A Novel Small Molecule with Epigenetic Activity Prolongs Survival in a Mouse Model of Multiple Myeloma Refractory to Standard Drugs

Abstract Abstract 5121 Multiple myeloma refractory to bortezomib, IMiDs™, and conventional therapies represents an unmet medical need. An increasing number of patients progress to this stage since treatment related mortality has decreased. To test promising compounds for activity in this setting we established an NSG mouse xenograft model with serial transplantation by tail vein injection of myeloma cells from a patient with IgG kappa myeloma relapsed and refractory to all standard drugs. Eight days after tail vein injection monoclonal human IgG can be detected in serum. Bone marrow engraftment in young (6–12 weeks) NSG mice after sublethal radiation (275cGy) is close to 100% (n=32). Untreated mice die within less than 2 months, usually with liver and spleen metastasis (anti-human CD138 flow cytometry). In a drug screen that used a novel method developed in our lab, chromatin condensation PCR, we identified a non nucleoside compound (4I3) that potently (1mM) reactivated expression of epigenetically silenced genes and displayed cancer-specific growth and survival inhibition in myeloma cell lines but not normal cells. Normal bone marrow cells continued to divide at doses 10x higher than required to kill 80% of myeloma cells. 4I3 suppressed DNMT1 protein but rapid cell kill (within 1–2 days) suggested additional mechanisms which we currently investigate. Given IV to mice after documentation of engraftment by IgG serum immunoblots, it prolonged survival in an ongoing experiment. Updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Impaired Lysophosphatidic Acid Receptor 3 Signaling in Mesenchymal Stromal Cells Promotes Multiple Myeloma Progression through Cellular Senescence and Transdifferentiation into Tumor-Associated Fibroblasts

Blood ◽

10.1182/blood.v126.23.1764.1764 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1764-1764

Author(s):

Masahiko Kanehira ◽

Tohru Fujiwara ◽

Shinji Nakajima ◽

Yoko Okitsu ◽

Yasushi Ohnishi ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cellular Senescence ◽

Lysophosphatidic Acid ◽

Research Funding ◽

Xenograft Model ◽

Myeloma Cells ◽

Mouse Xenograft ◽

Mouse Xenograft Model

Abstract Introduction Multiple myeloma (MM) is one of the hematologic malignancies characterized as accumulation of monoclonal tumor of plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma and acquire drug resistance. Stroma is known to secrete soluble factors and express some adhesion molecules advantageous for progression of MM. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) serve as a component of stroma and support growth and drug resistance of myeloma cells. Although the exact etiology has not been clearly defined, it has been suggested that the incidence of MM increases with aging. In this study, we tried to explore the relationship between MM progression and cellular senescence in MSC. Methods Human myeloma cell lines used in this study were IM-9, OPM-2, and RPMI-8226. Human MSC used in this study were obtained from Texas A&M Health Science Center for the Preparation and Distribution of Adult Stem Cells. To establish a mouse xenograft model of human MM, 1.0 x 106 of IM-9 cells were co-injected with 4.0 x 105 of human MSC subcutaneously into the flank of BALB/c nude mice. Visible and palpable tumors were formed in all animals after 20 days. Tumor specimens were resected surgically and diagnosed pathologically as MM based on marker expression. For in vivo bioimaging, luciferase-expressing IM-9 (IM-9-luc+) cells were co-injected with human MSC subcutaneously into BALB/c-nude mice, and photon emission was detected with a sensitive CCD camera 10, 15 and 20 days later. Results In our earlier research, we have demonstrated that the signaling of lysophosphatidic acid (LPA), a bioactive lipid mediator, modulates cellular senescence in MSC (Kanehira et al. PLoS One. 2012). MSC produced autotaxin (ATX), a key enzyme in LPA synthesis, in response to myeloma cells via Toll-like receptor 4 (TLR4)/NF-kappaB-dependent pathway. To determine the LPA receptor responsible for cellular senescence in MSC, six LPA receptors (LPAR1-6) were individually knocked down using siRNA for each receptor. In BrdU incorpotaion assay, LPAR3 gene-silenced MSC (siLPAR3-MSC) were less proliferative than control MSC, and in cell cycle analysis with 7-AAD and Pyronin Y, siLPAR3-MSC was arrested in G1 phase. Interestingly, siLPAR3-MSC exhibited some characteristics of cellular senescence, such as up-regulation of senescence-associated beta-galactosidase activity, increased cell size, and flattened morphology. In a mouse xenograft model of MM, siLPAR3-MSC promoted progression of MM and also tumor-associated angiogenesis. In in vitro study, we confirmed that siLPAR3-MSC easily transdifferentiated into alpha-SMA+ tumor-associated fibroblast and secreted FGF2 in response to myeloma cells. The MM promoting effect and elevated tumor-associated angiogenesis observed in siLPAR3-MSC co-injection were both completely cancelled by FGF2 gene-silencing in siLPAR3-MSC (Figure 1 and 2). Conclusion and discussion In this study, we verified the impairment of LPAR3 signaling may accelerate cellular senescence in MSC. And senesced MSC can provide an advantageous microenvironment for MM progression by FGF2-dependent formation of tumor-stroma milieu. Here, we provide the possibility that LPAR3 signaling could be promising as a therapeutic target in MM. Disclosures Fujiwara: Chugai Pharmaceutical CO., LTD: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding.

