bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5766: Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A
Authors: Friman Tomas , Fontaine Benjamin M. , Chernobrovkin Alexey , Molina Daniel Martinez , Brehmer Victoria , Lundgren Stina , Castaldi Paola M. , Verdine Gregory L.
Published: March 2024

Fungal genomes encode a vast collection of chemical diversity that has evolved over millions of years to engage specific targets and modulate diverse biological processes. These Genetically Encoded small Molecules (GEMs) constitute a largely untapped source of potential therapeutics. Although fungal GEMs such as cyclosporine, lovastatin, and penicillin have revolutionized modern medicine, these GEMs were discovered serendipitously through bioactivity-guided fractionation of fungal extracts. LifeMine is redefining GEM discovery through the deployment of proprietary genomic search algorithms that enable GEM target prediction a priori. Our strategy facilitates focused search-and-retrieval campaigns to rationally discover molecules selected by Nature to modulate the desired target protein. These GEM drug leads often exhibit unique structural and mechanistic features, necessitating comprehensive deconvolution of the human target profile and mechanism of action during the development process from GEM to clinical candidate. As a proof-of-concept study, we leveraged CETSA Explore to study the biological mechanism of the fungal polyketide lactone Brefeldin A (BFA) in human THP-1 cells. This GEM, originally isolated from the filamentous fungus Penicillium brefeldianis, inhibits protein trafficking between the endoplasmic reticulum and the Golgi complex, eliciting antiviral, antifungal, and anticancer activity. Our CETSA profiling study confirmed the Golgi-associated guanine nucleotide exchange factor GBF1 as the primary target of BFA, validating the established mechanism of this GEM as an inhibitor of GBF1-mediated activation of the small GTPase ARF1. These results demonstrate the utility of integrating the LifeMine GEM discovery engine with downstream proteomic profiling technologies to facilitate the mechanistic characterization of fungal GEMs en route to novel therapeutics.

Citation Format: Tomas Friman, Benjamin M. Fontaine, Alexey Chernobrovkin, Daniel Martinez Molina, Victoria Brehmer, Stina Lundgren, Paola M. Castaldi, Gregory L. Verdine. Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5766.
Abstract 5767: DNA binding mechanism of prostate cancer target ONECUT2
Authors: Chatterjee Avradip , Gallent Brad , Katiki Madhusudhanarao , Qian Chen , Harter Matthew R. , Freeman Michael R. , Murali Ramachandran
Published: March 2024

Transcription factors (TF) are essential determinants of gene regulation and their aberrant expression frequently drive cancers. The binding of TFs to cognate DNA is central to their role in gene regulation. An understanding of their DNA binding mechanism is, therefore, of fundamental and therapeutic relevance. Generally, TFs bind DNA through defined DNA binding domains. The CUT and homeodomain are two such evolutionarily conserved elements found paired in multiple transcription factor families including ONECUT (OC), POU, CUX and SATB. However, the mechanism of cross-talk between the CUT and homeodomain to coordinatedly bind DNA remains unknown. Here, we report an integrative DNA binding analyses of the transcription factor OC2, a promising target for treatment of aggressive prostate cancer. We demonstrate that the homeodomain of OC2 thermodynamically stabilizes the DNA binding through allosteric modulation of CUT. The binding energetics is further dependent on base interactions by evolutionarily conserved amino acids in both CUT and homeodomain. In addition, we have discovered a unique arginine pair in the homeodomain, conserved within the ONECUT family, but not across POU and SATB families. We show that one arginine of this pair binds distinctly to DNA in respective complexes formed by OC2 and OC1. Notably, the above base-interactions are crucial for OC2-driven disease progression in a prostate cancer cell-line model. Taken together, our studies show that base-interactions by the evolutionarily conserved residues determine the basic DNA binding functionality through establishing favorable thermodynamics, while interactions by the arginine motif impart family-specific attributes. These findings also establish the homeodomain as a key regulator of DNA binding by OC2 and provide a mechanism for cooperative DNA binding by the CUT-homeodomain module in general. Importantly, these insights reveal vulnerabilities for therapeutic targeting of OC2.

Citation Format: Avradip Chatterjee, Brad Gallent, Madhusudhanarao Katiki, Chen Qian, Matthew R. Harter, Michael R. Freeman, Ramachandran Murali. DNA binding mechanism of prostate cancer target ONECUT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5767.
Abstract 5770: Inhibition of KRAS G12D mutant with small molecules
Authors: Lee Dae-Hee , Lee Ji Yoon
Published: March 2024

Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types.

Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19.

Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity.

Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers.

Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University

Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5770.
Abstract 5775: Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation
Authors: Han Yichen , Dickerhoff Jonathan , Yang Danzhou
Published: March 2024

Overexpression of the receptor tyrosine kinase PDGFR-β promotes cancer growth and metastasis. PDGFR-β is a validated cancer target but PDGFR-β kinase inhibitors suffer from lack of selectivity and side effects. G-quadruplexes are an exciting class of non-B DNA secondary structures with functional importance. The G-quadruplex formed in the PDGFR-β promoter is a transcription repressor and targetable by small molecules. Therefore, the PDGFR-β promoter G-quadruplex is an attractive molecular target for anticancer drugs. Structural information of the PDGFR-β promoter G-quadruplex is crucial for understanding its cellular function and rational drug design. Herein, we used nuclear magnetic resonance (NMR) spectroscopy and biophysical methods to determine the structure and dynamics of the major G-quadruplex formed in the human PDGFR-β promoter. Unlike canonical G-quadruplexes formed by four runs of three continuous guanines (G), the major PDGFR-β G-quadruplex adopts a novel broken-strand structure where a two-G segment is continued by a non-adjacent guanine. The broken-strand G-quadruplex has a vacancy-G-tetrad (vG4) which is filled-in intramolecularly by a distal guanine. Interestingly, we discovered that the major PDGFR-β G-quadruplex is a novel mixture of two equilibrating broken-strand G-quadruplexes. Both structures adopt overall parallel-stranded folding with all 1-nt chain-reversal loops. However, a different 3’-filled-in guanine is delivered by a lateral loop, which forms structurally similar hairpin with a unique capping G-G base-pair. The formation of the equilibrating broken-strand G-quadruplexes and unique capping structures are both specific to the PDGFR-β promoter sequence. Intriguingly, the observed dynamic equilibrium between the two co-existing structures with different intramolecular fill-in guanines indicates a novel vG4 intermediate, which could be filled-in by cellular guanine metabolites such as cGMP. Therefore, our study provides critical insights into how the dynamics of the major PDGFR-β G-quadruplex lead to vG4 formation and potential regulation of gene transcription by cellular guanine metabolites. The determined molecular structures of the novel broken-strand G-quadruplexes provide a structural basis for rational design of small molecules to specifically target the major PDGFR-β promoter G-quadruplex for cancer therapeutics.

Citation Format: Yichen Han, Jonathan Dickerhoff, Danzhou Yang. Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5775.
Abstract 5793: Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling
Authors: Wang Linya , Han Zhen , Yang Marisa , Hernandez Ana G. Lujan , Prabhu Vishwas , Hu Cameron F. , Htoy Tammy , Villalta Mouna , Yuan Tom Z. , Giang Hoa , Sato Aaron
Published: March 2024

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the β-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. DKK1 has been shown to be a potent inhibitor of Wnt signaling via competing Wnt binding to LRP5/6. DKK1 is tumorigenic in multiple cancer types and also immunosuppressive via MDSCs and NK cells. Emerging evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. With Twist’s precision DNA writing technologies, we have created phage display libraries with diversity greater than 1 × 1010, and utilized machine learning model for optimal discovery. In this study, we discovered anti-DKK1 antibodies bind to different cysteine-rich domain (CRD) of hDKK1 by epitope binning and mapping, and block the binding of DKK1 to the receptor. Binding of the antibodies to different CRDs of hDKK1 lead to different activation effect. The in vitro functional assays showed that the interaction of Wnt to its receptor was restored in the presence of anti-DKK1 antibodies binding to DKK1 CRD2, resulting in TCF/LEF signaling upregulation. Moreover, anti-DKK1 antibodies binding to DKK1 CRD1 induced immune cell activation and led to tumor cell cytotoxicity. In vivo study indicated anti-DKK1 antibody lead is potent in tumor regression. Our anti-DKK1 lead shows promising efficacy in cancer immunotherapy without influencing Wnt canonical pathway.

Citation Format: Linya Wang, Zhen Han, Marisa Yang, Ana G. Lujan Hernandez, Vishwas Prabhu, Cameron F. Hu, Tammy Htoy, Mouna Villalta, Tom Z. Yuan, Hoa Giang, Aaron Sato. Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5793.
Abstract 5807: Bicycle Toxin Conjugates®for the treatment of solid tumors
Authors: Walsh Stephen J. , Lahdenranta Johanna , Huxley Philip , Mudd Gemma , Bennett Gavin , Brown Amy , an Rietschoten Katerine , Chen Liuhong , Scott Heather , Ivanova-Berndt Gabriella , Dzionek Katarzyna , Rigby Mike , Burenkova Olga , Jeffrey Phil , Beswick Paul , Skynner Michael , Keen Nicholas
Published: March 2024

