Akihiro Kitadate

Histone deacetylase inhibitors, such as vorinostat, show promise as treatment for T‐cell lymphomas including cutaneous T‐cell lymphoma. However, the emergence of resistance ultimately leads to disease relapse. To elucidate the underlying mechanisms and identify potential countermeasures, we established histone deacetylase inhibitor‐resistant cutaneous T‐cell lymphoma cell lines by prolonged exposure to vorinostat. We then comprehensively profiled gene expression in these cell lines by using microarrays and in silico analytical approaches. We identified 83 genes that were significantly upregulated in the resistant cell lines. Subsequent enrichment analyses using ChIP‐Atlas and Enrichr revealed that these genes are regulated by particular transcription factors, including RELA/p65, GATA3, and EP300, of which RELA (p65) exhibited the highest composite score. RELA is a key subunit of the NF‐κB complex, which is involved in inflammation, cell survival, and proliferation. We demonstrated marked upregulation and nuclear enrichment of p65 and pronounced NF‐κB pathway activation in the histone deacetylase inhibitor‐resistant cells. The mechanism involved acetylation‐mediated inhibition of p65 ubiquitination, which resulted in protein stabilization and enhanced transcriptional activity. Histone deacetylase inhibitor‐resistant cell lines displayed heightened sensitivity to inhibition of the NF‐κB pathway by bortezomib and dimethyl fumarate. These findings implicate aberrant NF‐κB activation as a central driver of the emergence of histone deacetylase inhibitor resistance in cutaneous T‐cell lymphoma. Ultimately, our results provide a strong rationale for exploring NF‐κB inhibition as a therapeutic strategy to restore or enhance the efficacy of histone deacetylase inhibitor‐based therapies, overcome histone deacetylase inhibitor resistance, and improve outcomes for patients with cutaneous T‐cell lymphoma.

Summary: Multiple myeloma (MM) with chromosome 1q21 gain/amplification (1q+) has been reported to respond poorly to daratumumab. We aimed to explore the mechanism of daratumumab resistance in 1q+ MM. Our findings revealed significantly lower CD38 expression in patients with 1q+ MM than in those with 1q wild type (WT) MM. Next, we focused on the interleukin‐6 receptor (IL6R) located in the 1q21 region because a previous report shows that interleukin‐6 (IL‐6) reduces CD38 expression via the IL‐6/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway activation in MM. Indeed, IL6R expression was significantly higher in 1q+ MM than in 1q WT MM. We verified that the 1q+ human myeloma cell lines (HMCLs) expressed higher IL6R levels than the 1q WT HMCLs. IL‐6 treatment induced CD38 downregulation in both the 1q+ HMCLs and primary bone marrow (BM) samples but not in their 1q WT HMCLs and BM samples. IL‐6 also resulted in the upregulation of phosphorylated STAT3 in 1q+ HMCLs but not in the 1q WT HMCLs. Furthermore, inhibition of the IL‐6/JAK/STAT pathway by treatment with ruxolitinib or tocilizumab restored CD38 expression in the 1q+ HMCLs and BM samples. These findings elucidate the mechanisms underlying daratumumab resistance in 1q+ MM and provide insights for future therapeutic strategies.

Various tubular diseases in patients with multiple myeloma (MM) are caused by monoclonal immunoglobulin light chains (LCs). However, the physicochemical characteristics of the disease‐causing LCs contributing to the onset of MM‐associated tubular diseases remain unclear. We herein report a rare case of MM‐associated combined tubulopathies: non‐crystalline light chain proximal tubulopathy (LCPT) and crystalline light chain cast nephropathy (LCCN). The patient's urinary κ‐LC (Bence–Jones proteins, BJP‐κ PT‐CN) was detected through immunofixation. Renal biopsy revealed cytoplasmic vacuoles in swollen proximal tubular cells and distal tubular casts. Immunohistochemistry showed proximal tubular reabsorption granules and distal tubular casts positively stained with an anti‐κ‐LC antibody. Electron microscopy identified vacuolation and an increased number of lysosomes in proximal tubular epithelial cells without crystalline structures. Distal tubular casts comprised numerous crystals with both rod‐shaped and needle‐like configurations and tube‐shaped materials. To elucidate the molecular mechanisms underlying tubular toxicity, we performed the following physicochemical analyses of BJP‐κ PT‐CN: N‐terminal amino acid sequencing, cDNA cloning, size‐exclusion chromatography, thermal shift assays, and X‐ray crystallography. The variable segment of BJP‐κ PT‐CN was derived from the IGKV1‐39 gene. The characteristic features of BJP‐κ PT‐CN were a positively charged surface patch, concentration‐dependent monomer‐dimer equilibrium, and the R61G mutation. This is the first biochemical and structural characterization of disease‐causing BJPs in MM‐associated LCPT and crystalline LCCN. The results obtained suggest that these characteristic features enhance protein binding to negatively charged sites on brush‐border membranes in proximal tubules and promote the formation of organized casts in distal tubular lumens.

