G9a (IC50 = 8 nM); EHMT1/GLP/KMT1D (IC50 = 2 nM); DNMT1 (IC50 = 382 nM); DNMT3A (IC50 = 85 nM); DNMT3B (IC50 = 1200 nM)
CM-272 targets G9a (EHMT2, Histone-Lysine N-Methyltransferase) and DNMT1 (DNA Modification Methylase) ; it is a reversible dual inhibitor of G9a and DNMTs (DNMT1) [1]

Treatment with CM-272 (100-1000 nM; 12-72 hours; CEMO-1, MV4-11, and OCI-Ly10 cell lines) suppresses cell growth in a time- and dose-dependent fashion [1]. Cell cycle progression is blocked by CM-272 treatment (100-1000 nM; 24-hour; CEMO-1, MV4-11, and OCI-Ly10 cell lines) [1]. In ALL, AML, and DLBCL cell lines, CM-272 (100–1000 nM; 12-72 hours; CEMO-1, MV4-11, and OCI-Ly10 cell lines) treatment causes apoptosis in a time- and dose-dependent manner[1]. 48 hours following treatment of the CEMO-1 acute lymphoblastic leukemia (ALL), MV4-11 acute myeloid leukemia (AML), and OCI-Ly10 cell line, the CM-272 diffuse large B-cell lymphoma (DLBCL) cell line showed reduced overall levels of H3K9me2 and 5mC (GI50 values of 218 nM, 269 nM, and 455 nM, respectively [1]. The processing activity of CM-272 is dependent on the early generation of type I interferon responses in tumor cells, which may cause the cells to undergo cell-autonomous immunogenic death [1].

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1. Anti-proliferative activity: CM-272 inhibits cell proliferation in hematological malignancy cell lines, with GI₅₀ values for CEMO-1 (ALL)、MV4-11 (AML) and OCI-Ly10 (DLBCL) cell lines (exact numerical GI₅₀ values not specified in the available content, only pGI₅₀ values in radar plot); treatment with CM-272 at GI₂₅, GI₅₀ and GI₇₅ concentrations for 12, 24, 48 and 72 hours dose-dependently reduces cell proliferation in CEMO-1, MV4-11 and OCI-Ly10 cell lines [1]
2. Epigenetic modulation: Treatment of CEMO-1 (ALL) cells with different doses of CM-272 for 48 hours reduces H3K9me2 (dimethylated histone H3 lysine 9) and 5mC (5-methylcytosine) levels (H3 total as loading control); treatment of MV4-11 (AML) cells with different doses of CM-272 for 48 hours also reduces H3K9me2 and 5mC levels (H3 total as loading control); treatment of OCI-Ly10 (DLBCL) cells with different doses of CM-272 for 48 hours reduces H3K9me2 levels (H3 total as loading control) [1]
3. Cell cycle and apoptosis effects: Treatment of CEMO-1, MV4-11 and OCI-Ly10 cell lines with CM-272 at GI₂₅, GI₅₀ and GI₇₅ concentrations for 24 hours alters cell cycle distribution (representative data from three experiments shown); treatment with CM-272 at the same concentrations for 12, 24, 48 and 72 hours dose-dependently and time-dependently promotes apoptosis in these three cell lines [1]
4. Immune-related effects: CM-272 treatment for 48 hours induces enrichment of interferon-stimulated genes (ISGs) in CEMO-1, MV4-11 and OCI-Ly10 cell lines (validated by GSEA); qRT-PCR confirms upregulation of ISGs in CEMO-1 and MV4-11 cells after 48-hour treatment with CM-272; qChIP-PCR analysis shows altered epigenetic regulation of ISGs in CEMO-1 (250 nM CM-272) and MV4-11 (270 nM CM-272) cells after 48-hour treatment; treatment with CM-272 at GI₂₅ and GI₅₀ concentrations for 48 hours induces calreticulin exposure (measured by FACS) and HMGB1 secretion (measured by ELISA) in CEMO-1, MV4-11 and OCI-Ly10 cells, indicating induction of immunogenic cell death [1]
5. Enzymatic selectivity: CM-272 (10 μM) shows high selectivity against a panel of 37 epigenetic enzyme targets from different families, with primary activity against G9a and DNMT1 [1]

Treatment with CM-272 (2.5 mg/kg; intravenously; daily; for 28 days; female Rag2/γc/mouse) considerably extended the CEMO-1 cell xenogeneic model [1].

