bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 514: Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice
Authors: Chen Yue , Zhai Ning , Zhu Yinsong , Yue Peibin , Verma Nagendra , Brotherton-Pleiss Christine , Fu Wenzhen , Nakamura Kayo , Chen Weiliang , Tius Marcus , Lopez-Tapia Francisco J. , Turkson James
Published: April 2023

Unlike classical Signal transducer and activator of transcription (Stat) signaling, which is transient in non-transformed cells, the aberrant activation of the family member, Stat3, occurs in malignant transformation and is implicated in breast, ovarian and many other human cancers. Thus, Stat3 remains a validated and important target for the discovery of novel anticancer drugs. Despite this, the discovery and development of potent Stat3 small molecule inhibitors has proven to be a significant challenge, and no drug is available yet in the market. We have discovered novel Stat3 irreversible azetidine inhibitors with unprecedent submicromolar potency through systematic medicinal chemistry structure activity relationship (SAR). Mechanism of action of most potent azetidine inhibitors depend on whether they are salicylic acids or not, i.e., salicylic acids preferably bind irreversibly to Stat3 Cys426 site in the DNA-binding domain (DBD), and non-salicylic acids, e.g., current lead H182, bind to Stat3 Cys468 DBD site. As expected, the inhibition of Stat3 DNA-binding activity was time dependent, with IC50 in the range of 0.27-0.87 µM at one hour incubation with active Stat3. On the other hand, azetidine salicylic acids also bind to the SH2 domain, although reversibly and at much weaker affinity, as determined in fluorescent polarization (FP) assay, with IC50 of 10-16 µM at one hour incubation, while non-salicylic acids, e.g., lead H182, present no binding affinity up to 600 µM for the SH2 domain. Despite that H182 does not bind to SH2 domain, it still inhibits its phosphorylation. Though lead compound H182 presents significant mouse in vivo efficacy, mouse in vivo pharmacokinetics shows very low plasma AUC, which correlates with quite high mouse in vitro hepatocyte CLint of 138 µL/min/106 cells; however, human in vitro hepatocyte assay gives much better results and is in the middle stability range (CLint of 14.6 µL/min/106 cells). The in vivo PK in other species, e.g., rat, is being determined. H182 represents a plausible molecule for further development.

Citation Format: Yue Chen, Ning Zhai, Yinsong Zhu, Peibin Yue, Nagendra Verma, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco J. Lopez-Tapia, James Turkson. Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 514.
Abstract 5144: Spatial T-cell atlas in more than 100 different tumor entities using BLEACH&STAIN
Authors: Huang Zhihao , Bady Elena , Müller Jan H. , Mandelkow Tim , Lurati Magalie C. , Simon Ronald , Bernreuther Christian , Jacobsen Frank , Sauter Guido , Möller Katharina , Luebke Andreas , Hinsch Andrea , Clauditz Till S. , Burandt Eike , Blessin Niclas C.
Published: April 2023

Background: The composition and functional state of T-cell subpopulations can highly impact patient’s outcome and response to immune checkpoint therapy. However, only little is known about the spatial interplay of most rare T-cell subpopulations.

Design: To assess the density, composition, degree of immune checkpoint expression, and spatial interplay of T-cell subpopulations in 5989 tumor samples from more than 100 tumor entities, two different types of tissue microarrays (0.6 mm and 4 mm in diameter) were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, FOXP3, CD56, CD45RA, CD45RO, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis.

Results: For identification and definition of immune cell subpopulations unsupervised X-shift clustering and 2D/3D t-distributed stochastic neighbor embedding (t-SNE) using the “Rtsne” package (Rtsne (RRID:SCR_016342)) were applied and revealed 102 T-cell subpopulations at certain functional state. Within these subpopulations, the well-characterized expression profiles were visually matched with single T-cell expression profiles and documented as digital images. This process resulted in 12 main T-cell subsets that were further subclassified according to their functional state (proliferation, immune checkpoint expression) and studied according to their spatial orchestration. Interestingly, the vast majority of T-cell subsets were found in all analyzed tumor entities. However, their spatial orchestration, immune checkpoint expression profile was highly variable between different tumor entities.

Conclusion: This study provides a comprehensive overview of rare T-cells subpopulations and its spatial orchestration in more than 100 different tumor entities.

Citation Format: Zhihao Huang, Elena Bady, Jan H. Müller, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Christian Bernreuther, Frank Jacobsen, Guido Sauter, Katharina Möller, Andreas Luebke, Andrea Hinsch, Till S. Clauditz, Eike Burandt, Niclas C. Blessin. Spatial T-cell atlas in more than 100 different tumor entities using BLEACH&STAIN. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5144.
Abstract 5174: PEGylated liposomal doxorubicin improves oral cancer response to radio-immunotherapy
Authors: Devkota Laxman , Bhavane Rohan , Badachhape Andrew , Veeramachaneni Ratna , Menon Renuka , Bhandari Prajwal , Cortes Sofia , Da Costa Fabio Henrique Brasil , Ghaghada Ketan , Young Simon , Sikora Andrew G. , Annapragada Ananth V.
Published: April 2023

Introduction: The immunosuppressive tumor microenvironment (TME) in oral cancer inhibits response to conventional treatment (surgery +/- chemoradiotherapy) and check-point inhibitor (αPD1) immunotherapy. The chemotherapeutic agent doxorubicin and its PEGylated liposomal nanoparticle formulation (PLD) have been shown to target myeloid derived suppressor cells (MDSCs) in the TME. In this preclinical work, we investigated whether PLD improves response to radio-immunotherapy in a highly aggressive and immunologically ‘cold’ oral cancer model.

