WO2021237609A1

WO2021237609A1 - Novel use of oridonin or oridonin derivative

Info

Publication number: WO2021237609A1
Application number: PCT/CN2020/093017
Authority: WO
Inventors: Jianwei Wang; Min Liao
Original assignee: Tsinghua University
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2021-12-02

Abstract

A method of treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof is provided and the method comprises administrating a therapeutically effective amount of oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof to the subject.

Description

Novel Use of Oridonin or Oridonin Derivative

FIELD

The present invention relates to the medical field, more particularly to the novel use of oridonin or oridonin derivative in treating, preventing or lessening a DNMT3A R882H mutation related disease and to a method for screening drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.

BACKGROUND

Hematopoietic stem cell (HSC) generates all blood cells throughout lifespan and certain mutations drive clonal hematopoiesis with aging, which is a risk factor for many diseases, especially for blood malignancies. Aging-elevated DNMT3A R882H-driven clonal hematopoiesis is a risk factor for myeloid malignancies remission and overall survival. Even though some studies were performed to investigate this phenomenon, the exact mechanism is still under debate.

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SUMMARY

The following just summarizes some aspects of the invention, but are not limited to these. These aspects and other parts will be described more completely later. All references of this specification are incorporated herein by reference in their entirety. Where there are differences between disclosure of the present specification and cited references, the disclosure of the present specification controls.

The present disclosure is completed based on the following founding: disclosing the molecular mechanism behind R882H-driven clonal hematopoiesis is a crucial step to uncover the internal link between aging and myeloid malignancies. Moreover, finding small molecule (s) that can inhibit DNMT3A R882H-carrying hematopoietic cells will be of great clinical significance. The present inventors surprisingly observed that Dnmt3a R878H HSCs (human allele: DNMT3A R882H, hereafter named “R878H HSCs” for mouse and “R882H HSCs” for human) displayed enhanced reconstitution capacity compared to wild-type HSCs by preventing the activation of RIPK1-RIPK3-MLKL mediated necroptosis induced by aging inflammatory milieu. The inventors then performed small molecule screen and found that oridonin selectively inhibits hematopoietic and leukemic cells carrying Dnmt3a R878H/DNMT3A R882H in vitro and in vivo at low micromolar concentration (IC ₅₀ = 2.1μM) by activating necroptosis. Briefly, the inventors elucidated the molecular mechanism driving DNMT3A R882H-based clonal hematopoiesis and identified oridonin selectively inhibiting hematopoietic and leukemic cells carrying Dnmt3a R878/DNMT3A R882 mutation, which raises clinical value for treating DNMT3A R882H-driven clonal hematopoiesis and myeloid malignancies with aging.

Then in one aspect of present disclosure, a method of treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof is provided, and in some embodiments of present disclosure, the method comprises administrating a therapeutically effective amount of oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof to the subject.

In another aspect, provided is a pharmaceutical composition for treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof, comprising: oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof.

In another aspect, provided is use of oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof in the manufacture of a medicament for treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof.

In another aspect, provided is a method for screening drug for treating, preventing or lessening a DNMT3A R882H mutation related disease, comprising: mixing a mutant cell carrying DNMT3A R882H mutation and a wild type cell at predetermined number ratio of the wild type cell to the mutant cell to obtain a cell mixture; contacting the cell mixture with a candidate compound; and determining a number ratio after contacting of the wild type cell to the mutant cell to obtain, wherein the number ratio after contacting higher than the predetermined number ratio is an indication that the candidate compound may be a drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.

The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These aspects and other aspects and embodiments are described more fully below.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows (A) Schematic illustration for the design of the DNMT3AR882H/R882H allele to produce a K562 cell line with DNMT3A R882H mutation (K562-R882H) . (B) An EGFP and tdTomato reporter gene were, respectively, inserted into adeno-associated virus integration site 1 (AAVS1) allele of K562-WT and K562-R882H cell line to generate K562-WT-EGFP (WT) and K562-R882H-tdTomato (R882H) cell lines. (C) Shows the experimental procedure designed for high-throughput screening to obtain small molecules selectively inhibit cell lines with DNMT3A R882 mutations.

Fig. 2 shows preliminary active compound from the first round screening is exhibited in the histogram (The y-axis is a ratio of WT to R882H cells, NT: non-treatment control) .

Fig. 3 shows six active small molecules were obtained after additional validation.

Fig. 4 shows the inhibition rate of oridonin by a 48-hour treatment at different concentrations, all data are presented as mean ± SD and compared to NT; *p < 0.05, **p < 0.01, and ***p < .001.

Fig. 5 shows that Necroptosis-associated genes (RIPK1, RIPK3 and MLKL) were determined by qRT-PCR in OCL-AML2 or OCL-AML3 cells treated with oridonin (data are presented as mean ± SD with two biological replicates) .

Fig. 6 shows that oridonin selectively inhibits Dnmt3a R878H HSC in vivo. Fig. 6A shows the schematic diagram of the experiment designed for oridonin treatment in vivo. WT or R878H BM cells (CD45.2) were mixed with WT competitor BM cells (CD45.1) at a ratio of 1: 1 (3×10 ⁵ : 3×10 ⁵ ) and transplanted into lethally irradiated recipient mice (CD45.1/2) . Five weeks ost-transplant, the chimeric mice were exposed to continuous treatment with oridonin (10 mg/kg injected i.p. every day for 15 days) or vehicle accordingly. Chimerism in PB was analyzed monthly. Fig. 6B to 6F show that the frequencies of donor derived cells in the PB of WT ±oridonin (B) or R878H ± oridonin (C) recipients at indicated time point and frequencies of B cells, T cells, myeloid within donor-derived PB at 20 weeks post-transplantation were evaluated (D) . Gating strategy with representative flow cytometry plots and the frequency of donor-derived (CD45.2+) HSC (Lineage–Sca1+ c-Kit+ CD34–CD150+ ) in WT ± oridonin (E) or R878H ± oridonin (F) recipients are shown in the histograms (n = 3–8 mice/group from 3 independent experiments) .

