Suppression of tumor growth by a heterologous antibody directed against multiple myeloma dominant CD38 antigen in SCID mice injected with multiple myeloma cells

Abstract Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in many human tumors including multiple myeloma. We have shown previously using animal models that heparanase promotes robust myeloma tumor growth and spontaneous metastasis to bone. In the present study, the role of heparanase in promoting myeloma bone disease was investigated. CAG human myeloma cells expressing either high or low levels of heparanase (heparanase-high or heparanase-low cells) were directly injected into the marrow cavity of human fetal long bones implanted subcutaneously in SCID mice (SCID-hu model). A second, non-injected human fetal bone was implanted on the contralateral side. Seven weeks after injection of myeloma cells into the primary bone, mice were euthanized and the osteolytic disease of both implanted bones was evaluated. Both X-ray and microCT analysis revealed marked osteolysis in the primary bones injected with heparanase-high cells, with little osteolytic disease detected in the bones injected with heparanase-low cells. Surprisingly, the non-injected, contralateral bones of the animals bearing heparanase-high tumors were also extensively degraded. Immunohistolochemical analysis of these contralateral bones revealed that osteolysis occurred in the absence of detectable tumor cells in the bone. Consistent with this osteolytic phenotype, TRAP staining of the primary and contralateral human bones harvested from mice bearing heparanase-high tumors showed a significant increase in osteoclast numbers, as compared to bones harvested from animals bearing heparanase-low tumors. In a second approach using heparanase-high or heparanase-low cells injected into the tibia of SCID mice, heparanase again enhanced osteolysis at the site of tumor injection as well as at distal sites, in the absence of resident tumor cells. These findings parallel our previously published observation that heparanase expressing breast cancer cells implanted in the mammary fat pad induced an increase in bone resorption in the absence of tumor cells within bone. The evidence in vivo suggested the release from heparanase-high cells of factor(s) that increase osteoclast formation. To test this idea, in vitro osteoclastogenesis assays were used to test the conditioned medium from heparanase-high cells. The conditioned medium from heparanase-high cells significantly enhanced osteoclastogenesis compared to conditioned medium from heparanase-low cells. Interestingly, conditioned medium derived from CAG cells expressing heparanase mutants lacking enzymatic activity failed to enhance osteoclastogenesis. Together, these data demonstrate for the first time that expression of heparanase is a major determinant of the osteolytic phenotype in myeloma. Increased osteolysis is the result of increased osteoclastogenesis that requires active heparanase enzyme and can occur in bones distal to the primary tumor prior to any subsequent metastasis. Thus, we hypothesize that therapies designed to block heparanase function will not only inhibit tumor growth, but may also protect bone from tumor-related bone destruction and possibly disrupt the metastasis of tumor to bone.

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Biallelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells

Cancer Science ◽

10.1111/cas.14386 ◽

2020 ◽

Vol 111 (5) ◽

pp. 1663-1675 ◽

Cited By ~ 1

Author(s):

Jo Kanasugi ◽

Ichiro Hanamura ◽

Akinobu Ota ◽

Sivasundaram Karnan ◽

Vu Quang Lam ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

Akt Signaling ◽

Myeloma Cells

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Bortezomib-Induced Proinflammatory Macrophages Contribute to Multiple Myeloma Cell Aggressiveness

Blood ◽

10.1182/blood.v126.23.5367.5367 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5367-5367

Author(s):

