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Mdm2 and its homologue MdmX form an E3-ligase complex that is best understood as the major regulator of p53. Yet Mdm2 and MdmX have functions in cells that are independent of their ability to degrade p53. Amongst the functions regulated by Mdm2 is cell migration, although the molecular mechanism(s) involved have not been well characterized. We show, in a p53 null model, that either siRNA knockdown of Mdm2 or MdmX as well as pharmacological inhibition of the Mdm2/X complex E3-ligase can reduce migration of cells grown as monolayer or invasion of cells from pre-formed spheroids into collagen-based matrices. This is consistent with our observation that Mdm2 ablation or inhibition leads to decreased cell spreading and attachment of cells to the extracellular matrix. In line with these findings, we found that modulation of Mdm2, MdmX or the Mdm2/X complex impacts focal adhesion (FA) formation, a main step in cell attachment, spreading and migration. Physiologically, Mdm2 silencing leads to decreased metastatic burden in mouse models. Mechanistically, we have discovered that Mdm2 modulates the RNA levels of Sprouty4 and that Sprouty4 is needed for the effects of Mdm2 knockdown on cell migration, FA formation and metastasis. Taken together, we have discovered a pathway by which Mdm2, through the activity of the Mdm2/X complex, mitigates FA formation, migration and ultimately metastasis by regulation of Sprouty4 independently of p53. Our findings suggest that blocking Mdm2 or the Mdm2/X complex might be a potential target to prevent metastasis. Citation Format: Rafaela Muniz de Queiroz, Gizem Efe, Asja Guzman, Naoko Hashimoto, Yusuke Kawashima, Tomoaki Tanaka, Anil K. Rustgi, Carol Prives. Mdm2 regulates metastasis and associated cellular processes through modulation of Sprouty4 in a p53-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB160.

Introduction: HER2 (encoded by the ERBB2 gene) is a member of the epidermal growth factor receptor (EGFR) family that exerts its activity through homo- or hetero-dimerization with other HER proteins. While multiple HER2-targeted therapies are FDA-approved for breast cancer, the clinical utility of targeting HER2 in bladder cancer patients remains undefined. We leveraged a prospective sequencing initiative and a new collection of patient-derived organoid (PDO) and xenograft (PDX) models to explore the prevalence of HER2 alterations in bladder cancers, its biologic role in bladder cancer pathogenesis and the potential clinical utility of HER2-targeted therapies. Methods: To define the landscape of HER2 alterations in bladder cancer patients, we analyzed data generated by The Cancer Genome Atlas (TCGA) and patients enrolled in the prospective MSK-IMPACT sequencing cohort. To study the biology of HER2 alterations in a bladder cancer context, we generated PDO and PDX bladder cancer models, including ERBB2 amplified, ERBB2 hotspot mutated and ERBB2 wildtype models. Patient-derived models were characterized using a multiplatform approach and were used to study HER2 oncogenic dependence and sensitivity to multiple anti-HER2 targeted agents. Results: The MSK-IMPACT assay revealed that ERBB2 alteration is common in bladder cancer, with a mutation frequency of 10.4% (breast cancer: 2.8%) and amplification frequency of 7.8% (breast cancer: 11.8%). ERBB2 alterations were more common in higher grade and stage bladder cancers. Among the HER2-altered PDOs, we identified some models with HER2 pathway dependence, similar to that observed in HER2 amplified breast cancer cell lines. However, most models had less dependence of downstream effector pathways such as AKT and ERK on HER2 signaling as compared to breast cancers. HER2-altered PDX models were significantly more sensitive to the HER2-targeted antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) than to HER kinase inhibitor neratinib. We also observed a complete response of a HER2+ bladder cancer patient treated with T-DXd (based on PET at 7 weeks). Sensitivity to the T-DXd payload, an exatecan derivative (DXd), was found to play a key role in determining sensitivity of bladder cancer models to T-DXd. Conclusion: Bladder cancer has higher ERBB2 mutation frequency than breast cancer, and HER2 alterations are more common in high grade and stage bladder cancers than in low-grade and non-invasive tumors, suggesting a role for HER2 in invasion and metastatic progression. The greater sensitivity of HER2 altered bladder cancer models to HER2-targeted ADC T-DXd and the complete response to T-DXd in a HER2+ bladder cancer patient provide justification for further clinical trials of HER2-targeted ADCs in bladder cancer. The sensitivity to ADC cytotoxic payload may be an important factor in determining patient response. Citation Format: Xinran Tang, Ziyu Chen, Jasmine Thomas, Karan Nagar, John Christin, Naryan Rustgi, Sizhi Gao, Carissa Chu, Elisa De Stanchina, Michael F. Berger, Jonathan A. Coleman, Michael M. Shen, Hikmat A. Al-Ahmadie, Gopakumar V. Iyer, Kwanghee Kim, David B. Solit. HER2 as a therapeutic target in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 472.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death worldwide. The 5-year survival rate is 90% in patients with localized tumors, but the survival rate drastically decreases to 14% in patients with distant metastasis. Therefore, it is imperative to elucidate the molecular mechanisms underlying CRC metastasis. The RNA-binding protein LIN28B is overexpressed in over 30% of patients with CRC and is associated with poor prognosis. Indeed, our previous work revealed that LIN28B promotes colorectal polyps and tumors in a genetic mouse model (PMID: 24142874) and metastasis in a subcutaneous xenograft model of CRC (PMID: 21512136). In the present study, we investigated the mechanism by which LIN28B promotes colorectal tumor progression and metastasis. To assess LIN28B-mediated biological changes, we established CRC cells (DLD-1 and LoVo) with overexpression of LIN28B. LIN28B overexpression increased the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in CRC cells. Knockdown of claudin-1 (CLDN1) or NOTCH3 expression in LIN28B-overexpresed CRC cells, or treatment of the cells with the pan-Notch inhibitor DAPT (γ-secretase inhibitor), revealed that the PI3K/Akt pathway works downstream of the tight junction protein CLDN1 and the transmembrane receptor NOTCH3. Furthermore, cell-cell contact of CRC cells was required for the activation of the NOTCH3 and PI3K/Akt signaling pathways, suggesting for an important role of CLDN1 as a tight junction protein upstream of NOTCH3 and PI3K. Inhibition of PI3K by either the pan-PI3K inhibitor LY294002 or PI3K-α-specific inhibitor Alpelisib (BYL719) suppressed LIN28B-mediated CRC cell migration and wound healing rate. Using a mouse model of metastatic CRC, we show that Alpelisib treatment significantly reduced LIN28B-induced liver metastases formation. Interestingly, the inhibitory effect of Alpelisib was even more pronounced when Alpelisib was given in combination with the pan-Notch inhibitor DAPT, resulting in complete inhibition of visible liver metastases formation. Immunohistochemical staining revealed that Alpelisib did not affect proliferation, apoptosis, or angiogenesis of the liver metastases, whereas DAPT may inhibit angiogenesis. Taken together, our results indicate that LIN28B promotes cell migration and liver metastasis formation through CLDN1 and NOTCH3-mediated activation of PI3K signaling. Development of new therapies that target the PI3K/Akt pathway may provide an effective strategy for preventing metastases in CRC patients. This work is supported by NIH 9R01CA277795-22, NIH 5P30CA0113696 and the American Cancer Society. The authors declare no conflicts of interest. Citation Format: Alice E. Shin, Kensuke Sugiura, Kensuke Suzuki, Yasunori Masuike, Christopher J. Lengner, Anil K. Rustgi. The role of the PI3K/Akt signaling pathway on LIN28B-mediated colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2484.

