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The advent of receptor tyrosine kinase inhibitors (RTKi) are important contributions to treat NSCLC patients harboring genetic aberration effectively. Nevertheless, the emergence of acquired or intrinsic RTKi resistance by other RTK activation bypass finally limits their efficacy. First of all, in this study, we identified MED12 mutation in TKI-resistant NSCLC cells based-on NGS analysis. Although MED12 was known to controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling reportedly, we additionally found that MED12 mutation activates not only TGF-beta receptor not also other ones, such as EGFR, meaning that blocking TGF-beta receptor or EGFR alone may not effective to overcome MED12 mutation-induced RTKi resistance. To overcome the RTKi resistance in NSCLC harboring MED12 mutation, we elaborated the two downstream signaling of RTK in MED12 knock-out NSCLC cells and observed that PI3K/AKT signaling is downregulated but MEK/ERK signaling is upregulated. We observed that only MEK inhibitor (MEKi), not PI3K/mTOR inhibitor, shows effective anti-cancer effect. Therefore, we explored why MEKi alone is effective in RTKi resistant NSCLC cells harboring MED12 mutation and presented that MED12 mutation directly suppressed activation of YAP via blocking a large mediator complex of MED12, MED13, CDK8 and CCNC, thereby increasing PTEN expression by inhibition of miR-29 expression. Increased PTEN inhibits reactivation of PI3K/AKT pathway. In conclusion, we first identified that MED12 mutation induces RTKi resistance though activating other RTKs, such as TGF-beta receptor or EGFR, and blocks PI3K/AKT pathway via dysregulation of YAP/PTEN/AKT interaction. Therefore, among two downstream signaling of RTK, activated MEK/ERK pathway can be a definitive target enough to overcome RTKi resistant NSCLC patients harboring MED12 mutation and MEKi could be a primary treatment option for resistant patients harboring MED12 mutation among NSCLC patients with repetitive recurrence of RTKi resistance by another RTK activation bypass. Citation Format: Hyun-Min Ryu, Deokhoon Kim, Shinkyo Yoon, Ho-Su Lee, Eunjin Lee, Chang Hoon Lee, Wanlim Kim, Seyoung Seo, Kyung Hae Jung, Sook Ryun Park, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. RTK inhibitor resistance in NSCLC harboring MED12 mutation is overcome by only blocking MEK signaling, not AKT due to mutated MED12-induced YAP-PTEN dysregulation [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 424.

Background: Lymphoma is the most common hematological malignancy with non-Hodgkin B-cell lymphomas (NHL), including those with high unmet clinical need such as relapsed/refractory Diffuse Large B Cell Lymphoma (DLBCL), and Mantle-Cell Lymphoma (MCL), representing 90% of all lymphoma cases. CD19 is an attractive target for antibody-directed lymphoma therapies as it is expressed in most B cell malignancies, with normal tissue expression limited to B-cells thus reducing any on-target toxicity concerns. Antibody Drug Conjugates (ADCs) are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less toxicity than standard of care chemotherapies. IKS03 is an ADC comprised of an anti-CD19 antibody conjugated to a proprietary DNA-crosslinking PBD prodrug and includes a tumor-selective glucuronide-trigger technology for payload release and activation. ADC activity requires processing by beta-glucuronidase, a lysosomal enzyme often upregulated in tumor cells, while normal tissues with low levels of the enzyme are less able to process the ADC and are differentially spared. Methods: IKS03 was generated via chemo-enzymatic bioconjugation at defined sites on the antibody yielding an ADC with a drug to antibody ratio of 2. In vivo efficacy was evaluated in CD19-expressing cell line-derived xenograft models in mice including Farage (DLBCL, Germinal Centre B-cell subtype), OCI-LY10 (DLBCL, Activated B-Cell subtype), Granta-519 (MCL) and Ramos (Burkitt’s lymphoma). Activity was compared to benchmark ADCs known to have demonstrated clinical efficacy. Efficacy was also assessed in low passage DLBCL patient-derived xenograft models of known genomic profile. Toxicology studies were conducted in cynomolgus monkeys with immunophenotyping by flow cytometry included to quantify the level of normal B-cells following IKS03 administration. Results: IKS03 is highly effective in causing tumor regressions in DLBCL models at doses that are well tolerated. Complete regressions were observed with a single dose of 0.1 mg/kg in the Farage xenograft model. IKS03 is also highly active in the Granta-519 MCL model, with complete regressions observed with a single dose of 0.1 mg/kg. Tumor regression was observed in a high-grade triple-hit lymphoma PDX model with a single 0.3 mg/kg dose. IKS03 was tolerated in monkeys at the highest dose tested of 1.5 mg/kg (single dose). IKS03 cross reacts with monkey CD19 and reduced B-cell depletion was observed in relation to comparator agents, even at doses much greater (1.5 mg/kg) than that needed for complete responses in B-cell lymphoma mouse models (0.1 mg/kg). Conclusion: Preclinical data demonstrates that IKS03’s advanced ADC design results in an increased therapeutic margin compared to traditional ADCs with DNA-crosslinking payloads, with increased efficacy and decreased toxicity. Citation Format: Jenny Thirlway, Adam Lodge, Daniel J. Williamson, Justyna Mysliwy, Jutta Deckert, Xavier Chauchet, Laura Cons, Yun-Hee Park, Hyun-Min Ryu, Na Ra Han, Ho Young Song, Chul-Woong Chung, Robert J. Lutz. IKS03, a CD19-targeted antibody drug conjugate with enhanced efficacy and tolerability for treatment of B-cell lymphomas [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 1755.

