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Extracellular vesicles (EVs) have emerged as immunomodulatory regulators during tumor progression. These small vesicles encapsulate a variety of molecules, including DNA, RNA, and proteins. When EVs come in contact with recipient cells, the EVs transmit various physiological characteristics; for example, proteins on the surface of EVs act as ligands. Immune checkpoint blockade targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has shown promise in a subset of cancer patients. PD-L1 on EVs acts as a key immunomodulator. Suppression of EV secretion enhances the efficacy of immunotherapy using immune checkpoint blockade antibodies. In addition to immune checkpoint blockade therapy, chimeric antigen receptor T (CAR-T) cell therapy has also been used to successfully eliminate cancer cells. Interestingly, CAR-T-cell-derived EVs express CAR on their surface. Compared with CAR-T cells, CAR-expressing EVs do not express PD1, so their antitumor effect cannot be weakened. In this review, we describe the current understanding of EVs in cancer immunity and summarize their crucial roles in immunomodulation.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC. Significance: Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Supplementary Figure from Aberrant Activation of Cell-Cycle–Related Kinases and the Potential Therapeutic Impact of PLK1 or CHEK1 Inhibition in Uterine Leiomyosarcoma

Supplementary Data from Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity and Molecular Features of Ductal Carcinoma In Situ

Purpose:Uterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates.Experimental Design:Transcriptome analysis was performed using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis (IPA) was used to identify potential therapeutic target genes for uterine leiomyosarcoma. Afterward, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using SK-UT-1, SK-LMS-1, and SKN cell lines.Results:We identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The IPA revealed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI-2536 or prexasertib) were found to exert a superior anticancer effect against cell lines at low nanomolar concentrations and induce cell-cycle arrest. In SK-UT-1 tumor-bearing mice, BI-2536 monotherapy remarkably suppressed tumorigenicity. Moreover, the prexasertib and cisplatin combination therapy inhibited tumor proliferation and prolonged the time to tumor progression.Conclusions:We identified upregulated expressions of PLK1 and CHEK1; their kinase activity was activated in uterine leiomyosarcoma. BI-2536 and prexasertib demonstrated a significant anticancer effect. Therefore, cell-cycle–related kinases may present a promising therapeutic strategy for the treatment of uterine leiomyosarcoma.

Supplementary Table from Aberrant Activation of Cell-Cycle–Related Kinases and the Potential Therapeutic Impact of PLK1 or CHEK1 Inhibition in Uterine Leiomyosarcoma

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC.Significance:Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Supplementary Figure from Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity and Molecular Features of Ductal Carcinoma In Situ

Purpose: Uterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates. Experimental Design: Transcriptome analysis was performed using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis (IPA) was used to identify potential therapeutic target genes for uterine leiomyosarcoma. Afterward, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using SK-UT-1, SK-LMS-1, and SKN cell lines. Results: We identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The IPA revealed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI-2536 or prexasertib) were found to exert a superior anticancer effect against cell lines at low nanomolar concentrations and induce cell-cycle arrest. In SK-UT-1 tumor-bearing mice, BI-2536 monotherapy remarkably suppressed tumorigenicity. Moreover, the prexasertib and cisplatin combination therapy inhibited tumor proliferation and prolonged the time to tumor progression. Conclusions: We identified upregulated expressions of PLK1 and CHEK1; their kinase activity was activated in uterine leiomyosarcoma. BI-2536 and prexasertib demonstrated a significant anticancer effect. Therefore, cell-cycle–related kinases may present a promising therapeutic strategy for the treatment of uterine leiomyosarcoma.

Cancer cells secrete extracellular vesicles (EVs) to regulate cells in the tumor microenvironment for their own benefit to grow and survive in the patient’s body. Although emerging evidence has demonstrated the molecular mechanisms of EV release, regulating cancer-specific EV secretion remains challenging. In this study, we applied a microRNA (miRNA) library to reveal the universal mechanisms of EV secretion in cancer cells. Here, we identified miR-891b and its direct target gene, phospho-aminotransferase 1 (PSAT1), which promotes EV secretion through the serine synthesis pathway. Inhibition of PSAT1 affected EV secretion in multiple types of cancer, suggesting that the miR-891b/PSAT1 axis shares a common mechanism of cancer EV secretion. Interestingly, PSAT1 also controlled cancer metastasis via EV secretion. Our data provide insights into the PSAT1-controlled EV secretion mechanism and its potential as a therapeutic target in multi types of cancer.

PurposeUterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates.Experimental DesignTranscriptome analysis was carried out using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis was then used to identify potential therapeutic target genes for uterine leiomyosarcoma. Moreover, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using the SK-UT-1, SK-LMS-1, and SKN cell lines.ResultsWe identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The Ingenuity Pathway Analysis showed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI 2536 or prexasertib) were found to exert a superior anti-cancer effect against cell lines at low nanomolar concentrations and induced cell cycle arrest. In SK-UT-1 tumor-bearing mice, BI 2536 monotherapy demonstrated a marked tumor regression. Moreover, the prexasertib and cisplatin combination therapy also reduced tumorigenicity and prolonged survival.ConclusionWe identified the upregulated expression of PLK1 and CHEK1; their kinase activity was considered to be activated in uterine leiomyosarcoma. BI 2536 and prexasertib demonstrate a significant anti-cancer effect; thus, cell cycle-related kinases may represent a promising therapeutic strategy for treating uterine leiomyosarcoma.Translational relevanceThe development of next-generation sequencing has had an immense impact on cancer research. However, the biological features of uterine leiomyosarcoma are not fully understood. Hence, no effective treatment strategies have been established based on its molecular background. In this research, we were able to assess the transcriptional profiles of 46 patients with uterine leiomyosarcoma using three independent datasets and through the assistance of our cohort. The integrative transcriptional analysis showed that the upregulation and activation of cell cycle-related genes were the dominant features of uterine leiomyosarcoma. Afterward, we demonstrated that PLK1 or CHEK1 inhibition induced cell cycle arrest and caused DNA damage, which resulted in cell death in leiomyosarcoma-derived cell lines. Moreover, these drugs had a more significant anti-cancer effect in the mice model. These data suggest that cell-cycle-dependent kinases represent novel therapeutic targets and could potentially improve the outcome for patients with uterine leiomyosarcoma.

Multiple myeloma (MM) is a hematopoietic malignancy whose prognosis has improved with the development of new agents such as lenalidomide over the last decade. However, long-term exposure to drugs induces the acquisition of resistance by MM cells and leads to treatment failure and poor prognosis. Here, we show the molecular and cellular mechanisms of lenalidomide resistance in MM. In a comparison between lenalidomide-resistant cell lines and the parental cell lines, extracellular vesicle (EV) secretion and adherence abilities were significantly elevated in the resistant cells. Whole-transcriptome analysis revealed that the SORT1 and LAMP2 genes were key regulators of EV secretion. Silencing of these genes caused decreased EV secretion and loss of cell adhesion in the resistant cells, resulting in increased sensitivity to lenalidomide. Analysis of publicly available transcriptome data confirmed the relationship between genes related to EV secretion and cell adhesion and patient prognosis. Together, our findings reveal a novel mechanism of lenalidomide resistance in MM mediated by EV secretion and cell adhesion via SORT1 and LAMP2.

Sarcoma is a rare cancer with several subtypes; therefore, our understanding of the pathogenesis of sarcoma is limited, and designing effective treatments is difficult. Circulating microRNAs (miRNAs), including exosomal miRNAs, have attracted attention as biomarkers in cancer. However, the roles of miRNAs and exosomes in sarcoma remain unclear. The present analysis of tissue and serum miRNA expression in osteosarcoma, Ewing's sarcoma and dedifferentiated liposarcoma (DDLPS) identified miR-1246, -4532, -4454, -619-5p and -6126 as biomarkers for DDLPS. These miRNAs were highly expressed in human DDLPS cell lines and exosomes, suggesting that they are secreted from DDLPS tissues. The present results suggested that specific miRNAs may be used as biomarkers for early diagnosis or treatment targets in DDLPS.

