Abstract IA004: Overcoming traditional design limitations with AI-based discovery

The majority of secreted and transmembrane (TM) proteins, including growth factors and TM oncogenes, require translocation through Sec61 into the ER for further processing. Sec61, therefore, represents a unique drug discovery opportunity through blockade of typically “undruggable” targets and modulation of protein homeostasis. Multiple inhibitors of protein secretion have been described that directly target Sec61 and have anti-tumor activity but lack adequate pharmaceutical properties and/or tolerability to be considered clinical candidates. We aimed to discover novel, selective inhibitors of Sec61 with increased tolerability as a potential treatment for various malignancies. Sec61 inhibitors were identified using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Selective anti-tumor activity was assessed via HEK293 reporter cell viability in comparison to tumor cell lines with known levels of sensitivity to published Sec61-modulating compounds. An initial screen resulted in KZR-4152 (4152), which blocked secretion of several target proteins but had no impact on tumor cell viability. Following a medicinal chemistry campaign, KZR-8834 (8834) was identified, which exhibited a 60-fold increase in potency against target proteins relative to 4152. On-target activity of 8834 was demonstrated via protein secretion assays in cells overexpressing a previously described Sec61 mutant with resistance to published compounds. In addition, 8834 did not affect the viability of HEK293 cells or endotoxin- or TCR-stimulated PBMCs. 8834 induced cell death in H929 multiple myeloma cells in vitro (IC50 = 98 nM) with rapid activation (≤8 hours) of caspase 3/7 via dual activation of caspases 8 and 9. When assessed in a panel of 346 human cancer cell lines across 25 different tumor types, head and neck squamous cell carcinoma, acute lymphocytic leukemia, non-Hodgkin’s lymphoma, melanoma, multiple myeloma, and prostate cancer were among the most sensitive tumor types. Weekly and twice-weekly administration of 8834 was effective in an H929 mouse xenograft model at doses that resulted in >90% tumor growth inhibition without significant body weight loss or clinical signs of toxicity. Anti-tumor activity was >50% at doses 1/3rd of the maximum tolerated dose, suggesting a broad therapeutic index. 8834 was also effective in the B16F0 melanoma model, showing tumor growth inhibition similar to that of anti-PD1 therapy. KZR-8834 is a novel, small molecule inhibitor of Sec61-dependent translocation of multiple Sec61 client proteins. 8834 exhibits a broad anti-tumor profile in vitro and therapeutic activity in multiple mouse tumor models, demonstrating a potential new therapy for the treatment of malignant diseases. Citation Format: Eric Lowe, Janet L. Anderl, Andrea R. Fan, Jing Jiang, Henry W. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Jack Taunton, Jennifer A. Whang. KZR-8834: A novel, small molecule inhibitor of Sec61-dependent protein secretion with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3087.

Preclinical assessment of direct drug-target engagement and analysis of binding kinetics within the cellular environment is essential for development of safer and more effective therapeutics. Infusion of such critical data into early drug discovery would significantly reduce the failure rate of new drug candidates, accelerate drug discovery, and validate repurposing of existing drugs. Described here is a novel drug-target engagement technology that can sensitively interrogate direct binding of drugs to cellular, bacterial, or viral protein targets within the physiological environment. Micro-Tag cell target engagement technology is based on complementation of a small 15-amino acid subunit with a large subunit into an active RNA-processing enzyme. The small subunit can be cloned to any drug target for transient or stable expression using existing tools such as CRISPR. Upon complementation, the active enzyme cleaves a FRET-based oligonucleotide substrate resulting in rapid generation of fluorescent signal that can be quantified in real time. The Micro-Tag technology enables in-cell quantitation of drug target levels using qPCR systems. This is the next-generation of target engagement technology that allows for real-time monitoring of drug-target interaction in the cell. We show here data demonstrating direct engagement of several reference compounds and novel small molecules with high-profile cancer targets such as K-RAS, MTH1, EGFR, and UBE2N. Selectivity of these drugs to the targets in the cell is further delineated using their mutant counterparts as well as inactive stereoisomer compounds. The Micro-Tag cell target engagement technology provides the power of cell target engagement to a large family of target proteins. It can be employed for high-throughput screens directed at initial on-target ranking or med-chem optimization of drug candidates. It is amenable for interrogation of drug candidates across various modalities: small molecules, peptides, antibodies and PROTACs. Importantly, this next-generation target engagement technology seamlessly integrates with qPCR systems, fluorescence microscopy, live-cell microscopy, FACS analysis, and offers multiplexing capability, thus allowing for further mechanistic insight. Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. Real-time cellular target engagement and protein quantification for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2759.

