Amy S. Farrell

The Axin1 scaffold protein promotes formation of a degradation complex for c‐Myc

Expression of the c‐Myc proto‐oncoprotein is tightly regulated in normal cells. Phosphorylation at two conserved residues, threonine58 (T58) and serine62 (S62), regulates c‐Myc protein stability. In cancer cells, c‐Myc can become aberrantly stabilized associated with altered T58 and S62 phosphorylation. A complex signalling cascade involving GSK3β kinase, the Pin1 prolyl isomerase, and the PP2A‐B56α phosphatase controls phosphorylation at these sites. We report here a novel role for the tumour suppressor scaffold protein Axin1 in facilitating the formation of a degradation complex for c‐Myc containing GSK3β, Pin1, and PP2A‐B56α. Although knockdown of Axin1 decreases the association of c‐Myc with these proteins, reduces T58 and enhances S62 phosphorylation, and increases c‐Myc stability, acute expression of Axin1 reduces c‐Myc levels and suppresses c‐Myc transcriptional activity. Moreover, the regulation of c‐Myc by Axin1 is impaired in several tested cancer cell lines with known stabilization of c‐Myc or loss of Axin1. This study provides critical insight into the regulation of c‐Myc expression, how this can be disrupted in three cancer types, and adds to our knowledge of the tumour suppressor activity of Axin1.

Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer

Significance Increased kinase activity and suppressed phosphatase activity are hallmarks of oncogenic signaling. The transcription factor c-MYC, a master driver of human cancer, is stabilized and activated by persistent serine 62 phosphorylation. The tumor suppressor protein phosphatase 2A (PP2A) targets this site and negatively regulates c-MYC. Here, we show that two cellular inhibitors of PP2A, the SET oncoprotein and cancerous inhibitor of PP2A (CIP2A), are overexpressed in breast cancer, and depletion or inhibition of SET or CIP2A reduces c-MYC expression and activity and decreases the tumorigenic potential of breast cancer cells. These findings strongly suggest that inhibiting SET or CIP2A to reactivate PP2A may be an effective therapeutic strategy for targeting c-MYC in breast cancer. The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50–60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.

Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression

High expression of the oncoprotein Myc has been linked to poor outcome in human tumors. Although MYC gene amplification and translocations have been observed, this can explain Myc overexpression in only a subset of human tumors. Myc expression is in part controlled by its protein stability, which can be regulated by phosphorylation at threonine 58 (T58) and serine 62 (S62). We now report that Myc protein stability is increased in a number of breast cancer cell lines and this correlates with increased phosphorylation at S62 and decreased phosphorylation at T58. Moreover, we find this same shift in phosphorylation in primary breast cancers. The signaling cascade that controls phosphorylation at T58 and S62 is coordinated by the scaffold protein Axin1. We therefore examined Axin1 in breast cancer and report decreased AXIN1 expression and a shift in the ratio of expression of two naturally occurring AXIN1 splice variants. We demonstrate that this contributes to increased Myc protein stability, altered phosphorylation at S62 and T58, and increased oncogenic activity of Myc in breast cancer. Thus, our results reveal an important mode of Myc activation in human breast cancer and a mechanism contributing to Myc deregulation involving unique insight into inactivation of the Axin1 tumor suppressor in breast cancer.

Pin1 Regulates the Dynamics of c-Myc DNA Binding To Facilitate Target Gene Regulation and Oncogenesis

ABSTRACT The Myc oncoprotein is considered a master regulator of gene transcription by virtue of its ability to modulate the expression of a large percentage of all genes. However, mechanisms that direct Mycs recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl isomerase enhances recruitment of serine 62-phosphorylated Myc and its coactivators to select promoters during gene activation, followed by promoting Mycs release associated with its degradation. This facilitates Mycs activation of genes involved in cell growth and metabolism, resulting in enhanced proproliferative activity, even while controlling Myc levels. In cancer cells with impaired Myc degradation, Pin1 still enhances Myc DNA binding, although it no longer facilitates Myc degradation. Thus, we find that Pin1 and Myc are cooverexpressed in cancer, and this drives a gene expression pattern that we show is enriched in poor-outcome breast cancer subtypes. This study provides new insight into mechanisms regulating Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in the regulation of transcription factors, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and Myc.

