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Background: Emerging HER2-targeted drugs especially antibody–drug conjugates (ADCs) are promising and provide more options for breast cancer management. Current assessment of HER2 status and treatment decisions are mainly dependent on immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on the primary tumor tissues. With the disease progression, the molecular status of the tumor may evolve and become discordant with the primary site. However, longitudinal monitoring of HER2 status is limited by infeasible repeated sampling of the tumor tissues. A non-invasive and accurate approach to obtaining real-time samples for measuring HER2 alterations is thus an unmet need for surveillance and guiding treatment selection in breast cancer. Detecting HER2 aberration in cell-free DNA (cfDNA) can allow repeated sampling and avoid effects from tumor heterogeneity of tissue biopsy. Previous approaches for HER2 status determination using liquid biopsy were mostly dependent on the detection of copy number changes in cfDNA, but the limited signal-to-noise ratio poses a great challenge to the accuracy and robustness of the tests. In this study, we identified a group of DNA methylation markers for determining HER2 status in cfDNA for breast cancer. Methods: Genome-wide DNA methylation sequencing was conducted in tissue (25 HER2-positive and 35 HER2-negative) and plasma (32 HER2-positive and 107 HER2-negative) samples to identify specific methylation markers for HER2 status. HER2-positive samples were defined by IHC 3+ and 2+ with FISH positive, while HER2-negative ones were IHC 0/1+ and 2+ with FISH negative. Candidate markers were verified in another two sets of plasma samples (1. 30 HER2-positive and 40 HER2-negative; 2. 33 HER2-positive and 53 HER2-negative) by using quantitative methylation-specific PCR (qMSP). The performance of the markers was estimated by the Wilcoxon test, receiver operating characteristic curve, and logistic regression modelling. Results: 36 HER2 status-specific markers were discovered from genome-wide DNA methylation sequencing. Based on the qMSP results, 11 markers were verified by the performance analyses. The individual area under the curve (AUC) of these markers was from 0.58 to 0.68. From logistic regression modelling and 2-fold cross-validation, a 7-marker diagnostic model was built and validated on plasma samples, with the highest AUC of 0.812. Conclusion: cfDNA methylation detection inferring HER2 overexpression is a novel and non-invasive option for monitoring HER2 status in breast cancer patients, with a potential application in response prediction of HER2-targeted treatments. Further validation of the test is undergoing in large multi-centre cohorts in China. Citation Format: Xianyu Zhang, Shiyao Lu, Hui Li, Xin Liu, Jun Wang, Liuhong Zeng, Zhipeng Lu, Siyu Liu, Yanling Yin, Marina Bibikova, Zhiwei Chen, Jian-Bing Fan, Da Pang. Liquid Biopsy for HER2 Status Assessment in Breast Cancer Using Surrogate DNA Methylation Markers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-27.

