Megakaryocyte Potentiating Factor Research Articles

Characterization of mesothelin gene expression in dogs and overexpression in canine mesotheliomas.

Canine mesotheliomas are uncommon malignant tumors typically detected late. Minimally invasive diagnostic biomarkers would facilitate diagnosis at earlier stages, thereby improving clinical outcomes. We hypothesized that mesothelin could be used as a reliable diagnostic biomarker for canine mesotheliomas since it has been used as a cancer biomarker for human mesothelioma. We aimed to explore and characterize mesothelin gene expression in dogs and assess its use as a diagnostic biomarker for canine mesotheliomas. We quantified expressed canine mesothelin transcripts via reverse transcription polymerase chain reaction (RT-PCR) and sequenced them using ribonucleic acid (RNA) extracted from a canine mesothelioma cell line. After confirming mesothelin expression, we assessed its levels in major organ tissues and compared them with those in the mesothelioma tissues using quantitative PCR (qPCR). Mesothelin overexpression in mesotheliomas was detected, and we further compared its levels using qPCR between mesotheliomas and non-mesotheliomas using tumor tissues and clinical sample effusions, confirming its significance as a diagnostic biomarker for canine mesothelioma. Mesothelin complementary deoxyribonucleic acid (cDNA) was amplified via RT-PCR, yielding a single band of expected upon DNA electrophoresis. Sequence analyses confirmed it as a predicted canine mesothelin transcript from the genome sequence database. Comparative sequence analysis of the deduced amino acid sequence of the expressed canine mesothelin demonstrated molecular signature similarities with the human mesothelin. However, the pre-sequence of canine mesothelin lacks the mature megakaryocyte potentiating factor (MPF) portion, which is typically cleaved post-translationally with furin. Mesothelin expression was quantified via qPCR revealing low levels in the mesothelial and lung tissues, with negligible expression in the other major organs. Canine mesothelin exhibited significantly higher expression in the canine mesotheliomas than in the noncancerous tissues. Moreover, analysis of clinical samples using qPCR demonstrated markedly elevated mesothelin expression in canine mesotheliomas compared to non-mesothelioma cases. Canine mesothelin exhibits molecular and biological characteristics akin to human mesothelin. It could serve as a vital biomarker for diagnosing canine mesotheliomas, applicable to both tissue- and effusion-based samples.

B-347 Performance Validation of an In-house Assay for Soluble Mesothelin Related Peptides

Abstract Background Mesothelin is a glycosylated phosphatidylinositol glycoprotein located on the surface of mesothelial cells. Mesothelin is released from the cell in a form denoted as Soluble Mesothelin Related Peptides (SMRP), that can be measured in the blood. Although mesothelin is produced by normal cells, studies have shown an overexpression in individuals suffering malignant mesothelioma, with an increase in serum SMRP concentration associated with the disease. The in-house ARUP MESOMARK® ELISA is a 96-well microtiter plate formatted sandwich immunoassay for the quantitative measurement of SMRP in human serum. The ELISA incorporates two separate monoclonal antibodies specific for SMRP (including megakaryocyte potentiating factor); one (4H3) bound to the microtiter well for capturing SMRP and the second (OV569), enzyme labeled with horseradish peroxidase (HRP), for bound SMRP detection. After several incubation and wash steps to remove unbound materials, color is produced by HRP turnover of a chromogenic substrate during a final incubation step. A direct relationship between the color intensity and the SMRP concentration of the specimen is generated, with results expressed as nmol/L SMRP. Methods Deidentified residual serum specimens sent to ARUP Laboratories for routine testing, as well as serum specimens from healthy volunteers, were collected under University of Utah’s Institutional Review Board approved protocols. Commercial kit (Fujirebio Diagnostics, Inc., Malvern, PA) testing was completed according to manufacturer’s protocol. ARUP ELISA measurements were conducted according to an established in-house protocol. ARUP ELISA performance characteristics evaluated included a method comparison against the commercial assay, linearity, recovery, precision, analytical sensitivity, and dilution studies. A reference limit was also verified. Results A method comparison study against the commercial SMRP ELISA generated a slope of 0.989, intercept of 0.0422, r2 of 0.998 and bias of -1.2% (n = 45, Deming regression, Bland-Altman analysis). Clinical agreement near the 1.5 nmol/L cutoff was 100%. The limit of detection was 0.3 nmol/L (20 determinations each of a blank and low-level serum pool). Linearity was established by combining serum specimens with high and low SMRP concentrations at different ratios, creating 10 specimens of varying SMRP concentrations. Each specimen was tested in triplicate. Regression analysis (measured vs expected concentration) produced a slope of 1.002, intercept of -0.739 and r2 of 0.998, with percent recoveries ranging 86.1 to 100.0%. Precision was determined from two serum pools of differing SMRP concentrations tested over 5 days, four replicates per pool per day. Repeatability and within-laboratory CVs were 3.0 and 5.0% at 1.6 nmol/L, and 1.7 and 1.7% at 17.3 nmol/L, respectively. Back-calculated results from a 10-fold dilution study of four serum specimens were within -9.8 to -8.1% of their corresponding neat values. The previously established reference limit of 1.5 nmol/L was verified for the ARUP ELISA, with 95% of results below the cutoff value (n = 20, sera from healthy volunteers). Conclusions The in-house ARUP MESOMARK ELISA demonstrates acceptable performance for quantifying SMRP in human serum and compares favorably to a well-established commercial ELISA. A reference limit of 1.5 nmol/L was also verified. Overall, the ARUP MESOMARK ELISA is acceptable for serum SMRP testing.

