PTK7 Expression Research Articles

Abstract 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2616.

Evaluation of the prevalence of MET and protein tyrosine kinase 7 expression in non-small cell lung cancer to evaluate overlap of ADC antigens.

e21105 Background: Biomarker-targeted antibody-drug conjugates (ADCs) have shown promise in treating lung cancer. Telisotuzumab vedotin (Teliso-V; ABBV-399) is a first-in-class MET (also known as c-Met)-directed ADC comprising the monoclonal antibody telisotuzumab (ABT-700) conjugated to a cytotoxic microtubule inhibitor, monomethyl auristatin E (MMAE), via a cleavable dipeptide linker. In the phase 2 LUMINOSITY study (NCT03539536), Teliso-V demonstrated promising anticancer activity in previously treated patients with MET-overexpressing (OE), non-squamous epidermal growth factor receptor wild type ( EGFR WT) non-small cell lung cancer (NSCLC; 52.2% overall response rate in MET OE high group, 36.5% in all MET OE cohort [intermediate and high]) and an acceptable safety profile (Camidge et al. J Clin Oncol. 2022;40:16 suppl, 9016). These data provide proof of concept that biomarker-selected MMAE-based ADCs could be beneficial to patients with NSCLC. Cofetuzumab pelidotin (ABBV-647), an ADC with a similar composition to Teliso-V, contains an anti-protein tyrosine kinase 7 (PTK7) monoclonal antibody (hu6MO24) conjugated to a microtubule-inhibiting cytotoxin, Aur0101 auristatin, via a cleavable cysteine-reactive linker. PTK7 expression and oncogenic functions have been reported in several cancers. We sought to establish the co-prevalence of MET OE and PTK7 in NSCLC to understand if both these antigens targeted by ADCs are co-expressed or present in distinct tumors. Methods: Antigen prevalence was analyzed in tumor samples from patients at the City of Hope National Medical Center with non-squamous NSCLC and EGFR WT or unknown status. Immunohistochemistry (IHC) assays used were MET (SP44) Assay for MET OE (Roche Tissue Diagnostics; positive if ≥25% cells at 3+ intensity) and an AbbVie-developed assay for PTK7 expression (positive if ≥90% cells at ≥2+ intensity). Results: A total of 148 patients (median age 67 years, 52% stage ≥III at diagnosis, 68% ECOG 0–2) were screened (data shown in table). About 24% of the patients were MET IHC positive, whereas 11% were PTK7 IHC positive. Further, MET OE and PTK7 had complementary prevalence with only 3% of the patients co-expressing both antigens. Conclusions: MET OE and PTK7 expression by IHC were found to be complementary in patients with non-squamous EGFR WT/unknown NSCLC. About 32% of patients were positive for MET OE or PTK7 expression or both. Data represent patients from a single center and more pre-treatment tissue samples that were viable and available. These IHC findings suggest that MET OE and PTK7 are indeed complementary NSCLC biomarkers. [Table: see text]

Abstract LB216: Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of metastatic breast cancer incidence, and remains an area of unmet clinical need due to the low rates of overall survival. Recently, the TROP2-targeting ADC sacituzumab govitecan has received an accelerated approval from the FDA for adult patients with metastatic TNBC, as more than 85% of TNBC is marked by TROP2 overexpression. However, the clinical efficacy of ADC therapies targeting TROP2 alone is limited by its on-target toxicity. In an effort to offer therapeutic alternatives that limit this toxicity, we sought to identify other targets to combat metastatic TNBC in combination with TROP2. PTK7 is highly expressed in breast cancer; notably, PTK7 expression is higher in TNBC than non-TNBC, and is correlated with worse prognosis, tumor metastasis and TNBC progression. PTK7 has also been demonstrated to be enriched in tumor-initiating cells (TICs) in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). We generated fully human anti-human PTK7 x TROP2 bispecific antibodies (bsAbs) from RenLite® mice, which harbor the complete human heavy chain immunoglobulin variable domain with a common human kappa light chain for subsequent bispecific antibody assembly. These bsAbs demonstrated reactivity to human, monkey, and dog antigens, and showed enhanced internalization in vitro compared with parental PTK7 antibodies. In addition, these bsAbs showed favorable tumor cell selectivity, as there was minimal internalization of the monovalent antibodies. These bsAbs were than conjugated with Monomethyl auristatin E (MMAE) to generate anti-PTK7 x TROP2 bispecific ADC (BCG033) candidates. BCG033 candidates showed potent anti-tumor activity in several cell line-derived xenografts including TNBC xenografts, indicating that BCG033 has strong therapeutic potential in TNBC and other PTK7/TROP2 co-expressing cancers. Patient-derived TNBC xenografts with co-expression of PTK7 and TROP2 have been screened for future in vivo drug efficacy screening. In summary, BCG033 has the potential to exert anti-tumor efficacy in TNBC and other solid tumors co-expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer [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 LB216.