Bicycle Therapeutics is developing a unique class of chemically synthesized medicines. Based on its proprietary bicycle peptide (Bicycle®) phage display platform, Bicycles are a unique class of highly constrained peptides, which have demonstrated utility in the targeted delivery of different classes of payloads (for example cytotoxic agents, radioisotopes, immune modulators) into tumors. Bicycles are currently being explored in the clinic as Bicycle Toxin Conjugates® (BTCs) for targeted delivery of cytotoxic payloads into tumors. BTCs consist of a bicyclic peptide that is conjugated to a cytotoxic payload via a cleavable linker, which allows payload release in the tumor microenvironment or within the tumor cell. BTCs were developed to address the shortcomings of antibody drug conjugates (ADCs) in several ways. First, the small size of BTCs (∼4 kDa) compared to large biologic entities such as monoclonal antibody (mAb)-based conjugates (∼150 kDa) allows rapid distribution to tissues and extensive tumor penetration, which enables rapid delivery of payload into the tumor. Second, the peptidic nature of BTCs results in relatively short, yet tunable, duration of systemic exposure and liver-sparing renal elimination. These properties limit the body’s exposure to payload and should therefore minimize damage to normal tissue. In this body of work, we used in vitro cytotoxicity and cell uptake assays and mouse and rat cell line derived xenograft models for 1) cytotoxicity and anti-tumor activity evaluation, and 2) Bicycle and toxin uptake and biodistribution evaluation. Here, we show that BTCs targeting a number of different tumor antigens can deliver toxins to tumor tissue producing durable responses in a range of preclinical in vivo models, spanning several solid tumor indications.

Citation Format: Stephen J. Walsh, Johanna Lahdenranta, Philip Huxley, Gemma Mudd, Gavin Bennett, Amy Brown, Katerine an Rietschoten, Liuhong Chen, Heather Scott, Gabriella Ivanova-Berndt, Katarzyna Dzionek, Mike Rigby, Olga Burenkova, Phil Jeffrey, Paul Beswick, Michael Skynner, Nicholas Keen. Bicycle Toxin Conjugates®for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5807.
Abstract 584: Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity
Authors: Edmondson Jacob L. , Reed Megan R. , Fil Daniel , Heflin Billie , Koss Brian , Tackett Alan J.
Published: March 2024

Although immune checkpoint blockade (ICB) marked a breakthrough in melanoma treatment, half of patients fail to respond, underscoring the crucial need for complementary adjuvant and neo-adjuvant therapies. Due to the low overall response rate and the stark contrast in outcomes between patients that respond and those that fail to respond, identifying the defining characteristics of immune checkpoint blockade (ICB) response is essential for enhancing the efficacy of melanoma treatments. To recapitulate and investigate the ICB-responsive phenotype in vitro, melanoma cells were metabolically adapted to an environment lacking glucose which successfully drove the dependence on oxidative metabolism and restored MHC-I expression, two established characteristics of ICB responsive tumors. Further, metabolic adaptations significantly enhance the tumor sensitivity to T-cell killing. Surprisingly even in JAK1 and IFNAR1 deficient cells, metabolic adaptation restored MHC I antigen presentation, indicating an interferon-independent mechanism of MHC-I regulation. Proteomic analysis of three metabolically conditioned melanoma cells lines (human and mouse) identified downregulation of the histone methyltransferase EZH2 and the activating transcription factor ATF6 as key corollaries of MHC-I expression. Interestingly, EZH2 and ATF6 activity were found to be inversely related in metabolically adapted cells and patient tumor samples. These studies mechanistically explore the epigenomic control of antigen presentation machinery and the transcriptional targets of ATF6 controlling sensitivity T cell killing. Here, through in vitro and in vivo studies, we demonstrate that constitutive activation of ATF6, and thereby activation of the adaptive unfolded protein response (UPR), sensitizes melanoma to immune control. These data suggest that EZH2 and ATF6 act synergistically to balance immune mediated killing during metabolic stress whereby loss of EZH2 increases MHC-I antigen presentation and activation of ATF6 sensitizes tumor cells to T cell-mediated control. Ongoing therapeutic studies combining EZH2 inhibition, ATF6 activation, and ICB in pre-clinical models further suggest targeting the synergy between EZH2 and ATF6 is a putative adjuvant or neo-adjuvant target to improve the clinical response of ICB.