Background Breast cancer is one of the most common cancers worldwide with the highest incidence among females in 2020 [1]. The application of breast ultrasound benefits the diagnosis of breast cancer at the early stage, but also leads to over-diagnosis. Patients with breast nodules detected by ultrasound at BI-RADS 4a or higher are usually advised to have a fine needle biopsy or surgery, although the PPVs of BI-RADS 4a and 4b categories are low (6% and 25%, respectively) [2]. Methods In this study, AnchorIRIS™ assay was used for analyzing the methylated status of cfDNA. Target enrichment was performed using an AnchorDx breast cancer-specific methylation panel consisting of 129,794 methylated markers. One hundred and twelve pairs of breast tissue and plasma samples (Malignant: Benign= 56: 56) and 40 leukocyte samples (Malignant: Benign = 20: 20) were used to identify reliable breast cancer-specific methylation markers with low noise background. Finally, a methylation model trained on 307 plasma samples (train set: test set = 214: 93) was selected for differentiating benign from malignant nodules, which was validated by two independent sets (Validation-1: Malignant: Benign = 42:46; Validation-2: Malignant: Benign = 62: 46).Results This methylation model exhibited a powerful performance on differentiating benign from malignant nodules with an AUC of 0.837 (95% CI: 0.757-0.918) in the test set, and maintained a stable predictive power with AUCs of 0.820 and 0.801 in two independent validation sets, respectively. In addition, this methylation model can reflect the difference in methylation signals between metastatic and non-metastatic cancers three years in advance. In contrast to ultrasound, the prediction rate of breast cancer is more accurate across the different age groups using the methylation model, especially for younger women less than 40 years old. Under the ultrasound BI-RADS 4 category, the accuracy (ACC) of the methylation model (IndVal-1, 73.02%; IndVal-2, 78.31%) is on average 22% higher than ultrasound (IndVal-1, 55.56%; IndVal-2, 51.81%). In both of the independent validation sets, the overall accuracy (78.7%) and specificity (SP) (58.7%) at the sensitivities above 95% of the combined model is greater than applying either ultrasound (ACC: 65%; SP: 26.1%) or the methylation model (ACC: 70.6%; SP: 52.2%) alone. Conclusion This methylation model has great potential for the diagnosis of early-stage breast cancer. It improves the diagnostic accuracy of the indeterminate breast nodules, which may assist in decreasing the unnecessary biopsies or surgeries of patients with benign lesions. The methylation model also has the potential in predicting metastatic and non-metastatic breast cancers that is valuable for patient surveillance and risk prediction. References: [1]. Siegel RL, Miller KD, Jemal A: Cancer statistics, 2020. CA Cancer J Clin 70:7-30, 2020 [2]. Spinelli Varella MA, Teixeira da Cruz J, Rauber A, et al: Role of BI-RADS Ultrasound Subcategories 4A to 4C in Predicting Breast Cancer. Clin Breast Cancer 18:e507-e511, 2018 Citation Format: Xianyu Zhang, Zhujia Ye, Yanling Yin, Liuhong Zeng, Jun Wang, Shan Lei, Marina Bibikova, Zhiwei Chen, Jian-Bing Fan, Da Pang. Improving the Performance of Early Breast Cancer Diagnosis by a Model Combining Breast Ultrasound with Methylation Markers in Non-Invasive Circulating Tumor DNA [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-11.

Figure Legends and Supplementary Methods

Supplemental Figure 1. TPO171 transgenic thyroid tissues are stained with CD3-specific and cleft caspase 3 (ClC3)-specific antibodies following treatment with active LMB100 itox (left set of images) vs. LMB100 inactive mutant variant (right set of images).

In vivo BLI of mice inoculated IP with Luc/GFP+ KLM1 Mock or KO#2 on day 3 (D3), 7 (D7) and 14 (D14) post-injection.

Supplemental Figure 2. A) KPCdelMsln+hMSLN cells were treated with increasing concentrations of LMB-100 for 72 hours then viability was measured by colorimetric assay. B) IHC characterization of tumors that form following orthotopic implantation of KPCdelMsln and KPCdelMsln+hMSLN tumor cells into C57Bl/6 syngeneic mice. In each set of three images, the scale bars are: 7mm (whole tumor images for top two sets and bottom left sets) and 8mm (a whole tumor image for bottom right set); 600 �m (intermediate magnification insets in all four sets); 100 �m (high magnification insets in all four sets). C) KPCdelMsln+hMSLN cells were inoculated IP into Msl mice. Mice were euthanized at various time points and all tumor tissue was dissected from the peritoneal cavity and weighed to determine total tumor burden. Blood collected at time of euthanasia was assessed for serum hMSLN by ELISA. Correlation was assessed by Pearson method.

Examination of involvement of NF-κB pathway in MSLN IP pro-tumorigenicity.

Examination of protein signaling pathways.

Legends for the three Supplementary Figures.

MSLN sequence showing positioning of the Exon 3 gRNA site.