Bladder cancer (BLCA) is the sixth most common cancer in the U.S with 83,730 new cases, accounting for 17,200 deaths in the year 2021 and 212,536 deaths worldwide. EP300, a histone acetyltransferase gene, is mutated in ~17% of BLCA and in other cancer types such as endometrial cancer. The mechanisms by which EP300 mutations contribute to tumorigenicity of BLCA is not currently understood. To determine the phenotypic effects of loss-of-function EP300 mutations, we generated isogenic BLCA cells with EP300 knocked out (KO) using CRISPR/CAS9. Conspicuous changes in phenotypes consistent with more aggressive forms of cancer were observed in EP300-null BLCA clones, including luminal-basal histotype plasticity, anchorage-independent growth, enhanced in vitro and in vivo cell proliferation and enhanced invasion in Boyden chamber assays. To identify mechanisms whereby EP300 KO promotes increased oncogenicity, we performed phospho Tandem Tag Mass Spectrometry (TMT MS) using lysates from parental and EP300 KO cells. A notable finding was significantly increased expression of phosphorylated STAT3 (pSTAT3), which we subsequently confirmed by western blot. siRNA knockdown and selective inhibitor experiments indicated JAK1 as the upstream activator of pSTAT3 in BLCA cells. Studies of conditioned media (CM) from the EP300 KO cells using ELISA and neutralizing antibodies demonstrated that secreted IL-6 ligand and soluble IL-6R in the CM drove STAT3 activation through the IL-6 family common signaling subunit, gp130, i.e. trans-signaling. BLCA RT112 cells express an oncogenic FGFR3-TACC3 fusion and are sensitive to the FDA-approved FGFR kinase inhibitor erdafitinib. pSTAT3 expression was not dependent on FGFR3 signaling in EP300 KO RT112 cells, and EP300 KO was sufficient to confer erdafitinib resistance in a BLCA context. In sum, our results uncover that EP300 loss enhances IL-6/JAK/STAT3 signaling which promotes BLCA tumorigenesis and FGFR inhibitor resistance. Our findings elucidate a role for cytokine induced sterile inflammation in EP300-mediated tumorigenesis in bladder cancers and suggest that targeting the IL-6/JAK/STAT3 axis may represent a novel therapy strategy to overcome FGFR3 inhibitor resistance. Citation Format: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit. EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 330.

INTRODUCTION/METHODS: The multistep process of the tumor invasion-metastasis cascade is a major barrier to effective therapy. Mutations of the tumor suppressor p53 are frequently detected in esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq, ChIP-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer, L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP, in which we have identified macrophage colony stimulating factor 1 (CSF1) to be a direct gene target of p53-R172H. We have targeted CSF1/CSF1R signaling to assess its pro-tumorigenic roles. In addition to studying tumor intrinsic mechanisms, we have established multiplex immunofluorescence and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Overall, this study aims to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote lung metastasis. RESULTS/DISCUSSION: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors and this is dependent upon p53 mutation status, which is reinforced by TCGA data and patient-derived tissue microarrays (TMAs). The TMA analysis also suggested that CSF1 expression is higher in tumors that are poorly differentiated. Furthermore, through the proximity ligation assay (PLA), we have identified that bromodomain and extra-terminal (BET) protein BRD4, which binds to acetylated histones to regulate transcription interacts with p53-R172H at higher levels compared to wild-type p53 to induce Csf1 expression. We show that the BRD4/CSF1/CSF1R signaling axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in lungs in a mutant p53 background. In accordance, targeting CSF1/CSF1R signaling reduces phosphorylation of STAT3 in the metastatic tumors, which suggests a mechanism for increased invasiveness and metastasis. In parallel, upon inhibiting this axis, we have identified decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages, as well as CD31+ endothelial cells, and increased number of CD3+CD8+ cytotoxic T-cells at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of squamous cell carcinoma (SCC) datasets reveals that specific p53 mutations are associated with differential survival rates and CSF1 expression. CONCLUSION: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other SCCs. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine M. Cunningham, Qiaosi Tang, Kensuke Sugiura, Karen Dunbar, Kensuke Suzuki, Lois Resnick-Silverman, Alison M. Taylor, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1289.

