The Humanized Anti-CD40 Antibody SGN-40 Inhibits Tumor Growth in LAGκ-1A, a CD40+ Mouse Model of Human Multiple Myeloma.

Abstract CD40 is a TNF receptor found on the cell surface of mature B cells (B lymphocytes) and most B-cell malignancies including multiple myeloma (MM). SGN-40 is a high-affinity, humanized monoclonal antibody that targets the CD40 antigen. Recently, it has been shown that SGN-40 decreases the proliferation of malignant B cells by partial agonistic signaling and effector functions in vitro. In this study, we examined the anti-MM effects of SGN-40 in vivo using a CD40+ SCID-hu murine model of human myeloma, LAGκ-1A. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A tumor fragment into the left hind limb muscle. The tumor was allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum. Mice were then randomly assigned to one of four SGN-40 treatment groups (6 mice per treatment group). SGN-40 was administered via intraperitoneal injection twice per week at doses of 0.1, 0.3, 1, and 3 mg/kg. Control mice were given a control IgG antibody (3 mg/kg) using the same schedule. Mice receiving the higher doses of SGN-40 showed marked inhibition of tumor growth (0.3 mg/kg, P < 0.02; 1 mg/kg, P < 0.03; and 3 mg/kg, P < 0.04) and reduction of paraprotein levels (1 mg/kg, P < 0.05; and 3 mg/kg, P < 0.03) compared to mice receiving control antibody. At the lowest dose of SGN-40 evaluated (0.1 mg/kg) a slight inhibition of tumor growth was observable, but there was no effect on human paraprotein. Treatment with SGN-40 was not associated with any observed toxicity. Based on these data with SGN-40 monotherapy, we are currently investigating the antitumor activity of SGN-40 plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine model. These data for single-agent SGN-40 are encouraging and support testing SGN-40 both alone and in combination regimens to treat MM patients.

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PK11195, a Ligand of the Peripheral Benzodiazepine Receptor, Inhibits Myeloma Cell Growth In Vitro and Chemosensitizes Myeloma Cells In Vivo in SCID-hu Models of Human Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3459.3459 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3459-3459

Author(s):

Richard A. Campbell ◽

Eric Sanchez ◽

Haiming Chen ◽

Lauren Turker ◽

Olivia Trac ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Tumor Growth ◽

Single Agent ◽

Benzodiazepine Receptor ◽

Peripheral Benzodiazepine Receptor ◽

Once Daily ◽

Inhibition Of Tumor Growth

Abstract The peripheral benzodiazepine receptor (mPBR) appears to be a potential target to induce apoptosis in tumor cells. The expression of this receptor has been linked to a poor prognosis in cancer patients. PK11195 may represent a new, well-tolerated potent chemosensitizing agent that affects multiple resistance mechanisms within malignant cells. We have evaluated whether PK11195 inhibits multiple myeloma (MM) cell growth in vitro; and, furthermore, whether this drug can chemosensitize a melphalan resistant human MM tumor, LAGλ-1 (Campbell et al, International Journal of Oncology 2006), to arsenic trioxide (ATO) and melphalan using an in vivo SCID-hu model. The MM cell lines RPMI8226 and U266 were treated with varying concentrations of PK11195 (1 – 100 mM). After incubating with PK11195 for 24 hours, cell growth was measured by MTT assay. Those cells treated with PK11195 showed decreased proliferation at concentrations as low as 1 mM compared to the untreated cells. Next, we investigated the chemosensitizing effects of PK11195 using an in vivo model of human MM. To accomplish this, each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGλ-1 tumor fragment into the left superficial gluteal muscle. The tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum or when tumors became palpable (21 days) and mice were blindly assigned into treatment groups. PK11195 (10, 50 and 100 mg/kg) was administered via oral gavage once weekly when combined with melphalan and once daily five times per week when combined with ATO. Melphalan (3 mg/kg) was administered once weekly via intraperitoneal (i.p.) injection. ATO (1.25 mg/kg) was administered once daily five times per week via i.p. injection. Mice receiving the combination of PK11195 and melphalan (3 mg/kg) showed marked inhibition of tumor growth (PK11195 10 mg/kg, P = 0.03; PK11195 50 mg/kg, P = 0.02; PK11195 200 mg/kg, P < 0.01) compared to mice receiving no therapy. Animals treated with melphalan, as a single agent, did show minimal tumor growth inhibition and reduced paraprotein levels whereas mice treated with single agent PK11195 showed tumor growth similar to the control mice. Mice receiving the combination of PK11195 and low dose ATO (1.25 mg/kg) also showed inhibition of tumor growth (PK11195 200 mg/kg, P < 0.01) whereas treatment with either single agent PK11195 or ATO demonstrated growth similar to the control groups. Treatment with the highest dose of PK11195 (200 mg/kg) was not associated with any observed toxicity suggesting that high doses can be safely administered and are well tolerated. In this study, we showed PK11195 inhibits MM cell growth in vitro at very low concentrations and can chemosensitize drug resistant tumor cells in vivo at doses that have no observable toxicity. We are further evaluating PK11195 as a single agent and in combination therapy both in vitro and in vivo..

