Katherine M. Morgan

p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53β are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8+ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28-CD57+) with decreased Δ133p53 and increased p53β expression. Human lung tumor-associated CD8+ T lymphocytes also harbored senescent cells. Cultured CD8+ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8+CD28- cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53β overexpression in CD8+CD28+ cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53β in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Patient-Derived Xenograft Models of Non-Small Cell Lung Cancer and Their Potential Utility in Personalized Medicine

Traditional preclinical studies of cancer therapeutics have relied on the use of established human cell lines that have been adapted to grow in the laboratory and, therefore, may deviate from the cancer they were meant to represent. With the emphasis of cancer drug development shifting from non-specific cytotoxic agents to rationally designed molecularly targeted therapies or immunotherapy comes the need for better models with predictive value regarding therapeutic activity and response in clinical trials. Recently, the diversity and accessibility of immunodeficient mouse strains has greatly enhanced the production and utility of patient-derived xenograft (PDX) models for many tumor types, including non-small cell lung cancer (NSCLC). Combined with next-generation sequencing, NSCLC PDX mouse models offer an exciting tool for drug development and for studying targeted therapies while utilizing patient samples with the hope of eventually aiding in clinical decision-making. Here, we describe NSCLC PDX mouse models generated by us and others, their ability to reflect the parental tumors’ histomorphological characteristics, as well as the effect of clonal selection and evolution on maintaining genomic integrity in low-passage PDXs compared to the donor tissue. We also raise vital questions regarding the practical utility of PDX and humanized PDX models in predicting patient response to therapy and make recommendations for addressing those questions. Once collaborations and standardized xenotransplantation and data management methods are established, NSCLC PDX mouse models have the potential to be universal and invaluable as a preclinical tool that guides clinical trials and standard therapeutic decisions.

Asymmetric segregation of template DNA strands in basal-like human breast cancer cell lines

Background and methodsStem or progenitor cells from healthy tissues have the capacity to co-segregate their template DNA strands during mitosis. Here, we set out to test whether breast cancer cell lines also possess the ability to asymmetrically segregate their template DNA strands via non-random chromosome co-segregation, and whether this ability correlates with certain properties attributed to breast cancer stem cells (CSCs). We quantified the frequency of asymmetric segregation of template DNA strands in 12 human breast cancer cell lines, and correlated the frequency to molecular subtype, CD44+/CD24-/lo phenotype, and invasion/migration ability. We tested if co-culture with human mesenchymal stem cells, which are known to increase self-renewal, can alter the frequency of asymmetric segregation of template DNA in breast cancer.ResultsWe found a positive correlation between asymmetric segregation of template DNA and the breast cancer basal-like and claudin-low subtypes. There was an inverse correlation between asymmetric segregation of template DNA and Her2 expression. Breast cancer samples with evidence of asymmetric segregation of template DNA had significantly increased invasion and borderline significantly increased migration abilities. Samples with high CD44+/CD24-/lo surface expression were more likely to harbor a consistent population of cells that asymmetrically segregated its template DNA; however, symmetric self-renewal was enriched in the CD44+/CD24-/lo population. Co-culturing breast cancer cells with human mesenchymal stem cells expanded the breast CSC pool and decreased the frequency of asymmetric segregation of template DNA.ConclusionsBreast cancer cells within the basal-like subtype can asymmetrically segregate their template DNA strands through non-random chromosome segregation. The frequency of asymmetric segregation of template DNA can be modulated by external factors that influence expansion or self-renewal of CSC populations. Future studies to uncover the underlying mechanisms driving asymmetric segregation of template DNA and dictating cell fate at the time of cell division may explain how CSCs are maintained in tumors.

Activation of the Notch1 Stem Cell Signaling Pathway during Routine Cell Line Subculture