Anti-PTK7 Monoclonal Antibodies Exhibit Anti-Tumor Activity at the Cellular Level and in Mouse Xenograft Models of Esophageal Squamous Cell Carcinoma.

PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.

Abstract P4-01-07: Insights into the compensation mechanism behind targeting PI3K and Wnt in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is a devastating disease accompanied with substantial morbidity and mortality with very few tools in the clinical armamentarium to improve outcomes. The PI3K pathway is activated in the majority of TNBCs. However, single-agent inhibition of PI3K has seen limited clinical efficacy. We have previously demonstrated that the Wnt pathway is upregulated in response to certain PI3K inhibitors as a compensatory resistance mechanism. Dual targeting of the PI3K and Wnt pathways leads to a synergistic anti-tumor effect that has been observed preclinically and in clinical trial (NCT03243331). Recently, we have found that several isoform-specific PI3K inhibitors predictably induce PTK7. PTK7 is a tyrosine kinase protein in the noncanonical Wnt pathway found to be overexpressed in various cancers, including TNBC, and is target of an antibody drug conjugate (ADC) currently in clinical trials. Herein, we sought to determine the PI3K isoforms that are responsible for PTK7 induction to help guide rational drug combination design. Methods: Twelve PI3K inhibitors including alpelisib, buparlisib, idelalisib, duvelisib, GSK2636771, GSK2292767, and CAY10505 were tested. Each inhibitor targets different isoforms individually or combinations of PI3K isoforms. Using four TNBC cell lines with different PI3K pathway mutational status and PTK7 expression (MDA-MB-231, MDA-MB-453, MDA-MB-468, and Hs578T), IC50s were initially measured using a viability assay. Using these IC50s, cells were treated in triplicate and RNA was isolated after 72 hours of treatment. RT-PCR was then performed using vehicle controls looking at the change in PTK7 expression. PI3K δ and λ isoform inhibition was also performed using siRNAs individually and in combination for validation of PI3K inhibitors. Protein expression of PI3K isoforms and PTK7 was also observed. Endogenous levels of PTK7 to assess induction after PI3K inhibitor treatment was assessed using both RNA and protein expression. Results: Testing multiple PI3K inhibitors covering various combinations of isoforms, we found that inhibition of PI3K δ and λ or pan-PI3K inhibition caused significant increases in RNA and protein levels of PTK7. We also found that individually targeting α and β in these cell lines did not have any effect on PTK7. We validated this induction further using siRNA inhibition to rule out any off targets of PI3K inhibitors. Additionally, after PI3K inhibition, the fold change in PTK7 expression was observed to be significantly higher in those cells with low or no PTK7 expression, whereas those with high baseline PTK7 expression observed little or no increase, maintaining pre-treatment levels. Conclusion: Refining clinical trial combinations and therapies to optimize drug efficacy while decreasing toxicity is a vital part of precision medicine. Here we have sought to optimize our previous clinical experience of dual targeting of PI3K and PTK7 and found two potential FDA approved therapies that may provide better efficacy and toxicities. Taking the previous trial and this preclinical data, future trials are warranted in TNBC. Citation Format: Jeffrey P Solzak, Chao Wang, Milan Radovich. Insights into the compensation mechanism behind targeting PI3K and Wnt in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-07.

Dual-Aptamer-Targeted Immunomagnetic Nanoparticles to Accurately Explore the Correlations between Circulating Tumor Cells and Gastric Cancer.