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Establishment of a Novel CNS Infiltrated Xenograft Model through Engraftment of Patient-Derived Acute Lymphoblastic Leukemic Cells Into NOD/SCID/γc Null Mouse

Blood ◽

10.1182/blood.v116.21.3248.3248 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3248-3248

Author(s):

Itaru Kato ◽

Akira Niwa ◽

Megumu Saito ◽

Hisanori Fujino ◽

Satoshi Saida ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Hematologic Malignancy ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Leukemic Cells ◽

Null Mouse ◽

Immunodeficient Mice ◽

Mouse Xenograft Model ◽

Nog Mice

Abstract Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.

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Clinical Study of Fludarabine, Mitoxantrone, and Dexamethasone in the Treatment of Refractory or Relapsed Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.5187.5187 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5187-5187

Author(s):

Shao-Kai Luo ◽

Juan Li ◽

Wen-De Hong ◽

Ying Zhao ◽

Xiu-Zhen Tong

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Study ◽

Serum Calcium ◽

Post Treatment ◽

High Recurrence Rate ◽

Treatment Level ◽

Myeloma Cells ◽

Number Of Patients ◽

Pre Treatment

Abstract BACKGROUND & OBJECTIVE: Until today, multiple myeloma is still an incurable malignancy by conventional therapy; it has low complete remission rate and high recurrence rate. Recurrence or relapse of the disease is almost inevitable for most of the patients after several cycles of combined chemotherapy. A safe and effective therapy for the treatment of relapsed and refractory multiple myeloma is urgently needed in clinical practice. This clinical study was designed to compare the safety and efficacy of the fludarabine-based regimen (fludarabine, mitoxantrone and dexamethasone [FMD]) with that of pirarubicin, vincristine and dexamethasone (VAD) in refractory or relapsed multiple myeloma. METHODS: The clinical data were retrospectively analyzed. The following indices were assessed before, during, and after the treatment in FMD-arm and VAD-arm: the partial remission (PR) rate, overall response (OR) rate, time to achieve PR, the number of patients and time to achieve the following: a decline in the serum M-component of more than 50% of the pre-treatment value, the ratio of myeloma cells in bone marrow drop to less than 50% of the pre-treatment value, the ratio of myeloma cells in bone marrow drop to less than 5% or drop more than 80% than pre-treatment level, the hemoglobin level increased more than 20 g/L, the white blood cell and platelet count of the peripheral blood, serum calcium, creatinine, β2-microglobin and ALT level, adverse events. RESULTS: The PR rate and OR rate are significantly higher in the FMD-arm than in the VAD-arm (PR rates were 46.2% vs 22.7% and OR rates were 61.5% vs 31.8%, P <0.05, respectively). The median time to achieve PR was 88 days in the FMD-arm, compared to 68 days in the VAD-arm (P <0.05). Approximately 40.0% of patients in the FMD-arm had >50% decrease in M-component or >20 g/L elevation in hemoglobin, compared to 22.7% and 18.2% in the VAD-arm respectively (P <0.05). The median times to achieve >50% decrease in M-component, decline of >80% of myeloma cells in the bone marrow than the pre-treatment level or ratio drop to less than 5%, or >20g/L elevation in hemoglobin were 62 days, 57 days, and 70 days, respectively. There were no significant differences between groups in serum calcium, creatinine, and ALT level pre-and post-treatment. The level of serum β2-microglobin was (1042.8±72.3 mg/L) post-treatment in the FMD-arm, which was lower than that observed before treatment (2350.2±184.0 mg/L; P <0.05). Most patients (76.9%) in the FMD-arm occurred III/IV grade leukocytopenia while 81.8% patient were I/II grade in the VAD-arm. The incidences of fever and cough were higher in FMD-arm comparing to VAD-arm (P <0.05). The progression free survival (PFS) on 1st and 2nd year and overall survival (OS) on 2nd year were 46.2%, 30.8% and 53.8% in the FMD-arm, while they were 50%, 27.3% and 40.9% respectively in the VAD-arm, which had no significant statistic difference (P >0.05). CONCLUSION: The PR rate and OR rate of FMD-arm are significantly higher than in the VAD-arm in the treatment of refractory or relapsed multiple myeloma, but it took longer time to achieve PR. The regimen of FMD shows no significant renal or hepatic toxicity, it’s a safe and effective regimen in the treatment of refractory or relapsed multiple myeloma.