Liquid biopsy is indispensable for the resolution of current medical issues, such as the cost of developing new drugs and predicting responses of patients to drugs. In this sense, not only the technology for liquid biopsy but also the target biomolecules for biomarkers need to be identified. Extracellular vesicles (EVs), which contain various proteins, including membrane-bound proteins, and RNAs, including mRNA and long/short noncoding RNAs, have emerged as ideal targets for liquid biopsy. These complex biomolecules are covered by a lipid bilayer, which can protect them from degradation. In this review, we review current topics regarding EVs as cancer biomarkers and introduce technologies used for these recently emerged biomolecules., Cancer diagnosis: good things come in small packages Extracellular vesicles (EVs), small packets of proteins and RNAs secreted by cells and carried in the bloodstream, are promising diagnostic markers for cancer. Developing new markers that can be collected non-invasively can dramatically improve detection and treatment of cancer. EVs secreted by cells, including tumor cells, send messages to distant cells. EVs collected from blood samples can potentially be excellent disease markers. Takahiro Ochiya and Nobuyoshi Kosaka at Tokyo Medical University in Japan and co-workers have reviewed available EV technology. They report that tumors reliably secrete diagnostic EVs, revealing the origin and state of the tumor. Some EVs can even be used to predict whether a cancer might spread to another tissue. With improvement of methods for isolation and standardization, EVs could provide sensitive markers for early diagnosis of many cancers.

The tenacity of late recurrence of estrogen receptor (ER)‐positive breast cancer remains a major clinical issue to overcome. The administration of endocrine therapies within the first 5 years substantially minimizes the risk of relapse; however, some tumors reappear 10–20 years after the initial diagnosis. Accumulating evidence has strengthened the notion that long noncoding RNAs (lncRNAs) are associated with cancer in various respects. Because lncRNAs may display high tissue/cell specificity, we hypothesized this might provide new insights to tumor recurrence. By comparing transcriptome profiles of 24 clinical primary tumors obtained from patients who developed distant metastases and patients with no signs of recurrence, we identified lncRNA NR2F1‐AS1 whose expression was associated with tumor recurrence. We revealed the relationship between NR2F1‐AS1 and the hormone receptor expressions in ER‐positive breast cancer cells. Gain of function of NR2F1‐AS1 steered cancer cells into quiescence‐like state by the upregulation of dormancy inducers and pluripotency markers, and activates representative events of the metastatic cascade. Our findings implicated NR2F1‐AS1 in the dynamics of tumor recurrence in ER‐positive breast cancers and introduce a new biomarker that holds a therapeutic potential, providing favorable prospects to be translated into the clinical field., We identified the long noncoding RNA NR2F1‐AS1 in the dynamics of tumor recurrence in ER‐positive breast cancers. NR2F1‐AS1 steers cancer cells into quiescence‐like state by the upregulation of dormancy inducers and pluripotency markers, and activates the metastatic cascade. Its expression is mediated by the PR/ER transcriptional complex bringing favorable prospects for developing new therapeutic strategies and introducing a new biomarker.

MicroRNA-based screening identifies the novel regulators of EV secretion from cancer cells., Extracellular vesicles (EVs) are involved in intercellular communication during cancer progression; thus, elucidating the mechanism of EV secretion in cancer cells will contribute to the development of an EV-targeted cancer treatment. However, the biogenesis of EVs in cancer cells is not fully understood. MicroRNAs (miRNAs) regulate a variety of biological phenomena; thus, miRNAs could regulate EV secretion. Here, we performed high-throughput miRNA-based screening to identify the regulators of EV secretion using an ExoScreen assay. By using this method, we identified miR-26a involved in EV secretion from prostate cancer (PCa) cells. In addition, we found that SHC4, PFDN4, and CHORDC1 genes regulate EV secretion in PCa cells. Furthermore, the progression of the PCa cells suppressing these genes was inhibited in an in vivo study. Together, our findings suggest that miR-26a regulates EV secretion via targeting SHC4, PFDN4, and CHORDC1 in PCa cells, resulting in the suppression of PCa progression.

