HDAC Inhibition Induces CD26 Expression on Multiple Myeloma Cells via the c-Myc/Sp1-mediated Promoter Activation.

CD26 is ubiquitously and intensely expressed in osteoclasts in patients with multiple myeloma, whereas its expression in plasma cells of patients with multiple myeloma is heterogeneous because of its cellular diversity, immune escape, and disease progression. Decreased expression levels of CD26 in myeloma cells constitute one of the mechanisms underlying resistance to humanized anti-CD26 mAb therapy in multiple myeloma. In the current study, we show that histone deacetylase inhibition (HDACi) with broad or class-specific inhibitors involves the induction of CD26 expression on CD26neg myeloma cells both transcriptionally and translationally. Furthermore, dipeptidyl peptidase Ⅳ (DPPⅣ) enzymatic activity was concomitantly enhanced in myeloma cells. Combined treatment with HDACi plus CD26mAb synergistically facilitated lysis of CD26neg myeloma cells not only by antibody-dependent cellular cytotoxicity but also by the direct effects of mAb. Of note, its combination readily augmented lysis of CD26neg cell populations, refractory to CD26mAb or HDACi alone. Chromatin immunoprecipitation assay revealed that HDACi increased acetylation of histone 3 lysine 27 at the CD26 promoter of myeloma cells. Moreover, in the absence of HDACi, c-Myc was attached to the CD26 promoter via Sp1 on the proximal G-C box of myeloma cells, whereas, in the presence of HDACi, c-Myc was detached from Sp1 with increased acetylation of c-Myc on the promoter, leading to activation of the CD26 promoter and initiation of transcription in myeloma cells. Collectively, these results confirm that HDACi plays crucial roles not only through its anti-myeloma activity but by sensitizing CD26neg myeloma cells to CD26mAb via c-Myc/Sp1-mediated CD26 induction, thereby augmenting its cytotoxicity. SIGNIFICANCE: There is a desire to induce and sustain CD26 expression on multiple myeloma cells to elicit superior anti-myeloma response by humanized anti-CD26 mAb therapy. HDACi upregulates the expression levels of CD26 on myeloma cells via the increased acetylation of c-MycK323 on the CD26 promoter, leading to initiation of CD26 transcription, thereby synergistically augments the efficacy of CD26mAb against CD26neg myeloma cells.

Rapid Progress in Immunotherapies for Multiple Myeloma: An Updated Comprehensive Review.

Despite rapid advances in treatment approaches of multiple myeloma (MM) over the last two decades via proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies (mAbs), their efficacies are limited. MM still remains incurable, and the majority of patients shortly relapse and eventually become refractory to existing therapies due to the genetic heterogeneity and clonal evolution. Therefore, the development of novel therapeutic strategies with different mechanisms of action represents an unmet need to achieve a deep and highly durable response as well as to improve patient outcomes. The antibody-drug conjugate (ADC), belanatmab mafadotin, which targets B cell membrane antigen (BCMA) on plasma cells, was approved for the treatment of MM in 2020. To date, numerous immunotherapies, including bispecific antibodies, such as bispecific T cell engager (BiTE), the duobody adoptive cellular therapy using a dendritic cell (DC) vaccine, autologous chimeric antigen (CAR)-T cells, allogeneic CAR-natural killer (NK) cells, and checkpoint inhibitors have been developed for the treatment of MM, and a variety of clinical trials are currently underway or are expected to be planned. In the future, the efficacy of combination approaches, as well as allogenic CAR-T or NK cell therapy, will be examined, and promising results may alter the treatment paradigm of MM. This is a comprehensive review with an update on the most recent clinical and preclinical advances with a focus on results from clinical trials in progress with BCMA-targeted immunotherapies and the development of other novel targets in MM. Future perspectives will also be discussed.

Monoclonal Antibody Therapies in Multiple Myeloma: A Challenge to Develop Novel Targets.

