Myeloma Xenograft Models Research Articles

Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047

Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

Combined Treatment with the Mek Inhibitor PD0325901 and Arsenic Trioxide Has Potent Antitumor Activity in Vivo against Human Multiple Myeloma Xenograft Model.

Despite recent advances in therapy, Multiple myeloma (MM) remains incurable because of the high resistance to apoptosis and both intrinsic and acquired drug resistance. Therefore, new therapeutic strategies are needed to improve patient outcome. We recently demonstrated that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD0325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in MM cells through a multiple modulation of apoptotic regulatory proteins, including p53 family proteins, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, several Bcl-2 family proteins and caspases, that depend on the functionality of the p53 pathway (Blood prebublished on line june 26, 2008). Furthermore, we also demonstrated that PD plus ATO treatment induces early tumor (volume approximately 200 mm3) regression, prolongs survival and is well tolerated in vivo in a human plasmacytoma xenograft model. The aim of this study was to investigate whether the combined treatment with PD and ATO is effective in animals with more advanced tumors; thus we used a murine model in which MM RPMI 8226 cells were injected subcutaneously into NOD-SCID mice and when the tumors reached approximately 1000mm3, mice were randomized (n=6/group) to receive vehicle or PD0325901 at 10 mg/kg administered by oral gavage or ATO (3.75 mg/kg) injected intraperitoneally or PD/ATO on a 5-days-a-week schedule for 3 consecutive weeks. Treatment of RPMI 8226 MM-advanced tumor-bearing mice with PD0325901 (10mg/kg) significantly reduced MM-tumor growth as compared to control (P <.01 Tukey-Kramer test), ATO (3.75 mg/kg) had minimal effect on the growth of tumors, which increased as in control mice. Importantly, when PD (10mg/kg) was combined with ATO (3.75mg/Kg), there was a significant reduction in tumor size and growth rate relative to untreated or PD treated mice (P<.001 for PD/ATO versus control, and P<.01 for PD/ATO versus PD Tukey-Kramer test). The combination of PD and ATO (3.75mg/Kg) significantly prolonged survival compared with treatment with either drug alone and was well tolerated in vivo because no differences in body weight and general appearance was noted in mice during the treatment. We next investigated the in vivo effects of the drug combination on proliferation and apoptosis; whole tumor-cell tissues and tumor lysates from mice treated for five days (n=2/group) were subjected to immunohistochemical staining and immunoblotting to assess in vivo phosphorylation of ERK, the proliferative antigen, Ki-67, and cleaved caspase-3. Tumor tissues from PD0325901 (10mg/kg) treatments resulted in profound p-ERK inhibition compared with tumor tissues from vehicle control or ATO-treated animals. In agreement with these data, a significant decrement in the number of Ki-67 positive plasma cells was noted in tumor sections from PD-treated mice relative to tumors from mice receiving either vehicle control or ATO (3.75mg/Kg) treatment alone thereby confirming the tumors growth retardation observed in PD-treated mice. Either PD (10mg/kg) or ATO (3.75mg/Kg) alone did not increase caspase activation compared with tumors from control cohorts. However, the combination PD/ATO dramatically activated caspase-3 in advanced tumors. Notably, consistent with our previous in vitro study demonstrating the involvement of the Bim pathway in MM PD/ATO-induced apoptosis, immunoblotting of MM tumors form PD plus ATO-treated mice showed an elevated ratio of proapoptotic Bim to antiapoptotic Mcl-1 compared with treatment with either drug alone. Collectively, our previous and present findings suggest that combining PD with ATO induces both cytostatic and cytotoxic responses in vivo, resulting in regression of early or advanced tumors, prolongs survival in vivo, and is well tolerated in vivo. In conclusion, our preclinical in vivo studies provide the framework for testing PD0325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

Efficacy of selective JAK inhibition in models of leukemia, lymphoma, myeloma, and JAK2V617F dependent malignancy