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CM-272 shows anti-leukaemic effects in vivo. [1]
ALL-derived CEMO cells (10 × 106) were injected i.v. in immunodeficient Rag2−/−γc−/− mice, which were treated with 2.5 mg kg−1 of CM-272 administered daily, starting 3 days after injection and continued during 28 days. Control animals received saline solution under the same protocol. CM-272 therapy induced a statistically significant increase in overall survival (OS) in mice in comparison with control animals (median OS; 92±5.7 days versus 55±10.5 days; P=0.0009) (Fig. 4a). Global H3K9me2 and 5mC levels were measured in the extract from total liver. Tumour infiltration was analysed in liver homogenates by flow cytometry (FACS) analysis and showed an infiltration of 60–80% of human cells (hCD45+). Both marks were reduced in leukaemic cells obtained from animals after 1 week of treatment (Supplementary Fig. 10f). No significant weight loss was observed in treated animals (Supplementary Fig. 10g). We obtained similar results in a second in vivo replicate with CEMO-1 cells (Supplementary Fig. 11a). To analyse the dose-dependent efficacy of CM-272 in vivo, we repeated the same study administering 1 mg kg−1 of CM-272 (Supplementary Fig. 12). We did not observe differences in the body weight of the animals (Supplementary Fig. 12a) nor significant changes in haematological parameters (Supplementary Fig. 12b) between mice treated with 1 mg kg−1 or 2.5 mg kg−1 of CM-272 and the control group. As expected, CM-272 plasma concentration was greater in the mice group treated with 2.5 mg kg−1 of CM-272 (Supplementary Fig. 12c). However, treatment with 1 mg kg−1 of CM-272 was not able to prolong survival of the mice unlike what was observed when the 2.5 mg kg−1 of CM-272 was used (Supplementary Fig. 12d). These results demonstrate dose-dependent efficacy of CM-272 and that a dose of 2.5 mg kg−1 of CM-272 is adequate to demonstrate the positive anti-tumour efficacy. In a second xenogeneic model, 10 × 106 of AML-derived MV4-11 cells were injected i.v. in Rag2−/−γc−/− mice, and 14 days latter animals were treated with 2.5 mg kg−1 of CM-272 for 28 days. As in ALL cells, CM-272 therapy prolonged OS in mice (median OS for treated versus untreated mice, 78±12 days versus 57±0.9 days; P=0.0005) (Fig. 4b). We obtained similar results in a second in vivo replicate with MV4-11 cells (Supplementary Fig. 11b), without any sign of toxicity (Supplementary Fig. 10h). Finally, 2.5 × 106 cells from the OCI-Ly10 activated B-cell DLBCL cell line were similarly i.v. injected into Rag2−/−γc−/− mice. Treatment with CM-272 at the same dose during 8 weeks also prolonged OS of treated mice in comparison to control animals (median OS; 59±8 days versus 49±6 days; P=0.010) (Fig. 4c). We obtained similar results in a second in vivo replicate with OCI-Ly10 cells (Supplementary Fig. 11c), without any sign of toxicity (Supplementary Fig. 10i). Although the effect on lymphoma cells was statistically significant, the effect was less robust than in the case of AML and ALL cells. These results show that CM-272 exerts a potent anti-tumour activity in vivo against different types of haematological malignancies by inhibiting the methyltransferase activity of both G9a/GLP and DNMTs. In addition to the information described above, minimal promiscuity versus other SAM-dependent epigenetic enzymes (Supplementary Tables 4a and 4b), further off-target selectivity profiling against other drug targets in cancer (a panel of 97 kinases, Supplementary Tables 12–14) confirmed G9a (and GLP) and DNMTs as primary main targets for CM-272.

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1. Antitumor efficacy in xenograft models: CM-272 treatment significantly prolongs survival of mice engrafted with ALL-derived CEMO-1 cells, AML-derived MV4-11 cells and DLBCL-derived OCI-Ly10 cells (Kaplan–Meier survival curves show statistically significant differences vs. control group, P values assessed by log-rank test); CM-272 reduces infiltration levels of tumor cells in liver, spleen and bone marrow of xenograft mice [1]

G9a and DNMT1 enzyme activity assays [1]
G9a and DNMT1 activities were measured using a time-resolved fluorescence energy transfer (TR-FRET). For G9a, TR-FRET is observed when biotinylated histone monomethyl-H3K9 peptide is incubated with cryptate-labelled anti-dimethyl-histone H3K9 antibody and streptavidin XL665 after enzymatic reaction of G9a. For DNMT1, TR-FRET is observed when antibody specific to S-adenosylhomocysteine labelled with Lumi4-Tb (donor) is incubated with d2-labelled S-adenosylhomocysteine (acceptor), using the EPIgeneous methyltransferase assay. Details are provided in Supplementary Information. The radioligand binding assay against G9a, DNMT1 and GLP was performed by Reaction Biology Corporation (http://www.reactionbiology.com).

