bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5288: Development of lead mutant-p53 reactivating peptides towards clinical application
Authors: Tal Perry , Oren Moshe , Palgi Orna , Rotter Varda , JEONG KWANGIL
Published: June 2022

p53 is the most extensively studied tumor suppressor. It serves as a major barrier against malignant transformation and the p53 pathway is inactivated in almost all human tumors. A large percentage of tumors inactivate p53 by point mutations in the DNA binding domain (DBD) of the protein. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, improperly folded form. Previously, we reported the use of phage display technology to select for peptides that reactivate mutant p53 by stabilizing a wt protein conformation, taking advantage of a unique combination of selection steps (Tal P, at al Oncotarget. 2017 Jan 3;8(1):164-178). We now describe further progress in the development of one lead peptide, pCAP-250, which contains 9 amino acids and a myristic fatty acid at its N-terminus. The pCAP-250 sequence bears 100% homology to RAD9, a known p53 interacting protein. We show physical interaction between pCAP-250 and the p53DBD. Characterization of this interaction by NMR reveals a significant chemical shift of amino acids located in the p53DBD L1 loop and Helix-2, two regions critical for p53 transcriptional activity and interaction with DNA. We also report the optimization of our lead sequence by rational design to yield more potent derivatives with higher efficacy in cancer cell elimination and longer in vivo half-life. RNA-seq analysis shows that our lead peptides induce robust activation of p53 target genes. Furthermore, we report that in addition to p53 activation, pCAP-250 also has a p53-independent anti-cancer activity. Overall, our lead peptides cause specific cell death in a wide spectrum of mutant p53 expressing cell lines, with an IC50 of 0.1-12µM, while having no effect on non-transformed cells at these concentrations. The pharmacokinetic profile of pCAP-250 and its derivatives shows that they are completely stable in human serum and have plasma half-life of 1.5-10h in vivo when administered IV or subcutaneously, presumably due to interaction with albumin through the fatty acid moiety. Remarkably, our lead peptides elicit remarkable regression of very aggressive tumors in several preclinical mouse models of renal, colon, pancreatic and ovarian cancer. In vivo efficacy can be achieved through several routes of administration, including intra-tumor injection as well as systemic subcutaneous continuous slow release and subcutaneous bi-daily injections. Thus, such peptides might serve as novel therapeutic agents for p53-mutated human cancer.

Citation Format: Perry Tal, Moshe Oren, Orna Palgi, Varda Rotter, KWANGIL JEONG. Development of lead mutant-p53 reactivating peptides towards clinical application [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 5288.
Abstract 5304: A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy
Authors: Lee Youl-Nam , Han Yun-hee , Kim Won-Sam , Lee Cheol-Min , Moon Eun-Joung , Kim Hae-Jin
Published: June 2022

Cytotoxic chemotherapy is initially effective in patients with metastatic triple-negative breast cancer (mTNBC). However, most of the TNBC patients have high risk of recurrence after chemotherapy and metastatic tumor recurrence indicates rapid tumor proliferation and drug resistance. In TNBC cells, treatment with the chemotherapeutic drug paclitaxel (PTX) induces Transforming growth factor (TGF)-β signaling pathway. TGF-β is a master regulator of tumor angiogenesis and epithelial-mesenchymal transition (EMT), which are limiting the efficacy of cancer therapies and suppressing the anti-tumor immune response. TGF-β induces autophagy in tumor cells and autophagy is closely associated with cancer cell survival and contributing to resistance to therapies. The targeting of TGF-β pathway is possibly an effective strategy to inhibit drug resistance and increase the sensitivity of cancer treatment. Charis 1000 (C1K), which is currently under development, is a synthetic peptide that targets TGF-β1. As C1K partially inhibits TGF-β1-induced Smad2 phosphorylation in TNBC cells, there is no risk of side effects unlike small molecules or antibodies that completely inhibit the TGF-β1 signaling pathway. Combination therapies including chemotherapy are actively investigated as effective treatments for various cancers. PTX, one of frequently used drugs in combination therapies, increases phosphorylation of Smad2, a TGF- β1 downstream signaling pathway and induces autophagy in TNBC cells, but PTX in combination with C1K increases apoptosis while reducing Smad2 phosphorylation and autophagy. The combination of C1K with PTX in the mouse TNBC model showed significant tumor size reduction in comparison to the PTX alone group. In addition, the combination of C1K with PTX confirmed the increased apoptosis and decreased autophagy in the tumor tissues. Furthermore, the combination of C1K with PTX increased the survival rate of mice that was reduced due to the side effects of PTX. These data indicate that the drug resistance induced by TGF-β signaling pathway can be suppressed by C1K combination through TGF-β signaling modulation. C1K may be a promising combination partner for cancer treatment by reducing drug resistance and increasing apoptotic potential of anticancer drugs. Currently, a phase1 clinical trial of the C1K combination therapy with PTX is ongoing and the preclinical studies of the C1K combination with immune checkpoint inhibitors (ICIs) are actively investigated as well.

