Wendi V. Rodrigueza

Cellular Cholesterol Efflux Mediated by Cyclodextrins DEMONSTRATION OF KINETIC POOLS AND MECHANISM OF EFFLUX

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol (Kilsdonk, E. P. C., Yancey, P., Stoudt, G., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256). In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-β-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1/2) ∼19-23 s) and a slow pool with t1/2 of 15–30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.

Remodeling and Shuttling Mechanisms for the Synergistic Effects Between Different Acceptor Particles in the Mobilization of Cellular Cholesterol

In normal physiology, cells are exposed to cholesterol acceptors of different sizes simultaneously. The current study examined the possible interactions between two different classes of acceptors, one large (large unilamellar phospholipid vesicles, LUVs) and one small (HDL or other small acceptors), added separately or in combination to Fu5AH rat hepatoma cells. During a 24-hour incubation, LUVs of palmitoyl-oleoyl phosphatidylcholine at 1 mg phospholipid (PL) per milliliter extracted approximately 20% of cellular unesterified cholesterol (UC) label and mass in a slow, continuous fashion (half-time [t1/2] for UC efflux was approximately 50 hours) and human HDL3 at 25 micrograms PL per milliliter extracted approximately 15% cellular UC label with no change in cellular cholesterol mass (t1/2 of approximately 8 hours). In contrast, the combination of LUVs and HDL3 extracted over 90% of UC label (t1/2 of approximately 4 hours) and approximately 50% of the UC mass, indicating synergy. To explain this synergy, specific particle interactions were examined, namely, remodeling, in which the two acceptors alter each others composition and thus the ability to mobilize cellular cholesterol, and shuttling, in which the small acceptor ferries cholesterol from cells to the large acceptor. To examine remodeling, LUVs and HDL were coincubated and reisolated before application to cells. This HDL became UC depleted, PL enriched, and lost a small amount of apolipoprotein A-I. Compared with equivalent numbers of control HDL particles; remodeled HDL caused faster efflux (t1/2 approximately 4 hours) and exhibited a greater capacity to sequester cellular cholesterol over 24 hours (approximately 38% versus approximately 15% for control HDL), consistent with their enrichment in PL. Remodeled LUVs still extracted approximately 20% of cellular UC. Thus, remodeling accounted for some but not all of the synergy between LUVs and HDL. To examine shuttling, several approaches were used. First, reisolation of particles after an 8-hour exposure to cells revealed that HDL contained very little of the cellular UC label. The label was found almost entirely with the LUVs, suggesting that LUVs continuously stripped the HDL of cellular UC. Second, bidirectional flux studies demonstrated that LUVs blocked the influx of HDL UC label into cells, while the rate of efflux of cellular UC was maintained. These kinetic effects explained the massive net loss of cellular UC to LUVs with HDL. Third, cyclodextrin, an artificial small acceptor that does not acquire PL and hence does not become remodeled, exhibited substantial synergy with LUVs, supporting shuttling. Thus, the presence of large and small acceptors together can overcome intrinsic deficiencies in each. Small acceptors are efficient at extracting cellular cholesterol because they approach cell surfaces easily but have a low capacity, whereas large acceptors are inefficient but have a high capacity. When present simultaneously, where the small acceptor can transfer cholesterol quickly to the large acceptor, high efficiency and high capacity are achieved. The processes responsible for this synergy, namely, remodeling and shuttling, may be general phenomena allowing cooperation both during normal physiology and after therapeutic administration of acceptors to accelerate tissue cholesterol efflux in vivo.