Programmed cell death ligand-1 (PD-L1) overexpressed on cancer cells has emerged as a key inhibitor that maintains the immunosuppressive microenvironment through its interaction with the PD-1 receptor in cancer. Here, we demonstrated that miR-424-5p delivery via extracellular vesicles (EVs) derived from adipose tissue-mesenchymal stromal cells (AT-MSCs) partly promotes proinflammation and enhances antitumor cytotoxicity in vitro and in vivo. Triple negative breast cancer (TNBC) exhibits increased expression of PD-L1, and PD-L1 is positively correlated with the overall survival of patients with TNBC. PD-L1 shows relatively higher expression in MDA-MB-231 (MM231) cells and can be downregulated by miR-424-5p. Furthermore, miR-424-5p transported by EVs can increase the secretion of proinflammatory cytokines, decrease the secretion of anti-inflammatory cytokines and promote the apoptosis of tumor cells. The intratumoral administration of miR-424-5p-EVs significantly slowed tumor growth. In conclusion, these results demonstrate that EVs may serve as a delivery system for novel immunotherapies for TNBC through the miR-424-5p/PD-L1 pathway.

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells and is the second most common hematological cancer. MM frequently occurs in the elderly population with the median age as the middle sixties. Over the last 10 years, the prognosis of MM has been dramatically improved by new therapeutic drugs; however, MM is still incurable. The pathogenesis of MM is still unclear, thus greater understanding of the molecular mechanisms of MM malignancy is desirable. Recently, microRNAs (miRNAs) were shown to modulate the expression of genes critical for MM pathogenesis. In addition, miRNAs are secreted via extracellular vesicles (EVs), which are released from various cell types including MM cells, and these miRNAs are involved in multiple types of cell-cell interactions, which lead to the malignancy of MM. In this review, we summarize the current knowledge regarding the role of miRNA secretion via EVs and of EVs themselves in MM development. We also discuss the potential clinical applications of EVs as promising biomarkers and new therapeutic targets for improving the outcome of MM, resulting in a brighter future for aging societies.

Multiple myeloma (MM) is a plasma cell malignancy that develops by the accumulation of various genetic abnormalities. In recent years, the prognosis of MM has improved by the development of novel drugs including immunomodulatory drugs (IMiDs), proteasome inhibitors, and antibody drugs. However, MM cells acquired drug resistance by long-term exposure to these therapeutic drugs. MM is a multiclonal disease, and various clone subtypes develop within the bone marrow microenvironment. It has been suggested that drug resistant phenotype could transmit from resistant clones to sensitive ones, and also to immune cells, or mesenchymal stem cells, resulting in the change of bone marrow microenvironment suitable for MM cell survival. It has been shown that extracellular vesicles (EVs) are one of the means of information transmission. EVs are secreted from almost all cells, and the amount of EV secretion is particular high from cancer cells. It was already known that cancer-derived EVs transmitted information associated with cancer progressions such as angiogenesis, metastasis, and drug resistance to the surrounding cells. Thus, EVs were proposed to play an important role in acquisition of drug resistance even though the mechanisms have not been fully understood in MM. In order to understand the mechanism of drug resistance in MM mediated by EVs, lenalidomide resistant cell lines were established by long-term exposure of lenalidomide. The amount of EVs was measured by ExoScreen, which is ultra-sensitive detection method of EVs by measuring surface protein of EVs, such as, CD9 and CD63, and by the nanoparticle tracking analysis. We found that lenalidomide resistant cell lines in KMS21R, KMS27R and KMS34R cell lines secreted about twice more EVs than their parental cell lines, and the amount of EV secretion was correlated with the drug sensitivity of lenalidomide. Suppression of EV secretion by knockdown of TSG101, which is known for EV secretion-associated protein, did not affect lenalidomide resistance. We could suppress the EV secretion to two-thirds, however cell proliferation and caspase activity were not change. From these results, we postulated the two possibilities; 1) EV secretion pathway other than TSG101 is associated with drug resistance via EVs; 2) EV derived from lenalidomide resistant cells can affect the cells exist in bone marrow microenvironment. From these hypotheses, we have done the following experiments. Firstly, to identify the genes which involved in EV secretion pathway associated with drug resistance, RNA sequence among the drug-resistant cell lines and their parental cell lines was performed. Drug resistant cell lines had some genetic abnormality, for instance immune system or angiogenesis. Now, we are detecting the EV secretion associated genes in drug resistant cell lines. Secondly, EV derived from the drug-resistant cell lines and EV derived from the parent cell lines were added to drug sensitive MM cell lines, then lenalidomide is added after 24hr. The cell proliferation and apoptosis assay were evaluated after 48hr. EV derived from the drug-resistant cell lines in KMS34R cell line significantly inhibited cell death measured by MTS assay and apoptosis assay compared with those from the drug sensitive KMS21 and KMS34 cell lines. EVs from KMS34R cell line, which is the most progressed cell line we established, could more transmit drug resistance than those from other cell lines. These results suggested that drug resistance was transmitted from drug-resistant cell lines to non-resistant cell lines via EVs. Now, we are analyzing the component of EV from drug-resistant MM cells by proteome analysis to identify the molecules associated with lenalidomide resistance in MM. In addition, we are investigating the molecules which associated with the secretion of EVs from drug-resistance MM. These results prompted us to hypothesize that attenuating the function of a molecule responsible for EV secretion could lead to the inhibition of cancer development such as drug resistance. It is expected that EVs will be novel therapeutic targets in refractory or relapsed MM. Disclosures Hattori: IDAC inc.: Research Funding; Takeda: Research Funding.