Phase 2 randomized trial of neoadjuvant or palliative chemotherapy with or without immunotherapy for peritoneal mesothelioma (Alliance A092001).

TPS8603 Background: Peritoneal mesothelioma is a rare and poorly studied disease with few treatment options. For patients (pts) who are not surgical candidates, treatment recommendations for systemic therapy have been extrapolated from clinical trials for pleural mesothelioma that commonly exclude pts with peritoneal mesothelioma. Recently, the combination of the PD-1 inhibitor nivolumab and the CTLA-4 inhibitor ipilimumab received FDA-approval for the frontline treatment of non-resectable pleural mesothelioma. Additionally, a prospective, non-randomized phase 2 trial demonstrated activity with combined PD-L1 (atezolizumab) and VEGF (bevacizumab) blockade in peritoneal mesothelioma. In parallel, encouraging activity with combined chemo-immunotherapy has been reported in pleural mesothelioma. Given the benefits observed with immunotherapy, and the potential to improve upon those with chemotherapy and VEGF inhibition, we seek to determine whether the addition of the PD-L1 inhibitor atezolizumab improves outcomes with chemotherapy and bevacizumab in pts with newly diagnosed peritoneal mesothelioma. Methods: Alliance for Clinical Trials in Oncology A092001 is a prospective, randomized phase 2 clinical trial. All pts with newly diagnosed peritoneal mesothelioma will be randomized 1:1 using a dynamic allocation Pocock-Simon procedure to receive carboplatin, pemetrexed and bevacizumab, with or without atezolizumab, every 21 days for four cycles. Patients who are eligible to proceed with surgery after four cycles of therapy will then do so. Pts who are not eligible to proceed with surgery may receive maintenance bevacizumab and atezolizumab, or second-line atezolizumab with bevacizumab until progression of disease or toxicity. The primary objective is to determine if frontline treatment with carboplatin, pemetrexed, bevacizumab and atezolizumab results in a superior best response rate (RR) to carboplatin, pemetrexed and bevacizumab as determined by RECIST. With 31 eligible pts per arm (62 eligible total), this randomized design has 80% power to detect an improvement in the RR from 20% to 45%, with a 1-sided significance level of 0.10 where an interim futility analysis will be conducted after 32 pts are enrolled. Stratification factors include eligibility for cytoreductive surgery at diagnosis and histologic subtype. Secondary endpoints include progression-free survival, overall survival and adverse events. As integrated biomarkers, we will determine if soluble mesothelin-related peptides and megakaryocyte potentiating factor correlate with responses. This trial was recently approved by the National Cancer Institute Central IRB and is activating at sites across the country. Clinical trial information: NCT05001880 .