The tumor microenvironment (TME), at both the primary tumor and sites of metastases, is a key determinant in the success of metastatic colonization. Cancer-associated fibroblasts (CAFs) are a predominant cell type in the TME that, through deposition and remodeling of extracellular matrix (ECM) components, can mediate the TME and promote cancer progression. In addition to cancer progression and metastasis, CAFs have also been shown to mediate resistance to chemotherapy and radiotherapy. Therefore, there is a need to identify the factors which activate and recruit CAFs to solid tumors. Though cytokine arrays, we identified CC motif chemokine ligand 5 (CCL5) as a secreted factor which is increased upon co-culture of esophageal cancer cell lines and CAFs. Using CRISPR/Cas9 technology, we determined that CCL5 is predominantly tumor-cell derived and loss of tumor-cell derived CCL5 reduced esophageal carcinoma cell proliferation in vitro and in vivo. We propose that this reduction in cell proliferation is mediated by ERK1/2 signaling as loss of tumor-cell derived CCL5 reduces expression of p-ERK1/2 and cyclin D1, an ERK1/2 target gene which is essential for cell cycle progression. Interestingly, the loss of tumor-derived CCL5 also reduced the percentage of CAFs, identified by αSMA positivity, recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists called Maraviroc. We treated xenografts generated from esophageal carcinoma cell lines or patient derived tumor fragments with Maraviroc. Maraviroc treated xenografts had reduced tumor volume, CAF recruitment and ERK1/2 signaling. Thus, mimicking the effects of the genetic loss of CCL5. Analysis of the TCGA database, identified that high CCL5 or CCR5 expression is associated with poorer prognosis in low grade esophageal carcinomas. Together, we propose that this data highlights the importance of CCL5 in esophageal carcinomas with regards to CAF recruitment and tumor cell proliferation, and that the maraviroc in vivo experiments suggest that targeting the CCL5-CCR5 axis may have therapeutic potential in esophageal carcinomas. Citation Format: Karen J. Dunbar, Qiaosi Tang, Tatiana A. Karakasheva, Anil K. Rustgi. Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B013.

Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

INTRODUCTION: Changes in the tumor microenvironment arbitrated by a stiffened ECM are associated with tumor aggression and enable increased propensity towards metastasis. For instance, in vitro (2D) studies have implicated ECM properties in EAC progression. However, these studies are limited by the lack of 3D intercellular interactions, underscoring the need for physiologically relevant 3D culture models, such as patient-derived organoids (PDOs), that better recapitulate human cancer and its microenvironment to elucidate underlying mechanisms. Engineered hydrogels are an evolving and important component of 3D organoid culture systems, especially to introduce tunable physicochemical matrix signals that have been investigated in tumor progression and metastasis. Furthermore, PDOs have become an attractive pre-clinical in vitro model to study cancer biology and evaluate response to therapeutics. METHODS: We have engineered a visible light-mediated hydrogel platform that supports the development of patient derived Barrett's esophagus (BE) organoids, a precursor to esophageal adenocarcinoma (EAC), as well as EAC organoids. This synthetic biomaterial platform allows control over hydrogel stiffness to better recapitulate the mechanically dynamic esophageal cancer microenvironment, and may help identify therapeutic targets in EAC organoids. RESULTS: Our preliminary data have demonstrated that BE and EAC organoid density, size and proliferation can be controlled by synthetic ECM biomechanical properties. Furthermore, our data show that increased matrix stiffness promotes changes in the transcriptional profiles of EAC organoids, as observed via Principal Component Analysis, and gene set enrichment analysis of upregulated genes reveals enrichment of anti-apoptotic pathways. This suggests that the synthetic ECM facilitates activation of mechanotransduction pathways in EAC organoids and that matrix mechanics have a significant role in activation of canonical anti-apoptotic signaling pathways. Ongoing studies involve identifying matrix stiffness-activated therapeutic targets via small molecule inhibition of upregulated genes that are considered prospective biomarkers in GI cancer. SUMMARY: Our work is significant because it establishes a biomaterial platform that overcomes the limitations of current 3D organoid culture methods to elucidate the role of the tumor microenvironment in EAC tumorigenesis and to identify disease-relevant therapeutic targets. This work will also provide an opportunity to further establish the engineered biomaterial as a platform to potentially elucidate the mechanisms of, and therapy targets for, other human adenocarcinomas in the context of changes in matrix biomechanics.FUNDING: NCI P01-CA098101, U54 CA-163004 and Charles H. Revson Senior Fellowships in Biomedical Science. Citation Format: Ricardo Cruz-Acuña, Secunda W. Kariuki, Claudia Loebel, Tatiana Karakasheva, Joel T. Gabre, Jason A. Burdick, Anil K. Rustgi. Engineered hydrogel elucidates contributions of matrix mechanics to esophageal adenocarcinoma and identify matrix-activated therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3838.

Introduction: Esophageal cancer is a highly aggressive cancer and is the 6th leading cause of cancer mortality worldwide, with esophageal squamous cell carcinoma (ESCC) being the most prevalent subtype. Mutations in the tumor suppressor TP53 (human homolog of mouse Trp53), which encodes p53, are detected frequently in ESCC that in turn correlate with poor survival and high metastatic rates. We have conducted RNA-Seq and cytokine array on primary and metastatic tumor cells harvested from our mouse models with Trp53R172H mutation. We have identified colony stimulating factor 1 (CSF1) to be upregulated. Our data suggest the existence of CSF1-CSF1R autocrine signaling, and its functional relationship to the promotion of metastasis in ESCC, recognizing that CSF1 is known to be critical in the polarization of macrophages to M2-like tumor associated macrophages. Our goal is to investigate the role and mediators of CSF1/CSF1R signaling by which missense TP53 mutations can promote invasion and lung metastasis in ESCC. Methods: We used L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and 2D/3D cell culture systems to model ESCC. To assess the functional role of CSF1, we established a CRISPR approach to knockout Csf1in our cells introducing ribonucleoprotein (RNP) Cas9/gRNA complex via nucleofection and overexpressed Csf1. We also utilized a small molecule inhibitor of CSF1R, GW2580. We conducted TCGA analysis on the association of distinct TP53 mutations with ESCC prognosis and Csf1 expression, as well as Csf1 expression in metastatic tumors. Based on this, we modeled ESCC by introducing various DNA contact and conformational p53 mutations into esophageal cells with wildtype p53 via base editing. Results: TCGA data and our models demonstrate that metastatic ESCCs have increased Csf1 expression compared to primary tumors and it is dependent upon p53 mutation status. In addition, analysis of ESCC patients’ datasets reveal that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. We show that Csf1 function is related to tumor cell invasion, primary tumor growth and metastatic tumor burden in a mutant p53 background. We have identified both bromodomain and extra-terminal motif (BET) protein BRD4 and transcription factor nuclear factor kappa B (NF-κB) as proteins that interact with Trp53R172H at higher levels compared to wildtype p53 to induce Csf1 expression. In addition, inhibition of CSF1R with small molecule GW2580 downregulated JAK-STAT3 pathway mediating epithelial-mesenchymal transition (EMT) mechanisms that would contribute to increased invasiveness and metastasis. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R autocrine signaling and its mediators in promoting lung metastasis from ESCC that may be applicable to other squamous cell cancers. We believe this mechanism can be targeted therapeutically. Citation Format: Gizem Efe, Qiaosi Tang, Ricardo Cruz-Acuna, Kensuke Suzuki, Kensuke Sugiura, Joel Gabre, Uma Sachdeva, Wei Gu, Carol L. Prives, Anil K. Rustgi. Elucidation of CSF1 autocrine signaling mediated by mutant p53 in metastatic esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2421.

Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment. Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1. Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation. Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 788.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral–upper-GI cancers. Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957–70. ©2018 AACR.

Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

Introduction: Pancreatic acinar cells can de-differentiate after acute injury (acute pancreatitis) to a progenitor-like cell type with ductal characteristics in a process termed acinar-to-ductal metaplasia (ADM). In the absence of oncogenic mutation, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), ADM lesions can evolve to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC). Our comprehensive and unbiased approach previously identified the Paired-Related homebox1 (Prrx1) as the most up-regulated transcription factor during pancreatic development, regeneration and evolution of PanIN. We showed that Prrx1 expression is upregulated in both ADM and PanIN lesion. Here, we explore the role of Prrx1 in ADM and PanIN formation using novel mouse models, ex vivo acinar culture systems, and human pancreatitis tissue microarrays (TMA). Methods: We generated novel Ptf1acre-ERT;Prrx1fl/fl;Rosa26YFP/YFP (Prrx1 KO) mice, in which Prrx1 is deleted and YFP is expressed exclusively in adult pancreatic acinar cells upon tamoxifen induction of Cre recombinase activity. Intraperitoneal caerulein injection was administered to induce acute pancreatitis. Human pancreatitis TMAs were utilized for the comparison of Prrx1 expression between pancreatitis and normal human pancreas. We also generated Pdx1Cre;LSL-KrasG12D/+; Prrx1fl/fl;Rosa26YFP/YFP (KCYPrrx1KO) mice to evaluate the function of Prrx1 in oncogenic Kras mutation induced pancreatic PanIN. Quantification of ADM regions was performed through histological examination by a pathologist (blinded to the genetic basis of the tissues) and automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. We also established a novel method for the induction of Prrx1 overexpression in dissociated acinar cells via nucleofection. Results: IF staining revealed that Prrx1 expression is efficiently deleted in ADM cells of Prrx1 KO mice 3 days post-caerulein treatment, a timepoint at which we see peak ADM region formation. Additionally, areas of ADM lesions and amylase loss were significantly reduced in Prrx1 KO mice compared with Prrx1 WT mice at day 3. We also found that Prrx1 overexpression promotes ADM formation in ex vivo acinar cell cultures. In human TMAs, pancreatitis tissues had higher expression of Prrx1 than in normal pancreas. In the presence of oncogenic Kras mutation, loss of Prrx1 significantly attenuates ADM formation in ex vivo culture. Moreover, 5-month-old KCYPrrx1KO mice have very limited ADM/PanIN region compared to 5-month-old KCY mice, which have a pancreas that has predominantly replaced the ADM/PanIN. Conclusions: Prrx1 promotes ADM formation in both adaptive ADM and oncogenic ADM. Our preliminary data suggest that Prrx1 facilitates PDAC progression through PanIN formation. Citation Format: Kensuke Suzuki, Alina Li, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Kensuke Sugiura, Rohit Chandwani, Anil K. Rustgi. The elucidation of the role of Prrx1 for acinar to ductal metaplasia in response to acute injury of pancreas in the novel mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-075.

Introduction: The multistep process of the tumor cell invasion-metastasis cascade, which involves the spread of cancer cells from primary tumor sites to colonization into distant organs, is a major barrier to effective therapy. Both intrinsic factors such as genomic instability and epigenetic alterations and extrinsic factors, such as microenvironmental cues, have been implicated in contributing to the metastatic proclivity of cancer cells. The crosstalk between tumor cells and cells within the heterogenous tumor microenvironment (TME) is a critical driver of tumorigenesis. This can nurture tumor cell migration and invasion into the stroma, providing a foundation for eventual metastasis. Using our mouse models of PDAC, we have identified Colony stimulating factor 1 (Csf-1; also known as M-Csf) to be differentially and significantly upregulated. CSF-1 is secreted by tumor cells to recruit and polarize macrophages into M2-like tumor associated macrophage (TAM) phenotype through binding to its cognate tyrosine kinase receptor colony-stimulating factor 1 receptor (CSF-1R). However, the role and mechanisms of CSF-1/CSF-1R pathway in modulating other elements of the PDAC TME that contributes to invasion and metastasis has yet to be investigated. Methods: We use Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H; Rosa26LSL-YFP (KPCY) mice and 2D/3D cell culture systems. We overexpressed Csf-1 and established a CRISPR system to knockout Csf-1 in our cells using ribonucleoprotein (RNP) Cas9/gRNA complex via nucleofection. These engineered cells are used to model syngeneic primary and metastatic tumor formation. We have established a novel quantitative multiplex immunofluorescence (qmIF) staining approach to characterize changes in the TME upon modulating Csf-1 expression, specifically focusing on macrophages, myeloid-derived suppressor cell (MDSC), T-cell, B-cell, fibroblast and nerve fiber markers. Results: Our data from in vivo studies suggested that Csf-1 overexpression leads to an increase in primary tumor growth and metastasis, while the depletion of Csf-1 reduces metastatic burden. The automated quantification and analysis of our unbiased IF approaches has yielded the following: CSF-1 overexpression leads to increased tumor infiltration of F4/80+CD163+CD206+ M2-polarized macrophages and decreased number of CD3+CD8+ cytotoxic T-cells, generating an immunosuppressive TME. Furthermore, our preliminary data demonstrated that Csf-1 is also upregulated in cancer associated fibroblasts (CAFs), which potentially synergizes with the epithelial compartment to attract immunosuppressive immune cells and promote immune evasion. Finally, the TCGA data reveals that metastatic PDACs have increased Csf-1 expression compared to primary tumors and overexpression of Csf-1 is associated with reduced survival. Conclusion: We have demonstrated a novel role of CSF-1 upregulation in reprogramming the TME in PDAC and fostering increased metastatic capacity. We believe this can be exploited therapeutically. Citation Format: Gizem Efe, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Alina L. Li, Anil K. Rustgi. The extrinsic and modulatory effects of CSF-1/CSF-1R signaling in generating an immunosuppressive pancreatic cancer tumor microenvironment and promoting metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-106.

Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial, and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Kensuke Suzuki, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-010.

Pancreatic cancer initiation features abundant rewiring of the normal acinar cell,. but little is known about the chromatin specification of pancreatic cell types and the epigenetic dysregulation of normal acinar cells in tumor initiation. To address these questions, we employed a lineage-traced autochthonous mouse model to examine systematically perturbed acinar cells. We coupled the spatiotemporal control of inflammation (via caerulein injections) with inducible oncogene activation (mutant Kras) in the adult mouse along with bulk RNA-seq and ATAC-seq to sorted acinar and acinar-derived cells. In addition, we generated Ptf1a-TdTomato mice to reliably sort pancreatic progenitors at e10.5 and e15.5. We observe that Kras activation alone does not disturb acinar cell chromatin nor the histologic appearance of the pancreas. By contrast, caerulein alters chromatin significantly in metaplasia and even in regeneration, with putative enhancers derepressed despite normal histology. In the context of Kras activation and caerulein administration, we find a broad and stable reorganization of chromatin, reflecting cooperativity between oncogenic stress and an inflammatory insult. We also find that in PanIN, the chromatin state bears few, if any, ductal, progenitor, or islet features and instead reflects a largely novel cell fate. To understand the dependencies of these findings on an inflammatory insult and mutant Kras, we leveraged temporal resolution of pancreatitis and the iKras system to define the reversibility of this epigenetic rewiring. Notably, neither resolution of inflammation nor withdrawal of mutant Kras expression are sufficient to revert an acinar cell to its initial chromatin state. Analysis of the acinar-derived cells undergoing the transition to PanIN reveals the induction of specific proliferative and progenitor master transcription factors and activation of associated transcriptional programs. In these data we also observe a specific unveiling of the AP-1 isoform Fra-1 (Fosl1) transcript, protein, and binding sites in chromatin. To address if Fra-1-associated alterations to chromatin are bona fide requirements for tumorigenesis, we coupled conditional Fra-1 knockout alleles with the iKras system, finding nearly complete ablation of PanIN in the absence of Fra-1. Together, our findings suggest that (1) loss of acinar cell identity is resistant to oncogenic stress and is susceptible to inflammation; (2) the acquired acinar cell fate reflects neither ‘pure’ metaplasia nor transdifferentiation nor dedifferentiation events, and (3) acinar cell regeneration is incomplete. In contrast to recent studies, we demonstrate that pancreatic tumorigenesis does not re-establish a progenitor cell fate, but hijacks the AP-1 transcription factors for tumor-specific genomic locations, with Fra-1 emerging as a dependency in tumorigenesis. Our data thus highlight the complexity of cell fate decisions in the preneoplastic pancreas and reveal key regulators of acinar cell identity. Citation Format: David Falvo, Jason Pitarresi, Alexa Osterhoudt, Adrien Grimont, Ben Stanger, Steven D. Leach, Anil K. Rustgi, Rohit Chandwani. Chromatin dynamics in vivo define coordinate functions of inflammation and mutant Kras in pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-005.

Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason Robert Pitarresi, Robert J. Norgard, Anna M. Chiarella, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification KRAS-PTHrP drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2861.

Introduction: Approximately 90% of cancer-related deaths are due to tumor metastasis. Metastasis is a very complex process with several steps including cancer cell motility, invasion, circulation, extravasation, and metastatic colonization/outgrowth. For these diverse processes to take place, a cancer cell needs to acquire and maintain plasticity. Of relevance, a hallmark of metastatic initiation is the loss of membranous E-cadherin and the induction of the Epithelial to Mesenchymal Transition (EMT). During EMT, cancer cells not only exhibit protein expression changes, such as in E-cadherin, but also undergo dramatic reprogramming evident at the chromatin level. Elucidating mechanisms underlying metastasis is especially important in the context of pancreatic ductal adenocarcinoma (PDAC), as this lethal disease typically presents at metastatic stages. Using a PDAC mouse model whereby we endow the cells to undergo EMT, we find that metastatic capability is significantly augmented. We have utilized RNA sequencing from this model to elucidate underlying mechanisms of action and have prioritized CD73/Nt5e. CD73 is involved in the sensing and conversion of extracellular AMP to adenosine. The extracellular adenosine pathway has been shown to create an immunosuppressive environment, yet investigation of this pathway in the context of pancreatic cancer is lacking. Methods: To study the extracellular adenosine pathway in the context of PDAC, we use several different model systems including patient-derived 3D organoids, an autochthonous mouse model of pancreatic cancer (Pdx1-cre; LSL-KrasG12D/+; LSL-Trp53R172H; Rosa26LSL-YFP herein KPCY), and 2D cell culture. Results: We have been investigating the role of CD73 in PDAC and have uncovered novel cancer cell intrinsic and cell extrinsic roles. We demonstrate that CD73 is overexpressed in PDAC, and its overexpression is significantly associated with poor overall survival. Using our 3D organoids, we show that CD73 inhibition induces cell death. In addition, our CRISPR-Cas9 CD73 knockout lines have decreased metastatic abilities in vivo. Our preliminary work also suggests synergistic efficacy of anti-CD73 therapy when combined with inhibition of the adenosine receptors, Adora2a and Adora2b, which are related to CD73 biology. SUMMARY. Using a mouse model of PDAC with increased metastatic capability, we have unveiled the importance of CD73 expression and function in the context of PDAC. Our recent data suggests that CD73 activity has important roles, not only in the cancer cells but in other cells within the tumor microenviroment, and that targeting CD73 be very effective therapeutically. Citation Format: Anna M. Chiarella, Jason R. Pitarresi, Gulam Manji, Anil K. Rustgi. Extracellular adenosine signaling in pancreatic cancer: New therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2691.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma which plays a critical role in the progression and therapeutic resistance of PDAC. The stroma is composed of extracellular matrix proteins, mainly deposited by the cancer-associated-fibroblasts (CAFs) and various types of immune cells. Cancer-associated fibroblasts display a high degree of interconvertible states including quiescent, inflammatory and myofibroblastic phenotypes. However, the mechanisms by which this plasticity is achieved are poorly understood. Here, we demonstrate that CAF plasticity promotes PDAC cell aggressiveness through multiple mechanism, particularly promoting Epithelial-to-Mesenchymal Transition and immune cell infiltration. Methods: To manipulate fibroblast plasticity in PDAC, we generated genetically engineered mouse models (GEMMs) in which CAF plasticity is modulated by genetical depletion of the transcription factor Prrx1 in fibroblasts by using orthotopic implantation models (Sm22-CreERT, Prrx1fl/fl, Rosa26mTmG) as well as dual recombinase-driven GEMMs (Pdx-Flp, FSF-KrasG12D/w t, p53fr/wtt, Sm22-CreERT, Prrx1fl/fl). To characterize the impact of CAFs on tumor differentiation, immune cell infiltration and response to chemotherapy various in vivo and in vitro co-culture experiments were performed. Results: Our in vivo results demonstrate that restraining CAF plasticity by Prrx1-depletion leads to more differentiated tumors, disrupts systemic tumor dissemination, including circulating tumor cells as well as metastases. Interestingly in tumors with Prrx1-deficient stroma, infiltration of macrophages and lymphocytes was increased. Specifically, we observed more B-cells as well as cytotoxic T-cells. Gene expression profiling of primary murine fibroblast samples revealed that Prrx1-deficient CAFs express myofibroblastic gene signatures characterized by ECM secretion phenotype. Indeed, on a functional level Prrx1-deficient CAFs secret more collagen and are highly migratory. Additionally, co-culture experiments of tumor cells and CAFs revealed that Prrx1-driven CAF-derived hepatocyte growth factor confers to a more invasive PDAC cell phenotype and resistant to therapy-induced apoptosis by inducing EMT in vitro. Importantly, in line with our in vitro and in vivo findings, compartment specific-gene expression analysis of human data revealed that pancreatic cancer patients with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC. Conclusions: Here, we define that the Prrx1 transcription factor is critical for CAF plasticity, allowing a dynamic switch between different states. This work demonstrates that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance. Citation Format: Karin Feldmann, Carlo Maurer, Katja Peschke, Steffen Teller, Kathleen Schuck, Katja Steiger, Thomas Engleitner, Rupert Öllinger, Aristeidis Papargyriou, Rim Sabrina Jahan Sarker, Wilko Weichert, Anil K. Rustgi, Roland M. Schmid, Roland Rad, Günter Schneider, Dieter Saur, Maximilian Reichert. Fibroblast plasticity driven by Prrx1 interferes the tumor cells - tumor microenvironment crosstalk towards a more aggressive pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR003.

Pancreatic cancer is a deadly disease that is expected to kill over 45,000 people in the United States this year alone. More than 80% of pancreatic cancer patients begin treatment after the disease has already metastasized. We recently published on an autochthonous mouse model of pancreatic cancer (Pdx1-cre; LSL-KRASG12D/+; LSL-Trp53R172H; Rosa26LSL-YFP herein KPCY), whereby we studied the molecular consequences of losing the tumor suppressor P120-CATENIN (encoded by the gene Ctnnd1). We demonstrated that pancreas-specific Ctnnd1 heterozygous deletion (herein P120-het) in KPCY mice dramatically increased metastatic potential relative to KPCY control animals. Upon further investigation with co-immunofluorescence, we found that the metastatic lesions were largely devoid of any P120 protein. The metastatic cells with complete P120 loss appear more mesenchymal, a phenotype associated with tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and with increased metastatic potential. To determine the underlying processes downstream of P120 loss that may be driving EMT and metastasis, we performed RNA-sequencing on P120-WT (nonmetastatic) and P120-null (highly metastatic) tumor cells. Compared to the P120-WT tumor cells, the P120-null tumor cells revealed significant upregulation of several genes involved in extracellular adenosine signaling, Enpp3, Nt5e, and Adora2B. More recently, we confirmed upregulation of ENPP3, NT5E, and ADORA2B at the protein level, compared to healthy pancreas tissue, using immunohistochemistry (IHC). In the highly metastatic cells, Enpp3 (Ectonucleotide pyrophosphatase/phosphodiesterase-3) expression was increased 57-fold. ENPP3 is a type II glycoprotein involved in binding to and converting extracellular ATP into AMP. Nt5e/CD73 was increased 9-fold and also has ecto-nucleotidase activity to convert AMP to adenosine. Increases in extracellular adenosine can activate ADORA2B (a cell membrane receptor that binds adenosine and initiates downstream intracellular signaling), which was activated 4-fold. While this pathway has been shown to be important and therapeutically relevant in other cancers, it has yet to be fully characterized in the context of pancreatic cancer. In summary, using a model with increased metastatic capability, we have unraveled a novel panel of upregulated and functionally related genes that are related to increasing and sensing extracellular adenosine levels. Our recent data suggest that extracellular adenosine signaling may be a previously undiscovered mechanism for cells to acquire metastatic ability. Citation Format: Anna M. Chiarella, Rustgi K. Rustgi. Elucidation of the extracellular adenosine pathway in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A06.

Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the PTHrP gene drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-057.