Fig. 6G shows OCL-AML2 or OCL-AML3 cells were subcutaneously inoculated into the right flank of nude mice to generate mouse xenograft model. 20 mg/kg oridonin or related volume of vehicle were intraperitoneal injection into the left flank of tumor inoculated mice when tumors were measurable. Fig. 6H shows the volume of tumors was recorded in each groups at the indicated time points during treatment (H) . (n = 4-6 animals per cohort from two independent experiments) .

DESCRIPTION OF THE DISCLOSURE

It should be noted that the expression of “DNMT3A R882H” means that the amino acid No. 882 in wild type DNMT3A is Arginine (Arg or R) , and the Arginine in the mutant may be replaced with Histidine (His or H) . And the person skilled in the art may understand that the numbering of the amino acid is based on the wild type human allel of DNMT3A with the sequence as follows (the underline R is R882) :

Then the person skilled in the art may understand that DNMT3A may have many variants in some organisms other than human or some cells, the person skilled in the art may obtain the corresponding amino acid site of R882 by alignment, for example, the for mouse, the real numbering is “R878H” .

As described above, it was surprisingly found by the inventors that oridonin selectively inhibits hematopoietic and leukemic cells carrying Dnmt3a R878H/DNMT3A R882H in vitro and in vivo at low micromolar concentration (IC ₅₀ = 2.1μM) by activating necroptosis.

In details, it was revealed that aging-induced TNFα impairs HSCs through ERK-ETS1-IL27Ra signaling. In this study, the inventors found that aged microenvironment and LPS challenge increased the repopulation capacity of R878H cells, suggesting that inflammatory factors facilitates Dnmt3a R878H-driven clonal hematopoiesis. Another study exhibited that chronic inflammation is able to accelerate clonal hematopoiesis driven by Tet2 12 deficiency. Mechanically, the inventors found that RIPK1-RIPK3-MLK-mediated necroptosis pathway is restrained in mouse HSPCs carrying DNMT3A mR878H/hR882H in response to aging-elevated inflammatory insult and this results in age-related clonal hematopoiesis. In this study, the inventors also identified that oridonin selectively inhibits mR878H/hR882H cells by activating necroptosis. A previous study revealed that DOT1L inhibitor exhibited selective inhibition effect on AML cells carrying DNMT3A R882 mutation. The inventors measured the IC ₅₀ of DOT1L inhibitors SGC0946 and EPZ5676 using experimental conditions performed in this study, and the results show that the IC ₅₀ of SGC0946 for OCL-AML2 and OCL-AML3 cells are 25.1 μM and 22.0 μM separately, the IC ₅₀ of EPZ5676 for OCL-AML2 and OCL-AML3 cells are 101 μM and 79.2 μM . While the IC ₅₀ of oridonin for OCL-AML2 and OCL-AML3 cells are 4.6 μM and 2.1 μM, which is significantly lower than DOT1L inhibitor and the selectivity is much better (Figures 5I) . AML patients with DNMT3A R882 mutation have poor outcome when treated with anthracycline. It will be interesting for us to explore whether oridonin can improve the overall survival of AML patients carrying DNMT3A R882 mutation. Oridonin is mainly extracted from a Chinese herbal medicine named Donglingcao which has been used as traditional medicine to treat inflammation and cancer for hundreds of years, and its analog called HAO472 has entered into Phase I human clinical trial (CTR20150246; www. chinadrugtrails. org. cn) in China for targeting AML1-ETO (Hu et al., 2019) . All these data suggest oridonin may be a promising drug candidate or a good lead compound for treating AML with DNMT3A R882 mutation.

In some examples, the DNMT3A R882H mutation related disease involves clonal hematopoiesis. In some examples, the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease, inflammatory disease, and diabete.

In some examples, the oridonin derivative comprises a 14-O-Acyl derivative of oridonin . In some examples, the 14-O-Acyl derivative of oridonin comprises 14-O-Dodecanoyl derivative of oridonin, 14-O-tetradecanoyl derivative of oridonin, and 14-O-hexandecanoyl derivative of oridonin.

The structure and carbon atom numbering of oridonin is shown below:

In some examples, the DNMT3A R882H mutation related disease involves clonal hematopoiesis.

In some examples, wherein the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease.

In some examples, the oridonin derivative comprises a 14-O-Acyl derivative of oridonin .

In some examples, the 14-O-Acyl derivative of oridonin comprises 14-O-Dodecanoyl derivative of oridonin, 14-O-tetradecanoyl derivative of oridonin, and 14-O-hexandecanoyl derivative of oridonin.

The compound of the invention may be typically formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols, solutions, and dry powders; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

In some examples, the compound (oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof ) may be formulatedd into solid dispersion, injection, for examples nanosuspension, liposomes.

It will also be appreciated that certain of the compounds of present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative or a prodrug thereof. According to the present invention, a pharmaceutically acceptable derivative or a prodrug includes, but is not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need thereof is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof.

In one embodiment, the compounds disclosed herein can be prepared to oral dosage forms. In one embodiment, the compounds disclosed herein can be prepared to inhalation dosage forms. In one embodiment, the compounds disclosed herein can be prepared to dosage forms of nasal administration. In one embodiment, the compounds disclosed herein can be prepared to transdermal dosage forms. In one embodiment, the compounds disclosed herein can be prepared to dosage forms of topical administration.