Experimental Procedure: Studies were performed in the MOC2 syngeneic mouse model of oral cancer. Mice were randomized to one of seven treatment groups: αPD1, Radiation (XRT), αPD1+XRT, PLD, PLD+αPD1, PLD+XRT, PLD+αPD1+XRT. High-resolution nanoparticle contrast-enhanced computed tomography (nCECT) imaging was performed, and whole-body T2-weighted magnetic resonance imaging (MRI) used to monitor primary tumor response and development of metastases. Response evaluation criteria in solid tumors (RECIST) were used to evaluate treatment outcomes.

Results: Tumors in all non-PLD groups were treatment resistant and grew progressively, consistent with the immunologically ‘cold’ MOC2 model. In contrast, all PLD groups showed tumor regression with 25-38% complete responders (CR) and 12-88% partial responders (PR) while all animals in non-PLD groups showed progressive disease (PD). MR imaging identified a high incidence of regional and distant metastases in the non-PLD groups while the PLD groups showed decreased regional and no distant metastases (Table 1). Presence of metastases was confirmed by nCECT imaging and histopathological analysis.

Conclusion: PLD improved response to radio-immunotherapy in a highly aggressive immunologically ‘cold’ mouse model of oral cancer while simultaneously preventing disease progression and metastasis.

Citation Format: Laxman Devkota, Rohan Bhavane, Andrew Badachhape, Ratna Veeramachaneni, Renuka Menon, Prajwal Bhandari, Sofia Cortes, Fabio Henrique Brasil Da Costa, Ketan Ghaghada, Simon Young, Andrew G. Sikora, Ananth V. Annapragada. PEGylated liposomal doxorubicin improves oral cancer response to radio-immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5174.
Abstract 580: A novel drug suppresses pancreatic cancer growth through immunomodulation
Authors: Gaikwad Shreyas Ramchandra , Srivastava Sanjay K.
Published: April 2023

Drug repurposing is an effective strategy that is being used to explore new potential treatments for various diseases. In the current study, utilizing drug repurposing approach, we have identified anti-cancer activity of an anti-parasitic compound (MB). We evaluated the anti-cancer activity of MB in pancreatic ductal adenocarcinoma (PDAC). The average IC50 of MB in several human and murine PDAC cells was 4-6µM. The mode of cell death was apoptosis and which was confirmed using Annexin-V assay. Oral administration of MB suppressed tumor growth in both human and murine subcutaneous and orthotopic models at a dose of 5mg/kg. In the syngeneic orthotopic model, MB showed immune modulation wherein it increased infiltration of CD8 T-cells in the tumor microenvironment as compared to control groups. Based on this, we combined MB with anti-PD1 therapy to evaluate the combinatorial effect. Our results showed significant PDAC tumor growth suppression when a combination of MB and anti-PD1 therapy was used in an orthotopic PDAC tumor model. In conclusion, MB has a promising anti-cancer effect against PDAC tumors and we are currently exploring the molecular mechanism of action in PDAC cell lines and also evaluating if MB has any direct effect on immune cell populations.

Citation Format: Shreyas Ramchandra Gaikwad, Sanjay K. Srivastava. A novel drug suppresses pancreatic cancer growth through immunomodulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 580.
Abstract 5801: Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression
Authors: Anand Sumyuktha V. , Lee Min K. , Skorput Alexander G. , Fox Isabella B. , Young Alison L. , Christensen Brock C. , Gulledge Allan , Havrda Matthew C.
Published: April 2023

Primary gliomas arising within the brain are the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation and/or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass responsible for disease progression. Understanding mechanisms responsible for maintaining GSCs and developing strategies to deplete the GSC population could improve glioma treatment and prognosis. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), which are observed in neural development and the adult brain. OPCs are prone to malignant transformation and are believed to be a cell of origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We hypothesized that cholinergic signaling may also maintain the primitive state of OPC-like GSCs. We analyzed publicly available single nuclei RNASeq data of patient glioblastoma samples and observed high expression of CHRM3, encoding the M3 mAChR, in OPC-like cells of the proneural subtype of glioma. Studies in mouse OPC-like GSCs confirmed high levels of CHRM3 expression and demonstrated that ACh generated voltage changes and rapid (< 1 second) increases in cytosolic calcium from internal calcium stores. Exposure to a brain permeant FDA-approved anti-muscarinic drug targeting M3mAChR suppressed proliferation, evoked calcium release, and the activation of intracellular second messengers PKC and ERK. Pharmacologic inhibition of mAChRs in established patient derived glioma grafts prevented re-initiation of tumors in subsequent host animals compared with vehicle treated tumors. These studies suggest that the cholinergic microenvironment maintains GSCs in a manner similar to normal OPCs and provides a platform for repositioning available small molecule mAChR antagonists for treatment of glioma.

