Timothy Nichols

[18F]FLT–PET Imaging Does Not Always “Light Up” Proliferating Tumor Cells

Purpose: [18F]FLT (3′-Fluoro-3′ deoxythymidine)–PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [18F]FLT–PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. Experimental Design: In exponentially growing xenografts, factors that could impact the outcome of [18F]FLT–PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [18F]FLT tracer avidity was compared with other proliferation markers. Results: In a panel of proliferating xenografts, [18F]FLT or [3H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [18F]FLT–PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [18F]FLT–PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. Conclusions: Tumor thymidine level is one of the factors that impact the correlation between [18F]FLT uptake and tumor cell proliferation. With careful validation, [18F]FLT–PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies. Clin Cancer Res; 18(5); 1303–12. ©2011 AACR.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells To Result In Sustained Tumor Regressions

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure- initiator mass spectrometry (NIMS) imaging

BackgroundTissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3′-deoxy-3′-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses.MethodsNIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt’s lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy.ResultsNIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism.ConclusionsNIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and provides new spatial and functional dimensions to studies of cancer metabolism without the need for radiotracers or tissue extraction. These findings should prove useful for in vitro and pre-clinical studies of cancer metabolism, and aid the optimization of metabolism-based cancer therapies and diagnostics.

CDK4/6 Inhibition on Glucose and Pancreatic Beta Cell Homeostasis in Young and Aged Rats

Genetic deletion of cyclin-dependent kinase 4 (Cdk4) is associated with pancreatic beta cell loss and glucose dysregulation in rodents. Palbociclib, one of the first selective CDK4/6 inhibitors approved for the treatment of advanced breast cancer, is currently being investigated as an adjuvant treatment in patients with early-stage breast cancer and in a variety of cancers covering a wide-range of patient populations. Hence, longer chronic toxicity studies were necessary to further examine its safety profile. The effects of different doses and duration of palbociclib administration on glucose and beta cell homeostasis in young (two months) versus aged (12 months) rats was compared. Glucose dysregulation, due to pancreatic beta cell degeneration, was observed in young rats administered the highest dose of palbociclib for 6 months. Abnormal pancreatic islet histology and activation of the endoplasmic reticulum stress response in beta cells were detected after shorter administration with high-dose palbociclib in young rats. To test the hypothesis that palbociclib-associated inhibition of beta cell proliferation will more profoundly affect younger animals that have not achieved replicative quiescence, we administered high-dose palbociclib to aged rats for 6 months. In contrast to the young rats, despite equivalent exposures to palbociclib, no evidence of impaired glucose tolerance, hypoinsulinemia, beta cell vacuolization, or beta cell loss was seen in aged rats. Palbociclib administration induces beta cell failure in young but not aged rats. Implications: Although adult humans receiving palbociclib have not displayed detectable adverse effects on glucose metabolism, the risk of beta cell failure in children remains unexplored. Mol Cancer Res; 15(11); 1531–41. ©2017 AACR.

Abstract 5425: An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression

Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [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 5425. doi:10.1158/1538-7445.AM2015-5425

Abstract 3518: Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways

Small cell lung cancer (SCLC) is an aggressive malignancy with a high propensity for early metastasis. Response rates to first-line chemotherapy are typically high, but short lived. We describe an epigenetic-based mechanism for targeting SCLC using inhibitors for the LSD1 histone lysine demethylase. Mechanistically reversible and irreversible LSD1 inhibitors (GSK690 and OG-86) demonstrate induction of either cell cycle arrest or apoptosis in 54% of SCLC cell lines (12/22) that becomes apparent upon continuous treatment for 7-to-10 days. Maximal rates of growth inhibition in sensitive cell lines vary from 50-to-95% and plateaus between 18-to-21 days of LSD1 inhibitor treatment. Thus, heterogeneous sensitivity to LSD1 inhibitors exists between cell line models as well as within subpopulations of cells in the same cell line. To understand the mechanisms underlying LSD1 inhibitor activity in SCLC we have performed RNA-seq and ChIP-seq experiments coupled with bioinformatic analysis of expression signatures in sensitive and resistant models. Our data indicate that although LSD1 is over-expressed in SCLC cell lines and patient samples relative to non-small cell lung cancers, high LSD1 expression does not predict sensitivity to LSD1 inhibitors. Pathway analysis demonstrates that LSD1 inhibition modulates the expression of genes involved in cell adhesion and axon guidance including members of the Ephrin and Semaphorins families. At LSD1 target genes, we demonstrate site-specific H3K4me2 histone methylation changes overlapping LSD1 binding sites, however no global changes in H3K4me2 were observed. Interestingly we find morphological and cell adhesion changes in sensitive cell lines that coincide with expression changes in markers of neuroendocrine differentiation of SCLC such as GRP, NCAM, and NEUROD1. Based on these data, we propose a model that LSD1 inhibition modulates the neuroendocrine differentiation program of SCLC cells promoting tumor inhibition in sensitive SCLC models. Citation Format: Thomas A. Paul, Shikhar Sharma, Jill Hallin, Tao Xie, Timothy Nichols, Mike Greig, James Hardwick, Martin Wythes, Dominique Verhelle. Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways. [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 3518. doi:10.1158/1538-7445.AM2015-3518