Citation Format: Jacob L. Edmondson, Megan R. Reed, Daniel Fil, Billie Heflin, Brian Koss, Alan J. Tackett. Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 584.
Abstract 5850: Multiomics analysis unravels tumor metabolic reprogramming and potential resistance mechanism(s) to curative therapy in TNBC
Authors: Ren Zuen , Kurmi Kiran , Morris Robert , Joshi Shakchhi , Zaniewski Eric , Kreuzer Johannes , Gioia Gabrielle Elena , Bossuyt Veerle I. , Dontchos Brian N. , Wang Gary X. , Chou Shinn-Huey S. , Spring Laura M. , Haas Wilhelm , Haigis Marcia C. , Ellisen Leif W.
Published: March 2024

Drug resistance is the leading cause of treatment failure to completely eliminate viable tumor cells in ~40% of patients with triple negative breast cancer (TNBC). Making a major impact on the treatment and prognosis for TNBC patients requires a better understanding of drug resistance mechanism(s). Combining clinical data with high-throughput metabolomic and proteomic analyses, we probed the molecular basis for chemotherapy resistance in treatment-naive TNBC primary tumors, reflected by pathological complete response (pCR) and non-pCR after neoadjuvant chemotherapy (NAC). Metabolic profiling uncovered that glycolysis and oxidative phosphorylation (OXPHOS) co-exist in TNBC primary tumors compared to benign lesions regardless of pCR status, indicating metabolic reprogramming. However, non-pCR tumors were more enriched in OXPHOS and less in glycolysis relative to pCR tumors, pointing to the potential importance of OXPHOS in drug resistance. Moreover, parallel proteomic profiling of these same TNBC tumors supported the results from metabolic profiling, showing that OXPHOS was indeed highly enriched at the protein level, and may be regulated by SIRT5-mediated sirtuin signaling. In alignment with these findings, non-pCR tumors were abundantly enriched in immune-inhibitory metabolites that may be due to the products of SIRT5-mediated mitochondrial metabolism. Thus, these findings led me to hypothesize that SIRT5 may play an important role in chemoresistance through regulation of mitochondrial metabolism and ROS detoxification. Together, the metabolite resulted from SIRT5-mediated mitochondrial metabolism led to the suppression of immune cell infiltration in non-pCR tumors. Understanding SIRT5-mediated chemoresistance mechanism may uncover a metabolic and therapeutic vulnerability to resistant TNBCs.

Citation Format: Zuen Ren, Kiran Kurmi, Robert Morris, Shakchhi Joshi, Eric Zaniewski, Johannes Kreuzer, Gabrielle Elena Gioia, Veerle I. Bossuyt, Brian N. Dontchos, Gary X. Wang, Shinn-Huey S. Chou, Laura M. Spring, Wilhelm Haas, Marcia C. Haigis, Leif W. Ellisen. Multiomics analysis unravels tumor metabolic reprogramming and potential resistance mechanism(s) to curative therapy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5850.
Abstract 5911: In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer
Authors: Elliott Mitchell J. , Mcguire Meghan , Silvester Jennifer , Tobin Chantal , Ghamrasni Samah El , Lui Francine , Zhai Andrew , Yoluk Ozge , Broom Aron , Stone Tracy , Butterfoss Glenn , Popa Serban , Lu Tianyu , Ing Chris , White David , Cescon David W.
Published: March 2024

Introduction: Antibody and peptide-conjugated drugs are rapidly entering the clinic for the treatment of patients with early and advanced breast cancer. These drugs are designed to achieve more targeted delivery of cytotoxic agents to tumor cells with robust clinical activity. ARB-1-6, a sortilin (SORT1) targeted peptide conjugated to the cytotoxic agent MMAE, has demonstrated pre-clinical activity and tolerability in cell line xenografts of breast cancer. To further characterize its spectrum of antitumor activity and biomarker correlates we evaluated ARB-1-6 in a collection of clinically and genomically annotated breast cancer patient-derived xenografts (PDX).

Methods: Breast cancer PDX models were generated and propagated under IRB approved protocols [14-8358]. ARB-1-6 was dosed in tumor-bearing SCID mice by tail vein injection at 3 mg/kg weekly x 4 doses. Tumor volume was measured twice weekly until day 35 or until models reached humane endpoints, after which tumors were collected. Animals were weighed regularly and assessed for treatment-related toxicity.

Results: 7 models (5 TN and 2 HR) have completed efficacy assessment to date. 3/7 models were derived from treatment naïve patients while 4/7 were derived from pre-treated patients (Table 1). Complete response (mRECIST) was seen in 5/7 (71.4%) models with stable disease occurring in 2/7 (28.6%). ARB-1-6 was well tolerated with no weight loss observed. Complete tumor regression was seen in a PDX derived from a patient with clinical resistance to a TROP2 antibody drug conjugate (ADC). Antitumor activity was observed in models with low SORT1 RNA expression; SORT1 IHC and other correlatives studies are underway.

Conclusion: ARB-1-6 demonstrates substantial preclinical activity in PDX models at doses that are well tolerated in mice. Treatment of additional models is ongoing. Full results and correlative analyses including relationships between antitumor activity and target protein expression will be presented.

