Small-molecule HDAC and Akt inhibitors suppress tumor growth and enhance immunotherapy in multiple myeloma

Background Multiple myeloma (MM) is an incurable disease. The acquisition of resistance to drugs, including immunomodulatory drugs (IMiDs), has a negative effect on its prognosis. Cereblon (CRBN) is a key mediator of the bioactivities of IMiDs such as lenalidomide. Moreover, genetic alteration of CRBN is frequently detected in IMiD-resistant patients and is considered to contribute to IMiD resistance. Thus, overcoming resistance to drugs, including IMiDs, is expected to improve clinical outcomes. Here, we examined potential mechanisms of a histone deacetylase (HDAC) inhibitor and Akt inhibitor in relapsed/refractory MM patients. Methods We established lenalidomide-resistant cells by knocking down CRBN with RNAi-mediated downregulation or knocking out CRBN using CRISPR-Cas9 in MM cells. Additionally, we derived multi-drug (bortezomib, doxorubicin, or dexamethasone)-resistant cell lines and primary cells from relapsed/refractory MM patients. The effects of HDAC and Akt inhibitors on these drug-resistant MM cells were then observed with a particular focus on whether HDAC inhibitors enhance immunotherapy efficacy. We also investigated the effect of lenalidomide on CRBN-deficient cells. Results The HDAC inhibitor suppressed the growth of drug-resistant MM cell lines and enhanced the antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by upregulating natural killer group 2D (NKG2D) ligands in MM cells. CRBN-deficient cells showed lenalidomide-induced upregulation of phosphorylated glycogen synthase kinase-3 (p-GSK-3) and c-Myc phosphorylation. Moreover, HDAC and Akt inhibitors downregulated c-Myc by blocking GSK-3 phosphorylation. HDAC and Akt inhibitors also exhibited synergistic cytotoxic and c-Myc-suppressive effects. The dual HDAC and PI3K inhibitor, CUDC-907, exhibited cytotoxic and immunotherapy-enhancing effects in MM cells, including multi-drug-resistant lines and primary cells from lenalidomide-resistant patients. Conclusions The combination of an HDAC and an Akt inhibitor represents a promising approach for the treatment of relapsed/refractory MM. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-021-01909-7.


Background
Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological malignancies [1][2][3]. Currently, there is no curative therapy for this disease, which is accompanied by symptoms including renal failure, anemia, hypercalcemia, and skeletal destruction [2]. However, the response rate in patients with MM has been improved by the introduction of various novel drugs available for MM treatment, including monoclonal antibodies (mAbs) such as daratumumab [4] and elotuzumab [5]. In addition, proteasome inhibitors (PIs), such as bortezomib [6] and immunomodulatory drugs (IMiDs), including thalidomide [3], lenalidomide [7], and pomalidomide [8], have shown efficacy in MM patients. Notably, the combined use of lenalidomide with PIs [9] and/or mAbs [10] is a key approach in current MM therapy [11].
Most patients, including those that responded to these drugs, developed drug resistance; therefore, it is essential to overcome this issue to improve survival. Among the novel drugs described, IMiDs are supposed to be key drugs as partners of PIs and mAbs for use in combination therapy [2]. Cereblon (CRBN) is a component of the CUL4 E3 ligase complex that serves as the primary target of IMiDs [12][13][14]. Reduced CRBN expression is thought to be associated with IMiD resistance [14][15][16]. IMiDs selectively bind to CRBN, which mediates the recruitment of Ikaros family zinc finger 1 (IKZF1; Ikaros) or 3 (IKZF3; Aiolos) to E3 ubiquitin ligase for subsequent degradation, resulting in the downregulation of interferon regulatory factor-4 and c-Myc [17]. DNA samples from the bone marrow of IMiD and PI doublerefractory MM patients have been examined using nextgeneration sequencing, and CRBN pathway-related mutations were identified in 32.5% of the patients [18]. Other reports have shown that most patients treated with lenalidomide have downregulated CRBN and upregulated IKZF1 gene expression [19]. CRBN mutations are also associated with pomalidomide resistance in MM [20]. A recent article on whole-genome sequencing (WGS) data from 455 patients and RNA sequencing (RNASeq) data from 655 patients, which included newly diagnosed, lenalidomide-refractory, and pomalidomiderefractory cohorts, reported an increase in the frequency of CRBN aberrations [20]. These aberrations include point mutations, copy losses/structural variations, and a specific variant transcript (exon 10 spliced), with progressive IMiD exposure.
Histone deacetylase (HDAC) inhibitors are known to activate innate immunity [21]. Meanwhile, HDAC inhibition reverses aberrant epigenetic changes contributing to tumor proliferation and enhances tumor immunogenicity [22][23][24]. HDAC inhibitors also enhance the expression of major histocompatibility complex class I-related chain A and B (MICA and MICB), both of which serve as key natural killer group 2D (NKG2D) ligands [25][26][27], in leukemic cell lines [28]. NK cells are activated by NKG2D receptor crosslinking on effector cells with NKG2D ligands expressed on tumor cells [25,29]. In addition to MICA and MICB, NKG2D ligands include UL16-binding proteins (ULBp) [25][26][27]. Activation of these NKG2D ligands enhances the antibody-dependent cellular cytotoxicity (ADCC) response mounted during mAb therapy for MM [30]. Hence, we proposed a strategy to induce activation of anti-tumor immunity with HDAC inhibitors to overcome resistance in lenalidomide relapse/refractory MM patients.