Discovering drug candidates that exhibit durable on-target pharmacology upon oral dosing has been extremely challenging for the pharmaceutical industry. The introduction of a covalent bond between a small molecule drug and its pharmacological target has the potential to achieve prolonged target inhibition with minimal systemic drug exposure. Several irreversible covalent inhibitors employing this strategy have been approved or are in late stage clinical development for the treatment of various cancer indications such as multiple myeloma (carfilzomib), lymphoma (ibrutinib), lung cancer (afatinib) and breast cancer (neratinib). However, the discovery process for covalent drugs presents unique challenges. Identification and optimization of lead molecules require screening assays that measure the degree of covalent engagement with the target, both in vitro and in vivo. In addition, the reactive chemistries often employed for covalent bonding can impart poor ADME properties, requiring IV administration or high oral doses. Principia Biopharma, a pioneer in covalent drug discovery and development, has overcome many of these challenges in the discovery of PRN1371, a covalent irreversible inhibitor of fibroblast growth factor receptor (FGFR) 1-4 with long target residence time. Aberrant signaling of the FGF/FGFR pathway is an oncogenic driver in many solid tumors. Early reports from Phase 1/2 studies with reversible ATP competitive inhibitors have demonstrated encouraging efficacy from FGFR inhibition in the treatment of bladder, lung, and breast cancers. However, some results are difficult to interpret due to the poor selectivity of the drugs used. Aberrant pathway activation may be a consequence of FGF ligand overexpression or changes to the FGF receptor including mutations, translocations/fusions, and protein overexpression. Such genetic alterations in one or more isoforms of FGFR have been reported in many solid tumors including bladder, breast, cervical, endometrial, gastric, hepatocellular, lung, and ovarian cancers. There remains a compelling medical need to identify a highly selective inhibitor of all four FGFR family members, FGFR1-4, to improve clinical responses in patients while minimizing off-target toxicity. PRN1371 is the culmination of a drug discovery program focused upon the creation of an oral, low dose, highly selective, potent, and irreversible inhibitor of FGFR1-4. Starting from a 7-oxopyridopyrimidine core, a series of covalent inhibitors were designed to incorporate a Michael acceptor appropriately positioned to engage Cys 488 in a covalent bond. Extensive SAR was generated using a FRET-based biochemical occupancy assay to measure the durability of drug binding over time. Molecules were optimized for prolonged FGFR residence time and potent inhibition of pERK in HUVEC cells upon FGF stimulation. Advanced leads with suitable cellular potency were studied in vivo, using an FGF-induced CCL2 readout in mice. The relationship between plasma drug concentration (PK) and pharmacological inhibition of CCL2 production (PD) in mice provided essential guidance in selecting optimal drug candidates. To assess the ADME liabilities of the Michael acceptor, in vitro microsomal stability and thiol reactivity assays were used. The final selection of PRN1371 as a clinical candidate was based upon the ideal combination of exquisite selectivity, potent and prolonged pharmacodynamic inhibition of FGFR1-4 in vivo and high predicted human oral bioavailability. PRN1371 is now evaluated in a Phase 1/2 open-label, multicenter clinical trial for solid tumors (ClinicalTrials.gov: NCT02608125). Citation Format: Ken A. Brameld. Discovery of the highly selective covalent FGFR1-4 inhibitor PRN1371, currently in development for the treatment of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY30-01.