Abstract 5144: Epigenetic reprogramming by tumor-derived EZH2 gain of function mutants leads to aggressive 3D-cell morphologies in both epithelial and melanoma cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In addition to numerous genetic changes underlying cellular transformation, cancer cells are also characterized by epigenetic changes that are likely to play important roles in disease progression. EZH2 is an epigenetic repressor that plays well-established roles in development. In addition to widespread overexpression in a variety of tumors, the discovery of gain of function (GOF) mutations of EZH2 in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and melanoma strongly suggests an important function for this histone methyltransferase in cancer. To ascertain the function of elevated EZH2 catalytic activity, we expressed either wild-type EZH2 (WT) or EZH2 GOF mutants in both non-tumorigenic (immortalized epithelial cells) and tumorigenic (melanoma cells) settings. In both systems, EZH2 GOF mutants greatly increased global levels of H3K27me3 and decreased H3K27me2 levels, similar to the epigenetic pattern seen in DLBCL cell lines with endogenous EZH2 GOF mutations. In epithelial cells, expression of an EZH2 GOF mutant caused striking changes in 3D-morphology and gene changes that are indicative of cells that have undergone an epithelial to mesenchymal transition. In the disease relevant melanoma cells, several distinct EZH2 GOF mutants (but not EZH2 WT) caused prominent branching morphology in 3D-culture. Interestingly, these GOF mutants did not affect 2D-cell morphology or proliferation of melanoma cells. Furthermore, catalytic inhibition of EZH2 GOF mutants with a commercially available tool compound attenuated the 3D-phenotype. Importantly, EZH2 inhibition in melanoma cells expressing an endogenous GOF mutation also caused similar changes in 3D-morphology. RNA-seq analysis revealed genes involved in processes such as cell adhesion and axonal guidance that were down-regulated by EZH2 GOF mutants. Finally, melanoma cells expressing ectopic EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. Collectively, these results suggest that EZH2 GOF mutants may alter the interaction of tumor cells with their microenvironment and in this way provide a selective advantage to such tumors. Citation Format: Robert A. Rollins, Anthony M. Barsotti, Michael Ryskin, Wenyan Zhong, Wei-Guo Zhang, Andreas Giannakou, Christine Loreth, Veronica Diesl, Maximillian T. Follettie, Jonathon Golas, Michelle Lee, Timothy Nichols, Conglin Fan, Gary Li, Stephen Dann, Paul A. Rejto, Kim T. Arndt, Dominique Verhelle. Epigenetic reprogramming by tumor-derived EZH2 gain of function mutants leads to aggressive 3D-cell morphologies in both epithelial and melanoma cells. [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 5144. doi:10.1158/1538-7445.AM2014-5144