Abstract 93: Soluble mesothelin acts as a signaling molecule

Abstract 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.

Phase 2 randomized trial of neoadjuvant or palliative chemotherapy with or without immunotherapy for peritoneal mesothelioma (Alliance A092001).

TPS8598 Background: Peritoneal mesothelioma is a rare and poorly studied disease with few treatment options. For patients who are not surgical candidates, treatment recommendations for systemic therapy have been extrapolated from clinical trials for pleural mesothelioma that commonly exclude patients with peritoneal mesothelioma. Recently, the combination of the PD-1 inhibitor nivolumab and the CTLA-4 inhibitor ipilimumab received FDA-approval for the frontline treatment of non-resectable pleural mesothelioma. Additionally, a prospective, non-randomized phase 2 trial demonstrated activity with combined PD-L1 (atezolizumab) and VEGF (bevacizumab) blockade in peritoneal mesothelioma. In parallel, encouraging activity with combined chemo-immunotherapy has been reported in pleural mesothelioma. Given the benefits observed with immunotherapy, and the potential to improve upon those with chemotherapy and VEGF inhibition, we seek to determine whether the addition of the PD-L1 inhibitor atezolizumab improves outcomes with chemotherapy and bevacizumab in patients with newly diagnosed peritoneal mesothelioma. Methods: A092001 is a prospective, randomized phase 2 clinical trial. All patients with newly diagnosed peritoneal mesothelioma will be randomized 1:1 using a dynamic allocation Pocock-Simon procedure to receive carboplatin, pemetrexed, and bevacizumab, with or without atezolizumab, every 21 days for four cycles. Patients who are eligible to proceed with surgery after four cycles of therapy will then do so. Patients who are not eligible to proceed with surgery may receive maintenance bevacizumab and atezolizumab, or second-line atezolizumab with bevacizumab until progression of disease or toxicity. The primary objective is to determine whether frontline treatment with carboplatin, pemetrexed, bevacizumab and atezolizumab results in a superior best response rate (RR) to carboplatin, pemetrexed and bevacizumab as determined by RECIST. With 31 eligible patients per arm (62 eligible total), this randomized design has 80% power to detect an improvement in the RR from 20% to 45%, with a 1-sided significance level of 0.10 where an interim futility analysis will be conducted after 32 patients are enrolled. As stratification factors we have included eligibility for cytoreductive surgery at diagnosis, and histologic subtype. Secondary endpoints include assessment of progression-free survival, overall survival, and adverse events. As integrated biomarkers, we will determine if soluble mesothelin-related peptides and megakaryocyte potentiating factor correlate with responses. This trial was recently approved by the National Cancer Institute Central IRB and is activating at sites across the country. Support: U10CA180821, U10CA180882. Clinical trial information: NCT05001880.

Exploiting mesothelin in thymic carcinoma as a drug delivery target for anetumab ravtansine.

Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.

