ALBERT

Description: Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-κB. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10−5M – 5x10−9M and melphalan concentrations ranging from 3x10−5M – 3x10−9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10−5 M – 5x10−10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAGλ–1 intramuscular (IM) tumor, LAGλ–1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAGλ–1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAGλ–1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by 〉50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.

Description: Inorganic arsenics like arsenic trioxide (ATO) are novel anti-cancer drugs active in acute promyelocytic leukemia (APL) and multiple myeloma (MM). ATO induces apoptosis of plasma cells by several mechanisms including down-regulation of BCL-2 expression and inhibition of DNA-binding by NF-κB. The amount of ATO that can be safely given is low because of QTc-prolongation. ZIO-101, a new organic arsenic, is in phase-1/-2 trials. ZIO-101 can be safely given at much higher doses than ATO. We evaluated the in vivo anti-myeloma activity of ZIO-101 in a SCID-hu mouse model of human myeloma. LAGλ-1 was developed from a person with melphalan-resistant and LAGλ-1B from a person with bortezomib-resistant myeloma. Each severe combined immunodeficient (SCID) mouse was implanted with a 2–4 mm3 fragment of LAGλ-1B or LAGλ-1 into the left superficial gluteal muscle. Fragments were allowed to grow for 14 d when human IgG was first detectable. ZIO-101 was given IV 1 or 2 times/d using three different schedules: one day/w, 3 days/w or 5 days/w at doses of 50– 200 mg/kg/d. Tumor volume and human IgG were assessed weekly. Doses up to 200 mg/kg were well-tolerated. Anti-myeloma effects were observed in both models at doses of 100 – 200 mg/kg on all 3 schedules. Mice receiving 100 mg/kg twice daily thrice weekly and those receiving 200 mg/kg once weekly showed marked anti-myeloma activity (100 mg/kg, P = 0.03; 200 mg/kg, P = 0.001) and reduced human IgG levels (100 mg/kg, P 〈 0.001; 200 mg/kg, P = 0.01) compared to controls. We are evaluating ZIO-101 in other SCID-hu models of human myeloma and exploring different doses and schedules of ZIO-101 alone or combined with other anti-myeloma agents. In summary, these data show activity of ZIO-101 in human myeloma in vivo. These studies provide the bases for future clinical trials.

Description: 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.

Description: The mammalian target of rapamycin (mTOR) is an intracellular protein that acts as a central regulator of multiple signaling pathways (IGF, EGF, PDGF, VEGF, amino acids) that mediate abnormal growth, proliferation, survival and angiogenesis in cancer. mTOR is a critical component of the PI3K/Akt pathway, a key cell survival pathway that is dysregulated in many cancers including multiple myeloma (MM). mTOR is an important therapeutic target because it is a “rate-limiting” bottleneck in the key signaling pathway that regulates cell survival, proliferation, and angiogenesis. RAD001 (everolimus) is a novel oral mTOR pathway inhibitor. Recent data suggests that RAD001 has direct effects on tumor cell proliferation and may have antiangiogenic activity due to inhibition of tumoral VEGF production and effects on vascular endothelial and smooth muscle cell biology. We first evaluated the in vivo effects of single agent RAD001 in mice bearing the human MM tumor LAGλ-1. Tumor-bearing mice receiving RAD001 at 3, 10, or 30 mg/kg once daily five times per week (M-F) via oral gavage showed marked inhibition of tumor growth at all doses (P

Description: Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. We previously evaluated the effects of single agent mouse/human anti-VEGF antibody G6.31 (Campbell et al, Blood (ASH Annual Meeting Abstracts), Nov 2006) in several of our mouse models of human MM. In this study, we evaluated the effects of the same anti-VEGF antibody in combination with bortezomib or lenalidomide. Severe combined immunodeficient (SCID) mice were implanted into the left superficial gluteal muscle with either a 2.0 – 4.0 mm3 fragment from a patient when she was bortezomib-sensitive, LAGκ-1A, or resistant, LAGκ-1B. The tumors were allowed to grow for 21 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups (n=10 mice/group). Treatment groups consisted of a control IgG antibody or anti-VEGF antibody administered via i.p. injection twice weekly at a dose of 2 mg/kg, bortezomib administered via intravenous injection at a dose of 0.25 or 0.5 mg/kg twice weekly, lenalidomide administered via i.p. injection at a dose of 50 mg/kg daily × 5 per week, anti-VEGF antibody (2 mg/kg) + bortezomib (0.25 or 0.5 mg/kg), and anti-VEGF (2 mg/kg) + lenalidomide (50 mg/kg). Mice receiving the combination therapy of anti-VEGF + bortezomib (0.5 mg//kg) antibody showed marked inhibition of tumor growth and reduction of paraprotein levels compared to mice receiving control antibody. Notably, this combination also produced much more marked anti-MM effects compared to bortezomib treatment alone, anti-VEGF antibody alone, or vehicle alone. This combination was well tolerated. In contrast, mice receiving the combination of anti-VEGF antibody + lenalidomide showed no significant differences in tumor volume or hIgG levels compared to single agent treatment or vehicle alone. The markedly improved anti-MM effects of the combination of bortezomib and anti-VEGF antibody compared to single agent treatment in this in vivo study of human MM is promising, and these results have provided the preclinical rationale for an ongoing randomized multi-center Phase II trial.