14564 Background: The increased kinase activity frequently observed in cancer is implicated in tumorigenesis and resistance to therapy. Genetic and epigenetic changes resulting in aberrant activation of the Janus kinase (JAK) family of tyrosine kinases is common in numerous hematological malignancies, including myeloproliferative disorders. As such, JAK inhibition may be a promising therapeutic strategy for the treatment of hematological diseases associated with JAK activation. Methods: Potent (IC50 < 10nM) and selective (∼100-fold against >30 kinases) JAK inhibitors were utilized to treat a number of engineered and spontaneous cancer cell lines derived from hematological malignancies in vitro and in vivo. The impact of JAK inhibition on downstream signaling pathways (e.g. STAT) and growth and survival of malignant cells was evaluated. Results: Constitutively phosphorylated STAT3/5 (pSTAT) was observed in nearly all cell lines tested. JAK inhibitors reduced pSTAT levels by > 80% in most cell lines. In some cell lines (e.g. INA-6, TF-1, BaF3JAK2V617F) viability was reduced by > 90% and was accompanied by apoptosis. Following oral administration of JAK inhibitors, pSTAT levels and tumor growth were inhibited (> 90%) in a mouse xenograft model of multiple myeloma. Moreover, JAK inhibition improved the therapeutic responses to a number of therapeutics in this model. Additionally, we demonstrate that JAK activation, through clinically relevant cytokine signaling pathways, conveys resistance ( > 50 fold) to imatinib in a Bcr-Abl expressing cells. Importantly, we show that this resistance is reversed with JAK inhibition in vitro and in vivo, the latter resulting in increased survival, suggesting this mechanism of resistance is surmountable. Conclusions: Aberrant STAT activation has been observed in a number of hematological malignancies. Here we have shown that selective JAK inhibition is sufficient to reduce pSTAT levels and inhibit the growth and survival of malignant cells. Moreover, we have demonstrated that JAK inhibition restores sensitivity to Abl kinase inhibition in CML cells rendered resistant to treatment. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Incyte Incyte

ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor

Overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) is commonly associated with tumor maintenance, progression, and chemoresistance. We previously reported the discovery of ABT-737, a potent, small-molecule Bcl-2 family protein inhibitor. A major limitation of ABT-737 is that it is not orally bioavailable, which would limit chronic single agent therapy and flexibility to dose in combination regimens. Here we report the biological properties of ABT-263, a potent, orally bioavailable Bad-like BH3 mimetic (K(i)'s of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioavailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulation. ABT-263 disrupts Bcl-2/Bcl-xL interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours posttreatment. In human tumor cells, ABT-263 induces Bax translocation, cytochrome c release, and subsequent apoptosis. Oral administration of ABT-263 alone induces complete tumor regressions in xenograft models of small-cell lung cancer and acute lymphoblastic leukemia. In xenograft models of aggressive B-cell lymphoma and multiple myeloma where ABT-263 exhibits modest or no single agent activity, it significantly enhances the efficacy of clinically relevant therapeutic regimens. These data provide the rationale for clinical trials evaluating ABT-263 in small-cell lung cancer and B-cell malignancies. The oral efficacy of ABT-263 should provide dosing flexibility to maximize clinical utility both as a single agent and in combination regimens.

Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy

Drug resistance remains a critical problem in the treatment of patients with multiple myeloma. Recent studies have determined that Notch signaling plays a major role in bone marrow (BM) stroma-mediated protection of myeloma cells from de novo drug-induced apoptosis. Here, we investigated whether pharmacologic inhibition of Notch signaling could affect the viability of myeloma cells and their sensitivity to chemotherapy. Treatment with a gamma-secretase inhibitor (GSI) alone induced apoptosis of myeloma cells via specific inhibition of Notch signaling. At concentrations toxic for myeloma cell lines and primary myeloma cells, GSI did not affect normal BM or peripheral blood mononuclear cells. Treatment with GSI prevented BM stroma-mediated protection of myeloma cells from drug-induced apoptosis. The cytotoxic effect of GSI was mediated via Hes-1 and up-regulation of the proapoptotic protein Noxa. In vivo experiments using xenograft and SCID-hu models of multiple myeloma demonstrated substantial antitumor effect of GSI. In addition, GSI significantly improved the cytotoxicity of the chemotherapeutic drugs doxorubicin and melphalan. Thus, this study demonstrates that inhibition of Notch signaling prevents BM-mediated drug resistance and sensitizes myeloma cells to chemotherapy. This may represent a promising approach for therapeutic intervention in multiple myeloma.

Homoharringtonine inhibits the AKT pathway and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells

Involvement of phosphatidylinositol 3-kinase/Akt-1 in cell survival and proliferation of multiple myeloma (MM) has been well established. In this study, we demonstrate that homoharringtonine (HHT), an antileukemic drug first isolated from the Chinese evergreen Cephalotaxus harringtonia, induces significant cytotoxicity in dexamethasone-sensitive and -resistant and chemotherapy-sensitive MM cell lines in a time and dose-dependent manner. HHT also triggers apoptosis in chemotherapy-resistant patient's myeloma cells. Contrary to dexamethasone, the cytotoxicity of HHT on myeloma is independent of interleukin-6. The mechanism of HHT cytotoxicity is related to down-regulation of Akt phosphorylation/activation and various target genes of Akt including nuclear factor κ B, XIAP, cIAP and cyclin D1. Moreover, in vivo antitumor activity of HHT is demonstrated in RPMI8226 myeloma xenograft model. Importantly, an additive effect of antitumor is confirmed in the myeloma cells treated with HHT and bortezomib concomitantly with inhibition of phosphorylated Akt. Together, these findings obtained with HHT should give useful insights into a novel antimyeloma chemotherapy.

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Modulation of Gene Expression Profile and In Vivo Anti-Myeloma Activity Induced by Valproic Acid, a Histone Deacytylase Inhibitor.

Valproic acid (VPA) is a well-tolerated anticonvulsant drug that has been recently recognized as powerful histone deacetylase (HDCA) inhibitor. VPA induces hyperacetylation of histone H3 and H4 and inhibits both class I and II HDCACs. Recently it has been shown that VPA exerts in vitro and in vivo anti-tumor activity against solid cancers and its in vitro anti-Multiple Myeloma (MM) activity has been previously reported. However, the molecular mechanisms are still unclear. Here we have investigated molecular changes induced by VPA as well as its in vivo activity in murine models of MM. We first studied the in vitro activity of VPA against IL-6 independent as well as IL-6 dependent MM cells. A time- and dose-dependent decrease in proliferation and survival of MM cell lines was observed (IC50 in the range of 1–3 mM). Gene expression profile following treatment with VPA at 2 and 5 mM showed down-regulation of genes involved in cell cycle regulation, DNA replication and transcription as well as up-regulation of genes implicated in apoptosis and chemokine pathways. The signaling pathway analysis performed by Ingenuity Systems Software identified the cell growth, cell cycle, cell death as well as DNA replication and repair as the most important networks modulated by VPA treatment. We next evaluated the in vivo activity of VPA using two xenograft models of human MM. A cohort of SCID mice bearing subcutaneous MM1s or OPM1 were treated i.p. daily with VPA (200 mg/kg, and 300 mg/kg, n=5 mice, respectively), or vehicle alone (n=5 mice) for 16 consecutive days. Tumors were measured every 2 days, and survival was calculated using the Kaplan Mayer method. Following VPA treatment, we found a significant (p=0.006) inhibition of tumor growth in mice bearing subcutaneous MM-1s cells treated with VPA at 200 mg/kg compared to control group, which translated into a significant (p= 0.002) survival advantage in the VPA treated animals. Similar results were obtained in animals bearing subcutaneous OPM1 cells. Flow cytometry analysis performed on retrieved tumor tissues from animals showed reduction of G2-M and S phase in tumor specimens following VPA treatment, versus untreated tumors, strongly suggesting in vivo effects of VPA on cell cycle regulation. Taken together, our data demonstrate the in vitro and in vivo anti-tumor activity of VPA, delineate potential molecular targets triggered by this agent and provide a preclinical rationale for its clinical evaluation, both as a single agent or in combination, to improve patient outcome in MM.

Preclinical Pharmacology and Toxicology of Intravenous MV-NIS, an Oncolytic Measles Virus Administered With or Without Cyclophosphamide

MV-NIS is an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS). Here, we report the results of preclinical pharmacology and toxicology studies conducted in support of our clinical protocol "Phase I Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or Refractory Multiple Myeloma." Dose-response studies in the KAS-6/1 myeloma xenograft model demonstrated a minimum effective dose of 4 x 10(6) TCID50 (tissue culture infectious dose 50)/kg. Toxicity studies in measles-naive squirrel monkeys and measles-susceptible transgenic mice were negative at intravenous doses up to 10(8) and 4 x 10(8) TCID50/kg, respectively. Abundant viral mRNA, maximal on day 8, was detected in cheek swabs of squirrel monkeys, more so after pretreatment with cyclophosphamide. On the basis of these data, the safe starting dose of MV-NIS for our clinical protocol was set at 1-2 x 10(4) TCID50/kg (10(6) TCID50 per patient).

Antitumor activity of fludarabine against human multiple myeloma in vitro and in vivo

Fludarabine, a nucleoside analogue, plays a major role in the treatment of B-cell lymphocytic leukemia, hairy cell leukemia, and indolent lymphomas. There is a controversy about antitumor activity of fludarabine in multiple myeloma (MM). The aim of this study was to evaluate the activity of fludarabine against human myeloma cells both in vivo and in vitro. We demonstrated that myeloma cell line RPMI8226 was efficiently inhibited by fludarabine, concomitantly with decreased phosphorylation of Akt, down-regulation of the inhibitor of apoptosis proteins (IAP) family, including XIAP and survivin, and induction of apoptosis related to activation of caspase cascade. Contrary to dexamethasone, the effect of fludarabine on RPMI8226 cells was independent of interleukin-6. Fludarabine also induced cytotoxicity in dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) cells at 48 h with IC50 of 13.48 microg/mL and 33.79 microg/mL, respectively. In contrast, U266 cells were resistant to fludarabine. Moreover, RPMI8226 myeloma xenograft model was established using severe combined immunodeficient mice. The tumors treated with fludarabine at 40 mg/kg increased less than 5-fold in 25 d comparing with approximately 10-fold in the control tumors, demonstrating the antitumor activity of fludarabine in vivo. These results suggest that fludarabine may be an important therapeutic option for MM patients who are resistant to dexamethasone.

A Single-Chain Fv Diabody against Human Leukocyte Antigen-A Molecules Specifically Induces Myeloma Cell Death in the Bone Marrow Environment

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma.

2-Methoxyestradiol in Combination with Velcade® Results in an Augmentation of NFκB Inhibition, In Vitro Antiproliferative Activity and In Vivo Antitumor Efficacy in the RPMI-8226 Myeloma Model.