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Epigenetics selectivity panel[1]
Selectivity of CM-272 and CM-579 against 37 epigenetic enzyme targets including Bromodomain-containing enzymes (ATAD2A, ATAD2B, BAZ2B, BRD1, BRD2(BD1+BD2), BRD4(BD1+BD2), BRDT(BD1), CREBBP, TRIM24, TAF1), Histone methyltransferases (EZH1, EZH2, GLP, PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT8, SETD2, SET7/9, SUV39H1, SUV39H2 and MLL-WARD), DNA methyltransferases (DNMT3A and DNMT3B) and histone demethylase (JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD2E, JMJD3, JMJD1A, LSD1, Jarid1A, Jarid1B and Jarid1C) was performed by BPS Bioscience (http://www.bpsbioscience.com/index.ph).

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HDAC1 and HDAC6 enzyme activity assays[1]
HDAC1 and HDAC6 enzyme activities were measured with a specific fluorescence-labelled substrate, containing an acetylated lysine side chain, after its deacetylation by HDACs. Details are provided in Supplementary Information.

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Kinase selectivity profiling[1]
The selectivity profiling of CM-272 against a selected panel of 97 kinases distributed through the kinome (out of which 90 are non-mutant kinases) was performed at DiscoverRx (http://www.discoverx.com/home) using the KINOMEscan screening platform at a test concentration of 10 μM.

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Direct binding analysis[1]
MicroScale Thermophoresis (MST) was performed to quantify biomolecular interactions between CM-579 and DNMT1 (full length). The MST analysis was performed using the Monolith NT.115 instrument.

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ADME profiling[1]
The following ADME studies: CYP inhibition on five human cytochrome P450s (1A2, 2C9, 2C19, 2D6 and 3A4 at 10 μM) in human liver microsomes, plasma protein binding, kinetic solubility, Pampa permeability and human and mouse liver microsomal stability were performed by Wuxi (http://www.wuxi.com/).

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hERG blockade assay[1]
The effect of the compound on hERG potassium channels was determined using the PredictorTM hERG fluorescence polarization commercial assay kit.

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1. G9a enzymatic activity assay: Recombinant G9a protein is incubated with SAM cofactor, histone peptide substrate (PepMe1) and serial concentrations of CM-272; enzymatic activity is measured by detecting methylated histone products; competition assays are performed by varying concentrations of SAM cofactor (to assess competition with SAM binding pocket) and histone peptide (PepMe1) (to assess competition with histone binding groove); molecular docking simulation is used to predict the binding mode of CM-272 into the histone binding groove of G9a (the 7-(3-pyrrolidin-1-yl) propoxy side chain interacts with the lysine binding channel, and CM-272 does not bind to the SAM binding pocket) [1]
2. DNMT1 enzymatic activity assay: Recombinant DNMT1 protein is incubated with SAM cofactor, DNA substrate and serial concentrations of CM-272; enzymatic activity is measured by detecting methylated DNA products; competition assays are performed by varying concentrations of SAM cofactor (to assess competition with SAM binding pocket) and DNA substrate (to assess competition with DNA binding groove); molecular docking simulation predicts the binding mode of CM-272 into the DNA binding groove of DNMT1 (the 7-(3-pyrrolidin-1-yl) propoxy side chain overlays with DNA cytosine, occupies the catalytic pocket and interacts with catalytic glutamate E1269 of mouse DNMT1, and CM-272 does not bind to the SAM binding pocket) [1]
3. Epigenetic enzyme selectivity assay: CM-272 (10 μM) is incubated with 37 different epigenetic enzyme targets (covering multiple families); enzymatic activity of each target is measured using target-specific substrates and detection methods; the selectivity profile is generated by comparing relative enzymatic activity in the presence vs. absence of CM-272 [1]

Cell Proliferation Assay[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4- 11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Inhibited cell proliferation in a dose- and time-dependent manner.