Mesothelin Is a Novel Disease Marker and Potential Therapeutic Target in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a significant therapeutic challenge in children with only 50% cure rate with highly intensive cytotoxic chemotherapy. Further intensification is limited by acute and late effects, including severe infectious and cardiac toxicities; therefore, alternative therapeutic strategies are needed. As part of the NCI/COG TARGET AML Initiative, using transcriptome sequencing (RNA Seq) in 635 children and 178 adult AML, we demonstrated that mesothelin (MSLN) was highly-overexpressed in over one-third of pediatric AML. This transcript is not expressed in normal hematopoiesis, making it an ideal target for therapeutic intervention. MSLN is a cell-surface protein normally found in pleura and peritoneum and overexpressed in a variety of solid tumors, such as mesothelioma. The protein product of the MSLN gene exists in 4 forms in the body, including cell surface MSLN, soluble MSLN, and soluble megakaryocyte potentiating factor (MPF), which is the N-terminus of the MSLN pro-protein. Elevated serum soluble MSLN is an established diagnostic test for mesothelioma, with an FDA-approved ELISA-based assay (Mesomark™).Three different AML RNA Seq data sets totaling 813 cases, including TARGET validation cohort (n= 475), TARGET discovery phase (n= 160), and TCGA AML (n= 178), were interrogated for expression of MSLN, with normal marrow cases as a control. MSLN transcript expression at >5 transcripts per million (TPM) was observed in 268 patients (range 0-1225 TPM) with observed prevalence of 48% in TARGET discovery set, 33% in TARGET validation and 15% in adult TCGA set with a collective prevalence of 33% in the entire cohort. MSLN expression was enriched in patients with t(8;21), inv(16), and KMT2A fusions and rare in those with common AML-associated mutations (FLT3- ITD, NPM1, CEBPa mutations). MSLN was not expressed in 20 normal marrow samples (<1 TPM in all cases).We conducted orthogonal validation of the transcriptome data by evaluating diagnostic specimens from an independent cohort of 323 newly diagnosed pediatric AML patients for the presence of MSLN using quantitative RT-PCR and ELISA. RNA extracted from leukemic blasts or plasma obtained from diagnostic specimens were tested for MSLN expression by quantitative RT-PCR or sandwich ELISA, respectively. Optimized q-RT-PCR identified 36% of patients with MSLN expression. Similarly, sandwich ELISA (similar to Mesomark™) confirmed expression of soluble MSLN in 34% of patients tested, with strong concordance between the two tests (p <0.0001). We have also optimized diagnostic multi-dimensional flow cytometry (MDF) and MPF ELISA for evaluation of MSLN.Mesothelin is a cell surface protein highly expressed in AML blasts but absent in other hematopoietic cells, making it an attractive target for immunotherapy. We sought to test the principle of MSLN targeting in leukemia with antibody-drug conjugate (ADC) anetumab ravtansine (AR; BAY94-9343), comprised of an anti-MSLN antibody conjugated to tubulin inhibitor DM4. MSLN -overexpressing cells were created by lentivirally-transducing MSLN into K562 cells (K562+ MSLN). K562+ MSLN exhibited exquisite sensitivity to AR in vitro with an IC50 of 1.4 nM, compared to 360 nM in the non-transduced K562 and 250-260 nM in isotype control conditions (Fig. 1). In vitro cytotoxicity of AR in two primary pediatric MSLN + AML samples also demonstrated MSLN -dependent killing. Additionally, mice bearing K562+ MSLN xenografts were treated in vivo with AR, which demonstrated significant MSLN -dependent efficacy, resulting in median survival of 87 days compared to 32-41 days in control conditions, including chemotherapy with daunorubicin and cytarabine (Fig. 2).MSLN is a cell-surface protein uniquely overexpressed in one-third of pediatric AML, as well as a subset of adult AML. We have shown that MSLN can be detected through several sensitive diagnostic mechanisms including MDF, q-RT-PCR and serum soluble MSLN, making it an optimal marker for disease monitoring. The leukemic expression of MSLN and absence from normal hematopoietic tissue make it an ideal target for immunotherapeutic targeting, and our findings demonstrate that MSLN can be exploited for therapeutic benefit using ADCs. Further studies in MSLN + AML on optimal detection and immunotherapeutic strategies are needed, as MSLN shows great promise for enhanced disease monitoring and leukemia-specific therapeutic targeting. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Pardo:Hematologics Inc: Employment.

Zika M Oligopeptide ZAMP Confers Cell Death-Promoting Capability to a Soluble Tumor-Associated Antigen through Caspase-3/7 Activation.

