Immunomodulatory Effect of Proteasome Inhibitors via the Induction of Immunogenic Cell Death in Myeloma Cells.

Several anti-cancer drugs are known to have immunomodulatory effects, including immunogenic cell death (ICD) of cancer cells. ICD is a form of apoptosis which is caused by the release of damage-associated molecular patterns (DAMPs), the uptake of cancer antigens by dendritic cells, and the activation of acquired immunity against cancer cells. ICD was originally reported in solid tumors, and there have been few reports on ICD in multiple myeloma (MM). Here, we showed that proteasome inhibitors, including carfilzomib, induce ICD in myeloma cells via an unfolded protein response pathway distinct from that in solid tumors. Additionally, we demonstrated the potential impact of ICD on the survival of patients with myeloma. ICD induced by proteasome inhibitors is expected to improve the prognosis of MM patients not only by its cytotoxic effects, but also by building strong immune memory response against MM cells in combination with other therapies, such as chimeric antigen receptor-T cell therapy.

GTN057, a komaroviquinone derivative, induced myeloma cells' death in vivo and inhibited c-MET tyrosine kinase.

OBJECTIVE: Despite the development of newly developed drugs, most multiple myeloma (MM) patients with high-risk cytogenetic abnormalities such as t(4;14) or del17p relapse at anin early stage of their clinical course. We previously reported that a natural product,komaroviquinone (KQN), isolated from the perennial semi-shrub Dracocephalum komarovi, i.e., komaroviquinone (KQN) and its derivative GTN024 induced the apoptosis of MM cells by producing reactive oxygen species (ROS), but both exhibited significant hematological toxicity. Aim of this study is to clarify anti-tumor activity, safety and pharmacokinetics of GTN057, an optimization compound of KQN in vivo. METHODS: ICR/SCID xenograft model of KMS11, a t(4;14) translocation-positive MM cell line, was used for in vivo study. Mice pharmacokinetics of GTN057 and the degradation products were analyzed by LC-MS/MS. RESULTS: Herein, our in vitro experiments revealed that GTN057 is much less toxic to normal hematopoietic cells, induced the apoptosis of both MM cell lines andpatient samples, including those with high-risk cytogenetic changes. A xenograft model of a high-risk MM cell line demonstrated that GTN057 significantly delayed the tumor growth with no apparent hematological or systemic toxicities in vivo. The pathological examination of GTN057-treated tumors in vivoshowed revealed apoptosis of MM cells and anti-angiogenesis. In addition to the production of ROS, GTN057 inhibited the downstream signaling of c-MET, a receptor tyrosine kinase a receptor forand hepatocyte growth factor (HGF) receptor. Thus, GTN057 is less toxic and is able tomay be a candidate drug for treating MM patients, via multifunctional mechanisms. We have also extensively studied the pharmacologyical analysis of GTN057. The metabolites of GTN057, (e.g.,such as GTN054), may also have anti-tumorantitumor activity. CONCLUSION: Natural products or and their derivatives can could be good sources of antineoplastic drugs even for high-risk cancer.

[PAX5-positive plasma cell leukemia presenting as lymphocytosis].

An 82-year-old Japanese male patient was initially diagnosed with lymphocytosis. His complete blood count revealed a white blood cell count of 30.9×109/l with 81% abnormal lymphocytes. The abnormal lymphocytes included monoclonal clones of CD38+ and CD138+cytoplasmic κ+ and IgG-κ M-protein, which led to the final diagnosis of plasma cell leukemia (PCL). Bortezomib and dexamethasone therapy was initiated, but the patient succumbed to the disease on the 8th day of hospitalization. A cytogenetic examination revealed a t (9;14)(p13;q32) translocation and the Western blotting confirmed high PAX5 expression. Similar to our present case, PCL cases with "lymphocytosis" have been widely reported, which some speculating the involvement of PAX5 overexpression in the pathogenesis. Such cases, including ours, may be classified as a unique group of disorders (PCL presenting as "lymphocytosis"), which requires accurate differential diagnosis and subsequent urgent multidisciplinary intensive treatment.

Novel Treatment Strategies Utilizing Immune Reactions against Chronic Myelogenous Leukemia Stem Cells.

Introduction of tyrosine kinase inhibitors (TKIs) has improved the prognosis of patients with chronic myelogenous leukemia (CML), and treatment-free remission (TFR) is now a treatment goal. However, about half of the patients experience molecular relapse after cessation of TKIs, suggesting that leukemic stem cells (LSCs) are resistant to TKIs. Eradication of the remaining LSCs using immunotherapies including interferon-alpha, vaccinations, CAR-T cells, and other drugs would be a key strategy to achieve TFR.

[Immunogenic cell death in hematological malignancies].