Quantitative reverse transcription PCR (qRT-PCR)
MM cell lines (RPMI8226, U266) were exposed to HDAC inhibitors (ACY-1215, CUDC-907, panobinostat, and romidepsin) and control agents for 0 h, 1 h, 2 h, or 4 h. After treatment, total RNA was extracted from MM cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany. 74,104). cDNA was synthesized from total RNA using the SuperScript III First-Strand Synthesis System (Thermo Fisher, Waltham, Massachusetts, USA. 18,080,051). Real-time PCR was performed for mRNA obtained from MM cell lines. RNA extraction, cDNA synthesis, and qRT-PCR were performed, as previously reported [35]. All qRT-PCR assays were performed in triplicate and repeated at least twice.

ADCC assay and NK assay
Luciferase-expressing MM cell lines were exposed to HDAC inhibitor or DMSO for 24 h. MM cells were then treated with 0.001, 0.01, 0.1, 1, or 10 μg/mL daratumumab, elotuzumab, or control (IgG) and coincubated with NK cells. NK cells were extracted from PBMCs obtained from healthy donors using a human NK Cell Isolation Kit (Miltenyi Biotec, Nordrhein-Westfalen, Germany). Cell counting was performed with a hemocytometer (Erma Inc., Tokyo, Japan). NK cells were collected in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin. Fresh NK cells were added at a ratio of 10:1 to MM cells. Cell death was calculated from the decrease in luciferase activity, which was detected by Steady Glo (Promega, Madison, Wisconsin, USA) or PicaGene (TOYO INK, Tokyo, Japan). Luciferase luminescence in the samples was evaluated using a Nivo spectrophotometer (Perkin Elmer, Massachusetts, USA). ADCC and NK assays were repeated at least twice.

Methyl thiazolyl tetrazolium assay
Each MM cell line was seeded in a 96-well plate and incubated with HDAC inhibitors, Akt inhibitor, GSK-3 inhibitor, and PI for 48-72 h. Cells were exposed to lenalidomide and pomalidomide for 5 days, doxorubicin for 5 h, or dexamethasone for 1 week. Methyl thiazolyl tetrazolium (MTT) assays were performed after staining with a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) for 50 min. Plates were read using a Nivo spectrophotometer. Each assay was performed in quintuplicates, and the experiment was repeated at least twice.

In vivo animal experiments
Six-week-old C.B-17/Icr-scid/scidJcl mice were purchased from Japan CLEA (Tokyo, Japan). Mice were subcutaneously injected with OPM-2 CRBN KO (OPM-2 CRBN-knockout cells) to generate SCID mice. After tumor cell injection, SCID mice with tumors over 10 mm in length along the major axis were treated with vehicle or CUDC-907 (50 mg/kg body weight) three times per week for 2 weeks. For the experiments, eight mice per study group (vehicle and CUDC-907 treated) were used. The outcome was a change in tumor size compared to the day treatment started. Mice were observed for 14 days after administration. This animal experiment was approved by the Animal Experiment Committee at the Institute of Medical Science of the University of Tokyo (see Study approval). The description was based on The ARRIVE guidelines 2.0 [36].

