Michael Woolliscroft

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 4321: A four-pronged imaging approach to characterization of bone metastasis in mouse models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: The process of bone metastasis involves both osteoblastic and resorptive components and is a common feature of both prostate and breast cancers. The ability to visualize and quantify the early stages of bone involvement in mouse models of bone metastasis would provide a platform for development of new agents targeted at inhibition or treatment of bone metastases. Two approaches that stand to enable this are 18F-NaF PET and optical imaging using biphosphonate fluorescent probes such as Osteosense, through targeting of hydroxyapatite (HA), a biomarker for osteoblastic activity. Additionally, bioluminescence imaging using luciferase expressing tumor lines and micro-CT imaging of the skeleton can enable anatomic characterization of tumor burden and development of bone lesion in bone metastasis models. Methods: MDA-MB-231-luc-D3H2LN human mammary adenocarcinoma cells (105 cells in 100µl) were injected into the left ventricle of female nu/nu mice on Day 0 approximately 6-7min after IP injection of luciferin (150mg/kg). Bioluminescence scans were used to determine successful injection by distribution of light throughout the body. On Day 14, all tumor pool mice were imaged using bioluminescence and enrolled on study based on incidence of luciferase signals at bone sites. After staging, serial bioluminescence imaging was used to localize tumor signals and determine incidence and growth of bone metastases and micro-CT was used to image the extent of bone lesions associated with the metastases. Additionally, whole body PET scans were used at multiple time points after 90min uptake of 18F-NaF. Images were reconstructed using a 2D OSEM method and localized bone sites segmented and analyzed for standardized uptake value (SUV). Fluorescence imaging was also used 24h after administration of Osteosense 750 to characterize localization of the tracer at the bone sites of interest. After background correction, the bone sites of interest were segmented and analyzed for average efficiency. The 18F-NaF and Osteosense endpoints were correlated with bioluminescence determined tumor burden and location. Results and Discussion: Both 18F-NaF PET imaging and fluorescent imaging using Osteosense highlighted localized bone signals that could be correlated with bioluminescent signals and micro-CT visualized bone lesions from approximately Day 17. Both the PET and fluorescence imaging approaches showed early indication of bone involvement, presumably through HA, indicating osteoblastic activity. This multi-endpoint approach enabled non-invasive determination of both soft tumor and bone components of metastatic bone disease. An approach like this may enable more quantitative characterization of the early stages of bone metastasis in mouse models, and facilitate development of bone targeted treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4321.

Abstract 5245: Regional analysis of DCE MR images to unravel tumor growth and response heterogeneity in SW-620 human colon adenocarcinoma xenografts

Introduction: Dynamic Contrast-Enhanced (DCE) MRI, a clinically relevant technique used to assess response to anti-angiogenic therapies, relies on imaging of a T1 modifying contrast agent to characterize vascular response by the dynamics of tracer exchange between the vessels and the interstitial space. The parameter K trans is a measure of the tumor vascular permeability, vascular surface area and blood flow, and is normally assessed as mean over the entire tumor volume. Generally, decreasing tumor mean K trans occurs after effective vascular targeting. However, localized tumor growth based necrosis/under-perfusion can also lower K trans , leading to uncoupling of the endpoint from the effect. Techniques that can overcome confounding effects of heterogeneity may improve K trans sensitivity and correlation with outcome. We applied a thresholding technique to K trans data to analyze regional K trans variations in subcutaneous SW-620 tumors treated with the vascular targeted agent, bevacizumab or vehicle. Materials and Methods: Female nu/nu mice were implanted with SW-620 tumor fragments on Day 0. On Day 14, mice were distributed into groups with matching tumor burden. DCE-MRI images were acquired 24 hours before treatment (vehicle or bevacizumab; IP; 5mg/kg; Q3Dx4), and one week after treatment. The data were fitted to a Tofts-Kermode kinetic model to generate K trans over the entire tumor volume. Thresholds we determined by pooling all the K trans values from the pre-treatment DCE MRI scans. The K trans values for each image were distributed into low, mid and high K trans fractions and the percentage of voxels in each fraction was calculated. Results: Bevacusimab resulted in moderate tumor growth inhibition (T/C = 43% on Day 22). Tumor mean K trans one week post-treatment start did not correlate with this effect, with K trans (T/C) = 131%. Analysis of location of voxels in the low, mid and high K trans fractions in the pre-treatment images showed that lower K trans values were generally confined to the tumor core, while higher K trans values were generally confined to the tumor periphery. These regions presumably related to under-perfused (core) and well perfused (periphery) tissue. A comparison of the high K trans volume fractions one week post-treatment (treated vs control) showed a correlation with the growth based treatment effect (high K trans volume fraction (T/C) = 44%). K trans thresholding may therefore provide a means to increasing sensitivity to treatment-based vascular inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5245.