Mosquito-borne Zika virus (ZIKV) is an emerging flavivirus of medical concern associated with neurological disorders. ZIKV utilizes apoptosis as a mechanism of cell killing. The structural M protein may play a role in flavivirus-induced apoptosis. The death-promoting capability of M has been restricted to an oligopeptide representing the residues M-32/40. Here, we evaluated the apoptosis inducing ability of the residues M-31/41 of ZIKV. The ZIKV M oligopeptide was associated to a soluble form of GFP (sGFP) and the resulting sGFP-M31/41 construct was assessed in Huh7 cells. Expression of sGFP-M31/41 can trigger apoptosis in Huh7 cells through caspase-3/7 activation. The translocation of sGFP-M31/41 in the endoplasmic reticulum was a prerequisite for apoptosis induction. The residues M-33/35/38 may play a critical role in the death-promoting activity of sGFP-M31/41. The effect of ZIKV M oligopeptide defined as ZAMP (for Zika Apoptosis M Peptide) on expression of a tumor-associated antigen was assayed on megakaryocyte-potentiating factor (MPF). Expression of MPF-ZAMP construct resulted in caspase-associated apoptosis activation in A549 and Huh7 cells. ZIKV has been proposed as an oncolytic virus for cancer therapy. The ability of the Zika M oligopeptide to confer death-promoting capability to MPF opens up attractive perspectives for ZAMP as an innovative anticancer agent.

Новые однодоменные антитела к мезотелину и клеточные модели для разработки таргетной терапии рака молочной железы

Most triple negative breast cancers (TNBC) are characterized by elevated expression of mesothelin (MSLN), a cell surface antigen and one of the preferred targets for the therapy of solid tumors. Most continuous TNBC cell lines are MSLN-negative, which obstructs the development of MSLN-targeted therapy for TNBC. The aim of this study was to identify TNBC cell lines with MSLN hyperexpression and to obtain single-domain antibodies (nanobodies) capable of recognizing MSLN in TNBC cells. Mesothelin expression levels were measured in the panel of TNBC cell lines by real-time reverse-transcription PCR. PCR results were verified by measuring concentrations of the megakaryocyte potentiating factor (the secreted fragment of the mesothelin precursor) using sandwich ELISA. Immune phage-display VHH fragment libraries were prepared from mononuclear cells of Vicugna pacos using a modified library enrichment protocol. Two nanobody variants with high specificity for the target and Kd of about 140 and 95 nmol, respectively were obtained. Two MSLN+ and three MSLN– cell lines were identified in the TNBC cell lines panel. The nanobodies demonstrated the ability to recognize the target antigen in MSLN+ cells and had the low ability to bind to MSLN– cells. Thus, we found a convenient MSLN+ TNBC cell model for MSLN-targeted therapy testing. The new single-domain antibodies can be used as targeting components of chimeric antigen receptors.

Abstract 1776: Exploring anetumab ravtansine in a preclinical model of thymic carcinoma

Abstract Thymic epithelial tumors (TETs) are a group of rare tumors (0.13 per 100,000 persons), comprised of thymomas and thymic carcinoma, arising from the epithelial cells of the thymus. The biology of these tumors is not well understood, and there are no approved targeted therapies against TETs. One promising drug target is mesothelin (MSLN), which is a surface glycoprotein that is cleaved to produce two proteins: mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF). In normal physiology, expression of MSLN is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Its expression is also present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. In this study, we investigate the ability of the MSLN targeting, fully human monoclonal antibody-drug conjugate (ADC) anetumab ravtansine (ARav) to inhibit growth of thymic carcinoma in in-vitro and in-vivo models. We utilized immunoblotting and RT-qPCR to characterize MSLN expression in human thymoma and thymic carcinoma derived cell lines. Binding of anetumab, the antibody moiety in ARav, to thymoma and thymic carcinoma cell lines was assessed by flow cytometry. In-vitro, ARav's effect on cell proliferation was tested against MP57 cells (thymic carcinoma), stably transfected with MSLN, utilizing CellTiter-Glo. ARav's activity in-vivo as a monotherapy, and as a combination therapy with cisplatin, was tested against MSLN expressing T1889 xenograft tumors in athymic nude mice and compared to vehicle, isotype control ADC, and standard-of-care (SoC) cisplatin. Using the anetumab antibody, thymoma (T1682) and thymic carcinoma (T1889) cell lines were shown to express MSLN on the cell surface. In-vitro, ARav was effective at inhibiting growth of MP57 cells stably transfected with mMSLN. In-vivo, 15 mg/kg ARav inhibited T1889 xenograft tumor growth more efficaciously than vehicle, the isotype control ADC (all given every 2 weeks), and 4 mg/kg cisplatin (given every week). Combining ARav at a dose of 7.5 mg/kg with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumor growth. MSLN is expressed in thymoma and thymic carcinoma derived cell lines. Anetumab ravtansine inhibits growth of MSLN positive thymic carcinoma in our preclinical model; when combined with SoC cisplatin, ARav confers enhanced tumor growth inhibition. Citation Format: Vincent Chen, Anette Sommer, Christoph Schatz, Sabine Zitzmann-Kolbe, In-Kyu Kim, Giuseppe Giaccone. Exploring anetumab ravtansine in a preclinical model of thymic carcinoma [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 1776.