The pharmaceutical compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach. Enteric-coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.

The pharmaceutical compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di (lower alkyl) acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxy groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the patient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6) , 318 (1986) .

The pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

The pharmaceutical compositions provided herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

The pharmaceutical compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

In some examples, the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease, inflammatory disease, and diabete.

In some examples, the oridonin derivative comprises a 14-O-Acyl derivative of oridonin.

In another aspect, provided is a method for screening drug for treating, preventing or lessening a DNMT3A R882H mutation related disease, comprising:

mixing a mutant cell carrying DNMT3A R882H mutation and a wild type cell at predetermined number ratio of the wild type cell to the mutant cell to obtain a cell mixture;

contacting the cell mixture with a candidate compound; and

determining a number ratio after contacting of the wild type cell to the mutant cell to obtain, wherein the number ratio after contacting higher than the predetermined number ratio is an indication that the candidate compound may be a drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.

In some examples, the mutant cell further carries a first reporter gene, the wild type cell further carries a second reporter gene, and the first reporter gene is different from the second reporter gene.

In some examples, one of the first reporter gene and the second reporter gene is EGFP, and the another one of the first reporter gene and the second reporter gene is red fluorescent protein.

In some examples, the number ratio is determined by signals from the first reporter gene and the second reporter gene.

In some examples, the number ratio after contacting being at least 50%higher than the predetermined number ratio is an indication that the candidate compound may be a drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.

Examples

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

Dnmt3aflox-R878H mice were obtained from Cyagen Biosciences Inc. (Guangzhou, China) . Targeting vectors were kindly provided by Professor Saijuan Chen from Shanghai Jiao Tong University (Figure 1A; Dai et al., 2017) . The Dnmt3a R878H knock-in construct was generated via the recombineering method. Briefly, approximately 17.5 kb of genomic DNA surrounding Dnmt3a exon 23 was subcloned into targeting vector (Figure 1A) . The linearized vector was subsequently delivered to ES cells (C57BL/6) via electroporation, followed by drug selection, PCR screening, and Southern Blot confirmation. After gaining 99 drug-resistant clones, 4 potentially targeted clones were confirmed, 4 of which were expanded for Southern Blotting. After confirming correctly targeted ES clones, some clones were selected for blastocyst microinjection, followed by chimera production. F0 generation were confirmed as germline-transmitted via crossbreeding with wile-type. Dnmt3aflox-R878H and Dnmt3a+/+littermate mice were genotyped by PCR with primers Dnmt3a-FP (5’-GCCAGTATAGATGCCTGTGAGGT-3’) and Dnmt3a-RP (5’-TGCCTCTTGGATGTGCTCTACAG -3’) using the following parameters: 94℃ for 3 min, followed by 35 cycles of 94℃ for 20 sec, 60℃ for 20 sec, 72℃ for 20 sec and 72℃ for 5 min to final extension. PCR product for WT and Mutant band is 260 bp and 316 bp, respectively. Expression of the R878H mutation (that results in an arginine to histidine substitution at amino acid position 878, Dnmt3a R878H) was confirmed by cDNA sequencing using the following primer: 5’-GGAGTGTGAATCTCAAAGCTGGGAT-3’.

When Dnmt3aflox-R878H mice exposed to Cre recombinase leads to inversion of the mutant exon inserted and then with an excision reaction to remove the WT exon and one loxP site, and another loxP site, fixing the inversion in the right place (Figure 1A) . To achieve hematopoietic-specifically mutant mice, Dnmt3aflox-R878H mice crossed to Vav-iCre mice. In all experiments, Vav-iCre knockin mice were heterozygous for both the Dnmt3aR878H allele and the Vav-iCre allele (Dnmt3aR878H/WTVav-iCre+ or Dnmt3aR878H/WT) . For xenograft AML mouse models, four-week-old female BALB/c nude mice were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd and all mice were allowed to acclimate for 1 week in the Laboratory Animal Research Center at Tsinghua University before tumor cells inoculated. All mice were housed in specific-pathogen-free, AAALAC-accredited animal care facilities at the Laboratory Animal Research Center, Tsinghua University and all procedures were approved by Institutional Animal Care and Use Committee of Tsinghua University.

Cell Lines

K562 cells and OCL-AML2 cells were purchased from DSMZ. OCL-AML3 cells were kindly gifted by Professor Min Xiao from Huazhong University of Science and Technology. K562-R882H cells (K562 cell line with DNMT3A R882H mutation) were generated by replacing the 882th amino acid (Arginine) in DNMT3A gene with Histidine through CRISPR-Cas9 gene editing system. A gene fragment editing a red fluorescent protein (tdTomato) was inserted into AAVS1 gene site of K562-R882H cells to obtain K562-R882H-tdTomato cells and K562-WT-EGFP cells were developed by inserting a gene fragment editing an enhanced green fluorescent protein (EGFP) into AAVS1 gene site of WT K562 cells. DNMT3A R882 mutations was confirmed in every batch of cells at least once by Sanger sequencing. All cell lines were grown in RPMI-1640 medium supplemented with 10–15%fetal bovine serum and penicillin/streptomycin at 37℃ with 5%CO ₂.