Abstract 5473: The sensitivity of targeting genomic BCL2 by PNT2258 is linked to chromosomal rearrangements and proliferative rate of tumor types

Background: The BCL2-mediated anti-apoptotic phenotype is a contributor to the genesis and maintenance of a broad variety of tumors. BCL2 is also implicated in the regulation of the cell cycle by playing a role in the transition between quiescence and the cycling state. Further, chromosomal rearrangements, including t(14;18), up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. PNT2258 is a DNA interference (DNAi) therapeutic targeted against BCL2 that is undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single-stranded phosphodiester DNA oligodeoxynucleotide, encapsulated in protective liposomes. Material and Methods: Lymphomas cell lines with distinct genetic characteristics, namely, WSU-FSCCL, characterized by t(14;18) BCL2 and t(8;14) CMYC rearrangements, WSU-DLCL2, characterized by t(14;18) BCL2 rearrangement, and WSU-WM, characterized as lacking t(14;18) BCL2, but having t(8;14) CMYC and t(12;17) rearrangements were tested for cell viability at 24, 48, 72, and 96 hours post-exposure to PNT2258 at concentrations of 2.5, 5, and 10 μM. Normalized maximum cell kill at 96 hours was used in each cell line to adjust for differences in growth rates and to calculate sensitivity. Results: Dose-dependent effects were observed across all three cell lines. WSU-FSCCL was the most proliferative cell line, and correspondingly most sensitive to the effects of PNT2258, with a control doubling time of 29.6h and 1% of viable control cells remaining at 96h at 10 μM. The next most sensitive cell line was WSU-DLCL2, having a 60.6h control doubling time and 11% of the viable control cells remaining at 96h at 10 μM. WSU-WM exhibited a control doubling time of 35.5h and 21% of viable control cells remaining at 96h at 10 μM. These findings parallel the single agent activity of PNT2258 against xenograft tumor models containing BCL2 or CMYC chromosomal rearrangements (AACR Meeting Abstracts, Apr 2007; 2007: 4889). Conclusions: The effects of the clinical therapeutic PNT2258 against cell lines with well-characterized growth and genetic drivers were examined. Findings show lymphoma cell lines harboring the t(14;18) rearrangement (WSU-FSCCL and WSU-DLCL2) were most sensitive to PNT2258 and in this context a higher proliferative rate is linked to sensitivity. These data suggest that commonly used clinical assays of proliferation and tumor “aggressiveness,” such as Ki-67 and PET/CT standardized uptake values (SUVs), may be useful in conjunction with cytogenetic analysis (e.g. for t(14;18) and/or t(8,14) by fluorescence in situ hybridization (FISH), to select patients with tumors that may be responsive to the effects of PNT2258. Citation Format: Michael J. Woolliscroft, Abdul-Shukkur Ebrahim, Richard A. Messmann, Shari K. Gaylor, Mina P. Sooch, Ayad Al-Katib, Wendi V. Rodrigueza. The sensitivity of targeting genomic BCL2 by PNT2258 is linked to chromosomal rearrangements and proliferative rate of tumor types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2014-5473

Abstract 3529: Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans.

Background: The dysregulation of apoptosis is a defining characteristic of malignant cells where excessive concentration of BCL2 protein contributes to the anti-apoptotic phenotype, driving development and subsequent resistance to therapy. Chromosomal translocations, including the t(14;18) rearrangement, up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. A new class of therapeutic agents, called DNA interference (DNAi) drugs, exert their therapeutic effect by selectively blocking the transcription of oncogenes. PNT2258 is the lead DNAi drug currently undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single stranded, native sequence oligonucleotide targeted against BCL2, which is delivered to cancer cells in a protective liposomal transport system. We present data on the anti-tumor and immunomodulatory effects of PNT228 in mice compared with that observed in patients with advanced solid tumors treated with escalating doses of PNT2258. Material and Methods: Twenty-two patients received PNT2258 doses ranging from 1 to 150 mg/mˆ2 as part of a Phase I dose-escalation study in patients with advanced, treatment refractory solid tumors. Patient plasma specimens were analyzed using a 61-marker multiplex immunoassay. Balb/c and WSU-DLCL2 xenograft mice received PNT2258 or an encapsulated scrambled control (oligonucleotide) sequence at a dose of 20 mg/kg by intravenous infusion. Murine plasma was analyzed with a 37-marker multiplex immunoassay. Results: In the murine xenograft model, PNT2258 demonstrated sequence-specific anti-tumor and anti-BCL2 activity not observed with the scrambled control. Xenograft mice demonstrated a strong innate immune response that was very similar in magnitude for both PNT2258 and scrambled control. Balb/c mice exhibited a broad and relatively weak immune response to PNT2258 and a stronger response to the scrambled control suggesting activation of both adaptive and innate immune responses. In patients, PNT2258 did not produce clinical signs of immune stimulation. Multiplex immunoassay revealed a lack of significant drug-induced modulation of inflammatory biomarkers following treatment. PNT2258 did induce statistically significant dose-dependent changes in IP-10, leptin, MIP-1β, MCP-1, IL-17F, and IL-1RA consistent with a mechanistic response to BCL2 suppression. Conclusions: Biomarker response profiles of encapsulated human sequence-specific oligonucleotide therapeutics in mice are not predictive of responses in humans. PNT2258 is safe and well tolerated in patients with no evidence of an innate (TLR) response. The observed biomarker profile provides mechanistic confirmation of drug-induced BCL2 suppression at all doses tested. Leptin represents a unique biomarker that may be used to monitor PNT2258 anti-BCL2 activity in the clinic. Citation Format: Elzbieta Izbicka, Robert Streeper, Michael J. Wick, Drew Rasco, Amita Patnaik, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Shari Gaylor, Michael J. Woolliscroft, Richard A. Messmann, Wendi V. Rodrigueza. Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2013-3529

