Wenyan Zhong

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.

Evolving Strategies for Target Selection for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) represent a promising modality for the treatment of cancer. The therapeutic strategy is to deliver a potent drug preferentially to the tumor and not normal tissues by attaching the drug to an antibody that recognizes a tumor antigen. The selection of antigen targets is critical to enabling a therapeutic window for the ADC and has proven to be surprisingly complex. We surveyed the tumor and normal tissue expression profiles of the targets of ADCs currently in clinical development. Our analysis demonstrates a surprisingly broad range of expression profiles and the inability to formalize any optimal parameters for an ADC target. In this context, we discuss additional considerations for ADC target selection, including interdependencies among biophysical properties of the drug, biological functions of the target and strategies for clinical development. The TPBG (5T4) oncofetal antigen and the anti-TPBG ADC A1-mcMMAF are highlighted to demonstrate the relevance of the target’s biological function. Emerging platform technologies and novel biological insights are expanding ADC target space and transforming strategies for target selection.

Breast cancer cells respond differentially to modulation of TGFβ2 signaling after exposure to chemotherapy or hypoxia.

Intratumoral heterogeneity helps drive the selection for diverse therapy-resistant cell populations. In this study, we demonstrate the coexistence of two therapy-resistant populations with distinct properties that are reproducibly enriched under conditions that characterize tumor pathophysiology. Breast cancer cells that survived chemotherapy or hypoxia were enriched for cells expressing the major hyaluronic acid receptor CD44. However, only CD44(hi) cells that survived chemotherapy exhibited cancer stem cell (CSC) phenotypes based on growth potential and gene expression signatures that represent oncogenic signaling and metastatic prowess. Strikingly, we identified TGFβ2 as a key growth promoter of CD44(hi) cells that survived chemotherapy but also as a growth inhibitor of cells that survived hypoxia. Expression of the TGFβ receptor TGFβR1 and its effector molecule SMAD4 was required for enrichment of CD44(hi) cells exposed to the chemotherapeutic drug epirubicin, which suggests a feed-forward loop to enrich for and enhance the function of surviving CSCs. Our results reveal context-dependent effects of TGFβ2 signaling in the same tumor at the same time. The emergence of distinct resistant tumor cell populations as a consequence of prior therapeutic intervention or microenvironmental cues has significant implications for the responsiveness of recurring tumors to therapy.

Upregulation of RNA Processing Factors in Poorly Differentiated Lung Cancer Cells.

Intratumoral heterogeneity in non–small cell lung cancer (NSCLC) has been appreciated at the histological and cellular levels, but the association of less differentiated pathology with poor clinical outcome is not understood at the molecular level. Gene expression profiling of intact human tumors fails to reveal the molecular nature of functionally distinct epithelial cell subpopulations, in particular the tumor cells that fuel tumor growth, metastasis, and disease relapse. We generated primary serum-free cultures of NSCLC and then exposed them to conditions known to promote differentiation: the air-liquid interface (ALI) and serum. The transcriptional network of the primary cultures was associated with stem cells, indicating a poorly differentiated state, and worse overall survival of NSCLC patients. Strikingly, the overexpression of RNA splicing and processing factors was a prominent feature of the poorly differentiated cells and was also observed in clinical datasets. A genome-wide analysis of splice isoform expression revealed many alternative splicing events that were specific to the differentiation state of the cells, including an unexpectedly high frequency of events on chromosome 19. The poorly differentiated cells exhibited alternative splicing in many genes associated with tumor progression, as exemplified by the preferential expression of the short isoform of telomeric repeat-binding factor 1 (TERF1), also known as Pin2. Our findings demonstrate the utility of the ALI method for probing the molecular mechanisms that underlie NSCLC pathogenesis and provide novel insight into posttranscriptional mechanisms in poorly differentiated lung cancer cells.

Cell Index Database (CELLX): a web tool for cancer precision medicine.

The Cell Index Database, (CELLX) (http://cellx.sourceforge.net) provides a computational framework for integrating expression, copy number variation, mutation, compound activity, and meta data from cancer cells. CELLX provides the computational biologist a quick way to perform routine analyses as well as the means to rapidly integrate data for offline analysis. Data is accessible through a web interface which utilizes R to generate plots and perform clustering, correlations, and statistical tests for associations within and between data types for ~20,000 samples from TCGA, CCLE, Sanger, GSK, GEO, GTEx, and other public sources. We show how CELLX supports precision oncology through indications discovery, biomarker evaluation, and cell line screening analysis.

