bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3011: Development of mitochondrial fission inhibitors in inducing non-apoptotic cell death in triple-negative breast cancer
Authors: Malla Saloni , Kumari Shikha , Terrero David , Raman Dayanidhi , Tiwari Amit K.
Published: June 2022

Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Presently used neoadjuvant chemotherapy against TNBC, such as taxanes, and/or anthracyclines show an initial treatment response but follows with drug resistance, tumor relapse, side effects and high proclivity to develop metastases. Recent studies point out that targeting dysregulated mitochondrial dynamics, that is now linked to the tumor initiation, progression, and metastasis in TNBC, as an effective therapeutic strategy. In particular, mitochondrial fission factors such as dynamin-related protein 1 (Drp1) is upregulated in TNBC and is known to affect mitochondrial energetics and alter metabolism which favors tumor progression. We have designed and developed small molecule inhibitors to specifically target Drp1 as a viable therapeutic strategy for metastatic TNBC (mTNBC). We recently identified a new class of structurally constrained thienopyridine (TPH) probes, and particularly a new analog TPH104c that showed higher binding affinity in silico to Drp1 as compared to Mdivi-1, a weak inhibitor of Drp1. Surface plasmon resonance studies suggest that TPH104c binds with Drp1 with high affinity. Similarly, mechanistic studies using Western blotting and immunofluorescence analysis confirmed that TPH104c significantly downregulated the levels of total and phosphorylated form of Drp1 at serine 616 in MDAMB-231 and BT-20 TNBC cells. Interestingly, Drp1 inhibition by TPH104c led to a hitherto unreported form of caspase- and reactive oxygen species (ROS)-independent non-apoptotic form of cell death. Rather than shrinking in size, or formation of apoptotic blebs which are classical features of apoptosis, TPH104c treated TNBC cells were enlarged, rounded, and swollen in size. These cells eventually lost their proliferative ability and were significantly less confluent than control cells. Additionally, in TPH104c-treated cells, neither the mitochondrial membrane potential was lost, nor the caspases were activated as also indicated by a lack of rescue of cell death when incubated with pan-caspase inhibitor. Further studies are in progress to understand the role of Drp1 inhibition in the induction of non-apoptotic cell death which will further help to optimize an effective lead compound for the future pre-clinical development of promising anticancer agents targeting mTNBC.

Citation Format: Saloni Malla, Shikha Kumari, David Terrero, Dayanidhi Raman, Amit K. Tiwari. Development of mitochondrial fission inhibitors in inducing non-apoptotic cell death in triple-negative breast 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 3011.
Abstract 3053: Using individual or a consortium of bacteria for immuno-oncology research
Authors: Maubant Sylvie , Leblanc Marie , Bertrand Elisabeth , Bertaux Audrey , Morgand Loic , Ramelet Maxime , Lux Marie , Duchamp Olivier , Vivani Fabrice
Published: June 2022

Preclinical and clinical studies have shed light on the beneficial role of bacteria for cancer therapy. Indeed, these studies have demonstrated that these microorganisms have beneficial properties that allow them to selectively colonize tumor and that they could also be considered as predictive drug efficacy biomarkers. Based on these results, bacteria are now used for delivering therapeutic components or for shaping the gut microbiota. Ultimately these approaches lead to the activation of an immune response against the tumor. Owing to our scientific and technological expertise in manipulating microbes, we propose tailor-made strategies for investigating the efficacy of bacteria-based treatments and/or the effect of therapies on the microbiota both in vitro and in vivo. Different methods/analyses can be used for culturing, detecting, quantifying, identifying and localizing live bacteria (e.g. counting of CFUs, PMA-qPCR, 16 rRNA gene sequencing, mass spectrometry, bioluminescence) in simple or complex samples (e.g. from culture of single bacterial species to rodent/human stools or other tissues). In addition, a continuum of assays allows us to evaluate the impact of bacteria or derived products directly on tumor and/or immune cells (e.g. immune infiltrate and phenotyping, cytokine/chemokine profiling, tumor burden). We will highlight some results obtained in a cancer context such as the immunostimulatory properties of bacteria or their derivatives, the selective colonization of tumor tissue by bacteria, the benefits in delivery of therapeutic proteins or antigens by bacteria, the impact of tumor engraftment/growth on gut microbiota, the effects of chemotherapeutic agents on intestinal microflora, the consequences of supplementation with bacteria or antibiotics treatment on the response to immune checkpoint inhibitors. Altogether these data demonstrate that bacteria are now allies in the treatment of cancer and that our comprehensive platform is suitable for evaluating both in vitro and in vivo therapeutics developed from bacteria (individuals, consortium or derived products; native or modified). All these technologies can be also applied to develop novel therapeutic strategies for inflammatory and infectious diseases known to increase the risk of cancer development.

