells from AML patients and received FB23-2 or DMSO treatment, were transplanted into NSGS mice irradiated at 2.5 Gy. 8 weeks post transplantation, PB cells were collected for FACS analysis using anti-human-CD45 and anti-mouse-CD45 and the mice were continued to monitor for survival.

FB23-2 displays a favorable pharmacokinetic profile [1]
Next, a single dose of 3 mg/kg FB23-2 was i.p. administrated to Sprague Dawley (SD) rats for the pharmacokinetic profile (Figure 7B and Table S7). The Cmax and Tmax value of FB23-2 were 2421.3 ± 90.9 ng/ml and 0.08 hr, respectively. FB23-2 elimination half-life, T1/2 was 6.7 ± 1.3 hr, and the AUC0–24 was 2184 ± 152 hr × ng/ml. Meanwhile, FB23 was also detected, with Cmax and Tmax as 142.5 ± 26.1 and 0.4 ± 0.1 hr, respectively. The metabolic stability of FB23-2 in the SD rat liver microsome was also determined, with an estimated T1/2 of 128 min, and an intrinsic clearance of 19.7 ml/min/kg. Lastly, we measured the degree of protein binding by FB23-2. Nearly 100% FB23-2 inhibitor was bound to plasma proteins. In summary, FB23-2 displayed a favorable pharmacokinetic profile for in vivo study.

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Pharmacokinetics [1]
Inhibitor FB23-2 was formulated in DMSO at 3 mg/ml. SD rat (male, 7 – 8 weeks old, n = 3) were treated intraperitoneally with 1 ml/kg formulated compound. Blood samples were collected by retro-orbital bleeding at 5 min, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hr after the intraperitoneal administration. Blood was collected into EDTA-containing tubes and plasma was obtained by centrifugation at 2,000 g for 5 min. FB23-2 and its hydrolysis metabolite FB23 concentrations in plasma were quantitated by LC-MS/MS method. Noncompartmental analysis with Phoenix 1.4 (Pharsight, USA) was used for all analytical measurements. Area under the concentration-time curve (AUC) was calculated using trapezoidal method. AUC0−∞ = AUC0-t + Ct/ke, ke is elimination rate constant. Elimination half-life (T1/2) = 0.693/ke, mean residence time (MRT) = AUMC/AUC.

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Quantitation of FB23-2 in plasma [1]
Calibration curve concentrations ranged from 1.00 to 500 ng/ml for FB23-2 and FB23. 50 μl of rat plasma was precipitated by adding 150 μl acetonitrile immediately and vortexed to stabilize FB23-2 at each sample collection. 50 μl of study sample supernatant, 25 μl internal standard solution (probenecid and estrone-3-sulfate: 400/100 nmol/l), and 50.0 μl of 5 mM ammonium acetate solution (containing 0.1% formic acid) were added to a 1.5 ml polypropylene tube, then vortexed and centrifuged at 11,000 × g for 10 min, the supernatant was injected to LC-MS/MS. A LC-30AD liquid chromatographic system coupled to a Triple Quad 5500 mass spectrometer was used for acquiring LC-MS/MS data. Analytes were separated on an Eclipse Plus C18 column (100 mm × 4.6 mm I.D., 3.5 μm;). The mobile phases used for isocratic elution were 25% (A) 5 mM ammonium acetate-formic acid (100/0.1, v/v) and 75% (B) acetonitrile. The flow rate was 0.6 ml/min. The mass spectrometer was operated in the negative MRM mode. The parent-to-product transitions were m/z 390.2→318.0 for FB23-2, m/z 283.9→239.9 for probenecid (internal standard of FB23-2), m/z 375.2→298.2 for FB23, m/z 349.2→269.2 for estrone-3-sulfate (internal standard of FB23). The collision energy was set at −16, −30, −28, and −43 eV. The dwell time for each transition was set at 100 ms.

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Microsomal stability assay [1]
The assay was conducted as the previously reported (Di et al., 2006). Briefly, 3 μM FB23-2 was incubated with 0.5 mg/ml rat liver microsomal protein at 37 °C in the presence of 1 mM NADPH cofactor. After incubation for 0, 5, 15, 30 and 60 min, respectively, cold acetonitrile was added to terminate the reactions. The solution was centrifuged, and the supernatants were analyzed using LC–MS/MS method similar with the quantitation of FB23 and FB23-2 in AML cells. The parent-to-product transition of probenecid, internal standard of FB23-2, was m/z 283.9→239.9. The formula for calculation was T1/2 = −0.693/k, and the inherent clearance rate CLint = (0.693/in vitro T1/2) × (incubation volume/mg of microsomal protein) × (mg of microsomal protein/gram of liver) × (gram of liver/kg body weight). Each time-point group includes two repeats.