Immunohistochemistry
Tumors of OPM-2 CRBN KO (CRBN knockout cells) were isolated when the SCID mice were sacrificed 13 days after starting the treatment with the vehicle or CUDC-907 (the next day after all oral administrations are completed). Formalin-fixed paraffin-embedded sections (3 μm) were deparaffinized and hydrated with graded ethanol. After heat-induced antigen retrieval for 10 min at 120°C, slides were incubated in the following primary antibodies: a 1:300 dilution of cleaved caspase-3 rabbit polyclonal antibody (Cell Signaling Technology, #9661, Massachusetts, USA), a 1:200 dilution of GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) rabbit polyclonal antibody (Abcam, ab75745, Cambridge, UK), a 1:100 dilution of c-Myc rabbit monoclonal antibody (clone Y69, Abcam, ab32072) for 30 min at 37°C respectively. Labeling of secondary antibody was performed with Histofine Simple Stain Mouse MAX-PO(R) (Nichirei Bioscience, Tokyo, Japan) for 30 min at 37°C. To visualize the antigen-antibody complex, the ImmPACT DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) was used, and then sections were counterstained with hematoxylin. A section from a breast cancer tissue placed on the same slide glass was used as the positive control, and sections treated with PBS instead of the primary antibodies were used as negative controls.

In vitro experiments using patient samples or healthy donors
Bone marrow samples were collected from MM patients (newly diagnosed or relapsed/refractory myeloma). The primary cells used were derived from 5 patients with newly diagnosed MM and seven patients with relapsed/ refractory MM. Upon patient consent, an additional 5 mL of bone marrow fluid was collected during a routine bone marrow examination. MM cells were then sorted using FACS Aria (BD Biosciences). The primary cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. For the control sample, 20 mL peripheral blood was collected from healthy donors after obtaining consent. Then, peripheral blood mononuclear cells (PBMNCs) were isolated using Lymphoprep (Cosmo Bio, Tokyo, Japan) and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin and 2 μg/mL phytohemagglutinin-L (PHA-L; Sigma-Aldrich, Missouri, USA).

Statistics
For each analysis, P-values < 0.05 were considered to represent statistically significant differences.
For the FACS experiments, the expression level relative to DMSO were tested by one-sample t-test with null hypothesis of the ratio = 1. As for some multiple comparisons, we performed ANOVA with post hoc Dunnett's test for the data from the qRT-PCR, and Kruskal-Wallis followed by Mann-Whitney with Bonferroni correction for MTT assays. Statistical methods were selected according to the data distributions assessed by appearance in the plots. Synergistic effects were evaluated by a generalized linear model including the statistical interaction term of two drugs (distribution: normal. Link function: log). Effects on ADCC activity and cell viability were evaluated by a general linear regression model with the statistical interaction term of drugs and log-transformed dose or effector/target (E/T) ratio. Repeated measurements in mice were evaluated by a linear mixed regression model with the statistical interaction term of drugs and time (days), considering intraindividual correlation by including random effects. Pvalues were calculated for the interaction terms in the regression models, which were interpreted as the . c-f MICA, MICB, IKZF1/3 mRNA levels in RPMI8226 cells exposed to ACY-1215. Experiments were performed in triplicate (n = 3). (*p < 0.05, "ns" indicates no significant difference). g-i ADCC and NK cell assay of daratumumab and elotuzumab in the presence of ACY-1215. Experiments were repeated three times (n = 3). Error bars represent the SD. (**p < 0.01) j Prognostic value of MICA expression based on MM patient clinical trial data difference in slope of dose−/time-dependent change between groups.