The treatment options in multiple myeloma (MM) has changed dramatically over the past decade with the development of novel agents such as proteasome inhibitors (PIs); bortezomib and immunomodulatory drugs (IMiDs); thalidomide, and lenalidomide which revealed high efficacy and improvement of overall survival (OS) in MM patients. However, despite these progresses, most patients relapse and become eventually refractory to these therapies. Thus, the development of novel, targeted immunotherapies has been pursued aggressively. Recently, next-generation PIs; carfilzomib and ixazomib, IMiD; pomalidomide, histone deacetylase inhibitor (HDADi); panobinostat and monoclonal antibodies (MoAbs); and elotuzumab and daratumumab have emerged, and especially, combination of mAbs plus novel agents has led to dramatic improvements in the outcome of MM patients. The field of immune therapies has been accelerating in the treatment of hematological malignancies and has also taken center stage in MM. This review focuses on an overview of current status of novel MoAb therapy including bispecific T-cell engager (BiTE) antibody (BsAb), antibody-drug conjugate (ADC), and chimeric antigen receptor (CAR) T cells, in relapsed or refractory MM (RRMM). Lastly, investigational novel MoAb-based therapy to overcome immunotherapy resistance in MM is shown.

Novel Antibody-Drug Conjugate with Anti-CD26 Humanized Monoclonal Antibody and Transcription Factor IIH (TFIIH) Inhibitor, Triptolide, Inhibits Tumor Growth via Impairing mRNA Synthesis.

Here, we report a novel antibody drug conjugate (ADC) with the humanized anti-CD26 monoclonal antibody YS110 and triptolide (TR-1). YS110 has an inhibitory activity against the CD26-positive tumor growth via the immunological and direct pathway, such as intra-nuclear transportation of CD26 and YS110, and suppressed transcription of RNA polymerase II (Pol II) subunit POLR2A. The ADC conjugated with YS110 and an antitumor compound triptolide (TR-1), which is an inhibitor for TFIIH, one of the general transcription factors for Pol II was developed. YS110 and triptolide were crosslinked by the heterobifunctional linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and designated Y-TR1. Antitumor efficacy of Y-TR1 against malignant mesothelioma and leukemia cell lines were assessed by the in vitro cell viability assay and in vivo assay using xenografted mouse models. Y-TR1 showed significant cytotoxicity against CD26-positive cell lines but not CD26-negative counterparts in a dose-dependent manner via suppression of mRNA synthesis by impairment of the Pol II activity. The tumors in xenografted mice administered Y-TR1 was smaller than that of the unconjugated YS110 treated mice without severe toxicity. In conclusion, the novel compound Y-TR1 showed antitumor properties against CD26-positive cancer cell lines both in vitro and in vivo without toxicity. The Y-TR1 is a unique antitumor ADC and functions against Pol II.

CD26 is a potential therapeutic target by humanized monoclonal antibody for the treatment of multiple myeloma.

CD26, a 110-kDa transmembrane glycoprotein that is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis: however, little is known regarding its role in multiple myeloma (MM). Recently, we identified CD26 expression on human osteoclasts (OCs) and demonstrated that humanized IgG1 monoclonal antibody targeting CD26, huCD26mAb, inhibits human OC differentiation. Herein, we show that CD26 expression was present on plasma cells in the bone marrow tissues of MM patients. In vitro immunostaining studies revealed that although CD26 expression was low or absent on MM cell lines cultured alone, it was intensely and uniformly expressed on MM cell lines co-cultured with OCs. The augmented CD26 expression in MM cells was exploited to enhance anti-MM efficacy of huCD26mAb via a substantial increase in antibody-dependent cytotoxicity (ADCC) but not complement-dependent cytotoxicity (CDC). Moreover, huCD26mAb in combination with novel agents synergistically enhanced huCD26mAb induced ADCC activity against CD26+ MM cells compared with each agent alone. huCD26mAb additionally reduced the ratio of the side population (SP) fraction in CD26+ MM cells by ADCC. Finally, huCD26mAb significantly reduced the MM tumor burden and OC formation in vivo. These results suggest that CD26 is a potential target molecule in MM and that huCD26mAb could act as a therapeutic agent.