It has been acknowledged that circulating tumor cells (CTCs) are promising biomarkers in liquid biopsy for cancer diagnosis and prognosis. However, the relationship between the CTC number and gastric cancer has scarcely been quantitatively investigated. Moreover, the single criterion of epithelial cell adhesion molecule (EpCAM) antibody/aptamer to specifically recognize epithelial CTCs cannot be universally applied for clinical applications, as it fails to recognize EpCAM-negative CTCs. Herein, we propose simple, low-cost, dual-aptamer (EpCAM and PTK7)-modified immunomagnetic Fe3O4 particles (IMNs) for efficient capture of heterogeneous CTCs and downstream analysis in gastric cancer patients. High PTK7 expression and a significant negative correlation between PTK7 and EpCAM expression were observed in primary gastric cancer tissues. Taking MGC-803 and BGC-823 cells as CTC models, the obtained dual-targeting IMNs could distinguishably recognize these cells with both high or low EpCAM and PTK7 expressions, which enhanced the accuracy of CTC recognition in gastric cancer. More than 95% of these two kinds of cells could be captured within 20 min of incubation, which was significantly more efficient than that of single EpCAM- or PTK7-modified IMNs. With this strategy, as low as five CTCs could be captured from phosphate-buffered saline (PBS), a cell mixture containing THP-1 cells, and lysed blood mediums. Moreover, the obtained CTCs can be used for subsequent gene analysis. Finally, the fabricated IMNs were successfully applied for CTC capture in 1.0 mL of peripheral blood samples from patients with gastric cancer. The detected CTC numbers in 72 participants were found to have close relationships with chemotherapy sensitivity, diagnosis, stage, and distant metastasis of patients. This work provides important references for further investigations on CTC-related diagnosis and individualized treatment.

Abstract 3034: Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma

Abstract Anti-GD2 antibody therapy significantly improved high-risk neuroblastoma (NB) patient survival yet is associated with toxicity due to GD2 expression on pain fibers. Also, GD2 may be downregulated in NB following chemotherapy and relapse. It is paramount to identify novel tumor-associated antigens that are upregulated on the cell surface throughout therapy with minimal normal tissue expression. We utilized a novel cell surface capturing technology to define the NB cell surfaceome before and after chemotherapy and nominate potential targets for cell-mediated immunotherapy. Two NB patient derived xenografts (PDXs) and one cell line xenograft (IMR5) were treated with topotecan/cyclophosphamide in vivo. Treated and untreated tumors were disaggregated and N-glycoproteins on intact cells were oxidized, selectively labeled with biocytin-hydrazide, lysed, proteins digested, and cell surface peptides enriched using NeutrAvidin beads, followed by LC-MS analysis. We prioritized cell surface proteins that were abundant on all NB models and did not change under the selective pressure of chemotherapy. Our top hit was confirmed for cell surface expression by flow, evaluated for primary tumor and normal cell expression using gene expression datasets, and evaluated for functional relevance using CRISPR/CAS9 and immunotherapy targeting via chimeric antigen receptor (CAR). In two replicates, 292 cell surface proteins were identified as abundant and unchanged following chemotherapy. 34/292 proteins had high abundance of ≥70 a.u. across all xenografts, including NB surface proteins ALK and NCAM1. One protein identified was PTK7, a member of the WNT signaling pathway that is overexpressed in adult tumors. PTK7 is highly expressed in primary NB tumors with very low normal tissue expression. PTK7 protein is present on the cell surface in 17/17 NB cell lines and PDXs. While high PTK7 expression in primary NB is associated with poor patient prognosis, CRISPR/CAS9 knockout of PTK7 shows no functional effect on cell survival, proliferation or chemotherapy response. We developed a codon-optimized, second-generation CAR construct specific to PTK7. Our CAR-engineered T cells specifically bound to recombinant human-PTK7 Fc chimera. PTK7 CAR Jurkat T cells became activated in the presence of PTK7-expressing KELLY, IMR-5, and NB-1643 NB cells, and activation was not seen in the presence of PTK7 negative CMK leukemia cells or NB-1643 PTK7 knockout cells, confirming activation is specific to the CAR recognizing PTK7. Primary T cell PTK7 CAR studies are ongoing to assess cytotoxicity against NB. Multiple surface proteins remain stable on NB tumors after chemotherapy treatment. PTK7 is a potential cell surface antigen that could be utilized for immunotherapy approaches in NB while minimizing damage to normal tissues. PTK7 is robustly expressed in other pediatric and adult cancers, suggesting broader clinical relevance. Citation Format: Arhanti Sadanand, Victor Maximov, Jasmine Lee, Suttipong Suttapitugsakul, Jenny Shim, Chris Doering, Ronghu Wu, Robert Schnepp, Trent Spencer, Kelly Goldsmith. Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma [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 3034.

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)–dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences.

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles

ObjectiveSporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in...