PDX Model Characteristics and Patient Treatment PDX Subtype Pt. Treatment 1 TN AC-T 2 TN FEC-D, Gem+Carbo 3 TN AC-T 4 TN No 5 TN No 6 HR No 7 HR TC, ET, Cape, Gem+Cis, TROP2-ADC

Citation Format: Mitchell J. Elliott, Meghan Mcguire, Jennifer Silvester, Chantal Tobin, Samah El Ghamrasni, Francine Lui, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Lu, Chris Ing, David White, David W. Cescon. In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5911.
Abstract 5934: Ferroptosis induction therapy for castration resistant prostate cancer
Authors: Awolowo Mojisoluwa , Dike Precious Elechi , Odero-Marah Valerie
Published: March 2024

Prostate cancer (PCa) is the most common non-skin cancer in men, and second leading cause of death. The fundamental treatment for treating PCa is androgen deprivation therapy which is an effective form of treatment initially. Nevertheless, the disease reoccurs in most men; this is referred to as castration-resistant PCa (CRPC), for which the standard treatment is enzalutamide. However, some develop resistance to this enzalutamide anti-androgen agent. Ferroptosis is an iron-dependent lipid peroxidation form of regulated cell death being investigated as a novel potential therapy for PCa using ferroptosis inducers such as RSL3; they have shown promise in selectively triggering cell death in cancer cells while sparing healthy tissues. We hypothesize that RSL3- induced ferroptosis will overcome enzalutamide resistance in PCa cells. To test this, we utilized C4-2B MDVR cells which represents a cell model for aggressive PCa that metastasized to the bone and is resistant to both castration therapy and the enzalutamide drug treatment. C42B-MDVR cells were treated with increasing concentrations of RSL3 followed by analysis of cell viability with MTS assay. A dose dependent reduction in cell viability was observed which was reversed by ferroptosis inhibitor, ferrostatin-1. Western blot analysis revealed a decreased expression of the antioxidant enzyme glutathione peroxidase 4 (GPX4) with ferroptosis induction indicating a mechanism of the RSL3-induced cell death. The limitations to our research are that the research was performed in vitro and have not been tested on mouse models to visualize the effects of the treatments in animals. In conclusion, ferroptosis inducer RSL3 decreases GPX4 expression and decreases proliferation of PCa cells while ferrostatin-1 reverses the effects of the RSL3 treatment. Thus, ferroptosis inducers present a potential novel therapeutic approach for CRPC patients who have developed resistance to conventional androgen therapies. NIH NIGMS RISE 5R25GM058904, NIH/NIMHD U54MD007590; U54MD013376

Citation Format: Mojisoluwa Awolowo, Precious Elechi Dike, Valerie Odero-Marah. Ferroptosis induction therapy for castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5934.
Abstract 5971: F3 targeted near-infrared photoimmunotherapy: The ideal therapeutic approach for numerous malignancies
Authors: Takao Seiichiro , Furusawa Aki , Fukushima Hiroshi , Okuyama Shuhei , Kano Makoto , Yamamoto Hiroshi , Choyke Peter L. , Kobayashi Hisataka
Published: March 2024

Introduction: F3 is a cell surface protein that plays a role in blood clotting, but recent research has revealed its implication in cancer proliferation, metastasis, angiogenesis, and immune escape. TCGA cohort analysis has shown that elevated F3 is a common feature of many cancers, and generally correlates with poor patient survival. Therefore, the potential of F3 as a therapeutic target in cancer is worth exploring. Tisotsumab-vedotin, an antibody-drug conjugate (ADC) that targets F3, was approved by the FDA for cervical cancer and is undergoing clinical trials in some solid cancers. However, ADCs may give rise to concerns about drag resistance following cellular internalization. Herein, we developed and tested an F3 targeted near-infrared photoimmunotherapy (NIR-PIT) to overcome some of these resistance mechanisms.

Methods: We created a conjugate of tisotumab as an F3 antibody and IR700. The efficacy of F3-targeted NIR-PIT was investigated using multiple cancer cell lines (A431; epidermoid carcinoma, HPAF-II; pancreatic adenocarcinoma, HSC-2; oral carcinoma, HT-1376luc; bladder carcinoma, MDAMB231; breast adenocarcinoma, and SKOV-3; ovarian serous cystadenocarcinoma) in vitro. In vivo, the efficacy of F3-targeted NIR-PIT was evaluated in HPAF-II tumor using xenograft mouse models. In addition, the pathological change after NIR-PIT was also evaluated in A431, HPAF-II, HSC-2, HT-1376luc, MDAMB231, and SKOV-3 tumors.

