Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:

The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.

Methods:

The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.

Results:

We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.

We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.

We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.

Conclusions:

Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy.