ABSTRACT

1. Combination Therapy with Novel Nanoparticle, SNS01-T, and Lenalidomide Triggers Synergistic Cytotoxicity in Vitro and in Vivo in Multiple Myeloma and Mantle Cell Lymphoma Catherine Taylor1, Terence Tang1, Zhongda Liu1, Sarah Francis1, Qifa Zheng1, Richard Dondero2, John Thompson1,2 1 Department of Biology, University of Waterloo, Waterloo, ON, Canada 2 Senesco Technologies, Bridgewater, New Jersey, USA NH2 NH2 CH2 CH2 CH2 CH2 NH2(CH2)3NH CH2 CH2CH2CH2CH2NH2 H-C-OH CH2 CH2 NH NH NH2 (CH2)4 (CH2)4 (CH2)4 NH-CH-CO NH-CH-CO NH-CH-CO ABSTRACT SNS01-T and Lenalidomide In Vivo Drug Combination Study in RPMI 8226 MM and SUDHL6 DLBCL Xenograft Models RESULTS SNS01-T: 2x per week; i.v. Lenalidomide: 5x per week; i.p. INTRODUCTION: SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and B-cell lymphomas. SNS01-T is comprised of a plasmid encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL). SNS01-T and its preclinical precursors have been studied extensively in multiple myeloma and B cell lymphoma tumor models. In this study we tested the in vitro and in vivo anti-cancer activity of SNS01-T in combination with the immunomodulatory drug lenalidomide. METHODS: In vitro cell viability (XTT) assays were performed by incubating RPMI 8226 MM cells with SNS01-T, lenalidomide, or both at a constant drug ratio and the combination index was calculated using CompuSyn software. To determine whether SNS01-T treatment increases the anti-myeloma activity of lenalidomide in vivo, 0.375 mg/kg SNS01-T (2x per week; intra-venous) was combined with 50 mg/kg lenalidomide (5x per week; intra-peritoneal) in a RPMI 8226 xenograft model of multiple myeloma. Mice were dosed for two cycles of treatment for a total of 11 weeks of dosing. Mice with no measurable tumor at the end of the first cycle of treatment did not receive treatment in the second cycle but were monitored closely for tumor recurrence. A two-week observation period at the end of the study allowed monitoring of tumor growth after the cessation of the second cycle of treatment. The effectiveness of 0.375 mg/kg SNS01-T in combination with 15 mg/kg lenalidomide (6 weeks dosing) was also examined in a JVM-2 MCL xenograft model . RESULTS: Tumor growth was inhibited by 84 % (p < 0.0001), 34 % (p = 0.05), and 98.1 % (p << 0.0001) in animals treated with SNS01-T, 50 mg/kg lenalidomide, and SNS01-T plus 50 mg/kg lenalidomide, respectively, at the end of the second cycle of dosing in the RPMI 8226 xenograft study. Complete tumor regression (undetectable tumor) was achieved in 40% of mice treated with SNS01-T, 0% of mice treated with 50 mg/kg lenalidomide, and 83% of mice treated with the combination therapy of SNS01-T and 50 mg/kg lenalidomide. Complete regression of tumors treated with the combination therapy was maintained for more than 8 weeks without treatment until the end of the study in 4 of 6 (67%) of treated mice. Combining SNS01-T treatment with 50 mg/kg lenalidomide inhibited tumor growth more effectively than either drug alone and prolonged survival with 100% of mice surviving to the end of the 102-day study. Combination therapy with SNS01-T and 15 mg/kg lenalidomide also demonstrated significant activity in a murine JMV-2 MCL xenograft model. Treatment of mice with the drug combination of SNS01-T and lenalidomide resulted in a statistically significant increase in survival compared to either SNS01-T (p = 0.002; logrank test) or lenalidomide (p = 0.007) alone. CONCLUSION: Collectively, these preclinical studies indicate that the combination therapy of SNS01-T and lenalidomide is well tolerated, has significant activity against MM and MCL, and provides a strong rationale to evaluate SNS01-T and lenalidomide combination therapy to improve patient outcome in MM and B cell lymphomas. Combination of SNS01-T and Lenalidomide Has Synergistic Effect on Viability of RPMI 8226 MM Cells *** ** ** Rest Observation Cycle 1 Cycle 2 * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to control group; Logrank test) Combination Index < 1 [Synergy] (Calculated using CompuSyn software using constant drug ratio; Chou-Talalay method) ** Constant