Results: These cell lines showed F3 expression in vitro and in vivo. Additionally, F3 expression was specific to cancer cells in HPAF-II tumor model. In vitro, F3-targeted NIR-PIT damaged the multiple cell lines in an NIR light dose-dependent manner. In vivo, F3-targeted NIR-PIT suppressed tumor growth and improved survival rates compared to the untreated group. Pathologically, A431, HPAF-II, HSC-2, HT1376-luc, MDAMB231 and SKOV-3 tumors after NIR-PIT contain large numbers of necrotic cells that are shown typically less dark nuclei and eosinophilic cytoplasm with some vacuolar degeneration than untreated tumors.

Conclusions: F3-targeted NIR-PIT holds the efficacy against multiple cancers expressing F3, regardless of the specific cancer type. Therefore, this modality exhibits significant promise as a potential NIR-PIT option.

Citation Format: Seiichiro Takao, Aki Furusawa, Hiroshi Fukushima, Shuhei Okuyama, Makoto Kano, Hiroshi Yamamoto, Peter L. Choyke, Hisataka Kobayashi. F3 targeted near-infrared photoimmunotherapy: The ideal therapeutic approach for numerous malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5971.
Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family
Authors: Maneval Daniel C. , Yang Cindy , Trevisiol Cynthia , Shepard H. Michael , Jin Pei
Published: March 2024

Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors.

Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5975.
Abstract 6020: GPC3-targeted radiopharmaceutical therapy for neuroendocrine prostate cancer
Authors: Clift Renee , Lin Fanching , Salvador Katrina , Guest Matt , Kim Daniel , Han Guangzhou , Li Gary
Published: March 2024

Introduction: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein with minimal expression in normal tissues. Besides hepatocellular carcinoma, upregulation of GPC3 protein has been observed in a significant subset of treatment-emergent neuroendocrine prostate cancer (NEPC), which accounts for to 30% of prostate cancer cases refractory to anti-androgen and other therapies. The differential expression of GPC3 between NEPC cells and normal tissues provides a theranostic opportunity for targeted radiopharmaceutical therapy (RPT).

Methods: The novel RPT agent RYZ-GPC3 comprises a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes. The expression of GPC3 in NEPC cells was assessed by flow cytometry and immunohistochemistry (IHC). The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. In vivo biodistribution of [177Lu]Lu-RYZ-GPC3 and anti-tumor therapy with [177Lu]Lu-RYZ-GPC3 or [225Ac]Ac-RYZ-GPC3 were performed in NEPC xenografts.

Results: The novel agent is a highly potent and selective peptide binder to GPC3 of human, mouse and NHP origins, and is compatible with multiple radiometal isotopes. In vivo biodistribution study of [177Lu]Lu-RYZ-GPC3 in GPC3-positive NEPC xenografts showed sustained tumor uptake of 10.8, 12.6, 5.1, and 2.2 %ID/g at 2, 24, 72 and 168 hours post dose, respectively, with fast renal clearance. Minimal or no uptake was observed in other normal tissues. A single injection of [225Ac]Ac-RYZ-GPC3 (11.1 kBq) resulted in significant tumor growth inhibition (TGI 109%) with durable regression and prolonged survival. All treatments were well tolerated.

Conclusion: Preclinical in vitro and in vivo data demonstrated the potential of the novel binder as a theranostic agent for the treatment of patients with GPC3+ NEPC.

Citation Format: Renee Clift, Fanching Lin, Katrina Salvador, Matt Guest, Daniel Kim, Guangzhou Han, Gary Li. GPC3-targeted radiopharmaceutical therapy for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6020.
Abstract 6031: Fasting associated microbiota contributes to small intestinal radioprotection by modulating the epigenome
Authors: Barrodia Praveen , Arslan Emre , Jeter-Jones Sabrina , Jenq Robert R. , Rai Kunal , Piwnica-Worms Helen
Published: March 2024

Normal tissue toxicity often restricts the administration of effective radiation doses for optimal tumor cell elimination. We identified a novel host-protective effect conferred by short-term fasting when treating mice with high dose radiation. We showed that fasting mice for 24h induces epigenetic and transcriptional changes in small intestinal epithelial cells (siECs) and confers protection to small intestinal stem cells from high dose radiation. Here we report that fasting induces changes in the gut microbiome favoring bacteria producing metabolites, specifically short-chain fatty acids, that induce epigenetic changes in siECs. 16S rRNA sequencing of feces isolated from the small intestines of fed and fasted mice revealed a marked enrichment of Akkermansia muciniphila (AKK) in fasted relative to fed mice. Specific depletion of AKK through antibiotic treatment impaired the ability of fasting to provide organismal radioprotection whereas both organismal and OLFM4+ stem cell protection was restored in fasted animals upon reintroduction of AKK. AKK produces short chain fatty acids (SCFAs) including propionate which is able to induce histone modifications and our metabolomic analysis of feces isolated from the ileums of mice demonstrated a significant increase in propionic acid levels in fasted mice compared to fed mice. Importantly, we showed that AKK conditioned medium as well as its metabolite (propionate) are able to induce histone acetylation in Lgr5+ stem cell enriched epithelial spheroids cultured in vitro. We are currently employing high-dimensional analysis techniques, including single cell analysis, Cut&Tag and RNAseq to determine how the epigenetic and transcriptional changes induced by fasting provide organismal and SI radioprotection.