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Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

Blood ◽

10.1182/blood.v122.21.5333.5333 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5333-5333

Author(s):

Hiroshi Ikeda ◽

Tadao Ishida ◽

Toshiaki Hayashi ◽

Yuka Aoki ◽

Yasuhisa Shinomura

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Contact ◽

Drug Induced ◽

Myeloma Cells

Abstract The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma　(MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

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A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish

Blood ◽

10.1182/blood.v126.23.915.915 ◽

2015 ◽

Vol 126 (23) ◽

pp. 915-915

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Dongdong Ma ◽

Jiantao Shi ◽

Yuji Mishima ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Zebrafish Embryos ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Myeloma Cells ◽

Cell Homing ◽

Qrt Pcr ◽

Hematopoietic Niche

Abstract Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos. We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells. We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR<0.25; P<0.05). Overall, these findings indicate that the changes observed in MM cells that have homed to the CHT mirror those that are seen in MM cells that are resident in the human BM. In order to investigate the functional relevance of the zebrafish model, we established CXCR4-, VLA-4- and FAK-silenced MM cells and compared their ability to home to CHT to that of control scrambled shRNA-transfected cells. DiO-labeled-CXCR4-silenced and DiO-labeled-scrambled-probe control MM cells were mixed in equal numbers and subsequently injected into recipient zebrafish. We found a significant reduction in the number of CXCR4-silenced MM cells homing to the CHT, compared to the control cells (P<0.00). We then examined VLA-4- and FAK-knock-down MM cells and observed that the homing of MM cells to CHT was impaired when either VLA-4 or FAK were silenced (P<0.001; Fig. 3C-D). Having demonstrated the role of CXCF4, FAK and VLA4 in MM cell homing to the CHT niche, we next performed qRT-PCR for those transcripts and confirmed that MM cells harvested from the CHT areas expressed higher levels of CXCR4, FAK and VLA4, compared to MM cells harvested from non-CHT areas. (P<0.05). To ascertain whether homing to zebrafish embryo CHT is occurs in other hematologic malignancies that are known to home to the human and murine BM, we used a cultured cell lined derived from a patient with Waldenstrom's Macroglobulinemia (WM). We injected either CXCR4-overexpressing or CXCR4-silenced WM cells and found that increased CXCR4 expression in WM cells led to enhanced CHT-homing of WM cells (P<0.001), while the homing of CXCR4-silenced WM cells to the CHT was reduced compared to scrambled control (P<0.001). These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. Disclosures No relevant conflicts of interest to declare.