HDAC inhibitors upregulate NKG2D ligands and enhance mAb effects in MM cell lines
In a previous study, HDAC inhibitors enhanced NKG2D ligand expression in hepatoma cells and sensitized them to NK-mediated cytolysis [37]. Panobinostat reportedly increased CD38 expression in MM cells in a timedependent manner, enhancing the efficacy of daratumumab [38]. However, it is unknown whether panobinostat alters NKG2D ligand expression on MM cells. We found that 24 h panobinostat treatment increased NKG2D ligand (MICA/B and ULBp-2/5/6) expression in RPMI8226 and U266 cells. Panobinostat increased CD38 expression The number below the band shows the density of CRBN normalized to that of actin. d-i MTT proliferation assay. Sensitivity of CRBN knockdown and CRBNknockout MM cell lines to lenalidomide and pomalidomide. DMSO vehicle control and the corresponding values of MTT cell viability were designated as 100% (0 μM). To show whether inhibitors compromised cell proliferation of CRBN-knockdown cells, the ratio (%) of MTT cell viability for each concentration of lenalidomide or pomalidomide used, compared with DMSO vehicle control, is indicated. Each experiment was performed in quintuplicate (n = 5). Results are shown as the mean ± SD. (*p < 0.05, **p < 0.01, "ns" indicates no significant difference). j-m MICA and IKZF1 mRNA levels in CRBN-deficient RPMI8226 cells exposed to ACY-1215. Experiments were performed in triplicate (n = 3). (*p < 0.05, **p < 0.01, "ns" indicates no significant difference) in both RPMI8226 and U266, but only slightly in RPMI8226 (Fig. 1a). Romidepsin enhanced surface marker MICA/B, ULBp-2/5/6, and CD38 expression in RPMI8226 and U266. ACY-1215 enhanced MICA/B expression of RPMI8226 and MICA/B, ULBp-2/5/6, and CD38 expression in U266 (Fig. 1b). Next, we tested whether MICA and MICB expression is altered under HDAC inhibition at the mRNA level. Panobinostat and ACY-1215 increased MICA mRNA expression in RPMI8226 and U266 cells (Fig. 1c, Supplementary Fig.   Fig. 3 HDAC and Akt inhibitor treatment can overcome c-Myc upregulation by lenalidomide in CRBN-deficient cells. a-b Western blot validation of the effects of lenalidomide (Len) on CRBN-deficient cells. c-Myc expression could not be detected in OPM-2 CRBN sh#395 cells exposed to the control drug and Len 10 μM. c-d Validation of the effects of Len on control H929 or OPM-2 cells via western blot analysis. e-f RPMI8226 and H929 cell lysates treated with ACY-1215 and/or afuresertib were immunoblotted with the indicated antibodies (against GSK-3, p-GSK-3, c-Myc, and actin). g Myc mRNA levels in RPMI8226 cells treated with ACY-1215. (**p < 0.01) h RPMI8226 cell lysates treated with ACY-1215 and/or afuresertib were immunoblotted with indicated antibodies (against caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, actin) 1A-C). MICB expression did not significantly increase after ACY-1215 treatment in RPMI8226 (Fig. 1d). Panobinostat slightly enhanced MICB in U266 cells (Supplementary Fig. 1D). Other results are summarized in Supplementary Fig. 1E.
As it has been suggested that HDAC inhibitors may act on NKG2D ligands of MM cells, we decided to investigate how the combined use of HDAC inhibitors on mAbs against MM cells affects ADCC activity. Based on previous reports [40,41], we aimed to verify the ADCC activity of daratumumab in RPMI8226 cells and that of elotuzumab in U266. We found that daratumumab ADCC was significantly upregulated by ACY-1215 treatment (Fig. 1g). Additionally, ACY-1215 tended to enhance the ADCC activity of elotuzumab (Fig. 1h) and the direct cytotoxicity of NK cells against MM cells (Fig. 1i).
Analysis of gene expression profiles from 414 newly diagnosed MM patients [42] deposited in an integrated gene expression and disease prognosis database (Geno-micScape, http://www.genomicscape.com) revealed that (*p < 0.05, **p < 0.01, "ns" indicates no significant difference). c-f MICA, IKZF1/3, Myc mRNA levels in RPMI8226 cells exposed to CUDC-907. Experiments were performed in triplicate (n = 3). (*p < 0.05, **p < 0.01, "ns" indicates no significant difference). g-h ADCC assay of daratumumab and elotuzumab. Experiments were performed in triplicate (n = 3), and data are presented as the mean ± SD. P-values were calculated for the interaction terms in the regression models, which are interpreted as the difference in slope of dose-dependent change between the groups (**p < 0.01). i Western blot validation of the effects of CUDC-907 on RPMI8226 cells higher MICA expression was significantly associated with better overall survival (OS; p = 0.0049, Fig. 1j). We also analyzed data from a clinical trial that compared the efficacy and safety of bortezomib treatment [43]. The data indicated longer OS of patients with high MICA and ULBp2 expression compared with that of patients with low MICA (p = 0.068, Supplementary Fig. 1G) and ULBp2 (p = 3.6e-05, Supplementary Fig. 1H) expression. We hypothesized that upregulation of NKG2D ligands, including MICA and ULBp2, is critical for enhanced MM cell recognition by NK cells and contributes to better clinical outcomes.