Successful reconstitution of WT or Y318A MSLN expression in KLM1 KO#2 and over-expression in MIA PaCa-2.

Supplemental Figure 3. A) Immunostaining with anti-hMSLN antibody in orthotopic KPCdelMsln+hMSLN tumors following LMB-100 treatment. Scale bar = 800 µm. B) IP and orthotopic KPCdelMsln+hMSLN tumors were stained with Maisson Trichrome and percent positive pixels was quantified by image analysis. Each point represents quantification from a separate tumor/animal.

Identifying a novel mediator for MSLN activity using qPCR.

The cell-surface glycoprotein mesothelin (MSLN) is overexpressed in pancreatic adenocarcinoma (PDAC) and is positively correlated with tumor aggressiveness. MSLN knock-out (KO) PDAC cells have delayed growth of peritoneal metastases in mice. Complementation with wild-type MSLN rescued the phenotype, although complementation with MSLN containing a Y318A point mutation to ablate interaction with MSLN binding partner MUC-16 could not. Mature MSLN is shed into the tumor microenvironment to generate soluble MSLN (sMSLN). We hypothesized that sMSLN would promote peritoneal metastasis. We engineered MSLN C-terminal truncation mutants (∆599-WT and ∆599-Y318A) that lack the membrane attachment site and exist only in soluble form. Parent and MSLN KO PDAC cells were transduced with the vector constructs. These cells were injected into athymic nude mice and peritoneal tumor growth was measured 6 weeks later. MSLN KO cells complemented with ∆599-WT MSLN exhibited poor peritoneal tumor growth, while ∆599-Y318A resulted in growth rescue. We considered that sMSLN might exert a dominant negative effect because it competes for MUC-16 binding with membrane-bound MSLN but cannot facilitate cell-to-cell interaction. Expression of ∆599-WT inhibited tumor growth in parent cells, while ∆599-Y318A failed to do so. The activity of ∆599-Y318A in KO cells remained unexplained. We hypothesized that levels of mircoRNA-198, a purported tumor suppressor regulated by MSLN, might be altered by ∆599 expression and correlate with phenotype rescue. However, no change in microRNA-198 expression was observed in ∆599-expressing cells. We are continuing to investigate the mechanism for a novel activity of sMSLN that is unrelated to MUC-16 binding. Citation Format: Neel M. Panchwagh, Theressa Ewa, Leela Rani Avula, Sarah Joseph, Chin-Hsien Tai, Samantha Sevilla, Vishal Koparde, Xianyu Zhang, Christine Alewine. Role of soluble mesothelin in peritoneal 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 1280.

Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(−) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. Implications: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.

ProAgio is a therapeutic cytotoxin that targets and kills integrin αvβ3-expressing cells, resulting in reduced tumor collagen, decreased formation of new blood vessels in the tumor microenvironment (TME), and prolonged survival in mouse models of pancreatic ductal adenocarcinoma (PDAC). ProAgio is currently being tested in a Phase 1 dose-finding study in patients with solid tumors including pancreas cancers. The cell subsets within the PDAC TME that express integrin αvβ3 and could be subject to ProAgio-mediated killing have not been previously studied. Using the KPC genetically engineered mouse model of PDAC, we found that ProAgio (20 mg/kg daily, x7 days) restrained tumor growth by 20% compared to vehicle treatment without changing the cancer cell proliferation rate. We subsequently re-analyzed publicly available scRNA-seq PDAC datasets to identify cells present in human and mouse PDAC tumors that co-express integrins αv and β3 and could be targets of ProAgio. We found that specific subsets of cancer-associated fibroblasts (CAFs), monocytes and macrophages expressed both integrins. Flow cytometry was performed on cells dissociated from orthotopically implanted KPC-derived tumors treated for 15 days with ProAgio or vehicle to identify TME subsets affected by ProAgio treatment. There was no significant difference in the myofibroblast (myCAF) subset, but an increase in the percentage of inflammatory CAFs (iCAFs) was seen. ProAgio treatment also significantly increased the percentage of conventional dendritic cells which play a pivotal role in antigen recognition and T cell priming. T cell abundance was unchanged, but a decrease in B cells was observed. These data demonstrate that ProAgio treatment modifies specific immune and fibroblast subsets within the PDAC TME. Ongoing studies seek to further delineate the cell subtypes affected by ProAgio and the cytokine and chemokine mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Zhi-ren Liu, Serguei Kozlov, Christine Alewine. Remodeling the tumor microenvironment by targeting integrin alpha V beta 3 (αvβ3) expressing cells in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C058.

