Non-small Cell Lung Cancer Xenograft Models Research Articles

Abstract B025: The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title>

Abstract KRAS is the most frequently mutated RAS family member. Within non-small cell lung cancer (NSCLC), G12C is the most prevalent KRAS mutation (~13%) and the KRAS G12C inhibitors (G12Ci) sotorasib and adagrasib are now FDA approved. However, it has been shown that simultaneous targeting of multiple nodes in the MAPK pathway may be optimal for deeper and more durable responses. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on sotorasib or adagrasib, altogether supporting the need for clinical combinations with G12Ci. Based on the clinical success of G12Ci, several KRAS G12D inhibitors (G12Di) are now being developed as G12D is the most prevalent KRAS mutation across human cancers including pancreatic (~28%) and colorectal (~11%) cancers. Avutometinib (VS-6766; avuto) is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avuto to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Here, we tested whether addition of avuto to G12Ci or G12Di could improve MAPK pathway blockade and anti-tumor efficacy. Avuto was synergistic with sotorasib and adagrasib in reducing viability of a panel of KRAS G12C cancer cell lines in 3D culture, and the combination of avuto + G12Ci showed improved depth and duration of MAPK pathway inhibition, stronger inhibition of cell cycle/proliferation markers and stronger activation of pro-apoptotic markers relative to G12Ci alone. In mouse models, avuto enhanced efficacy of G12Ci in KRAS G12C NSCLC xenograft models. Furthermore, avuto potentiated G12Ci efficacy in a KRAS(+/G12C);p53null NSCLC GEMM model with avuto + G12Ci combination inducing complete response in 25% of the mice. To evaluate efficacy of avuto against KRAS mutations that have been observed clinically upon acquired resistance to G12Ci, proliferation of Ba/F3 cells expressing these mutations was assessed. While avuto potently inhibited (IC50 < 15 nM) proliferation of cells bearing KRAS G12C/R68S, G12C/H95D or G12C/Y96C mutations, adagrasib showed diminished potency (IC50 ≥ 85 nM) against all 3 of these mutations and sotorasib showed diminished potency (IC50 ≥ 150 nM) against G12C/R68S and G12C/Y96C. Accordingly, avuto strongly inhibited tumor growth of an in vivo model expressing KRAS G12C/Y96D, while sotorasib was ineffective. Similarly, avuto was found to be synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D cancer cell lines, and avuto enhanced anti-tumor efficacy of MRTX1133 in KRAS G12D colorectal (CR3300) and pancreatic (PA1252) cancer PDX models. These results support ongoing clinical studies of avutometinib in combination with sotorasib (NCT05074810) and adagrasib (NCT05375994) for patients with KRAS G12C NSCLC. Additionally, these preclinical data support combination of avutometinib with a G12D inhibitor for patients with KRAS G12D colorectal or pancreatic cancers. Citation Format: Silvia Coma, Monica Musteanu, Cristina Caffarra, Alessia Mira, Enrico Patrucco, Julien Dilly, Andrew J. Aguirre, Mariano Barbacid, Chiara Ambrogio, Jonathan A. Pachter. The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title> [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B025.

An Aptamer against MNK1 for Non-Small Cell Lung Cancer Treatment.

Lung cancer is the leading cause of cancer-related death worldwide. Its late diagnosis and consequently poor survival make necessary the search for new therapeutic targets. The mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) is overexpressed in lung cancer and correlates with poor overall survival in non-small cell lung cancer (NSCLC) patients. The previously identified and optimized aptamer from our laboratory against MNK1, apMNKQ2, showed promising results as an antitumor drug in breast cancer in vitro and in vivo. Thus, the present study shows the antitumor potential of apMNKQ2 in another type of cancer where MNK1 plays a significant role, such as NSCLC. The effect of apMNKQ2 in lung cancer was studied with viability, toxicity, clonogenic, migration, invasion, and in vivo efficacy assays. Our results show that apMNKQ2 arrests the cell cycle and reduces viability, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes in NSCLC cells. In addition, apMNKQ2 reduces tumor growth in an A549-cell line NSCLC xenograft model. In summary, targeting MNK1 with a specific aptamer may provide an innovative strategy for lung cancer treatment.

Abstract 5000: MYTX-011: A novel cMET-targeting antibody drug conjugate (ADC) engineered to increase on-target uptake in and efficacy against cMET expressing tumors