Targeting Inhibitors of the Tumor Suppressor PP2A for the Treatment of Pancreatic Cancer

Pancreatic cancer is a deadly disease that is usually diagnosed in the advanced stages when few effective therapies are available. Given the aggressive clinical course of this disease and lack of good treatment options, the development of new therapeutic agents for the treatment of pancreatic cancer is of the upmost importance. Several pathways that have shown to contribute to pancreatic cancer progression are negatively regulated by the tumor suppressor protein phosphatase 2A (PP2A). Here, the endogenous inhibitors of PP2A, SET (also known as I2PP2A) and cancerous inhibitor of PP2A (CIP2A), were shown to be overexpressed in human pancreatic cancer, contributing to decreased PP2A activity and overexpression and stabilization of the oncoprotein c-Myc, a key PP2A target. Knockdown of SET or CIP2A increases PP2A activity, increases c-Myc degradation, and decreases the tumorigenic potential of pancreatic cancer cell lines both in vitro and in vivo. Moreover, treatment with a novel SET inhibitor, OP449, pharmacologically recapitulates the phenotypes and significantly reduces proliferation and tumorigenic potential of several pancreatic cancer cell lines, with an accompanying attenuation of cell growth and survival signaling. Furthermore, primary cells from patients with pancreatic cancer were sensitive to OP449 treatment, indicating that PP2A-regulated pathways are highly relevant to this deadly disease. Implications: The PP2A inhibitors SET and CIP2A are overexpressed in human pancreatic cancer and are important for pancreatic cancer cell growth and transformation; thus, antagonizing SET and/or CIP2A may be an innovative approach for the treatment of human pancreatic cancer. Mol Cancer Res; 12(6); 924–39. ©2014 AACR.

The Tumor Suppressor Protein HBP1 Is a Novel c-Myc-binding Protein That Negatively Regulates c-Myc Transcriptional Activity

c-Myc is an important transcription factor that regulates cellular proliferation, cell growth, and differentiation. A number of transcriptional co-factors for c-Myc have been described that have binding sites within highly conserved regions of the c-Myc transactivational domain (TAD). Given the importance of the c-Myc TAD, we set out to identify new proteins that interact with this region using a yeast two-hybrid assay. HBP1 was identified in our screen as a protein that interacts with full-length c-Myc but not a c-Myc mutant lacking the TAD. HBP1 is a transcriptional repressor and has been shown to negatively regulate the cell cycle. A correlation between HBP1 under-expression and breast cancer relapse has been described, suggesting that HBP1 may be an important tumor suppressor protein. We have found that HBP1 binds c-Myc in cells, and expression of HBP1 inhibits c-Myc transactivational activity at least partly by preventing c-Myc binding to target gene promoters. c-Myc binds to the C terminus of HBP1, a region lost in some breast tumors, and some HBP1 mutants found in breast cancer weakly interact with and/or no longer negatively regulate c-Myc. This work adds to our understanding of c-Myc regulation and mechanisms of tumor suppression by HBP1.

MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.Neuroendocrine differentiation of epithelial tumor cells can contribute to cancer cell resistance and survival. Here, the authors show that dysregulated c-Myc promotes neuroendocrine differentiation in pancreatic ductal adenocarcinoma, leading to poor survival and chemoresistance.

Abstract B21: Protein phosphatase 2A (PP2A) activation functions synergistically with kinase inhibition in pancreatic cancer

The 5-year survival rate for pancreatic cancer patients is only 6%, the lowest of all major cancers, indicating a critical need for increased understanding of pancreatic cancer development and identification of new therapeutic targets. Several genetic mutations are associated with the progression of human pancreatic ductal adenocarcinoma, including KRAS, which is mutated in ~95% of all pancreatic ductal adenocarcinomas (PDAC). Targeted therapies, such as kinase inhibitors, provide a means to reduce oncogenic signaling pathways that drive pancreatic cancer; however, the presence of resistance mechanisms, through extensive feedback loops, reduces the clinical efficacy of single agent treatments. Protein Phosphatase 2A (PP2A) is a critical tumor suppressor that negatively regulates several key oncogenic pathways implicated in mediating therapeutic resistance, including the PI3K/Akt, RAS/ERK, and Myc pathways. The goal of this research is to identify the therapeutic benefit of combined phosphatase activation and kinase inhibition, as a means to attenuate resistance mechanisms, reduce pancreatic tumor growth, and improve therapeutic efficacy in pancreatic cancer patients. We have determined that SET, an endogenous inhibitor of PP2A, is overexpressed in pancreatic cancer cell lines and primary patient samples, suggesting that suppression of PP2A contributes to pancreatic oncogenic signaling. As a result, we have started investigating the therapeutic efficacy of two novel therapeutic agents that activate PP2A: OP449, a SET antagonist, and DT1154, a small molecule direct PP2A activator. Treatment of pancreatic cancer cell lines with these compounds results in increased PP2A activity, reduced MYC levels, and reduced tumorigenic potential both in vitro and in vivo. Since these compounds reduce oncogenic signals known to contribute to pancreatic cancer, we hypothesize that phosphatase activation will function synergistically with select kinase inhibitors, resulting in sustained attenuation of oncogenic signaling in pancreatic cancer cells. In order to identify pathways that function synergistically with PP2A activation, a panel of pancreatic cancer cell lines was plated with and without OP449 into a 384-well kinase inhibitor (KI) screen, which evaluates the efficacy of over 130 kinase inhibitors at seven serial dilutions spanning the predicted IC50. Several signaling nodes were found to be synergistic with OP449, with the PI3K/AKT/mTOR pathways being particularly susceptible. Specifically, the combination of OP449 with Ink128, an mTOR1/2 inhibitor, reduced pancreatic cancer cell survival, oncogenic signaling, and transformed phenotypes, over either drug alone. These results provide new insight into our understanding of the regulation of pancreatic oncogenic signaling by phosphatases and identify new combination therapies that reduce resistance mechanisms. Citation Format: Brittany L. Allen-Petersen, Amy S. Farrell, Zina P. Jenny, Colin J. Daniel, Zhiping Wang, Charles D. Lopez, Dale J. Christensen, Goutham Narla, Brett C. Sheppard, Rosalie C. Sears. Protein phosphatase 2A (PP2A) activation functions synergistically with kinase inhibition in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B21.

Abstract IA10: Post-translational regulation of MYC's oncogenic activity

In response to growth stimulatory signals in normal cells or in transformed cells with constitutive signaling, the c-Myc oncoprotein is post-translationally stabilized through enhanced phosphorylation at the conserved Serine 62 residue. In addition to increasing c-Myc stability, phosphorylation at Serine 62 (pS62-Myc), in conjunction with Proline 63 isomerization mediated by the Pin1 Proline Isomerase, also increases the rate of recruitment of newly synthesized c-Myc to its target genes involved in pro-proliferative phenotypes, enhancing their expression. In normal cells, phosphorylation of S62 primes phosphorylation at a second conserved residue, Threonine 58 (pT58), which then enhance c-Myc degradation by facilitating a second Pin1-mediated isomerization event, which promotes Protein Phosphatase 2A (PP2A-B56α)-mediated dephosphorylation of Serine 62 and recruits the E3 ubiquitin ligase, SCFFbw7. In cancer cell lines as well as patient tumor samples pS62-Myc is elevated relative to non-transformed cell lines or adjacent normal tissue. In order to study the role of pS62-Myc in vivo, we created c-Myc knock-in mice that express either Myc wild-type (WT) or the MycT58A or MycS62A phosphorylation mutant from the ROSA26 locus in response to Cre-recombinase. Analysis of these mice revealed increased tumorigenic potential of MycT58A, which has constitutive S62 phosphorylation as it is resistant to PP2A-mediated S62 dephosphorylation. Together, this research has revealed an important role for S62 phosphorylation and Pin1-mediated isomerization in the tumorigenic activity of c-Myc, which presents new strategies to target c-Myc by inhibiting these post-translational activation steps. Thus, we are currently testing the therapeutic efficacy of PP2A activation and Pin1 inhibition in mouse models of c-Myc-driven tumorigenesis. To create physiologically relevant c-Myc-driven tumor models that engage post-translational activation of c-Myc, we have crossed our ROSA-LSL-MycWT mice with mice carrying organ-relevant oncogenic signaling transgenes. Thus far, we have generated mouse models of Her2+ and Triple Negative breast cancer, and pancreatic cancer. We have demonstrated dramatic tumor growth inhibition with a novel, orally available small molecule PP2A activator drug in these mouse models. Citation Format: Rosalie Sears, Amy Farrell, Xiaoyan Wang, Juan Liang, Mahnaz Janghorban, Yulong Su, Brittany Allen-Petersen, Michael Ohlemeyer, Narla Goutham. Post-translational regulation of MYC9s oncogenic activity. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA10.