Citation Format: Praveen Barrodia, Emre Arslan, Sabrina Jeter-Jones, Robert R. Jenq, Kunal Rai, Helen Piwnica-Worms. Fasting associated microbiota contributes to small intestinal radioprotection by modulating the epigenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6031.
Abstract 6035: Fluoride effects on thermal properties of irradiated dentin and enamel
Authors: Kulchar Rachel J. , Kakiuchi Yusuke , Sanchez-Gamarra Vania , Deng Jie , Mante Francis
Published: March 2024

Aim: Radiation therapy to treat oral cancer introduces various harms, including decreased hardness of dentin and enamel. Fluoride treatments, including silver diamine fluoride (SDF), have been used as a tool to combat these harms, but its influences on the thermal properties of dentin and enamel prior to and after radiation therapy are not well understood. Thus, this study analyzed how SDF influences the thermal stability of dentin and enamel that has been exposed to radiation therapy intended for head and neck cancer patients.

Methods: Forty human posterior teeth were collected, cleaned of debris and soft tissue, and then mounted into acrylic resin molds. The teeth were divided into two equal groups, with one group being the control (no treatment) and the other group receiving SDF treatment. Using an X-Rad 320ix biological irradiator, all teeth were subjected to two grays of radiation therapy every day for six weeks. Afterwards, the dentin and enamel were pulverized a SPEX freezer mill. Samples were heated in a NETZSCH Thermal Analysis instrument, and the amount of carbon dioxide and water released was determined. All teeth were heated to 1429 °C from room temperature.

Results: The SDF-treated dentin released the most carbon dioxide (49,611.82 ppm/mg), followed by irradiated dentin (47,672.76 ppm/mg) and untreated dentin (45,249.66 ppm/mg). In contrast, the SDF-treated enamel released less carbon dioxide (7,367.62 ppm/mg) than the control enamel (8,172.01 ppm/mg). The pattern of release of water was different from that of carbon dioxide. The control dentin group released the most water (45.56 ppt/mg), followed by the SDF-treated (39.72 ppt/mg) and irradiated dentin (23.51 ppt/mg). Similarly, the control enamel also released the highest amount of water (0.83 ppt/mg), as well as the highest amount of carbon dioxide. The SDF-treated enamel released around 65% of the water compared to the control enamel group (0.54 ppt/mg). Furthermore, the irradiated dentin had the largest heat reversal temperatures for carbon dioxide (85.19 °C) and water (86.57 °C).

Conclusions: The impacts of SDF treatment and radiation vary between dentin and enamel. Comparatively, dentin has a higher content of combustible material than enamel. Radiation treatment reduces the water content of both enamel and dentin, which may make teeth more susceptible to fracture. Therefore, regular dental check-ups and frequent fluoride treatment to increase the strength of the teeth are recommended for patients undergoing radiation therapy.

Citation Format: Rachel J. Kulchar, Yusuke Kakiuchi, Vania Sanchez-Gamarra, Jie Deng, Francis Mante. Fluoride effects on thermal properties of irradiated dentin and enamel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6035.
Abstract 6054: UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform
Authors: Ha Kyungsik , Song Woojeung , Choi Woochang , Lee Gi Bbeum , Ryu Soohee , Lee Jun Kyu , Lee Hwajin
Published: March 2024

