Class 1 <scp>HDAC</scp> and <scp>HDAC</scp>6 inhibition inversely regulates <scp>CD</scp>38 induction in myeloma cells via interferon‐α and <scp>ATRA</scp>

Abstract

CD38 levels on the surface of myeloma (MM) cells appear to be a major determinant of MM cell sensitivity to daratumumab (Nijhof et al, 2016). Therefore, augmentation of CD38 expression on MM cells is required to improve the therapeutic efficacy of daratumumab, especially for patients whose MM cells express low levels of CD38. Nijhof et al (2015) previously reported that all-trans retinoic acid (ATRA) increased CD38 expression on MM cells, which subsequently enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity by daratumumab in vitro. Consistent with the observation that the upstream sequence of the CD38 gene contains an interferon regulatory factor 1 (IRF1)-binding site, interferon (IFN)-γ is also accepted to upregulate CD38 expression via activation of the signal transducers and activators of transcription 1 (STAT1)-IRF1 pathway (Ferrero & Malavasi, 1997; Bauvois et al, 1999; Tliba et al, 2008). Of note, IFN-α was reported to enhance CD38 expression in adult T cell leukaemia cells more efficiently than IFN-γ, and synergistically upregulate their CD38 expression in combination with ATRA (Mihara et al, 2016). More recently, panobinostat was reported to upregulate CD38 expression in MM cell lines and primary MM samples, which improved the in vitro cytotoxic effects of daratumumab on MM cells (Garcia-Guerrero et al, 2017). We here examined the effects of histone deacetylase (HDAC) inhibitors in combination with IFN-α and/or ATRA on the regulation of CD38 expression in MM cells. The materials and methods are described in Data S1 and Table SI. MM cell lines expressed CD38 at various levels (Figure S1A). IFN-α and ATRA dose-dependently (Figure S1B) and cooperatively (Fig 1A) upregulated CD38 expression in KMS-11 and MM.1S cells, which express CD38 at low and high levels, respectively. CD38 expression on the surface of KMS-11 and MM.1S cells was more markedly enhanced by IFN-α than by IFN-γ, but IFN-α and IFN-γ upregulated PD-L1 (also termed CD274) at similar levels (Figure S1C), indicating preferential induction of CD38 expression by IFN-α. Furthermore, IFN-α and ATRA cooperatively enhanced CD38 expression on the surface of all MM cell lines tested (Fig 1B), and CD38 mRNA expression in KMS-11 and MM.1S cells (Figure S2). However, we found that panobinostat attenuated or mitigated the CD38 upregulation in these MM cells under treatment with IFN-α and ATRA in combination (Fig 1B). As panobinostat is a pan-HDAC inhibitor, we next investigated the effects of HDAC inhibitors specific for class 1 HDACs or HDAC6 on the CD38 upregulation by IFN-α and/or ATRA. The class 1 HDAC-specific inhibitor MS-275 alone increased CD38 expression more than panobinostat in all MM cell lines tested (Fig 1B). MS-275 further increased the CD38 expression upregulated by IFN-α or ATRA alone or in combination in all the MM cell lines, suggesting restoration of CD38 expression by class 1 HDAC inhibition in MM cells without interfering in CD38 upregulation by IFN-α and ATRA. In contrast to MS-275, both the HDAC6 inhibitor ACY-1215 and panobinostat only marginally increased the CD38 upregulation by IFN-α or ATRA in INA-6 and U266 cells, and instead reduced its expression in KMS11 and MM.1S cells (Fig 1B and Figure S2). Of note, ACY-1215 and panobinostat attenuated the CD38 expression that was cooperatively upregulated by IFN-α and ATRA in combination in all MM cell lines tested. These results suggest that HDAC6 inhibition antagonizes the upregulation of CD38 expression in MM cells by IFN-α and ATRA. We then further examined the effects of HDAC inhibition on the downstream signalling of IFN-α or ATRA in MM cells. IFN-α promptly and sustainably phosphorylated STAT1 in KMS-11 cells, which was followed by the upregulation of IRF1 protein levels 1 h later (Figure S3A). The upregulation of IRF1 protein levels in KMS-11 cells by IFN-α or ATRA was reduced by ACY-1215, but not by MS-275 (Fig 2A, top panel). Given that the CD38 gene is known to be a target of the transcription factor IRF1, the reduction of IRF1 protein levels may be involved in the downregulation of CD38 by ACY-1215. However, ACY-1215 did not reduce IRF1 mRNA levels, which were upregulated by IFN-α and/or ATRA (Fig 2A, bottom panel), suggesting IRF1 protein degradation. IRF1 and HDAC6 are known to be client proteins of the molecular chaperone heat shock protein 90 (HSP90, also termed HSP90AA1). HSP90 chaperone activity is regulated by reversible acetylation and controlled by HDAC6 (Bali et al, 2005; Rao et al, 2008). Treatment with the HSP90 inhibitor 17-allylamino-demothoxy geldanamycin (17-AAG) inhibits chaperone function of HSP90, thereby inducing polyubiquitylation and proteasomal degradation of HSP90 client proteins (Rao et al, 2008). The synergism between the inhibition of both HDAC6 and HSP90 was previously demonstrated (Bali et al, 2005; Rao et al, 2008). Thus, HSP90 and HDAC6 interact with each other to maintain their functions and activity, and are suggested to be critical modulators of the activity and stability of IRF1. Consistent with these observations, CD38 upregulation in MM cells by IFN-α and/or ATRA was reduced by ACY-1215 or 17-AAG alone and cooperatively by both in combination (Fig 2B). IFN-α activates multiple signalling pathways other than the JAK/STAT signalling pathway, including the MAPK/activator protein 1 (AP1) pathway. The AP1 inhibitors, SR11302 and T-5224, reduced the CD38 upregulation in KMS-11 cells by IFN-α but not by ATRA (Figure S3B). Furthermore, IRF1 gene silencing reduced CD38 expression in KMS-11 cells without stimulation; however, IFN-α was able to induce CD38 expression in the KMS-11 cells even with IRF1 gene silencing (Fig 2C), indicating IRF1-dependent and independent CD38 upregulation by IFN-α. The AP1 inhibitor SR11302 further reduced CD38 upregulation in the IRF1 gene-silenced KMS-11 cells in the presence of IFN-α (Fig 2C), suggesting the role of AP1 activation in the IRF1-independent CD38 upregulation by IFN-α. These results collectively demonstrate that IFN-α and ATRA cooperatively enhance CD38 expression in MM cells, and suggest that IRF1 and HDAC6 function in the CD38 upregulation by IFN-α and/or ATRA although class 1 HDACs repress CD38 expression. Therefore, the present study provides a rationale for treatment using therapeutic anti-CD38 antibodies for MM with IFN-α and/or ATRA, and a caveat against further addition of HDAC inhibitors with anti-CD38 antibodies in combination with IFN-α and/or ATRA. This work was supported in part by MEXT/JSPS KAKENHI Grant Numbers JP18K08329, JP17K09956 and JP18K16118. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. A.B. and M.A. designed the research and conceived the project. PCR was performed by A.B., M.I., T.H., A.O., J.T., M.H., M.O., K.S. K.K. and S.Y.; flow cytometry by A.B., M.I., A.O., T.H., U.K., S.Y., K.A., I.E. and K.K.; immunoblotting by A.B., M.I., M.A., J.T., A.O., M.H., S.F., K.K., S.Y. and S.N.; transfection by A.B., M.I. and T.H.; and cell cultures by A.B., A.O., M.O., K.S., S.F., S.N., M.A., H.M., U.K., K.K., K.A. and I.E. A.B., S.N., M.I., T.H., A.O., J.T. and M.A. analysed the data. A.B., S.N., M.I., T.H. and M.A. wrote the manuscript. The authors have no potential conflicts of interest to declare. Figure S1. (A) Surface expression of CD38 on MM cells. Surface levels of CD38 in MM cell lines as indicated were analyzed by flow cytometry. Shaded histograms represent the staining with the mouse IgG1 isotype control. Figure S2. Effects of HDAC inhibitors on CD38 expression on MM cells. Figure S3. (A) Activation of the STAT1-IRF1 pathway in MM cells by IFN-α. Data S1. Materials and methods. Table SI. The list of primers. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.