Pancreatic ductal adenocarcinoma (PDAC) has an exceptionally poor prognosis, with about 50,000 new cases and 45,000 deaths each year. While novel chemotherapeutics targeted to driver pathways and immune responses have increased the overall survival rate of cancer patients in general, these improvements are rarely seen in PDAC. PDAC often metastasizes early, and metastases ultimately lead to death in many PDAC patients. A stronger understanding of disease mechanisms and new therapeutic modalities are desperately needed to improve outcomes for many patients. Previously, we showed that cancer cell lines of multiple types are “addicted” to constitutive Ca++ flow into mitochondria through the mitochondrial calcium uniporter (MCU) at endoplasmic reticulum-mitochondria contact sites. We hypothesized that mitochondrial Ca++ influx through MCU contributes to cancer cell development, proliferation, and metastasis in PDAC by promoting metabolic activity within mitochondria. We now show that mRNA expression of MCU is elevated in a subset of pancreatic cancer patients, and high MCU protein expression correlates with poor survival outcomes. In addition, MCU gene expression is associated with the expression of a number of genes which associate with metastasis and/or poor survival outcomes, including ADAM proteins, KRAS, and RAC1. To further examine the role of MCU in PDAC, we used the Pdx1cre; KrasLSL-G12D/+; p53fl/+, Rosa26LSL-YFP/LSL-YFP; Mcufl/fl (KPCY) murine model of PDAC. We generated KPCY-McuKO (knockout) animals and cell lines from their YFP-positive tissues for further analysis. Cell lines developed from KPCY-McuKO pancreatic tissues fail to take up Ca++ into mitochondria in a manner that is rescued by stable re-expression of MCU. This Ca++ uptake is associated with an increase in pyruvate dehydrogenase activation. In these cells, MCU expression is also associated with an increase in cell motility, self-renewal capacity, and cell proliferation. Re-expression of MCU in these cells is associated with a morphological change to a more fibroblastic morphology indicative of epithelial to mesenchymal transition (EMT), including decreased surface expression of e-cadherin. These findings suggest that MCU may contribute to growth and metastasis. Indeed, in an immunocompetent, syngeneic orthotopic model of murine PDAC using one of these cell lines, tumor growth and metastasis were greatly ablated. Such results suggest that MCU-mediated influx of mitochondrial Ca++ contributes to PDAC development and metastasis and may present a therapeutic target for cancer treatment. Citation Format: Jillian S. Weissenrieder, Jason R. Pitarresi, Emily Fernandez-Garcia, Ben Z. Stanger, Anil K. Rustgi, J. Kevin Foskett. The mitochondrial calcium uniporter contributes to PDAC development and invasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-026.

Introduction: Heavy alcohol (EtOH) drinking and tobacco smoking increase the risk for upper digestive (e.g. oral and esophageal) squamous cell carcinomas (SCC) in individuals with polymorphic mutations in aldehyde dehydrogenase 2 (Aldh2). Mutant Aldh2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and constituents of tobacco smoke, aka a major human carcinogen. The role of Aldh2 in tumor biology remains elusive. Methods: Single cell-derived three-dimensional (3D) organoids were generated from murine oral and esophageal preneoplastic and SCC lesions carrying wild-type Aldh2WT or mutant Aldh2E487K as well as human SCC cell lines with Aldh2E487K, the latter subjected to xenograft transplantation in immunodeficient mice. 3D organoids and xenograft tumors were analyzed by morphology and flow cytometry to determine CD44, autophagy (cyto-ID), proliferation (EdU), and reactive oxygen species (ROS) following EtOH exposure ex vivo (0.1-2% EtOH) or in vivo (10% EtOH in drinking water) along with or without inhibitors of alcohol metabolism (4-methylpyrazole; 4MP, siRNA against Adh1B and Cyp2E1), and autophagy flux (chloroquine; CQ or siRNA against Atg7). Flow cytometry was performed to determine cancer stem-like cells with high CD44 expression (CD44H cells). Results: EtOH stimulated xenograft tumor growth and increased highly proliferative intratumoral CD44H cells with increased autophagy. EtOH-stimulated tumor growth and induction of CD44H cells were sharply suppressed by concurrent 4MP treatment, indicating that EtOH is directly metabolized in SCC cells to promote tumor growth via CD44H cells. These EtOH-induced effects were recapitulated in 3D organoids where Aldh2E487K augmented EtOH-mediated induction of CD44H cells and organoid formation with increased cellular atypia. EtOH induced mitochondrial superoxide and ROS-dependent autophagy activation in CD44H cells. Inhibition of autophagy prevented EtOH from enriching CD44H cells in 3D organoids, suggesting that autophagy may limit alcohol-induced oxidative stress as a cytoprotective mechanism. Conclusions: These studies provide not only mechanistic insights into the role of Aldh2 mutation in oral and esophageal SCC pathogenesis but also establish the utility of the 3D organoid system to test the functional interplay between genetic and environmental factors which may subsequently be targeted for pharmacological intervention in the setting of personalized medicine. Citation Format: Masataka Shimonosono, Takashi Kijima, Hisatsugu Maekawa, Satoshi Takada, Koji Tanaka, Varun Sahu, Manti Guha, Andres J. Klein-Szanto, J. Alan Diehl, Adam J. Bass, Anil K. Rustgi, Elizabeth Philipone, Angela J. Yoon, Hiroshi Nakagawa. Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 501.

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell–mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctnf/f), a model of oral–esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor–derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38+ MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy. Cancer Res; 75(19); 4074–85. ©2015 AACR.

Metastasis and chemoresistance—the two main reasons for the high mortality of cancer—are associated with a form of cellular plasticity known as epithelial-to-mesenchymal transition (EMT). Cancer cells undergoing EMT become invasive, facilitating metastasis, and undergo a shift in their vulnerability to antineoplastic drugs. In recent work, it has been shown that EMT does not involve a single mechanism but rather a diversity of programs, yielding a continuum of cell phenotypes along the epithelial-mesenchymal spectrum. We previously developed a lineage-traced model of pancreatic ductal adenocarcinoma (PDA) to study EMT in the context of stochastically-arising tumors. As expected, epithelial-mesenchymal plasticity in some tumors involves transcriptional repression of the epithelial state, resulting in a “classical EMT” (C-EMT) phenotype. Surprisingly, however, epithelial-mesenchymal plasticity in the majority of tumors involves post-transcriptional repression of the epithelial state, resulting in a “partial EMT” (P-EMT) phenotype. These two plasticity programs are associated with other aspects of tumor biology as well, including distinct modes of cellular invasion. Here, we identify calcium signaling in pancreatic cancer cells as a regulator of the P-EMT phenotype. Prolonged calcium flux induces PDA cells to remove E-cadherin (ECAD) and other epithelial proteins from the surface and relocalize it intracellularly. This loss of the epithelial phenotype occurs without changes in the abundance of mRNAs for these proteins, reminiscent of the P-EMT phenotype observed in tumors in vivo. In addition, inhibition of the calcium-signaling protein calmodulin blunts this EMT-inducing effect. These results implicate calcium signaling as a mediator of partial EMT phenotypes. Citation Format: Robert J. Norgard, Ravikanth Maddipati, Nicole M. Aiello, David Balli, Jason R. Pitarresi, Derick N. Rosario-Berrios, Jinyang Li, Salina Yuan, Taiji Yamazoe, Yogev Sela, Allyson J. Merrell, Maximilian D. Wengyn, Kathryn Sun, Anil K. Rustgi, Ben Z. Stanger. Calcium signaling induces a partial EMT in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B38.