Abstract 1449: The molecular dissection of pro-tumorigenic activity of mesothelin in pancreatic cancer

Abstract 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.

Abstract 1382: Megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma

Abstract Objectives: Effective biomarkers for malignant mesothelioma (MM) are needed for clinical management and the development of cell surface mesothelin-targeted therapies. We evaluated serum megakaryocyte potentiating factor (MPF) as a biomarker predictive of clinical outcome in MM patients, for patient monitoring, and for developing mesothelin-targeted therapies. Patients and Methods: Serial serum samples from MM patients in two clinical trials of an anti-mesothelin immunotoxin were tested for serum MPF. For the purpose, we developed a new MPF assay and conducted both analytical and clinical validation. Correlative studies were performed to determine the test effectiveness in treatment monitoring and outcome prediction. MPF was further evaluated for an association with response to an anti-mesothelin therapy and for disease monitoring. Results: Our data showed that in MM patients, elevated serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (Log Rank P = 0.003; n = 95). In refractory MM patients, elevated MPF is a strong predictor of poor outcome with a HR = 6.12 (Log Rank P = 0.0007; n = 57). There was a significant reduction of serum MPF in patients with elevated baseline and radiological response, with an average change from -52% to -78% after 1 to 6 cycles. Using a -50% change as the cutoff, MM patients with a positive MPF-response had significantly improved progression-free survival (PFS) (P &amp;lt; 0.0001), with median extended from 1.9 to 11.3 months. These MPF-response patients further exhibited improved overall survival (OS) (P = 0.004), with median extended from 8.8 to 22.3 months. In refractory MM patients, there was an association between elevated pre-treatment serum MPF and radiological response to an experimental anti-mesothelin therapy (P = 0.033). Furthermore, in these response patients, serum MPF was monitored between 32.2 and 63.8 months and found to reflect the response and disease progression. Conclusion: In MM patients, elevated MPF was observed in 2/3rd of the patients and is a predictor for poor survival, throughout the course of the disease. Following systemic therapies, a &amp;gt; -50% reduction in MPF was associated with improved clinical outcome, both PFS and OS. An elevated baseline serum MPF may be a predictor of response to an experimental anti-mesothelin therapy in refractory MM patients, however, the finding needs to be confirmed in a larger study. Citation Format: Liang Cao, Yunkai Yu, Anish. Thomas, Jingli Zhang, Masanori Onda, Paul Meltzer, Raffit Hassan, Ira Pastan. Megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma [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 1382.

Elevated Serum Megakaryocyte Potentiating Factor as a Predictor of Poor Survival in Patients with Mesothelioma and Primary Lung Cancer.