METHOD DETAILS

Flow Cytometric Analysis and Cell Sorting

2 Cells were suspended in HBSS+ buffer (D-Hank's buffer containing 2%fetal bovine serum and 1%HEPES) and then stained with fluorochrome-labeled antibodies. Flow cytometric analysis data were collected with a BD LSRFortessa SORP flow cytometer (BD Biosciences) and analyzed using FlowJo ^TM Software (Becton, Dickinson and Company) . Cell sorting was performed by BD Influx (BD Biosciences) and target fraction was sorted into HBSS+ buffer. Non-lysed bone marrow (BM) cells were applied for analysis of HSPC (antibodies containing Lin-APC/Cy7 cocktail, c-Kit APC, Sca-1 PE/Cy7, CD150 PE, CD34 AF700, CD127 BV421, CD16/32 FITC and CD135 PE-CF594) and lineage (antibodies against Mac1, Gr1, B220, CD3) . Chimerism analysis in mature cells from peripheral blood (PB) were lysed by ACK buffer (NH ₄Cl 150 mM, KHCO ₃ 10 mM, Na ₂EDTA 0.1 mM, adjust the pH to 7.2–7.4) and subsequently subjected to flow cytometer after staining with fluorochrome-conjugated antibodies (antibodies containing Mac1, B220, CD3, CD45.1 and CD45.2) .

Competitive Bone Marrow Transplantation

Lethally irradiated (10 Gy) WT CD45.1/2 recipient mice (F1 generated by mating CD45.1 with CD45.2 mice, C57BL/6J mice) were co-injected intravenously (i.v. ) with 3×10 ⁵ pooled WT or Dnmt3a R878H (CD45.2, C57BL/6J mice) whole BM cells and 3×10 ⁵ WT (CD45.1, C57BL/6J mice) whole BM competitor cells for the competitive bone marrow transplantation assay and oridonin treatment assay. The repopulating capacity of the test bone marrow populations was analyzed by FACS in the peripheral blood of recipient monthly post-transplantation. For the young and old recipient transplantation assay, 1×10 ⁵ either WT or R878H (CD45.2, C57BL/6J mice) total BM cells were transplanted into 2 months and 15 months lethally irradiated (10 Gy) WT recipients (CD45.1, C57BL/6J mice) together with 5×10 ⁵ WT competitor cells (CD45.1/2, C57BL/6J mice) and the chimera in peripheral blood was evaluated every month until the 4th month. In the LPS-treated assay, a mixture of 5×10 ⁵ whole BM cells from WT or Dnmt3a R878H (CD45.2, C57BL/6J mice) mice for treatment with PBS or 3 LPS combined with 5×10 ⁵ WT (CD45.1, C57BL/6J mice) whole BM competitor cells were transplanted into lethally irradiated (10 Gy) WT recipients (CD45.1/2, C57BL/6J mice) and the chimera were evaluated monthly.

Small Molecules Screening

An FDA-approved Drug Library containing 2572 drugs purchased from Selleck Chemicals and two natural products libraries (Yaodu with 936 compounds and TargetMol with 409 compounds) were employed for DNMT3A R882H selectively inhibitors screening. All chemicals in 384-well plates were dissolved in DMSO and stored at -20℃ as 10 mM stock solutions until use. 1×10 ⁴ (K562-WT-EGFP cells: K562-R882H-tdTomato cells = 1: 1) cells were seeded into 96-well plates before different chemicals were added into each well (final concentration to 5μM) by an

550 Liquid Handler (Labcyte Inc. ) and equal volume of DMSO were used as control. Then, cell suspension was subjected to a BD LSRFortessa SORP with a BD ^TM High Throughput Sampler (HTS) for analysis after 72 h. Frequency of GFP+ and RFP+ cells were represent DNMT3A WT and R882H cells respectively. Preliminary candidates were identified with a criteria of inhibition ratio no less than 1.5, and the following formula was used to calculate inhibition ratio: Inhibition ratio = (WT/R882H) treatment / (WT/R882H) control. Subsequently, preliminary candidates were identified in triplicate to exclude false positive candidates and obtained 6 compounds. OCL-AML2 and OCL-AML3 cells were applied to further evaluate the selectivity of 6 candidates by CCK-8 assay described above and identified oridonin as a potential DNMT3A R882 mutations inhibitor from both Selleck and TargetMol library.

In vivo Treatments

For clonal hematopoiesis mouse model, 1×10 ⁵ total BM cells either from WT or R878H mice (CD45.2, C57BL/6J mice) were transplanted into lethally irradiated recipients (CD45.1/2, C57BL/6J mice) mixed with 5×10 ⁵ competitor cells (CD45.1, C57BL/6J 4 mice) . One month later, LPS (0.8 mg/kg) was administrated to the recipients intraperitoneally every two days for 15 doses, and the chimera of peripheral blood was evaluated every month until the 5th month. In the acute inflammatory insult experiment, WT or R878H mice were intraperitoneally injected with LPS (0.8 mg/kg) or PBS for a single dose and mature cells from peripheral blood together with HSPC from BM of treated mice were analyzed 48 h later. While 5 doses (every other day) of LPS (0.8 mg/kg) or PBS injection were administrated to WT or R878H mice intraperitoneally for the chronic inflammatory challenge experiment, and the hematologic fraction was evaluated at the 10th day. For the Dnmt3a R878H-driven clonal hematopoiesis rescue assay, chimeric mice generated as in Figure 6A were injected intraperitoneally with oridonin (10 mg/kg) or vehicle (2%DMSO + 20%PEG300 + 78%PBS) once a day for 15 days, and donor-derived peripheral blood was evaluated every four weeks before analyzing donor-derived HSPCs until the 20th weeks.