【 Background and purpose 】Introduction of novel drugs such as immunomodulatory drugs (IMIDs), and proteasome inhibitors has significantly prolonged survival of multiple myeloma (MM). However, MM harboring high-risk cytogenic changes such as del 17 and t(4;14) is still significantly associated with short survival. Immunotherapy including monoclonal antibodies against MM cells or chimeric antigen receptor expressing T cell (CAR-T) therapy has also showed excellent response to high-risk MM, although the cost of these treatments is expensive. Some of conventional anti-cancer drugs, such as anthracyclines, have been reported to cause induction of immunomodulatory effects by cell surface expression of calreticulin (CRT) followed by release of high mobility group box 1 (HMGB 1) and ATP from cancer cells. Dendritic cells (DCs) recognize CRT, HMGB1 and ATP through CD91, TLR4, P2X7 receptors respectively, and take up cancer cells. This series of events is called immunogenic cell death (ICD). However, there is substantially no information on ICD in MM. The purpose of this study is to investigate whether anti-MM drugs can induce ICD. 【 Methods 】Myeloma cell line harboring high-risk cytogenetic changes, MUM24, KMS34, and KMS21 were treated with anti-myeloma drugs at the IC50, including dexamethasone(320µM), melphalan(2µM), lenalidomide(3.5µM), bortezomib(3nM), carfilzomib(4nM), and panobinostat(6nM). Expression of cell surface CRT and HMGB1 release, which are indicators of ICD, were detected using flow cytometry 48hr after drug treatment and western blotting 24hr after treatment, respectively. We also checked CRT expression on CD138+ cells derived from bone marrow samples of myeloma patients (n=3). Furthermore, the phagocytosis of drug-treated MM cells by DCs generated from healthy human peripheral blood mononuclear cells was evaluated by flow cytometry. 【 Results / Discussion 】Bortezomib and carfilzomib significantly induced expression of CRT and release of HMGB1 in MM cell lines and bone marrow CD138+ cells obtained from MM patients compared with other anti-MM drugs. Especially carfilzomib induced CRT expression at lower concentration. It was also observed that MUM24 cells treated with bortezomib or carfilzomib were effectively taken up by DCs compared to cells treated with other drugs. 【 Conclusion 】Our results suggest that proteasome inhibitors could not only directly kill MM cells but also induce anti-myeloma immune response via ICD. In particular, these immune effects are expected to improve the prognosis of patients in continuous therapy setting after induction therapies by evoking immune memories against residual MM cells, which might contribute to decrease recurrence or formation of extramedullary disease. Moreover, synergistic effects with other cancer immunotherapy such as immune checkpoint inhibitors and CAR-T therapy can be expected. Figure. Figure. Disclosures Hattori: IDAC inc.: Research Funding; Takeda: Research Funding. Matsushita:Amgen: Research Funding.