Abstract MET alterations can act as an oncogenic driver in non-small cell lung cancer (NSCLC) and elevated cMET expression occurs in many cancers. Antibody drug conjugates targeting cMET (cMET-ADCs) have been developed as a strategy to treat cMET+ tumors irrespective of dependency on cMET signaling. cMET- ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients having the highest cMET levels, indicating tumor cMET levels may be limiting for efficacy. We sought to create an ADC with the potential to benefit a broader population of patients including those expressing moderate cMET levels. Here, we describe MYTX-011, an ADC incorporating the clinically validated vcMMAE linker-payload conjugated to a novel, pH-dependent anti-cMET antibody. We hypothesized that engineering the antibody to rapidly lose affinity at acidic endosomal pH would boost ADC uptake and efficacy in cMET+ tumor cells by avoiding non-productive ADC recycling. We conducted mutagenesis of anti-cMET antibodies, screening for variants that selectively lost binding under acidic conditions, and, in parallel, assessed antibody internalization in cell-based assays. The resulting lead humanized IgG1 antibody was conjugated to vcMMAE at engineered cysteine residues (DAR=2) to create MYTX-011, which exhibited rapid dissociation from cMET at pH5.4 but retained high affinity binding at pH7.4 and 6.4. MYTX-011 showed markedly higher (>3 fold) internalization in cMET+ tumor cells and broader, more potent cytotoxicity across a large panel of cMET+ cancer cell lines in vitro compared to a matched ADC based on the unmodified parent antibody lacking pH-dependent binding, or an in-house version of a clinical stage cMET ADC. These findings translated in vivo where MYTX-011 showed superior efficacy (>3 fold based on dose titration) in NSCLC xenograft models with high (EBC-1) or only moderate cMET expression levels (H1373, H1975) compared to the matched parent ADC or an in-house version of a clinical stage cMET ADC. PK and toxicity studies in cynomolgus monkeys revealed that MYTX-011 exhibited favorable PK characteristics, and a toxicity profile similar to previously described MMAE-based ADCs. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ malignancies than other cMET-ADCs. Citation Format: Nimish Gera, Kyle Fitzgerald, Vijay Ramesh, Purvi Patel, Lena Kien, Deepak Kanojia, Simon Aoyama, Federico Colombo, Amit Deshpande, William Comb, Thomas Chittenden, Brian Fiske. MYTX-011: A novel cMET-targeting antibody drug conjugate (ADC) engineered to increase on-target uptake in and efficacy against cMET expressing tumors. [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 5000.

Abstract 5737: Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate

Abstract AZD9592 is a bispecific antibody drug conjugate (ADC) designed to deliver a topoisomerase 1 inhibitor (TOP1i) cytotoxic payload (AZ14170133) to tumor cells. AZD9592 selectively binds to epidermal growth factor receptor (EGFR) and c-MET, two cell surface receptors highly expressed in solid tumors including non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Here we evaluate the pharmacodynamic activity of the TOP1i payload delivery by AZD9592 in an NSCLC-derived xenograft model using immunohistochemistry (IHC) approaches. Treatment-induced DNA double-strand breaks (DSB) and apoptotic cell death were measured using γH2AX, phospho-RAD-50 (pRAD50), and cleaved-caspase-3 (CC3) across increasing exposure to AZD9592. Furthermore, we report in vivo antitumor efficacy of AZD9592 in a panel of NSCLC and HNSCC patient-derived xenograft (PDX) models that were characterized for somatic tumor alterations, including oncogenic driver mutations in EGFR, tumor cell expression of EGFR and c-MET by IHC and deep-learning based image analysis, and targeted proteomics by mass spectrometry. Results demonstrate dose-dependent increases in pRAD50 and γH2AX upon treatment with AZD9592, signifying induction of DNA damage. Increased CC3 and reduced tumor volume (TV) in all treatment groups compared with control groups supports that AZD9592 induces tumor cell death due to formation of DNA DSB. In PDX experiments, tumor growth inhibition (TGI), defined as ≥30% reduction in TV from baseline after a single dose of AZD9592 8 mg/kg, was observed in 73% (16/22) of EGFR mutant NSCLC models. The models evaluated included tumors with or without prior exposure to EGFR tyrosine kinase inhibitors, and harboring diverse mutational profiles and heterogeneous expression levels of EGFR and c-MET. In EGFR wildtype NSCLC and HNSCC PDX, TGI was observed in 60% (12/20) and 44% (4/9) of models, respectively. IHC demonstrated an association of target expression and response to treatment, suggesting a potential predictive feature of response in tumors with elevated antigen expression. Targeted proteomics demonstrated an association between the expression of SLFN11, a known TOP1i sensitivity marker, and treatment response. Collectively, these results support the hypothesized mechanism of action of AZD9592: TOP1i induced tumor cell death due to formation of DNA DSB, and suggest opportunities in the treatment of tumors with a range of molecular features. AZD9592 is currently in a Phase 1 clinical trial in advanced solid malignancies. Citation Format: Lara McGrath, Ying Zheng, Simon Christ, Christian C. Sachs, Sihem Khelifa, Claudia Windmüller, Steve Sweet, Yeoun Jin Kim, Daniel Sutton, Michal Sulikowski, Arthur Lewis, Ivan Inigo, Nicolas Floch, Edward Rosfjord, Fernanda Arnaldez, Frank Comer. Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate. [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 5737.

Abstract 6311: ABBV-400: An ADC delivering a novel topoisomerase 1 inhibitor to c-Met-positive solid tumors