Cell Viability and Proliferation Assays

Cell viability of cells treated with small molecules was measured by CCK-8 assay using the Cell Counting Kit-8 (Dojindo, Cat #CK04-11) . Generally, OCL-AML2 or OCL-AML3 cells were seeded in 96-well plates at 1×10 ⁵/ml density with 100 μl medium and exposed to various concentrations of chemicals in triplicate for 72 h. Then, 10 μl of CCK-8 reagent was added to each wells by incubating at 37 ℃ for 4 to 6 h and the absorbance was measured by an Envision Multilabel Reader (PerkinElmer) spectrophotometry until with the maximum absorbance reached value of about 1 optical density (OD) at 450 nm. Values between the treatment group and vehicle controls were normalized, and measured data were submitted to GraphPad Prism 6 software to calculate the concentration of chemicals at which there was half cell death (IC ₅₀) .

In vivo Tumor Xenograft

Four-week-old female BALB/c nude mice bought from Beijing Vital River Laboratory Animal Technology Co., Ltd were subcutaneous inoculated with 4 × 10 ⁷ OCL-AML2 or OCL-AML3 cells into the right flank. When tumor was measurable, nude mice were treated with oridonin (20 mg/kg) or vehicle (2%DMSO + 20%PEG300 + 78%PBS) intraperitoneally once a day for 14 consecutive days. Tumor volumes and body weights were measured every day. Length and width of tumor were calipered and the following formula was utilized to calculate tumor volumes: tumor volumes = 4/3 × (width/2) ² × (length/2) . When tumors reached 2 cm or the mice became moribund, animals were killed and tumors were isolated for analysis. All procedures were performed in the Laboratory Animal Research Center and were approved by Institutional Animal Care and Use Committee of Tsinghua University.

Hematological Cell Counts

Hematologic parameters in PB after tail bleeding were analyzed by Auto Hematology Analyzer BC-5000 (MINDRAY) . BM cells were harvested from one femur and kept in HBSS+ buffer on ice before evaluating by Vi-CELL Cell Counter (Beckman) .

Western Blotting

5 × 10 ⁶ c-Kit+ BM cells purified from magnetic beads enrichment (Miltenyi Biotec) after staining with c-Kit-APC antibody or 1 × 10 ⁶ AML cell lines obtained after related treatment lysed with 150 μL NETN buffer (100 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM PMSF and 0.5%Nonidet P-40) on ice for 30 min and subsequently centrifuged at 14000 g for 5 min. The supernatant was combined with 2×loading buffer and then lysed by sonication before boiling for 6 min. Samples were resolved on 10%SDS-PAGE followed by transferring onto a PVDF membrane (BioRad) , and then membrane was blocked by 5%skim milk in TBST buffer before incubating with indicated primary antibodies.

Quantitative Real-time PCR

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions from 5 × 10 ⁴ donor-derived CD45.2 LSK cells sorted from BM of the fourth month recipient after whole BM transplantation or 2 × 10 ⁴ AML cell lines collected after treating with chemicals. Concentration of RNA was normalized and cDNA was synthesized by PrimeScript RT reagent Kit (Takara, Cat #RR047A) . Acquired cDNA was analyzed by PowerUp ^TM SYBR ^TM Green mix (Applied Biosystems, Cat #A25780) with indicated primers on a QuantStudio-3 Real-time PCR System (Applied Biosystems) .

Statistical Analysis

All data are shown as mean ± SD. Two-tailed unpaired Student’s t-test was used for statistical significance analysis after testing for normal distribution and data were

Example 1 Dnmt3a R878H HSCs Display Enhanced Self-renewal Capacity

To investigate the function of Dnmt3a R878H HSCs, the inventors generated Dnmt3afl-R878H/+ mice and confirmed the mutation by sequencing analysis of the genomic site, and then crossed them with Vav-iCre mice to generate Dnmt3a R878H heterozygous mice (Dnmt3aR878H/+) , wherein Dnmt3a R878H occurs mainly in hematopoietic cells (hereafter named R878H mice or R878H HSCs) . The inventors then analyzed R878H mice at the age of 2-4 months and observed that the cell counts in peripheral blood (PB) and the frequency of lineage cells (B cells, T cells and myeloid) in PB and bone marrow (BM) of R878H mice display no difference compared to wild-type (WT) mice. The frequency and absolute number of HSCs in R878H mice is significantly larger than WT mice, but the frequency and absolute number of CMP (common myeloid progenitor) , GMP (granulocytemacrophage progenitors) , CLP (common lymphoid progenitors) , MEP (megakaryocyte-erythroid progenitors) , ST-HSC (short-term HSC) and MPP (multipotent progenitor cell) of R878H mice holds static compared to WT mice.

To further evaluate the function of R878H HSCs, the inventors performed competitive transplantation assay by transplanting 3×10 ⁵ freshly isolated total bone marrow cells from either R878H mice or WT mice into lethally irradiated recipients together with 3×10 ⁵ competitor cells, and the chimera was investigated every month until the 4th month. The result shows that R878H HSCs exhibited significant growth advantage compared to age-matched WT HSCs and the distribution of mature hematopoietic lineages exhibits no difference between R878H and WT cells. These data indicate that R878H HSCs display enhanced self-renewal capacity at both steady and stressed condition.