Citation Format: Youl-Nam Lee, Yun-hee Han, Won-Sam Kim, Cheol-Min Lee, Eun-Joung Moon, Hae-Jin Kim. A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy [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 5304.
Abstract 5338: Pamiparib as a non-P-glycoprotein substrate PARP inhibitor can overcome ABCB1-mediated multidrug resistance in ovarian cancer cells
Authors: Deng Minjuan , Qin Kang , Li Yang , Jiang Yu , Jin Wei , Shen Zhirong
Published: June 2022

Ovarian cancer is commonly treated with debulking surgery followed by platinum-taxane based chemotherapy and targeted therapy (PARP inhibitors). However, a key challenge facing these therapies is drug resistance caused by various mechanisms, one of which is the P-glycoprotein (P-gp, encoded by the ABCB1 gene) mediated multidrug resistance. Tumor cells with high P-gp protein expression extrude P-gp substrate drugs from intracellular space and reduce drug efficacy. Since Paclitaxel and some PARP inhibitors such as Olaparib are P-gp substrates, their clinical benefit may be compromised in primary or acquired P-gp positive patients. Pamiparib, on the other hand, is PARP inhibitor that is not P-gp substrate, therefore, it is reasonable to hypothesize that Pamiparib will have better efficacy in P-gp high ovarian tumors.

To test this hypothesis, Paclitaxel or Olaparib acquired resistant cell lines were developed through prolonged in vitro culture of sensitive A2780 cells (P-gp negative) with stepwise increased concentrations of individual drug. Both Paclitaxel and Olaparib resistant cells (A2780pacR and A2780olaR, respectively) highly express P-gp protein and are cross-resistant to each other, indicating this acquired resistance is mediated by P-gp protein upregulation. Indeed, P-gp protein inhibitor Verapamil can largely restore sensitivity to Paclitaxel and Olaparib in A2780pacR and A2780olaR cells, while over-expression of P-gp protein into parental A2780 cell line recapitulated the resistant phenotype. In contrast to Paclitaxel and Olaparib, Pamiparib as non P-gp substrate, is equally sensitive to parental A2780, A2780pacR and A2780olaR cell lines and not affected by P-gp expression. Consistently, intracellular drug concentration of Pamiparib is equivalent in A2780 and A2780olaR cells, however, decreased cellular Olaparib was detected in resistant cell line. These findings have been further confirmed in Olaparib resistant CDX model, in which Pamiparib showed tumor growth inhibition while Olaparib did not. The results highly suggest that Pamiparib as a non-P-gp substrate PARP inhibitor can overcome ABCB1-mediated drug resistance in tumors and may provide additional clinical benefits to ovarian cancer patients.

Citation Format: Minjuan Deng, Kang Qin, Yang Li, Yu Jiang, Wei Jin, Zhirong Shen. Pamiparib as a non-P-glycoprotein substrate PARP inhibitor can overcome ABCB1-mediated multidrug resistance in ovarian cancer cells [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 5338.
Abstract 5346: WAVE3 phosphorylation promotes chemoresistance and cancer stemness in triple negative breast cancer through downstream of β-catenin oncogenic signaling
Authors: Wang Wei , Rana Priyanka , Alkrekshi Akram , Markovic Vesna , Sossey-Alaoui Khalid
Published: June 2022

Acquisition of the metastatic phenotype is responsible for the death of ~90% of breast cancer (BC) patients. Metastatic BC is the 2nd leading cause of cancer-related deaths in women in the United States, annually accounting for more than 40,100 deaths and 266,000 new cases of invasive BC. Amongst individual BC subtypes, those classified as TNBCs are especially lethal due to their highly metastatic behavior and propensity to recur rapidly. As a group, TNBCs lack expression of hormone receptors (ER-α and PR) and ErbB2/HER2, which has prevented the development of FDA-approved targeted drug therapies that are effective against this BC subtype. Likewise, recurrent TNBCs frequently acquire resistance to standard-of-care chemotherapeutic agents (e.g., doxorubicin, cyclophosphamides, and taxanes) through mechanisms that remain incompletely understood. We established Wiskott-Aldrich Syndrome Protein-3 (WAVE3) as a novel promoter of TNBC development and metastatic progression. Our recently published studies have shown that WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways. In this study, we show that WAVE3 phosphorylation promotes chemotherapy-resistance and cancer stemness in TNBC, both in vitro and in in vivo mouse models. CRISPR/Cas9-mediated inactivation of WAVE3 resulted in inhibition of 2D growth and 3D tumorspheres’ invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 or the pharmacologic inhibition of WAVE3 phosphorylation did not. Further studies revealed that dual blocking WAVE3 expression or phosphorylation with chemotherapy treatment inhibited the activity and expression of β-catenin. Importantly, a combination of WAVE3-deficiency and chemotherapy suppressed the oncogenic behavior of chemo-resistant TNBC cells, both in vitro and in vivo. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.