Citation Format: Sylvie Maubant, Marie Leblanc, Elisabeth Bertrand, Audrey Bertaux, Loic Morgand, Maxime Ramelet, Marie Lux, Olivier Duchamp, Fabrice Vivani. Using individual or a consortium of bacteria for immuno-oncology research [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 3053.
Abstract 3156: Tumor Treating Fields reduce cellular survival of human mesenchymal stromal cells via apoptosis and senescence induction
Authors: Rühle Alexander , Strack Maren , Grosu Anca-Ligia , Nicolay Nils H.
Published: June 2022

Background Tumor Treating Fields (TTFields), alternating electric fields with intermediate field intensity, have shown clinical efficacy in several tumor entities, leading to clinical approval for glioblastoma and mesothelioma treatment. As mesenchymal stromal cells (MSCs) belong to the tumor microenvironment and exhibit - depending on the tumor type and microenvironment - either pro-tumorigenic or anti-tumorigenic abilities, we aimed to study the effects of TTFields on human MSCs.

Methods Human MSCs were isolated from the bone marrow of three different healthy donors and were exposed to TTFields for 72 hours. MSCs’ clonogenic survival, proliferation and viability was analyzed after different TTFields frequencies. Boyden chamber assays were carried out to investigate the influence of TTFields on the migratory potential of MSCs. Flow cytometry analyses of caspase-3 and annexin V were conducted to determine MSCs’ apoptosis rates after TTFields treatment. TTFields-induced senescence was quantified using beta-galactosidase stainings. MSCs’ characteristic surface marker expression (CD73, CD90 and CD105) was studied after TTFields treatment.

Result TTFields led to significantly reduced clonogenic survival (70-90% reduction compared to controls) and cellular proliferation (approximately 50% reduction) in all analyzed MSC specimens. However, MSCs’ metabolic viability was largely unaffected after TTFields exposure. Within a range between 100 and 500 kHz, there was no frequency dependence of TTFields’ anti-proliferative effect on human MSCs. As determined by Bodyen Chamber assays, TTFields resulted in a considerable reduction of MSCs’ migratory potential. TTFields increased both cellular apoptosis and senescence in all MSC samples, whereas there was no alteration of MSCs’ characteristic surface marker pattern.

Conclusion As TTFields considerably reduce MSCs’ survival and migratory potential, TTFields could attenuate the pro-tumorigenic effects of tumor-associated MSCs that were recently described in glioblastoma patients. Co-culture experiments of MSCs and tumor cells after TTFields treatment as well as in vivo experiments are planned in the future.

Citation Format: Alexander Rühle, Maren Strack, Anca-Ligia Grosu, Nils H. Nicolay. Tumor Treating Fields reduce cellular survival of human mesenchymal stromal cells via apoptosis and senescence induction [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 3156.
Abstract 3259: TAS3351 is a 4th-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with EGFR common mutations
Authors: Kasuga Hidefumi , Kataoka Yuki , Yamamoto Fuyuki , Mizutani Takashi , Tsuji Shingo , Tanaka Sakiho , Mizuarai Shinji
Published: June 2022

Background: Common mutations (exon 19 deletion; ex19del or L858R point mutation; L858R) of the epidermal growth factor receptor (EGFR) are found in patients with non-small cell lung cancer (NSCLC) and are sensitive to EGFR-tyrosine kinase inhibitors (TKIs), including osimertinib, a third-generation, irreversible EGFR-TKI. Although osimertinib is widely utilized as the standard of care in first-line therapy of NSCLC, resistance inevitably occurs during the treatment. Among EGFR-dependent/on-target resistant mechanisms of osimertinib, the C797S mutation is reported to emerge as the most frequent alteration. The cysteine residue at position 797 of EGFR is covalently bound with osimertinib, and its alteration to serine causes an inability to form a covalent bond, making osimertinib resistant to NSCLC with a C797S mutation. While first-generation, reversible EGFR-TKIs (gefitinib and erlotinib) might be effective for patients with C797S mutation, the T790M mutation is reported to emerge in almost half of patients with EGFR common mutations during treatment with first-generation EGFR-TKIs. Here, we report TAS3351 as a novel, next-generation EGFR-TKI designed to overcome C797S and T790M-mediated resistance in NSCLC patients.

Methods: The inhibitory potency of TAS3351 on cellular phospho-EGFR was analyzed by In-cell western assay. In addition, inhibition of cell viability in Ba/F3 cells transformed by human wild type or mutated EGFR, as well as in human NSCLC cell lines whose EGFR harbors common mutations, was tested. Anti-tumor activity of TAS3351 was evaluated in NIH/3T3-EGFR allograft models and human NSCLC xenograft models.

Results: TAS3351 exhibited almost comparable inhibitory potency against cellular phosphorylation of EGFR harboring ex19del or L858R with or without C797S and/or T790M while sparing wild type EGFR activity. TAS3351 also showed similar trends in the cell viability assay for Ba/F3-EGFR cells and human NSCLC cell lines. Finally, TAS3351 demonstrated favorable anti-tumor activity in NIH/3T3 allografts transformed by human EGFR harboring ex19del/C797S/T790M.