2-Methoxyestradiol (2ME2, Panzem®) is an endogenous metabolite of estradiol with antiproliferative, proapoptotic, and antiangiogenic activity. Some of these activities are mediated through the downregulation of HIF1-α. Panzem® capsules have been evaluated in several oncology clinical trials, including multiple myeloma where some patients have been on therapy for more than 4 years with stable disease. A new formulation of 2ME2 with enhanced absorption, Panzem® NCD, is currently in Phase 1 and 2 clinical studies in oncology. In anticipation of combination studies with Panzem® NCD, we evaluated the effectiveness of 2ME2 alone or with the proteosome inhibitor, Velcade® (bortezomib), in the RPMI-8226 myeloma xenograft tumor model. Preliminary in vitro studies indicated significantly increased antiproliferative activity when 2ME2 (1.5μM) was combined with Velcade® (5nM) compared to Velcade® alone (p<0.05 ANOVA). Additionally, 2ME2 in combination with Velcade® also resulted in decreased nuclear NFκB in response to TNFα, as compared to either agent alone. Subsequent to these studies, female CB17 SCID mice were injected subcutaneously with 1 x 106 RPMI-8226 tumor cells. Starting 10 days after tumor cell inoculation when mean tumor volume was approximately 100–200 mm3, cohorts of mice (n=8/group) began treatment with (a) vehicle alone ad libitum in the drinking water; (b) 2ME2 300 mg/kg ad libitum in the drinking water; (c) Velcade® 1.5 mg/kg i.p., biw; or (d) 2ME2 (300 mg/kg) in combination with Velcade® (1.5 mg/kg). On study day 27, mean tumor volume in vehicle-treated, control mice was approximately 3400 mm3. Under these experimental conditions, treatment with either 2ME2 alone or Velcade® alone generated a treatment/control (T/C) value of 0.66 and 0.64, respectively. In comparison, 2ME2 in combination with Velcade® resulted in a T/C value of 0.35. Velcade® is a well recognized and commonly used antineoplastic agent in multiple myeloma. The degradation of HIF1-α is accomplished through the proteosome. These studies demonstrate that a combination strategy using Velcade® and 2ME2 can enhance the antitumor effectiveness of either agent alone in the treatment of a xenograft model of human multiple myeloma.

Update on Phase I Clinical Trial of IPI-504, a Novel, Water-Soluble Hsp90 Inhibitor, in Patients with Relapsed/Refractory Multiple Myeloma (MM).

Introduction: IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. In contrast, IPI-504 exists as a hydrochloride salt which is soluble in water in excess of 200 mg/mL. Therefore, IPI-504 is 4000-fold more soluble than 17-AAG. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium which has been observed in human clinical trials as well. In preclinical animal studies, IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma (MM).Methods: An open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in patients (pts) with relapsed/refractory MM. IPI-504 is administered to pts in 250cc of normal saline over thirty minutes. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. Levels of Hsp70 protein were measured as a marker of IPI-504's biologic activity in peripheral blood leukocytes. Toxicity is evaluated by NCI CTCAE (v3.0) and response by EBMT. An accelerated dose titration design was employed with dose escalation of 100% for the first three dose levels with 1 pt / cohort. The first three pts received 22.5, 45 and 90 mg/m2 respectively. Per protocol, once the 150 mg/m2 level was achieved, dose escalation converted to a modified Fibonacci schema with 3 pts per cohort. Subsequently dose escalation occurred in increments of 66%, 50%, 33% and 25%.Results: Currently, 15 pts (11 female / 4 male), average age 61.3 years, have been treated. IPI-504 has been well-tolerated at doses ranging from 90 to 300 mg/m2. Patients continue to be enrolled at the 400 mg/m2 dose level. Dose limiting toxicities (DLTs), defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment, have not been observed to date. Dose escalation will continue until a recommended phase 2 dose is identified. Analysis of PK, safety, tolerability, and biologic response to IPI-504 is ongoing. The above support the planning of further clinical exploration of IPI-504 including combination with agents such as bortezomib that have proven synergistic in animal models of MM.

Efficacy of an Antagonistic Anti-CD40 Monoclonal Antibody, HCD122 (CHIR-12.12), in Preclinical Models of Human Non-Hodgkin's Lymphoma and Hodgkin's Disease.

Non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt's lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt's lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.