The transcription factor and tumor suppressor protein p53 regulates cellular outcomes (e.g. cell cycle arrest, senescence or apoptosis) following DNA damage. Cellular outcomes can be influenced by modulating the temporal expression dynamics of p53, which in turn alters target gene mRNA expressions patterns. However, it is not clear how changes in such mRNA dynamics would eventually results in differences in temporal protein expression dynamics, which ultimately determines the cell outcome. By quantifying mRNA and protein by RNA-Seq and mass-spectrometry, respectively over time, we systematically categorized the different dynamics which could arise of p53 target mRNAs and proteins. We show that while p53 target proteins plateaus under pulsatile dynamics, the majority of target proteins are enhanced and accumulates under sustained dynamics including many regulators of terminal cell fates such as apoptosis and senescence. Next we derived synthesis and degradation rates based on empirical measurements to model how different p53 dynamics could lead to the assortment of subsequent target protein dynamics. We show that target protein dynamics are highly governed by protein degradation rate, implying the temporal selection of target gene expression is not pre-determined by the initiating stress signal, but rather through differences in the inherent protein degradation rates. Furthermore, we identify multiple unique signaling network motifs which results in target protein accumulation only under sustained (persistent) but not pulsatile (oscillatory) p53 dynamics. Our results highlight the diversity of mechanisms cells evolved to decode transcription factor dynamics as well as the role of p53 in governing cellular outcome, important both in understanding cellular information processing as well as therapeutic implications for cancer treatments. Citation Format: Dan Lu, Alba Jimenez, Ashwini Jambhekar, Galit Lahav. Oscillating p53 temporal dynamics enable proliferative recovery of cells following DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2159.

Calpain-1 and calpain-2 are heterodimeric calcium-dependent cysteine proteases composed of the catalytic subunits CAPN1 or CAPN2, respectively, and the common regulatory subunit CAPNS1. They are associated with cancer progression, metastasis, and treatment resistance in breast cancer. Here, we present novel insights into the therapeutic potential of calpain inhibition by combining genetic disruption and preclinical mouse tumor models and biosensor-based detection of calpain heterodimerization with ongoing efforts to discover small-molecule and peptide inhibitors. CRISPR-Cas9-mediated knockout of CAPN1, CAPN2, or CAPNS1 in MDA-MB-231 human triple-negative breast cancer (TNBC) cells revealed that disruption of both calpains, through CAPNS1 knockout, significantly reduced cell migration and inhibited spontaneous metastasis in a mouse orthotopic engraftment model by over 80%. Individual knockouts of CAPN1 or CAPN2 also reduced metastasis but to a lesser extent, highlighting the necessity of dual inhibition for optimal therapeutic effect. In parallel, we have been actively seeking small molecules and peptide inhibitors to target calpain through two approaches: inhibiting the PEF-PEF interaction that mediates heterodimerization using small molecules; and targeting the active site using a calpastatin (CAST)-based peptide. To identify small molecules capable of disrupting the PEF-PEF interaction, we conducted in silico screens of over 3.6 million compounds from several libraries. These compounds were evaluated based on their calculated binding affinity and potential to sterically hinder the conformational changes required for calpain activity. The CAST-based peptide inhibitor was designed based on the active site binding B-domain and tested on a purified calpain-2. These efforts lay the groundwork for novel therapeutic approaches targeting calpain-1 and calpain-2, which have emerged as promising targets for preventing metastasis in TNBC. We will present our progress in identifying and validating these small molecules and peptides for further development. Citation Format: Ivan Shapovalov, Pitambar Poudel, Shailesh K. Panday, Danielle Harper, Jung Yeon Min, Yan Gao, Kazem Nouri, Emil Alexov, Peter A. Greer. Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A016