Conclusion: TAS3351 is a fourth-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with common mutations. The present results support the clinical development of TAS3351 as monotherapy in NSCLC patients with EGFR mutations harboring C797S and/or T790M EGFR-TKI resistance mutations.

Citation Format: Hidefumi Kasuga, Yuki Kataoka, Fuyuki Yamamoto, Takashi Mizutani, Shingo Tsuji, Sakiho Tanaka, Shinji Mizuarai. TAS3351 is a 4th-generation EGFR-TKI overcoming T790M and C797S-mediated resistance in NSCLC with EGFR common mutations [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 3259.
Abstract 3287: Baseline CD166 expression determines CRISPR-mediated ALCAM gene knockout effects on migratory phenotype in metastatic melanoma cells
Authors: Kaminski Rafal , Luongo Francesca P. , Dabrowski Konrad , Bellizzi Anna , Mancuso Pietro , Zaidi M. Raza , McMillan Johanna , Patel Meha , Coleman Tyrone , Arosarena Oneida A.
Published: June 2022

Advanced stage melanoma is a mostly incurable, highly aggressive tumor whose incidence is rising. Metastatic dissemination of primary tumors involves the repeated crossing of tissues barriers, which requires dynamic modulation of tumor cell adhesion, presumably through mechanisms like those utilized by trafficking leukocytes. Activated leukocyte cell adhesion molecule (ALCAM/CD166) has been shown to play an important role in the metastatic process, including detachment from the primary tumor, adhesion to endothelium, and extravasation. ALCAM expression correlates with melanoma progression, and it has been identified as a marker of pluripotent stem cells. Furthermore, its expression in primary melanoma strongly correlates with tumor thickness and level of invasiveness. shRNA silencing of ALCAM in cells with high levels of ALCAM resulted in reduced motility and transmigration. In the current study, we tested the effect of CRISPR-mediated ALCAM knockout (KO) on migration and invasion of two human melanoma cell lines representing opposite phenotypic states of melanoma. MEL501 (proliferative type, SOX10/MITF-high and AP1/TEAD1-low) cells express low levels of ALCAM at baseline, while the Rosi cell line (invasive type, SOX10/MITF-low and AP1/TEAD1-high) expresses high baseline ALCAM levels. ALCAM KO promoted migration of MEL501 cells (p<0.000001) but inhibited migration of Rosi cells (p=0.005050) as compared to the parent cell lines. In addition, ALCAM KO promoted the invasion of MEL501 cells (p=0.019768) but had no impact on Rosi cell invasion. While ALCAM has been shown to regulate matrix metalloproteinase-2 (MMP-2) activation, zymograms revealed no difference in MMP-2 activity between MEL501 and Rosi ALCAM-KO cells and the parent cell lines. These data indicate that ALCAM may play a tumor-suppressive role in melanoma cells with low ALCAM expression and are consistent with a previous study which demonstrated that introduction of an amino-terminally truncated ALCAM in early stage melanoma cells promoted metastasis. Our data also provide in vitro evidence that gene therapy may reverse the migratory phenotype of melanoma cells with high ALCAM expression.

Citation Format: Rafal Kaminski, Francesca P. Luongo, Konrad Dabrowski, Anna Bellizzi, Pietro Mancuso, M. Raza Zaidi, Johanna McMillan, Meha Patel, Tyrone Coleman, Oneida A. Arosarena. Baseline CD166 expression determines CRISPR-mediated ALCAM gene knockout effects on migratory phenotype in metastatic melanoma 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 3287.
Abstract 3291: Anti-HER2 scFv antibody-directed lentiviral delivery of myrosinase for eradicating HER2+ cancer cells after dosing prodrug sinigrin
Authors: Tarar Ammar Ahmad , Peng Ching-An
Published: June 2022

Chemotherapy is widely used for the treatment of cancers, but it lacks selectivity and therefore results in collateral damage to healthy cells and drug resistance of cancer cells. Gene-directed enzyme prodrug therapy (GDEPT) has been adopted to mitigate these issues. In this study, we demonstrated the specific targeting of myrosinase-sinigrin paired GDEPT to treat HER2+ (human epidermal growth factor 2) cancer cells. Sinigrin excessively present in cruciferous vegetables can be catalyzed by myrosinase to produce cytotoxic ally isothiocyanate (AITC), which has shown anticancer effectiveness. The myrosinase gene was packed in biotinylated lentiviral vectors produced by co-transfection of HEK293T cells. The single chain variable fragment (scFv) gene sequence of anti-HER2 antibody and core streptavidin (coreSA) gene sequence were cloned in pET-30a(+) plasmid, which was then transformed in Lemo21 (DE3) competent cells for protein expression. The anti-HER2-coreSA chimeric protein was purified using immobilized metal affinity chromatography and characterized by SDS-PAGE and Western blot. The purified chimeric protein was tagged on biotinylated lentivirus via streptavidin-biotin binding. Our results showed that myrosinase-encoding lentivirus tagged with anti-HER2 selectively delivered myrosinase to HER2+ cancer cells. When treated with prodrug sinigrin, the targeted cancer cells underwent apoptosis and ensuing cell death. In summary, the targeted delivery of myrosinase to HER2+ cancer cells using lentivirus functionalized with anti-HER2 ScFv antibody can eradicate malignant cells when treated with enzymatically catalyzed prodrug sinigrin.