PurposeHeat shock protein-90 (HSP90) remains an important cancer target because of its involvement in multiple oncogenic protein pathways and biologic processes. Although many HSP90 inhibitors have been tested in the treatment of KRAS-mutant non–small cell lung cancer (NSCLC), most, including AUY922, have failed due to toxic effects and resistance generation, even though a modest efficacy has been observed for these drugs in clinical trials. In our present study, we investigated the novel mechanism of resistance to AUY922 to explore possible avenues of overcoming and want to provide some insights that may assist with the future development of successful next-generation HSP90 inhibitors.Materials and MethodsWe established two AUY922-resistant KRAS-mutated NSCLC cells and conducted RNA sequencing to identify novel resistance biomarker.ResultsWe identified novel two resistance biomarkers. We observed that both integrin Av (ITGAv) and β3 (ITGB3) induce AUY922-resistance via focal adhesion kinase (FAK) activation, as well as an epithelial-mesenchymal transition, in both in vitro and in vivo xenograft model. mRNAs of both ITGAv and ITGB3 were also found to be elevated in a patient who had shown acquired resistance in a clinical trial of AUY922. ITGAv was induced by miR-142 downregulation, and ITGB3 was increased by miR-150 downregulation during the development of AUY922-resistance. Therefore, miR-150 and miR-142 overexpression effectively inhibited ITGAvB3-dependent FAK activation, restoring sensitivity to AUY922.ConclusionThe synergistic co-targeting of FAK and HSP90 attenuated the growth of ITGAvB3-induced AUY922-resistant KRAS-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome acquired AUY922-resistance in KRAS-mutant NSCLC.

Anti-PD-1/PD-L1 immunotherapy, as a treatment for many tumors, has shown good efficacy. However, responses to immunotherapy did not always occur or last long., i.e. primary or acquired resistance, even tumors were PD-L1 positive. Several oncogenic pathways, including PI3K/AKT activation by PTEN loss and NF-κB activation, induce PD-L1 expression and PD-L1 inhibitor-resistance. They also induce expression of CCL2, an inhibitory chemokine that blocks T cell tracking into the tumor by binding to CCR2 on the T cell surface. In this study, we showed that transglutaminase 2 (TG2), a post-translational modification enzyme, induced ubiquitin-proteasome dependent degradation of tumor suppressors including PTEN and IκBα by peptide cross-linking, inducing CCL2 as well as PD-L1 expression via PI3K/AKT and NF-κB activation. It also induced PD-L1 inhibitor-resistance because CCL2 was expressed despite increased PD-L1, which was blocked by PD-L1 inhibitor. We also revealed that inhibition of TG2, instead of PD-L1, restored T cell-dependent killing effect by blocking expression of both PD-L1 and CCL2 in PD-L1(+) triple negative breast cancer (TNBC) cells. In addition, the TG2-expressing TNBC patient group showed higher PD-L1 expression incidence than did the TG2-negative TNBC patient group. In conclusion, TG2 induces primary PD-1/PD-L1 inhibitor-resistance by inducing CCL2 expression. TG2 blockade can be utilized as an excellent therapeutic strategy to overcome PD-L1 inhibitor-resistance in PD-L1(+) TNBC patients. Our study suggested that PD-L1 expression alone might not always be a predictive biomarker for PD-L1(+) TNBC, but TG2 could be a useful predictive marker to select PD-L1 inhibitor-resistant TNBC patients.