[Introduction] Recent development of novel drugs significantly improved prognosis of Multiple Myeloma (MM). Immunomodulatory drugs (IMiDs) and proteasome inhibitors play central roles in MM therapy. However expanded usage of lenalidomide (Len) has increased the number of Len-resistant patients. And limited information is obtainable with regand to Len-resistant mechanism, such as overexpression and genetic mutation of IMiDs-binding protein, celeblon (CRBN); thus, elucidating the Len-resistant mechanism and development of drugs overcoming the Len-resistance are very important for improving the outcome of MM. The purposes of this study are [1] to clarify the molecular mechanism of Len-resistance using Len-resistant MM cell lines [2] to confirm the mechanism of CRBN independent myeloma cell death by novel phthalimide-derivatives, TC11 and PEG(E)-TC11. [Method] [1] In our laboratory, Len-resistant cell lines, KMS21R, KMS27R and MUM24R have been established by long-term co-culture with low-dose Len. Using these cell lines, we examined expression of CRBN and the downstream molecules, IKZF1/3, IRF4 and c-MYC by western blotting. We also examined the mutation of CRBN in KMS27R. [2] We have originally developed a novel phthalimide-derivative, TC11 and PEG(E)-TC11 synthesized for improving water solubility. We examined whether TC11 and PEG(E)-TC11 induced cell death to Len-resistance MM or not. [Result] [1] First, we validated expression of CRBN and the down-stream molecules, which mediate pharmacological action of Len. Decreased expression of CRBN and subsequent up-regulation of down-stream IKZF1 were confirmed in KMS21R cell. In KMS27R cell, IKZF 1/3 expressions are increased without alteration of CRBN expression level. Thus, genetic mutation in CRBN or IKZF1 is suspected in KMS27R cells. In MUM24R cell, no significant change in the expression levels of the CRBN pathway molecules was confirmed, suggesting other molecular alternation than CRBN pathway. [2] TC11 significantly induced apoptosis of Len-resistant cells. We have previously reported that TC11 didn't bind to CRBN and TC11 directly bound to nucleophosmin1 (NPM1) and α-tubulin. It was found that TC11 induced G2/M arrest and subsequent apoptosis by inhibition of tubulin polymerization and NPM1 oligomerization. Fluorescence microscopy observation showed that TC11 treatment induced hyper duplication of centrosomes in MM cells. Water solubility and blood absorption of PEG(E)-TC11 were significantly improved compared with those of TC11. As a consequence, PEG-(E)TC11 significantly delayed tumor growth in xenograft model mice. [Discussion & Conclusion] [1] Our present data suggested diversity of Len-resistant mechanism in MM patients. For example, in KMS21R, decreased expression of CRBN was likely the cause of Len-resistance. In KMS27R, genetic mutation in CRBN-IKZF1 pathway caused inhibition of IKZF1 degradation. In MUM24R, the Len-resistant mechanism didn't relate to the CRBN pathway but to unknown molecular mechanism. Len-resistant cell lines are useful tools for studying Len-resistant mechanisms and developing drugs overcoming Len-resistance. [2]TC11 abrogated tubulin polymerization and NPM1 oligomerization, induced centrosome disruption and G2/M arrest. Since G2/M check point doesn't closely rely on p53. TC11 was able to induce apoptosis of MM cells with high-risk cytogenetic mutations such as deletion of TP53 gene. TC11 and PEG-(E)TC11 are expected as a candidate compound overcoming Len-resistance and high-risk MM. Disclosures Matsushita: Amgen: Research Funding. Hattori:Takeda: Research Funding; IDAC inc.: Research Funding.