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Plasma protein binding [1]
The plasma protein binding assay was conducted with the Rapid Equilibrium Dialysis (RED) (Wang and Williams, 2013). 3 μM FB23-2 was added to rat plasma, which were vortexed well before placing 100 μl in the red chamber of the RED device. 300 μl of PBS (pH 7.4) was added to the corresponding white chamber, the base plate was covered with a gas-permeable membrane, and then incubated in a CO2 incubator set at 250 rpm and 37 °C for 4 hr. Samples collected from both chambers were analyzed with LC-MS/MS to determine the compound concentrations (C0 - initial concentration, Cf - ultrafiltrate and Cp - plasma). Percentage of protein binding (PPB) using the RED device was calculated with the formula, PPB (%) = (1- Cf /Cp) × 100%. The experiments were performed in triplicate.

FB23-2 is safe in mice [1]
To determine if FB23-2 is safe for in vivo treatment, we examined the toxic effects of multi-doses of FB23-2 in BALB/c mice over a two-week time frame. The BALB/c mice (n = 5) were treated by way of intraperitoneal injection (i.p.) daily with 10, 20, 40, and 80 mg/kg FB23-2 respectively, for 14 days. Under a dosing scheme of 20 mg/kg FB23-2, we observed no evidence of body weight loss (Figures 7A and S5A); nor was any physical damage observed on different organs (Figures 7A, S5A, and S5B). Blood was collected, and further hematology and plasma biochemistry analysis showed that no significant difference was observed in hematopoiesis among the vehicle control and the 20 mg/kg inhibitor-treated mice (Tables S5 and S6). These data indicate that FB23-2 in a dosage of 20 mg/kg is safe for exploring in vivo efficacy.

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Toxicity study [1]
6 to 8-week old BALB/c mice weighed 20 ± 2 g were used. A veterinary health check was performed to select healthy BALB/c mice. Mice were randomly grouped and treated daily with either vehicle control or FB23-2 intraperitoneally for 14 days. The mice were housed five per ventilated polysulfone cage and maintained under constant temperature (18 – 26 °C), humidity (30 – 70%) and lighting conditions (12 hr light and 12 hr dark). After 14 days, animals were euthanized. Then PB samples were collected for complete blood content analysis and plasma biochemical analysis. The vital organs (heart, kidney, lung, liver, and spleen) were collected and weighed.

[1]. Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia. Cancer Cell. 2019 Apr 15;35(4):677-691.e10.

FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.[1]

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Few inhibitors for regulation of RNA methylation have been characterized, which exists in sharp contrast to factors of DNA and histone epigenetics. Here we report that through structure-based rational designs, we have successfully developed more effective small-molecule inhibitors of FTO. The MA-derived inhibitor FB23 displays significantly improved inhibitory activity on FTO demethylation of m6A-RNA in vitro. Next, we optimized the physicochemical property of FB23, thus leading to the identification of FB23-2 with a significantly improved ability to hinder the proliferation of a panel of AML cell lines, and also inhibits primary AML LSCs in PDX mice, thus suggesting that FTO might serve as a potential molecular target in LSCs in order to inhibit leukemogenesis. The discovery of FB23-2 and its anti-proliferative effects on AML would increase the current intense interest in RNA methylation, especially with regard to the pharmacology.[1]

Importantly, we tend to show our inhibitors target FTO and impair its demethylation, and by targeting FTO our inhibitor causes a significant biological impact. We validated that the effects of FTO inhibitors on AML are linked to certain downstream targets, e.g., MYC, CEBPA, RARA, and ASB2 RNA transcripts. It remains unknown whether FB23-2 impairs FTO’s binding to target transcripts in cells, however. The target engagement of current inhibitors needs further explorations with a more depth, which could show the potential for these inhibitors to help propel the field of epitranscriptomics forward.

In summary, we provide here a proof-of-concept that small-molecule targeting of oncogenic FTO demethylase may be an effective therapeutic strategy for the treatment of AML. Our study demonstrates the feasibility of attenuated FTO demethylation for the induction of differentiation of AML cells. This effect is likely achieved through specifically regulating expression of critical genes and signalling pathways as a result of elevated m6A levels in mRNA transcripts of these genes that are induced by FTO inhibitors. As FTO-mediated demethylation has also been linked to a variety of cancer types, our findings may have a broad impact on cancer therapy by targeting epitranscriptomic RNA methylation.

C18H15CL2N3O3

392.2360

391.05

C, 55.12; H, 3.85; Cl, 18.08; N, 10.71; O, 12.24

2243736-45-8

2243736-45-8

138454779

White to light yellow solid powder

4.9

3

5

4

26

486

0

ILHNIWOZZKIBNW-UHFFFAOYSA-N

InChI=1S/C18H15Cl2N3O3/c1-9-16(10(2)26-23-9)11-7-13(19)17(14(20)8-11)21-15-6-4-3-5-12(15)18(24)22-25/h3-8,21,25H,1-2H3,(H,22,24)

2-((2,6-dichloro-4-(3,5-dimethylisoxazol-4-yl)phenyl)amino)-N-hydroxybenzamide

FB 23-2; FB232; FB23-2; FB-23-2; FB 232; FB-232

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~25 mg/mL (~63.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (25.49 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)