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X. Li et al.
The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is involved in cellular prolif- eration, differentiation, and survival. It has been reported that 50-80% of AML patients have a constitutively active PI3K/mTOR pathway, which correlates with very poor prognosis.4,5 In addition, aberrant activation of the PI3K pathway is a feature of leukemia-initiating cells.6,7 Although PI3K inhibitors have been shown to target AML cells, including leukemia-initiating cells, clinical results have been disappointing, likely due to compensatory activation of other survival pathways.8-10 Thus, PI3K inhibitors must be used in combination to prevent compensation by other sur- vival pathways and ensure successful eradication of AML cells.
Preclinical testing has revealed promising anti-cancer activity for the combination of PI3K inhibitors with histone deacetylase (HDAC) inhibitors.11-13 This prompted the design and synthesis of the dual PI3K and HDAC inhibitor CUDC- 907 (fimepinostat).14 CUDC-907 has shown encouraging preclinical activity against multiple types of cancers and the drug is currently being tested in phase I and II clinical trials for the treatment of lymphoma, multiple myeloma, and advanced/relapsed solid tumors (www.clinicaltrials.gov).15-17 It has shown such promising clinical efficacy that the US Food and Drug Administration recently granted Fast Track desig- nation to CUDC-907 for the treatment of adults with relapsed or refractory diffuse large B-cell lymphoma (http://www.curis.com/). In this study, we investigated CUDC- 907 in AML cell lines, primary AML samples, and a cell line- derived xenograft AML model. We showed that CUDC-907 induces apoptosis in AML cell lines and primary AML sam- ples and this effect is, at least partially, mediated by Mcl-1, Bim, and c-Myc. Additionally, CUDC-907 treatment down- regulates CHK1, Wee1, and ribonucleotide reductase (RR) catalytic subunit M1 (RRM1) and induces DNA replication stress and damage. In vivo results show that CUDC-907 has potential for the treatment of AML.
Methods
A detailed description of the methods is given in the Online Supplementary Material.
Cell culture
The characteristics of the cell lines are presented in Online Supplementary Table S1.
Clinical samples
Diagnostic AML blast samples were obtained from patients at the First Hospital of Jilin University (Changchun, China). Written informed consent was provided according to the Declaration of Helsinki. The Human Ethics Committee of the First Hospital of Jilin University approved this study. Clinical samples were screened for gene mutations by polymerase chain reaction (PCR) amplification and automated DNA sequencing, and fusion genes by real-time reverse transcriptase PCR, as described previously.18, 19 The patients’ characteristics are presented in Online Supplementary Table S2. Samples were chosen based on availability of adequate material at the time the assay was performed.
Annexin V/propidium iodide staining
Apoptosis was determined using an Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Kit (Beckman Coulter; Brea, CA, USA), as described elsewhere.20,21 The
mean percentage (± standard error of mean) of annexinV+/PI- (early apoptotic) and annexin V+/PI+ (late apoptotic and/or dead) cells from one representative experiment is shown.
Colony formation assay
Cells were treated with CUDC-907 for 24 h, washed with phos- phate-buffered saline, plated in triplicate in MethoCult (Stem Cell Technologies, Cambridge, MA, USA) and incubated for 10-14 days, according to the manufacturer’s instructions. Colony-forming units were visualized using an inverted microscope and colonies contain- ing >50 cells were enumerated.
Leukemia xenograft model
Immunocompromised triple transgenic NSG-SGM3 female mice at 8 weeks of age [NSGS, JAX#103062; non-obese diabetic scid gamma (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ; Jackson Laboratory, Bar Harbor ME, USA] were injected with MV4-11 cells (1 x 106 cells/mouse; 0.2 mL/injec- tion) intravenously (day 0). Mice were randomly divided into three groups (5 mice/group; day 3): one group was the no treatment con- trol group, the other two groups were given 100 or 150 mg/kg CUDC-907 [3% ethanol (200 proof), 1% Tween-80 (polyoxyethyl- ene 20 sorbitan monooleate) and sterile water; all USP grade; v/v)]. Mice were treated daily for 8 days followed by 4 days off treat- ment, and then treated for a further 6 days. Body weights were recorded daily and condition assessed (at least twice daily) for the duration of the study. Mice were humanely euthanized when they presented with: >20% weight loss, decreased mobility limiting access to food and water, lymph node metastases, progressive ane- mia, or lateral recumbency. The percentage increase in lifespan (%ILS) was calculated: % ILS = [T-C/C] x 100 where “T” is the median day of death of treated mice and “C” is the median day of death of control animals. In vivo experiments were approved by the Institutional Animal Care and Use Committee at Wayne State University.
For the pharmacodynamics study, NSG mice were injected with MV4-11 cells (1 x 107 cells/mouse) intravenously. Twenty-one days later, mice were randomized (5 mice/group) and injected once with vehicle control, 100 or 150 mg/kg CUDC-907. The mice were sac- rificed 24 h later and bone marrow cells were collected. Human cells were enriched using the EasySep Mouse/Human Chimera Isolation Kit (Stem Cell Technologies).
Statistical analysis
Differences were compared using the pair-wise two-sample t- test (comparisons of apoptosis, colony-forming units, and %DNA in the tail) or the Mann-Whitney two-sample U test [comparison of CUDC-907 half maximal inhibitory concentration (IC50)]. The over- all survival probability was estimated using the Kaplan-Meier method and the statistical analysis was performed using the log- rank test. The statistical computations were conducted using GraphPad Prism 5.0. The level of statistical significance was set at P<0.05.
Results
CUDC-907 decreases viable cells and induces apoptosis in acute myeloid leukemia cell lines, and shows promise against a MV4-11-derived xenograft model in vivo
CUDC-907 IC50 for seven AML cell lines, as measured by MTT assays, ranged from 12.4 nM (for MOLM-13) to 73.7 nM (for CTS) (Figure 1A). Annexin V/PI staining and flow cytometry analysis revealed that treatment with
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haematologica | 2019; 104(11)