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Blocking Pleiotrophin Activity Inhibits Multiple Myeloma (MM) Cell Growth In Vitro and in a Severe Combined Immunodeficient (SCID)-hu Murine Model of Human MM.

Blood ◽

10.1182/blood.v106.11.114.114 ◽

2005 ◽

Vol 106 (11) ◽

pp. 114-114

Author(s):

Richard A. Campbell ◽

Haiming Chen ◽

Hee Jin Lee ◽

Howard S. Yeh ◽

Melinda S. Gordon ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Tumor Growth ◽

Cell Lines ◽

Murine Model ◽

Fluorescent Protein ◽

Pcr Analysis ◽

Mouse Serum

Abstract Pleiotrophin (PTN) is a heparin-binding growth factor that binds CD138 and stimulates angiogenesis, tumor growth and metastasis in some solid tumors. Recently, we have shown that this factor is highly produced by multiple myeloma (MM) cell lines including RPMI8226 and U266 and fresh malignant plasma cells, and is secreted into the culture medium following short-term culture of bone marrow from MM patients. We investigated the effects of PTN on MM growth in vitro and in vivo using a SCID-hu murine MM model. We determined the anti-proliferative effects of suppressing PTN by cloning a whole PTN sense or anti-sense cDNA construct containing the green fluorescent protein (GFP) gene into the MM cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with vector alone whereas the anti-sense-containing MM cells showed reduced cell numbers. In addition, we treated RPMI8226 and U266 cells with a polyclonal anti-PTN antibody and evaluated its effect on MM growth. These cells were cultured for 48 hours in the presence of the anti-PTN antibody at a concentration of 100 micrograms/ml or a control antibody, and effects on cell growth assessed with an MTT assay. Marked anti-MM effects were observed with the anti-PTN antibody compared to the control antibody in both cell lines [RPMI8226 (p < 0.01) and U266 (p < 0.001)]. In order to further define the importance of PTN in the growth of MM in a more clinically relevant in vivo setting, we determined whether this polyclonal anti-PTN antibody could suppress tumor growth and human paraprotein secretion using our SCID-hu murine model of human myeloma LAGλ-1. LAGλ-1 has been previously shown by our group to produce large amounts of PTN as measured in mouse serum by ELISA and by RT- PCR analysis on freshly isolated LAGλ-1 tumor cells. Thirty SCID mice (n = 5 mice/group) were implanted with a 0.4 – 0.6 cm3 LAGλ-1 tumor fragment into the left hind limb muscle. Fourteen days following implantation, mice were randomized into treatment groups, and received treatment intraperitoneally (IP) with anti-PTN antibody at doses of 0.1, 0.3, 1.0, 3.0 or 10 mg/kg or vehicle alone twice weekly. Mice receiving anti-PTN antibody at the highest doses (3.0 and 10 mg/kg) showed marked inhibition of tumor growth [3.0 mg/kg (p < 0.03), 10 mg/kg (p < 0.008)] as well as decreases in levels of human paraprotein [3.0 mg/kg (p < 0.004), 10 mg/kg (p < 0.003)]. Notably, immunohistochemical staining with an anti-CD138 antibody showed a marked reduction in cells with CD138 positivity in the LAGλ-1 tumors from animals treated with anti-PTN antibody compared to mice treated with vehicle alone. These in vitro and in vivo results demonstrate that PTN may be a potential new target for the treatment of MM. The effects of this therapy on angiogenesis and cell signaling are currently under investigation.

