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Multiple myeloma with t(11;14)‐associated immature phenotype has lower CD38 expression and higher BCL2 dependence

Abstract CD38 expression on myeloma cells is a critical factor affecting the early response to the anti‐CD38 antibody daratumumab. However, factors affecting CD38 expression in untreated multiple myeloma are not fully elucidated. In this study, we found that CD38 expression was significantly lower in myeloma patients with the translocation t(11;14)‐associated immature plasma cell phenotype, and particularly in those expressing B–cell‐associated genes such as PAX5 and CD79A. CD138, a representative marker of plasmacytic differentiation, was also significantly lower in these patients, suggesting that CD38 expression may be associated with the differentiation and maturation stages of myeloma cells. Furthermore, the BCL2/BCL2L1 ratio, a response marker of the BCL2 inhibitor venetoclax, was significantly higher in patients with the immature phenotype expressing B–cell‐associated genes. The BCL2/BCL2L1 ratio and CD38 expression were significantly negatively correlated. We also confirmed that patients with translocation t(11;14) expressing B–cell‐associated genes were indeed less sensitive to daratumumab‐mediated direct cytotoxicity but highly sensitive to venetoclax treatment in ex vivo assays. Moreover, all‐trans‐retinoic acid, which enhances CD38 expression and induces cell differentiation in myeloma cells, reduced B‐cell marker expression and the BCL2/BCL2L1 ratio in myeloma cell lines, leading to reduced efficacy of venetoclax. Venetoclax specifically induces cell death in myeloma with t(11;14), although why patients with translocation t(11;14) show BCL2 dependence is unclear. These results suggest that BCL2 dependence, as well as CD38 expression, are deeply associated with the differentiation and maturation stages of myeloma cells. This study highlights the importance of examining t(11;14) and considering cell maturity in myeloma treatment strategies.


| INTRODUC TI ON
Daratumumab, a fully human monoclonal antibody against CD38, has emerged as a promising agent for treating MM. 1 It has shown substantial clinical activity in several clinical trials when used in combination with PIs or IMiDs. [2][3][4][5] Various mechanisms of action have been reported for daratumumab. Daratumumab has classic Fc-dependent immune effector mechanisms, including CDC and ADCC. 6 A previous study has demonstrated a significant positive association between CD38 expression levels in myeloma cells and the efficacy of daratumumab monotherapy. 7 In addition, daratumumab targets CD38-expressing immunosuppressive cells, including CD38-positive (CD38 + ) regulatory T-cells (Tregs), thereby inducing immunomodulatory effects. It has been shown previously that cytotoxic T-cell number and clonality are increased after daratumumab treatment. 8,9 We recently showed that CD38 expression levels on myeloma cells and the frequency of circulating CD38 + Tregs are associated with the response to daratumumab. 10 In the study, we demonstrated that the pretreatment levels of CD38 on myeloma cells may be a predictive marker for an early response to daratumumab treatment, reflecting the direct cytotoxicity of daratumumab. A previous study showed that IMiDs such as lenalidomide upregulate CD38 expression and prime myeloma cells for daratumumab-mediated cytotoxicity by degrading the CD38 gene repressors, IKZF1 and IKZF3. 11 Therefore, prior exposure to IMiDs may cause heterogenous CD38 expression in previously treated patients with MM. Daratumumab was recently approved for treating newly diagnosed patients with MM. however the factors affecting CD38 expression in untreated MM remain unclear. It is important to obtain a rapid response by daratumumab during initial treatment, as several reports have suggested that immunomodulatory effects are less likely to be induced if debulking of the tumor is not achieved. 12,13 Therefore, it may be clinically relevant to identify the determinants of CD38 expression. Therefore, we investigated the factors affecting CD38 expression in newly diagnosed patients with MM.
It has been shown that MM produces tumors at various stages of differentiation and maturation. Particularly, cases with translocation t (11;14) frequently show an immature phenotype, such as lymphoplasmacytic morphology, 14,15 B-cell-associated gene expression, 16,17 low-concentration of monoclonal proteins, 18 and unmeasurable secretory status such as oligosecretory and nonsecretory types. 19 In this study, we show that CD38 expression is significantly lower in patients with myeloma and the t(11;14)-associated immature plasma cell phenotype. Moreover, we found that CD38 expression was negatively correlated with the BCL2/BCL2L1 ratio, and BCL2 dependence was significantly higher in patients with myeloma and the t(11;14)associated immature phenotype. These results suggest that BCL2 dependence, as well as CD38 expression, could be deeply associated with the differentiation and maturation stages of myeloma cells.