Citation Format: Sumyuktha V. Anand, Min K. Lee, Alexander G. Skorput, Isabella B. Fox, Alison L. Young, Brock C. Christensen, Allan Gulledge, Matthew C. Havrda. Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5801.
Abstract 5805: Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase
Authors: Seo Juyeon , Park Minsu , Ko Dongmi , Kim Seongjae , Park Soeun , Nam Kee Dal , Kang Yong Koo , Seuk So Ra , Park Jaeyoun , Jung Eunsun , Kim Yoon-Jae , Kim Ji Young , Seo Jae Hong
Published: April 2023

Triple-negative breast cancer (TNBC) exhibits an aggressive behavior associated with poor prognosis due to the absence of established molecular targets. Focal adhesion kinase (FAK) is a major determinant and participates in the acquisition of migration and invasion, as well as the maintenance of breast cancer stem cell (BCSC)-like traits in TNBC. We sought to investigate the effect of ebastine, a second-generation antihistamine on apoptosis, FAK activation, BCSC subpopulations and metastasis in TNBC in vitro and in vivo. TCGA dataset analysis revealed that mRNA levels of FAK were highly expressed in TNBC compared to other breast cancer subtypes. We found that ebastine binds to the tyrosine kinase domain of FAK, which blocks phosphorylation at the Y397 and Y576/577 residues and subsequent inactivation of SRC. Ebastine-induced apoptosis was associated with attenuation of JAK2/STAT3 and MEK/ERK signaling in TNBC cells. Kinetic analysis revealed a concentration-dependent impairment of cell migration in the presence of ebastine in MDA-MB-231 and 4T1 cells in vitro. Ebastine targets BCSC-like cell populations as evidenced by a sharp decline in the expression of the BCSC markers ALDH1, CD44 and CD49f and suppression of mammosphere-forming capacity. Ebastine administration led to a significant reduction in the growth ebastine of BCSC-enriched 4T1 mammospheres orthotopically injected into the mammary glands of Balb/c mice. Ebastine administration significantly impeded angiogenesis and distant metastasis while reducing MMP-2/-9 levels in circulating blood in vivo. Importantly, biochemical analysis in mice serum showed that ebastine had no effect on liver and kidney function. Our findings suggest that EBA may be an effective therapeutic repositioning candidate for the simultaneous targeting of multiple survival signaling pathways for the treatment of molecularly heterogeneous TNBC. Further investigation of ebastine as an anti-metastatic agent for the treatment of TNBC is warranted.

Citation Format: Juyeon Seo, Minsu Park, Dongmi Ko, Seongjae Kim, Soeun Park, Kee Dal Nam, Yong Koo Kang, So Ra Seuk, Jaeyoun Park, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5805.
Abstract 5807: mTOR regulates stem cells of glioblastoma by altering the self-renewal and proliferation
Authors: Gellerson Olivia , Bree Julie , Das Mohan , Kleinman George , Gandhi Chirag , Jhanwar-Uniyal Meena
Published: April 2023

The mechanistic target of rapamycin (mTOR: aka mammalian target of rapamycin), a serine/threonine kinase, functions by forming two multiprotein complexes termed mTORC1 and mTORC2. Glioblastoma (GBM) is a malignant and lethal brain tumor that remains incurable partly due to the existence of untreatable cancer stem cells (CSC). The pathogenesis of GBM is largely due to the loss of the tumor suppressor gene PTEN, which is implicated in the aberrant activation of the mTOR pathway. The major cause of tumor recurrence, growth, and invasion is the presence of a unique population of CSC. Resistance to conventional therapies appears to be caused by both extensive genetic abnormalities and dysregulation of the transcription landscape. Consequently, CSCs have emerged as targets of interest in new treatment paradigms. Evidence suggests that inhibition of the mTOR pathway can also be applied to target CSCs. In this study, we explored the role of the mTOR pathway in the regulation of stem cells of GBM. GBM stem cells were treated with various inhibitors of canonical PI3K/AKT/mTOR pathways such as rapamycin (mTORC1 inhibitor), PP242 (ATP binding mTORC1/2 inhibitor), LY294002 (PI3K inhibitor), and MAPK inhibitor, U0126. We previously demonstrated the presence of CSC in the peritumor area of GBM. A significant number of GBM tumors expressed stem cell marker nestin and activated mTOR (pmTORSer2448), with most tumor cells co-expressing both markers. The expression of stem cell marker NANOG was suppressed following rapamycin treatment, while U0126 induced translocation of NANOG from the nucleus to the cytoplasm. The neurospheres were disrupted following rapamycin and LY294002 treatments. Rapamycin or PP242 treatments given with differentiating agent All-trans-retinoic acid reduced stem cell proliferation. Treatment with novel small molecule dual inhibitors of mTORC1/2 demonstrated that Torin1 and Torin2 suppressed the proliferation of GBM CSC, while XL388 was less effective. Torin1 and XL388 delay the process of self-renewal as compared to controls, whereas Torin2 halted the self-renewal of GBM CSC. Additionally, Torin2 was able to eradicate tumor cells. Torin2 effectively targeted CSCs of GBM by halting self-renewal and inhibiting cell proliferation, underscoring the use of Torin2 in the treatment of GBM.