Abstract Antibody-drug conjugate (ADC) technology is founded on the premise that antigen-mediated payload delivery selectively targets tumor cells and spares normal tissue. While approval of several ADCs has proven this to be a successful therapeutic approach, there remain opportunities to generate optimized ADCs with tailored selection of antigen matched with payload highly active in the target indication. To this end, ABBV-400 has been developed as an ADC that targets c-Met and topoisomerase-1 (Top1), both of which are overexpressed in solid tumors. The antibody is directed to the cell surface tyrosine kinase receptor, c-Met, a key driver of tumor cell growth and development of resistance to EGFR-targeted and other targeted therapeutics. Supporting this, Teliso-V, a c-Met-targeted ADC with MMAE payload, has been granted Breakthrough Therapy Designation in non-small cell lung cancer (NSCLC). To extend activity of a c-Met-targeted ADC to patients with tumors expressing lower c-Met levels and to indications, such as colorectal cancer (CRC), where MMAE-based ADCs have not been effective, a novel topoisomerase-1 inhibitor linker-drug was developed. Screening over 400 linker-drugs identified a unique, potent Top1 inhibitor with a cleavable valine-alanine linker and stable bromoacetamide attachment designed to minimize systemic payload release. Preclinically ABBV-400 exhibits favorable pharmacokinetics, anti-proliferative activity in vitro, and compelling efficacy in NSCLC, gastroesophageal, and CRC xenograft models. Furthermore, ABBV-400 is well tolerated by cynomolgus monkeys with bone marrow and gastrointestinal tract toxicities common to other Top1 inhibitors. This pairing of a novel linker-drug and an antibody with demonstrated clinical activity provides an exciting opportunity to treat patients with c-Met expression in indications sensitive to Top1 inhibition. ABBV-400 has progressed through dose escalation, and dose expansion is currently ongoing in a Phase 1 FIH clinical study (NCT05029882). All authors were employees of AbbVie during this research. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication Citation Format: Regina M. Reilly, Cheng Ji, Ryan P. Matuszak, Mark G. Anderson, Lora Tucker, Nadezda Klunder, Adelyn L. Lazo, Erwin R. Boghaert, Marybeth A. Pysz, Aloma D’Souza, Laura Kreckler, Milan Bruncko, Brittney J. Mills, Matthew Ravn, George Doherty, Kevin J. Freise, Andrew C. Phillips. ABBV-400: An ADC delivering a novel topoisomerase 1 inhibitor to c-Met-positive solid tumors [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 6311.

Abstract 6256: Inhibition of class IIa HDACs potentiates MYC inhibitor-driven cytotoxicity by inducing MYC depletion and oxidative stress in non-small cell lung cancer

Abstract Lung cancer is the most common cause of cancer mortality worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Dysregulation and/or overexpression of MYC are widely implicated in NSCLC, which suggests that MYC may be a promising therapeutic target for the treatment of NSCLC. Recently, two direct MYC inhibiting agents with improved in vivo efficacy and tolerability, MYCi975 and Omomyc, have been developed. To define new drug strategies that could potentiate the therapeutic effects of MYC inhibitors, transcriptome datasets from MYCi975 and Omomyc studies were reanalyzed. These data revealed the induction of two class IIa histone deacetylases (HDACs), HDAC5 and HDAC9, upon MYC inhibition. Importantly, class IIa HDACs are involved in cancer proliferation, aggressiveness, and prognosis. In addition, novel agents that selectively inhibit class IIa HDACs have demonstrated promising anti-tumor efficacy; thus, suggesting potential therapeutic benefits from dual targeting of MYC and class IIa HDACs. We assessed the treatment efficacy of MYCi plus class IIa HDACi across 18 NSCLC cell lines, 10 of which demonstrated substantial reduction of cell viability upon combination treatment. Querying of driver mutation spectra associated with differential drug response in our cell line panel defined that EGFR mutant cell lines exhibited resistance, while STK11 or Ras mutant cell lines were sensitive to our drug paradigm. Gene set enrichment analysis comparing the transcriptomes of sensitive versus resistant cell lines revealed that MYC Targets, Oxidative Phosphorylation, and Reactive Oxygen Species (ROS) pathways were activated in combination treatment responsive cell lines. RNA sequencing on treated cell lines identified large-scale transcriptional shifts facilitated by combination treatment, including suppression of MYC, cell cycle, and mitochondrial pathways. Furthermore, G1/S arrest and elevated mitochondrial ROS levels in combination drug-treated cells were confirmed using flow cytometry. Concordant with the role of MYC as a key cell cycle regulator, MYC protein levels were decreased in the combination treatment group as compared to the vehicle or mono-treatment groups. Importantly, both MYC overexpression and ROS scavenger treatment partially rescued the reduction of cell viability by combination treatment, suggesting both MYC depletion and ROS elevation as contributors to therapeutic efficacy driven by combination treatment. Expanding these studies to in vivo models, we discern that combination MYC and class IIa HDAC inhibition significantly reduced tumor burden in a patient-derived xenograft model of human NSCLC. In summary, we define here a novel drug paradigm combining MYC and class IIa HDAC inhibitors, which potentiates anti-tumor efficacy in NSCLC via MYC depletion and oxidative stress. Citation Format: Jina Park, Ying-Yu Chen, Jennie Cao, Michelle Vaz, Ray-Whay C. Yen, Stephen B. Baylin, Michael J. Topper. Inhibition of class IIa HDACs potentiates MYC inhibitor-driven cytotoxicity by inducing MYC depletion and oxidative stress in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6256.