Ofrat Beyar Katz ◽

Neta Ben-Tsedek ◽

Irit Avivi ◽

Dror Alishekevitz ◽

Michael Timaner ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

Host Response ◽

De Novo ◽

Imaging System ◽

Conflicts Of Interest ◽

Scid Mice ◽

Tail Vein ◽

Inflammatory Macrophages

Abstract Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. Previous studies revealed that chemotherapy induces pro-tumorigenic host-mediated effects which could explain tumor re-growth and metastasis(Gingis-Velitski, Loven et al. 2011, Katz, Shaked 2014). Here we show that plasma from bortezomib-treated mice significantly increases migration, viability and proliferation of human MM cells in vitro, compared to plasma from control untreated mice. Comparable results were demonstrated with plasma obtained from patients with MM treated with bortezomib. Additionally, bortezomib induces the mobilization of pro-angiogenic bone marrow cells. Mice treated with bortezomib and subsequently intravenously injected with MM cells succumb to MM aggressiveness earlier than mice treated with the vehicle control(Figure 1). We show that pro-inflammatory macrophages contribute to MM cell aggressiveness in response to bortezomib treatment, in part by secreting interleukin-16(IL-16). Blocking IL-16 in conditioned medium obtained from bortezomib-treated macrophages generated reduced viability of MM cells in vitro. Accordingly, co-inoculation of MM cells with pro-inflammatory macrophages from bortezomib-treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti-tumor activity of bortezomib, this drug can induce host-driven pro-tumorigenic effects that may promote MM aggressiveness. Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system. Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system. Disclosures No relevant conflicts of interest to declare.

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A gp130 Interleukin-6 Transducer-Dependent SCID Model of Human Multiple Myeloma

Blood ◽

10.1182/blood.v91.12.4727.412k35_4727_4737 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4727-4737 ◽

Cited By ~ 1

Author(s):

Cosette Rebouissou ◽

John Wijdenes ◽

Patrick Autissier ◽

Karin Tarte ◽

Valerie Costes ◽

...

Keyword(s):

Multiple Myeloma ◽

Interleukin 6 ◽

Myeloma Cell ◽

Scid Mice ◽

Term Survival ◽

Myeloma Cells ◽

Tumoral Cells ◽

Human Myeloma Cells

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)–related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6–dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.

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Pre-Clinical Validation of Polo-Like Kinase 1 as a Therapeutic Target in Multiple Myeloma with the Selective Inhibitor BI2536.

Blood ◽

10.1182/blood.v110.11.2514.2514 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2514-2514

Author(s):

Roger Sidhu ◽

Tara Steffler ◽

Gail Hipperson ◽

Michelle Jung ◽

Joyce Fung ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Content ◽

Plasma Cells ◽

Annexin V ◽

Myeloma Cell ◽

Selective Inhibitor ◽

Scid Mice ◽

Mitotic Apparatus ◽

Myeloma Cells ◽

Bone Marrow Plasma

Abstract BACKGROUND: The centrosome is the cellular organelle that nucleates the mitotic spindle. Polo-like kinase 1 (Plk1), a centrosome-associated serine/threonine kinase, serves as a key regulator of multiple steps in mitosis. Plk1 is overexpressed in a broad spectrum of tumor types, and its expression often correlates with poor patient prognosis. We have previously observed centrosome amplification in myeloma, and have validated other components of the myeloma centrosome and mitotic apparatus as therapeutic targets. The present study explored the expression of Plk1 in myeloma and the effect of BI2536, a potent and selective inhibitor of Plk1, on myeloma cells in the pre-clinical setting. METHODS: Plk1 expression was assayed in a panel of myeloma cell lines (MMCL) and AutoMACS-purified CD138+ patient bone marrow plasma cells (BMPC). The anti-myeloma effects of BI2536, alone or in combination with conventional agents, were assayed on myeloma cells with proliferation (MTS) and apoptosis (Annexin V/propidium iodide) assays. The phenotype of treated cells was examined with DNA content analysis and immunofluorescence microscopy. The efficacy of BI2536 monotherapy was evaluated in NOD/SCID mice bearing RPMI8226 xenografts. RESULTS: Plk1 is ubiquitously expressed in myeloma to varying degrees in both MMCL and BMPC. BI2536 inhibited the proliferation of MMCL (RPMI 8226, U266, LP-1 and KMS-11) and patient BMPC at nanomolar concentrations. The addition of BI2536 was able to overcome resistance to dexamethasone. Bortezomib in combination with BI2536 had significantly increased anti-myeloma effects compared to the use of either agent alone. BI2536-treated MMCL accumulated 4N DNA content prior to undergoing apoptosis. The phenotype of BI2536-treated cells is consistent with inhibition of Plk1, showing prometaphase arrest and monopolar mitotic spindles in a dose-dependent fashion. BI2536 induces regression of human myeloma xenografts in NOD/SCID mice. Taken together, BI2536 is a promising new agent for the treatment of multiple myeloma. This work provides further evidence that Plk1 and the amplified myeloma centrosome are targets for therapy.