Abstract 2764: Effect of PNT2258 combinations with docetaxel, dacarbazine, or vemurafenib on the A375 melanoma xenograft

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Bcl-2 and bcl-XL are anti-apoptotic proteins that promote drug resistance and enhance tumor cell survival. Bcl-2 gene amplification occurs in 90% of cutaneous melanomas implicating it as a potential target. PNT100 is a 24-mer unmodified DNA oligonucleotide complementary to a non-coding region of bcl-2. PNT2258, is comprised of PNT100 encapsulated in a unique pH tunable nuclear-targeted liposomes termed SMARTICLES®. Preliminary studies demonstrated fluorescently-labeled PNT100 in a SMARTICLE® is delivered to the cell nucleus and that PNT2258 produces dramatic tumor regression in a diffuse large cell lymphoma xenograft, prolonging survival with a coincident decrease in bcl-2 protein. PNT2258 activity in the A375 melanoma model was assessed as a single agent or in combination in two separate studies. Study 1: PNT2258 alone, IV, 5 days/week for 3 weeks, to mice bearing A375 produced growth delays of 10.4, 7.8, and 7.8 days at 12.5, 8.3, and 5.6 mg/kg/dose, respectively. Docetaxel alone, IV weekly x3, produced growth delays of 19.4 (1/10 TFS), 14.8 (1/10 TFS), and 11.5 days at 30, 20, and 13.3mg/kg/dose. Combination therapy produced growth delays of 45.3 (5/10 TFS), 18.9, 28.1 (2/10 TFS), and 25.0 (3/10 TFS) days at 12.5+30, 12.5+20, 5.6+30, and 5.6+20 mg/kg/dose, respectively. Study 2: PNT2258 alone produced a growth delay of 12 days at 15mg/kg/dose. Dacarbazine alone was ineffective at 80 mg/kg/dose IP. Combination therapy was not superior to PNT2258 alone. In contrast, vemurafenib alone (37.5 mg/kg/dose, PO, BIDx18 days) was highly active producing a growth delay in excess of 30 days, as did the combination of PNT2258+vemurafenib. PNT2258 is currently being evaluated for safety and tolerability in a phase Ia clinical trial. Preliminary mouse and human pharmacokinetics suggest that exposure levels in humans are similar to those producing anti-tumor efficacy in mice. Overall, 1) PNT2258+docetaxel produced a greater than additive effect compared to single agents, 2) PNT2258+dacarbazine was not superior to PNT2258 alone, 3) PNT2258+vemurafenib while highly active, was not superior to vemurafinib therapy alone, and 4) therapeutic PNT2258 exposures have been attained in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2764. doi:1538-7445.AM2012-2764