Abstract 3243: Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors

Abstract Protein Tyrosine Kinase 7 (PTK7), also known as colon cancer carcinoma kinase 4 (CCK-4), is a highly conserved catalytically inactive tyrosine kinase with inherent signal transduction activity. PTK7 is a member of the Wnt signaling pathway and thought to function in cell proliferation, adhesion, migration and apoptosis. PTK7 has been shown to be highly expressed in certain cancer types including colon, breast, lung, pancreatic, renal and ovarian. Downregulation of PTK7 by shRNA has been shown to reduce tumor growth and metastases development in xenograft mouse models. Furthermore, high PTK7 expression in triple negative breast cancer and NSCLC patients has been linked to poor prognosis. PTK7 therefore appears to be an attractive target for solid cancer therapeutic intervention and has been targeted clinically with an Antibody Drug Conjugate (PF-06647020). However, the protein is also expressed to a lesser degree in some normal adult tissues, particularly in the stromal cells of the ovary, placenta, uterus and lung. We have developed a human/murine cross-reactive anti-PTK7 CAR construct and assessed the ability of allogeneic anti-PTK7 CAR-T cells bearing that construct to target cancer cells and elicit toxicity in mice. These anti-PTK7 CAR-T cells were shown to be efficacious in vitro and in immunocompromised mouse xenograft models of breast, lung, colon, pancreatic and ovarian cancer. However, consistent in all xenograft models studied, a drop in body weight was observed shortly after injection from which all mice rapidly recovered to above baseline. Latent toxicity that was more variable amongst the xenograft experiments was also observed. Understanding and developing appropriate mitigation strategies to address the observed toxicities may be required to develop further a safe and efficacious allogeneic anti-PTK7 CAR-T cell therapy for solid tumor cancers. Citation Format: Katie Levitsky, Zejun Li, Minh Thu Pham, Kelly Maeng, Luke Hanley, Jason Sagert, Jon Terrett, Jui Dutta-Simmons, Melanie Allen. Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors [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 3243.

Identification of PTK7 as a promising therapeutic target for thyroid cancer.

To evaluate the possible involvement of PTK7 in the progression of human thyroid cancer and assess its potential effects on the proliferation and apoptosis of thyroid cancer. Immunohistochemical (IHC) assays and clinical significance analysis were performed to explore the correlations between PTK7 expression and clinical characteristics of patients with thyroid cancer. Quantitative PCR assays and Immunoblot assays were performed to detect the expression of PTK7 in control or PTK7 shRNA plasmids transfected thyroid cancer cells. MTT assays were performed to detect the effects on the proliferation of thyroid cancer cells. Flow cytometry (FCM) assays were performed to assess the changes in cell apoptosis of thyroid cancer. Additionally, the effects of PTK7 on tumor growth were detected through in vivo tumor growth assays. PTK7 is highly expressed in human thyroid cancer tissues, and its expression levels are associated with the clinical characteristics, including TNM stage (p=0.015*), and intraglandular dissemination (p=0.024*) of patients with thyroid cancer. PTK7 ablation inhibits cell proliferation and stimulates cell apoptosis of thyroid cancer in vitro. Additionally, PTK7 contributes to tumor growth of thyroid cancer cells in mice. We demonstrated the involvement of PTK7in the progression of thyroid cancer, and therefore provided a novel and promising therapeutic target for thyroid cancer treatment.

PTK7 promotes the malignant properties of cancer stem-like cells in esophageal squamous cell lines.

This study was performed to investigate the role of PTK7 in esophageal squamous cell carcinoma (ESCC) stem-like cells (CSCs). PTK7 expression in ESCCs identified by RT-qPCR, and CSC-like cells were isolated from populations of NEC and TE-1 cells. The CSC-like cells were verified by flow cytometric analyses performed using CD34 and CD133 antibodies, and RT-qPCR and western blot assays were used to examine the self-renewal capability of CSC-like cells. CSC-like cells treated with PTK7 siRNA or a P53-specific inhibitor (PFTα) were analyzed for their sphere formation capacity and their apoptosis and migration/invasion capabilities by sphere formation, flow cytometry, and transwell assay, respectively. Their levels of P53, MKK3, and cleaved caspase 3 expression were examined by western blot analysis. Our results revealed that a majority of the isolated CSC-like cells were CD34+/CD133+ double positive cells. Nango, Sox2, and OCT4 were dramatically increased in the separated CSC-like cells, which had the pluripotency and self-renewal properties of stem cells. Additional, PTK7 was dramatically upregulated in the ESCC tissues and CSC-like cells. An investigation of the function of CSC-like cells revealed that knockdown of PTK7 reduced their sphere formation, promoted apoptosis, and suppressed their migration and invasion abilities, all of which could be significantly reversed by PFTα. Mechanistic studies showed that PFTα could attenuate the upregulation of P53, MKK3, and cleaved caspase 3 expression that was induced by PTK7 knockdown in CSC-like cells. PTK7 increased the malignant behaviors of CSC-like cells derived from ESCC cells by regulating p53. Therefore, this study suggests PTK7 as an underlying target for therapy against ESCC.