Cell culture is essential across cancer cell biology laboratories. Cancer cell lines that are attached to the culture vessel as monolayers are routinely passaged to new vessels in order to produce a large number of cells for carrying out various experiments. Cell detachment from the extracellular matrix can activate numerous cell signaling pathways. For example, enzymatic cell detachment using trypsin activates the HIPPO pathway, resulting in phosphorylation and inhibition of YAP within 10 min after cell detachment (1). Furthermore, Bcl2 protein levels can be down-regulated, whereas p53 and p21 protein levels are upregulated immediately after trypsinization (2). It is likely that many additional pathways that play key roles in cancer cell biology are also impacted during cell passaging. Having a full understanding of pathway activation during cell culture is essential for designing experiments and for accurate interpretation of results. Since its discovery in Drosophila melanogaster almost a century ago (3, 4), the Notch pathway has proven to regulate numerous developmental processes as well as tissue homeostasis in multicellular organisms (5). This conserved pathway has also been found to be dysregulated across a wide range of cancers (6). When Notch is activated by ligands of the Jagged or Delta family proteins, the intracellular domain of the Notch receptor is cleaved and translocates to the nucleus to activate transcription of downstream targets, including the Hes family of proteins. Notch undergoes crosstalk with a multitude of signaling pathways such as p53, EGFR, Kras, Wnt, TGFβ, sonic hedgehog, NFκB, and others (5, 7–12). Thus, dysregulation of Notch signaling can have global cellular consequences that modulate cell survival, behavior, and function.

Gamma Secretase Inhibition by BMS-906024 Enhances Efficacy of Paclitaxel in Lung Adenocarcinoma

Notch signaling is aberrantly activated in approximately one third of non–small cell lung cancers (NSCLC). We characterized the interaction between BMS-906024, a clinically relevant Notch gamma secretase inhibitor, and front-line chemotherapy in preclinical models of NSCLC. Chemosensitivity assays were performed on 14 human NSCLC cell lines. There was significantly greater synergy between BMS-906024 and paclitaxel than BMS-906024 and cisplatin [mean combination index (CI) value, 0.54 and 0.85, respectively, P = 0.01]. On an extended panel of 31 NSCLC cell lines, 25 of which were adenocarcinoma, the synergy between BMS-906024 and paclitaxel was significantly greater in KRAS- and BRAF-wildtype than KRAS- or BRAF-mutant cells (mean CI, 0.43 vs. 0.90, respectively; P = 0.003). Paclitaxel-induced Notch1 activation was associated with synergy between BMS-906024 and paclitaxel in the KRAS- or BRAF-mutant group. Knockdown of mutant KRAS increased the synergy between BMS-906024 and paclitaxel in heterozygous KRAS-mutant cell lines. Among KRAS- or BRAF-mutant NSCLC, there was a significant correlation between synergy and mutant or null TP53 status, as well as between synergy and a low H2O2 pathway signature. Exogenous overexpression of activated Notch1 or Notch3 had no effect on the enhanced sensitivity of NSCLC to paclitaxel by BMS-906024. In vivo studies with cell line– and patient-derived lung adenocarcinoma xenografts confirmed enhanced antitumor activity for BMS-906024 plus paclitaxel versus either drug alone via decreased cell proliferation and increased apoptosis. These results show that BMS-906024 sensitizes NSCLC to paclitaxel and that wild-type KRAS and BRAF status may predict better patient response to the combination therapy. Mol Cancer Ther; 16(12); 2759–69. ©2017 AACR.

Abstract 4544: FBW7 induces S-phase arrest caused by DNA double strand breaks through targeting SOX9 for proteasomal degradation

SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis or progression of many human tumors through increasing cell proliferation and epithelial-mesenchymal transition. We observed that, in response to UV irradiation or certain chemotherapeutic agents, SOX9 is actively and rapidly degraded by a ubiquitin pathway dependent mechanism across several different tumor types including lung cancer, colon cancer, osteosarcoma and melanoma, as well as normal human bronchial epithelial cells. We found that SOX9 is phosphorylated by GSK3β at Ser-236, facilitating the direct binding and degradation of SOX9 via the F box protein, FBW7α. We also determined that the de-ubiquitinase, USP28, stabilizes SOX9 under normal conditions by sequestering FBW7, but is released from FBW7 after UV irradiation, allowing FBW7 to bind SOX9 and target it for destruction. DNA damage-induced SOX9 degradation was independent of p53, ATM, ATR and MAPK pathways. Failure to deplete SOX9 attenuated the DNA damage-induced intra-S-phase checkpoint and increased long-term cell survival. Moreover, mutations within the FBW7 phosphodegron binding site of SOX9 prevented SOX9 degradation after DNA damage, and incurred resistance of non-small cell lung cancer (NSCLC) cells to cisplatin in vivo. We found that cancer patients with tumors expressing high Sox9 and low Fbw7 levels exhibit inferior survival. Our discovery reveals a novel function of SOX9 in the cellular response to DNA damage. Induced degradation of SOX9 may be part of the protection mechanisms to maintain genomic stability. This new regulatory mechanism of the FBW7-SOX9 axis in cancer could have diagnostic and therapeutic implications. Citation Format: Xuehui Hong, Wenyu Liu, Ruipeng Song, Hiroyuki Inuzuka, Hatem E. Sabaawy, Katherine M. Morgan, Jamie J. Shah, Samuel F. Bunting, Xing Feng, Chi-Kwan Tsang, Zhiyuan Shen, X. F. Steven Zheng, LianXin Liu, Sharon R. Pine. FBW7 induces S-phase arrest caused by DNA double strand breaks through targeting SOX9 for proteasomal degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4544.