Abstract A104: Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer

The 5-year survival rate for pancreatic cancer patients is only 6%, the lowest of all major cancers, indicating a critical need for increased understanding of pancreatic cancer development and identification of new therapeutic targets. Several genetic mutations are associated with the progression of human pancreatic ductal adenocarcinoma, including KRAS, which is mutated in ~95% of all pancreatic ductal adenocarcinomas (PDAC). Targeted therapies, such as kinase inhibitors, provide a means to reduce oncogenic signaling pathways that drive pancreatic cancer; however, the presence of resistance mechanisms, through extensive feedback loops, reduces the clinical efficacy of single agent treatments. Protein Phosphatase 2A (PP2A) is a critical tumor suppressor that negatively regulates several key oncogenic pathways implicated in mediating therapeutic resistance, including the PI3K/Akt, RAS/ERK, and Myc pathways. The goal of this research is to identify the therapeutic benefit of combined phosphatase activation and kinase inhibition, as a means to attenuate resistance mechanisms, reduce pancreatic tumor growth, and improve therapeutic efficacy in pancreatic cancer patients. We have determined that SET, an endogenous inhibitor of PP2A, is overexpressed in pancreatic cancer cell lines and primary patient samples, suggesting that suppression of PP2A contributes to pancreatic oncogenic signaling. Therapeutic inhibition of SET utilizing a novel SET antagonist, OP449, leads to increased PP2A activity and significant cytotoxic activity in pancreatic cancer cells in vitro and in vivo. Importantly, these results correlate with reduced activation of pathways known to contribute to therapeutic resistance, including c-Myc and Akt. I hypothesize that phosphatase activation will function synergistically with select kinase inhibitors, resulting in sustained attenuation of oncogenic signaling in pancreatic cancer cells. In order to identify pathways that function synergistically with PP2A activation, a panel of pancreatic cancer cell lines was plated with and without OP449 into a 384-well kinase inhibitor (KI) screen, which evaluates the efficacy of over 130 kinase inhibitors at seven serial dilutions spanning the predicted IC50. Several signaling nodes were found to be synergistic with OP449, with the PI3K/AKT/mTOR pathways being particularly susceptible. Specifically, the combination of OP449 with Ink128, an mTOR1/2 inhibitor, reduced pancreatic cancer cell survival, oncogenic signaling, and transformed phenotypes, over either drug alone. These results provide new insight into our understanding of the regulation of pancreatic oncogenic signaling by phosphatases and identify new combination therapies that reduce resistance mechanisms. Citation Format: Brittany L. Allen-Petersen, Amy S. Farrell, Colin J. Daniel, Zhiping Wang, Charles D. Lopez, Dale J. Christensen, Brett C. Sheppard, Rosalie C. Sears. Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A104.