Purpose: Early detection and intervention can decrease the mortality of breast cancer significantly. Assessments of genetic/genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of early-stage breast cancer. Experimental Design: In this study, 861 serial plasma and matched tissue specimens from 102 patients with early-stage breast cancer who need chemotherapy and 50 individuals with benign breast tumors were deeply sequenced via next-generation sequencing (NGS) techniques using large gene panels. Results: Cancer tissues in this cohort of patients showed profound intratumor heterogeneities (ITHGs) that were properly reflected by ctDNA testing. Integrating the ctDNA detection rate of 74.2% in this cohort with the corresponding predictive results based on Breast Imaging Reporting and Data System classification (BI-RADS) could increase the positive predictive value up to 92% and potentially dramatically reduce surgical overtreatment. Patients with positive ctDNA after surgery showed a higher percentage of lymph node metastasis, indicating potential recurrence and remote metastasis. The ctDNA-positive rates were significantly decreased after chemotherapy in basal-like and Her2+ tumor subtypes, but were persistent despite chemotherapy in luminal type. The tumor mutation burden in blood (bTMB) assessed on the basis of ctDNA testing was positively correlated with the TMB in tumor tissues (tTMB), providing a candidate biomarker warranting further study of its potentials used for precise immunotherapy in cancer. Conclusions: These data showed that ctDNA evaluation is a feasible, sensitive, and specific biomarker for diagnosis and differential diagnosis of patients with early-stage breast cancer who need chemotherapy.

Mesothelin (MSLN) is a cell surface glycoprotein that is expressed by most pancreatic ductal adenocarcinoma (PDAC) and is used as a target for anti-cancer therapy. MSLN expression appears to make pancreatic cancer more aggressive. Previously, we found that MSLN KO PDAC cells grow in culture and as subcutaneous tumors similarly to wild-type (WT) cells but form intraperitoneal metastases poorly. MSLN pre-cursor (pre-MSLN) is a 71-kD glycosylphosphatidylinositol (GPI)-anchored protein that is cleaved intracellularly to make the 31-kD secreted protein Megakaryocyte Potentiating Factor (MPF), and a 40-kD mature MSLN (mMSLN) that retains membrane attachment. The mMSLN can be shed from the cell surface by the action of specific proteases, resulting in release of soluble MSLN (sMSLN) to both the tumor microenvironment and serum. The goal of this study is to see if sMSLN promotes cancer cell aggressiveness. We transduced MSLN KO pancreas cancer cells to express: 1) a mutant, secreted-only pre-MSLN (includes WT MPF) with a C-terminal truncation (MSLNΔ599) that prevents GPI-linkage of MSLN to the membrane, and 2) truncated pre-MSLN consisting of MPF only. Results show that complementation of KO cells with MPF or MSLNΔ599 does not rescue the MSLN KO phenotype, while MSLNΔ599 bearing a Y318A point mutation that disrupts the binding of MSLN to MUC16/CA125 does restore growth of peritoneal metastasis. We performed RNA-deep sequencing on MSLN KO cells exposed to WT or Y318A sMSLN from conditioned medium. We identified 2000 genes that were differentially expressed across the treatment groups. Exposure to sMSLN resulted in changes in expression of cell adhesion and sterol biosynthetic synthesis pathway genes. Individual genes that were top hits were also identified and are under investigation. Exposure to sMSLN changes the transcriptomic program of pancreas cancer cells and may promote peritoneal colonization. Citation Format: Theressa Ewa, Leela Avula, Sarah Joseph, Michael Rudloff, Samantha Sevilla, Vishal Koparde, Xianyu Zhang, Christine Alewine. Soluble mesothelin acts as a signaling molecule [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 93.