Citation Format: Olivia Gellerson, Julie Bree, Mohan Das, George Kleinman, Chirag Gandhi, Meena Jhanwar-Uniyal. mTOR regulates stem cells of glioblastoma by altering the self-renewal and proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5807.
Abstract 5820: Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells
Authors: Hou Xiang-Ling , Koo Hui Min , Lin Chia-Wen , Huang Chien-Chi , Tseng Yu-Chi , Tseng Kai-Yu , Chao Wei-Ting
Published: April 2023

In has been demonstrated epithelial mesenchymal transition (EMT) is related to E-cadherin expression level. Recently, studies have been demonstrated the collective cancer cell migration which expresses high E-cadherin is a powerful mechanism of metastasis. Therefore, the role of E-cadherin in cancer metastasis is still not clear. Endocytosis was shown to regulate E-cadherin turnover on the membrane and the function of lysosome is also associated with cell vesicle trafficking. The past studies indicated Src can regulate vesicle trafficking. In this study, HT29 colon cancer cells were treated with Src inhibitor, Dasatinib (DST), to investigate the role of lysosome in regulating E-cadherin dynamics through vesicle trafficking in collective cell migration. In the result, the recycling protein Rab11 and lysosome marker Lamp1 were found localized with E-cadherin in the cell-cell contacts, however, Dasatinib sensitive (DSTS) cells showed less colocalization of Rab11 with E-cadherin. When cells were treated with lysosome inhibitor chloroquine, Rab11 were accumulated in the cell-cell contacts and reduced collective cell migration in Dasatinib resistant (DSTR) cells. These results suggest lysosome inhibition can suppress Rab11 mediated vesicle trafficking of E-cadherin transport for collective cell migration, and chloroquine has therapeutic potential in colon cancer treatment.

Citation Format: Xiang-Ling Hou, Hui Min Koo, Chia-Wen Lin, Chien-Chi Huang, Yu-Chi Tseng, Kai-Yu Tseng, Wei-Ting Chao. Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5820.
Abstract 5821: Synergism of FAK and ROS1 inhibitors in the treatment of CDH1-deficient cancers mediated by FAK-YAP-TRX signaling
Authors: Gao Jiaming , Yao Yunying , Wang Zaiqi , Zhang Baoyuan , Ren Ruibao
Published: April 2023

CDH1 deficiency is common in triple negative breast cancer and diffuse gastric cancer patients, both of which lack effective therapeutics. Although inhibiting ROS1 function results in synthetic lethality in CDH1-deficient cancers, adaptive resistance limits their potential clinical benefits. Hyperactive focal adhesion kinase (FAK) signaling contributes to adaptive drug resistance, and FAK inhibitors are partially effective against CDH1–deficient cancers. Thus, co-treatment with ROS1 and FAK inhibitors may provide stronger anti-cancer effects by targeting CDH1 deficiency and potentially overcome drug resistance. Here, we evaluated the effects and mechanisms of combined treatment with FAK inhibitor IN10018 and ROS1 inhibitors. Cell-based assays and in vivo animal experiments revealed that FAK and ROS1 inhibitor combination treatment showed stronger anti-cancer effects than either monotherapy. ROS1 inhibitors induced FAK-YAP-TRX signaling, decreasing oxidative stress–related DNA damage, and consequently reducing its anti-cancer effects. However, the combination treatment suppressed the aberrant FAK-YAP-TRX signaling, reinforcing the cytotoxicity of ROS1 inhibitor towards cancer cells. This mechanism of action was confirmed by inhibiting the activity of FAK-YAP-TRX downstream signaling molecules and by pretreatment with oxidative stress scavengers. These findings support the co-administration of FAK and ROS1 inhibitors as a therapeutic strategy in CDH1-deficient triple negative breast cancer and diffuse gastric cancer patients and support further clinical testing of this combination regimen.

Citation Format: Jiaming Gao, Yunying Yao, Zaiqi Wang, Baoyuan Zhang, Ruibao Ren. Synergism of FAK and ROS1 inhibitors in the treatment of CDH1-deficient cancers mediated by FAK-YAP-TRX signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5821.
Abstract 592: Therapeutical antibody against LILRB2 enhances antitumor immunity by reprogramming M2 macrophage
Authors: Su Chen , Feng Yanping , Zhao Hui , Zhang Meng , Luo Longtan , Hao Feng , Ning Jinying , Wu Guojin
Published: April 2023

M2 macrophage is critical in maintaining an immune-suppressive microenvironment inside the tumor. Here we developed a LILRB2 antagonist antibody KA-1769 for reprogramming M2 macrophage to M1 phenotype. KA-1769 binds to LILRB2 with higher affinity (KD=0.67nM) than MK-4830 (KD=7.4nM). And it also binds to LILRB1 with weak affinity (KD=41nM) but not to other LILR family members. KA-1769 blocks bindings of HLA-G to both LILRB1 and LILRB2. Macrophage derived from monocyte with M-CSF stimulation polarizes to M1 phenotype in the presence of KA-1769 by increasing expression of CD86, MHCII, and TNF-α and decreasing expression of CD14, CD206, CD163, and IL-10. KA-1769 accelerates T cell proliferation when co-cultured with macrophage. In a syngeneic Lewis lung carcinoma murine model with LILRB2-transgenic mice, injection of KA-1769 significantly suppressed tumor growth. It decreased the tumor's myeloid-derived suppressor cells (MDSC) and CD4+ CD25+ FoxP3+ Treg population. Based on these preclinical data, KA-1769 is an excellent therapeutical antibody for enhancing antitumor immunity.