Citation Format: Wei Wang, Priyanka Rana, Akram Alkrekshi, Vesna Markovic, Khalid Sossey-Alaoui. WAVE3 phosphorylation promotes chemoresistance and cancer stemness in triple negative breast cancer through downstream of β-catenin oncogenic signaling [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 5346.
Abstract 5368: Role of spatially distinct tumor fibroblast in erlotinib resistance
Authors: Bouchard Gina , Plevritis Sylvia K.
Published: June 2022

Metabolic reprogramming toward the hexosamine biosynthesis pathway (HBP), an understudied glucose pathway leading to O-glycosylation, is associated with poor survival in the lung adenocarcinoma (LUAD). We found that this metabolic switch happens primarily in Cancer-Associated Fibroblasts (CAFs), an “activated” phenotype associated with bad outcomes in cancer. We show that spatially distinct CAFs harvested from the tumor edge (Tumor-Adjacent-Fibroblasts; TAFs) of LUAD specimens vs the tumor core (Tumor Core Fibroblasts; TCFs) successfully protect cancer cells from erlotinib treatment, as measured by proliferation (ki67), migration (podoplanin) and resistance (Multidrug Resistance 1; MDR1) markers. However, while TCFs increase O-GlcNAcylation in cancer cells, a type of dynamic O-glycosylation, TAFs decrease O-GlcNAcylation in a subpopulation of cells. Interestingly, this subpopulation of cancer cells shows an increase of partial EMT features, as well as proliferation. In addition, we find this population of cancer cells to overexpress the MDR1 transporter, known to actively export erlotinib outside the cell. These results were reproduced in a novel in vitro assembloid model using patient-derived organoids and primary fibroblast mixtures that allow us to characterize cancer and spatially distinct stromal cells in response to erlotinib treatment. Overall, our preliminary results show that TAFs from the invasive leading edge of lung tumors decrease O-GlcNAcylation in cancer cells, which promotes erlotinib resistance. In summary, O-GlcNAcylation represents a potential avenue to mitigate resistance induced by tumor-stroma crosstalk and improve cancer treatment.

Citation Format: Gina Bouchard, Sylvia K. Plevritis. Role of spatially distinct tumor fibroblast in erlotinib resistance [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 5368.
Abstract 5381: Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth
Authors: Kushwaha Prem Prakash , Verma Shiv , Sarathy Gautham , Kumar Anmol , Gupta Sanjay , Ramaswamy Bhuvaneswari , Majumder Sarmila , Shankar Eswar
Published: June 2022

Treatment for Triple Negative Breast Cancer remains a challenge due to its aggressive behavior; poor clinical outcome and disposition to therapeutic resistance. The diverse clinical and molecular characteristics drives high level of heterogeneity in breast cancer. Overall change in the epigenome triggers oncogenic activity of enhancer of zeste homolog 2 (EZH2) causing changes in gene expression that leads to cancer progression. EZH2 is the catalytic member of the Polycomb group protein complex that provides the active site for covalent methylation reactions. High expression of EZH2 is strongly associated with aggressive TNBC phenotypes, compared to other non-TNBCs. Development of resistance to EZH2 inhibitors limit their therapeutic use. Studies have shown that dopamine receptor (DR) agonists and antagonists alter TNBC cell growth in both in vitro and in vivo models. Our objective was to investigate if a combination of a D1R agonist with an EZH2 inhibitor was effective to attenuate TNBC cell proliferation. Docking studies were performed using Schrodinger software and visualization software to determine the affinity of GSK126 and SKF38393 towards EZH2. TNBC cells were treated individually and combined with EZH2 inhibitor, GSK126, and D1 agonist SKF38393 for cell viability and immunoprecipitation. Insilco analysis using docking studies revealed that SKF38393 actively interacts in the same region where GSK126 binds to EZH2 i.e. the catalytic site. Combination treatment of TNBC cells with GSK126 and SKF38393 caused synergistic inhibition of cell viability, suppressed the H3K27 trimethylation activity, and inhibited invasion of TNBC cells. Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 dissociated the association between EZH2, EED, and SUZ12 in MDA-MB-231 cells. We report that combination of GSK126 and SKF38393 may have an enhanced efficacy in inhibiting the catalytic function of EZH2. The combined treatment of TNBC cells with GSK126 and SKF38393 inhibit TNBC proliferation by disrupting EZH2 functions. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar)

Citation Format: Prem Prakash Kushwaha, Shiv Verma, Gautham Sarathy, Anmol Kumar, Sanjay Gupta, Bhuvaneswari Ramaswamy, Sarmila Majumder, Eswar Shankar. Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth [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 5381.
Abstract 5450: New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells
Authors: Ramos-Inza Sandra , Encío Ignacio , Raza Asif , Sharma Arun , Sanmartín Carmen , Plano Daniel
Published: June 2022