Citation Format: Ammar Ahmad Tarar, Ching-An Peng. Anti-HER2 scFv antibody-directed lentiviral delivery of myrosinase for eradicating HER2+ cancer cells after dosing prodrug sinigrin [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 3291.
Abstract 3298: Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse melanoma
Authors: Purde Mette-Triin , Hartmann Fabienne , Cupovic Jovana , Schmidt Sarah , Bomze David , Stemeseder Felix , Lercher Alexander , Besse Lenka , Berner Fiamma , Tüting Thomas , Bergthaler Andreas , Schietinger Andrea , Kochanek Stefan , Ludewig Burkhard , Orlinger Klaus K. , Bald Tobias , Ring Sandra S. , Flatz Lukas
Published: June 2022

Breaking self-tolerance and inducing a robust immune response targeting tumor cell derived self-antigens by therapeutic vaccines has remained challenging. We have generated a replicating viral vector based on the arenavirus lymphocytic choriomeningitis virus expressing the melanoma tissue-specific self-antigen Trp2 (artLCMV-Trp2) and show that a single immunization of mice bearing established melanoma with this vector can break tolerance against this self antigen and induces high levels of tumor-infiltrating TRP2+ CD8+ T cells. These T cells express the effector cytokines IFNγ and TNFα, lack markers of T cell exhaustion, and their appearance coincides with marked inhibition of tumor growth and significant survival benefit. artLCMV-TRP2 induced a strong type I interferon response that resulted in transient type I-dependent Treg inhibition that was necessary for the generation of optimal antitumoral T cell responses. Moreover, artLCMV-TRP2 vaccination proved able to induce complete tumor remission when combined with infusion of exogenous TRP2-specific T cells into mice. In conclusion, replication competence is a key property of artLCMV-TRP2 that enabled effective anti-cancer immunity and therapeutic effect.

Citation Format: Mette-Triin Purde, Fabienne Hartmann, Jovana Cupovic, Sarah Schmidt, David Bomze, Felix Stemeseder, Alexander Lercher, Lenka Besse, Fiamma Berner, Thomas Tüting, Andreas Bergthaler, Andrea Schietinger, Stefan Kochanek, Burkhard Ludewig, Klaus K. Orlinger, Tobias Bald, Sandra S. Ring, Lukas Flatz. Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse 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 3298.
Abstract 3330: GB5121 is a novel highly potent and selective CNS-penetrant BTK inhibitor for CNS malignancies
Authors: Yusuf Isharat , Sahdeo Sunil , Ismaili Taylor , Naiman Zachary , Guimond David , Schiff Theodore , Clemons Bryan , Herman Krystal , Paulson Bryanna , Hou Kay , Meadows Kristen Taylor , Kennedy Michael , Rose Mark , Carter Laura
Published: June 2022

Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) penetrance in instances of CNS disease. GB5121 is an orally available, selective, irreversible small molecule BTK inhibitor. GB5121 was selected based on its potency, specificity, and high brain penetrance in preclinical models. We evaluated its pharmacodynamic properties using both cell-free enzymatic and cell-based functional assays, showing covalent BTK inactivation with a potency in the nM range. GB5121 furthermore demonstrated rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissue, a critical parameter for covalent irreversible inhibitors. In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating >50% inhibition at 1 µM; GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. When compared with other BTK inhibitors, GB5121 showed superior CNS target occupancy using a probe-based ELISA measuring free BTK in the brain of mice receiving 3 daily oral doses of 10 mg/kg. GB5121 also demonstrated significantly higher brain to plasma ratio in mice with an intact blood brain barrier. Specifically, GB5121 showed ~2-fold higher brain concentrations with lower plasma levels than ibrutinib. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses. Together, these features differentiate GB5121 from both FDA-approved BTK inhibitors as well as those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including in CNS lymphoma.

Citation Format: Isharat Yusuf, Sunil Sahdeo, Taylor Ismaili, Zachary Naiman, David Guimond, Theodore Schiff, Bryan Clemons, Krystal Herman, Bryanna Paulson, Kay Hou, Kristen Taylor Meadows, Michael Kennedy, Mark Rose, Laura Carter. GB5121 is a novel highly potent and selective CNS-penetrant BTK inhibitor for CNS malignancies [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 3330.
Abstract 3359: Association of Hippo signaling pathway in acute myeloid leukemia
Authors: Jang Heejun , Lee Sungyoung , Yun Hongseok , Koh Yongil , Yoon Sung-Soo
Published: June 2022