Citation Format: Chen Su, Yanping Feng, Hui Zhao, Meng Zhang, Longtan Luo, Feng Hao, Jinying Ning, Guojin Wu. Therapeutical antibody against LILRB2 enhances antitumor immunity by reprogramming M2 macrophage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 592.
Abstract 5930: Single day workflow for high-quality whole genome analysis of thousands of single cells
Authors: West Jay A. , Blackinton Jeff G. , Hukari Kyle , Dahl Joe
Published: April 2023

DNA sequencing technology provides the capability to tell the story of every cell in a heterogenous sample. ResolveDNA® generates uniform coverage of >95% of a single genome, enabling researchers to generate single-cell genomic data of unparalleled quality. Here, we report an enhancement to the ResolveDNA® workflow that increases the rate of data collection; the ability to reliably generate complete genomic information from up to 1536 cells in parallel from a single experiment. To achieve this, we have coupled a high-throughput automated process to a low volume reaction using the latest ResolveDNA® 2.5-hour chemistry. This establishes the potential for users to go from a single cell, or low-input DNA sample, to ready to sequence libraries in a single day for >3000 samples. As with earlier ResolveDNA® workflows, these reactions generate negligible amounts of contaminating and off target DNA during the reaction. A typical single-cell reaction yields ~ 85 ng of DNA. The single cell’s amplified DNA product is amenable to multiple post amplification library preparation processes. We utilized both BioSkryb ResolveDNA® and Watchmaker Library Preparation kits for Illumina sequencing. We did not identify significant differences in the performance of the two products. Libraries were assessed at a depth of ~ 2million reads and determined to be of high quality, with low rates of chimeric DNA formation and less than 1% mtDNA resulting in the reaction product. A subset of samples was probed at >200 million reads, with >85% of the genome covered at 1x. Consistent with earlier ResolveDNA® workflows, the libraries were characterized with >99% precision and >80% sensitivity. The story told from a single cell’s genome drives discovery and shines a light on the questions surrounding the causes of disease and cellular function. This workflow is designed to empower researchers to probe the genomic landscape of a heterogenous tissue sample.

Citation Format: Jay A. West, Jeff G. Blackinton, Kyle Hukari, Joe Dahl. Single day workflow for high-quality whole genome analysis of thousands of single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5930.
Abstract 5965: B2M loss of heterozygosity in melanoma is associated with increased numbers of activated NK cells and higher expression of the NK cell inhibitory ligand, HLA-G
Authors: Galvez Mildred , Campbell Katie M. , Medina Egmidio , Ribas Antoni
Published: April 2023

β2-microglobulin (B2M) is a critical subunit of antigen presenting major histocompatibility complex (MHC) class I molecules. Its genetic inactivation in tumor cells has been implicated in tumor-intrinsic immune escape and immunotherapy resistance. Currently, the impact of B2M dysregulation on tumor-immune dynamics requires furthers characterization. Using a clinical dataset of patients with melanoma treated with immune checkpoint blockade (ICB), whole-exome sequencing and bulk RNA-sequencing data from baseline tumor biopsies (n=295) were analyzed to identify somatic B2M alterations and evaluate correlative clinical outcomes, infiltrating immune cells, and gene expression patterns. No loss-of-function mutations in B2M were found; however, 50% of tumors had B2M alterations in the form of homozygous loss (n=2), loss of heterozygosity (LOH; n=56), or copy number gains (n=88). B2M LOH events were overrepresented in tumors from patients who progressed during ICB therapy compared to those who responded (34% versus 19%; Pearson chi-square test, p=0.035). Additionally, tumors with B2M LOH had lower B2M expression compared to B2M-unaltered tumors (Wilcoxon test, p=0.05). Gene set enrichment analysis revealed reduced expression of immune and inflammatory signatures in B2M-LOH tumors. Furthermore, immune cell deconvolution showed that B2M-LOH tumors had lower total immune cell counts, but greater numbers and proportions of activated natural killer (NK) cells (Wilcoxon test, numbers p=0.021, proportions p=0.032). Differential gene expression analysis revealed concordant higher expression of human leukocyte antigen G (HLA-G), an NK cell inhibitory ligand, in B2M-LOH tumors (Wilcoxon test, p=0.0035). Moreover, B2M-LOH progressive tumors had the highest median HLA-G expression and trended toward a positive correlation between increasing HLA-G expression and increasing numbers of activated NK cells (Pearson correlation, r=0.55, p=0.015). In conclusion, melanoma biopsies with B2M LOH were associated with non-response to ICB, had increased presence of activated NK cells, and demonstrated a compensatory increase in HLA-G. These results suggest that in melanoma B2M alterations may be implicated in modulating NK cell interactions and point toward HLA-G potentially playing a role in inhibiting NK cell anti-tumor activity.