Breast cancer is currently the most frequently diagnosed cancer and one of the leading causes of mortality among women worldwide. The introduction of a selenium atom (Se) in the structure of organic molecules has been demonstrated to be a valid approach in the design of novel chemotherapeutic agents with several biological effects, including antitumoral activity. In addition, several epidemiological, preclinical, and clinical studies support the chemopreventive and chemotherapeutic potential use of non-steroidal anti-inflammatory drugs (NSAIDs).On the basis of these findings, and taking into account the results of previous studies by our research group, we have developed a new series of twenty-five NSAID analogs bearing Se in the form of selenoesters modulated with different functional groups. The new compounds were tested in four cancer cell lines (HTB-54, DU-145, HT-29 and MCF-7) and two non-malignant cells (184B5 and BEAS-2B). The cytotoxicity of the five most potent and selective molecules was further studied in other cancer cell lines as well. Compounds 4a and 4d stood out as the most selective compounds with a selectivity index at least 5-fold higher in breast cancer cells. Compound 4d showed IC50 values below 10 µM and was found to be more potent than 4a in almost every cell line tested. Additionally, 4d effectively induced apoptosis in triple negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines. Taken together, this study supports the design of new derivatives containing Se as potential anticancer agents.

Citation Format: Sandra Ramos-Inza, Ignacio Encío, Asif Raza, Arun Sharma, Carmen Sanmartín, Daniel Plano. New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells [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 5450.
Abstract 5458: Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse models
Authors: Zheng Shansong , Zheng Qingmei , Dong Xin , Wang Xinmei , Chen Dongmei , Duan Huicheng , Ma Cuicui , Zhang Chenwei
Published: June 2022

Objective: To evaluate the antitumor activity of WX-0593 (anaplastic lymphoma kinase/ROS proto-oncogene 1 receptor tyrosine kinase [ALK/ROS1] tyrosine kinase inhibitor) combined with bevacizumab biosimilar in vivo.

Methods: Cell line-derived xenograft (CDX) model: NCI-H3122 cell suspension was subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 100-300 mm3, the mice were randomly divided into six groups (eight mice/group) and administered with solvent, WX-0593 1.5 or 5 mg/kg, bevacizumab biosimilar 1 mg/kg, or WX-0593 1.5 or 5 mg/kg + bevacizumab biosimilar 1 mg/kg, respectively. Patient-derived xenografts (PDX) model: LU-01-1443 tumor tissue blocks of 20-30 mm3 were subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 115 mm3, the mice were randomly divided into four groups (eight mice/group) and administered with solvent, WX-0593 2.5 mg/kg, bevacizumab biosimilar 1.5 mg/kg or their combination. Tumor diameter was measured twice a week. The tolerability was also assessed.

Results: CDX tumor model: There were significant differences between each group and the control group (all P <0.01). The combination of WX-0593 5 mg/kg and bevacizumab biosimilar 1 mg/kg was significantly more effective than WX-0593 5 mg/kg alone or bevacizumab biosimilar 1 mg/kg alone (both P <0.01). PDX tumor model: Compared with the control group, single-agent WX-0593 2.5 mg/kg and bevacizumab biosimilar 1.5 mg/kg delayed tumor growth. The tumor suppressive effect of WX-0593 combined with bevacizumab biosimilar was impressive (treated/control ratio =11.81%; tumor growth inhibition =92.19%) and significantly better than WX-0593 2.5 mg/kg or bevacizumab biosimilar 1.5 mg/kg alone (P =0.004, 0.003, respectively). Toxicity: There were no abnormalities in appearance and mental state in each group during the experiment, and the weight of the mice remained stable.

Conclusions: The combination of WX-0593 and bevacizumab biosimilar had obvious synergistic effect and showed potent tumor suppressive activity, which was significantly better than WX-0593 or bevacizumab biosimilar alone at the same dose. There was no drug-related toxicity in all groups, and no increase in toxicity after combined administration.

Citation Format: Shansong Zheng, Qingmei Zheng, Xin Dong, Xinmei Wang, Dongmei Chen, Huicheng Duan, Cuicui Ma, Chenwei Zhang. Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse models [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 5458.
Abstract 5503: Tumor-resilient T-cell signatures identify cellular therapy response biomarkers and reveal FIBP inhibition as an immunotherapy potentiator
Authors: Jiang Peng , Zhang Yu
Published: June 2022

Despite breakthroughs in cancer immunotherapy, most tumor-reactive T cells cannot persist in solid tumors due to an immunosuppressive environment. We developed Tres (Tumor-resilient T cell), a computational model and web server that utilizes single-cell transcriptomic data to identify signatures of T cells that are resilient to immunosuppressive signals, such as TGFβ, TRAIL, and PGE2. Analyzing transcriptomic data from pretreatment or pre-manufacture patient samples, Tres reliably predicts the clinical effectiveness of immune checkpoint blockade or adoptive cell transfer in melanoma, lung, and B-cell malignancies. Further, Tres identified FIBP as the top negative marker of tumor-resilient T cells across many cancers. FIBP knockouts in murine and human CD8 T cells significantly enhanced T-cell mediated cancer-killing and adoptive cell therapy by limiting cholesterol metabolism, which otherwise inhibits effector T-cell function. These results demonstrate Tres’s utility in identifying clinical biomarkers of T-cell effectiveness and potential therapeutic targets for enhancing immunotherapies in solid tumors.