Background: Multiple myeloma (MM) is a hematopoietic malignancy for which proteasome inhibitors have become available in recent years. However, many patients develop resistance to these drugs during treatment. Therefore, it is important to elucidate the mechanisms underlying resistance acquisition by proteasome inhibitors. Side population (SP) cells, which have a high drug efflux capacity and hypoxic responses in the microenvironment have both provided important insights into drug resistance in MM; however, little is known about the characteristics of SP cells in hypoxic microenvironments.Methods: We performed cDNA microarray analysis for SP and non‐SP obtained from RPMI‐8226 and KMS‐11 cell lines cultured for 48 h in normoxic and hypoxic conditions (1% O₂). Genes specifically upregulated in hypoxic SP were examined.Results: Our comprehensive gene expression analysis identified HMOX1, BACH2, and DUX4 as protein‐coding genes that are specifically highly expressed in SP cells under hypoxic conditions. We have shown that HMOX1/heme oxygenase‐1 (HMOX1/HO‐1) is induced by hypoxia‐inducible reactive oxygen species (ROS) and reduces ROS levels. Furthermore, we found that HMOX1 contributes to hypoxia‐induced resistance to proteasome inhibitors in vitro and in vivo. Excessive ROS levels synergistically enhance bortezomib sensitivity. In clinical datasets, HMOX1 had a strong and significantly positive correlation with MAFB but not MAF. Interestingly, hypoxic stimulation increased MAFB/MafB expression in myeloma cells; in addition, the knockdown of MAFB under hypoxic conditions suppressed HMOX1 expression.Conclusion: These results suggest that the hypoxia‐ROS‐HMOX1 axis and hypoxia‐induced MafB may be important mechanisms of proteasome inhibitor resistance in hypoxic microenvironments.

Despite the introduction of rituximab-containing regimens, approximately 20% of patients with follicular lymphoma (FL) still experience progression of disease within 24 months (POD24) and have poor overall survival. Therefore, a more accurate risk assessment tool is required. We investigated the predictive value of two new volume-based parameters determined from baseline 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT), baseline total metabolic tumor volume (TMTV) and total lesion glycolysis (TLG), in 45 patients with high-tumor-burden FL who underwent baseline PET/CT. We observed that high TMTV, high TLG, and poor initial treatment response (less than complete [metabolic] response [non-CR/CMR] at the end of induction therapy) independently predicted poor PFS. Notably, POD24-positive patients were more common in the high-TLG group than in the high-TMTV group, which suggests that TLG is a stronger predictor of outcomes than TMTV. Combining baseline TLG and initial treatment response showed that patients with both high TLG and non-CR/CMR experienced significantly poorer outcomes, with a 2 year PFS of 0% (hazard ratio 60.39, P = 0.000002). This combination had 56% sensitivity and 100% specificity for detecting patients who would experience POD24. Baseline TLG and initial treatment response can precisely identify patients at high risk of POD24.

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor‐suppressive miRNAs, such as miR‐16, miR‐29, miR‐150, and miR‐26, in advanced cutaneous T‐cell lymphoma (CTCL). Among these, the function of miR‐26 remains unclear. In this study, we aimed to reveal the function of miR‐26 in CTCL oncogenesis. First, we confirmed that the miR‐26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR‐26a‐transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor‐suppressive function. We performed gene expression assays and identified 12 candidate miR‐26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL‐22–STAT3–CCL20–CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR‐26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22‐transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR‐26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL‐22–STAT3–CCL20 cascade. Therefore, a IL‐22‐targeting therapy could be a novel therapeutic strategy for advanced CTCL.

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.