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Cell Cycle Analysis [1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells) ; 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 24 hrs (hours)
Experimental Results: Blocked cell cycle progression.

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Apoptosis Analysis[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Induced apoptosis in ALL, AML and DLBCL cell lines in a dose- and time-dependent manner.

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1. Cell proliferation assay: CEMO-1 (ALL), MV4-11 (AML) and OCI-Ly10 (DLBCL) cells are seeded in 96-well plates at appropriate densities; serial concentrations of CM-272 are added, and cells are incubated for 12, 24, 48 and 72 hours; cell proliferation is quantified using a standard cell viability assay (method not specified); GI₂₅, GI₅₀ and GI₇₅ values are calculated from dose-response curves (n=3 replicates) [1]
2. Western blot for epigenetic markers: CEMO-1, MV4-11 and OCI-Ly10 cells are treated with different doses of CM-272 for 48 hours; cells are lysed, and protein extracts are separated by SDS-PAGE; proteins are transferred to membranes, which are probed with antibodies against H3K9me2, 5mC and total H3 (loading control); immunoreactive bands are detected using standard chemiluminescence methods (n=3 replicates) [1]
3. Cell cycle analysis: CEMO-1, MV4-11 and OCI-Ly10 cells are treated with CM-272 at GI₂₅, GI₅₀ and GI₇₅ concentrations for 24 hours; cells are harvested, fixed, stained with a DNA-binding fluorescent dye (not specified), and analyzed by flow cytometry to determine cell cycle distribution (representative data from three independent experiments) [1]
4. Apoptosis assay: CEMO-1, MV4-11 and OCI-Ly10 cells are treated with CM-272 at GI₂₅, GI₅₀ and GI₇₅ concentrations for 12, 24, 48 and 72 hours; cells are harvested, stained with apoptosis-detecting reagents (not specified), and analyzed by flow cytometry to quantify apoptotic cells (n=3 replicates, error bars indicate s.d.) [1]
5. GSEA and gene expression analysis: CEMO-1, MV4-11 and OCI-Ly10 cells are treated with CM-272 for 48 hours; total RNA is extracted, and gene expression profiles are generated; GSEA is performed to analyze enrichment of ISG gene sets (published by Schoggins et al. and Sistigu et al.); qRT-PCR is used to validate expression of selected ISGs (primers not specified) with RNA from CEMO-1 and MV4-11 cells (n=3 replicates, error bars indicate s.d.) [1]
6. qChIP-PCR assay: CEMO-1 (250 nM CM-272) and MV4-11 (270 nM CM-272) cells are treated for 48 hours; chromatin is cross-linked, sheared, and immunoprecipitated with antibodies against epigenetic markers (not specified); DNA fragments are purified, and qPCR is performed using primers targeting ISG promoters (n=3 replicates) [1]
7. Immunogenic cell death assay: CEMO-1, MV4-11 and OCI-Ly10 cells are treated with CM-272 at GI₂₅ and GI₅₀ concentrations for 48 hours; calreticulin exposure on the cell surface is detected by FACS using specific antibodies (n=3 replicates); HMGB1 secretion into cell supernatants is quantified by ELISA (n=3 replicates, error bars indicate s.d.) [1]

Animal/Disease Models: Female BALB/Ca-Rag2−/−γc−/− mice (6–8weeks old) with CEMO-1 cells[1]
Doses: 2.5 mg/kg
Route of Administration: intravenous (iv) injection; daily; for 28 days
Experimental Results: Induced a statistically significant increase in overall survival (OS) in mice.

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PK study of CM-272 and CM-579 in plasma samples [1]
CM-272 and CM-579 were measured in plasma samples using a Xevo-TQ MS triple quadropole mass spectrometer with an electrospray ionization (ESI) source and an Acquity UPLC. CM-272 and CM-579 solutions were prepared by dissolving the solid in saline. A drug dosage of 1 mg kg−1 or 2.5 mg kg−1 (CM-272 ) or 1 mg kg−1 (CM-579) was administered as a single intravenous injection. Blood was collected at predetermined times over 24 h post injection (0.25, 2, 4, 6, 8 and 24 h for CM-272) and (0.25, 1, 2, 4 and 8 h for CM-579). Chromatographic separation was performed by gradient elution at 0.6 ml min−1 using an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm particle size). The PK parameters were obtained by fitting the blood concentration-time data to a non-compartmental model with the WinNonlin software. Details are provided in Supplementary Information.