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

63 Background: Worldwide, EBV is associated with ~10% of gastric cancer. Currently, no pharmaceutical-based therapies exist that selectively target EBV associated gastric cancers. EBV, in its latent oncogenic form, is dependent on the continuous expression of Epstein-Barr Nuclear Antigen 1 (EBNA1), a multifunctional dimeric protein critical for viral replication, genome maintenance and viral gene expression. Methods: The aim of this program is to advance the development of a New Chemical Entity (NCE) for latent infection of Epstein-Barr Virus (EBV) to treat EBV-associated gastric cancer. We have used structure-based drug design and medicinal chemistry methods to identify and develop a small molecule clinical candidate that selectively inhibits the DNA-binding activity of EBNA1. Results: The clinical candidate inhibits EBNA1 function with nanomolar potency in biochemical assays and low micromolecular activity in several cell-based assays. We demonstrate that our clinical candidate provides protection in 4 different xenograft models of EBV-driven tumor growth, including patient-derived xenografts. Furthermore, RNA analysis experiments confirm in vivo target engagement by the elimination of EBV in treated tumor tissue. The clinical candidate is selective, showing no activity in an EBV-negative xenograft experiments. The clinical candidate has met industry-accepted criteria for drug suitability, safety and toxicology including physicochemical properties, metabolic stability, selectivity in broad-based screens and bioavailability, and lack in vivoliabilities, including genotoxicity and 14-day toxicity studies. Conclusions: IND-enabling studies including safety pharmacology and toxicology and GMP manufacturing have begun with a projected First-In-Human clinical trial in Q4 2017. These data establish proof-of-concept for targeting EBNA1—a protein previously thought to be undruggable.

Nanotechnology offers unique advantages over small-molecule drugs by delivering a high payload of photosensitizers to the target tissue, providing flexibility in the excitation wavelengths, enabling longer retention in the target tissue, possessing regenerative reactive oxygen speciesproducing capability and stimulating the immune system.

The MYC oncogene, a pivotal regulator of various cellular processes, is deregulated in approximately 70 % of human malignancies. Mitotic Kinesin-like Protein 2 (MKLP2) plays a versatile role in both interphase and mitosis and has emerged as a significant prognostic indicator and therapeutic target in cancer. The undruggability of MYC and the scarcity of MKLP2 inhibitors, however, have impeded clinical translation. Our development of JMBI-001, a potent and orally bioavailable small-molecule compound, overcomes these barriers. JMBI-001 elicits loss of function phenotypes in MKLP2 and a synthetic lethal interaction with MYC overexpression. Extensive kinome and safety profiling have revealed no significant off-target effects, and the compound is well-tolerated in long-term animal studies. In preclinical models, JMBI-001 has demonstrated an average tumor growth inhibition rate of 75 % across more than 20 MYC-overexpressing tumor models, including those in the stomach, lung, colon, liver, breast, kidney, skin, and hematopoietic system. Its anti-tumor activity positively correlates with high MYC abundance, aligning with the selective eradication of cells with abundant MYC both in vitro and in vivo. Notably, JMBI-001 also robustly stimulates systemic anti-tumor immunity, enhancing NK and CD3+ T cell infiltration in tumors of syngeneic cancer models. Moreover, additive or synergistic effects have been observed when combined with anti-PD1 therapy even in tumors refractory to the immune checkpoint blockade (ICB) therapy. The dual therapeutic actions of JMBI-001 stem from its disruption of MKLP2 functionalities, leading to anomalies, such as Golgi fragmentation in interphase and multipolarity in pro-metaphase. These disruptions lead to two key outcomes: apoptosis and immunogenic cell death, marked by secreted ATP, released high mobility group protein B1 (HMGB1), and surface-exposed calreticulin. These abnormalities, primed and amplified by deregulated MYC, are not observed in non-transformed cells, suggesting their potential as pharmacodynamic markers for monitoring JMBI-001's activity in vivo. In conclusion, JMBI-001 represents a novel class of anticancer agents that simultaneously triggers MYC synthetic lethality and anticancer immunity by targeting MKLP2-mediated cellular processes. Its unique mechanisms of action, exceptional bioavailability, potency at low nanomolar concentrations, wide-spectrum efficacy, and favorable safety profile establish JMBI-001 as a promising clinical study-ready drug candidate for treating MYC-driven cancers. Citation Format: Ting Zhang, Qiong Shi, Julia Kalashova, Xumei Liu, Xiaohu Zhou, Chenglu Yang, Yan Long, Hongmei Li, Jinhua Li, Gang Lv, Duo Yu, Xuejiao Jiang, Shenqiu Zhang, Jing Zhang, Hong Liu, Dun Yang. An orally available small molecule JMBI-001 elicits MYC-synthetic lethality and anti-tumor immunity by disabling MKLP2-mediated cellular processes [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 5802.