Mesothelin (MSLN) is a cancer-specific antigen that is differentially expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinomas. The MSLN gene encodes a 71 kD precursor protein, that is reportedly cleaved by the intracellular protease furin to produce a 31 kD N-terminal fragment called megakaryocyte potentiating factor (MPF) and mature 40 kD MSLN C-terminal fragment. Mature MSLN is localized to the cell surface by a GPI-linkage. We synthesized a series of mutant MSLN constructs containing sequence modifications predicted to disrupt the canonical signaling sequences for furin cleavage and GPI-linkage. These constructs were then expressed in a MSLN knock-out pancreatic cancer cell line. We used flow cytometry to detect cell surface MSLN expression and immunoblotting for MSLN proteins to assess the effect of mutation. We expected that a 23aa C-terminal truncation that removed a predicted hydrophobic signaling sequence for GPI-linkage would eliminate plasma membrane insertion of MSLN. However, this truncation did not suppress cell surface expression. A deeper 31aa truncation which eliminated two potential anchoring sites at Ser599 and Ser606 was required to successfully eliminate surface expression. We are currently evaluating the growth of this pancreatic cancer cell line expressing GPI-truncated soluble MSLN in vitro and in vivo. Our results suggest that MSLN may have an alternate means for membrane insertion besides GPI-linkage. For the panel of furin site mutants, non-conservative mutations were made to disrupt the reported consensus recognition motif for furin 290-RPRFRR-295 (R290H, R292Q, RR294-5GG) or the entire motif region was deleted (289_315del). Mutants were also made to disrupt the putative furin cleavage site (R286A) and neighboring basic residues that were potential protease cleavage sites (R282A, K306Q, K307Q). These furin site mutants were expected to eliminate cleavage of the 71 kD precursor protein to 40 kD mature MSLN. We found that all mutants did increase levels of precursor but did not ablate formation of mature MSLN. Similarly, expression of WT full-length MSLN into the LoVo cell line, which lacks furin activity, still resulted in formation of 40 kD mature MSLN predominately. In conclusion, we found that precursor processing to mature MSLN still took place despite extensive mutation of the putative furin cleavage site and in the absence of active furin. These results suggest that additional proteases besides furin can perform MSLN precursor cleavage. Citation Format: Sarah Mary Joseph, Leela Rani Avula, Xianyu Zhang, Christine Campo Alewine. The molecular dissection of pro-tumorigenic activity of mesothelin in pancreatic cancer [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 1449.