There is an urgent need for a companion assay to work with mesothelin-targeted therapeutic agents and for noninvasive and accurate prognostication of malignant mesothelioma (MM) patients. We report the development and validation of a blood-based assay for megakaryocyte potentiating factor (MPF) and the evaluation of its effectiveness for prognosis in MM and lung cancer patients. Using electrochemiluminescence technology, we developed a sensitive MPF assay and performed both analytical and clinical validations. Further, the effectiveness of the MPF assay in predicting prognosis was evaluated for 95 MM and 272 lung cancer patients. We performed comprehensive analytical and clinical validation, including precision and accuracy, interference, preanalytical variables, sensitivity, and specificity for mesothelioma. In MM patients, increased serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (log-rank P = 0.003; n = 95). In refractory MM patients, increased MPF is a strong predictor of poor outcome with an HR = 6.12 (log-rank P = 0.0007; n = 57). In a lung cancer patient cohort, increased MPF is a predictor of poor survival, with an HR = 1.57 (log-rank P = 0.003; n = 272). The MPF assay has robust technical characteristics, with strong analytic and clinical validation. Clinical studies indicate that increased serum MPF is a predictor of poor survival for MM patients, throughout the course of the disease. Increased MPF is also associated with poor overall survival for patients with newly diagnosed lung cancer.

Abstract 104: Identification of the molecular components required for the protumorigenic effect of MSLN in pancreatic cancer

Abstract Mesothelin (MSLN) is a GPI-linked protein expressed in &amp;gt;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&amp;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.

Megakaryocyte Potentiating Factor as a Predictive Biomarker for Therapies Against Malignant Mesothelioma.

Effective biomarkers for malignant mesothelioma (MM) are needed for clinical management and the development of mesothelin-targeted therapies. We evaluated serum megakaryocyte potentiating factor (MPF) as a biomarker predictive of treatment outcome in patients with MM and for developing mesothelin-targeted therapies. Serial serum samples from patients with MM in two clinical trials of an antimesothelin immunotoxin were tested with our clinically validated MPF assay. Correlative studies were performed to determine the test effectiveness in treatment monitoring and outcome prediction. MPF was further evaluated for an association with response to an antimesothelin therapy and for disease monitoring. There was a significant reduction of serum MPF in patients with elevated baseline and radiologic response, with an average change from -52% to -78% after one to six cycles. Using a -50% change as the cutoff, patients with MM with positive MPF response had significantly improved progression-free survival (P < .001), with the median extended from 1.9 to 11.3 months. These patients with MPF response further exhibited improved overall survival (P = .004), with the median extended from 8.8 to 22.3 months. In patients with refractory MM, there was an association between elevated pretreatment serum MPF and radiologic response to an antimesothelin therapy (P = .033). Furthermore, in these response patients, serum MPF was monitored between 32.2 and 63.8 months and was found to reflect treatment response and disease progression. At a cutoff of -50% change after receiving systemic therapies, a reduction in MPF was associated with improved clinical outcome, both progression-free survival and overall survival. An elevated baseline serum MPF was associated with a response to an antimesothelin therapy in patients with refractory MM; however, this finding needs to be confirmed in another study.

Diagnostic and prognostic biomarkers for malignant mesothelioma: an update.

Malignant mesothelioma (MM) is an aggressive and lethal cancer, mostly related to inhalation of asbestos and erionite fibers. MM is associated with poor prognosis, because of its resistance to current therapies, even if higher survival occurs in patients diagnosed and treated when at stage I of the disease. However, these do not exceed 5% of the total number of cases, due to the inadequacy of the existing biomarkers for early and accurate diagnosis. Therefore, new effective biomarkers are needed for MM detection at earlier stages and to develop tailored therapies. Here we review the most promising biomarkers in MM to date: mesothelin, soluble mesothelin-related peptides (SMRPs), megakaryocyte potentiating factor (MPF), Osteopontin (OPN), Fibulin-3, high mobility group B1 (HMGB1), microRNAs (miRNAs), multiplex protein signatures. The validation of these biomarkers will allow their use, alone or in combination, for monitoring individuals from cohorts at risk of MM and attaining early detection of MM that is instrumental in improving patient survival.

