Inecalcitol Induces CD38 Expression in Multiple Myeloma Cell Lines More Efficiently Than All-Trans Retinoic Acid (ATRA)

Targeting CD38 has proven to be an effective strategy for the treatment of multiple myeloma (MM), with the therapeutic success of the first anti-CD38 monoclonal antibody: daratumumab. Interestingly, pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016, Blood). Therefore, increasing the levels of CD38 expression is a relevant pharmacological objective, which ATRA has already been shown to achieve in MM cell lines (Nijhof et al., 2015, Leukemia).

Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential (Okamoto R et al ., 2012, Int J Cancer; Ma Y et al ., 2013, Cell Cycle, Medioni et al . 2014, Clin Cancer Res), has also been shown to stimulate CD38 expression levels in several MM cell lines (ASH 2016, Abstract#3521). The aim of the present study was to further screen a large collection of 38 human MM cell lines. After culture for 72 hrs with or without 10 nM INE, cell surface CD38 labeling was measured with a fluorescent anti-human CD38 mouse FITC antiboby (eBiosciences, 11-0389). On 3 independent experimental days, cells from 3 different wells of either control (ethanol 0.1% v/v) or treated conditions were analyzed by flow cytometry to measure mean fluorescence intensity (MFI). Paired Student's t test was performed to check the unilateral hypothesis that INE had stimulated CD38 expression. Basal control CD38 labeling among the 38 MM cell lines was heterogeneous and ranged from minimal (MFI < 150) to very high (MFI > 10,000). The stimulation of CD38 expression by INE reached statistical significance in 37 out of the 38 (97%) tested MM cell lines, with a mean ± SEM of [4,75 ± 1.05]-fold increase in MFI. Only the U266 cell line remained insensitive to INE and was subsequently shown to entirely lack vitamin D receptors.

INE was then compared head-to-head with ATRA at 5 concentrations (0.3, 1, 3, 10 and 30 nM) for 72 hrs on 8 MM cell lines. At all tested concentrations on any cell line (40 experimental conditions), INE induced a higher increase in CD38 than ATRA, the difference reaching statistical significance in 36 out of the 40 bilateral paired Student's t test comparisons. At the threshold concentration of 0.3 nM, CD38 expression was significantly increased by INE on 7 out of 8 cell lines with a [3.7 ± 1.1]-fold stimulation, and on 6 cell lines by ATRA with a [1.4 ± 0.09]-fold increase. Maximal stimulation was achieved by INE at 3 nM, 10 nM and 30 nM on 2, 2 and 4 cell lines, respectively, with a [7.9 ± 3.3]-fold increase, ranging from 3.0 to 31. Maximal stimulation was observed only at 30 nM of ATRA in all cell lines with a [2.9 ± 0.5]-fold increase, ranging from 1.3 to 5.9. INE was on average 2.6 - 2.7 times more efficient than ATRA to induce CD38 in MM cell lines, both in terms of potency and efficacy.

These results suggest that inecalcitol could be a therapeutic agent of choice to potentiate the clinical response of a large majority of MM patients to anti-CD38 antibodies, such as daratumumab.