SMARCA2 and SMARCA4 are essential, redundant, catalytic subunits of the multi-subunit BRG1/BRM-associated factor (BAF) complex. This complex regulates gene expression and DNA repair through chromatin remodeling activity. The paralogues SMARCA2 and SMARCA4 bind acetylated histones through their highly conserved bromodomains and alter DNA-histone contacts using ATP-dependent helicase activity. Recently, mutations in specific components of the BAF complex have been identified. Of great interest are loss of function mutations in SMARCA4, found in 5-7% of all human cancers and occur with high prevalence (∼10%) in non-small cell lung cancer (NSCLC), making these SMARCA4-deficient tumor cells highly dependent on SMARCA2 for viability. This synthetic lethal relationship may provide a unique opportunity for SMARCA2-selective degraders to block the growth of SMARCA4 deficient tumors, while sparing normal tissues. To enable degrader discovery, we focused our efforts on a monovalent direct degrader approach, where small molecules are designed to bind the target protein and induce its degradation through the recruitment of an E3 ligase complex. Initial screening hits from distinct chemical series were optimized to produce potent and selective SMARCA2 degraders. Co-treatment with a proteasome inhibitor confirmed that degradation was mediated via the ubiquitin proteasome system (UPS), and a UPS-focused CRISPR screen identified the E3 ligase responsible for compound-induced degradation of SMARCA2. Cell based protein-protein interaction (PPI) studies advanced our understanding of compound-mediated E3 ligase recruitment and provided mechanistic insights into the observed selectivity for SMARCA2 over SMARCA4. Ternary complex dynamics were further investigated in vitro via surface plasmon resonance (SPR) studies. Using a combination of co-immunoprecipitation, mass spectrometry, and point mutant studies, specific amino acids that are required for compound-induced PPI and subsequent degradation of SMARCA2 were identified. Collectively, these results highlight the discovery and characterization of novel small molecule monovalent direct degraders of SMARCA2. The detailed description of the degradation mechanism presented here provides critical insights into an efficient approach to eliminating pathogenic proteins through targeted protein degradation. Citation Format: Julia Toth, Gregory Parker, Geoffray Leriche, Tayor Kampert, Aleksandar Jamborcic, Gabrielle Blanco, Kenneth Steadman, Duc Tran, Shu You, Luis Lopez, Farhana Barmare, Kyohei Hayashi, Gang Liu, Alex Campos, Meg McCarrick, Kevin Freeman-Cook, Peggy Thompson. Mechanistic characterization of selective monovalent direct degraders of SMARCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 404.