Citation Format: Mildred Galvez, Katie M. Campbell, Egmidio Medina, Antoni Ribas. B2M loss of heterozygosity in melanoma is associated with increased numbers of activated NK cells and higher expression of the NK cell inhibitory ligand, HLA-G [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5965.
Abstract 5966: Cathepsin S mediates interleukin 7 to sculpture the tumor immunity in head and neck cancer
Authors: Chang Yung-Chieh , Chen Shan-Hung , Chang Kwang-Yu , Chang Jang-Yang
Published: April 2023

Cathepsin S (CTSS) is a lysosomal enzyme that belongs to cysteine proteases. It is associated with progression of different types of cancer, mediating the antigen presentation, extracellular matrix degradation, angiogenesis, and metastasis. In recent studies, CTSS was suggested as a therapeutic target for cancer treatment. We found that, with a CTSS inhibitor RJW-58 treatment over tumor-bearing mouse, the serum showed an enhanced anti-tumor cytokine releasing of interleukin 7 (IL-7). In the mRNA array results of 40 pair head and neck patients’ sample, we found the negative correlation between CTSS and IL-7 comparing to the normal head and neck tissue. Moreover, we also found that the level of CD8 T cell infiltration is negatively correlated with the expression of CTSS in 20 head and neck cancer patients’ slides. In cell model, targeting CTSS by siRNA or RJW-58 promoted IL-7 secretion in different head and neck cancer cell lines. We demonstrated that cathepsin S decreased the secretion level of IL-7 through downregulating IL-7 receptor, a receptor that mediating IL-7 secretion, protein expression level resulting in activating lysosomal degradation pathway. Promoting cell proliferation of activated CD8 T cells was proved when CD8 T cells were treated with the condition medium from CTSS-knockdown head and neck cancer cells. In conclusion, this is the first study demonstrated that CTSS acted as a negative regulator in mediating IL-7 resulting in inducing an immune cell-suppressed microenvironment in head and neck cancer cells. (This work is supported by the NSTC grand number: MOST 110-2314-B-400-035)

Citation Format: Yung-Chieh Chang, Shan-Hung Chen, Kwang-Yu Chang, Jang-Yang Chang. Cathepsin S mediates interleukin 7 to sculpture the tumor immunity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5966.
Abstract 5972: CDK1 bridges NF-kB and b-catenin signaling in response to H. pylori infection in gastric tumorigenesis
Authors: Al-Mathkour Marwah M. , Zhu Shoumin , Cao Longlong , Khalafi Shayan , Chen Zheng , Poveda Julio , Peng Dunfa , Lu Heng , Soutto Mohammed , Hu Tianling , McDonalnd Oliver , Zaika Alexander , El-Rifai Wael
Published: April 2023

Background: Cell cycle dysregulation is a hallmark of cancer, resulting in unregulated cell proliferation and, eventually, tumor development. Cyclin-dependent kinase 1 (CDK1) is a cell cycle regulatory protein that is involved in cell cycle maintenance. CDK1 has been discovered to be substantially elevated in a several tumors and is linked to poor overall and relapse-free survival. The aim of this study is to understand the regulation role of Helicobacter Pylori infection and inflammation on CDK1 expression in gastric cancer.

Methods: Using TCGA data and our integrated comprehensive gene expression analysis, we found a significant overexpression of CDK1 in gastric cancer human and mouse tissues. We detected overexpression of CDK1 in human and mouse gastric glands in response to H. pylori infection. Our data demonstrated that H. pylori infection induced phosphorylation (S536) and activation of NF-kB in vitro and in vivo. Furthermore, H. pylori infection and TNF-α treatment increased the CDK1 mRNA and protein levels in gastric cancer cell lines. Using the ChIP assay, we detected direct biding of NF-κB on the CDK1 promoter regulating its transcription. CDK1 promoted activation of the β-catenin signaling pathway. Using the pTOP/pFOP luciferase reporter assays, as a measure of β-catenin/TCF transcription activity, we confirmed CDK1-dependent activation of β-catenin in response to H. pylori infection. Pharmacologic and genetic inhibition of CDK1 reversed these effects and decreased number and size of gastric tumors organoid from mouse and human.

Conclusion: Our findings demonstrate NF-kB-mediated induction of CDK1 expression in response to H. pylori infection with subsequent activation of tumorigenic β-catenin signaling. This novel regulatory link between infection, CDK1, and β-catenin suggests the importance of considering CDK1 inhibitors in gastric cancer.