Citation Format: Peng Jiang, Yu Zhang. Tumor-resilient T-cell signatures identify cellular therapy response biomarkers and reveal FIBP inhibition as an immunotherapy potentiator [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 5503.
Abstract 5566: Evaluation of the efficacy of ADC in vitro and in vivo
Authors: Huang Jun , Shi Xiaohe , Gu Qingyang , Ji Qunsheng
Published: June 2022

Antibody drug conjugate (ADC) is a promising complex with therapeutic potential that aimed to the treatment of solid tumor and hematological malignancies. Compared with the therapeutic monoclonal antibody, ADCs-derived monoclonal antibody is conjugated with cytotoxic agents which can deliver potent cellular toxins to targeted cancer cells specifically. According to the specialization of ADCs, we have established the state-of-the-art platform to support the evaluation of the efficacy of ADC or the combination strategy of ADC and other anti-cancer therapy in the process of pre-clinical drug discovery. such as target validation, efficient internalization, cytotoxic effects and in vivo efficacy testing.

In order to explore the mechanism of action and the functional effects of ADC for supporting the application of investigational new drug. Our flow cytometry platform (BD LSRFortessa X20) supports to evaluate the target validation and the process of internalization in a quick, reliable and reproducible way. Also we are able to construct customized antigen-specific overexpression or KI cells used for the development of ADCs which target on the specific antigens theoretically, for example, Claudin18.2 overexpressed MIA-PaCa-2 cell line. In terms of in vivo ADCs assessment, our abundant CDX and PDX resource facilitates the process of in vivo ADCs evaluation, and we have already assess the efficacy of ADC or the combination treatment of ADC and other anti-tumor drugs in several animal models. Last but not the least, our high-throughput MSD (MESO QuickPlex) and Luminex (Bio-Plex࣪ 200 system) platform boosts the evaluation of ADCs safety by measuring the extent of cytokine release syndrome.

Citation Format: Jun Huang, Xiaohe Shi, Qingyang Gu, Qunsheng Ji. Evaluation of the efficacy of ADC in vitro and in vivo [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 5566.
Abstract 5567: hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers
Authors: Tomar Sakshi , Zhang Jingli , Li Nan , Pastan Ira , Ho Mitchell , Hassan Raffit
Published: June 2022

Background and Hypothesis: Mesothelin is an attractive target for CAR-T cell therapy since it is highly expressed in many solid tumors but has limited expression on normal human tissues. However, anti-mesothelin CAR-T cells have shown limited efficacy in patients. Since most antibodies to mesothelin bind the membrane distal epitopes we generated antibodies that bind mesothelin in a membrane-proximal region. We hypothesize that CAR-T cells recognizing a membrane proximal epitope on mesothelin will be more efficacious.

Study Design: We developed CAR-T cells targeting a membrane-distal (SS1CAR-T) and a membrane-proximal epitope (hYP218 CAR-T) of mesothelin. Using mesothelin-expressing cancer cell lines, anti-tumor efficacy of CAR-T cells was assessed in vitro by measuring direct killing of tumor cells by the CAR-T cells and quantifying cytokine-release by the CAR-T cells. In vivo anti-tumor efficacy of CAR-T was determined in NSG mice using three different solid tumor models of high-mesothelin expression: ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived xenograft (NCI-Meso63). Blood and tumor infiltration of CAR-T cells in vivo was determined via flow cytometry analysis.

Results: Upon co-culture with tumor cells with high mesothelin expression (A431-H9, OVCAR-8, KLM-1, H226, NCI-Meso63, NCI-Meso29), hYP218 CAR-T cells killed cancer cells more efficiently than SS1 CAR-T cells, with a 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). Additionally, compared to SS1 CAR-T cells, hYP218 CAR-T cells secreted significantly higher level of cytokines (IL-2, TNFα, IFNγ) in a co-culture with tumor cells. In three solid tumor models, single treatment of tumor-bearing mice with hYP218 CAR-T cells lead to improved tumor response and survival compared to SS1 CAR-T, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR-T cells, higher hYP218 CAR-T cell infiltration was detected both in the blood and tumors isolated from hYP218 CAR-T treated mice seven days post-treatment.

Conclusion: We demonstrate that hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are more potent than similar CAR-T cells targeting a membrane distal epitope. Increased accumulation of hYP218 CAR-T cells in the tumor may explain their increased efficacy. These results support its clinical development to treat patients with mesothelin expressing cancers.