The Hippo signaling pathway, also known as the Salvador-Warts-Hippo pathway, is a well-known oncogenic signaling pathway associated with organ size regulation, cell proliferation, and apoptosis. In this study, we analyzed the association of Hippo signaling pathway genes in patients with acute myeloid leukemia (AML) using whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data. We used Seoul National University Hospital (SNUH) AML patient data (n=16) collected from December 2015 to January 2019 for analysis. Additionally, public datasets of AML patients, TCGA-LAML (n=179) and LeuceGene (n=110) were used. we performed hierarchical clustering using Hippo pathway gene expression from AML patient samples. As a result, we discovered a gene expression signature in which the expressions of tumor suppressor genes among the Hippo pathway genes were significantly decreased. We divided AML patients into the case group and the control group according to the presence or absence of the corresponding gene expression signature and attempted to identify differences in biological features between the two groups. First, in the process of comparing differences in genetic alteration, it was confirmed that the frequency of PML-RARA fusion was significantly higher in the case group. We further analyzed the data of the AML patients in the case group without PML-RARA fusion to discover other biological features. To discover the correlation between the Hippo pathway and other oncogenic signaling pathways on gene expression level, we performed a correlation analysis. As a result, it was confirmed that the expression of the Hippo pathway gene showed a high correlation with the gene expression of the Notch signaling pathway and the Myc signaling pathway in the patients of the case group. In addition, we performed DEGs analysis to find new therapeutic target candidate genes from the case group. As a result, we identified a significantly upregulated gene, LAMP5.In conclusion, we identified the gene expression signatures of the Hippo signaling pathway in AML patients. Also, we discovered a correlation between the Hippo pathway and other signaling pathways and suggested potential therapeutic candidate genes using gene expression signatures of the Hippo signaling pathway genes. And we will perform further analysis about validating therapeutic candidate genes related to the Hippo signaling pathway gene expression signature.

Citation Format: Heejun Jang, Sungyoung Lee, Hongseok Yun, Yongil Koh, Sung-Soo Yoon. Association of Hippo signaling pathway in acute myeloid leukemia [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 3359.
Abstract 3368: Biomarkers of precancerous colorectal cancer stages identified by transcriptome profiling
Authors: Siskova Anna , Vymetalkova Veronika , Vodicka Pavel , Ferrero Giulio , Naccarati Alessio , Hampel Chuanpit , Schneider-Stock Regine
Published: June 2022

A colorectal adenoma, an aberrantly growing tissue that arises from the intestinal epithelium is considered a precursor of colorectal cancer, the third most common cancer worldwide. In the presented study, we performed a whole transcriptome analysis of 18 fresh frozen colorectal adenomas and their adjacent mucosa from the Czech and Italian populations. Samples were matched for histology and sex. The libraries were sequenced through NovaSeq6000 with a paired-end approach (2 × 150 bp) and with 100 million reads per sample. The enrichment analysis showed that the most downregulated pathways belonged to immune responses and complement activation while the most upregulated pathways were enriched predominantly in proliferation and development-related Gene Ontology biological processes. In total 2,753 differentially expressed genes (DEGs) coherent between these two populations were identified. To determinate, the subset of discriminant DEGs for colorectal adenomas, the output of seven feature selection methods was combined highlighting a total of 321 DEGs scored at the top 95% of at least one method. Further from this set, 8 candidate genes AXIN2, ETV4, RNF43, CXCL12, FBLN1, GNG7, ZEB2, and SFRP1 were selected for validation on a larger population as well as for functional in vitro assays. The results will be presented at the annual meeting. We believe that the study could provide new candidate biomarkers for precancerous colorectal cancer stages. Acknowledgement: This study was financed by NV18-03-00199.

Citation Format: Anna Siskova, Veronika Vymetalkova, Pavel Vodicka, Giulio Ferrero, Alessio Naccarati, Chuanpit Hampel, Regine Schneider-Stock. Biomarkers of precancerous colorectal cancer stages identified by transcriptome profiling [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 3368.
Abstract 3386: Detection of TGFbR2 frameshift mutation
Authors: Eriksen Henrik K. , Eriksen Jon A.
Published: June 2022