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Autocrine Sonic Hedgehog Protects Multiple Myeloma Cells From Apoptosis and Enhances Drug Resistance

Blood ◽

10.1182/blood.v118.21.2904.2904 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2904-2904

Author(s):

Zhiqiang Liu ◽

Yuhuan Zheng ◽

Haiyan Li ◽

Yong Lu ◽

Donna M. Weber ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Tumor Growth ◽

Signaling Pathway ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Conflicts Of Interest ◽

Myeloma Cells ◽

Apoptosis Rate ◽

Chemotherapy Drugs

Abstract Abstract 2904 The secreted protein sonic hedgehog (SHH) and the hedgehog signaling are of great importance in proliferation and differentiation of cells in the hematopoietic system, and also play a vital role in oncogenesis of B cell malignance. However, the functions and mechanism of SHH signaling in multiple myeloma (MM) is mostly unknown. Thus far, aberrant activation of the hedgehog signaling in tumor growth promoting and/or survival capabilities as well as a paracrine model of SHH secretion have been demonstrated in MM. In the current study, we demonstrated a new autocrine SHH functioning manner in MM cells. The Shh mRNA and the SHH protein were highly expressed both in the MM cell lines and in purified CD138+ MM cells from patients using real-time PCR, Western Blot and immunohistochemistry analyses, respectively; and the SHH protein was also detected in the culture medium. Accordingly, the Hh ligand receptor PTCH1 and PTCH2 as well as the transcriptional factor GLI1 were all overexpressed in MM cells, indicating the activation of Hh signaling pathway. Autocrine SHH played a role in MM cells survival and protected MM cells from apoptosis in vitro, and autocrine SHH accelerated xenograft tumor growth in myeloma-SCID mouse model in vivo. Moreover, autocrine SHH enhanced drug resistance of MM cells, as SHH overexpressed CAG cells (SHH+CAG) had a significantly low apoptosis rate when treated with chemotherapy drugs dexamethasone or bortezomib, as compared with wild type cells (wt-CAG). On the contrary, SHH knockdown cells (SHH-CAG) had a dramatically higher apoptosis rate. Blocking autocrine SHH ligand and treating cells with dexamethasone or bortezomib significantly improved the drug killing effect. Finally, we found that upregulated BLC2 via SHH-Gli1signaling is the signaling pathway by which MM cells enhanced the drug resistance. Our study provides a new insight into the biologic function of the autocrine SHH in proliferation, survival and the drug resistance in the myeloma cells. Disclosures: No relevant conflicts of interest to declare.

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Targeting CD47/TNFAIP8 by miR-155 overcomes drug resistance and inhibits tumor growth through induction of phagocytosis and apoptosis in multiple myeloma

Haematologica ◽

10.3324/haematol.2019.227579 ◽

2019 ◽

Vol 105 (12) ◽

pp. 2813-2823 ◽

Cited By ~ 6

Author(s):

Nasrin Rastgoo ◽

Jian Wu ◽

Mariah Liu ◽

Maryam Pourabdollah ◽

Eshetu G. Atenafu ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Tumor Growth ◽

Myeloma Cell ◽

Therapeutic Modality ◽

Drug Resistant ◽

Myeloma Cells ◽

Advanced Stages

The mechanisms of drug resistance in multiple myeloma are poorly understood. Here we show that CD47, an integrin-associated receptor, is significantly upregulated in drug resistant myeloma cells in comparison with parental cells, and that high expression of CD47 detected by immunohistochemistry is associated with shorter progression free and overall survivals in multiple myeloma patients. We show that miR-155 is expressed at low levels in drug resistant myeloma cells and is a direct regulator of CD47 through its 3'UTR. Furthermore, low miR-155 levels are associated with advanced stages of disease. MiR-155 overexpression suppressed CD47 expression on myeloma cell surface, leading to induction of phagocytosis of myeloma cells by macrophages and inhibition of tumor growth. MiR-155 overexpression also re-sensitized drug-resistant myeloma cells to bortezomib leading to cell death through targeting TNFAIP8, a negative mediator of apoptosis in vitro and in vivo. Thus, miR-155 mimics may serve as a promising new therapeutic modality by promoting phagocytosis and inducing apoptosis in patients with refractory/relapsed multiple myeloma.