Abstract 6276: SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is overexpressed in various human malignancies and induces epigenetic and post-translational changes, which have a significant impact on cell growth, proliferation, apoptosis, and DNA damage repair. SKL27969 is a potent and selective brain-penetrating PRMT5 inhibitor demonstrating antitumor activity in the preclinical models of glioblastoma (GBM) and non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) brain metastasis models. In this study, we evaluated the antitumor activity of SKL27969 alone or in combination with DNA-damaging agents in various cancer models. Methods: Cell cycle analysis, apoptosis, proliferation, and invasion assays were conducted to determine in vitro profiles of SKL27969. In vivo efficacy of SKL27969 was evaluated in human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models. Immunohistochemistry and RNA-sequencing analysis in tumor samples from animal models were conducted to confirm the in vivo mechanism of action (MoA). In vitro synergistic effects of SKL27969 with combination partner drugs were analyzed using Combenefit software. Results: SKL27969 exhibits strong anti-proliferative effect by inducing cell cycle arrest and apoptosis and also inhibits invasion of cancer cells. An evaluation of SKL27969 in a panel of over 100 cancer cell lines revealed a broad spectrum of anti-proliferative activity in various cancer types, and an especially more sensitive response in cell lines harboring genetic alterations in the mitotic DNA damage checkpoint, DNA repair, or RNA processing pathways. Administration of SKL27969 demonstrated significant tumor growth inhibition, with a decrease in tumor symmetric dimethylarginine (SDMA) in NSCLC and TNBC subcutaneous xenograft models. Transcriptome and immunohistochemical analysis of SKL27969-treated tumors revealed that the cell cycle checkpoint pathways and DNA damage repair genes were downregulated, supporting the rationale for combination treatment with DNA-damaging agents such as chemotherapeutic agents. Robust antitumor effects were observed after treatment with SKL27969 in combination with paclitaxel or gemcitabine in NSCLC PDX or pancreatic cancer CDX models. Further ex vivo studies to confirm the in vivo MoA of this synergism, and in vivo combination studies of SKL27969 with standard chemotherapeutic agents in other solid cancer CDX models, are ongoing. Conclusion: This is the first preclinical study that suggests the therapeutic potential of SKL27969 in combination with currently approved DNA-damaging agents, and supports further clinical investigation of SKL27969’s role in improving the therapeutic benefit of standard therapies. Citation Format: Mijin Moon, Yongje Shin, Soyoung Ki, Jungtae Na, Ho Yeon Lee, Ilkyoo Koh, Beomjin Hong, Jiyeon Yun, Janice Laramy, Vijaykumar Vashi, Sook-Kyung Park. SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors. [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 6276.

Abstract 5484: Synergistic antitumor activity of nab-sirolimus in combination with KRAS inhibitors (KRASis) sotorasib and adagrasib in KRAS G12C NSCLC and bladder cancer xenografts

Abstract KRAS is frequently mutated in non-small cell lung cancer (NSCLC) and other tumor types, with KRAS G12C mutation representing ~12% of patients with NSCLC. Sotorasib (sot) is approved and adagrasib (ada) is under review for the treatment of KRAS G12C NSCLC. Mutations in KRAS may lead to mTORC1 activation, and mTOR may contribute to adaptive resistance to KRASis. nab-Sirolimus (nab-S) is a novel albumin-bound nanoparticle form of the mTOR inhibitor sirolimus approved for the treatment of locally advanced unresectable or metastatic malignant perivascular epithelioid cell tumors. This study investigated the antitumor activity of nab-s in combination with KRASis in KRAS G12C NSCLC and bladder xenograft models. Athymic mice bearing subcutaneous KRAS G12C- and STK11-mutant NCI-H2030 and NCI-H2122 NSCLC and KRAS G12C and PTEN-null UMUC3 bladder cancer were treated for 6 weeks with nab-s IV at 15 mg/kg/wk, sot or ada (for NCI-H2122 and UMUC3) orally at 30 mg/kg/d alone or in combination. Tumor samples were harvested for analysis of tumor drug levels and biomarkers. In all 3 models tested, single-agent (SA) nab-s or KRASis overall showed modest tumor growth suppression. In contrast, nab-s in combination with either sot or ada showed greater tumor growth inhibition and a higher meaningful tumor regression rate vs SA (Table). There was no significant difference in antitumor activity between combinations of nab-s with either sot or ada. High trough intertumoral sirolimus levels were observed in groups treated with nab-s alone or in combination with KRASis, indicating sustained presence of sirolimus in the tumor. nab-S in combination with either sot or ada showed synergistic antitumor activity compared to the SA. A multicenter, single-arm, open-label Phase 1/2 clinical study is planned to determine the recommended Phase 2 dose, safety, tolerability, and efficacy for the combination of ada and nab-s in patients with KRAS G12C tumors. Changes in TGI and Tumor Regression Tumor Model Combination Treatment TGI vs Single Agent (%) P Value vs Single Agent for Tumor Growth Curve (ANOVA) Tumor Regression Over 30% P Value vs Single Agents for Rate of Tumor Regression Over 30% (Chi-Square) vs nab-S vs KRASi vs nab-S vs KRASi NCI-H2030 nab-S + Sotorasib 129 111 0.01 0.001 3/6 0.03 NCI-H2122 nab-S + Sotorasib 112 106 0.001 <0.001 8/10 <0.001 nab-S + Adagrasib 101 100 0.001 <0.001 4/10 0.03 UMUC3 nab-S + Sotorasib 107 105 <0.001 <0.001 6/6 <0.001 nab-S + Adagrasib 107 107 <0.001 0.04 6/6 0.001 ANOVA, analysis of variance; KRASi, KRAS inhibitor; nab-s, nab-sirolimus; TGI, tumor growth inhibition. Citation Format: Shihe Hou, Andrew Kwon, Jorge Nieva, Neil Desai. Synergistic antitumor activity of nab-sirolimus in combination with KRAS inhibitors (KRASis) sotorasib and adagrasib in KRAS G12C NSCLC and bladder cancer xenografts. [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 5484.