The Fc Portion of UV3, an Anti-CD54 Monoclonal Antibody, Is Critical for its Antitumor Activity in SCID Mice With Human Multiple Myeloma or Lymphoma Cell Lines

UV3 is a monoclonal antibody that recognizes human CD54 (intercellular adhesion molecule-1), and it was generated for the therapy of human multiple myeloma. In a severe combined immunodeficient (SCID) xenograft model of human multiple myeloma, UV3 significantly prolonged the survival of mice with either early or advanced stages of disease. However, the mechanism by which UV3 exerted its antitumor effect remained unknown. As reported previously UV3 could mediate antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity in vitro. F(ab)'2 fragments of UV3 had therapeutic efficacy in vivo, suggesting that effector functions were not critical. The purpose of this study was to further define the importance of the Fc portion of UV3 for its antitumor activity in vivo. To this end, we examined the effect of an "ultrapure" preparation of UV3 F(ab)'2 to treat SCID mice xenografted with either ARH-77 cells, a human multiple myeloma cell line, or Daudi cells, a human Burkitt's lymphoma cell line. In addition, we evaluated different doses of UV3 immunoglobulin G (IgG) in these mice to determine the minimum amount of IgG that would produce a therapeutic effect. Data obtained from this study suggest that (1) the Fc portion of UV3 is critical for its antitumor activity in vivo, (2) low levels of UV3 IgG in a preparation of F(ab)'2 fragments account for all of its in vivo activity in multiple myeloma and most of its activity in lymphoma, and (3) UV3 IgG significantly prolongs the survival of SCID/ARH-77 mice as well as SCID/Daudi mice.

Antitumor activity of humanized monoclonal antibody against HM1.24 antigen in human myeloma xenograft models

A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.

Phase I Clinical Trial of IPI-504, a Novel, Water-Soluble Hsp90 Inhibitor, in Patients with Relapsed/Refractory Multiple Myeloma (MM).

IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. IPI-504, however, exists as a hydrochloride salt which is soluble in water in excess of 100 mg/mL. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium. When IPI-504 or 17-AAG is administered to mice, rats, or monkeys a dynamic equilibrium occurs in vivo in which both species are present in plasma as well as tissue. Thus, IPI-504 recapitulates all of the Hsp90 inhibitory properties and biological activity of 17-AAG without its formulation liabilities. In preclinical studies IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma. Therefore an open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in pts with relapsed/refractory MM. IPI-504 is infused in 250cc of normal saline over thirty minutes. Pts receive IPI-504 for one cycle of treatment. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. An accelerated titration design was utilized during the first three dose levels. Dose limiting toxicity (DLT) is defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment. Results demonstrate that the in vivo dynamic equilibrium of IPI-504 and 17-AAG is detectable in human plasma PK samples and the water-based formulation is well-tolerated. Data on PK, dose proportionality, safety, tolerability, PD markers, and anti-tumor activity of IPI-504 will be reported. In conclusion, clinical evaluation of this agent is ongoing to define the safety, tolerability and potential activity of IPI-504 to treat pts with advanced MM. These results may help clarify the effects of Hsp90 inhibition using a water-soluble, ansamycin-based Hsp90 inhibitor without the confounding effects of DMSO-based formulations.

Fully Retargeted Oncolytic Measles Virus Specific for the WUE-Ag Results in Specific Lysis of Multiple Myeloma Cells.