In Target Protein Degradation (TPD) field, conventional E3 ligases utilized in the clinic are limited to CRBN and VHL, which opens the need for exploration of novel E3 ligases for the advancement of TPD. To address this, we have developed a proprietary platform, UPPGRADER, for systematic discovery of novel E3 ligases based on -omics data including single cell RNA sequencing (scRNA-seq) and whole genome sequencing (WGS), in conjunction with proteomics data and bioinformatics tools.UPPGRADER enables systematic analyses on alterations occurred in all genomic areas including E3 ligase genes and potential target genes. For example, analyzing Pan-Cancer Analysis of Whole Genomes (PCAWG) data identified amino acid changes into Cysteine or Lysine, which can be targeted by covalent binders with cancer specificity. Through such analysis, we discovered several novel E3 ligases, including but not limited to FBXW7, with the categorization of the findings based on specific cancer types.At the single cell expression level, UPPGRADER provides the capability to assess gene expression levels of E3 ligases and target proteins by proprietary measurements based on gene detection rate. This system also allows assessment of E3 ligase-Target gene co-expression per single cell. In colorectal cancer, UPPGRADER has shown that the co-expression of E3 ligase FBXW7 and well-known colorectal cancer target (KRAS) increases from 1% (4 out of 334) in normal epithelial cells to 17% (2,967 out of 17,458) in cancerous epithelial cells, highlighting a significant change in their simultaneous expression patterns during cancer progression.UPPGRADER also identified E3 ligase X which showed high co-expression level with KRAS gene alongside universal expression properties and high cancer cell dependencies. For E3 ligase X, we developed novel small molecule binders (nM binding affinity) with bioavailability > 20% in animal pharmacokinetics. Bifunctional degraders utilizing these binders displayed BRD4 degradation in more than 20 cell lines with double to triple digit nM DC50 and DC90 values. Other targets (AURKA, CRBN and others) were potently degraded with anticipated mechanism of action (assessed by E3 ligase KO cell line and proteasomal inhibitors).To conclude, UPPGRADER is a comprehensive bioinformatics tool for discovering novel E3 ligases with biological validations. Through recent explosion of scRNA-seq and WGS datasets in different diseases and mechanism-based identification of additional molecular targets, UPPGRADER is poised to advance the field of TPD.

Citation Format: Kyungsik Ha, Woojeung Song, Woochang Choi, Gi Bbeum Lee, Soohee Ryu, Jun Kyu Lee, Hwajin Lee. UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6054.
Abstract 6064: Discovery of potent and selective EP300 degraders with anti-cancer activity
Authors: Zimmerman Mark , Adam Ammar , Ahmad Hafiz , Adams Benjamin , Adhikari Ketaki , Austin Wesley , Bullock Breanna , Bernardo Julie Di , Dixon Thomas , Daniels Danette , Dominici Claudia , Elliot GiNell , Ethell Brian , Gervais Anais , Hossain Md Imran , Huang David , Lahr Dave , Lin Mei Yun , Mayhew David , Mizeracka Karolina , Negretti Solymar , Nguyen Tyler , Prifti Olga , Sappal Darshan , Schiller Shawn , Sherbanee Brenna , Terry David , Ucisik Nihan , Wittenborn Elizabeth , Zhou Qianhe , La Bonte Laura
Published: March 2024

E1A binding protein (EP300) and CREB binding protein (CBP) are paralog histone acetyltransferases involved in many cellular processes via their activity as transcriptional co-activators. Dysregulation of one or both proteins has been implicated in various cancers, and functional genomic screens have demonstrated a bidirectional synthetic lethal relationship between the two genes in tumor cells. Due to the high homology between EP300 and CBP, identifying chemical matter that selectively targets EP300 or CBP has proven challenging. Here, we describe a potent, highly selective heterobifunctional degrader of EP300 with biological activity in CBP-deficient and EP300-dependent tumor cells. Targeted degradation of EP300 protein resulted in a stronger suppression of cell growth and survival than targeting the bromodomain or HAT activity of EP300/CBP with small molecule inhibitors. Anti-proliferative effects have been demonstrated in multiple cancer types, including malignant lymphomas and castration-resistant prostate tumors, highlighting the essential role of EP300 in mediating oncogenic transcription required for tumor cell growth and survival. Degradation of EP300 in vivo attenuated androgen-driven transcription and inhibited tumor growth in VCAP (AR+) prostate tumor xenografts. Importantly, no evidence of overt toxicity or thrombocytopenia was observed at therapeutically efficacious doses. These findings indicate that selective targeting of EP300 with targeted protein degradation is a safe and effective treatment strategy for advanced tumors.

Citation Format: Mark Zimmerman, Ammar Adam, Hafiz Ahmad, Benjamin Adams, Ketaki Adhikari, Wesley Austin, Breanna Bullock, Julie Di Bernardo, Thomas Dixon, Danette Daniels, Claudia Dominici, GiNell Elliot, Brian Ethell, Anais Gervais, Md Imran Hossain, David Huang, Dave Lahr, Mei Yun Lin, David Mayhew, Karolina Mizeracka, Solymar Negretti, Tyler Nguyen, Olga Prifti, Darshan Sappal, Shawn Schiller, Brenna Sherbanee, David Terry, Nihan Ucisik, Elizabeth Wittenborn, Qianhe Zhou, Laura La Bonte. Discovery of potent and selective EP300 degraders with anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6064.