Cyclovirobuxine D inhibits growth and progression of non‑small cell lung cancer cells by suppressing the KIF11‑CDC25C‑CDK1‑CyclinB1 G2/M phase transition regulatory network and the NFκB/JNK signaling pathway.

Lung cancer is the leading cause of cancer‑related mortality worldwide. Non‑small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer and is associated with low 5‑year overall survival rates. Therefore, novel and effective chemotherapeutic drugs are urgently required for improving the survival outcomes of patients with lung cancer. Cyclovirobuxine D (CVB‑D) is a natural steroidal alkaloid, used for the treatment of cardiovascular diseases in Traditional Chinese Medicine. Several studies have also demonstrated the antitumor effects of CVB‑D. Therefore, in the present study, the therapeutic effects of CVB‑D in lung cancer and the underlying mechanisms were investigated using the invivo xenograft model of NSCLC in nude mice and invitro experiments with the NSCLC cell lines. Bioinformatics analyses of RNA‑sequencing data, and cell‑based functional assays demonstrated that CVB‑D treatment significantly inhibited invitro and invivo NSCLC cell proliferation, survival, invasion, migration, angiogenesis, epithelial‑to‑mesenchymal transition and G2/M phase cell cycle. CVB‑D exerted its antitumor effects by inhibiting the KIF11‑CDK1‑CDC25C‑cyclinB1 G2/M phase transition regulatory oncogenic network and the NF‑κB/JNK signaling pathway. CVB‑D treatment significantly reduced the sizes and weights and malignancy of xenograft NSCLC tumors in the nude mice. In conclusion, the present study demonstrated that CVB‑D inhibited the growth and progression of NSCLC cells by inhibiting the KIF11‑CDK1‑CDC25C‑CyclinB1 G2/M phase transition regulatory network and the NF‑κB/JNK signaling pathway. Therefore, CVB‑D is a promising drug for the treatment of NSCLC patients.

EXTH-103. DISCOVERY AND PHARMACOLOGICAL CHARACTERIZATION OF SKL27969, A NOVEL BRAIN-PENETRATING PRMT5 INHIBITOR WITH STRONG ANTITUMOR ACTIVITY, IN GLIOBLASTOMA AND BRAIN METASTASIS PRECLINICAL MODELS

Abstract Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, regulates cellular processes such as survival, proliferation, and apoptosis by inducing symmetric arginine dimethylation of multiple cellular proteins involved in the regulation of cellular transcription and RNA splicing. Elevated tumor PRMT5 protein level has recently been correlated with poor prognosis and poor patient survival in various human cancers, including glioblastoma multiforme (GBM), lung cancer, breast cancer, and ovarian cancer. SKL27969 is a highly selective and potent PRMT5 inhibitor that is designed for brain penetration. It strongly binds to S-adenosylmethionine (SAM)-bound PRMT5/MEP50 (methylosome protein 50), leading to potent cellular symmetric dimethylarginine (SDMA) inhibition with an IC50 of 3.4 nM. Cell line panel profiling of SKL27969 revealed a broad spectrum of anti-proliferative activity in various cancer cell types, including CNS tumors, solid cancers prone to spread to brain, other solid cancers, and hematologic cancers. SKL27969 showed excellent brain exposure, with total and unbound partition coefficient (Kp and Kpuu) values of 6.57 and 1.08, respectively, and high tumor-to-plasma ratio, which resulted in significant survival benefit in orthotopic GBM xenograft models and metastatic brain cancer models such as intracranial xenograft models of non-small cell lung cancer (NSCLC) or triple-negative breast cancer (TNBC). Administration of SKL27969 showed strong tumor growth suppression in subcutaneous xenograft models of GBM, NSCLC, or TNBC. High correlation between tumor and plasma SDMA levels after SKL27969 treatment were observed in the preclinical models. Furthermore, SKL27969 showed potentiation of DNA damage when used in combination with DNA-damaging agents, which may suggest possible benefit as a combination therapy. Given the therapeutic target potential of PRMT5 in glioblastoma and other brain metastatic cancers, and the preclinical efficacy of SKL27969, these results have enabled the initiation of Phase 1/2 open-label, multicenter dose-finding study in patients with advanced solid tumors (NCT05388435).

Optimized thyroid transcription factor-1 core promoter-driven microRNA-7 expression effectively inhibits the growth of human non-small-cell lung cancer cells.

Targeted gene therapy has become a promising approach for lung cancer treatment. In our previous work, we reported that the targeted expression of microRNA-7 (miR-7) operated by thyroid transcription factor-1 (TTF-1) promoter inhibited the growth of human lung cancer cells in vitro and in vivo; however, the intervention efficiency needed to be further improved. In this study, we identified the core promoter of TTF-1 (from -1299 bp to -871 bp) by 5' deletion assay and screened out the putative transcription factors nuclear factor-1 (NF-1) and activator protein-1 (AP-1). Further analysis revealed that the expression level of NF-1, but not AP-1, was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer (NSCLC) cells. Moreover, the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter. Of note, we optimized four distinct sequences to form additional NF-1-binding sites (TGGCA) in the sequence of TTF-1 core promoter (termed as optTTF-1 promoter), and verified the binding efficiency of NF-1 on the optTTF-1 promoter by electrophoretic mobility shift assay (EMSA). As expected, the optTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro, accompanied by a reduced transduction of NADH dehydrogenase (ubiquinone) 1α subcomplex 4 (NDUFA4)/protein kinase B (Akt) pathway. Consistently, optTTF-1 promoter-driven miR-7 expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC. Finally, no significant changes were detected in the biological indicators or the histology of some important tissues and organs, including heart, liver, and spleen. On the whole, our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells, providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.