TGFβR2 a10→a9 frameshift mutation is an early occurring cancer driver mutation and mutant TGFβR2 cell free DNA (cfDNA) is therefore a potential biomarker for early detection of cancer (E.G. Lynch Syndrome) as well as for monitoring response to cancer treatment. The frameshift mutation is associated with micro satellite instability (MSI) and is found in 80% of MSI-colorectal cancers (MSI-CRC) and in over 90% of hereditary nonpolyposis colorectal cancer. Here we show that cell free mutant (a9) TGFβR2 DNA is present in liquid biopsies from MSI-CRC patients and that the a9 micro satellite of mutant TGFβR2 can be selectively detected by using specially designed PCR primers modelled after the a9 microsatellite. PCR and primers defining a fragment of TGFβR2 comprising the a10/a9 microsatellite was used to amplify cfDNA extracted from MSI-CRC and microsatellite stable-CRC (MSS-CRC) patients. The resulting PCR products were analysed and confirmed by DNA sequencing after gel electrophoresis. In addition, primers modelled after the shorter a9 microsatellite was designed and tested using synthetic DNA of both TGFβR2 variants. Despite the naturally established mismatch between the a10 sequence and primers modelled after the a9 sequence, conventionally designed primers could not differentiate enough for specific annealing to the a9 sequence. A further mismatch with the a10 sequence was introduced at the 3’ end of the primer, which, in combination with the already established mismatch, denied the primer tail-end annellation with the a10 sequence that is necessary for polymerase amplification of this sequence. Experiments to optimise PCR conditions and buffer solution were conducted prior to analysing the cfDNA extracted from the patient samples. It was found that TGFβR2 DNA was present in cfDNA from all (10/10) CRC patients tested and that TGFβR2 a10→a9 frameshift was present in all (5/5) MSI-CRC patients and none (0/5) of MSS-CRC patients tested, shown by sequencing. PCR using the selected a9 modelled primer yielded a PCR product that was clearly detectable on the electrophoresis gel only for cfDNA from the MSI-CRC patients (5/5). The results show that TGFβR2 a10→a9 frameshift DNA is present in cfDNA of liquid biopsies from MSI-CRC patients which establishes cell free TFGβR2 frameshift DNA as a potential biomarker for early detection of hereditary CRC (Lynch Syndrome) as well as for monitoring cancer progression and remission of sporadic MSI-CRC.

Citation Format: Henrik K. Eriksen, Jon A. Eriksen. Detection of TGFbR2 frameshift mutation [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 3386.
Abstract 3553: Harnessing soluble CD40L to enhance anti-tumor efficacy of Her2-LAMP DNA vaccine using UNITE࣪ platform
Authors: Shen Wei , Xu Renhuan , Kao Yun-Ting , Karkada Mohan , Heiland Teri
Published: June 2022

CD40 ligand (CD40L; CD154) is a transmembrane protein expressed on the surface of activated T cells, particularly on CD4 T cells, that stimulates CD40-dependent activation of antigen-presenting cells, resulting in enhancement of T cell and antibody responses. Soluble multimeric forms of CD40L (sCD40L) act as an effective adjuvant to enhance vaccine immunogenicity. We previously developed a Her2/Neu-LAMP DNA vaccine based on our proprietary UNITE࣪ (UNiversal Intracellular Targeted Expression) platform, which fuses a tumor-associated antigen with lysosomal associated membrane protein 1 (LAMP-1). To increase T cell responses induced by this vaccine, we developed a bicistronic DNA construct in which both the tumor antigen (Her2) fused with LAMP and sCD40L were expressed separately. Secretion of CD40L was confirmed by transient transfection of 293T cells with plasmid DNA followed by ELISA for sCD40L in the cell culture supernatant. Intradermal vaccination of mice with bicistronic Her2-LAMP-sCD40L elicited significantly enhanced Her2-specific T cell and antibody responses compared with mice immunized with Her2-LAMP DNA. Intracellular staining revealed that inclusion of sCD40L in the vaccine induced potent antigen specific T cell IFNγ production, primarily in CD4 T cells. Furthermore, in a murine TSA breast cancer model, Her2-LAMP-sCD40L significantly inhibited tumor growth and prolonged survival in a therapeutic vaccine setting, suggesting that this novel bicistronic vaccine is an effective strategy to promote anti-tumor efficacy in vivo. Ongoing studies are aimed at exploring vaccine-induced changes within the tumor microenvironment, including cytokine profiles, expression of PD-1/PD-L1, and effect on myeloid derived suppressor cells and NK cells. This new approach in bicistronic DNA vaccine design using the UNITE࣪ platform may provide future therapeutic benefit for multiple Her2-expressing cancer types.

Citation Format: Wei Shen, Renhuan Xu, Yun-Ting Kao, Mohan Karkada, Teri Heiland. Harnessing soluble CD40L to enhance anti-tumor efficacy of Her2-LAMP DNA vaccine using UNITE࣪ platform [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 3553.
Abstract 356: Pre-clinical investigations utilizing Bcl2 inhibitors in glioblastoma
Authors: Kala Srishti , Earp Leland , Aulakh Sonikpreet
Published: June 2022