Purpose: An unbiased approach to discover candidate cancer genes in pancreatic ductal adenocarcinoma (PDAC) identified the p120 catenin gene (Ctnnd1) as one of the top 20 driver genes, and further analysis revealed that P120CTN loss correlated with reduced survival. Results presented herein used genetically engineered mouse models (GEMMs) to show that P120CTN is a potent metastasis suppressor in PDAC. Methods: We employed orthotopic injection, tail vein metastasis assays, and mouse hospital preclinical trials to show that deletion or pharmacologic inhibition of Parathyroid Hormone Related Protein (PTHrP/PTHLH), a signaling element downstream of P120CTN, delayed tumor development and metastatic outgrowth. Finally, we generated a novel GEMM of Pthlh deletion to demonstrate in the autochthonous KPC model that loss of PTHrP phenocopies anti-PTHrP therapy by blocking both primary and metastatic tumor growth. Results: We have generated a mouse model to delete the gene Ctnnd1, whose gene product p120 catenin (P120CTN) is necessary for E-cadherin stability, resulting in enhanced epithelial-to-mesenchymal transition (EMT) and metastasis in KPC animals. Specifically, we show that KPC-p120ctncKO mice have a dramatically enhanced metastatic phenotype relative to KPC controls, suggesting that P120CTN is a critical factor in metastatic cell dissemination. An unbiased screen of tumor cells isolated from these mice identified misregulated calcium signaling through the Parathyroid Hormone Related Protein (PTHrP) as a previously unappreciated contributor to EMT and metastasis. Genetic deletion of the gene that codes for PTHrP in orthotopic transplantation experiments showed significantly reduced tumor growth and metastasis, establishing PTHrP as an oncogenic and prometastatic secreted peptide. Furthermore, treatment with anti-PTHrP monoclonal antibodies reduced tumor cell proliferation and migration in vitro, demonstrating that anti-PTHrP therapies may be of clinical benefit. Importantly, we generated KPC-PthlhcKO mice and showed that they have significantly reduced primary and metastatic tumor burden and increased survival relative to KPC controls. In parallel experiments, we treated KPC mice in a preclinical trial with anti-PTHrP neutralizing antibodies, which delayed both primary and metastatic tumor growth. Finally, analysis of human samples demonstrated that increased PTHLH expression is associated with significantly decreased survival, and that a subset of patients have PTHLH genomic amplifications. Conclusions: This novel work has demonstrated the importance of the previously unappreciated role that PTHrP-mediated calcium signaling plays in pancreatic cancer cellular plasticity and metastasis, and future studies will look to determine the efficacy of anti-PTHrP monoclonal antibodies with a view towards translation in human clinical trials. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Ben Z. Stanger, Anil K. Rustgi. p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of PTHrP-mediated calcium signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B43.

LIN28B is a homologue of LIN28 that induces pluripotency when expressed in conjunction with OCT4, SOX2, and KLF4 in somatic fibroblasts. LIN28B represses biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Recently, we have determined that LIN28B overexpression occurs in colon tumors. We conducted a comprehensive analysis of LIN28B protein expression in human colon adenocarcinomas. We found that LIN28B overexpression correlates with reduced patient survival and increased probability of tumor recurrence. To elucidate tumorigenic functions of LIN28B, we constitutively expressed LIN28B in colon cancer cells and evaluated tumor formation in vivo. Tumors with constitutive LIN28B expression exhibit increased expression of colonic stem cell markers LGR5 and PROM1, mucinous differentiation, and metastasis. Together, our findings point to a function for LIN28B in promoting colon tumor pathogenesis, especially metastasis. Cancer Res; 71(12); 4260–8. ©2011 AACR.

Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expression and translation. In contrast to normal adult tissue, IMP-1 is reexpressed and/or overexpressed in human cancers. We show that knockdown of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further show that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase- and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3′UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible proapoptotic protein CYFIP2 is upregulated in IMP-1 knockdown SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3- and PARP-mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knockdown. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (P ≤ 0.0001) and correlates with K-Ras expression (r = 0.35, P ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-Myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis. Cancer Res; 71(6); 2172–82. ©2011 AACR.

KRAS mutation is present in about 30% of human lung adenocarcinomas. While recent advances in targeted therapy have shown great promise, KRAS remains undruggable and concurrent alterations in tumor suppressors render KRAS mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS mutant tumors harboring these co-mutations are immunosuppressive mechanisms such as increased presence of suppressive Tregs in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. BET bromodomain inhibitors demonstrate clinical benefit in hematologic malignancies, and prior reports demonstrate their Treg-disruptive effects in a NSCLC model. Targeting PD-1 inhibitory signals through anti-PD-1 antibody blockade has also shown substantial therapeutic impact in lung cancer although these outcomes are still limited to a minor pool of patients. We therefore hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote robust anti-tumor response in lung cancer. In the present study, using Kras+/LSL-G12D; Trp53L/L (KP) mouse models of non-small cell lung cancer, we identified cooperative effects among JQ1 and anti-PD-1 that included reduced numbers of tumor-infiltrated Tregs and enhanced activation of tumor-infiltrating T cells, which exhibited a Th1 cytokine profile that favored their demonstrated improved effector function. Furthermore, lung-tumor-bearing mice under this combinatorial treatment regimen showed robust and long-lasting anti-tumor responses compared to either agent alone, culminating in substantial improvement in the survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Citation Format: Dennis O. Adeegbe, Shengwu Liu, Michaela Bowden, Peter S. Hammerman, James E. Bradner, Anil K. Rustgi, Adam J. Bass, Gordon J. Freeman, Huawei Chen, kwok-Kin Wong. BET bromodomain inhibition synergizes with PD-1 blockade to facilitate anti-tumor response in Kras-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4713.

Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate that is related to a propensity for invasion and metastasis. Here, we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor-invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor-invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain-of-loss and loss-of-function experiments. Inhibition of epidermal growth factor receptor signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting the interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (three-dimensional) culture can offer an important tool to discover novel biological effectors in cancer. Cancer Res; 70(13); 5281–92. ©2010 AACR.