Discovery of Novel Allosteric EGFR L858R Inhibitors for the Treatment of Non-Small-Cell Lung Cancer as a Single Agent or in Combination with Osimertinib

Addressing resistance to third-generation EGFR TKIs such as osimertinib via the EGFRC797S mutation remains a highly unmet need in EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we present the discovery of the allosteric EGFR inhibitor 57, a novel fourth-generation inhibitor to overcome EGFRC797S-mediated resistance in patients harboring the activating EGFRL858R mutation. 57 exhibits an improved potency compared to previous allosteric EGFR inhibitors. To our knowledge, 57 is the first allosteric EGFR inhibitor that demonstrates robust tumor regression in a mutant EGFRL858R/C797S tumor model. Additionally, 57 is active in an H1975 EGFRL858R/T790M NSCLC xenograft model and shows superior efficacy in combination with osimertinib compared to the single agents. Our data highlight the potential of 57 as a single agent against EGFRL858R/C797S and EGFRL858R/T790M/C797S and as combination therapy for EGFRL858R- and EGFRL858R/T790M-driven NSCLC.

Obesity accelerates immune evasion of non-small cell lung carcinoma via TFEB-dependent upregulation of Siglec-15 and glycolytic reprogramming

Although obesity contributes to tumor incidence and progression in various cancers, whether obesity impacts the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC) remains largely under-explored. We generated NSCLC xenograft model in diet-induced obese mice and identified that TFEB is critical to accelerate obesity-related NSCLC progression with mimic intrinsic functions on tumor biology. Mechanically, TFEB binds directly to Siglec-15 promoter to upregulate Siglec-15 expression and binds to Hk2 and Ldha promoters to enhance glycolytic flux in NSCLC cells, which restrain the expansion and cytotoxic function of CD8+ T cells while maintain suppressive Treg cells in TME, jointly promoting immune evasion of NSCLC cells in obesity. Blocking tumor TFEB improves the therapeutic efficiency of anti-PD-1 in obese mice. Altogether, our data identify essential roles of TFEB in remodeling immunosuppressive TME and promoting NSCLC development in obesity, providing scientific rational for TFEB as a potential biomarker to predict immune checkpoint blockade efficiency in obese NSCLC patients.

The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer.

Objective:The novel compound GCJ-490A has been discovered as a pan-histone deacetylase (HDAC) inhibitor that exerts potent inhibitory activity against HDAC1, HDAC3, and HDAC6. Because of the important roles of HDACs in lung cancer development and the high distribution of GCJ-490A in lung tissue, we explored the anti-tumor potency of GCJ-490A against non-small cell lung cancer (NSCLC) in vitro and in vivo in this study.Methods:The in vitro effects of GCJ-490A alone or combined with the EGFR inhibitor gefitinib against NSCLC were measured with proliferation, apoptosis, and colony formation assays. NSCLC xenograft models were used to investigate the efficacy of GCJ-490A combined with gefitinib for the treatment of NSCLC in vivo. Western blot assays, luciferase reporter assays, chromatin immunoprecipitation assays, quantitative real time-PCR, immunohistochemistry, and transcription factor activity assays were used to elucidate possible mechanisms.Results:GCJ-490A effectively inhibited NSCLC cell proliferation and induced apoptosis in vitro and in vivo. Interestingly, inhibition of HDAC1 and HDAC6 by GCJ-490A increased histone acetylation at the IKKα promoter and enhanced IKKα transcription, thus decreasing c-Met. Moreover, this c-Met downregulation was found to be essential for the synergistic anti-tumor activity of GCJ-490A and gefitinib.Conclusions:These findings highlight the promising potential of HDAC inhibitors in NSCLC treatment and provide a rational basis for the application of HDAC inhibitors in combination with EGFR inhibitors in clinical trials.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

BackgroundTyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.MethodsKINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic–pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.ResultsIn mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.ConclusionsTaken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial registrationClinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Design, synthesis, and biological evalution of bifunctional inhibitors against Hsp90-HDAC6 interplay

HDAC6 and Hsp90, existing as a cytosolic complex play an important role in maintaining the protein homeostasis. The interplay of HDAC6 and Hsp90 has attracted wide attention due to their important role and promise as therapeutic targets in malignant cancers. Therefore, the discovery of dual inhibitors targeting HDAC6 and Hsp90 is of high importance. In the present study, we describe the design, synthesis, and biological evaluation of bifunctional inhibitors against HDAC6 and Hsp90 interplay. In particular, compound 6e shows a significant inhibitory activity against both HDAC6 and Hsp90 with IC50 values of 106 nM and 61 nM, respectively. Compound 6e promotes the acetylation of HDAC6 substrate proteins such as α-tubulin and Hsp90 via HDAC6 inhibition, and also induces the degradation of Hsp90 clients such as Her2, EGFR, Met, Akt, and HDAC6 via Hsp90 inhibition. Compound 6e consequently furnishes potent antiproliferative effect on gefitinib-resistant H1975 non-small cell lung cancer (NSCLC) with a GI50 value of 1.7 μM. In addition, compound 6e successfully achieved significant tumor growth inhibition in H1975 NSCLC xenograft model without noticeable abnormal behavior, body weight changes, and apparent ocular toxicity. We conclude that compound 6e constitutes an excellent tool as well as a valuable lead for assessment of Hsp90 and HDAC6 dual inhibition with a single molecule.