Most monoclonal antibody-based therapeutics are specific for the human isoform of their targets and do not cross-react with orthologous mouse isoforms. This frequently limits pre-clinical modeling to human-derived tumors growing in immune compromised mice and precludes combination studies with immune modulating agents which rely on an active immune system for activity. Although introduction of human cDNA into mouse tumor cells will make these cells susceptible to targeting, a syngeneic mouse may reject or partially reject cells bearing the foreign transgene, impacting anti-tumor efficacy studies. Mesothelin (MSLN) is the target of many cell- and antibody-based therapies that have reached clinical trials due to its lack of expression in critical normal tissues and robust expression in many solid tumors. We have generated two distinct transgenic C57/Bl6 mouse models with compartment-limited expression of human MSLN (hMSLN) for use in pre-clinical testing of MSLN-targeted therapeutics: 1) TPO/hMSLN mice express full-length hMSLN from an insulated rat thyroid peroxidase gene promoter. Analysis by RT-PCR, immunoblot and IHC demonstrate strong thyroid-specific expression hMSLN. 2) In Nor/hMSLN mice, the hMSLN coding region was inserted as a full-length cDNA into the native mMSLN locus via a homologous recombination knock-in technique such that hMSLN is expressed in place of the mouse isoform under the control of endogenous murine MSLN transcription regulatory elements. IHC studies show expected expression of hMSLN in mouse pleura, pericardium and peritoneum. Shed hMSLN can be readily detected in the serum of both transgenic lines using ELISA, similar to human patients. Treatment of either model with MSLN-targeted immunotoxin LMB-100 at the maximum dose tolerated by non-transgenic C57/Bl6 mice resulted in no gross toxicity. Histologic analysis revealed subclinical pericarditis in Nor/hMSLN mice. No post-treatment thyroid damage was observed in TPO/hMSLN mice even upon detailed histologic examination of thyroid tissue. Syngeneic mouse pancreatic cancer cells expressing hMSLN transgene implanted orthotopically into TPO/hMSLN or Nor/hMSLN grew as well or better as those grown in non-transgenic C57/Bl6 mice. We have established two immunologically proficient transgenic mouse models expressing hMSLN that can be used for evaluating immune effect of hMSLN-targeted therapeutics as well as their on-target/off-tumor toxicities. Citation Format: Xianyu Zhang, Brendan Hagerty, Theresa Guerin, Norene O'Sullivan, Serguei Kozlov, Christine Alewine. Engineering of transgenic mice expressing human mesothelin for investigation of mesothelin-targeted therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 268.

Peritoneal metastases develop in 70-80% of nonresectable patients with pancreatic cancer, and the onset of clinically significant ascites is associated with rapid demise. Mesothelin (MSLN) is a GPI-linked cell-surface glycoprotein expressed in >90% of pancreatic ductal adenocarcinomas (PDAC) but not in healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, multiple therapeutics targeting MSLN have already reached clinic. Overexpression of MSLN has been implicated in increased aggressiveness of PDAC, both in laboratory models and in patients. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (MSLN KO) and have examined the growth of these cells in culture and in nude mice compared to mock deleted cells (WT). The cells were also transduced to express GFP/Luciferase(Luc) in order to allow for monitoring of tumor by bioluminescence imaging (BLI) in the mouse intraperitoneal (IP) cavity. MSLN KO and WT cells grew at the same rate in culture and as subcutaneous xenografts in nude mice. But when cells were inoculated IP, KO cells grew poorly compared to WT cells or did not grow at all. When MSLN KO cells grew, visible tumors were located primarily at the inoculation site. In contrast, WT cells grew extensive metastases throughout the IP cavity. GFP/Luc+ tumor cells adhered to the mesothelial lining of the peritoneum could be detected by BLI and histology as early as 24h post IP injections. By 72h, the MSLN KO tumors had significantly decreased microvascular density as determined by CD31 staining, lower metastatic dissemination and growth as detected by BLI and H&E staining, and decreased proliferation as determined by Ki67 quantification. These differences were maintained even at timepoints out to 2 weeks. Our results suggest that MSLN might enhance peritoneal carcinomatosis of PDAC by positively regulating vascular remodeling/angiogenesis and tumor cell proliferation within the IP cavity. The mechanism of MSLN action has been previously ascribed to activation of MAPK or NF-kB pathways or signaling through MUC16, the only known binding partner of MSLN. However, we found no changes in phosphorylated p38 or ERK, nor in total levels of the NF-kB target OCT-2 upon KO of MSLN expression. Since we also observed protumorigenic effect of MSLN in a MUC16 deficient pancreatic cancer cell line, it is unlikely that MUC16 is responsible for the protumorigenic activity of MSLN. We are currently performing comprehensive molecular studies to uncover key upstream and downstream signaling factors responsible for the protumorigenic effects of MSLN in peritoneal dissemination. Citation Format: Leela Rani Avula, Michael Rudloff, Salma El-Behaedi, Danielle Arons, Xianyu Zhang, Christine Alewine. Characterization of the protumorigenic role of MSLN in peritoneal metastases in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3767.