Example 2 Aged Bone Marrow Microenvironment Promotes Dnmt3a R878H-based Clonal Hematopoiesis

Given that the self-renewal capacity of R878H HSCs increases, and that DNMT3A R882H promotes clonal hematopoiesis in the elderly, and that aged bone marrow microenvironment displays elevated inflammatory stress, the inventors attempt to speculate that aged microenvironment might select R878H HSCs. To test this hypothesis, the inventors transplanted 1×10 ⁵ either R878H or WT total bone marrow cells into lethally irradiated young (2-month) and aged (15-month) recipients together with 5×10 ⁵ competitor cells and the chimera in peripheral blood was evaluated every month until the 4 ^th month . The result shows that the reconstitution capacity of R878H cells in aged recipients is significantly higher than that in young recipients (60.6%± 7.1%vs 36.0%± 17.8%) , while WT cells exhibit no difference between young and aged recipients (8.0%± 5.6%vs 4.0%± 2.5%) , indicating that aged bone marrow milieu fosters R878H cells. Moreover, the inventors observed significantly increased differentiation potential towards myeloid lineage of WT and R878H cells in aged recipients , which is consistent with a conclusion that aged bone marrow environment promotes the differentiation bias to myeloid lineage, and decreased potential towards T lineage of WT cells . This result indicates that inflammatory environment resulted in the enhanced self-renewal capacity and differentiation skewing of R878H HSCs. To further test whether inflammatory stress propagates R878H cells and lead to differentiation skewing, the inventors applied continuous LPS challenge to imitate chronic inflammation. The inventors transplanted freshly isolated 1×10 ⁵ total bone marrow cells from either WT or R878H mice into lethally irradiated young recipients together with 5×10 ⁵ competitor cells. One month later, the inventors injected the recipients with LPS (0.8 mg/kg) every two days intraperitoneally for one month, and the chimera of peripheral blood was evaluated every month until the 5 ^th month. The result shows that R878H cells significantly outcompeted the competitor cells upon LPS challenge compared to PBS-treated ones (70.0%±29.0%vs. 36.7%±26.2%) and the difference mainly stemmed from myeloid lineage. Similarly, the mature hematopoietic lineage distribution revealed significant differentiation skewing towards myeloid lineage of R878H cells in response to LPS challenge and this trend primarily originated from the significant increase of myeloid progenitors (CMP, GMP and MEP) . However, no difference was observed in WT group in response to LPS challenge. Moreover, HSC analysis shows that LPS-induced inflammation propagates R878H HSCs, but has no effect on WT HSCs. The above data suggest that R878H HSCs resist inflammatory stress and exhibit differentiation bias towards myeloid lineage.

Example 3: Dnmt3a R878H Prevents the Exhaustion of HSCs Induced by Chronic Inflammation

The above experimental results were generated by using bone marrow transplantation assay. In order to further verify our hypothesis, the inventors challenged WT and R878H mice by LPS intraperitoneally (0.8 mg/kg) and analyzed these mice 48 hours later. The result shows that the frequency of HSCs, ST-HSCs and MPP in both mutant and WT mice increase significantly upon inflammatory insult, While, the fold change of HSCs (7.2 vs1.4) , ST-HSC (10.4 vs. 5.0) and MPP (4.4 vs. 2.5) is significantly higher in WT mice compared to R878H mice in response to LPS challenge. The frequency of CMP, MEP and CLP of both WT and mutant mice decrease significantly upon the single-dose LPS challenge compared to PBS treated group respectively, while GMP holds static. Meanwhile, lineage cells (B cells, T cells and myeloid) in both BM and PB of WT and R878H mice showed similar trend upon LPS challenge. These data indicate that R878H mutation blunts the response of hematopoietic stem and progenitor cells, but not downstream progenitor and mature differentiated cells, upon acute inflammatory stress.

Repeated activation of HSCs out of their dormant state may provoke the attrition of normal HSCs , the inventors then speculate that R878H HSCs might withstand repeated activation. To test this hypothesis, the inventors injected LPS (0.8 mg/kg) every other day to WT and R878H mice for 5 times. At the 10th day, all mice were analyzed and the result shows that the frequency of HSC, ST-HSC and MPP in LPS-treated WT mice declined significantly compared to PBS-treated ones. While the frequency of HSCs increased significantly, and the frequency of ST-HSCs and MPPs holds static in LPS-treated R878H mice compared to PBS-treated ones. Further analysis revealed that WT HSCs, ST-HSCs and MPPs decreased by 50%, 30%and 40%upon consecutive LPS challenge, but R878H HSCs increased by 40%and the difference between WT and R878H mice is significant. Meanwhile, consecutive LPS stimulation in R878H or WT mice resulted in a decrease of CMP, MEP and CLP, but not GMP relative to PBS-treated group. However, the fold change of CMP, MEP and CLP displays no difference between WT and R878H mice. Moreover, no differences were observed for mature hematopoietic lineages in the bone marrow of R878H mice compared to WT mice after continuous LPS stimulation even though B cells, T cells and myeloid exhibited significant change in both WT and R878H mice. While T cells, but not B and myeloid cells, in the peripheral blood of R878H mice revealed compromised response compared to WT mice. These data indicate that R878H mutation prevent the attrition of HSC, ST-HSC and MPP compartments induced by chronic inflammation.

Example 4 Dnmt3a R878H Prevents the Activation of Necroptosis in HSCs upon Inflammatory Stress

Given our data shows that R878H HSCs withstand acute and chronic inflammatory stress, and necroptosis is a direct target in response to inflammatory insult, the inventors then set out to investigate the influence of R878H on necroptosis in HSCs. the inventors transplanted 5×10 ⁵ bone marrow cells freshly isolated from either R878H mice or wild-type mice into lethally irradiated recipients together with 5×10 ⁵ competitor cells and the inventors isolated donor-derived 5×104 LSK cells (Lineage-ScaI+ cKit+) at the 4th month after transplantation . The expression of Ripk1, Ripk3 and Mlkl was evaluated by qRT-PCR and the result showed that the expression of Ripk1, Ripk3 and Mlkl is significantly lower in R878H LSKs compared to WT, indicating that the necroptosis signaling is compromised in R878H cells in response to proliferation stress.

