Inecalcitol and Decitabine Synergize to Inhibit Cell Proliferation and to Induce Differentiation of Human Acute Myeloid Leukemia Cell Lines
Authors: Mouly Enguerran , Rousseau Emilie , Planquette Cecile , Delansorne Remi 
Published: October 2019

Abstract
Decitabine (DAC) is a hypomethylating agent indicated as front-line therapy for de novo or secondary acute myeloid leukemia (AML) in newly diagnosed patients aged 65 years or older unfit for standard induction chemotherapy (Kantarjian et al., 2012, Malik & Cashen, 2014, Nieto et al., 2016, He et al., 2017). Its mechanism of action at the DNA level mostly results in inhibition of cell proliferation. Cellular differentiation can also be involved in some extent in a fraction of the leukemic cell population, as reported in initial pharmacological studies (Creusot et al., 1982; Pinto et al., 1984). Overall survival advantage is nevertheless limited to several more months and the next challenge is to combine DAC with other drugs to improve it further (Kubasch & Platzbecker, 2018).
Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol) is a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating general properties on cancer cells and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), and especially on AML cell lines (AACR 2017, 2018). INE is currently being tested in combination with DAC in this category of elderly AML patients unfit for standard chemotherapy.
The aim of the present report was to look for synergies in vitro between DAC and INE on four non-APL human AML cell lines (MOLM-13, U-937, THP-1, OCI-AML2) both on inhibition of proliferation and induction of differentiation. After 72 hours of incubation, cells were counted and labeled for CD11b and CD14 at the cell surface as biomarkers of monocytic/macrophagic differentiation. The range of DAC concentrations had to adapted to each cell line to avoid maximal cytotoxicity: 1.2 nM to 100 nM on MOLM-13, 3 nM to 250 nM on U-937 and THP-1, and 31 nM to 500 nM on OCI-AML2. The same range of 0.12 to 10 nM INE concentrations was tested on each cell line. Each concentration of INE was tested in combination with each concentration of DAC. Synergy was calculated as the excess over the highest single agent (HSA) using the open source Combenefit software (Di Veroli et al., 2016).
The highest concentration of DAC alone (MOLM-13: 100 nM, U-937 and THP-1: 250 nM; OCI-AML2: 500 nM) induced a decrease in cell count of 30% of THP-1 cells, 50% of OCI-AML2 cells, 65% of U-937 cells and 80% of MOLM-13 cells. The highest concentration of INE alone (10 nM) induced a decrease in cell count of 20% of U-937 and THP-1 cells, 60% of OCI-AML2 cells and 70% of MOLM-13 cells. The antiproliferative effects of DAC and INE were at least additive in all combinations tested. Significant HSA synergy indexes were found for the decrease in cell number in all four cell lines, ranging from 12% to 23% depending on cell lines and combinations of concentrations.
The highest concentration of DAC alone had no (U-937, THP-1) or limited activity (<+12% of labeled MOLM-13 or OCI-AML2 cells) to induce either CD11b or CD14 on the cell surface. By contrast, the highest concentration of INE alone (10 nM) stimulated the expression of CD11b and CD14 in up to 70% to 95% of the cells depending on the cell line (except the CD14 labeling of U-937 cells which remained < 8%). The respective EC50 of INE for CD11b and CD14 induction was 1 and 3 nM on THP-1 cells, 4 and 3 nM on MOLM-13 cells, 3 and 3 nM on OCI-AML2 cells and 1 nM on U-937 cells (50% not reached for CD14). There was no antagonistic effect of DAC towards the pro-differentiating properties of INE. A significant HSA synergy index in the 16% to 26% range was observed for both CD11b and CD14 in MOLM-13 cells and for CD14 in OCI-AML2 cells. A very high HSA synergy index of 75% was observed for the stimulation of CD14 in U-937 cells.
In summary, DAC exerted more antiproliferative activity than INE which was more potent to induce monocytic/macrophagic differentiation of four non-APL human AML cell lines. The combination of DAC and INE systematically resulted in a synergy to inhibit cell proliferation, and the strong stimulation of cell differentiation induced by INE alone was in some cases boosted by DAC. These in vitro results provide the mechanistic basis for the potential interest of treating elderly AML patients with INE in addition to DAC in the ongoing double-blind placebo-controlled Phase II clinical trial (NCT02802267).

Disclosures
Mouly: Hybrigenics: Employment. Rousseau:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.