Effective cancer therapy with a minimum of undesired side effects in multiple myeloma (MM) requires specifity of the therapeutic agent against the neoplastic cells. One approach to reach this aim is virotherapy using oncolytic measles virus. Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer, lymphoma and glioma. The virus is selectively oncolytic, causing extensive lethal cell to cell fusion via CD46, which is more highly expressed on tumor cells than on normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM which are present on many different cell types. One approach to avoid this risk is to engineer the viral attachment protein to ablate its natural tropisms and at the same time, redirect its specificity to interact with alternative tumor specific receptors. The native measles Hemagglutinin (H) protein recognizes CD46 or SLAM resulting in membrane fusion and syncytia formation of cells. In this work the H protein was engineered to restrict and retarget membrane fusion through the display of a single-chain antibody (scFv) recognizing the Wue-1-antigen known to be highly specific for MM cells and abrogation of native measles binding domains for CD46 and SLAM by mutation. This modified H protein (chimeric H) was cloned in a full-length viral backbone including EGFP enabling to detect infected cells and syncytia formation under the UV light emitting green fluorescence (EGFP). On the basis of the parental measles virus expressing EGFP (MV-GFP) two different viruses were generated:non-ablated virus expressing the chimeric H protein including the scFv Wue-1 still competent to infect cells expressing CD46 and SLAM (MV-W1)with the chimeric H protein but ablated for the interactions with CD46 and SLAM (MV-W2).The genetically modified viruses propagated as recently described (Nat. Biotech., Vol.23, Nr.2, Feb.2005, pp.209–214.). To determine if the fully retargeted MV-Edm would be able to infect MM cell lines selectively a first array of infection assays was performed using the MM cell lines RPMI 8226 and ARH-77 expressing the Wue-1 antigen as expected targets and K562 and healthy CD40L activated CD19 positive B cells as controls. 24 to 96 hours after infection with MV-GFP, MV-W1 and MV-W2 we observed syncytia formation and expression of EGFP with MV-GFP and MV-W1 in all cells indicating that the modification of the virus with the scFv-Wue-1 doesn't alter the potential to infect and kill cells compared to the parental virus. In contrast MV-W2 was able to form only EGFP positive plaques with the Wue-1 antigen positive cells RPMI 8226 and ARH-77 but did not infect K562 or CD19 positve B cells both negative for Wue-1 antigen.These results indicate that the measles virus vaccine strain Edmonston B can be modified to express a scFv recognizing the Wue-1 antigen. In addition the native H protein can be mutated resulting in ablation of the natural tropism towards CD46 and SLAM positive cells. Viruses with these modifications can be rescued and propagated in vitro and selectively infected Multiple Myeloma cells without causing damage to normal B-cell progenitors.

The Telomerase Template Antagonist GRN163L in Combination with Chemotherapeutics Reduces Tumor Volume in Multiple Myeloma Xenograft Models.

Primary hematopoietic malignancies are characterized by short telomeres, suggesting that they would be acutely susceptible to telomerase inhibition. We have previously shown that treatment with GRN163L, a lipid-conjugated 13-mer thio-phosphoramidate oligonucleotide (Geron Corporation), inhibits the growth of human multiple myeloma (CAG) and ovarian carcinoma (OVCAR3) cell lines in vitro and in vivo (AACR 2005 Annual Meeting). The CAG multiple myeloma cell line (telomere length 2.7 Kb) was transfected with a retroviral vector encoding a triple fusion gene for HSV-TK, firefly luciferase and green fluorescence protein, and whole animal bioluminescence imaging was undertaken in either a subcutaneous or disseminated NOD/SCID model with extensive spinal cord and bone marrow tumor infiltration. In a subcutaneous model with CAG, GRN163L was administered three times per week (tiw) at 36 mg/kg for 4 weeks alone or in combination with either a single dose of melphalan (5 mg/kg) or 9 doses of Velcade (0.25 mg/kg tiw). Tumor mass was reduced 56% with GRN163L alone (p<0.001), 60% with melphalan alone (p<0.001), 89% with the combination of GRN163L and melphalan (p<0.001). At the dose used, Velcade alone showed no efficacy, but the combination of GRN163L with Velcade reduced the tumor mass by 68% (p<0.001) compared to controls. Data on the treatment with a mismatch control oligonucleotide in addition to dose optimization of GRN163L in combination with Melphalan and Velcade in subcutaneous and disseminated myeloma models (CAG, MM.1S) will be presented. Geron initiated a Phase I/II trial with GRN163L in chronic lymphocytic leukemia in July 2005. These data support the development of GRN163L as an effective and safe agent for therapy of hematopoietic malignancies.