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Gadolinium Containing Contrast Agent Promotes Multiple Myeloma Cell Growth: Implication for Clinical Use of MRI in Myeloma.

Blood ◽

10.1182/blood.v114.22.1809.1809 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1809-1809

Author(s):

Mariateresa Fulciniti ◽

Swaminathan Sundararaman ◽

Puru Nanjappa ◽

Samir B Amin ◽

Prajwal Chevireddy ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Growth ◽

Contrast Agent ◽

Conflicts Of Interest ◽

Bone Lesions ◽

Myeloma Cells ◽

Growth Promoting

Abstract Abstract 1809 Poster Board I-835 Bone marrow infiltration by myeloma cells and osteolytic bone lesions are the major features of Multiple Myeloma. Magnetic Resonance Imaging (MRI) has been used in MM not only to image bone marrow (BM) and to identify lytic bone disease but to also evaluate therapeutic response and prognosis. Gadolinium (Gd)-based contrast agents are frequently used to enhance MRI resolution. We evaluated effect of the most common Gd-containing agent, Omniscan, on myeloma cells. We observed that Omniscan induced both time and dose dependent MM cell growth in vitro (8-20 fold increase relative to control). Importantly, the presence of BMSC enhanced the effect of Omniscan on growth of both MM cell lines and primary MM cells. However, Omniscan was not able to overcome cytotoxic effects of conventional and novel agents in MM. This growth promoting effects were not observed on normal BM stromal cells. Evaluating the molecular mechanism of action of Omniscan on MM cells, we observed time dependent ERK1/2 phosphorylation as well as reversal of growth promoting effects of Omniscan by specific inhibition of ERK signaling; however, Omniscan had no effect on STAT3 and AKT signaling pathways. Next, we investigated in vivo effect of Omniscan in a murine xenograft model of MM. Following detection of tumor, mice were treated with either iv Omniscan or PBS. Treatment with Omniscan significantly induced MM tumor growth compared to control mice (1042 ±243 mm3 vs 502 ±137 mm3 respectively; p=0.0001). Finally in autopsies in 8 MM patients with repeated exposure to Omniscan, we quantified gadolinium in various tissues using Inductively-coupled mass spectrometry. We observed massive quantities of gadolinium accumulation in tissues of these MM patients regardless of their renal function. These results, confirming both in vitro and in vivo growth promoting effects of Gd-containing contrast agent on MM, suggest the need for further analysis of the mechanism of its action on myeloma cells and careful analysis of its clinical impact in MM patients undergoing MRI evaluation. Disclosures No relevant conflicts of interest to declare.

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The Novel ADCC-Optimized Human CD54 (ICAM-1) Antibody MSH-TP15e Has Potent Anti-Myeloma Activity

Blood ◽

10.1182/blood.v128.22.4471.4471 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4471-4471

Author(s):