Glioblastoma (GBM) is a lethal malignant cancer of the central nervous system. The multimodal management approach does not extend survival beyond 2 years. Due to diverse variability in subpopulations of patients with glioma, treatment benefits are not uniform with apoptosis evasion being a key attribute of treatment resistance in GBM. Antiapoptotic protein, Bcl-2, Bcl-xL, and Mcl-1, are frequently upregulated in acquired chemo resistant cancer cells. Here in, we investigated the anti-proliferative effects of disrupting “Bcl-2-regulated pathways” in GBM. Of note, accessibility of FDA approved Bcl-2 inhibitors with known safety profile, makes it a biologically relevant target where intra- and inter-patient heterogeneity can be overcome successfully. Western Blot (WB) analysis was performed to compare the Bcl-2 family expression in human GBM cell lines (LN229, T98G, DI321, SA21). RT-PCR data was obtained for Bcl-2 family mRNA expression profile in present GBM cell lines. IC50s were determined for afore mentioned cell lines using MTS assay for Temozolomide (TMZ, alkylating agent), Bcl-2 inhibitor ABT199, Bcl-xL inhibitor A1331852, Mcl-1 inhibitor AZD5991, and BTK inhibitor Ibrutinib. Further, synergistic activity of TMZ, ABT199, A1331852, AZD5991 and Ibrutinib in several permutations. All experiments were performed with an n of 3/5. Our data suggests upregulation of antiapoptotic Bcl-2 family proteins in GBM cells including GSCs (DI321) and patient derived cells (SA21). There was significant decrease in viability of these cells when treated with ABT199 and A-1331852 (p< 0.0001) as compared to TMZ as a single agent. Other combinations ABT199+AZD5991 (p< 0.0001), AZD5991+A-1331852 (p< 0.0001) and TMZ+ABT (p<0.0002) also shows anti-tumor activity. Alternatively, we observed increased sensitivity in TMZ-resistant GBM cells against these drugs using MTS assay. GBM is an aggressive malignancy with 100% recurrence over time. Our preclinical data suggests synergism of Bcl-2 protein inhibitors in GBM cells, both TMZ-naïve and TMZ-resistant. Our ongoing research is investigating the biological relevance of Bcl-2 in conjugation with other tumor suppressor proteins, in vitro and in vivo.

Citation Format: Srishti Kala, Leland Earp, Sonikpreet Aulakh. Pre-clinical investigations utilizing Bcl2 inhibitors in glioblastoma [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 356.
Abstract 3599: Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas
Authors: Gray Joshua P. , Bildik Gamze E. , Sutton Margie N. , Zhou Yong , Millward Steven , Hancock John F. , Lu Zhen , Bast Robert C.
Published: June 2022

The overall 5-year survival for pancreatic ductal adenocarcinoma (PDAC) has changed little over the past few decades, and PDAC is predicted to become the second leading cause of cancer-related mortality in the next decade in Western countries. Low grade serous ovarian cancer (LGSOC) is a slow-growing but generally chemo-resistant cancer with few effective treatments for recurrent disease. More than 90% of PDAC and up to 40% of LGSOC express mutationally activated KRAS that drives persistent cell division, anti-apoptosis, cell migration and metastasis. Preventing signaling from the KRAS oncoprotein has been challenging. While recent studies have shown promising results by targeting the binding pocket in Switch II for development of KRAS mutant-specific inhibitors for the G12C mutant and G12D mutant, targeting other mutations remains a work in progress. We have discovered that DIRAS3, a novel endogenous physiological RAS inhibitor, blocks KRAS activity by directly binding KRAS with high affinity, inhibiting KRAS dimerization/nanoclustering and blocking effector activation.

In this study we have developed drug-like, helical 10-mer stapled peptides derived from DIRAS3 α5 domain. Preliminary studies show that these lead compounds associate with KRAS with low nanomolar affinity, are largely resistant to serum proteases, and rapidly cross the cellular membrane. Functionally, DIRAS3 peptides—but not control peptides—block KRAS homodimers in ReBiL split luciferase assays of KRAS dimerization and disrupt nanoclustering by TEM analysis of GFP-KRAS(G12V)-labeled gold nanoparticles on the inner leaflet of the plasma membrane of cells. Moreover, DIRAS3 peptides significantly inhibit phospho-ERK, decrease cell viability and induce apoptosis in pancreatic and low-grade ovarian cancer cells with KRAS mutations. Finally, daily treatment with stapled DIRAS3-derived peptides inhibited growth of ASPC-1 (KRAS G12D) PDAC xenografts and improved survival. Thus, our study suggests that development of DIRAS3 stapled peptides may provide a novel therapeutic approach to target mutant KRAS-driven cancers.

Citation Format: Joshua P. Gray, Gamze E. Bildik, Margie N. Sutton, Yong Zhou, Steven Millward, John F. Hancock, Zhen Lu, Robert C. Bast. Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas [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 3599.
Abstract 3654: Development and application of the iHELP platform to facilitate the establishment of healthy habits for the prevention of pancreatic cancer
Authors: Ke Temin , HELP I , Lophatananon Artitaya , Nieroda Marzena , Muir Kenneth
Published: June 2022

Background: Pancreatic cancer is the 7 th leading cause of cancer-related mortality and the 12 th most common cancer globally. The survival rate from pancreatic cancer is very poor so prevention and early detection approaches are important. It is estimated that 37% of pancreatic cancer cases can be prevented. Despite advanced knowledge on risk factors, prevention by personalized approach remain to be explored. The iHelp platform is being developed based on combining people’s risk factors and can be used as a tool to help people develop new health habits.