Drug Ratio (1:21.5) Combination of SNS01-T and Lenalidomide Increases Apoptosis in RPMI 8226 MM Cells Spermidine * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to all other groups; Logrank test) Deoxyhypusine Hydroxylase (DHH) Deoxyhypusine Synthase (DHS) * * Mature eIF5A Growth/Cell survival Inflammation eIF5A Intermediate eIF5A precursor Pro-apoptotic mRNA shuttling/translation * Eukaryotic Translation Initiation Factor 5A CONCLUSIONS • eIF5A is the only known protein to undergo the post-translational modification of a conserved lysine to the unique amino acid hypusine • Hypusine50- eIF5A • is the dominant form of eIF5A in growing cancer cells and is a marker of neoplastic growth • is involved in inflammatory pathways and activation of NF-kB [1] • inhibition of eIF5A expression reduces pro-inflammatory cytokine production and increases survival of LPS-challenged mice [2] • Lysine50-eIF5A • accumulates during apoptosis due to decreased DHS activity • mutants of eIF5A that cannot be hypusinated (eIF5AK50R) induce apoptosis in numerous cancer cell lines, including multiple myeloma, and inhibit growth of multiple myeloma xenograft models [1] • Inhibition of hypusinated eIF5A sensitizes multiple myeloma cells to apoptosis induced by over-expression of eIF5AK50R [1] • SNS01-T • is a nanoparticle designed for treatment of multiple myeloma and B cell lymphomas and • consists of : • an siRNA that targets the native eIF5A that promotes growth/anti-apoptosis of cancer cells • a plasmid with a B cell-specific promoter/enhancer expressing a pro-apoptotic mutant of eIF5A • a synthetic polymer (polyethylenimine) that acts as a delivery vehicle • is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma, mantle cell lymphoma or diffuse large B cell lymphoma (ClinicalTrials.gov Identifier: NCT01435720) • In Vitro Data: • Combination of SNS01-T and lenalidomide causes synergistic reduction in viability of RPMI 8226 multiple myeloma cells and induced apoptosis in a higher percentage of cells than either drug alone.. • Combination of SNS01-T and bortezomib causes synergistic reduction in viability of RPMI 8226 multiple myeloma cells. • In Vivo Data: • In a xenograft model of multiple myeloma, 100 % of mice treated with SNS01-T and lenalidomide survived to the end of the 102-day study compared to 20 % of mice treated with lenalidomide and 60 % of mice treated with SNS01-T. • Complete tumor regression (undetectable tumor) was achieved in 40% of mice treated with SNS01-T and 67% of mice treated the combination therapy of SNS01-T and lenalidomide. These mice remained tumor-free to the end of the study. Complete regression was not observed in any of the mice treated only with lenalidomide. • At the end of the second cycle of dosing in the RPMI 8226 xenograft study, tumor growth was inhibited by 84 % (p < 0.0001), 34 % (p = 0.05), and 98.1 % (p << 0.0001) in animals treated with SNS01-T, 50 mg/kg lenalidomide, and SNS01-T plus 50 mg/kg lenalidomide, respectively. • In a xenograft model of mantle cell lymphoma, combination therapy with SNS01-T and lenalidomide was more efficacious than either drug alone and resulted in a statistically significant increase in survival compared to either SNS01-T (p = 0.002; logrank test) or lenalidomide (p = 0.007) alone. • Combination of SNS01-T with either lenalidomide or bortezomib caused a synergistic decrease in viability of RPMI 8226 multiple myeloma cells. • SNS01-T potentiates the anti-tumoral activity of the immunomodulatory drug lenalidomide in xenograft models of multiple myeloma and mantle cell lymphoma. Combination of SNS01-T and Bortezomib Has Synergistic Effect on Viability of RPMI 8226 MM Cells Hypusination of eIF5A Hypusine Combination Index < 1 [Synergy] When Fraction Affected > 0.75 (Calculated using CompuSyn software using constant drug ratio; Chou-Talalay method) Constant Drug Ratio (1:1.25) Conflict of Interest Statement: R. Dondero and J. Thompson are employees of Senesco Technologies; J. Thompson owns shares in Senesco Technologies. References: [1] Taylor et al. (2012) Mol Ther; 20:1305. [2] Moore et al. (2008) J Infect Dis; 198:1407.