Anticancer therapies using cytotoxic drugs, which had been considered to be immunosuppressive, could induce immunogenic cell death (ICD) in cancer cells. Anticancer drugs, tumor-lytic viruses, and radiation therapies can establish long-term immune memory via emmision of damage-associated molecular pattern from cancer cells, activating the acquired immunity. This phenomenon could prolong the survival of patients with cancer. ICD was originally reported in solid tumors; however, recent studies have shown that it also occurs in hematological malignancy. Several studies have been trying to combine ICD inducers and other immunotherapies, such as CAR-T-cell therapy. Therefore, treatment strategies should be considered based on drugs' ability to evoke ICD.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Characteristics of a Novel Target Antigen Against Myeloma Cells for Immunotherapy.

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells, and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

A phenylphthalimide derivative, TC11, induces apoptosis by degrading MCL1 in multiple myeloma cells.

To date, the prognosis of multiple myeloma (MM) in patients harboring cytogenetic abnormalities (CA) involving t (4; 14) and deletion of chromosome 17 remains poor despite recent advances in drug development that include the use of immunomodulatory drugs (IMiDs) such as lenalidomide for MM. To address this issue, we have developed a novel phenylphthalimide derivative, TC11, that is structurally related to IMiDs. It remains unclear how TC11 induces apoptosis of MM cells with high-risk CA. Here, we show that TC11 does not induce degradation of CRBN's substrates, IKZF1/3 and CK1α, and induces apoptosis of CRBN-silenced MM; this effect was independent of the cereblon (CRBN) pathway, which is involved in the mechanism of action of IMiDs used for the treatment of MM. We also revealed that TC11, in contrast to existing IMiDs, induced degradation of MCL1 and activation of caspase-9. Furthermore, inhibition of CDK1 by CGP74514A prevented TC11-induced MCL1 degradation, caspase-9 activation, and the subsequent apoptotic cell death. We showed that ectopic MCL1 expression rescued apoptosis of MM. These observations suggest that TC11 induces apoptotic death caused by degradation of MCL1 during prolonged mitotic arrest. Therefore, our findings suggest that TC11 is a potential drug candidate for high-risk MM.

Immunomodulatory Effects of Drugs for Effective Cancer Immunotherapy.

Recent advances in cancer immunotherapy, including immune checkpoint inhibitors or adoptive T cell therapies, have contributed to better outcomes in cancer patients. However, there are still many cancers with no cure. Therefore, combinations of several treatment strategies are being explored, and enhancing anticancer immunity will play an important role to combat the disease. There have been several reports on the immune-modulatory effects of commonly used drugs, namely, statin, metformin, and angiotensin receptor blockers (ARBs), which suggest that these drugs could enhance immunity against cancer cells. Other anticancer drugs, such as anthracyclines, thalidomides, lenalidomides, and hypomethylating drugs, could also strengthen the immune system to attack cancer cells at a relatively low dose. Hence, these drugs might contribute to better outcomes in cancer patients.

A novel derivative (GTN024) from a natural product, komaroviquinone, induced the apoptosis of high-risk myeloma cells via reactive oxygen production and ER stress.

New drugs have significantly improved the survival of patients with multiple myeloma (MM), but the prognosis of MM patients with high-risk cytogenetic changes such as t(4; 14), t(14; 16) or del17p remains very poor. A natural product, komaroviquinone (KQN), was originally isolated from the perennial semi-shrub Dracocephalum komarovi and has anti-protozoal activity against Trypanosoma cruzi, the organism causing Chagas' disease. Here we demonstrate that a novel KQN-derivative, GTN024, has an anti-MM effect both in vitro and in vivo. GTN024 induced the apoptosis of MM cell lines including those with high-risk cytogenetic changes. GTN024 produced reactive oxygen species (ROS) and increased phosphorylated eIF2α. The ROS production and subsequent endoplasmic reticulum (ER) stress are thought to play a key role in GTN024-induced apoptosis, as the apoptosis was completely abrogated by anti-oxidant treatment. In a mouse xenograft model, an intraperitoneal injection of 20 mg/kg of GTN024 significantly delayed tumor growth. Hematological toxicity and systemic toxicity as indicated by weight loss were not observed. These results suggest that the novel KQN-derivative GTN024 could become a candidate drug for treating high-risk MM.

Optimal Anti-cancer Drug Profiles for Effective Penetration of the Anti-cancer Drug Market by Generic Drugs in Japan.