This paper presents an efficient and eco-friendly soil stabilization method for binding the soil particles of rammed earth walls. The method uses a combination of a water-soluble acrylic acid–acrylic amide copolymer and an epoxy system containing a hardener. The results revealed that the unconfined compressive strength (UCS) of soil stabilized with the mixture of the copolymer and epoxy system was five times higher than that of soil treated with water and three times higher than that of stabilized soil (SS) with only the copolymer. The roles of the copolymer and epoxy system in UCS enhancement were determined based on morphology and apparent density (AD) and elucidated using proposed models. The soil stabilized with the copolymer and epoxy system exhibited the lowest AD and highest UCS. Further, its unique web-like morphology is consistent with the formation of an interpenetrating polymer network (IPN). The carboxylic and amide groups in the copolymer contribute to the formation of the IPN through interactions with the epoxy and amine groups of the hardener. The surface hydroxyl groups in the soil promote the coating of the polymer layer on the soil surface and the inter-particle cohesion, resulting in the formation of larger compacted lumps with a decreased AD and increased UCS. The hydrophilic copolymer acts as a stabilizer and plasticizer. The results confirmed that the epoxy system and copolymer significantly increased the UCS by forming an IPN. Thus, this study provides an efficient and eco-friendly water-soluble copolymer and epoxy system for enhancing the UCS of soil.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an oncofetal protein prevalently expressed in a variety of hematological and solid malignancies but largely absent in normal adult tissues, making it an attractive ADC target. CS5001/ABL202/LCB71 is an ADC composed of a human monoclonal antibody targeting ROR1, site-specifically conjugated with a proprietary cleavable β-glucuronide linker to a prodrug of PBD dimer. Both linker and prodrug are selectively cleaved by the lysosomal b-glucuronidase, which is overexpressed in many cancerous cells, to allow tumor-selective release of the DNA-crosslinking PBD dimer. In vitro and in vivo pharmacology of CS5001 were evaluated and benchmarked against CS5001-BMK1 (an MMAE-based ROR1 ADC). Binding specificity, affinity and internalization were determined by Octet, ELISA and flow cytometry. Cytotoxicity and predictive biomarkers were evaluated in 20 human cancer cell lines with various levels of ROR1. In vivo efficacy was studied in Jeko-1 mantle cell lymphoma and MDA-MB-231 triple-negative breast cancer xenografts. CS5001 bound to human ROR1, but not ROR2, with a KD value of 1.38 nM. CS5001 has cross reactivity against mouse, rat and cynomolgus ROR1 at similar affinities. Upon binding, CS5001 was rapidly internalized by ROR1-expressing cancer cells at 37°C. CS5001 demonstrated potent cytotoxicity towards ROR1 high expressing cell lines such as Jeko-1 and MDA-MB-231, with an IC50value of 0.161 and 0.900 nM, whereas the IC50 value of CS5001-BMK1 was 10.8 nM and 129 nM, respectively. The growth inhibition activity of CS5001 but not CS5001BMK was significantly correlated with ROR1 density (Pearson correlation= -0.697, P = 6.4 x 10-4). CS5001 potently induced DNA damage in MDA-MB-231 cells, and resulted in increased apoptosis and G2-M cell cycle arrest in a concentration-dependent manner, indicating a mechanism of cytotoxicity consistent with that of the PBD payload. CS5001 exhibited prominent antitumor activity in both Jeko-1 and MDA-MB-231 xenograft models in a dose-dependent manner. In Jeko-1, complete regression (CR) was observed after a single administration of CS5001 at 1mg/kg (approximate 1/5th the maximum tolerated dose (MTD) in mice), whereas no CR was achieved for CS5001-BMK1 at the highest dose (2.5mg/kg, QWx3). When both CS5001 and CS5001-BMK1 were administrated at 1/20th of their respective MTDs, CS5001 (0.25mg/kg, single dose) produced significantly superior efficacy to CS5001-BMK1 (2.5mg/kg, single dose) with tumor growth inhibition (TGI) of 60% and 38%, respectively (p=0.018). In MDA-MB-231, 106% and 65.7% of TGI were observed for CS5001 at 1mg/kg (single dose) and CS5001-BMK1 at 2.5mg/kg (QWx3) respectively. CS5001 exhibited potent and selective cytotoxicity to a variety of ROR1-expressing cell lines and showed remarkable in vivo antitumor activity. ROR1 cytometric density predicts sensitivity to CS5001 in vitro. CS5001 is a promising therapeutic candidate for ROR1-expressing hematological and solid malignancies with precision medicine potential. Citation Format: Fu Li, Yongwang Li, Lan Zhang, Clarence K. Zhang, Hui-Han A. Hu, Juan Zhang, Ying Pan, Jinwon Jung, Sang Hoon Lee, Hyun-Min Ryu, Yun-Hee Park, Haixiang Yu, Archie N. Tse. CS5001, a novel ROR1-targeting antibody drug conjugate (ADC) armed with tumor-cleavable β-glucuronide linkers and pyrrolobenzodiazepine (PBD) prodrugs for hematological and solid malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA008.