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CSF1R-Inhibition and Reduction of Macrophages Delays Multiple Myeloma Growth in a Non-T-Cell-Dependent Manner

Blood ◽

10.1182/blood.v124.21.5717.5717 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5717-5717

Author(s):

Fredrik H. Schjesvold ◽

Ole Audun Haabeth ◽

Bjarne Bogen ◽

Anders Tveita

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Tumor Growth ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

M1 Macrophages ◽

Myeloma Cells ◽

Tumor Killing ◽

Stimulating Factor

Abstract Macrophages are important tumor-promoting cells, and can constitute up to half of the tumor mass, orchestrating remodeling, inducing angiogenesis and suppressing the immune system to terminate local inflammatory responses. Colony stimulating factor (CSF-1), also known as macrophage colony-stimulating factor (M-CSF), is the most important cytokine involved in survival, proliferation and differentiation of tissue macrophages and their precursors. Previous experiments from our group has shown that amount of CSF-1 in the tumor bed correlates with successful rejection of cancer cells in a TCR-transgenic model where CD4+ T cells recognize a tumor specific antigen produced by multiple myeloma cells. We therefore tested if reduced signaling via CSF-1R, and thereby a decrease in tumor infiltrating macrophages, would results in decreased growth of multiple myeloma cells. . PLX3397, a tyrosine kinase inhibitor with specificity for CSF1R and KIT, has been demonstrated to delay tumor growth in a CD8+ T-cell-dependent manner after chemotherapeutic treatment in a murine model of mammary carcinogenesis. Here, we show that MOPC315 multiple myeloma cells implanted subcutaneously in matrigel plugs in SCID mice become infiltrated with M2 phenotype macrophages that enhances their growth. When given chow containing PLX3397, recruitment of macrophages is strikingly reduced, with significant delay in tumor growth. Importantly, this delay in tumor development is not T cell dependent, since the anti-tumor effect is seen in T cell deficient SCID-mice as well as in BALB/c mice. Importantly, treatment with PLX3397 does not abrogate efficient tumor killing, and does not affect survival, when tested in a TCR-transgenic model where CD4+ T cells via induction of M1 macrophages reject multiple myeloma. In conclusion, PLX3397 delays tumor growth by reducing amounts of tumor infiltrating M2 macrophages, while rendering effective tumor killing by M1 macrophages uninhibited. Thus, drugs that inhibit signaling of CSF-1 could be of value in treatment of multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

10.1101/575159 ◽

2019 ◽

Author(s):

Marita Westhrin ◽

Toril Holien ◽

Muhammad Zahoor ◽

Siv Helen Moen ◽

Glenn Buene ◽

...

Keyword(s):

Gene Therapy ◽

Multiple Myeloma ◽

Bone Formation ◽

Tumor Growth ◽

Trabecular Bone ◽

Bone Morphogenetic Protein ◽

Bone Morphogenetic Protein 4 ◽

Myeloma Cells ◽

Morphogenetic Protein ◽

Virus Serotype

AbstractMultiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre- and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4-treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2−/−γC−/−mice. Mice were treated with adeno-associated virus serotype 8 BMP4 vectors (AAV8-BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4-treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non-myeloma femurs by μCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8-CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent.

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A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cdk4/6.

Blood ◽

10.1182/blood.v108.11.369.369 ◽

2006 ◽

Vol 108 (11) ◽

pp. 369-369 ◽

Cited By ~ 1

Author(s):

Linda B. Baughn ◽

Maurizio Di Liberto ◽

Kaida Wu ◽

Peter Toogood ◽

Tracey Louie ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Tumor Growth ◽

Small Molecule ◽

Critical Role ◽

Cellular Transformation ◽

G1 Arrest ◽

Specific Inhibition ◽

Myeloma Cells ◽

Orally Active

Abstract Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow (BM). Inhibition of Cdk4 and Cdk6 by the Cdk inhibitor, p18(INK4c), is required for the generation of normal, functional plasma cells1. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of Cdk4-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of BM myeloma cells in vivo2. These data identify Cdk4 and Cdk6 as key determinants in the loss of cell cycle control in myeloma and suggest that Cdk4/6 may be effective targets for therapeutic intervention. Here we show that by specific inhibition of Cdk4/6, the orally active small molecule PD 0332991 potently induces G1 arrest in primary BM myeloma cells ex vivo, and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation, and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B cell cancers.

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