Given that phosphorylated MLKL (p-MLKL) acts as a final executioner of necroptosis, the inventors then sought to investigate whether R878H regulates the expression of p-MLKL. the inventors treated freshly isolated WT and R878H cKit+ bone marrow cells with TSZ (T, Tnfα; S, Smac mimetic; Z, z-VAD) for 3 hours, which is a frequently used approach to activate p-MLKL. The result revealed that the expression of p-MLKL in R878H cells is lower than WT upon TSZ stimulation, suggesting that R878H limits the activation of necroptosis in response to TSZ stimulation.

Given that necroptosis and apoptosis are the most important pathways leading to cell death, to determine whether R878H results in cell death through necroptosis, the inventors stimulated freshly isolated WT and R878H LSK cells by various combination of TSZ and the inventors observed that there is no difference between WT and R878H LSKs in non-treated group (NT) or TS-treated group (TS stimulation introduces apoptosis) . However, R878H LSKs exhibit significantly less cell death upon TSZ stimulation (TSZ activates necroptosis) . These data further confirms that hematopoietic stem cells carrying Dnmt3a R878H mutation compromised TSZ-induced necroptosis.

To further verify this observation in vivo, the inventors challenged two-month old WT, Dnmt3aR878H/+, Ripk1K45A, Ripk3Δ/Δ and Mlkl-/-mice with 3 doses of TNF α (5μg/mouse, intraperitoneal injection) every 12 hours and analyzed them 48 hours later. The results show that TNFα injection depletes HSCs in WT and Ripk1K45A mice to about half of the PBS-treated counterpart, while the frequency of HSCs in Dnmt3aR878H, Ripk3Δ/Δ and Mlkl-/-mice keeps static

However, all of progenitor cells, including CMP, GMP, MEP and CLP, and lineage cells remain unaffected in response to TNFα administration. All these data suggest that Dnmt3a R878H protected HSCs from inflammatory insult by modulating necroptosis signaling.

Example 5: Small Molecules Screening Targeting DNMT3A R882 Mutation

Given that human DNMT3A R882H (mouse R878H) is a driver mutation for clonal hematopoiesis and is one of the most risk factor for myeloid leukemia progression, it is of great importance to obtain a small molecule selectively inhibiting hematopoietic cells carrying DNMT3A R882H mutation. In order to screen such compound (s) , the inventors generated a K562 cell line with DNMT3A R882H mutation (hereafter named K562-R882H) and two reporter cell lines: K562-R882H-tdTomato, wherein a red fluorescent protein (tdTomato) was inserted into AAVS1 gene site of K562-R882H, and K562-EGFP, wherein an enhanced green fluorescent protein (EGFP) was inserted into AAVS1 gene site of WT K562 cell line. These construction of the above cells were confirmed by sequencing. Then, inventors mixed K562-R882H-tdTomato and K562-EGFP cells at the ratio of 1: 1 and screened three small molecule libraries (one FDA-approved drug library and two natural product libraries See Table 1) for small molecule (s) that can inhibitK562-R882H-tdTomato by detecting the ratio of tdTomato: EGFP. (See Fig. 1)

Table 1

A total of 30 active compounds showed inhibitory effect on R882H cells and further verification reduced the number to 6 (Fig. 2 and 3) . To determine the selectivity of these 6 active compounds, the inventors treated OCL-AML2 (DNMT3A R882 WT cell line) and OCL-AML3 (DNMT3A R882C cell line, which is frequently used as a model to study the function of DNMT3A R882 mutatio) cells with these 6 small compounds separately and measured cell viability using Cell Counting Kit-8 (CCK8) assay. The results show that only S2335, a natural product named oridonin, selectively inhibited OCL-AML3 cells (Fig. 4) and the IC ₅₀ of oridonin on OCL-AML2 and OCL-AML3 is 4.6 μM and 2.1 μM respectively. Similiarly, the IC ₅₀ of oridonin on K562-R882H-tdTomato and K562-EGFP cells is 3.7 μM and 6.4 μM respectively. Moreover, the inhibition efficiency of oridonin on R882H cells is in a concentration-dependent manner, but the selective inhibition effect decreases when the concentration is over 6 μM.

Given that R878H prevented the activation of necroptosis, and that oridonin selectively inhibits R882H leukemic cells, it is conceivable that oridonin might modulate necroptosis. To test this hypothesis, we then treated OCL-AML2 and OCL-AML3 cells with oridonin for 48h, and the expression of RIPK1, RIPK3 and MLKL was evaluated by qRT-PCR. The results show that the expression level of RIPK1, RIPK3 and MLKL in both OCL-AML2 and OCL-AML3 increase significantly in response to oridonin stimulation, but the increase is much larger in OCL-AML3 cells (Fig. 5) . Combining the aforementioned data that the R878H/R882H cells exhibited growth advantage through limiting necroptosis pathway and the efficiency of oridonin to activate necroptosis is significantly higher in OCL-AML3, it may be concluded that oridonin selectively inhibits OCL-AML3 by activating necroptosis signaling.

Example 6 Oridonin Selectively Inhibits Dnmt3a R878H HSC in vivo

To test the inhibitory effect of oridonin on Dnmt3a R878H HSCs in vivo, inventors transplanted 3×10 ⁵ total bone marrow cells either from WT or Dnmt3a R878H mice into lethally irradiated recipients together with 3×10 ⁵ competitor cells and the chimera of PB was evaluated every 4 weeks until the 5th month. One month after transplantation, the inventors treated the recipients with either vehicle or oridonin by i.p. injection for 15 days. The result shows that oridonin had no effect on the reconstitution efficiency of WT bone marrow cells (Fig. 6B) , while the percentage of R878H-derived cells declines after oridonin treatment, including B, T and myeloid lineages (Fig. 6C) . Moreover, the distribution of mature hematopoietic lineages exhibits no difference between oridonin-treated and vehicle-treated group in both WT and R878H cells (Fig. 6D) .