Abstract 4834: Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer (NSCLC) cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among NSCLC patients whose tumors are wildtype for TP53. Here, we characterized the interaction between BMS-906024, a clinically relevant gamma secretase inhibitor (GSI) that inhibits Notch activation, and front-line chemotherapy in preclinical models of NSCLC. MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy were performed on a panel of human NSCLC cell lines, most of which were derived from adenocarcinomas. Analysis of the drug effects with CalcuSyn yielded significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. Treatment of lung adenocarcinoma xenografts in NOD scid gamma mice confirmed enhanced antitumor activity for the combination treatment of BMS-906024 and paclitaxel by mechanisms currently under investigation. These results are a step toward identification of the optimal combination of the GSI BMS-906024 with standard chemotherapies, as well as potential biomarkers that could be used to predict patient response to Notch-targeted therapy. Citation Format: Katherine M. Morgan, Francis Lee, Erin Michaud, Bruce S. Fischer, Sharon R. Pine. Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4834.

Abstract 2535: Synergistic anti-tumor activity of the Notch gamma secretase inhibitor BMS-906024 and paclitaxel in the treatment of lung adenocarcinoma

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among non-small cell lung cancer patients whose tumors are wildtype for TP53. We set out to evaluate the combination of Notch-targeted therapy with front-line chemotherapy as an effective treatment for non-small cell lung cancer. Our study focused on lung adenocarcinoma, the most common histological subtype in lung cancer. To target Notch, we utilized the gamma secretase inhibitor (GSI) BMS-906024 which inhibits Notch activation. BMS-906024 is currently in Phase 1 clinical trials for patients with T-cell acute lymphoblastic leukemia and metastatic solid tumors, including lung cancer. Human cell lines representing the major genetic subtypes of lung cancer, most of which were derived from adenocarcinomas, underwent MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy. The dosing and timing for BMS-906024 administration were optimized by examination of maximal Notch1 inhibition. Analysis of the drug effects with CalcuSyn yielded Combination Index (CI) values, in which a CI of 0.5 or less was considered as strong synergism for the drug combination. We found that there were significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). We then grouped the cell lines by major genetic subtype (wildtype versus mutant or null for EGFR, Kras or TP53). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. These results are a step toward identification of potential biomarkers that could be used to predict patient response to Notch-targeted therapy, which could have a positive impact on the care of lung adenocarcinoma patients and be informative for treatment decisions. Citation Format: Katherine M. Morgan, Francis Lee, Erin Michaud, Joseph R. Bertino, Bruce S. Fischer, Sharon R. Pine. Synergistic anti-tumor activity of the Notch gamma secretase inhibitor BMS-906024 and paclitaxel in the treatment of lung adenocarcinoma. [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 2535. doi:10.1158/1538-7445.AM2015-2535

Abstract B17: Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma.

Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans. Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9. Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B17.

Abstract 1149: The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify upstream pathways that regulate Sox9 expression in lung cancer, as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known to induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling. Sox9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse Sox9 promoter are not conserved in humans. We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is downstream of Notch1 in lung adenocarcinoma cell lines. By ChIP we determined that Sox9 is a direct target of Notch1 and using luciferase reporter assays, we located the previously unidentified human RBP-Jκ binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the Sox9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Loss of Notch1 expression led to an induced MET phenotype, characterized by decreased cell invasion/migration, MET-like morphological changes, and increased E-cadherin expression, which were rescued by Sox9 overexpression. Our data also demonstrate that Sox9 contributes to Notch1-induced EMT in lung adenocarcinoma. These results establish Sox9 as a key Notch1 target gene mediating Notch1-induced EMT independent of TGF-β, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen M. Capaccione, Xuehui Hong, Katherine M. Morgan, Thaddeus D. Allen, Gregory D. Miles, Elke K. Markert, J. Michael Bishop, Sharon R. Pine. The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT. [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 1149. doi:10.1158/1538-7445.AM2014-1149