A recombinant polypeptide of the megakaryocyte potentiating factor is a potential biomarker in plasma for the detection of mesothelioma

Malignant mesothelioma (MM) is a fatal disease mostly associated with asbestos exposure and difficult to detect by non-invasive methods. This study aimed to use recombinant fragments of the megakaryocyte potentiating factor (MPF) for the development of cost-effective MPF ELISAs. Three polypeptides spanning the MPF region (MPF1-148, MPF 34-288, MPF/MSLN254-400) were produced in E.coli as maltose-binding protein hybrids. After isolation, Factor Xa digest, and purification, the polypeptides were used for the generation of rabbit antibodies and development of ELISAs. Forty-one MM patients with known histological subtype before tumor-specific treatment and 70 asbestos-exposed individuals free of any cancer were matched according to age, gender, and smoking. Plasma of all subjects was tested with the three newly developed polyclonal antibody-based ELISAs and a commercial mesothelin assay (MESOMARK™). The latter differentiated patients (median concentration 1.95 nM) from controls (median 1.07 nM, p < 0.0001) and showed an area under curve (AUC) of 0.77 in receiver operating characteristics (ROC) analysis. Of the MPF variants, exclusively the ELISA based on antibodies against the MPF34-288 fragment displayed significantly (p = 0.0002) higher values in patients than in controls (median 1.61 nM versus 0.88 nM; AUC = 0.72). The combination of the MPF34-288 and mesothelin displayed an improved ROC performance (AUC = 0.80). The MPF34-288 ELISA could be a cost-effective and minimal-invasive contribution to support a diagnosis of mesothelioma, especially in regions with a limited medical care.

Clinical impacts of mesothelin expression in gastrointestinal carcinomas.

Mesothelin, C-ERC/mesothelin is a 40-kDa cell surface glycoprotein that is normally present on normal mesothelial cells lining the pleura, peritoneum, and pericardium. Moreover, mesothelin has been shown to be overexpressed in several human cancers, including virtually all mesothelioma and pancreatic cancer, approximately 70% of ovarian cancer and extra bile duct cancer, and 50% of lung adenocarcinomas and gastric cancer. The full-length human mesothelin gene encodes the primary product, a 71-kDa precursor protein. The 71-kDa mesothelin precursor is cleaved into two products, 40-kDa C-terminal fragment that remains membrane-bound via glycosylphosphatidylinositol anchor, and a 31-kDa N-terminal fragment, megakaryocyte potentiating factor, which is secreted into the blood. The biological functions of mesothelin remain largely unknown. However, results of recent studies have suggested that the mesothelin may play a role of cell proliferation and migration. In pancreatic cancer, mesothelin expression was immunohistochemically observed in all cases, but absent in normal pancreas and in chronic pancreatitis. Furthermore, the expression of mesothelin was correlated with an poorer patient outcome in several human cancers. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. The present review discusses the expression and function of mesothelin in cancer cells and the utility of mesothelin as a target of cancer therapy.

Diagnostic values of soluble mesothelin-related peptides for malignant pleural mesothelioma: updated meta-analysis

ObjectiveAlthough the values of soluble mesothelin-related peptides (SMRPs), including mesothelin and megakaryocyte potentiating factor, in serum and/or pleural fluid for diagnosing malignant pleural mesothelioma (MPM) have been extensively studied, the...

Folate receptor alpha, mesothelin and megakaryocyte potentiating factor as potential serum markers of chronic kidney disease.

Renal disease is the eighth leading cause of death in the United States. Early diagnosis is usually based on the detection of proteinuria or elevated serum creatinine, a relatively poor biomarker that does not accurately predict renal disease progression. As a result, more predictive biomarkers of renal function are sought. We present preliminary data on three protein biomarkers, folate receptor alpha (FRA), mesothelin (MSLN), and megakaryocyte potentiating factor (MPF), currently being pursued for applications in oncology diagnostics, and evaluate serum and urine levels in subjects with renal disease. Compared to healthy subjects, a significant (P < 0.0001) increase in all three biomarkers in both serum and urine of subjects with renal disease was demonstrated. Further, serum levels of these three protein biomarkers increased with increasing stage of disease suggesting their potential value in predicting progression in subjects with renal disease and raising caution in interpretation of data in oncology applications.