Citation Format: Marwah M. Al-Mathkour, Shoumin Zhu, Longlong Cao, Shayan Khalafi, Zheng Chen, Julio Poveda, Dunfa Peng, Heng Lu, Mohammed Soutto, Tianling Hu, Oliver McDonalnd, Alexander Zaika, Wael El-Rifai. CDK1 bridges NF-kB and b-catenin signaling in response to H. pylori infection in gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5972.
Abstract 6085: Molecular profile by next generation sequencing in papillary thyroid carcinoma, Colombian Cohort at Fundacion Santa fe de Bogota University Hospital
Authors: Rodriguez Paula , González Sebastián , Cruz Sergio , Juez Yesith , Becerra David , Baldión Margarita , Perdomo Sandra , Beltran Angela , Hakim Jose , Gonzales Deyanira
Published: April 2023

Objective/aim: To describe the main molecular alterations in a Colombian Cohort of patients diagnosed with Papillary Thyroid Carcinoma (PTC) at Fundacion Santa Fe de Bogota University Hospital between 1993 and 2011

Methods: A total of 231 surgically intervened patients at our institution and diagnosed with PTC with available paraffin blocks were analysed. DNA and RNA extraction was performed from FFPE samples using Quick-DNA & RNA FFPE MiniPrep Kit (Zymo Research). Next Generation Sequencing analysis was performed using SOPHiA Solid Tumor Solutions Kit (SOPHiA GENETICS, Saint Sulpice, Suiza), which detects 42 DNA genes and 137 fusion genes with the platform MiSeq System (Illlumina, San Diego, California). Data underwent QA and QC and variants were analysed through Sophia commercial pipeline for somatic aberrations.

Results: Among 231 patients, 82% were women, average age 45 +/- 13 y-o, 97% from Colombia, with institutional pathologic diagnosis of PTC. The most frequent histological subtypes were Classic PTG (33%), Follicular Infiltrative (29%) and Tall cell (23%). Identification of clinically relevant molecular alterations was done (see table 1). The most frequent SNV mutations were BRAF-V600E (72%), TP53 (9%) and IDH1-R132H (8%). The most frequent insertions or deletions (INDELS) were found in KIT (75%). We identified 8 fusion genes (most frequent were RET-CCD6 and ETV6-NTRK3). Interestingly, we detected 3 MSI positive cases.

Conclusions: PTC molecular profile by NGS can allow us to improve molecular understanding of the disease and identify possible prognostic factors and treatment targets.

Table 1. Molecular alterations detected by next-generation sequencing in Papillary carcinoma Molecular alterations Gene n % INDEL (n=20) KIT 15 75,0 NRAS 1 5,0 PTPN11 1 5,0 TP53 3 15 SNV (n=226) BRAF 163 72,1 FGFR1 1 0,4 HRAS 2 0,9 IDH1 19 8,4 KIT 2 0,9 KRAS 2 0,9 NRAS 9 4,0 PDGFRA 1 0,4 PIK3CA 3 1,3 SMAD4 4 1,8 TP53 20 8,8 Fusion genes (n=6) CCDC6-RET 2 25,0 ETV6-NTRK3 2 25,0 GOLGB1-TACC3 1 12,5 MYH14-BICC1 2 25,0 GOPC-RET 1 12,5 MSI (n=231) MSI-L 3 1,3 Negative 228 98,7

Citation Format: Paula Rodriguez, Sebastián González, Sergio Cruz, Yesith Juez, David Becerra, Margarita Baldión, Sandra Perdomo, Angela Beltran, Jose Hakim, Deyanira Gonzales. Molecular profile by next generation sequencing in papillary thyroid carcinoma, Colombian Cohort at Fundacion Santa fe de Bogota University Hospital [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6085.
Abstract 6227: Tumor cell heterogeneity may cause treatment failure of GPA33-targeted therapy in colorectal cancer
Authors: Börding Teresa , Janik Tobias , Morkel Markus , Sers Christine , Horst David
Published: April 2023

Purpose: Glycoprotein A33 (GPA33) is a membrane protein almost exclusively expressed in intestinal epithelia and in more than 95% of colorectal cancers (CRCs). This renders GPA33 a promising antigen for targeted therapy, but trials did not meet pre-specified response rates. We investigated intratumoral heterogeneity as a reason for low treatment efficacy, GPA33 regulation and the potential for pharmacological induction of GPA33 expression in vitro and in vivo to enhance treatment response.

Methods: We assessed intratumoral heterogeneity of GPA33 in tissue specimens of 223 stage II CRCs using immunohistochemistry and immunofluorescence, and obtained scRNAseq transcriptomic analysis from 12 primary CRCs. We then manipulated WNT and PPARγ signaling pathways and transcription factor expression in colon cancer cells using specific inhibitors, siRNAs or inducible dominant negative TCF4 (dnTCF4), and analyzed the effects on GPA33 protein and RNA expression. Furthermore, we investigated the effects of WNT inhibition on GPA33 in vivo in NOD/SCID xenografts. Additionally, we developed anti-GPA33 CAR-T cells and measured their activation after co-cultivation with colon cancer cells using flow cytometry.

Results: In the CRC cohort we found that GPA33 expressing cells had a differentiated phenotype and low WNT activity. GPA33-negative subpopulations were located at the infiltrative tumor edge and showed increased nuclear β-catenin. Downregulation of WNT pathway activity using inhibitors, siRNAs or inducible dominant negative TCF4 (dnTCF4), as well as activation of PPARγ signaling induced GPA33 expression in vitro. Forced expression of dnTCF4 also enhanced GPA33 expression in vivo in previously GPA33-negative tumor cell subpopulations in colon cancer xenografts. siRNA mediated knockdown of CDX1 and KLF4, two transcription factors that were previously implicated in GPA33 regulation, only moderately reduced GPA33 expression levels in vitro. Anti-GPA33 CAR-T cells activated in response to co-culture with GPA33-expressing CRC cells in vitro.