Study aim: To further develop and evaluate the i-Help risk assessment and support platform that utilizes big data management and Artificial Intelligence (AI) approaches to establish person-centric early risk prediction, prevention, and intervention measures.

Study design: The iHelp platform is an AI-based Scalable Big Data Platform which provides the necessary performance and features for the management and analysis of the heterogeneous data. The data source are derived from various sources including patients’ historical medical data as well as self-reported lifestyle habits and behaviors, social media interactions, integrated in the form of a Holistic Health Record (HHRs). Through the utilization of the iHelp platform we are implementing innovative AI algorithms and techniques to accomplish multi-modal causal analysis, real-time anomaly detection to estimate risk prediction, forecasting of risk trajectories, and detection of suggested behavioral changes. AI techniques will draw adaptive learning models that provide the decision support based on the risk predictions as well as personalized prevention and intervention measures. Users of the platform are supported to make appropriate lifestyle changes and monitored by using mobile or wearable devices.

Results: We will present the design of the platform together with feedback from end users on key modules. Ultimately we propose that the outcomes of iHelp components will not only integrate data from multiple sources, but they will also provide informative and motivational individualized pancreatic cancer risk reduction recommendations.

Citation Format: Temin Ke, I HELP, Artitaya Lophatananon, Marzena Nieroda, Kenneth Muir. Development and application of the iHELP platform to facilitate the establishment of healthy habits for the prevention of pancreatic 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 3654.
Abstract 3719: Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in cancer
Authors: Prado Gabriel , Bennett Richard L. , Licht Jonathan D.
Published: June 2022

In mammals, the histone methyltransferase KMT2C is one of the major regulators of gene expression and is one of the most commonly lost epigenetic regulators across all cancers. Studies in mice demonstrated that Kmt2c is a haploinsufficient tumor suppressor in AML and inoculation of mammary fat pads with breast cancer cells that lack KMT2C had significantly larger tumor growth and reduced survival compared to mice injected with isogenic wild-type cells. Analysis of cBioportal’s curated set of non-redundant cancer patient studies indicates that loss of function KMT2C mutations significantly co-occur with RAS gain of function mutations. Our lab previously discovered that cells expressing oncogenic RAS displayed altered activation of enhancer gene regulator elements. We will test how loss of KMT2C cooperates with oncogenic Ras by: 1) characterizing the impact of KMT2C loss, with or without expression of oncogenic RAS, on biological properties of cells and global chromatin architecture and 2) characterizing how KMT2C loss may cooperate with oncogenic RAS to alter hematopoiesis and the global enhancer landscape in animal models. MEFs expressing heterozygous loss of Kmt2c have a proliferative advantage relative to WT littermate controls. Interestingly, WT MEFs demonstrate enhanced migration in a wound assay, but heterozygotes demonstrate an advantage in a transwell membrane assay. Heterozygotes further display greater EdU incorporation, indicating a greater propensity for entering S-phase and undergoing cell-division. RNA-seq experiments have indicated an upregulation of MAPK & Ras signaling pathway genes in heterozygotes, whilst simultaneously downregulating gene pathways critical for proper extra-cellular matrix organization. Therefore, loss of Kmt2c disrupts biological cell phenotypes, in a manner relatable to the hallmarks of cancer, demonstrating how Kmt2c loss may promote malignancy. Further studies must now be performed to elucidate the molecular mechanisms altered by loss of KMT2C and how they cause changes in cellular phenotypes.

Citation Format: Gabriel Prado, Richard L. Bennett, Jonathan D. Licht. Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in 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 3719.
Abstract 3739: EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA
Authors: Williams Louise
Published: June 2022

The study of DNA methylation from cell-free DNA (cfDNA) and DNA extracted from formalin fixed paraffin embedded (FFPE) tissues are notoriously difficult. The use of these DNA inputs is becoming increasingly important in the field of cancer diagnostics. The cytosine modifications, 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC), are key epigenetic factors that play an important role in cellular processes and their mis-regulation results in diseased states like cancer. Advances in the study of these epigenetic factors in combination with improvements in sample preparation have enabled cancer biomarker identification based on methylation profiling.

The historical gold standard for detecting cytosine methylation has been bisulfite sequencing. This method uses chemical conversion of cytosines into uracils and has been used for both targeted and whole genome methylation analysis. However, bisulfite conversion leads to DNA damage which results in shorter DNA insert sizes as well as biases in the data. It is challenging to make bisulfite libraries from cfDNA and FFPE DNA as typically the DNA is of low quantity and quality.

We developed an enzyme-based methylation detection technology (EM-seq) to address the flaws inherent to bisulfite sequencing. This method, primarily due to the intact nature of DNA after conversion, results in better quality libraries with longer insert sizes, lower duplication rates and minimal GC bias. EM-seq libraries prepared from cfDNA and lung FFPE DNA had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to bisulfite libraries. EM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. EM-seq’s superior sequencing metrics establish it as a standard method for robust methylation profiling, particularly for these types of challenging DNA samples.

Citation Format: Louise Williams. EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA [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 3739.