Targeted protein degradation (TPD) technology has been developed showing the therapeutic potential to modulate historically ‘undruggable’ disease-associated proteins. Currently, proteolysis targeting chimeras (PROTACs) using small-molecule binders represent the TPD field, and PROTAC-induced proximity between target proteins and E3 ligases enables specific degradation of target proteins. However, substantial amounts of clinically relevant cancer targets such as transcription factors lack active sites or allosteric binding pockets for small molecules which challenges the development of classical PROTACs for these targets. SOX2 is one of the pluripotency transcription factors and has been well-known for its crucial role in tumor development through promoting cancer cell survival, metastasis, stemness, and drug resistance. Numerous studies have also reported SOX2 amplification and overexpression in various tumor tissues. Thus, SOX2 has been validated as an attractive anticancer target, but directly targeting SOX2 has been proved to be difficult since SOX2 is an “undruggable” transcription factor. EPD Biotherapeutics develops EPDegTM, a next-generation engineered protein degrader platform in order to overcome the limitations of small molecule-based PROTAC. Here we report a series of SOX2-targeting bioPROTACs, being composed of a nanobody against SOX2 with high affinity and several ubiquitin E3 ligases with deletion of natural substrate binding domain to allow highly selective degradation of SOX2 protein. To evaluate the in vitro efficacy, SOX2 bioPROTACs were generated by mRNA IVT with CleanCap ® and modified uridine. First, robust degradation of SOX2 with a half-maximal degradation concentration (DC50) of the picomolar range was observed with the treatment of SOX2 bioPROTACs in multiple SOX2-overexpressing cancer cell lines. The degradation was ubiquitin- and proteosome-dependent manner, and neither nanobody nor ubiquitin E3 ligase alone affected SOX2 protein level. Then, SOX2 bioPROTACs led to significant cancer cell growth inhibition caused by apoptotic cell death with a half-maximal growth inhibition concentration (GI50) of the picomolar range. Notably, chemo-resistant cancer cell lines expressing SOX2 protein in high levels were sensitized to chemotherapy in combination with our SOX2 bioPROTACs. Furthermore, i.v. injection of one of our lead candidates formulated with LNP significantly inhibited tumor growth in the A549 xenograft model, proving the in vivo anti-tumor efficacy of SOX2 degraders for the first time in the world. Taken together, EPD’s SOX2 bioPROTACs show potent in vitro and in vivo anti-tumor efficacy as first-in-class SOX2 degraders and provide new insights for the therapeutic potential of transcription factor-degrading strategies. Citation Format: Jaehyun Choi, Saeyi Lim, Xuelian Bai, Hyerin Song, Yeonji Oh, Yeryoung Yong, Seunghyun Lee. Novel SOX2-targeting bioPROTACs for the treatment of cancers [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 3256.

Targeting PRMT5 in MTAP-deleted tumors in a synthetic lethal approach represents a promising antitumor strategy across many tumor types. Metabolic gene MTAP is localized at the 9p21 chromosome in the close proximity to CDKN2A tumor-suppressor locus. Co-deletion of MTAP may be observed in 80-90% of all tumors harboring homozygous deletion of CDKN2A, which represents 10-15% of all human tumors. MTAP deletion results in a massive accumulation of methylotioadenosine (MTA) in cells. MTA in high concentrations is a selective inhibitor of PRMT5 type II methyltransferase. PRMT5 conjugated with WD-repeat containing protein (WDR77) builds methylosome, which regulates essential cellular functions via symmetric demethylation (SDMA) of target proteins involved in regulation of gene expression, RNA splicing, signal transduction, metabolism and other functions. Accumulation of MTA in cells with MTAP deletion causes a partial inhibition of the methylation activity of PRMT5, which in turn reduces the level of symmetric arginine dimethylation of the whole proteome, and thus an increased sensitivity of cells to modulation of the methylosome activity. Therapeutic targeting of PRMT5 in homozygous MTAP-deleted cancers constitute a promising strategy of selective killing of genetically defined cancer cells. Currently available clinical stage PRMT5 small molecule inhibitors are not MTA-cooperative and therefore are not selective in tumors harboring MTAP deletion. Here we present MTA-cooperative PRMT5 inhibitors, which selectively inhibit the growth of MTAP deleted cancer cells. Ryvu has identified a series of MTA-cooperative PRMT5 inhibitors which have good drug-like physicochemical properties and block methyltransferase activity with nanomolar IC50 values. Ryvu compounds selectively inhibit growth of MTAP deleted cancer cells in prolonged 3D culture, which strongly correlates with inhibition of PRMT5-dependent protein symmetric demethylation (SDMA) in those cells. Selectivity between effects observed in MTAP deleted and WT cells exceeds 100-fold both for SDMA and growth inhibition. The DMPK profile of these compounds allows for oral administration, which enables testing dose-dependent antitumor activity in MTAP null tumor xenograft-bearing mice. Overall, these studies provide rationale for further optimization of chemical series towards clinical candidate. Citation Format: Oleksandr Levenets, Anna Bartosik, Marta Sowińska, Karol Zuchowicz, Sujit Sasmal, Klara Korta-Piątek, Adam Radzimierski, Paulina Niedziejko, Oleksandr Popika, Mateusz Świrski, Agata Stachowicz, Maciej Mikulski, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Kinga Michalik, Kamil Kuś, Monika Madej, Adrian Podkowa, Karolina Gluza, Grzegorz Satała, Ewelina Cieluch, Dobrosława Krzemień, Andrzej Gondela, Grzegorz Ćwiertnia, Marek Wronowski, Nicolas Boutard, Aleksandra Więckowska, Joanna Zezula, Justyna Jabłońska, Mirosława Gładysz, Igor Tomczyk, Jacek Faber, Marcin Serocki, Eliza Drwal, Kamila Kozłowska-Tomczyk, Marta Skoda, Martin Swarbrick, Krzysztof Brzózka, Mateusz Nowak. Discovery of novel MTA-cooperative PRMT5 inhibitors as a targeted therapeutics for MTAP deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1806.