Katja Klausz ◽

Renate Burger ◽

Christian Kellner ◽

Andreas Guenther ◽

Matthias Peipp ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Cell Lines ◽

Plasma Cells ◽

Xenograft Model ◽

The Novel ◽

Single Chain ◽

Myeloma Cells ◽

Number Of Patients

Abstract Background: Monoclonal antibodies directed against various target antigens have proven efficacy in cancer therapy including hematological malignancies. Recently, daratumumab and elotuzumab became the first antibodies approved for the treatment of multiple myeloma (MM). Due to the substantial number of patients still in need for long-term disease control, the investigation of additional target structures and potent molecule formats for antibody-based therapy of MM remains important. Methods: Human synthetic single-chain fragment variable (scFv) phage display libraries were subjected to a cellular screening approach. The MSH-TP15 clone was selected based on its binding to various MM cell lines and patient-derived CD138-positive malignant plasma cells. Intercellular adhesion molecule-1 (ICAM-1/CD54), known to be important for the interaction of malignant plasma cells with the bone marrow microenvironment, was identified as target antigen. Based on the scFv sequence, fully human IgG1 antibody variants with selected mutations in the Fc domain were constructed to generate wild-type, antibody-dependent cell-mediated cytotoxicity (ADCC)-optimized and Fc-knockout variants. First, these variants were analyzed for their capacity to kill MM cells in vitro. Apoptosis induction and growth inhibition were tested by flow cytometry and in MTS proliferation assays. ADCC and complement-dependent cytotoxicity were investigated in chromium-release assays using human serum, peripheral blood mononuclear cells or purified NK cells of healthy donors. In vivoefficacy of an ADCC-optimized variant was analyzed in the INA-6 myeloma xenograft model. Results: The MSH-TP15 antibodies target an epitope on the N-terminal part of the extracellular domain of human ICAM-1/CD54. The antibody variants showed no anti-proliferative effects on patient-derived bone marrow stromal cells, nor directly induced apoptosis or inhibited proliferation of myeloma cells. While complement-dependent cytotoxic activity was generally absent, the Fc-engineered antibody variant MSH-TP15e significantly triggered ADCC against various MM cell lines and freshly isolated patient myeloma cells. The recruitment of human NK cells was crucial for the cytotoxic effects observed in vitro. Importantly, an ADCC-optimized variant completely prevented tumor engraftment in the INA-6 xenograft model. Conclusions: The novel ADCC-optimized fully human antibody MSH-TP15e directed against ICAM-1/CD54 exerts potent anti-myeloma activity in vitro and in vivo. Therefore, it has promising characteristics and will be further evaluated for MM immunotherapy. Disclosures Guenther: Novartis: Consultancy, Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria.

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Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.3265.3265 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3265-3265

Author(s):

Mridul Roy ◽

Long Liang ◽

Xiaojuan Xiao ◽

Yuanliang Peng ◽

Yuhao Luo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Single Agent ◽

Xenograft Model ◽

Hmgb1 Expression ◽

Resistant Cells

Abstract Multiple myeloma (MM) is the second most prevalent hematologic malignancy, characterized by the infiltration of malignant plasma cells into bone marrow. In spite of current efficient therapeutic regimens, which have significantly increased patients overall survival, the major features inevitably present in MM are the intrinsic and acquired resistance with nearly universal relapse. In addition, the diverse heterogeneous characteristics of this largely incurable disease emphasize the importance of innovative therapies and identification of more effective drugs. Autophagy removes defective cellular organelles, protein aggregates, and intracellular microbes and is associated with cell survival and tumor maintenance. Inhibition of autophagy enhances sensitivity of a number of anticancer agents and induces cell death in MM. High-mobility group box-1 (HMGB1) protein plays an important subcellular localization-dependent role during autophagy. The importance of HMGB1 for induction of autophagy and tumor development has made this protein as a novel target for cancer therapy. Lycorine is a natural alkaloid with significant anti-cancer activity. While previous studies mainly showed lycorine as a potential apoptosis inducer, recent studies stated that apoptosis is not the primary underlying anti-proliferative mechanism of this compound. This led the interest to investigate the role of lycorine on other cell maintenance systems, such as autophagy. In addition single-agent efficacy of lycorine or in combination with other anti-MM agents has not been evaluated in vivo. Herein we investigated the anti-MM effect of lycorine and the role of this natural agent on regulation of autophagy in vitro and in vivo. We found that lycorine inhibits proliferation and induces apoptosis in MM cells with less sensitivity to the normal B-cell at the same concentrations. We also found that lycorine promisingly inhibits autophagy, the mechanism that MM cells use to survive and defeat treatment. We identified HMGB1, an important regulator of autophagy, as the most aberrantly expressed protein after lycorine treatment. Furthermore, we characterized HMGB1 as a critical mediator of lycorine activity against MM. Gene expression profiling (GEP) analysis showed that higher expression of HMGB1 is linked with the poor prognosis of MM. We further confirmed this correlation in human bone marrow CD138+ primary myeloma cells and MM cell lines. Mechanistically, by activating the proteasomal degradation of HMGB1, lycorine induces a rapid turnover of HMGB1. This led to decreased Bcl-2 phosphorylation by MEK-ERK pathway and increased association of Bcl-2 with Beclin-1 resulting in autophagy inhibition and growth attenuation. In addition, we observed higher HMGB1 expression in bortezomib resistant cells. The combination of bortezomib plus lycorine was highly efficient against MM cells and MM cells grown in bone marrow micro-environment. Lycorine showed the capability of inhibiting bortezomib induced autophagy as well as re-sensitizing resistant cells to bortezomib. In agreement with our in vitro observations, in vivo study using human MM xenograft model showed that lycorine is well tolerated, inhibits HMGB1 expression and thereby autophagy and induces enhanced bortezomib activity. These observations indicated lycorine as an effective autophagy inhibitor and revealed that lycorine alone or in combination with bortezomib is a potential therapeutic strategy. Our study supports the feasibility of lycorine in anti-MM treatment and provides a preclinical framework for combining lycorine with bortezomib in clinical setting. Disclosures No relevant conflicts of interest to declare.