Citation Format: Sakshi Tomar, Jingli Zhang, Nan Li, Ira Pastan, Mitchell Ho, Raffit Hassan. hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers [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 5567.
Abstract 5570: Development of piggyBac transposon-mediated HER2-CAR-T cells for the treatment of solid tumors
Authors: Shioya Koichiro , Matsumura Tomio , Ohira Yuta , Komatsuzaki Naomi , Shinagawa Manaka , Tanaka Miyuki , Yagyu Shigeki , Nakazawa Yozo , Zhang Lilin
Published: June 2022

Background: Although chimeric antigen receptor (CAR)-T therapies have achieved remarkable success in the treatment of hematologic malignancies, the outcome for patients with solid tumors remains poor. There are several reasons behind this, including exhaustion of CAR-T cell, poor homing and penetration in the tumor, and the lack of persistence in the immunosuppressive tumor microenvironment. To solve these problems, we have developed HER2-CAR-T cells (BP2301) using the piggyBac (PB) transposon-based gene transfer system.

Methods and Results: Second generation HER2-CAR construct plus PB transposase were introduced into autologous peripheral blood mononuclear cells (PBMC) by electroporation. These cells were activated with UV-inactivated genetically-engineered antigen presenting autologous PBMC (AP cells) expressing HER2, CD80, and 4-1BBL, and propagated for 14 days to obtain the final product (BP2301). BP2301 exhibited dominant fraction of less exhausted stem cell memory-like T cells (Tscm), which could be associated with antitumor efficacy. BP2301 showed robust in vitro killing activity against HER2+ tumor cells even after multiple rounds of cancer cell challenge, suggesting the long-term functionality without immune-exhaustion. In a xenograft murine model of rhabdomyosarcoma cell line (SJCRh30), a single intravenous administration of BP2301 (5 × 106 CAR-positive cells) eradicated the tumor, and mice treated with BP2301 rejected the second tumor establishment owing to the in vivo expansion of BP2301.

Conclusion: BP2301 has demonstrated potent anti-tumor activity in vitro and in vivo. We have established a GMP manufacturing process for BP2301, completed non-clinical safety and efficacy studies, and plan to launch an investigator-initiated phase 1 clinical trial to evaluate the safety and efficacy of BP2301 in HER2-expressing sarcoma and gynecologic malignancies in the second quarter of 2022.

Citation Format: Koichiro Shioya, Tomio Matsumura, Yuta Ohira, Naomi Komatsuzaki, Manaka Shinagawa, Miyuki Tanaka, Shigeki Yagyu, Yozo Nakazawa, Lilin Zhang. Development of piggyBac transposon-mediated HER2-CAR-T cells for the treatment of solid tumors [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 5570.
Abstract 5624: PD-L1 mediates IFNγ-regulation of glucose but not of tryptophan metabolism in clear cell renal cell carcinoma
Authors: Garige Mamatha , Ghosh Susmita , Norris ALexis , Li Guangyuan , Sourbier Carole
Published: June 2022

The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed cell death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors. Recently the intrinsic role of PD-L1 and interferon gamma (IFNγ) signaling have been studied in several types of tumor cells, yet it remains unclear how they affect the metabolism and signaling pathways of ccRCC. Using metabolomics, metabolic assays and RNAseq, we showed that IFNγ enhanced aerobic glycolysis and tryptophan metabolism in ccRCC cells in vitro and induced the transcriptional expression of signaling pathways related to inflammation, cell proliferation and cellular energetics. These metabolic and transcriptional effects were partially reversed following PD-L1 transient silencing. Aerobic glycolysis as well as signaling pathways related to inflammation were not induced by IFNγ when PD-L1 was silenced, however tryptophan metabolism and activation of Jak2 and STAT1 were maintained. Our data demonstrate that PD-L1 expression is required to mediate some of IFNγ’s effect in ccRCC cells and highlight the importance of PD-L1 signaling in regulating the metabolism of ccRCC cells in response to inflammatory signals.

Citation Format: Mamatha Garige, Susmita Ghosh, ALexis Norris, Guangyuan Li, Carole Sourbier. PD-L1 mediates IFNγ-regulation of glucose but not of tryptophan metabolism in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5624.
Abstract 5628: Immune repertoire sequencing facilitates gamma delta T cell clonal determination
Authors: Song Chen , Erijman Ariel , Langhorst Bradley W. , Liu Pingfang , Dimalanta Eileen T. , Davis Theodore B.
Published: June 2022

Gamma-delta T cells are a small fraction of T lymphocytes with only 1-5% of the overall T cell population, but they are an important subset that have unique contributions to both innate and adaptive immunity. Gamma-delta T cells can recognize a broad range of antigens to provide rapid responses to pathogens, and can also interact with both immune cells and non-immune tissue cells triggering regulatory and cytotoxic responses. These unique features make them ideal candidates that could be targeted to induce durable immunity in the context of different pathologies. There has been growing interest in understanding the contributions of gamma-delta T cells to immunology and developing efficient gamma-delta T-cell-based therapies for cancer, infectious disease, and autoimmune disease. In this study, we have implemented T cell repertoire sequencing for high throughput characterization. Gamma-delta T cells were enriched from tissues and peripheral blood mononuclear cells (PBMCs). RNA was extracted and used to generate full length TCR libraries. Unique molecular identifiers (UMIs) were incorporated to discretely barcode each mRNA molecule, enabling absolute quantitative ranking of TCR clone abundance. Full length immune repertoire sequencing facilitates the detection of distinct and shared clones in tissue and blood samples, enabling the identification of disease specific clones to evaluate immunotherapy effects. In addition, RNA sequencing was performed for gene expression analysis of gamma-delta T cells to identify cell phenotypes.