Oncofusions, cancer-causing chimeric proteins formed by chromosomal rearrangements, are key drivers in approximately 16% of all cancer cases. These fusion proteins are associated with a wide range of malignancies, including varieties of leukemia, lung cancer, and sarcoma. Thousands of unique oncofusions have been identified in recent years, yet most of these fusion proteins lack mechanistic annotation. Many oncofusions dysregulate chromatin architecture in complex ways that are not yet fully elucidated. Investigating these chromatin alterations is both labor-intensive and costly, limiting the feasibility of large-scale studies. This lack of mechanistic insight has hindered the development of targeted therapies, with only a handful of efficacious small-molecule inhibitors directed toward oncofusions currently in clinical trials or approved for patient use. To address this critical gap, we adapted our recently developed PROD-ATAC method into a high-throughput, scalable platform to systematically elucidate the effects of dozens of candidate inhibitory small molecules on the chromatin dysregulation profiles induced by a pooled library of diverse oncofusions. In the PROD-ATAC scheme, a pooled protein-coding variant library is expressed in a single disease-agnostic cell line, and scATAC-seq (single-cell assay for transposase accessible chromatin) catalogs each variant’s effect on the chromatin landscape. We applied PROD-ATAC to simultaneously assay the chromatin regulatory impact of >40 small molecule inhibitors against a panel of tens of oncofusions (including the majority of patient-identified fusion sequences causal for Ewing sarcoma) in a single assay, generating genome-wide chromatin accessibility datasets representative of >1,000 unique drug - fusion interactions. In this manner, we characterized several small molecules as previously unidentified inhibitors of multiple oncofusions. Certain inhibitors were found to abrogate the chromatin dysregulation caused by an oncofusion either globally or in a targeted cell signaling pathway-specific manner. By profiling multiple rare fusions causal for the same cancers, we identified separate instances of homogenous and heterogenous modes of chromatin dysregulation associated with distinct fusion proteins. In this work, PROD-ATAC chromatin accessibility readouts coupled the identification of novel oncofusion inhibitors with the characterization of each inhibitor’s impact on the epigenetic landscape of oncofusion-expressing cells. Citation Format: James E. Corban, Max Frenkel, Srivatsan Raman. High-throughput characterization of inhibitor impacts on chromatin dysregulation induced by oncofusions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A024.

