Eva Tonsing-Carter

Abstract 5196: Histologic and whole slide quantitative image analysis of lung metastases in a mouse breast cancer model using carboplatin and Nutlin 3A alone and in combination

Breast cancer will affect 1 in 8 women in their lifetime. Approximately 20-30% of all breast cancer cases become metastatic. This study was designed to evaluate the effect of tumor met knockdown with individual and combination drug therapy. In this mouse model, with 3 per group, we used the following chemotherapeutic agents: a vehicle control, carboplatin (20mg/kg i.p.), Nutlin-3a (200mg/kg p.o.) and a combination group with the same doses. Nutlin-3 is a small-molecule inhibitor that binds to the p53-binding pocket of MDM2, and activates the p53 pathway. This has been shown to be effective in mouse models of cancer that have a wild-type p53, including prostate, leukemia and multiple myeloma. The lungs were removed and examined for metastatic lung lesions, fixed in 10% neutral buffered formalin (NBF), processed into a paraffin block, microtomed, and slides were stained with HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2015-5196

Abstract A26: Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells

A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p in vivo with an acceptable toxicity profile. Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

Abstract 1680: Modulation of MDM2 in context of DNA damage enhances cell death in a metastatic breast-to-lung xenograft model

Metastatic breast cancer is highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. In metastatic disease, inhibiting key protein-protein interactions with the murine double minute 2 (MDM2) could be beneficial for developing new treatment modalities since this signaling pathway is a critical regulatory point in cancer progression. Inhibition of protein binding to the hydrophobic pocket of MDM2 by Nutlin-3a can activate pro-apoptotic proteins such p73 and E2F1 as well as decrease pro-angiogenic Hif-1α. Since the DNA damaging agent carboplatin is currently being studied in clinical trials of triple-negative breast cancers (TNBCs), our objective was to evaluate the effects of carboplatin and Nutlin-3a in combination in TNBC in a mutant p53 background. Using a TNBC cell line TMD231 derived from the MDA-MB-231 human breast cancer cell line, we performed combination studies using different ratios of carboplatin to Nutlin-3a in vitro to evaluate the range of carboplatin-mediated DNA damage required to obtain synergism with inhibition of MDM2 function. A fixed ratio of 1:1 carboplatin:Nutlin-3a was strongly synergistic with a combination index of Citation Format: Eva Tonsing-Carter, Harlan E. Shannon, Barbara J. Bailey, Anthony L. Sinn, Kacie M. Peterman, Lindsey D. Mayo, Karen E. Pollok. Modulation of MDM2 in context of DNA damage enhances cell death in a metastatic breast-to-lung xenograft model. [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 1680. doi:10.1158/1538-7445.AM2014-1680

Abstract 4309: Whole slide image analysis quantification in a mouse lung metastasis model

Digital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power fields, which can be commonly used to evaluate the tissue. Many of these systems have a whole slide software image analysis system. The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2014-4309

Abstract 4639: Blockade of MDM2-mediated signaling in context of DNA damage increases E2F1 expression and enhances cell death in triple-negative breast cancer cells.

Metastatic breast cancer is highly refractive to current treatment strategies and new multi-targeted treatments need to be elucidated. In metastatic disease, blocking key protein-protein interactions with the murine double minute 2 (MDM2) could be beneficial for developing new treatment modalities since this signaling pathway is a critical regulatory point in cancer progression. Inhibition of protein binding to the hydrophobic pocket of MDM2 by Nutlin-3a can result in increases in pro-apoptotic proteins such as p53, p73, and E2F1. Since the DNA damaging agent carboplatin is being studied in clinical trials of triple-negative breast cancers (TNBCs), our objective was to evaluate the effects of carboplatin and Nutlin-3a in combination in TNBC cancer in a mutant p53 background. We utilized TMD231 breast cancer cells derived from the MDA-MB-231 human TNBC cancer line. TMD231 cells are highly metastatic in vivo and readily metastasize from the primary tumor to the lung. Combination studies were performed using different ratios of carboplatin to Nutlin-3a to evaluate the range of carboplatin-mediated DNA damage required to obtain synergism with inhibition of MDM2 function. A fixed ratio of 1:1 carboplatin:Nutlin-3a was strongly synergistic with a combination index of Citation Format: Eva Tonsing-Carter, Harlan E. Shannon, Barbara J. Bailey, Lindsey D. Mayo, Karen E. Pollok. Blockade of MDM2-mediated signaling in context of DNA damage increases E2F1 expression and enhances cell death in triple-negative breast cancer cells. [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 4639. doi:10.1158/1538-7445.AM2013-4639

Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells

Hematopoietic stem cells (HSCs) are a rare population of pluripotent cells that predominantly reside in the bone marrow. Under the appropriate microenvironmental cues, HSCs can undergo self-renewal, expansion, and differentiation into all types of progenitor and terminally differentiated blood cells required for survival of the host (Figure 1). Due to the importance of this cell population for survival, protection of its genome from endogenous and exogenous genotoxic insults is a necessity. However, the intracellular molecular signaling network in hematopoietic cells that control surveillance of the genome as well as maintain genome stability is still largely unexplored. As more is learned regarding how these cells detect a genotoxic event and seek to repair the damaged nucleotides (i.e. DNA adducts), it will become even more feasible to design strategies to protect these life-sustaining cells when the host is exposed to a genotoxic event. Maintenance of genome stability in both the hematopoietic stem and progenitor cell (HSPC) populations is essential for the sustainment of normal hematopoiesis. For example, transient depletion of bone-marrow derived HSC induced by irradiation or chemotherapy can induce these primitive cells to expand so that the bone marrow can be fully reconstituted; blood-cell development can then continue with minimal disruption. However, once therapy-mediated DNA damage is too high, a DNA-damage threshold is reached resulting in subsequent cell death, myelosuppression, and if not treated, life-threatening bone-marrow failure (Figure 1). With the basal level of DNA repair relatively low in these cells, this does present a challenge to maintain normal hematopoiesis in individuals exposed to prolonged or high levels of genotoxic stress. The reduced ability to repair DNA damage in HSPCs that give rise to multiple mature blood-cell lineages can cause detrimental and long-lasting effects to the host resulting in abnormal cell function, cell death, cellular transformation, and eventually leukemogenesis (Figure 1). Numerous studies have shown that HSPCs are intrinsically more sensitive than other cell types and tissues mostly due to intrinsic limitations in DNA-repair capacity. Buschfort-Papewalis et al previously demonstrated that when human HSPCs (phenotypically defined as CD34+ cells) or differentiated cells (phenotypically defined as CD34cells) from the same donor were exposed to alkylating agents, an overall decrease in repair capacity of the more primitive CD34+ cells compared the more differentiated cells CD34cells was observed. When human CD34+ cells were exposed to a variety of chemotherapeutic drugs, single-strand DNA breaks as well as DNA adducts were found at higher levels and persisted for longer time periods than in CD34cells (Buschfort-Papewalis et al., 2002), providing evidence that the kinetics of DNA repair are slower overall in the