Bone-targeted agents in multiple myeloma.

Osteolytic bone disease, characterized by bone pain, increased risk of pathologic fractures, tumor-induced hypercalcemia known as skeletal-related events (SREs), is a frequent complication of patients with multiple myeloma (MM) and persists even in the absence of active disease, resulting in a major cause of morbidity and mortality. The interaction between myeloma cells and their surrounding cells in the bone marrow (BM) microenvironment promotes both myeloma cell growth and bone destruction and forms the vicious cycle of MM bone disease. Therefore, therapeutic strategies targeting the interaction between myeloma cells and cellular components including osteoclasts (OCs), stromal cells and osteoblasts (OBs) in the BM is crucial not only to attain tumor regression but also to prevent or delay the incidence of SREs, which leads to improve survival and quality of life in affected patients. Recently, several novel targets which act on components of the cycle for treating MM-associated bone disease have been identified in addition to current treatments including nitrogen-containing bisphosphonates. This review focuses on the overview of pathophysiology in MM-associated bone disease and summarizes its current clinical management. Several novel bone-targeted agents in preclinical setting will be also discussed.

Primary isolated bone marrow diffuse large B cell lymphoma with long-term complete remission.

Secondary bone marrow involvement of non-Hodgkin's lymphoma (NHL) is relatively common. However, primary isolated bone marrow involvement in NHL which was successfully treated and remains in complete remission (CR) for a long-term duration without any relapse is extremely rare. We herein report a patient of primary bone marrow diffuse large B cell lymphoma (PBML/DLBCL) who presented a prolonged high-grade fever and systemic purpura due to severe thrombocytopenia. The patient was successfully treated with systemic chemotherapy by R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) and remains in CR at 8 years after the initial diagnosis. Review of the literature in PBML/DLBCL cases are also shown.

A humanized anti-CD26 monoclonal antibody inhibits cell growth of malignant mesothelioma via retarded G2/M cell cycle transition.

BACKGROUND: Malignant Mesothelioma (MM) is a highly aggressive tumor with poor prognosis. Multimodal treatments and novel molecular targeted therapies against MM are in high demand in order treat this disease effectively. We have developed a humanized monoclonal antibody YS110 against CD26 expressed in 85 % of MM cases. CD26 is thought to be involved in tumor growth and invasion by interacting with collagen and fibronectin, or affecting signal transduction processes. METHODS: We evaluated the direct anti-tumor effect of YS110 against MM cell lines, NCI-H2452 and JMN, and investigated its effects on cell cycle and on the cell cycle regulator molecules. In addition, we investigated synergistic effects of YS110 and anti-tumor agent pemetrexed (PMX) against MM cell line both in vitro and in vivo. RESULTS: YS110 suppressed the proliferation of NCI-H2452 cells by approximately 20 % in 48 h. Based on cell cycle analysis, percentage of cells in G2/M phase increased 8.0 % on the average after YS110 treatment; in addition, cell cycle regulator p21 cip/waf1 was increased and cyclin B1 was decreased after YS110 treatment. Inhibitory phosphorylation of both cdc2 (Tyr15) and cdc25C (Ser216) were elevated. Furthermore, activating phosphorylation of p38 MAPK (Thr180/Tyr182) and ERK1/2 (Thr202/Tyr204) were augmented at 24 h after YS110 treatment. PMX rapidly induced CD26 expression on cell surface and the treatment with both YS110 and PMX inhibited in vivo tumor growth accompanied by a synergistic reduction in the MIB-1 index. CONCLUSION: This is a first report of a novel anti-proliferative mechanism of the humanized anti-CD26 monoclonal antibody YS110, which resulted in G2/M cell cycle delay through regulation of quantity and activity of various cell cycle regulating molecules.

[Isolated central nervous system relapse in type II enteropathy-associated T cell lymphoma].