| Flow cytometric analysis of bone marrow samples from patients
CD38 and CD138 expression levels were determined as MFI using flow cytometry. Flow cytometry was performed using the DURAClone RE PC antibody panel on a Navios cytometer, and the data were analyzed using Kaluza analysis software (all from Beckman Coulter). CD38 and CD138 MFI were accessed in the neoplastic plasma cell population (CD38 + /CD138 + /CD56 + or CD56 − /CD19 − ) as previously described. 10

| Cell viability assay
Cell viability assays were carried out using an MTT-based In Vitro Toxicology Assay Kit, in accordance with the manufacturer's protocol (Sigma-Aldrich).

| Reagents
Venetoclax was purchased from Selleck Chemicals. Daratumumab was obtained from Janssen Pharmaceuticals. ATRA was purchased from Sigma-Aldrich.

| Statistical analysis
Mann-Whitney U tests, Kruskal-Wallis test and Student t tests were used to examine significance. Correlations between variables were identified using the Spearman's rank correlation coefficient. P-values below .05 were considered as significant. All statistical analysis was performed using GraphPad Prism 8 software (GraphPad, Inc).

| CD38 expression is significantly lower in t(11;14) myeloma, particularly in patients with the immature plasma cell phenotype and B-cellassociated gene expression
The clinical characteristics of the patients are shown in Table 1.
First, we confirmed that CD38 MFI was heterogeneous in the newly diagnosed cases, as it is in relapsed/refractory cases ( Figure 1A). 7,10 As previously reported, CD38 expression was not significantly associated with clinical parameters such as age, sex, lactate dehydrogenase levels, β2-microglobulin levels, and the International Staging System stage (data not shown). 7 In contrast, we found that CD38 MFI was significantly lower in cases with lymphoplasmacytoid morphology, which was identified by Hoyer et al 14 ( Figure 1B). In these patients, the plasma cells were small, with scant cytoplasm and without prominent nucleoli, suggesting a less differentiated plasma cell phenotype. This lymphoplasmacytoid morphology was reportedly associated with t(11;14). 14,15 Indeed, lymphoplasmacytoid morphology occurred only in patients with t(11;14) (in 50% of such cases), which was consistent with previous reports. 14,15 Furthermore, CD38 MFI was significantly lower in patients with t(11;14) compared with those without t(11;14) ( Figure 1C).
CD38 and CD138 expression levels increase during plasma cell development. 22 Notably, CD138 MFI was also significantly lower in patients with lymphoplasmacytoid morphology and with t(11;14) ( Figure 1D). Furthermore, CD38 and CD138 expression levels were significantly positively correlated ( Figure 1E). However, not all patients with t(11;14) showed lower CD38 and CD138 MFI.
Some patients with t(11;14) have been reported to express B-cellassociated genes such as PAX5 and CD79A. 16,17 Silencing of PAX5 is essential for the terminal differentiation of B-cells to plasma cells. However, in our study, PAX5 expression was detected more frequently in patients with t(11;14) ( Figure 1F) and in those with The BCL2/BCL2L1 ratio was inversely correlated with CD38 MFI, and was significantly higher in t(11;14) patients expressing B-cell-associated genes. Asterisks denote significant changes (*.01 ≤ P < .05, **.001 ≤ P < .01, and ***P < .001); ns, not significant.  Figure 1G). Importantly, PAX5 high cases showed significantly lower CD38 expression compared with PAX5 low cases in t(11;14) patients ( Figure 1G). Furthermore, CD38 MFI was significantly lower in patients with high CD79A expression ( Figure 1G). PAX5 high and CD79A high mostly occurred together in patients, although some divergence was observed. PAX5 is a transcription factor of CD19, and therefore, we examined the surface expression of CD19. All our patient samples were negative for surface expression of CD19. We also examined the expression of CD19 mRNA. However, the expression of CD19 mRNA and CD38 MFI and PAX5 mRNA expression did not show a significant correlation. This may be because the expression of PAX5 in t(11;14) myeloma patients is relatively lower than that in normal B-cells, and therefore it does not have sufficient ability to induce the expression of CD19.
In the process of plasma cell development, the expression of CD38, CD138, IRF4, XBP1, and PRDM1 increases, whereas that of CD19, CD20, BCL6, PAX5, and CD79A decreases. 23 Therefore, we also examined other differentiation markers such as CD20, BCL6, XBP1, IRF4, and PRDM1; however, none of these genes was significantly correlated with CD38 MFI. These results suggested that CD38 expression is affected by the t(11;14)-associated immature plasma cell phenotype, particularly when B-cell-associated genes such as PAX5 and CD79A are also expressed.