Mesothelin (MSLN) is a GPI-linked protein expressed in >90% of pancreatic adenocarcinomas (PDAC) but not in healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, multiple antibody-based therapies are targeting MSLN. The physiologic role of MSLN is yet unknown. The MSLN gene encodes for a precursor protein which is cleaved to produce mature MSLN protein and soluble Megakaryocyte Potentiating Factor (MPF), both of which can be secreted or shed. Previous reports suggested that overexpression of MSLN precursor protein increases tumorigenicity and metastatic potential of PDAC. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but ΔMSLN had significantly impaired growth when inoculated into the mouse intraperitoneal (IP) cavity as well as when injected orthotopically into the pancreas. Histological analysis (H&E, Ki67 and Trichrome) showed no significant gross differences between ΔMSLN and Mock IP tumors. RNA deep sequencing analysis comparing ΔMSLN and Mock cell lines showed strong upregulation of MUC-4 in KO cells. Restoration of WT MSLN expression in ΔMSLN cells restored IP growth, however, transduction of a Y318A point mutant could not. Point mutation of Y318 diminished ability of shed MSLN to interact with KLM-1 cells, presumably by impairing association with MUC-16, the only known binding partner of MSLN. To determine the role of mature MSLN and MPF in tumorigenicity, we expressed these proteins separately in ΔMSLN cells and found that neither could restore IP growth, although mature MSLN was appropriately expressed on the cell surface and MPF secreted. This suggests either that both proteins are required for tumorigenicity or that when synthesized individually they are incorrectly processed by the cell. To examine the role of MSLN in tumorigenesis, MSLN KO mouse were bred into the KPC genetically engineered mouse model of PDAC, which spontaneously develops autochthonous tumors. Unlike PDAC patients, KPC typically die from complications of their primary tumors. Loss of MSLN had no effect on the survival of these mice. Effect of MSLN loss on metastasis in this model is currently being evaluated. In summary, we have demonstrated that MSLN enhances the IP growth of PDAC and defined key regions of MSLN important for this activity. Citation Format: Leela Rani Avula, Michael Rudloff, Danielle Arons, Salma El-Behaedi, Xianyu Zhang, Theresa Guerin, Serguei Kozlov, Christine Alewine. Identification of the molecular components required for the protumorigenic effect of MSLN in pancreatic 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 104.

The mesothelin (MSLN) protein is expressed in at least 85% of pancreatic adenocarcinomas but not on cells of the healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, MSLN has been used as a target for various antibody-based treatments and cancer vaccines. The physiologic role of MSLN is unknown. The MSLN gene encodes for a precursor protein, which is cleaved to produce the mature MSLN protein. Previous reports have suggested that overexpression of MSLN precursor protein may increase tumorigenicity and metastatic potential of pancreatic adenocarcinoma. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but had significantly impaired growth when inoculated into the mouse intraperitoneal cavity. Lentivirus transduction was used to restore expression of MSLN in ΔMSLN cells. Transduction of full-length WT MSLN, but not a Y318A mutant, restored intraperitoneal growth. Interestingly, reintroduction of mature MSLN instead of the precursor protein did not restore growth even though it was appropriately expressed on the cell surface. In summary, we have demonstrated that MSLN enhances the growth of pancreatic cancer within the peritoneal cavity and defined key regions of MSLN important for this activity. Citation Format: Christine Campo Alewine, Michael Rudloff, Danielle Arons, Salma El-Behaedi, Xianyu Zhang, Leela Avula. Deletion of mesothelin impairs intraperitoneal growth of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B029.