Citation Format: Chen Song, Ariel Erijman, Bradley W. Langhorst, Pingfang Liu, Eileen T. Dimalanta, Theodore B. Davis. Immune repertoire sequencing facilitates gamma delta T cell clonal determination [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 5628.
Abstract 5641: Tumor cell intrinsic factors in colorectal cancer immune evasion
Authors: Wangmo Dechen , Zhao Xianda , Yuan Angelo , Gates Travis , Subramanian Subree
Published: June 2022

The poor response of most colorectal cancer (CRC) patients to immunotherapy is a major unmet clinical need. Although early detection through screening has reduced CRC incidence, the prognosis for metastatic CRC is poor. In the past decade, immune checkpoint inhibitors (ICIs) have been a major clinical breakthrough with promising clinical benefits in many cancer types, but its efficacy is limited in most (~85%) CRC patients. ICIs preferentially have robust responses from tumors with pre-existing T cell infiltration. However, most CRC lacks robust T-cell infiltration due to immune evasion mechanisms. Therefore, it is crucial to characterize factors shaping immune evasion to improve the response to ICIs. We hypothesize that targeting tumor-cell intrinsic factors can overcome T cell exclusion and will sensitize the tumor to ICIs. To elucidate the factors underlying ICI resistance, it is critical that preclinical models faithfully recapitulate the tumor physiology and genetics of the human disease. Therefore, we developed a tumor organoid (tumoroids) model system adapted from Jacks lab that harbors shApc, KrasG12D, p53-/- and Smad4-/- (shAKPS) mutations, which are co-mutated with high frequency and associated with poor prognosis. We demonstrated that tumoroids with only shApc and KrasG12D mutations can form a solid tumor in two weeks. Addition of p53-/- and Smad4-/- mutations drives invasiveness and metastasis. The shAKPS tumoroids present key features of advanced CRC that are unresponsive to ICIs including invasive tumor, metastasis, and T cell exclusion. Using CRISPR/Cas9 screening and integrative immune score analysis, we identified potential immune evasive genes. To define their expression profile in the tumor, we performed TCGA data analysis in fourteen different cancer types. Furthermore, we performed correlation tests to determine their role in immune cell infiltration and T cell dysfunction. Our results show that these immune suppressive genes are overexpressed in tumor tissues, and negatively correlate with CD8+ T cell infiltration. Our findings suggest that overexpression of immune suppressive genes in cancer cells may cause T cell dysfunction and exclusion in tumors. Furthermore, the functional role of these immune suppressive genes will be characterized using shAKPS mutant organoids and in vivo orthotopic CRC tumor models.

Citation Format: Dechen Wangmo, Xianda Zhao, Angelo Yuan, Travis Gates, Subree Subramanian. Tumor cell intrinsic factors in colorectal cancer immune evasion [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 5641.
Abstract 5652: A novel anti apoptotic function of CTLA4 in melanoma
Authors: Kazmi Hasan Raza , Mo Xuan , Zhou Bo , Preston-Alp Sara , Gross Scott , Wajeeh Hassaan , Merali Carmen , Soboloff Jonathan , Merali Salim , Zaidi M. Raza
Published: June 2022

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells.

Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [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 5652.
Abstract 5660: Anti-proliferation effect of polo-like kinase-4 inhibitor in ovarian cancer via AKT/mTOR/p70S6K pathway
Authors: Tse Ka Yu , Chan Wai Sun , Mo Moxil Xuetang , Lee Horace Hok Yeung , Ip Philip Pun Ching , Mak Tak Wah
Published: June 2022

Background: Chromosomal instability is a hallmark in epithelial ovarian cancer. Polo-like kinases (PLK) play a vital role in cell cycle regulation. In particular, PLK4 is crucial in centriole duplication. PLK4 overexpression causes defect in cytokinesis, which in turn leads to aneuploidy and mitosis catastrophe. Previous studies showed that PLK4 inhibitors are effective against cell growth in different cancers like breast cancer and lung cancer. We hypothesize that the AKT/mTOR and p70S6K pathway is involved in this mechanism.

Methods: The importance of cell proliferation of PLK4 was assessed by its inhibition in ovarian cancer cell lines and patient-derived organoids by CFI-400945. RNA sequencing was performed. The effects of PLK4 inhibition on the AKT/mTOR and p70S6K pathway was demonstrated by qPCR and western blot. Correlation of the mRNA expression level with the IC50 of CFI-400945 in ovarian cancer cell lines.

