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CUDC-907 combined with venetoclax in AML
enhances DNA damage induced by DNA damaging agents in AML cells.1,4,6 Therefore, we hypothesized that simultaneously downregulating Mcl-1, upregulating Bim, and inducing DNA damage can maximally enhance vene- toclax-induced cell death.
CUDC-907 (Fimepinostat) is an oral, dual inhibitor of PI3K and histone deacetylases (HDAC) presently under investigation in multiple phase I and II clinical trials in the context of multiple myeloma, solid tumors, and lym- phoma (www.clinicaltrials.gov) – in the latter, it carries Fast Track designation from the FDA for use in adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) (www.curis.com).7-10 CUDC-907 also shows prom- ising antileukemic activity against preclinical models of
11 AML, as demonstrated in our most recent studies. This
activity appears to be at least partially mediated by the downregulation of key proteins involved in the cellular response to DNA damage such as CHK1 and Wee1,12-14 as well as ribonucleotide reductase catalytic subunit M1 (RRM1) and c-Myc.11,15 CUDC-907 also decreases Mcl-1 protein and increases Bim protein – findings that have been shown to contribute to this agent’s efficacy in lym- phoma16 and AML.11 Importantly, these changes occur fol- lowing downregulation of c-Myc, suggesting that early c- Myc downregulation may be the inciting event in the subsequent alterations in protein expression.9,11 Elevated or aberrant c-Myc expression and activation has been shown to be a key factor in AML leukemogenesis.17 Further, c-Myc has recently been identified as a highly statistically significant prognostic marker in AML, with notably shortened overall survival, event-free survival, and relapse-free survival in those with elevated levels of expression.18 Therefore, CUDC-907 would be an ideal compound to combine with venetoclax to enhance its antileukemic activity against AML.
To this end, we investigated the combination of CUDC-907 and venetoclax in preclinical in vitro and in vivo models of AML. The combination synergistically induces apoptosis in AML cell lines and primary AML patient samples ex vivo. CUDC-907 treatment enhances veneto- clax activity in AML cells by altering Bim and Mcl-1 pro- tein levels, downregulating c-Myc, and reducing DNA damage response proteins Wee1, CHK1, and RRM1. In vivo results show that CUDC-907 enhances venetoclax efficacy in an AML cell line derived xenograft model, sug- gesting that this combination has potential for the treat- ment of AML.
Methods
See the Online Supplementary Appendix for a detailed description of the methods.
Clinical samples
Diagnostic blast samples were obtained from the First Hospital of Jilin University. Written informed consent was provided accord- ing to the Declaration of Helsinki. This study was approved by the Human Ethics Committee of The First Hospital of Jilin University. Clinical samples were screened for gene mutations by PCR ampli- fication and automated DNA sequencing, and screened for fusion genes by real-time RT-PCR, as described previously.19,20 Patient characteristics are shown in the Online Supplementary Table S1. Samples were chosen based on availability of adequate sample at the time the assay was performed.
Annexin V/propidium iodide staining
Apoptosis was determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis Kit (Beckman Coulter, Brea, CA, USA), as described.21,22 Mean per- centage of AnnexinV+/PI- (early apoptotic) and Annexin V+/PI+ (late apoptotic and/or dead) ± standard error of the mean (SEM) from one representative experiment is shown.
Colony formation assay
Colony formation assays were carried out as previously described.11,23,24 Cells were treated with venetoclax and CUDC- 907, alone or in combination, for 24 hours. Cells were washed three times with PBS, plated in MethoCult (catalog number 04434; Stem Cell Technologies, Vancouver, Canada) and incubated for 10‐14 days, according to the manufacturer’s instructions. Colony forming units (CFU) were visualized utilizing an inverted micro- scope. Colonies containing over 50 cells were counted.
Leukemia xenograft model
Eight-week old immunocompromised triple transgenic NSG- SGM3 female mice (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 intravenously with MV4-11 cells (1x106 cells/mouse; 0.2 mL/inj.; day 0). On day 3, mice were randomized into No Rx control, 100 mg/kg/inj CUDC-907, 85 mg/kg/inj venetoclax, and 100 mg/kg/inj CUDC-907 + 85 mg/kg/inj venetoclax cohorts (5 mice/cohort). Drugs were prepared in 3% ethanol (200 proof), 1% Tween-80 (polyoxyethylene (20) sorbitan monooleate) and sterile water; all United States Pharmacopeia grade; v/v and administered orally. Mice were treated daily x 8 days given 4 days off and then treated x 6 days. Body weight and condition were assessed 1-2 times a day for the duration of study. Experimental endpoint and efficacy response was determined based on the median day for development of leukemic symptoms (hindleg weakness, >15% weight loss, metastatic spread to internal organs). All mice were provided food and water ad libitum, given supportive fluids and supplements as needed, and housed within an Association for Assessment and Accreditation of Laboratory Animal Care Internationa accredited animal facility with 24/7 veterinary care. In vivo experiments were approved by the Institutional Animal Care and Use Committee at Wayne State University.
Statistical analysis
Differences between treatment groups and/or untreated cells (comparison of the sum of Annexin V positive cells) were com- pared by pair-wise two-sample t-test or 1-way ANOVA with Bonferroni post hoc test (when comparing differences between three or more groups). Overall survival probability was estimated (Kaplan-Meier method) and statistical analysis was performed using the log-rank test. Statistical analyses were performed utiliz- ing GraphPad Prism 5.0. Error bars represent ± SEM; significance level was set at P<0.05.
Results
CUDC-907 synergizes with venetoclax to induce apoptosis in acute myeloid leukemia cells and shows in vivo efficacy in an acute myeloid leukemia xenograft mouse model
In order to determine if CUDC-907 has the capacity to enhance venetoclax-induced cell death, six AML cell lines were treated with venetoclax and CUDC-907, alone or combined, to determine the extent of the antileukemic
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