| The BCL2/BCL2L1 ratio is inversely correlated with CD38 MFI and significantly higher in t(11;14) patients expressing B-cell-associated genes
Venetoclax, a first-in-class BCL2 inhibitor, has shown clinical activity in MM, particularly in patients with t(11;14), who often show high BCL2 expression and low expression of MCL1 and BCL2L1 (encoding Bcl-xL). 24 However, not all t(11;14) patients showed high BCL2 expression, and it remains unclear why t(11;14) patients showed BCL2 dependence. Interestingly, BCL2 expression was significantly higher in PAX5 high cases than in PAX5 low cases among our t(11;14) patients (Figure 2A). In a previous study, it was shown that the expression of BCL2 family members in MM cells is heterogeneous, and upregulation of either MCL1 or BCL2L1 can confer resistance to venetoclax. 25 Indeed, in a recent clinical study, high BCL2/BCL2L1 and/or BCL2/MCL1 mRNA ratios showed a good correlation with the response to venetoclax. 24 Therefore, we analyzed these ratios in our patient samples. Importantly, the BCL2/BCL2L1 and BCL2/MCL1 ratios were also significantly higher in the PAX5 high t(11;14) cases ( Figure 2B). Furthermore, we confirmed that the BCL2/BCL2L1 ratio was significantly higher in the CD79A high cases than in the CD79A low cases ( Figure 2C). Notably, the BCL2/BCL2L1 ratio, but not the BCL2/MCL1 ratio, showed a significant negative correlation with CD38 MFI ( Figure 2D). These results suggested that BCL2 dependence, as well as CD38 expression, is deeply associated with the differentiation and maturation stages of myeloma cells. Patients with t(11;14) expressing B-cellassociated genes are less sensitive to daratumumabinduced direct cytotoxicity but highly sensitive to venetoclax

|
In Figures 1 and 2 Figure 3A). In contrast, the PAX5 high t (11;14) patients were highly sensitive to venetoclax and more so than 11;14) and other patients ( Figure 3B). A similar trend was obtained when t(11;14)-positive cases were divided by cell morphology, because almost all cases with PAX5 expression showed a lymphoplasmacytoid morphology ( Figure S1). These results suggested that the t(11;14)-associated immature phenotype can affect sensitivity to daratumumab and venetoclax.

| ATRA induces CD38 expression and cell differentiation in MM cell lines but decreases the BCL2/BCL2L1 ratio and venetoclax efficacy
ATRA is known to enhance CD38 expression and induce cell differentiation in MM cells. 26 expression levels ( Figure 4C). We confirmed that the sensitivity to daratumumab was increased by ATRA in KMS12BM and NCU-MM1 cells ( Figure S2). In these cell lines, ATRA treatment downregulated PAX5 and CD79A expression ( Figure 4D). Moreover, these cell lines showed a high baseline BCL2/BCL2L1 ratio, whereas ATRA exposure resulted in a lower ratio ( Figure 4E). Consistent with these results, pretreatment with ATRA significantly reduced the efficacy of venetoclax in the KMS12BM and NCU-MM1 cell lines ( Figure 4F). These results indicated that the differentiation and maturation stages of myeloma cells, CD38 expression, and BCL2 dependence are closely related.

| D ISCUSS I ON
Our findings suggested that it may be difficult to obtain rapid direct effects using daratumumab in patients with the t(11;14)-associated immature phenotype, because of their decreased CD38 expression.
Indeed, in the ALCYONE study, a randomized phase 3 trial of bortezomib, melphalan, and prednisone, with or without daratumumab, in patients with newly diagnosed MM, addition of daratumumab failed to improve survival in the non-IgG type. 4 It has been reported that the frequency of IgG type is relatively lower, whereas that of the non-IgG type is relatively higher in t(11;14) cases. 18,28 Therefore, the t(11;14)-associated immature phenotype may be more abundant in the non-IgG type in the ALCYONE study, which may lead to a poor early response to daratumumab. However, the long-term followup data in the ALCYONE study showed improved efficacy for the non-IgG type, suggesting that the durable response, reflecting the immunomodulatory effects, may be induced for cases with t(11;14)associated immature phenotype, as well. Therefore, we recommend the combination of daratumumab and IMiDs, rather than daratumumab, and PIs, for patients with the immature phenotype. This is because PIs are less effective and IMiDs are more effective in immature cases, 29 and IMiDs enhance CD38 expression on myeloma cells and the immunomodulatory effect of daratumumab. 8,9,11  In conclusion, we found that CD38 expression was significantly lower in patients with MM with the t(11;14)-associated immature plasma cell phenotype, particularly those expressing B-cellassociated genes such as PAX5 and CD79A. Furthermore, the BCL2/ BCL2L1 ratio was negatively correlated with CD38 expression and higher in patients with MM with the immature phenotype accompanied by B-cell-associated genes. Our findings may be useful for determining the optimal combination in daratumumab-based treatment, as well as for predicting the treatment response to venetoclax.
Despite being the most frequent chromosomal abnormality in MM, t(11; 14) has not been widely examined. Our findings highlight the importance of evaluating t (11;14) and considering the MM differentiation and maturation stages in treatment strategies.