Results: PLK4 inhibition led to reduced cell proliferation in ovarian cancer cells based on XTT and colony formation assays. The anti-proliferative effect was further confirmed in 12 patient-derived organoids using ATP bioluminescent assay. RNA sequencing showed that PLK4 inhibition led to downregulation of the AKT/mTOR/p70S6K pathway. It was found that the most sensitive cell line had the highest mRNA expression of mTOR and p70S6K, while those less sensitive had lower mTOR and p70S6K mRNA expression. Treatment with CFI-400945 showed a reduction of total and phosphorylated forms of AKT, mTOR and p70S6K in a dose-dependent level. Combined treatment with metformin, a drug that had mTOR inhibition effect, showed a synergistic effect in one ovarian cancer cell line.

Conclusion: PLK4 inhibition caused reduced cell proliferation in in vitro and ex vivo assays, and this was accompanied by a downregulation of the AKT/mTOR/p70S6K pathway. This suggested that this pathway might be one of the downstream effector pathways of PLK4.

Citation Format: Ka Yu Tse, Wai Sun Chan, Moxil Xuetang Mo, Horace Hok Yeung Lee, Philip Pun Ching Ip, Tak Wah Mak. Anti-proliferation effect of polo-like kinase-4 inhibitor in ovarian cancer via AKT/mTOR/p70S6K pathway [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 5660.
Abstract 5664: Characterization of OASEP1 as a biomarker and therapeutic target for oral cancer
Authors: Takano Atsushi , Yoshitake Yoshihiro , Shinohara Masanori , Daigo Yataro
Published: June 2022

This study aims to identify new biomarkers and therapeutic targets for oral cancer. Our strategies are as follows: i) Identification of overexpressed genes in oral cancers by gene expression array analysis, ii) Validation of clinicopathologic significance of their protein expression by tissue microarray, iii) Examination of their growth effect on cancer cell by siRNAs. We identified a secreted protein, OASEP1 (oral cancer-associated serum protein 1) as a candidate. Immunohistochemical staining showed that OASEP1 protein expression was observed in 120 (75.4%) of 159 oral cancers that had undergone curative surgery. High level of OASEP1 expression was associated with poor prognosis for oral cancer patients (P = 0.0054, by log-rank test). Multivariate analysis revealed that strong OASEP1 expression was an independent prognostic factor. Suppression of OASEP1 expression by siRNA specific for OASEP1 significantly inhibited the growth of oral cancer cell lines through apoptosis as detected by apoptosis assays and time lapse imaging. In addition, siRNAs for the receptor of OASEP1 also significantly inhibited the growth of oral cancer cells. Enforced OASEP1 expression significantly enhanced the growth of cancer cells. Addition of OASEP1 protein significantly increased the growth of oral cancer cells in a dose dependent manner, suggesting that the interaction between OASEP1 and its receptor regulated the growth of oral cancer cells. Microarray analysis coupled with siRNAs for OASEP1 demonstrated various OASEP1-related oncogenic pathways in oral cancer cells. Our data suggest that OASEP1 is a possible prognostic biomarker and therapeutic target for oral cancer.

Citation Format: Atsushi Takano, Yoshihiro Yoshitake, Masanori Shinohara, Yataro Daigo. Characterization of OASEP1 as a biomarker and therapeutic target for oral cancer [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 5664.
Abstract 5666: MyD88 in myofibroblasts regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation
Authors: Yuan Qi , Zhang Jinhua
Published: June 2022

Hepatic stellate cells (HSCs) and cancer-associated fibroblasts (CAFs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). MyD88 controls the expression of several key modifier genes in liver tumorigenesis; however, whether and how MyD88 in myofibroblasts contributes to the development of fibrosis-associated liver cancer remain elusive. Here, we used an established hepatocarcinogenesis mouse model involving apparent liver fibrogenesis, in which MyD88 was selectively depleted in myofibroblasts. Myofibroblast MyD88-deficient (Fib-MyD88 KO) mice developed significantly fewer and smaller liver tumor nodules. MyD88 deficiency in myofibroblasts attenuated liver fibrosis and aerobic glycolysis in hepatocellular carcinoma tissues. Mechanistically, MyD88 signaling in myofibroblasts increased the secretion of CCL20, which promoted aerobic glycolysis in cancer cells. This process was dependent on the CCR6 receptor and ERK/PKM2 signaling. Furthermore, liver tumor growth was greatly relieved when the mice were treated with a CCR6 inhibitor. Our data revealed a critical role for MyD88 in myofibroblasts in the promotion of hepatocellular carcinoma by affecting aerobic glycolysis in cancer cells and might provide a potential molecular therapeutic target for HCC.

Citation Format: Qi Yuan, Jinhua Zhang. MyD88 in myofibroblasts regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation [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 5666.