Description: Histone deacetylase (HDAC) inhibitors represent a new mechanistic class of anti-cancer therapeutics that inhibit HDAC enzymes and have been shown to have anti-proliferative effects in cancer cells (including drug resistance subtypes), induce apoptosis, inhibit angiogenesis, and sensitize cancer cells when combined with other available anti-cancer therapies. PXD101 is a novel investigational small molecule drug that selectively inhibits HDAC enzymes. In recent preclinical studies, PXD101 has been shown to have the potential to treat a wide range of solid and hematological malignancies either as a monotherapy or in combination with other active agents. In this study, we evaluated the activity of PXD101 on multiple myeloma samples when used as monotherapy or in combination with the proteasome inhibitor bortezomib. In vitro experiments indicated that PXD101 pretreatment (20 mM; 3h) sensitized RPMI-8226 human multiple myeloma cells to subsequent bortezomib exposure (5 nM; 72h). To examine PXD101 and bortezomib in vivo, two mouse models of human multiple myeloma were utilized (LAGλ-1 and LAGκ-1B). LAGλ-1 was generated from a patient resistant to melphalan therapy and LAGκ-1B from a patient who progressed on bortezomib treatment (Campbell et al, International Journal of Oncology 2006). SCID mice were implanted with LAGλ-1 or LAGκ-1B tumor fragments into the left superficial gluteal muscle. Tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum, and mice were randomly assigned into treatment groups. Groups consisted of Vehicle only, PXD101 alone (40 mg/kg), bortezomib alone (0.5 mg/kg), or PXD101 (40 mg/kg) + bortezomib (0.5 mg/kg). In one cohort, PXD101 and bortezomib were administered twice weekly (M, Th) and in another cohort PXD101 was administered 5 days a week (M-F) and bortezomib twice weekly (M, Th). When administered, PXD101 was given i.p twice daily and bortezomib once daily intravenously. The results of these animal experiments will provide preclinical information on the activity of PXD101 monotherapy and PXD101/bortezomib combination therapy on drug-resistant myeloma samples, and may help to define the optimal schedule for potential clinical evaluation of this drug combination.

Description: 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..

Description: Glutathione levels have previously been shown to be associated with the development of resistance to a variety of anti-myeloma therapies. Ascorbic acid (AA) depletes intracellular glutathione levels which, in turn, should increase the sensitivity of tumor cells to anti-myeloma agents such as arsenic trioxide (ATO) and melphalan. To determine the synergistic effects of combining AA, with ATO and/or melphalan, we evaluated the effects of these combinations with MTT assays on myeloma cell lines in vitro and using our severe combined immunodeficient (SCID)-hu murine myeloma models. We determined the synergistic effects of combining AA with ATO and/or melphalan on the myeloma cell lines RPMI8226, 8226/dox, U266, and U266/dox in vitro. MTT assays demonstrated marked synergistic anti-proliferative effects of AA at 10 mM when added to these cell lines in the presence of ATO concentrations ranging from 5x10−5 M – 5x10−9 M, and melphalan concentrations ranging from 3x10−5 M – 3x10−9 M. In order to provide further evidence for the clinical relevance of these synergistic effects of AA, we investigated the potential of AA to increase the efficacy of current anti-myeloma therapies in our SCID-hu murine model of human myeloma LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Twenty-eight days following implantation, mice then received treatment intraperitoneally (IP) with either AA (300 mg/kg) daily x5/week, ATO (1.25 mg/kg) daily x5/week, or melphalan (3.0 mg/kg) x1/week, or the combination of these agents. AA, ATO, and melphalan alone have no anti-myeloma effects at these doses, whereas AA+melphalan results in significantly decreased tumor burden and paraprotein levels. The most profound anti-myeloma effects were observed in animals treated with all three drugs together. These data show not only the additional synergistic anti-myeloma effects of AA on both ATO and melphalan in vitro but for the first time suggest that these effects are also present in vivo. This provides the rationale for combining AA with these agents in myeloma patients with resistant disease. In support of this, early results of clinical trials using the combination of AA, ATO and low doses of oral melphalan are promising.

Description: Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. Based on these findings, we evaluated a new mouse/human anti-VEGF antibody using our SCID-hu mouse models of human MM. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A 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, and mice were blindly assigned into one of two treatment groups. In one group, anti-VEGF antibody was administered via intraperitoneal injection twice per week at a dose of 5 mg/kg. In the other cohort, control mice were given a control IgG antibody (5 mg/kg) on the same schedule. Mice receiving the anti-VEGF antibody showed marked inhibition of tumor growth (P = 0.0005) and reduction of paraprotein levels (P = 0.0002) compared to mice receiving control antibody. On day 42, LAGκ-1A-bearing mice receiving the anti-VEGF antibody showed a 70% reduction in human paraprotein levels and an 80% decrease in tumor volume compared the control antibody treated animals. Treatment with the anti-VEGF antibody was not associated with any observed toxicity. We are currently evaluating this anti-VEGF antibody in several of our mouse models of human MM and plasma cell leukemia. Based on these data with anti-VEGF monotherapy, we are currently investigating the anti-tumor activity of anti-VEGF antibody plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine models. Preliminary results are encouraging with single agent anti-VEGF antibody and additional studies may be used to direct the clinical development of anti-VEGF antibody treatment alone and in combination regimens for patients with relapsing or refractory MM.

Description: 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.