Cardiac Glycosides Induce Immunogenic Changes in Myeloma Cells and Enhance the Efficacy of Monoclonal and Bispecific Antibody Therapy
Authors: Nakamura Tomoyuki , Ri Masaki , Ebina Toru , Marumo Yoshiaki , Oshima Yoshiko , Sasaki Hirokazu , Asano Arisa , Kinoshita Shiori , Suzuki Tomotaka , Narita Tomoko , Sanda Takaomi , Komatsu Hirokazu , Inoue Yasumichi , Hayashi Hidetoshi , Iida Shinsuke 
Published: December 2024

Background: Cardiac glycosides (CGs) are naturally-occurring compounds with a characteristic sugar structure that enhance myocardial contractility by inhibiting Na/K ATPase in cardiomyocytes. Several CGs, such as digoxin and digitoxin, can be used to treat heart failure and arrhythmias.
According to epidemiological studies of cancer patients, taking CGs was associated with lower recurrence rates and longer 5-year survival. However, the mechanisms responsible remain unknown.
Previously, we reported that periplocin, a type of CG, exerts its cytotoxic effects by inhibiting the unfolded protein response in multiple myeloma (MM) cells (M. Tokugawa et al. Sci Rep 2021). This agent causes increased expression of several genes encoding immunologically-important molecules, such as ICAM-1 and 4-1BBL, and a therapeutic target molecule, SLAMF7, in MM cells (M. Tokugawa et al. J Biochem 2023). ICAM1 is a ligand for LFA1 and is involved in lymphocyte adhesion and activation of immune responses. 4-1BBL binds to the 4-1BB receptor and is involved in T cell activation and proliferation, and is considered a potent enhancer of CAR-T therapy. Therefore, the 4-1BB agonist Utomilumab is being tested in combination with CAR-T therapy (NCT 03704298).
Here, to develop improved immune cell therapies for MM, we evaluated whether CGs enhances the anti-myeloma activity of monoclonal and bispecific antibodies by augmenting immune responses.
Methods: Alterations of ICAM1 and 4-1BBL or SLAMF7 at the protein level were evaluated on two MM cell lines, AMO1 and KMS12PE, by flow cytometry after exposure to the CGs, i.e., digoxin, periplocin, ouabain, or hellebrin at minimally toxic concentrations.
Treated MM cells were then co-cultured with immune effector cells from healthy donors together with elotuzumab (anti-SLAMF7 antibody) or teclistamab (BCMA bispecific antibody) in the presence or absence of low doses of CGs for 48 h. Thereafter, cytotoxic effects and effector cell activity were evaluated. Isolated NK cells were tested with elotuzumab, whereas PBMCs were used with teclistamab.
Cytotoxic effects were also evaluated using the LFA1/ICAM1 interaction blocker A286982 to assess how ICAM1 affects immunogenic cell death in the presence of CGs.
Results: Two MM cell lines cultured with low concentrations of CGs for 48 h (AMO1 with digoxin at 10-20 nM, periplocin 10-20 nM, ouabain 10-20 nM, hellebrin 2-5 nM; and KMS12PE with digoxin at 30-50 nM, periplocin 30-50 nM, ouabain 30-50 nM, hellebrin 30-50 nM). We confirmed that all CGs caused increased expression of ICAM1,4-1BBL and SLAMF7 protein in both cell lines at low levels of cytotoxicity.
Furthermore, we observed that periplocin, a representative CG, increased antibody-dependent cell-mediated cytotoxicity by Elotuzumab and T-cell dependent cellular cytotoxicity (TDCC) by Teclistamab at concentrations of 10 nM for AMO1 and 30 nM for KMS12PE. For example, in AMO1, the addition of 10 nM periplocin dramatically increased the TDCC by Teclistamab from 21.55% to 34.06% (p=0.004). Similar results were also obtained with digoxin.
For both antibodies, the inclusion of periplocin also tended to increase the expression of CD25, a lymphocyte activation marker, on effector cells.
Next, to confirm the significance of elevated ICAM for the efficacy of antibody activities, A286982 was added to AMO1 cultured together with PBMCs, Teclistamab and periplocin. Adding A286982 attenuated the effect of periplocin on increased TDCC (33.61% without A286982 vs. 26.53% with A286982 p=0.025). Thus, up-regulation of ICAM1 expression by CGs may contribute to enhancing the efficacy of monoclonal and bispecific antibody therapies in MM.
Conclusions: CGs may enhance the efficacy of antibody therapy by inducing immunogenic changes in MM cells at relatively low concentrations and thereby facilitate immune responses. Digoxin, a drug that can be used in clinical practice, also caused immunogenic changes in MM cells and enhanced the efficacy of antibody therapy. Similar effects of CGs would be expected to enhance the efficacy of immuno-cell therapies such as CAR-T cell therapy.