Abstract 3600: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models

Abstract Mutationally-activated RAS oncoproteins are detected in approximately 19% of newly diagnosed human cancers and have been targets for drug discovery for over 40 years. Recently, the FDA approved sotorasib, a covalent inhibitor of KRASG12C, for the treatment of non-small cell lung cancer (NSCLC). Although sotorasib demonstrated clear clinical benefits, the emergence of drug resistance has led to drug combination approaches with the goal of deepening and sustaining the durability of patient responses. One potential mechanism of drug resistance is the induction of autophagy, which cancer cells use to survive during periods of stress such as treatment with pathway-targeted therapies. Indeed, KRAS-mutated cancer cells have been shown to exhibit constitutive autophagy for survival. Additionally, such cells further increase autophagy when treated with pathway-targeted inhibitors of RAS>RAF>MEK>ERK MAP kinase signaling as a resistance mechanism. Hence, the combination of a KRASG12C inhibitor with a specific and potent autophagy inhibitor could lead to deeper and more sustained clinical responses. DCC-3116 is an investigational, potent and selective pharmacological inhibitor of the protein kinases ULK1 and ULK2, which are critical initiating components of the autophagy pathway. Treatment of KRASG12C non-small cell lung cancer (NSCLC) cell lines with sotorasib induced autophagy by ~2-fold via activation of ULK kinases as measured by an increase in ULK-mediated phosphorylation of ATG13. Sotorasib-mediated ULK kinase activation, and resulting autophagic flux, was inhibited by DCC-3116 in a dose-dependent manner with IC50 values of 81-160 nM in NSCLC cell lines. These effects translated to in vivo efficacy. In the Calu-1 and H358 KRASG12C NSCLC xenograft models, the combination of DCC-3116 and sotorasib resulted in tumor regression whereas single treatment arms afforded only inhibition of tumor growth. A NSCLC KRASG12C patient derived xenograft (PDX) model further supported the DCC-3116 and sotorasib combination with increased tumor growth inhibition compared to sotorasib alone. These data demonstrate a compelling rationale to study DCC-3116 in combination with KRASG12C inhibitors such as sotorasib in NSCLC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with documented KRAS, NRAS or BRAF mutations (NCT04892017). Citation Format: Martin McMahon, Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Phaedra Ghazi, Yu Zhan, Bryan D. Smith, Conan G. Kinsey, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models [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 3600.

Abstract 3232: Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation

Abstract INTRODUCTION: Epithelial ovarian cancer (EOC) represents the most lethal gynecological disease, with a 5-year relative survival rate of 46% after the diagnosis. The standard treatment of advanced EOC is based on surgery, followed by platinum (Pt)-based chemotherapy. However, the development of platinum resistant disease could occur and strongly impact on the survival of EOC patients for whom we still do not have valid therapeutic options. By using a proteomic approach followed by a bioinformatic analysis, we previously validated the role of HSP90 in the mechanism of platinum-resistance. Here, we propose a novel therapeutic strategy based on the combined pharmacologic inhibition of HSP90 and mTOR to further potentiate Pt-based chemotherapy and to revert Pt-resistance in EOC and non-small-cell lung cancer (NSCLC) models. METHODS: TOV-112D parental and Pt-resistant cells were characterized by phosphoproteomics followed by functional analysis and proteins validation by western blot. Synergistic anti-tumor effect was evaluated in Pt-sensitive and Pt-resistant EOC and NSCLC cell lines by calculating combination indexes (CI), colony formation assay, apoptosis and DNA damage, as well as on 3D in vitro microtissues obtained by co-culturing cancer cells with normal fibroblasts and in vivo EOC and NSCLC xenograft models. RESULTS: 542 differentially phosphorylated expressed proteins were identified in Pt-resistant TOV-112D compared with parental cells, and mTOR and the transcription factor HSF1 emerged as the most enriched pathways. The up-regulation of the phosphorylated form of PDK1, AKT, mTOR and rpS6 was observed in Pt-resistant compared to parental cells. Moreover, we also demonstrated the up-regulation of the activity of HSF1 along with the elevation of its targets such as heat shock proteins HSP90, HSP70 and HSP40, crucial components of chaperone complex machinery. Interestingly, among the differentially expressed proteins, we identified the kinase DYRK2 able to phosphorylate HSF1, supporting its transactivation. Accordingly, the combination of HSP90 inhibitor ganetespib and the mTOR inhibitor temsirolimus plus cisplatin, synergistically reduced colony formation, cancer cells and microtissues cell growth in vitro by increasing DNA-damage and apoptosis and in vivo by enhancing mouse survival. Mechanistically, the triple combination treatment, impaired the proteins involved in mTOR signalling and HSF1 transactivation. Notably, all these data were confirmed in Pt-resistant NSCLC models, supporting the possibility that the same mechanism is present in different tumor types. CONCLUSIONS: Our findings identify a promising new antitumor strategy based on the combination of HSP90 and mTOR inhibitors to revert Pt-resistance that that warrant further clinical evaluation. Citation Format: Rita Lombardi, Laura Addi, Biagio Pucci, Maura Sonego, Maria Serena Roca, Francesca Capone, Federica Iannelli, Francesca Bruzzese, Gustavo Baldassarre, Alfredo Budillon. Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation [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 3232.