BACKGROUND: The increased use of generic drugs is a good indicator of the need to reduce the increasing costs of prescription drugs. Since there are more expensive drugs compared with other therapeutic areas, "oncology" is an important one for generic drugs. The primary objective of this article was to quantify the extent to which generic drugs in Japan occupy each level of the Anatomical Therapeutic Chemical (ATC) classification system. METHODS: The dataset used in this study was created from publicly available information obtained from the IMS Japan Pharmaceutical Market database. Data on the total amount of sales and number of prescriptions for anti-cancer drugs between 2010 and 2016 in Japan were selected. The data were categorized according to the third level of the ATC classification system. RESULTS: All categories of the ATC classification system had increased market shares in Japan between 2010 and 2016. The barriers to market entry were relatively low in L01F (platinum anti-neoplastics), L01C (plant-based neoplastics), L02B (cytostatic hormone antagonists), and L01D (anti-neoplastic antibiotics) but were high in L02A (cytostatic hormones), L01H (protein kinase inhibitors), and L01B (anti-metabolites). CONCLUSIONS: Generic cancer drugs could bring savings to Japanese health care systems. Therefore, their development should be directed toward niche markets, such as L02A, L01H, and L01B, and not competitive markets.

Anticancer Drug Prescription Patterns in Japan: Future Directions in Cancer Therapy.

BACKGROUND: Despite their benefits, the rapid development of new cancer treatments has been a significant driver of increasing health care expenditures in the face of limited health care budgets. In this study, we analyzed the prescribing trends for anticancer drugs from 2010 through 2016 in Japan and sought to identify unique trends that could provide a basis for future medical economic research aiming to develop more efficacious and cost-effective cancer therapies. METHODS: We used publicly available marketing data for anticancer drugs in Japan for 2010-2016. The drugs selected for this research were categorized according to the Anatomical Therapeutic Chemical Classification System. We investigated the overall anticancer drug market size, the number of anticancer drugs, the top 30 selling anticancer categories, sales and prescription volumes, and changes in sales and prescription volumes between 2010 and 2016 in the country. RESULTS: The anticancer agent market expanded each year from 2010 to 2016, with sales exceeding 1 trillion yen in 2015. The proportion of molecular targeted drugs (antineoplastic mAbs and protein kinase inhibitors) among the top 30 selling anticancer categories has continued to increase, and both the sales and prescription volumes of these drugs exceeded those of drugs in other categories, suggesting that these treatments play a dominant role in cancer pharmacotherapy. CONCLUSION: The availability and increasing use of innovative but more expensive targeted therapies were major drivers of increases in pharmaceutical expenditures for cancer treatment in Japan. Therefore, the effective use of genetic testing can mitigate these rising costs.

A novel phenylphthalimide derivative, pegylated TC11, improves pharmacokinetic properties and induces apoptosis of high-risk myeloma cells via G2/M cell-cycle arrest.

Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.

Synthesis and biological evaluation of the natural product komaroviquinone and related compounds aiming at a potential therapeutic lead compound for high-risk multiple myeloma.

Alternatives of treatments for multiple myeloma (MM) have become increasingly available with the advent of new drugs such as proteasome inhibitors, thalidomide derivatives, histone deacetylase inhibitors, and antibody drugs. However, high-risk MM cases that are refractory to novel drugs remain, and further optimization of chemotherapeutics is urgently needed. We had achieved asymmetric total synthesis of komaroviquinone, which is a natural product from the plant Dracocephalum komarovi. Similar to several leading antitumor agents that have been developed from natural compounds, we describe the antitumor activity and cytotoxicity of komaroviquinone and related compounds in bone marrow cells. Our data suggested that komaroviquinone-related agents have potential as starting compounds for anticancer drug development.

An Empirical Study of the Prescription Pattern of Drugs for Hematological Malignancies in Japan from 2010-2014.

The number of elderly patients with hematologic malignancies has been steadily increasing with the aging of society. However, little research has been conducted to evaluate the prescription status of drugs for such diseases in Japan. Therefore, the aims of this study were to identify the patient population currently being prescribed drugs for hematologic malignancies in Japan and the direction of drug development. To examine the prescription pattern of drugs for the treatment of hematological malignancies in Japan from 2010-2014, we used the IMS Japan Pharmaceutical Market database and the Japanese Society of Hematology Clinical Practice Guidelines, and for drug development status, we used ClinicalTrials.gov and the University Hospital Medical Information Network Clinical Trials Registry. We found a significant upward trend in prescriptions for molecular-targeted agents, which are typically prescribed over the long term, and a significant downward trend in chemotherapeutic agents, which are usually prescribed for the short term. We also found that recent drug development in hematological malignancies has focused on molecular-targeted agents. These results suggest that drug development should be directed toward anti-tumor agents in hematological malignancies that can help maintain and improve patients' QOL.

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies.

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2(271-279)). The CTLs induced by PEPP2(271-279) recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2'-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2'-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 µM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.