Inecalcitol Induces CD38 Expression in Multiple Myeloma and Myeloid Cell Lines
Authors: Benjamin Susan , Planquette Cecile , Delansorne Remi 
Published: November 2019

Abstract
Targeting CD38 has proven to be an effective strategy for the treatment of relapsed refractory multiple myeloma (MM), with the recent approval of the first therapeutic monoclonal antibody directed against human CD38: daratumumab (Sanchez L et al., 2016, J Hematol Oncol; van de Donk NWC et al., 2016, Immunol Rev). Interestingly, the 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 IS et al., 2016, Blood). Therefore, increasing the levels of CD38 expression is the next logical pharmacological step to enhance the efficacy of anti-CD38 therapy.
In this context, we tested inecalcitol (INE: 14epi-, 19nor-, 23yne-, 1,25dihydroxy-cholecalciferol) on the MM.1S cell line (from ATCC, CRL-2974) in culture and found a reproducible concentration-dependent increase in CD38 expression levels after 3 days of treatment in 4 independent experiments. CD38 levels were measured at the surface of the cells by flow cytometry (ACEA NovoCyte flow cytometer) after one hour of incubation at 4°C with a fluorescent anti-human CD38 mouse FITC antiboby (eBioscience, 11-0389) or with the matched isotype control mouse IgG1 K (eBioscience, 11-4714). More than a 2-fold increase in labelling intensity was already observed with 0.1 nM INE and a maximal 5-fold stimulation was achieved at 3 nM INE. The half-maximal effective concentration (EC50) of INE was 0.47 ± 0.23 nM (n=4).
Next, we tested INE on L-363 cells, another MM cell line, purchased from DSMZ (ACC 49). In a single experiment, the L-363 cells were also shown to be responsive to INE, but about 10 times less sensitive than the MM.1S cells: a similar maximal 5-fold increase in CD38 levels on cell surface was also reached but at the much higher concentration of 30 nM INE and the threshold 2-fold stimulation of fluorescent labelling was obtained with 1 nM INE. The EC50 of INE in this single experiment was 4 nM.
In normal cell culture conditions, both MM cell lines showed a mix of CD38-positive (CD38+) and CD38-negative (CD38-) cells, as illustrated by the distribution spectrum of intensities of individual cell labelling. Without any treatment by INE, the CD38+/CD38- ratio was higher among the MM.1s cells (80/20) than the L-363 cells (30/70). The effect of INE resulted in both a higher proportion of CD38+ cells (>93% CD38+ MM.1S cells treated from 0.3 nM and higher INE concentrations; only 58% CD38+ L-363 cells at 3 nM INE), and a higher mean intensity of individual cell labelling.
Finally, we studied the HL-60 and U-937 cells classically described as representative of myeloid hematological malignancies. In normal culture conditions, both cell lines were CD38-. A concentration-dependent increase in CD38 expression levels on the surface of U-937 cells (from ATCC, CRL-1593,2) was obtained after 3 days of treatment with INE in the range of 0.1 to 10 nM in two independent experiments (EC50 = 0.47 nM and 0.76 nM). A concentration-dependent increase in CD38 expression levels on the surface of HL-60 cells (from ECACC, 98070106) was obtained after 3 days of treatment with INE in the range of 0.1 to 3 nM in two independent experiments (EC50s = 0.33 nM and 0.38 nM). All HL-60 or U-937 cells were CD38+ after 3 days of treatment with INE 0.3 nM or 1 nM, respectively.
Inecalcitol is a vitamin D receptor agonist already 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), allowing its administration at high oral doses to human cancer patients (Medioni J et al, 2014, Clin Cancer Res). The present findings suggest that INE could be tested in MM patients to increase the percentage of CD38+ MM cells and the density of CD38 at their surface, thereby potentially increasing the clinical response to anti-CD38 antibodies such as daratumumab. Furthermore, HL-60 and U-937 cells, all initially CD38-, became CD38+ after 3 days of in vitro treatment by INE, suggesting that CD38- myeloid hematological malignancies could potentially represent new indications for daratumumab when used after "priming" or in combination with INE.

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