Abstract 3263: Preclinical characterization of PRT3789, a potent and selective SMARCA2 targeted degrader

Abstract SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to nonsense mutations, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have identified PRT3789, a potent and selective SMARCA2 targeted degrader, that selectively inhibits proliferation of SMARCA4-deleted cancer cells. Here, we describe the potential mechanism of action for PRT3789 at the molecular level and the in vitro and in vivo anti-tumor activity in SMARCA4-deleted cancer cells. To further elucidate the SMARCA2 degradation selectivity of PRT3789, we performed mass spectrometry to identify the selective SMARCA2 lysine residues ubiquitinated following treatment with PRT3789. This data, in combination with site-directed mutagenesis against these SMARCA2-specific ubiquitinated residues, has revealed important insights into the mechanism of action of PRT3789. In addition, to further understand the specific vulnerability of SMARCA2 in SMARCA4-deleted cells, we investigated whether PRT3789 affected the integrity of the residual SWI/SNF complex. Coimmunoprecipitation of SMARCC1 revealed that PRT3789 disrupts specific SWI/SNF complex subunits, including ACTL6A (BAF53). Functional genome-wide experiments are ongoing to evaluate the impact of this finding and the residual activity of the SWI/SNF complex. Furthermore, treatment with PRT3789 demonstrated robust inhibition of cell proliferation of SMARCA4-deleted non-small cell lung cancer (NSCLC) cells in vitro and NSCLC PDX tumors ex vivo, but not SMARCA4 WT cancer cells, in a concentration-dependent manner. Lastly, PRT3789 shows favorable pharmacokinetic properties in vivo, which correlate to its pharmacodynamics effects as evidenced by reduced SMARCA2 protein and KRT80 mRNA levels in tumor tissues. In subcutaneous cell-line derived xenograft (CDX) models of NSCLC, administration of PRT3789 demonstrated significant dose-related inhibition of SMARCA4-deleted NSCLC growth at tolerated doses, but no effect on the growth of SMARCA4 WT cancers. In summary, consistent with our previous validation studies and genomic perturbation analyses, our potent and selective SMARCA2 targeted degrader PRT3789 induces strong synthetic lethality in SMARCA4-deleted cancers in vitro and in vivo. Citation Format: Michael Hulse, Anjana Agarwal, Min Wang, Jack Carter, Monisha Sivakumar, Brian Vidal, Justin Brown, Andrew Moore, Alexander Grego, Neha Bhagwat, Joseph Rager, Liang Lu, Corey Basch, Klare Bersch, Chaofeng Dai, Philip Pitis, Andrew Combs, Bruce Ruggeri, Kris Vaddi, Peggy Scherle, Koichi Ito. Preclinical characterization of PRT3789, a potent and selective SMARCA2 targeted degrader [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 3263.

Abstract 5284: M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression

Abstract Mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are co-expressed at high prevalence in multiple cancer indications such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma, triple negative breast cancer and ovarian cancer. Tumor-associated MUC1 is hypoglycosylated and exposes peptide epitopes within its extracellular domain, which are not accessible to antibodies in normal epithelial tissue. MUC1 co-localizes with EGFR as a result of loss of cell polarity in tumor cells. M1231 is a bispecific antibody-drug conjugate (ADC) based on a SEED (strand-exchange engineered domain)-antibody scaffold targeting tumor-associated MUC1 and EGFR. Sutro Biopharma’s cell free expression system Xpress CF+™ enables incorporation of non-natural amino acids at precise positions in the SEED-antibody for site-specific linker-payload conjugation. The cytotoxic payload of M1231 (SC209) is a hemiasterlin-related microtubule inhibitor. The cleavable linker (ValCit-PABA) maintained ADC stability in human plasma in vitro, ensuring low systemic payload release. Pre-clinical activity of M1231 was assessed in vitro in cancer cell lines and in vivo in NSCLC and ESCC patient-derived xenograft (PDX) models. M1231 inhibited tumor cell viability in vitro at sub-nanomolar IC50 values. The bispecific MUC1xEGFR antibody has demonstrated superior internalization and lysosomal trafficking compared to monospecific bivalent antibodies. Efficient ADC uptake resulted in superior antitumor activity of M1231 in PDX models compared to monospecific bivalent ADCs. M1231 demonstrated durable and dose-dependent antitumor activity in PDX models. Strong antitumor activity with complete regression was observed in NSCLC and ESCC PDX models following a single treatment of M1231. M1231 antitumor activity was associated with high target expression in NSCLC PDX models and was observed across a range of target expression levels in ESCC models. In summary, efficient payload delivery to tumor cells and strong antitumor activity of M1231 indicate that M1231 may have the potential to deliver an efficacy benefit to patients with tumors co-expressing MUC1 and EGFR. Citation Format: Christine Knuehl, Lars Toleikis, Julia Dotterweich, Jianguo Ma, Seema Kumar, Edith Ross, Claudia Wilm, Martina Schmitt, Hans Juergen Grote, Christiane Amendt. M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression [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 5284.