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Multiple Myeloma Derived Mitochondrial Damps Induce Inflammation in the Bone Marrow Adipose Tissue Which Promotes Tumour Development

Blood ◽

10.1182/blood-2021-149191 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2654-2654

Author(s):

Aisha Jibril ◽

Charlotte Hellmich ◽

Jamie A Moore ◽

Jayna J Mistry ◽

Stuart A Rushworth ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Mitochondrial Dna ◽

Blood Serum ◽

Disease Progression ◽

Conflicts Of Interest ◽

Secretory Cells ◽

Serum Samples ◽

Myeloma Cells ◽

Nsg Mice

Abstract Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, characterized by the localisation and accumulation of tumour cells in the bone marrow (BM) microenvironment. The mitochondrial DNA (mtDNA) belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients, highlighting a need to explore the functional purpose of mtDNA in the BM microenvironment. Moreover, myeloma relies on the BM microenvironment for survival, proliferation and drug resistance. BM adipocytes are highly secretory cells that support myeloma cells through the release of various factors. It has been shown that myeloma cells induce a shift from osteoblast progenitors towards adipogenesis. Here, we hypothesise that MM cells release mtDAMPs that leads to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion by inducing adipocyte inflammation. First, we engrafted immunocompromised NSG mice with human myeloma cell line MM1S, blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA. We found elevated levels of human mtDNA in the serum of engrafted mice correlating to disease progression. Next, we showed that mtDNA was higher in serum patients with MM compared to healthy controls, moreover, we discovered that BM serum from MM patients had significantly higher levels of mtDNA than blood serum. Furthermore, this observation was recapitulated in myeloma engrafted NSG mice. Next, we examined the effect of mtDNA on various cell populations of the BM. We found that in vitro both BM derived macrophages and BM derived adipocytes had increased inflammatory signatures, including upregulation of IL-6, when treated with mtDNA from myeloma cells. MtDNA induced IL-6 expression was attenuated upon blocking of the TLR9 receptor with antagonist ODN 2088. To understand the role of BM adipocyte induced inflammation in MM, we treated adipocytes with mtDNA before culturing them with MM. We found that MM cells had enhanced proliferation on mtDNA activated adipocytes. We further show that MM cells cultured with mtDNA activated adipocytes have increased uptake of free fatty acids and seahorse analysis confirmed an increase in b-oxidation derived metabolism. Here we establish that MM releases mtDNA into the microenvironment promoting the disease progression via a pro-inflammatory BM. We also show that both BM macrophages and BM adipocytes play a supportive role in multiple myeloma expansion and contribute to the pro-inflammatory bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

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Multiple Myeloma Cells Adhere to Netrin-1 Via Heparin-Sulphate Moieties

Blood ◽

10.1182/blood.v120.21.3952.3952 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3952-3952

Author(s):

Jeesun Park ◽

David R Fooksman ◽

Amitabha Mazumder ◽

Michael L Dustin

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Adhesion ◽

Conflicts Of Interest ◽

Cell Spreading ◽

Myeloma Cell ◽

Axonal Guidance ◽

Intercellular Adhesion Molecule ◽

Myeloma Cells ◽

Guidance Cue

Abstract Abstract 3952 A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells. We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50μM), and VCAM-1(50μM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60–90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well. Disclosures: No relevant conflicts of interest to declare.

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