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In vivo experiments[1]
The human ALL CEMO-1 (control group with saline solution n=6; treated group with CM-272 , n=6), AML MV4-11 (control group with saline solution n=8; treated group with CM-272 n=8) and DLBCL OCI-Ly10 (control group with saline solution n=6; treated group with CM-272 n=6) xenograft mice models were generated by i.v. injection of cells diluted in 100 μl of saline solution in the tail vein of a 6–8-week-old female BALB/cA−Rag2−/−γc−/− mice as described in Supplementary Information. CM-272 administration was detailed in Supplementary Information. Statistical results were calculated using the statistical software medcalc.

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CM-272 toxicity assay: haematological and liver parameters[1]
After treating Rag2−/−γc−/− mice with daily i.v. 2.5 mg kg−1 of CM-272 during 4 weeks, followed by a 7 days washout period, haematological and liver parameters were measured as described in Supplementary Information.

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1. Xenograft model establishment and treatment: BALB/c female mice (strain specified as inbred BALB C) are engrafted with CEMO-1 (ALL), MV4-11 (AML) or OCI-Ly10 (DLBCL) tumor cells (engraftment method not specified); after tumor engraftment, mice are randomized into control and treatment groups; control group receives saline solution (diluent of CM-272), and treatment group receives CM-272 (dosage, administration route and frequency not specified); survival time of mice is monitored, and Kaplan–Meier survival curves are generated (log-rank test for P values); at the end of the experiment, mice are euthanized, and liver, spleen and bone marrow tissues are collected to assess tumor cell infiltration levels [1]

Prior to conducting pharmacokinetic studies, the researchers determined the maximum tolerated dose (MTD) of CM-272 to be 2.5 mg kg−1 (IV); however, for CM-579, doses higher than 1 mg kg−1 (IV) could not be administered. Pharmacokinetic studies conducted using the MTD dose (Supplementary Tables 9-11) showed a clearance of 5.7 lh−1 kg−1 for CM-579 and 0.91 lh−1 kg−1 for CM-272.

Before evaluating the in vivo efficacy of our dual inhibitors, the researchers examined the therapeutic window and pharmacokinetic (PK) parameters of both molecules. Therefore, we investigated the toxicity of CM-272 and CM-579 using the non-tumor hepatocyte cell line THLE-2 (LC50 of 1.78 and 1.30 μM, respectively) and peripheral blood mononuclear cells (PBMCs) from healthy donors (LC50 of 1.90 and 7.39 μM, respectively) (Supplementary Table 2) and compared it with the activity of in vitro antitumor cell lines (Supplementary Table 3). The researchers found that CM-272 and CM-579 had acceptable therapeutic windows (approximately 1 log unit). Prior to conducting pharmacokinetic studies, we determined the maximum tolerated dose (MTD) of CM-272 to be 2.5 mg kg−1 (IV); however, for CM-579, the researchers were unable to administer doses exceeding 1 mg kg−1 (IV). Pharmacokinetic studies using MTD doses (Supplementary Tables 9-11) showed that the clearance of CM-579 was 5.7 lh−1 kg−1 and that of CM-272 was 0.91 lh−1 kg−1. [1] Next, the researchers investigated the potential toxicity of CM-272 in Rag2−/−γc−/− mice. After daily intravenous injection of CM-272 at a dose of 2.5 mg kg−1 for 4 consecutive weeks, followed by a 7-day washout period, the mice did not lose body weight (Supplementary Fig. 10b), and no other abnormalities were observed in the disease signs or hematological parameters (Supplementary Fig. 10c). In addition, no abnormalities were observed in liver histology and liver parameters in the CM-272-treated mice compared with the control group (Supplementary Fig. 10d, e). In summary, these results indicate that CM-272 is a safe molecule for mice. [1]

[1]. Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies. Nat Commun. 2017 May 26;8:15424.

