Mark Leonard Elliott

An Improved Method of Chlorinating 2-alkoxynicotinic Acids

Abstract A novel chlorination process for preparing 5-chloro-2-alkoxynicotinic acids has been developed using commercial 5.25% sodium hypochlorite (Clorox®) as a chlorinating agent.

Abstract 1958: Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML. Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [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 1958. doi:10.1158/1538-7445.AM2014-1958

Gemtuzumab Ozogamicin (GO) Inclusion to Induction Chemotherapy Eliminates Leukemic Initiating Cells and Significantly Improves Survival in Mouse Models of Acute Myeloid Leukemia

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117− and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

Abstract 2476: Bispecific redirected T-cell immunotherapy targeting P-cadherin expressing tumors

Introduction: Strong evidence exists supporting the important role T-cells play in the immune response against tumors. Still, the ability to initiate tumor specific immune responses remains a challenge. We have developed a Dual-Affinity Re-Targeting (DART®) protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. We designate this bispecific redirecting T-cell molecule as P-cadherin LP-DART. P-cadherin up-regulation has been reported in various tumors, including breast, gastric, endometrial, colorectal and pancreatic cancers, and is correlated with poor survival of patients. Methods: The P-cadherin LP-DART was stably expressed by CHO cells and purified to homogeneity via standard antibody-purification methods. Functional in vitro studies were performed with a panel of human cancer cell lines and human T cells isolated from healthy donors. In vivo tumor growth inhibition studies were performed in immunodeficient athymic nude or NOD-scid IL2Rgamma null (NSG) mice bearing human tumor cell line- or patient derived-xenografts and human T-cells, and treated with P-cadherin LP-DART. Results: P-cadherin LP-DART exhibited binding to a broad panel of cancer cell lines expressing various levels of endogenous cell surface P-cadherin, and comparable binding to a number of human donor derived T-cells expressing CD3. In the presence of T-cells, this bispecific molecule elicited P-cadherin expression level dependent cytotoxic T-lymphocyte (CTL) responses against the different tumor cell lines, and induced antigen dependent T-cell activation and cytokine release. P-cadherin LP-DART also demonstrated potent in vivo anti-tumor activity against implanted tumor xenografts. Significant tumor growth inhibition was observed across a range of potential indications that express P-cadherin. Measurement of in vivo pharmacodynamic markers support P-cadherin LP-DART mediated CTL infiltration and killing as the mechanism of tumor inhibition. Conclusions: P-cadherin LP-DART displays potent in vitro and in vivo redirected T-cell activity against a broad panel of cancer cell lines expressing a range of cell surface P-cadherin levels. These data support further investigation of P-cadherin LP-DART as a potential novel therapeutic treatment for cancers expressing P-cadherin. Citation Format: Timothy S. Fisher, Adam Root, Bryan Peano, Allison Rohner, Justin Lucas, Mark Elliott, Konstantinos Tsaparikos, Hui Wang, Jonathan Golas, Maria Gavriil, Susan Benard, Tao He, Tracey Clark, Nahor Haddish-Berhane, Ralph Alderson, Yinhua Yang, Syd Johnson, Paul Moore, Lioudmila Tchistiakova, Hans-Peter Gerber, Chad May. Bispecific redirected T-cell immunotherapy targeting P-cadherin expressing tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2015-2476

Abstract 2841: A simplified approach to measuring thymidine kinase activity in cells, tumors, and blood as a biomarker of tumor proliferative potential

Understanding tumor size and growth is essential for preclinical oncology drug development, but costs and technical hurdles can prevent accurate, repeated measurements of cell proliferation and tumor growth. Many investigators instead measure the accumulation of nucleosides and analogs (e.g., [ 18 F]-FLT; [ 3 H]-Thy; BrdU) in cells and tissues, a manifestation of thymidine kinase 1 activity (TK1 a ). These methods can be costly and laborious, and can generate significant hazardous waste, promoting the use of even less-direct measures of cell division. Seeking further insights into the role of TK1 in cancer, we developed rapid, robust, non-isotopic methods that measure TK1 a in tissue extracts and blood. In this approach, samples are exposed to FLT in vivo or in vitro, promoting its phosphorylation. Subsequently, nucleosides and their phosphorylated metabolites are isolated for analysis by a simple organic extraction. Using these methods, TK1 a was measured in primary and transformed cells, and showed excellent comparability with established proliferation assays including 3 H-thymidine incorporation, ATP generation, and fluorescent dye dilution flow cytometry. In drug screens, our method was as sensitive as commercial viability assays, and typically detected drug effects much sooner than those less-specific measures. We also demonstrated chemotherapy-induced alterations in TK1 a in xenografted tumor tissue that were comparable to results obtained using radioisotopes. Finally, this approach was adapted to measure TK1 a in blood samples. Consistent with published reports, circulating TK1 a correlated well with tumor burden in solid tumor models and primary canine hematological cancer. These methods require only small sample volumes ( 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 2841. doi:1538-7445.AM2012-2841