Background & Objective The therapy of multiple myeloma (MM) has improved greatly over the last decade. However, the rise in health care costs has become a big problem. Therefore, it is necessary to consider the cost-effectiveness including Quality of Life (QoL). In this study, we assessed the real-world cost-effectiveness of MM treatment. Methods & Patients A total of 209 MM patients who were newly diagnosed and treated only at Japanese Red Cross Medical Center (JRC-MC) from January 2006 to December 2015 were registered for this retrospective research. Those patients who were treated at other institute than JRC-MC were excluded. All of the costs for MM treatment in hematology department were analyzed. The costs were calculated based on the health care costs spent in hematology department. Overall survival was evaluated by the Kaplan-Meier method. All of the costs were calculated in US dollars based on average value from 2006 to 2015 (1USD = 100.294 Japanese yen). Result First, the cost paid for treatment of 84 patients who died from symptom onset to death was about 0.14 million USD. We also calculated the total annual health care cost for their patients from 2006 to 2015. Annual cost of all MM patients at 2006 was 0.454 million USD, and the cost at 2015 was 7.505 million USD. The cost/patient-year of 2006, when novel agents were not obtainable was 21,656 USD, and that cost of 2015, when three new agents were available was 55,608 USD. These results showed that the costs of MM treatment became 2.56 times higher in the past 10 years. Individual costs for bortezomib, thalidomide, and lenalidomide is shown in Fig.1. Since 2011 to 2014, the ratio of cost of novel agents exceeded 50%. Secondly, overall survival was evaluated by statistical analysis. The median survival from diagnosis was 56 (range, 0-117) months 95% CI (45.12-66.88). We divided patients into 5 subgroups, according to the time of diagnosis. (1)2006: 21 patients, (2)2007-2008: 26 patients, (3)2009-2010: 38 patients, (4)2011-2012: 42 patients, (5)2013-2015: 82 patients. In Japan, the novel agents such as bortezomib, thalidomide, lenalidomide were approved the year 2006, 2008, 2010 respectively. Median survival of subgroup (1), (2), and (3) were 56 months 95% CI (42.66-69.33), 55 months 95% CI (19.12-90.87) and 54 months 95% CI (36.42-71.57) respectively. Median survival of subgroup (4) and (5) have not been reached. Then we investigated the treatment costs in these groups. In each subgroup the average annual cost was 332, 518, 507, 588 and 631 USD respectively. Compared to subgroup (1), the cost of other groups were significantly high. These results showed that the impact of three novel agents on medical cost were significant. We also investigated the relationship between health care costs and the length of hospital stay or autologous stem cell transplantation. There were no statistically significant differences among them, however, there was tendency that the length of hospital stay shortened by time; Average length of total hospital stay the subgroup (1), (2), and (3) were 205, 125 and 56 days respectively. Subgroup (4) and (5) showed also shortened length of hospital stays, although observation years subgroup (4) and (5) were short (date was not shown). Among 5 subgroups the cases of received autologous stem cell transplantation were not stastically differences (total 56 patients). Conclusion We revealed the real-world costs of MM treatment in a single-institute, by following up individual patients sequentially. Health care costs for treatment of MM increased drastically, by introduction of three novel agents. We also showed that the introduction of three novel agents shorten hospital stay, which leads to improve QoL. Therefore, bortezomib, thalidomide and lenalidomide had the significant impact of QoL. After the further approval of novel agents in Japan, health care costs will be even higher but on the other hand, they might bring positive impacts on OS and QoL. Our study clearly indicated the importance of considering the balance of cost and effectiveness for the MM treatment is important. Character count: 3477(excluding spaces) Disclosures No relevant conflicts of interest to declare.

The fluctuating pressures on two circular cylinders arranged side-by-side to the approaching flow is experimentally investigated at a Reynolds number of 1.87×104. The transverse spacing G between the circular cylinders is 0.5D to 3.25D, where D is the cylinder diameter. Strouhal numbers and dominant components-magnitude and phase-of fluctuating pressures on the cylinders are measured, and the fluctuating drag and lift forces acting on the cylinder are calculated by integrating pressure distributions. The results indicate that the fluctuating forces are very sensitive to spacing, but Strouhal numbers are nearly constant. At the nondimensional spacing G/D=1.25, the amplitude of the fluctuating resultant force is 40 percent greater than that on a isolated cylinder.