CM-272 belongs to the aminoquinoline class of compounds, with its quinoline derivatives having 5-methylfuran-2-yl, (1-methylpiperidin-4-yl)amino, methoxy, and 3-(pyrrolidone-1-yl)propoxy substitutions at positions 2, 4, 6, and 7, respectively. It is a dual G9a/DNA methyltransferase inhibitor with antitumor activity. It inhibits G9a, DNMT1, DNMT3A, DNMT3B, and GLP (IC50 values of 8 nM, 382 nM, 85 nM, 1200 nM, and 2 nM, respectively). It exhibits apoptosis-inducing, ferroptosis-inducing, antitumor, EC 2.1.1.43 (enhancer Zeste homolog 2), and EC 2.1.1.37 (DNA (cytosine-5-)methyltransferase) activities. It is an N-alkylpyrrolidine compound, belonging to the furans, aminoquinolines, aromatic ethers, piperidines, tertiary amines, secondary amines, and diethers. Epigenetics plays an undeniable role in cancer, and the reversibility of epigenetic alterations has facilitated the development of epigenetic drugs. This study designed and synthesized a highly efficient, novel, selective, and reversible chemical probe that simultaneously inhibits the activity of G9a and DNMTs methyltransferases. In vitro experiments showed that the lead compound CM-272 inhibited the proliferation of hematologic malignancies (acute myeloid leukemia-AML, acute lymphoblastic leukemia-ALL, and diffuse large B-cell lymphoma-DLBCL) cells and promoted apoptosis, inducing interferon-stimulated gene expression and immunogenic cell death. CM-272 significantly prolonged the survival of xenograft models of AML, ALL, and DLBCL. Our results represent the first discovery of a dual G9a/DNMTs inhibitor and demonstrate that this series of compounds holds promise as an effective tool for addressing unmet needs in the treatment of hematologic malignancies. [1] Transcriptome analysis after CM-272 treatment consistently showed that AML, ALL, and DLBCL cells all exhibited tumor type I interferon responses, accompanied by ISG expression and induction of immunogenic cell death (ICD), suggesting a common anti-tumor mechanism. Although immunogenic cell death (ICD) has not been described as the mechanism of action of epigenetic drugs, these results may at least be partially predictable, as recent studies have shown that the expression of interferon-stimulating genes (ISG) is epigenetically regulated by H3K9me2 (reference 30), which supports the role of G9a inhibition in activating type I interferon responses and ICD. Therefore, we speculate that the use of immunodeficient mice that cannot generate anti-tumor immune responses may underestimate the efficacy of CM-272 against tumor cells, and therefore, immune-normal models need to be evaluated to explore the full potential therapeutic effects of our compound. Based on recent studies showing that type I interferon response helps improve the efficacy of chemotherapy drugs³², the combination of CM-272 with such drugs and/or immunomodulators (e.g., checkpoint inhibitors) may be an attractive treatment strategy. In summary, CM-272 is a potent, novel, first-in-class dual reversible inhibitor that inhibits G9a (GLP) and DNMTs, which prolongs survival in in vivo models of hematologic malignancies at least in part by inducing immunogenic cell death. These compounds represent a new approach to safely and effectively targeting cancer, paving the way for the treatment of a variety of poorly prognostic human tumors. [1]

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1. CM-272 is a first-in-class reversible dual small molecule inhibitor that inhibits G9a and DNMTs (DNMT1), designed and synthesized as an epigenetic drug candidate for hematologic malignancies. [1]
2. CM-272 binds non-competitively to SAM cofactors (does not bind to the SAM binding pocket), but competitively to histone peptide (G9a) and DNA substrate (DNMT1)[1]
3. CM-272 exerts antitumor effects in hematologic malignancies (AML, ALL, DLBCL) through multiple mechanisms: inhibiting cell proliferation, inducing apoptosis, epigenetically regulating gene expression, inducing interferon-stimulated gene expression, and immunogenic cell death[1]
4. CM-272 shows good therapeutic potential in addressing unmet medical needs in hematologic malignancies, as demonstrated in xenograft models of AML, ALL, and DLBCL, where it can significantly prolong survival[1]

C28H38N4O3

478.64

478.294

C, 70.26; H, 8.00; N, 11.71; O, 10.03

1846570-31-7

1846570-31-7;1846570-32-8 (TFA);

118607432

Light brown to brown solid powder

1.2±0.1 g/cm3

631.9±55.0 °C at 760 mmHg

336.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.601

5.41

1

7

9

35

640

0

RLQLKZTYUYIWDB-UHFFFAOYSA-N

InChI=1S/C28H38N4O3/c1-20-7-8-26(35-20)25-18-23(29-21-9-14-31(2)15-10-21)22-17-27(33-3)28(19-24(22)30-25)34-16-6-13-32-11-4-5-12-32/h7-8,17-19,21H,4-6,9-16H2,1-3H3,(H,29,30)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)