Abstract 2669: Targeting the tumor vasculature with antibody drug conjugates

Clinical use of vascular targeting antibody drug conjugates (VT-ADCs) has not yet been explored. Herein, we preclinically demonstrate utility, mechanisms and advantages of VT-ADCs as targeted cancer therapeutics. A monoclonal human IgG 1 antibody selective for murine VEGFR2 (B3) is non-VEGF-A neutralizing and traffics to the lysosome. B3 was conjugated to a proprietary auristatin microtubule inhibitor with a non-cleavable maleimidocaproyl linker, resulting in the proof-of-concept VT-ADC, B3-mcMTI. In vitro, free-payload and anti-VEGFR2-mcMTI selectively inhibit proliferating endothelial cells (ECs) with pM and low nM IC 50 s - suggesting that even with a broadly expressed, low-level target, a VT-ADC should be potent and tumor specific. To test this in vivo, we treated three cell line xenografts (CLX, HT29, Ls174T, A498) and a colorectal carcinoma patient derived xenograft (CRC PDX) with B3-mcMTI at 3 mg/kg q4d, resulting in tumor stasis in four models. In the HT29 colon carcinoma CLX and the CRC PDX model, B3-mcMTI was superior to VEGF-neutralizing antibody G6-31 (anti-VEGF mAb). In standard-of-care (SOC) chemotherapy (irinotecan and 5-FU) combination studies in HT29, B3-mcMTI had improved anti-tumor activity over SOC alone, and, moreover, was as effective in inhibiting tumor growth as a single agent ADC as anti-VEGF mAb combined with SOC. In the 4T1 orthotopic anti-VEGF resistant breast carcinoma setting, B3-mcMTI outperformed anti-VEGF mAb, and when combined with SOC (paclitaxel) demonstrated improved activity over single agent without an increase in metastases. B3-mcMTI internalization in endothelial cells is VEGFR2-mediated, with antibody binding and active payload releasing in normal and tumor tissues at concentrations above the in vitro IC 50 s; however, payload activity as measured by quantitative image analysis of pharmacodynamic biomarkers such as phospho-Histone H3 and cleaved caspase 3 is overwhelmingly localized to tumor ECs. Targeted vessels are smooth muscle invested, suggesting that VT-ADCs may target vasculature that contributes to anti-VEGF resistance. In conclusion, VT-ADCs are effective both as single agents and when combined with SOC, and VT-ADCs may overcome resistance mechanisms to standard anti-angiogenics. Taken together, these data suggest that payloads selective for proliferating ECs enable ADC-mediated targeting of widely expressed EC surface proteins, supporting the clinical pursuit of VT-ADCs. Citation Format: Andrea T. Hooper, Chao-Pei Betty Chang, Kimberly Marquette, Jonathon Golas, Justin Lucas, Timothy Nichols, Judy Lucas, Gavriil Maria, Edward Rosfjord, Anton Xavier, Nathan Scott, Sadhana Jain, Wei Cao, Mauricio Leal, Andreas Maderna, Magali Guffroy, Xiang Zheng, Lioudmila Tchistiakova, Frank Loganzo, Hans-Peter Gerber, Chad May. Targeting the tumor vasculature with antibody drug conjugates. [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 2669. doi:10.1158/1538-7445.AM2014-2669

Abstract 2059: In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer

Antibody drug conjugates (ADCs) are clinically validated as a modality for targeted therapy of solid and hematological cancer due to advancements in target selection, conjugation chemistry and linker technology. However, much about mechanism of action (MoA) is yet to be fully understood. Our goal was to interrogate ADC pharmacokinetics and pharmacodynamics establishing proof of mechanism (PoM) of drug action with a diverse panel of ADCs. Herein, we describe the development of novel immunohistochemical (IHC) methods for in situ visualization of ADCs binding to target expressing cells and their cognate downstream biomarkers of response in formalin fixed paraffin embedded cells/tissues. We demonstrate specific binding of 4 different ADCs spanning 2 solid tumor targets and an endothelial cell target using IHC with anti-human IgG in human tumor xenograft models expressing the respective targets. ADC binding to target is observed as early as 20 minutes after a single dose of ADC at 3 mg/kg. Utilizing an anti-microtubule inhibitor (MTI) payload-specific antibody we additionally detect ADC binding to tumor cells by monitoring the cytotoxic payload. The cell type where the antibodies and payload localized was identified by double and triple IHC. Pharmacodynamic biomarkers of response for two payload classes (DNA damaging agents and MTIs) were detected with antibodies against phospho-Histone H2AX and phospho-Histone H3, respectively - confirming the expected ADC MoAs. Downstream apoptosis of target cells was detected with cleaved caspase 3 IHC. The kinetics of biomarker response and downstream cellular impact was quantified via image analysis with biomarkers evident as early as 24 hours after a single dose for both tumor cell and vascular targets. Furthermore, we observed a correlation between biomarkers of response and efficacy of the ADCs as measured by statistically significant tumor growth inhibition for the 4 ADCs we studied. These data suggest that IHC interrogations of drug action should be used to further the clinical development of ADCs via demonstration of pharmacodynamic activities at the cellular level, establishing PoM data, and enabling predictive preclinical oncology models in order to reduce clinical attrition of ADCs. Citation Format: Jonathon Golas, Andrea T. Hooper, Justin Lucas, Heather Jones, Timothy Nichols, Kiran Khandke, Manoj Charati, Roger Conant, Michael Cinque, Judy Lucas, Marc Damelin, Ken Geles, Caiazzo Teresa, Frank Loganzo, Puja Sapra, Hans-Peter Gerber, Chad May. In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer. [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 2059. doi:10.1158/1538-7445.AM2014-2059