PTK-7 Expression in Gastric Cancer: A Prognostic Determinant

Background:Protein tyrosine kinase-7, a regulatory protein in the Wnt signaling pathway, was highly overexpressed in various cancer types and assumed to be related to prognosis.Aims:The purpose of this study is to assess whether protein tyrosine kinase-7 expression status in curatively resected gastric carcinoma would independently identify patients with a high risk of recurrence and death.Study Design:Retrospective cohort study.Methods:We included patients who were at least 18 years of age and diagnosed with gastric cancer. The exclusion criterion was a metastatic disease at the time of diagnosis or operation. Data on clinicopathological prognostic determinants and clinical courses, including the date of disease relapse and survival status, were collected with the use of medical records. Surgically removed tumor tissue specimens were examined by two independent pathologists at the pathology department of our institution. Protein tyrosine kinase-7 expression status was assessed with immunohistochemical processing and stratified on a scale ranging from 0 to +3 according to the extent of stained tumor cells. It was then further categorized into two groups, one being + (positive), including +1, +2, and +3 scores, another was-(negative), including-and +/− scores.Results:A total of 114 patients were analyzed. Protein tyrosine kinase-7 expression was present in 66.7% of the surgical tumor specimens. There was no statistically significant difference in almost all relevant parameters between the protein tyrosine kinase-7 positive and negative groups. The estimated median survival in the protein tyrosine kinase-7 positive group was significantly better than the protein tyrosine kinase-7 negative group (60 vs 22 months, p<0.001). Disease-free survival was found to be 55 months in the protein tyrosine kinase-7 positive group, whereas it was 21 months in the negative group (p=0.015). In the multivariate analysis, along with negative protein tyrosine kinase-7 expression, poor performance status, and advanced stage were significantly associated with the risk of death (p<0.001 for each).Conclusion:Compared to patients with negative PTK-7 expression, patients with positive PTK-7 expression have better disease-free survival and overall survival rates. Efforts should be made to enhance this finding and translate it into clinical practice.

ENST00000489707.5 Is a Preferred Alternative Splicing Variant of PTK7 in Adrenocortical Cancer and Shows Potential Prognostic Value.

BackgroundThis study aimed to explore the transcript preference of PTK7 in adrenocortical cancer (ACC), the prognostic value, and the potential underlying genetic alterations.Material/MethodsData from the Cancer Genome Atlas-Adrenocortical Cancer (TCGA-ACC) and the Genotype-Tissue Expression (GTEx)-normal adrenal gland were used for analysis.ResultsA non-canonical alternative transcript, ENST00000489707.5, which only encodes an extracellular immunoglobulin (Ig)-like domain and an intracellular kinase domain, is the dominant isoform of PTK7 in both ACC and normal adrenal gland. Its expression percentage was significantly higher in ACC than in normal adrenal gland. ACC tissues showed preferred expression of this transcript compared with other cancers with known PTK7 expression. Prognostic analysis showed that ENST00000489707.5 had independent prognostic value in progression-free survival (PFS) (HR: 1.227, 95%CI: 1.077–1.398, p=0.002) and disease-specific survival (DSS) (HR: 1.419, 95%CI: 1.154–1.745, p=0.001) after adjustment of other risk factors. cg20819617 methylation was negatively correlated with both PTK7 and ENST00000489707.5 expression.ConclusionsENST00000489707.5 is a preferred alternative splicing product of PTK7, with a significantly increased proportion in ACC compared with other cancers. Its expression shows potential prognostic value in terms of PFS and DSS in ACC patients. The methylation status of cg20819617 might play a critical role in modulating PTK7 transcription and ENST00000489707.5 expression.

In silico DNA methylation analysis identifies potential prognostic biomarkers in type 2 papillary renal cell carcinoma.