A 75-year-old male presented with progressive lower abdominal discomfort. CT scan demonstrated hypertrophy of the intestinal wall, small bowel dilatation, and masses in the descending colon. Biopsy specimens of the jejunum and descending colon revealed widespread distribution of medium-sized atypical lymphocytes with an immunophenotype, positivity for CD3, CD8, CD56, TAI-1, granzyme B and TCRß, but negativity for CD4, CD5, CD20, CD30 and EBER-ISH. Type II enteropathy-associated T cell lymphoma (EATL; Lugano, stage IIE) was diagnosed. Subsequently, he received 6 cycles of chemotherapy with 2/3 dose CHOP and obtained complete remission. However, 18 months after the initial presentation, he presented with rapidly progressive mental deterioration. Gadolinium enhanced T1-weighted brain MR images showed multiple masses with mild heterogeneous enhancement. Brain biopsy revealed necrotic tumors composed of medium-sized atypical lymphocytes, positive for CD3, CD8, CD56, TIA-1, granzyme B and TCRß, but negative for CD4, CD20, and EBER-ISH. CT scan disclosed no evidence of systemic lymphoma relapse, indicating central nervous system relapse of EATL. Despite immediate high-dose chemotherapy with methotrexate, he died of disease progression. EATL is a rare disease with a very poor outcome, for which a validated standard treatment is still lacking. Further studies are needed to identify innovative therapies for treating EATL.

Optic nerve involvement of Waldenström's macroglobulinemia: with autopsy findings.

Waldenström's macroglobulinemia (WM) is an indolent chronic lymphoproliferative disorder within the spectrum of lymphoplasmacytic lymphoma (LPL), characterized by a proliferation of plasmacytoid lymphocytes and the production of monoclonal IgM. Although, peripheral neurologic complications commonly occurs due to hyperviscosity in WM, central nervous system (CNS) involvement is very rare. Herein, we present the case of a 67-year-old man who initially presented with progressive visual loss and was diagnosed as WM/LPL with a very aggressive clinical course. He underwent chemotherapy with high dose methotrexate (MTX) plus cytarabine (Ara-C). However, he died and findings of a subsequent autopsy revealed the presence of lymphoplasmacytoid cells in the optic nerve.

Blockade of CD26 signaling inhibits human osteoclast development.

Bone remodeling is maintained by the delicate balance between osteoblasts (OBs) and osteoclasts (OCs). However, the role of CD26 in regulating bone remodeling has not yet been characterized. We herein show that CD26 is preferentially expressed on normal human OCs and is intensely expressed on activated human OCs in osteolytic bone alterations. Macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (sRANKL) induced human OC differentiation, in association with CD26 expression on monocyte-macrophage lineage cells. CD26 expression was accompanied by increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which is crucial for early human OC differentiation. The humanized anti-CD26 monoclonal antibody, huCD26mAb, impaired the formation and function of tartrate-resistant acid phosphatase (TRAP)/CD26 positive multi-nucleated (nuclei > 3) OCs with maturation in the manner of dose-dependency. It was revealed that huCD26mAb inhibits early OC differentiation via the inactivation of MKK3/6, p38 MAPK and subsequent dephosphorylation of microphthalmia-associated transcription factor (mi/Mitf). These inhibitions occur immediately after RANKL binds to RANK on the human OC precursor cells and were demonstrated using the OC functional assays. huCD26mAb subsequently impaired OC maturation and bone resorption by suppressing the expression of TRAP and OC fusion proteins. In addition, p38 MAPK inhibitor also strongly inhibited OC formation and function. Our results suggest that the blockade of CD26 signaling impairs the development of human functional OCs by inhibiting p38 MAPK-mi/Mitf phosphorylation pathway and that targeting human OCs with huCD26mAb may have therapeutic potential for the treatment of osteolytic lesions following metastasis to alleviate bone destruction and reduce total skeletal-related events (SREs).

Nuclear localization of CD26 induced by a humanized monoclonal antibody inhibits tumor cell growth by modulating of POLR2A transcription.