Introduction: Activating mutations of EGFR are well known as oncogenic driver mutations in lung adenocarcinoma. Currently, EGFR TKIs including Gefitinib and Erlotinib are used as the first line therapy in NSCLC patients harboring EGFR activating mutations. However, drug resistance caused by T790M mutation limits the efficacy of these 1st generation EGFR TKIs. Currently, some of the next generation EGFR TKIs are under investigation for the treatment of lung cancer patients having T790M mutation. In our current presentation, to obtain HM61713, an EGFR mutant selective inhibitor, as a clinical candidate and the evaluation of HM61713 for mutant EGFR cancer model will be introduced. Method: Novel analogues were designed and synthesized to find active compounds for the T790M mutation as well as EGFR activating mutations with good selectivity over wild- type EGFR. Finally, HM61713 was selected as a clinical candidate through multi-optimization processes including both in vitro and in vivo pharmacologcal studies. Results: HM61713 was designed as an irreversible kinase inhibitor having a Michael acceptor, which covalently binds to a cysteine residue near the kinase domain of mutant the EGFR. In a cell wash out test, HM61713 inhibited phospho-EGFR for a long duration with a half-life of over 24 hours. From in vitro study, HM61713 showed potent activities for H1975 (L858-T790M) and HCC827 (exon 19 del.) with GI50 values of 9.2 nM and 10 nM, respectively. Instead, it showed low potency for H358 (wild type EGFR NSCLC) with GI50 of 2,225 nM. In xenograft studies using H1975 and HCC827, HM61713 resulted in good efficacy without showing any side effects. Conclusion: HM61713 showed excellent in vitro and in vivo activities for H1975 harboring L858R-T790M mutation as well as HCC827 having exon 19 deletion mutation with selectivity over wild-type EGFR. Currently, HM61713 is undergoing phase I study (NCT01588145) for NSCLC patients after the failure of 1st generation EGFR TKIs in Korea. Citation Format: Kwang-Ok Lee, Mi Young Cha, Mira Kim, Ji Yeon Song, Jae-Ho Lee, Young Hoon Kim, Young-Mi Lee, Kwee Hyun Suh, Jeewoong Son. Discovery of HM61713 as an orally available and mutant EGFR selective inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2014-LB-100

Mesothelin (MSLN) is a cell surface glycoprotein widely expressed in a variety of cancers, with prevalence levels by IHC of 85% in ovarian, 75% in pancreatic and 45% in mesothelioma, and normal expression limited to the mesothelia, suggesting it could be an ideal target for antibody-drug conjugate therapy of these cancers. We have generated a high affinity (subnanomolar), humanized antibody to MSLN and conjugated it to auristatin anti-mitotic drugs (monomethylauristatin E and F, Seattle Genetics) via an uncleavable linker (anti-MSLN-mc-MMAF) or a cathepsin-cleavable valine-citrulline linker (anti-MSLN-mc-vc-PAB-MMAE/F) for comparison. The in vivo efficacy obtained in an ovarian transplant model was superior with the anti-MSLN-MMAE ADC. Anti-MSLN-MMAE was specifically internalized by MSLN-expressing cells in vitro, resulting in cell death compared to control ADCs. Pancreatic, ovarian and mesothelioma tumor cell lines endogenously expressing physiological levels of MSLN were identified and established as xenografts in mice. A single dose of anti-MSLN ADC was sufficient to inhibit or shrink tumor growth in models of each of the three indications in vivo, as well as inducing complete regressions in primary human pancreatic models, even those expressing low levels of MSLN typical of most human pancreatic tumors. Additionally, anti-MSLN-MMAE (at suboptimal doses) appeared to synergize with gemcitabine at clinically relevant doses in an HPAC xenograft model. Furthermore, anti-MSLN-MMAE was well tolerated in non-clinical toxicity studies (see accompanying abstract by Gupta et al.). Our data suggest that anti-MSLN-vc-MMAE is a promising clinical candidate for the treatment of several types of mesothelin-positive cancers. Citation Format: Suzie J. Scales, Nidhi Gupta, Glenn Pacheco, Ron Firestein, Dorothy M. French, Josefa Chuh, Yin Zhang, Leanne Berry, Jenny Bostrom, Elizabeth Luis, Aimee Fourie O'Donohue, Katherine R. Kozak, Sarajane Ross, Mark S. Dennis, Jay Tibbitts, Susan D. Spencer. A clinical candidate anti-mesothelin-MMAE antibody-drug conjugate (ADC) for therapy of mesothelin-expressing cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2014-4494