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Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

Acta Pharmacologica Sinica ◽

10.1038/s41401-021-00728-y ◽

2021 ◽

Author(s):

Yu-bo Zhou ◽

Yang-ming Zhang ◽

Hong-hui Huang ◽

Li-jing Shen ◽

Xiao-feng Han ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Targets ◽

Single Agent ◽

Hdac Inhibitors ◽

Preclinical Study ◽

Parp Cleavage ◽

Rpmi 8226 ◽

Ongoing Phase

AbstractHDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

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Bcl-2 antagonist apogossypol (NSC736630) displays single-agent activity in Bcl-2–transgenic mice and has superior efficacy with less toxicity compared with gossypol (NSC19048)

Blood ◽

10.1182/blood-2007-09-113647 ◽

2008 ◽

Vol 111 (6) ◽

pp. 3211-3219 ◽

Cited By ~ 58

Author(s):

Shinichi Kitada ◽

Christina L. Kress ◽

Maryla Krajewska ◽

Lee Jia ◽

Maurizio Pellecchia ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

Parent Compound ◽

Single Agent ◽

Maximum Tolerated Dose ◽

Low Grade ◽

Altered Expression ◽

Cell Counts

Abstract Altered expression of Bcl-2 family proteins plays central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. In this study, we compared the toxicity and efficacy in mice of natural product gossypol and its semisynthetic derivative apo-gossypol, compounds that bind and inhibit antiapoptotic Bcl-2 family proteins. Daily oral dosing studies showed that mice tolerate doses of apogossypol 2- to 4-times higher than gossypol. Hepatotoxicity and gastrointestinal toxicity represented the major adverse activities of gossypol, with apogossypol far less toxic. Efficacy was tested in transgenic mice in which Bcl-2 is overexpressed in B cells, resembling low-grade follicular lymphoma in humans. In vitro, Bcl-2–expressing B cells from transgenic mice were more sensitive to cytotoxicity induced by apogossypol than gossypol, with LD50 values of 3 to 5 μM and 7.5 to 10 μM, respectively. In vivo, using the maximum tolerated dose of gossypol for sequential daily dosing, apogossypol displayed superior activity to gossypol in terms of reducing splenomegaly and reducing B-cell counts in spleens of Bcl-2–transgenic mice. Taken together, these studies indicate that apogossypol is superior to parent compound gossypol with respect to toxicology and efficacy, suggesting that further development of this compound for cancer therapy is warranted.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma

Blood ◽

10.1182/blood-2008-11-191577 ◽

2009 ◽

Vol 114 (2) ◽

pp. 371-379 ◽

Cited By ~ 264

Author(s):

Mariateresa Fulciniti ◽

Pierfrancesco Tassone ◽

Teru Hideshima ◽

Sonia Vallet ◽

Puru Nanjappa ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Murine Model ◽

Severe Combined Immunodeficiency ◽

Neutralizing Antibody ◽

Nuclear Factor Κb ◽

Bone Lesions ◽

Wnt Inhibitor

Abstract Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

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Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽

10.1182/blood-2007-08-105601 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1654-1664 ◽

Cited By ~ 138

Author(s):

Dharminder Chauhan ◽

Ajita Singh ◽

Mohan Brahmandam ◽

Klaus Podar ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitors ◽

Xenograft Model ◽

Er Stress Response ◽

Synergistic Cytotoxicity ◽

Proteolytic Activities ◽

Dose Combination ◽

Inhibition Of Tumor Growth

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

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Inhibition of tumor growth induced by histamine: In vivo and in vitro studies

Agents and Actions ◽

10.1007/bf01996451 ◽

1993 ◽

Vol 38 (3-4) ◽

pp. C175-C177 ◽

Cited By ~ 16

Author(s):

G. P. Cricco ◽

C. A. Davio ◽

R. M. Bergoc ◽

E. S. Rivera

Keyword(s):

Tumor Growth ◽

In Vitro Studies ◽

Inhibition Of Tumor Growth

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Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.1566.1566 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1566-1566

Author(s):

Tiffany Khong ◽

Janelle Sharkey ◽

Andrew Spencer

Keyword(s):

Multiple Myeloma ◽

Murine Model ◽

Dna Methyltransferase ◽

Myeloma Cell ◽

Apoptotic Pathway ◽

P16 Gene ◽

Induced Apoptosis ◽

The Impact

Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

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Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽

10.1182/blood.v108.11.3409.3409 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3409-3409

Author(s):

Paola Neri ◽

Pierfrancesco Tassone ◽

Masood Shammas ◽

Mariateresa Fulciniti ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Growth ◽

Gene Expression Profile ◽

Expression Profile ◽

Murine Model ◽

Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

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