HDAC and Akt inhibitors are potential treatments for lenalidomide-resistant cells
Enhanced p-GSK-3 α/β expression was previously reported in MM cell lines following extended exposure to lenalidomide, in turn compromising lenalidomide sensitivity [34]. Thus, we examined how treatment with 10 and 30 μM lenalidomide for 1 month would alter p-GSK-3 α/β levels [34] in H929 and OPM-2 cells when CRBN was downregulated to a variable extent. We observed a phosphorylation-dependent mobility shift of GSK-3 α/β and an increase in p-GSK-3 α/β and c-Myc levels in H929 cells after lenalidomide exposure (Fig. 3a). Increased p-GSK-3 α/β and c-Myc levels were also observed in OPM-2 cells (Fig. 3b). These changes were exacerbated when cells were exposed to higher lenalidomide concentrations, implying that CRBN disruption in MM cells could further increase c-Myc levels after extended lenalidomide treatment. Previous report showed that CRBN-deficient activated mouse CD8 + T cells and human CD8 + T cells treated with immunemodulating compounds exhibited increased Myc levels [46]. The mechanism behind this is unknown, but it is possible that a similar reaction was caused by lenalidomide in MM cells. We also exposed control H929 and OPM-2 cells to 10 or 30 μM lenalidomide, but only for 5 days as both concentrations were shown to kill the cells (Fig. 2e-f). In control H929, p-GSK-3 β and c-Myc was increased (Fig. 3c), however, p-GSK-3 and c-Myc expression was not significantly increased in control OPM-2 cells (Fig. 3d). These results indicate that the increase in p-GSK3 α/β and c-Myc due to lenalidomide treatment was more prominent in CRBN-low cells than in control cells.
Since GSK-3 and c-Myc are located downstream of the PI3K/Akt pathway, it was speculated that Akt inhibitors might also be useful for MM therapy. We, therefore, assessed the effects of HDAC inhibitors, Akt inhibitors, and the combination of HDAC inhibitors and Akt inhibitors. Oral Akt inhibitor afuresertib has been clinically tested in patients with advanced MM. Both phase I [47] and II [48] trials of afuresertib were performed in relapsed/refractory malignant lymphoma patients. However, the efficacy of combined HDAC and Akt inhibitor treatment in MM is currently unknown. We measured GSK-3 α/β, p-GSK-3 α/β, and c-Myc protein levels following HDAC inhibitor and/or Akt inhibitor treatment. ACY-1215 and/or afuresertib decreased p-GSK-3 α/β and c-Myc protein levels in RPMI8226 and H929 cells   Fig. 2A). ACY-1215 also downregulated c-Myc mRNA in RPMI8226 cells (Fig. 3g). According to clinical trial data comparing the efficacy and safety of treatment with and without bortezomib [43], patients with high GSK-3 α/β expression had better OS than those with low GSK-3 α (p = 1.9 × 10 − 9 , Supplementary Fig. 2B)/β (p = 0.02, Supplementary Fig. 2C) expression. Further, patients with high Myc expression had poorer OS than those with low expression (p = 0.0043, Supplementary  Fig. 2D). These results are suggestive of the negative  Table 1 effect of GSK-3 α/β inactivation-related c-Myc stabilization on MM patient prognosis. Finally, we investigated the proapoptotic effect of ACY-1215 and afuresertib alone and in combination. Both inhibitors induced apoptosis, and combined treatment had an even stronger effect (Fig. 3h, Supplementary Fig. 2E).

Efficacy of CUDC-907 in primary cells and drug-resistant MM cells
We examined the efficacy of CUDC-907 on parental MM cell lines, in addition to lenalidomide-resistant, doxorubicin-resistant, bortezomib-resistant, and dexamethasone-resistant cells ( Fig. 7a-g, Supplementary Fig. 3A-H), as well as in primary cells from MM patients (Table 1) sensitive to lenalidomide (S1-S6) (Fig. 8a-f) and six patients refractory to lenalidomide (R1-R6) (Fig. 8g-l). Refractory patients were defined as patients with disease that had no response while on lenalidomide-based therapy or experienced progression within 60 days of their last therapy, as per The International Myeloma Working Group criteria [54]. Lenalidomide (5 μM and 10 μM) showed no cytotoxicity in primary cells from R1 (Fig. 8g) and R2 (Fig. 8h) patients. Thus, we confirmed that cells derived from R1 and R2 patients were resistant to lenalidomide even in vitro. On the other hand, most cells, including R1 and R2 cells, were sensitive to CUDC-907 after 48 h of treatment (Fig. 8a, c-k). Although, primary cells from Patient S2 and R6 were not significantly affected by CUDC-907 (Fig. 8b, l).
Finally, we examined how HDAC and Akt inhibitors, as well as CUDC-907, affect PBMNCs of healthy donors to survey normal cellular functions. We found that the HDAC inhibitor, the Akt inhibitor, and CUDC-907 did not show cytotoxicity toward PBMNCs from healthy donors ( Supplementary Fig. 3I).