There are currently no effective treatments for advanced‐stage papillary renal cell carcinoma (PRCC). The goal of this study is to define potential DNA methylation‐based markers and treatment targets for advanced‐stage type 2 PRCC. Progressive DNA methylation changes and copy number variation (CNV) from localized to advanced‐stage type 2 PRCC are analyzed by using methylation data generated by TCGA's kidney renal papillary cell carcinoma (TCGA‐KIRP, 450k array) project. Survival analyses are performed for the identified biomarkers and genes with CNV. In addition, expression of the corresponding genes is investigated by RNA‐seq analysis. Progressive methylation changes in several CpGs from localized to advanced‐stage type 2 PRCC are observed. Four CpGs (cg00489401, cg27649239, cg20555674, and cg07196505) in particular are identified as markers for differentiating between localized and advanced‐stage type 2 PRCC. Copy number analysis reveals that copy gain of PTK7 mostly occurs in advanced‐stage type 2 PRCC. Both the four CpG methylation changes and PTK7 copy number gain are associated with patient survival. RNA‐seq analysis demonstrates that PTK7 copy gain leads to higher PTK7 expression relative to tumors without copy number gain. Moreover, PTK7 is significantly upregulated from localized to advanced‐stage type 2 PRCC and is linked to cancer cell invasion. In conclusion, DNA methylation markers that differentiate between localized and advanced‐stage type 2 PRCC may serve as useful markers for disease staging or outcome, while PTK7 copy gain represents a potential treatment target for advanced‐stage type 2 PRCC. Stepwise methylation changes and copy number gain also associate with disease stage in PRCC patients.

Abstract 1784: High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis. Due to the frequent distant metastasis and lack of successful targeted therapies, the overall survival rate for the patients with TNBC is significantly lower than estrogen receptor positive and HER2 positive breast cancers. To identify potential druggable targets, previously, we performed a gene expression analysis and identified protein kinases that are highly expressed in ER-negative breast cancers. Through these studies, we discovered that the expression of pseudo kinase-7 (PTK-7) is highly elevated in TNBCs and correlated with p53 mutation status and disease progression. We hypothesize that high expression of PTK-7 is required for the growth and invasiveness of TNBC cells. Experimental design and methods: In this study, we analyzed the expression status of PTK-7 in TNBC and the association of PTK-7 expression with p53 status and disease prognosis using publicly available datasets. Protein levels of PTK-7 were determined through western blotting analysis. The association of p53 and PTK-7 expression was determined using Oncomine analysis. Using specific siRNAs against PTK-7, we determined whether PTK-7 is required for the growth of TNBC cells. The role of PTK-7 in migration and invasion of TNBC cells was determined using Boyden trans-well assays. Results: Analysis of publicly available datasets reveals that PTK-7 expression is elevated in TNBCs compared with normal breast and ER-positive breast cancers. High PTK-7 expression was found to be associated with p53 mutations. Knockdown of PTK-7 using siRNA significantly reduced the growth of TNBC cells in vitro. In vitrotrans-well migration and invasion studies also demonstrated that knockdown of PTK-7 significantly reduced the migration and invasion of TNBC cells. In contrast knockdown of PTK-7 did not affect the growth and invasiveness non-TNBC cells. Conclusion: Our results suggest that the expression of PTK-7, a critical regulator of growth and invasion of TNBCs. These results suggest that PTK-7 is a promising target for the treatment of women with TNBC. This work was supported by Susan G Komen Promise Grant (PB), SAB Komen grant (PB) and Young Foundation grant (PB). Citation Format: Lakshmi Reddy Bollu. High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells [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 1784.

Abstract OT3-06-02: An initial safety study of gedatolisib plus PTK7-ADC for metastatic triple-negative breast cancer