Conclusion: Our analysis highlights a heterogenous expression of GPA33 in vivo as a limiting factor for anti-GPA33 therapy. WNT inhibition or PPARγ activation induced GPA33 expression in vitro and in vivo. Depletion of CDX1 and KLF4 had moderate effects on GPA33 only, indicating a complex regulation in colon cancer. We suggest that pharmacological induction of GPA33 expression may increase the efficacy of anti-GPA33 targeted therapy in patients with CRC.

Citation Format: Teresa Börding, Tobias Janik, Markus Morkel, Christine Sers, David Horst. Tumor cell heterogeneity may cause treatment failure of GPA33-targeted therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6227.
Abstract 6232: Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma
Authors: Hwang Jae Ryoung , Cho Young-Jae , Ryu Ji-Yoon , Choi Ju-Yeon , Choi Jung-Joo , Lee Jeong-Won
Published: April 2023

Purpose: Ulipristal acetate (UPA) is used for the treatment of uterine leiomyoma as a selective progesterone receptor modulator. Until now, there has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models.

Experimental Design: The cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1) by MTT and caspase-3 activity assays. A migration assay was done using a Transwell chamber, and MMP-2 was measured with its specific ELISA kit. Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. The in vivo efficacy of UPA was examined in uterine sarcoma cell line- and patient-derived xenograft mice models.

Results: UPA inhibited cell growth and increased apoptosis in cell lines including uterine sarcoma (MES-SA) and leiomyosarcoma (SK-UT-1 and SK-LMS-1) in a dose-dependent manner. UPA also inhibited cell migration and the expression of MMP-2. cDNA microarray analysis revealed that CCL2 was commonly down-regulated by UPA in investigated cell lines. The UPA-mediated inhibition of CCL2 expression was confirmed by ELISA. The phosphorylation and the total expression of STAT3, downstream from the CCL2 signaling pathway, was also inhibited by UPA. We also found that UPA reduced cell growth and increased caspase-3 activity in primary culture cells from leiomyosarcoma patient-derived xenografts (PDX-C). Moreover, we found that STAT3/CCL2 inhibition by UPA was independent of the expression of progesterone receptor. In vivo studies with MES-SA and SK-UT-1 cell line-derived xenografts and a PDX model with leiomyosarcoma demonstrated that UPA significantly decreased tumor growth.

Conclusions: UPA had significant anti-tumor effects in uterine sarcoma through the STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease.

Citation Format: Jae Ryoung Hwang, Young-Jae Cho, Ji-Yoon Ryu, Ju-Yeon Choi, Jung-Joo Choi, Jeong-Won Lee. Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6232.
Abstract 6753: Non-HIV cryptococcal patients with hematological malignancies: A retrospective analysis of a ten-year study from a Peruvian oncologic center
Authors: Meza Kelly , Cuellar Luis E.
Published: April 2023

Background Globally, cryptococcal infection has become a public health challenge. However, relatively little is known about cryptococcal infection in people with hematological disorders.

Methods We developed a retrospective cohort study that includes hematological patients who had cryptococcosis infection seen in the Instituto Nacional de Enfermedades Neoplasicas between 2007 and 2017.

Results During the 11-year study period, a total of 21 cases of cryptococcosis (incidence rate of 13,4 per 1000 hematological malignancies). The time of diagnosis of cryptococcosis since hematologic malignancy diagnosis was 42 (18-161) days. Non-Hodgkin lymphoma (NHL) was the most frequent malignancy (47.6%), followed by leukemias (23.8%), multiple myeloma in (19.1%), and HL (9.5%). Three cases of adult T-cell leukemia/lymphoma associated with HTLV-1 infection were identified in the NHL. Cryptococcemia was the most frequent diagnosis (85.7%) followed by pulmonary (52.4%) and CNS (38.1%) involvement. Regarding clinical presentation, f ever was the most common symptom in meningeal cryptococcosis (76.2%) and dyspnea in pulmonary cryptococcosis (61.9%). The use of immunosuppressant chemotherapy within a month before the diagnosis of cryptococcosis was the most common risk factor. The main characteristics between patients with cryptococcosis and type of neoplasms (NHL vs other hematological neoplasms) such as age at admission, site of presentation of cryptococcosis, and lymphocyte count at admission were different significantly between these groups. In the NHL group, there was less involvement of the lungs. Overall survival (OS) in 15 and 120 days were 70% (95%CI: 0.43-0.86) and 51% (95%CI: 0.26-0.72), respectively. OS for patients with NHL was 57% at 15 days (95% CI: 0.17-0.83) and 42% at 120 days (95%CI:0.09-0.73). OS for other hematological malignancies was 90% at 15 days (95%CI:0.47-0.98) and 60% at 120 days (95%CI: 0.25-0.82).

Conclusions CNS cryptococcal infection was more frequent in patients with NHL.

Citation Format: Kelly Meza, Luis E. Cuellar. Non-HIV cryptococcal patients with hematological malignancies: A retrospective analysis of a ten-year study from a Peruvian oncologic center. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6753.