Discussion
In the current study, we investigated whether smallmolecule HDAC and Akt inhibitors could overcome lenalidomide resistance through suppression of tumor growth and enhancement of immunotherapy in MM. HDAC inhibitors reduced tumor growth by downregulating c-Myc in a CRBN-independent manner, while upregulating MICA expression and, thus, enhancing the efficacy of immunotherapy. In particular, HDAC inhibitors enhanced the ADCC activity of daratumumab and elotuzumab.
Lenalidomide is currently used for induction and maintenance therapy in MM treatment. However, the development of lenalidomide resistance worsens the prognosis of MM patients. Thus, overcoming this challenge is important for improving clinical outcomes. CRBN is the primary target of IMiDs [12][13][14], and low CRBN expression is associated with resistance to lenalidomide and pomalidomide monotherapy [15]. Unlike lenalidomide, pomalidomide exhibited cytotoxicity in some CRBN-knockdown cells.
Surprisingly, CRBN-knockdown and -knockout cells cultured with lenalidomide showed an upregulation of p-GSK-3 and c-Myc. These results are consistent with the significant increase in c-Myc following the development of lenalidomide resistance, when compared to its levels in bone marrow samples collected from MM patients at diagnosis [16]. Our data indicate that c-Myc is an important therapeutic target in CRBN-deficient MM cells.
As GSK-3 and c-Myc are located downstream of the PI3K/Akt pathway, we explored the effects of Akt inhibition. Akt inhibitor afuresertib downregulated p-GSK-3 α/β and c-Myc at the protein level. Furthermore, combined HDAC and Akt inhibitor treatment induced stronger downregulation of c-Myc than either inhibitor alone. Thus, combined HDAC and Akt inhibition could suppress the proliferation of lenalidomide-resistant cell lines by overcoming c-Myc upregulation.
Based on the synergetic suppression of MM cell proliferation by combined HDAC and Akt inhibition, we evaluated dual HDAC and PI3K inhibitor CUDC-907. CUDC-907 enhanced daratumumab and elotuzumab ADCC, while also upregulating NKG2D ligand expression even in CRBN-knockout cells and suppressing IKZF1, IKZF3, p-GSK-3, and Myc expression. We also confirmed the efficacy of CUDC-907 in SCID mice injected with CRBN-knockout cells. It is difficult to assess how activation of NKG2D ligand by CUDC-907 contributes to tumor suppression in the xenograft model. Previous reports on NK cells during breast cancer cell growth and metastasis in SCID mice have shown that these mice have certain residual immunity, which included an NK cell-mediated response [55]. Thus, there exists a possibility that the tumor transplanted in SCID mice was attacked by residual NK cells, in addition to the direct effect of CUDC-907. Additionally, CUDC-907 was cytotoxic to primary cells from MM patients, including those resistant to various lenalidomide treatment regimens. According to the data from a pharmacokinetics study involving CUDC-907 [52], the in vitro concentration of CUDC-907 used in the current study was similar to that detected in the plasma of six patients (0-20 ng/mL, 0-39.3 nM). This concentration was observed on day 15 after receiving 60 mg of oral CUDC-907 daily for 5 days, followed by a 2-day intermittent break [52]. Furthermore, Phase I trial data indicated that the most common adverse effects of CUDC-907 were diarrhea, fatigue, nausea, and thrombocytopenia. However, these effects were relatively rare [thrombocytopenia (18%), neutropenia (7%), hyperglycemia (7%), and diarrhea (5%)] [52].

Conclusions
HDAC inhibitor treatment downregulated IKZF1 and IKZF3, inducing Myc downregulation and MICA upregulation. In turn, MICA upregulation led to enhanced ADCC activity of mAbs and NK cell activity. Moreover, HDAC or Akt inhibitors downregulated p-GSK-3, which functions to stabilize c-Myc. In turn, p-GSK-3 suppression caused c-Myc downregulation. Meanwhile, the dual PI3K and HDAC inhibitor, CUDC-907, enhanced MICA and suppressed Myc (Fig. 9). In summary, HDAC and Akt inhibitors, as well as CUDC-907, are promising drugs for cases of relapse/refractory MM, including lenalidomide resistance.