Abstract Background: The PI3K pathway is dysregulated in the majority of triple-negative breast cancer (TNBCs). Contrary to the theory of oncogene addiction, single agent inhibition of the PI3K pathway in TNBC has had only modest activity. Our group has demonstrated preclinically that when PI3K is inhibited, an immediate compensatory up-regulation of the Wnt pathway occurs. The Wnt pathway is known known for its role in cancer metastases and can confer resistance to initial PI3K inhibition. Simultaneous dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We have initiated a Phase I clinical trial using Gedatolisib (PI3K/mTOR inhibitor) and PTK7-ADC (Wnt pathway) for patients with metastatic TNBC (NCT03243331). Gedatolisib is a pan-class I isoform PI3K/mTOR inhibitor, and PTK7-ADC is an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway co-receptor) with an Auristatin payload. PTK7 is an attractive second target due to its up-regulation after PI3K inhibition and its known overexpression in TNBC. Further data has shown that the PTK7-payload, Auristatin, is in itself synergistic with Gedatolisib. The combination of using both of these drugs suggests a unique concept of “double synergy”. Where Gedatolisib increases the expression of the target of PTK7-ADC leading to one mechanism of synergy, and the Auristatin payload on PTK7-ADC is synergistic with Gedatolisib providing a second mechanism. Study Design: This is an open-label, Phase I, dose-escalation study with a 3 + 3 cohort design. The trial will enroll 12-18 patients. 3 cohorts of at least 3 patients will receive Gedatolisib (weekly) &amp; PTK-ADC (q3w) at 110mg+1.4mg/kg, 180mg+1.4mg/kg, and 180mg+2.8mg/kg dose levels. Eligibility Criteria: This trial enrolls patients with metastatic triple negative (ER-, PgR-, HER2-) or low estrogen expressing (ER and PgR &amp;lt;5%, HER2-) breast cancer. Patients must have received at least one prior chemotherapy for advanced disease and have adequate hematologic, renal, and hepatic function. Patients with previously treated CNS involvement are eligible. Patients with uncontrolled diabetes are excluded, given the potential for hyperglycemia with Gedatolisib. Patients must have disease amenable and consent to biopsy for correlative endpoints. Objectives: The primary objective is to evaluate the safety of Gedatolisib plus PTK7-ADC. The secondary objective is to evaluate efficacy as determined by objective response rate, clinical benefit at 18 weeks, and progression free survival (PFS). Exploratory objectives will evaluate efficacy in patients with genomic aberrations in the PI3K pathway; and association of tumor DNA, RNA, plasma and circulating tumor cell sequencing with clinical efficacy to identify putative biomarkers. Correlative Sciences: We are collecting matched pre-/post-treatment tumor biopsies and serial blood samples to determine biomarkers of clinical response to inform subsequent trials. We plan to evaluate: 1) PI3K activity; 2) genomic aberrations in the PI3K pathway; 3) baseline PTK7 expression; 4) PTK7 upregulation after Gedatolisib treatment; and 5) mutations in plasma circulating tumor DNA. Supported by the BCRF, 100 Voices of Hope, Catherine Peachey Foundation, and Pfizer. Citation Format: Radovich M, Solzak JP, Hancock BA, Storniolo AMV, Schneider BP, Miller KD. An initial safety study of gedatolisib plus PTK7-ADC for metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-06-02.

PTK7 is a novel oncogenic target for esophageal squamous cell carcinoma

BackgroundOverexpression of PTK7 has been found in multiple cancers and has been proposed to serve as a prognostic marker for intrahepatic cholangiocarcinoma. Its role in esophageal cancer, however, remains to be clarified. We hypothesize that PTK7 positively regulates tumorigenesis of esophageal cancer.MethodsWe examined PTK7 expression pattern in human esophageal squamous carcinoma by Oncomine expression analysis and by immunohistochemistry (IHC) staining. We knocked down PTK7 in two esophageal squamous cell carcinoma cell lines, TE-5, and TE-9, by siRNA, and evaluated cell proliferation, apoptosis, and migration ofPTK7-defective cells. Expressions of major apoptotic regulators and effectors were also determined by quantitative real-time PCR in PTK7-defective cells. We further overexpressed PTK7 in the cell to evaluate its effects on cell proliferation, apoptosis, and migration.ResultsBoth Oncomine expression and IHC analyses showed that PTK7 is overexpressed in clinical esophageal squamous cell carcinoma tumors. PTK7 siRNA suppressed cell growth and promoted apoptosis of TE-5 and TE-9. PTK7-defective cells further displayed reduced cellular migration that was concomitant with upregulation of E-cadherin. Conversely, overexpression of PTK7 promotes cell proliferation and invasion, while apoptosis of the PTK7-overexpressing cells is repressed. Notably, major apoptotic regulators, such as p53 and caspases, are significantly upregulated in siPTK7 cells.ConclusionsPTK7 plays an oncogenic role in tumorigenesis and metastasis of esophageal squamous carcinoma. PTK7 achieves its oncogenic function in esophageal squamous cell carcinoma partially through the negative regulation of apoptosis.