CD26 is a type II glycoprotein known as dipeptidyl peptidase IV and has been identified as one of the cell surface markers associated with various types of cancers and a subset of cancer stem cells. Recent studies have suggested that CD26 expression is involved in tumor growth, tumor invasion, and metastasis. The CD26 is shown in an extensive intracellular distribution, ranging from the cell surface to the nucleus. We have previously showed that the humanized anti-CD26 monoclonal antibody (mAb), YS110, exhibits inhibitory effects on various cancers. However, functions of CD26 on cancer cells and molecular mechanisms of impaired tumor growth by YS110 treatment are not well understood. In this study, we demonstrated that the treatment with YS110 induced nuclear translocation of both cell-surface CD26 and YS110 in cancer cells and xenografted tumor. It was shown that the CD26 and YS110 were co-localized in nucleus by immunoelectron microscopic analysis. In response to YS110 treatment, CD26 was translocated into the nucleus via caveolin-dependent endocytosis. It was revealed that the nuclear CD26 interacted with a genomic flanking region of the gene for POLR2A, a subunit of RNA polymerase II, using a chromatin immunoprecipitation assay. This interaction with nuclear CD26 and POLR2A gene consequently led to transcriptional repression of the POLR2A gene, resulting in retarded cell proliferation of cancer cells. Furthermore, the impaired nuclear transport of CD26 by treatment with an endocytosis inhibitor or expressions of deletion mutants of CD26 reversed the POLR2A repression induced by YS110 treatment. These findings reveal that the nuclear CD26 functions in the regulation of gene expression and tumor growth, and provide a novel mechanism of mAb-therapy related to inducible translocation of cell-surface target molecule into the nucleus.

Primary B-cell lymphoma of the cauda equina, successfully treated with high-dose methotrexate plus high-dose cytarabine: a case report with MRI findings.

Primary malignant lymphoma of the cauda equina is an extremely rare disease. Previously, there have been only 12 reported cases of malignant lymphoma of the cauda equina, and most cases relapsed early in the clinical course. So, the optimal treatment strategy for this condition has not been established yet and the prognosis is thought to be poor. We experienced a case of B-cell malignant lymphoma of the cauda equina, with rapid progression of the muscle weakness of the bilateral lower extremities, successfully treated with high-dose methotrexate plus high-dose cytarabine (Ara-C) chemotherapy, followed by radiotherapy and in complete remission without any recurrence signs, 1.5 years after the initial diagnosis. Intrathecal chemotherapy with MTX, Ara-C, and predonisolone was simultaneously performed. We should carefully continue to monitor the clinical course of our case, with the examinations of magnetic resonance imaging and cerebrospinal fluid in order not to overlook any subtle neurological changes or other clinical symptoms.

Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia.

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9(+) cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9(-) cells. CD9(+) cells were also serially transplantable in immunodeficient mice, indicating that CD9(+) cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9(+) population. Moreover, CD9(+) cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells.

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.

CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells.

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9(+) cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.

Stem cell properties and the side population cells as a target for interferon-alpha in adult T-cell leukemia/lymphoma.

The cancer stem cell theory suggests that chemoresistance and recurrence of tumors are often due to the similarity of stem cell properties between normal and cancer cells. Adult T-cell leukemia/lymphoma (ATLL) has poor prognosis, suggesting that ATLL cells possess common stem cell properties. We analyzed side population (SP), a characteristic stem cell phenotype, and CD markers in ATLL cell lines. We found that several lines contained SP with expressions of some hematopoietic stem cell markers. On the other hand, treatment with interferon (IFN)-alpha is sometimes effective in ATLL, particularly combined with other drugs. We examined its effect on ATLL cells and found that IFN-alpha significantly reduced the SP proportion. Moreover, CD25-positive cells and phosphorylation of STAT1/5 and ERK were upregulated during this process. These data suggest that their stem cell properties render ATLL cells therapy-resistant, and IFN-alpha exerts its clinical effect through a reduction of the SP cell population.