When the inventors analyzed donor-derived HSCs (Lineage-ScaI+ cKit+ CD34-CD150+) , it was observed that the percentage of WT donor-derived HSCs remains unchanged (42.9 ± 8.4 vs 38.6 ±8.6) between oridonin-treated and vehicle-treated group (Fig. 6E) . The percentage of R878H HSCs dominated in the vehicle-treated group, but it declines significantly after oridonin treatment (Fig. 6F) . These data suggest that oridonin specifically inhibit hematopoietic stem cells carrying Dnmt3a R878H mutation in vivo.

Inventors then evaluated the inhibitory effect of oridonin on DNMT3A R882 mutation in vivo. A mouse xenograft model was established by subcutaneous inoculation of 4×10 ⁷ OCL-AML2 or OCL-AML3 cells into the right flank of nude mice. When the recipients developed a measurable tumor at day 7, half of mice in each group were treated intraperitoneally with oridonin (20 mg/kg) and the rest were administrated with equal volume of vehicle. Volume of tumors was measured every day using published method (Zhou et al., 2007) for 14 consecutive days (Fig. 6G) and body weights of all recipients were recorded throughout the study. The results showed that the volume of the OCL-AML3 tumors were significantly reduced after treating with oridonin compared with the control group, whereas the tumor volume of OCL-AML2 showed no difference between the oridonin and control group (Figure 6H) . Notably, oridonin did not affect body weight of mice indicating no obvious toxicity under this treatment paradigm. These data suggests oridonin selectively inhibits leukemic cells with DNMT3A R882 mutation in vivo. In brief, oridonin selectively restricts leukemic cells or hematopoietic stem cells carrying DNMT3A R882H/C or mouse R878H mutation and it may be a promising candidate for treating DNMT3A R882 mutation related clonal hematopoiesis or leukemia.

Reference throughout this specification to "an embodiment, " "some embodiments, " "one embodiment" , "another example, " "an example, " "a specific examples, " or "some examples, " means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as "in some embodiments, " "in one embodiment" , "in an embodiment" , "in another example, " in an example, " "in a specific examples, " or "in some examples, " in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Claims

A method of treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof, comprising:

administrating a therapeutically effective amount of oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof to the subject.
The method of claim 1, wherein the DNMT3A R882H mutation related disease involves clonal hematopoiesis.
The method of claim 2, wherein the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease, inflammatory disease, and diabete.
The method of claim 1, wherein the oridonin derivative comprises a 14-O-Acyl derivative of oridonin.
The method of claim 4, wherein the 14-O-Acyl derivative of oridonin comprises 14-O-Dodecanoyl derivative of oridonin, 14-O-tetradecanoyl derivative of oridonin, and 14-O-hexandecanoyl derivative of oridonin.
A pharmaceutical composition for treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof, comprising:

oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof.
The pharmaceutical composition of claim 6, wherein the DNMT3A R882H mutation related disease involves clonal hematopoiesis.
The pharmaceutical composition of claim 7, wherein the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease, inflammatory disease, and diabete.
The pharmaceutical composition of claim 6, wherein the oridonin derivative comprises a 14-O-Acyl derivative of oridonin.
The pharmaceutical composition of claim 9, wherein the 14-O-Acyl derivative of oridonin comprises 14-O-Dodecanoyl derivative of oridonin, 14-O-tetradecanoyl derivative of oridonin, and 14-O-hexandecanoyl derivative of oridonin.
Use of oridonin or oridonin derivative, a stereoisomer, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof in the manufacture of a medicament for treating, preventing or lessening a DNMT3A R882H mutation related disease in a subject in need thereof.
The use of claim 11, wherein the DNMT3A R882H mutation related disease involves clonal hematopoiesis.
The use of claim 11, wherein the DNMT3A R882H mutation related disease comprises at least one selected from a group comprising: cancer, acute myeloid leukemia, cardiovascular disease, inflammatory disease, and diabete.
The use of claim 11, wherein the oridonin derivative comprises a 14-O-Acyl derivative of oridonin.
The use of claim 11, wherein the 14-O-Acyl derivative of oridonin comprises 14-O-Dodecanoyl derivative of oridonin, 14-O-tetradecanoyl derivative of oridonin, and 14-O-hexandecanoyl derivative of oridonin.
A method for screening drug for treating, preventing or lessening a DNMT3A R882H mutation related disease, comprising:

mixing a mutant cell carrying DNMT3A R882H mutation and a wild type cell at predetermined number ratio of the wild type cell to the mutant cell to obtain a cell mixture;

contacting the cell mixture with a candidate compound; and

determining a number ratio after contacting of the wild type cell to the mutant cell to obtain, wherein the number ratio after contacting higher than the predetermined number ratio is an indication that the candidate compound may be a drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.
The method of claim 14, wherein the mutant cell further carries a first reporter gene, the wild type cell further carries a second reporter gene, and the first reporter gene is different from the second reporter gene.
The method of claim 15, wherein one of the first reporter gene and the second reporter gene is EGFP, and the another one of the first reporter gene and the second reporter gene is tdTomato.
The method of claim 15, wherein the number ratio is determined by signals from the first reporter gene and the second reporter gene.
The mehtod of claim 15, wherein the number ratio after contacting being at least 50%higher than the predetermined number ratio is an indication that the candidate compound may be a drug for treating, preventing or lessening a DNMT3A R882H mutation related disease.