Detecting expression of 5T4 in CTCs and tumor samples from NSCLC patients

The fetal oncogene 5T4 is a cell surface protein, with overexpression observed in a variety of cancers as compared to normal adult tissue. The ability to select patients with tumors that express high levels of 5T4 may enrich a clinical trial cohort with patients most likely to respond to 5T4 targeted therapy. To that end, we developed assays to measure 5T4 in both tumors and in circulating tumor cells (CTCs). We identified the presence of 5T4 in both adenocarcinoma and squamous cell carcinoma of lung, in all clinical stages and grades of disease. CTCs were identified in peripheral blood from the majority of patients with NSCLC, and 5T4 was detectable in most samples. Although 5T4 was present in both CTCs and tumors in most patients, there was no concordance between relative amount in either sample type. Clinical response rates of patients treated with the therapies directed against 5T4 in early stage clinical trials, as determined by these assays, may provide important insights into the biology of 5T4 in tumors and the mechanisms of action of 5T4-targeting therapy.

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 3487: CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines

Tumor-Initiating Cells (TICs), or Cancer Stem Cells (CSCs), are considered a subpopulation of cells within a tumor that are particularly aggressive because when isolated, these cells can form secondary tumors. Along these lines, experiments in which fragments of tumors, derived from primary patient samples, are re-implanted into nude mice, the majority of cells within the fragment die. However, day 3 after re-implantation, a small subpopulation emerges that is able to seed a new tumor. We hypothesized that these cells are unique and rare within the original tumor and consequently present an opportunity to identify novel markers that can identify these aggressive tumor cells. After sorting live human cells from tumor fragments three days after passaging, we used single cell PCR to determine the extent of heterogeneity within the fragment population. We, in fact, found that there is a subpopulation that highly expresses markers of proliferation and stemness. This is in contrast to the major population within the fragment that is enriched for genes associated with hypoxia and stress response. Interestingly, we found that the hypoxia responsive gene, CA9, does not correlate with other markers of hypoxia, such as VEGF, when analyzing cells derived from the fragment. Instead, CA9 was highly expressed among cells that were also enriched for markers such as Ki67, survivin, and Lgr5. To confirm if CA9 does identify a more proliferative, stem-like population, we have sorted CA9+ and CA9- cells from three day PDX fragments and analyzed using DNA microarrays and real-time PCR. We have consistently found, among several different PDX lines, that passaging of tumors followed by sorting CA9+ cells enriches for cells with increased levels of Lgr5, ASCL2, CD133, and Ki67. This is also validated through IHC analysis in which the majority of the cells that are positive for Ki67 are also positive for CA9. We are continuing to investigate the tumorigenicity of these cells by reinjecting CA9+ and CA9- cells from fragments back into immune-compromised mice. Furthermore, we are building a gene list derived from the microarrays to inspect by single cell PCR if any of these genes associate with Lgr5 in a grown tumor. Thus, we believe this is a functionally relevant and novel method for identify novel TIC markers. Citation Format: Julia Friedman, Wenyan Zhong, Christine Loreth, Veronica Diesl, Xin Han, Justin Lucas, Andrea Hooper, Vlad Buklan, Edward Rosfjord, Danielle Leahy, Judy Lucas, Maximillian Follettie, Kim Arndt. CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines. [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 3487. doi:10.1158/1538-7445.AM2014-3487

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 1968: Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms

Cancer relapse following treatment remains a significant barrier to achieving cures for many patients. An emerging framework for addressing this problem focuses on cancer stem cells (CSCs). Clinical and preclinical data suggest that CSCs survive chemotherapy and antiangiogenic therapy. In breast cancer, CSCs are marked by the cell surface expression of CD44, the major receptor for hyaluronic acid. The SKBR3 cell line was previously shown to be a clinically relevant model of breast CSCs in vivo, and we have further developed this model to enable mechanistic studies of tumor relapse in vitro. We found that chemotherapy and hypoxia both enriched for CD44 hi populations in SKBR3, but surprisingly, the populations were phenotypically distinct. CD44 hi cells from chemotherapy but not hypoxia exhibited increased tumor cell growth and increased sensitivity to the CSC-specific inhibitor salinomycin, compared to CD44 lo cells. To examine these CD44 hi and CD44 lo populations further, we performed transcriptional profiling with sorted cells. We found that the growth factor TGF beta-2 was upregulated in chemotherapy-treated CD44 hi cells and also enhanced the growth of these cells, which suggests that TGF beta-2 autocrine/paracrine signaling can promote the growth of surviving cells. We also observed an increase in xCT (SLC7A11) expression upon chemotherapy treatment and identified the CD44v8-10 variant expressed in the CD44 hi SKBR3. CD44v could therefore stabilize xCT and promote survival by lowering intracellular reactive oxygen species (ROS). We have also established a functional role for CD44 in SKBR3 cell growth: CD44 knockdown prevented colony formation, and conversely the CD44 ligand hyaluronic acid enhanced colony formation. These findings indicate that CD44 not only marks CSC populations that arise in response to chemotherapy but also functions in their survival. Our work suggests that mechanisms of tumor relapse vary based on the particular therapy, and defining these mechanisms will allow for the development of novel therapeutic strategies to enable long-term responses in the clinic. Citation Format: Siobhan O9Brien, Liang Chen, Wenyan Zhong, Douglas Armellino, Maximillian Follettie, Marc Damelin. Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms. [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 1968. doi:10.1158/1538-7445.AM2014-1968