Cell-derived Xenograft Models Research Articles

The FLRT3-UNC5B Pathway is a Novel Regulator of T Immunosurveillance

Abstract Our understanding of molecular cell-surface interactions that control T cell responses in cancer remains limited despite many advances in the field. We hypothesized that tumor cells take advantage of developmental protein-protein interactions such as axon guidance interactions that could inhibit or repel tumor-immune interactions in the TME to evade T cell anti-tumor immunity. We used gain-of-function screening to evaluate a gene-set of axon guidance proteins for inhibitory function in human T cells and identified and validated Fibronectin and Leucine-Rich Transmembrane protein-3 (FLRT3) as an inhibitor of T cell activity. FLRT3 inhibited human T cell function and promoted tumor growth in humanized tumor cell-derived xenograft (CDX) models. FLRT3 inhibited T cells through interaction with Unc-5 Netrin Receptor B (UNC5B) expressed on activated T cells. A FLRT3 monoclonal antibody designed to specifically block FLRT3 interactions with UNC5 family proteins reduced tumor growth in CDX models that was dependent on T cells. FLRT3 blockade also enhanced CAR-T and BiTE + T cell activity and infiltration in humanized zebrafish tumor models. These studies support the notion that aberrant axon guidance protein expression in the TME act as T cell checkpoints, and the FLRT3:UNC5B pathway can be targeted therapeutically to improve T cell-based immunotherapy activity and infiltration.

AHNAK2 Promotes the Progression of Pancreatic Ductal Adenocarcinoma by Maintaining the Stability of c-MET.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and rapidly progresses. The overall response rate of PDAC to current treatment methods is still unsatisfactory. Thus, identifying novel targets and clarifying the underlying mechanisms associated with PDAC progression may potentially offer additional treatment strategies. AHNAK2 is aberrantly expressed in a variety of tumors and exerts pro-tumorigenic effects. However, the biological role of AHNAK2 in PDAC remains poorly understood. The expression of AHNAK2 in PDAC and paired non-tumor tissues was detected by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). Lentivirus knockdown was performed to investigate the impact of AHNAK2 on the biological function of pancreatic cancer cells. The subcutaneous cell-derived xenograft (CDX) model and the KPC spontaneous mouse model with AHNAK2 silencing were used to observe the effects of AHNAK2 on tumor growth and prognosis. The expression of c-MET at protein level in response to HGF treatment was assessed using western blot. Our results demonstrated that AHNAK2 was highly expressed in PDAC clinical samples and associated with poor prognosis. Knockdown of AHNAK2 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells. AHNAK2 knockdown or knockout resulted in tumor growth suppression and prolonged survival in mice with PDAC. In addition, AHNAK2 and c-MET expression levels showed a significant positive correlation at the post-transcriptional level. Mechanistically, AHNAK2 promoted tumor progression by preventing c-MET degradation and persistently activating the HGF/c-MET signaling pathway. Overall, our study revealed that AHNAK2 plays an important role in PDAC progression by modulating the c-MET signaling pathway, and targeting AHNAK2 may be an effective therapeutic strategy for PDAC.

Metabolic difference between patient-derived xenograft model of pancreatic ductal adenocarcinoma and corresponding primary tumor

BackgroundPatients-derived xenograft (PDX) model have been widely used for tumor biological and pathological studies. However, the metabolic similarity of PDX tumor to the primary cancer (PC) is still unknown.MethodsIn present study, we established PDX model by engrafting primary tumor of pancreatic ductal adenocarcinoma (PDAC), and then compared the tumor metabolomics of PC, the first generation of PDX tumor (PDXG1), and the third generation of PDX tumor (PDXG3) by using 1H NMR spectroscopy. Then, we assessed the differences in response to chemotherapy between PDXG1 and PDXG3 and corresponding metabolomic differences in drug-resistant tumor tissues. To evaluate the metabolomic similarity of PDX to PC, we also compared the metabolomic difference of cell-derived xenograft (CDX) vs. PC and PDX vs. PC.ResultsAfter engraftment, PDXG1 tumor had a low level of lactate, pyruvate, citrate and multiple amino acids (AAs) compared with PC. Metabolite sets enrichment and metabolic pathway analyses implied that glycolysis metabolisms were suppressed in PDXG1 tumor, and tricarboxylic acid cycle (TCA)-associated anaplerosis pathways, such as amino acids metabolisms, were enhanced. Then, after multiple passages of PDX, the altered glycolysis and TCA-associated anaplerosis pathways were partially recovered. Although no significant difference was observed in the response of PDXG1 and PDXG3 to chemotherapy, the difference in glycolysis and amino acids metabolism between PDXG1 and PDXG3 could still be maintained. In addition, the metabolomic difference between PC and CDX models were much larger than that of PDX model and PC, indicating that PDX model still retain more metabolic characteristics of primary tumor which is more suitable for tumor-associated metabolism research.ConclusionsCompared with primary tumor, PDX models have obvious difference in metabolomic level. These findings can help us design in vivo tumor metabolomics research legitimately and analyze the underlying mechanism of tumor metabolic biology thoughtfully.

An oncolytic virus delivering tumor-irrelevant bystander T cell epitopes induces anti-tumor immunity and potentiates cancer immunotherapy

Tumor-specific T cells are crucial in anti-tumor immunity and act as targets for cancer immunotherapies. However, these cells are numerically scarce and functionally exhausted in the tumor microenvironment (TME), leading to inefficacious immunotherapies in most patients with cancer. By contrast, emerging evidence suggested that tumor-irrelevant bystander T (TBYS) cells are abundant and preserve functional memory properties in the TME. To leverage TBYS cells in the TME to eliminate tumor cells, we engineered oncolytic virus (OV) encoding TBYS epitopes (OV-BYTE) to redirect the antigen specificity of tumor cells to pre-existing TBYS cells, leading to effective tumor inhibition in multiple preclinical models. Mechanistically, OV-BYTE induced epitope spreading of tumor antigens to elicit more diverse tumor-specific T cell responses. Remarkably, the OV-BYTE strategy targeting human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory efficiently inhibited tumor progression in a human tumor cell-derived xenograft model, providing important insights into the improvement of cancer immunotherapies in a large population with a history of SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Abstract 4592: Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor

Abstract Background: PRMT5 inhibitors are considered as a promising approach for cancer therapy due to their crucial role in the initiation and progression of many types of malignancies. Nevertheless, the crucial involvement of PRMT5 in controlling hematopoiesis, even in normal cells, resulted in significant on-target-off-tumor hematological toxicity when using first-generation, non-selective PRMT5 inhibitors in clinical settings, which hampered the clinical development of these drugs. The MTAP gene is deleted in around 10-15% of all human malignancies. As a result, the loss of MTAP leads to the buildup of MTA, which can bind to PRMT5 and partially inhibit PRMT5 activity. Inhibitors targeting the PRMT5-MTA complex become a promising strategy for treating MTAPnull cancer in a synthetically lethal manner. Herein, we report the preclinical feature of ATG-042-198, a novel MTAPnull-selective PRMT5 inhibitor. Methods: The in vitro activity and MTAP selectivity of ATG-042-198 on cell proliferation and cellular symmetric dimethylarginine (SDMA) expression were profiled using HCT116 MTAP wild type (wt) cells and HCT116 MTAP knock out (ko) cells, as well as in five MTAPnull cell lines. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with HCT116 MTAP wt cells, HCT116 MTAP ko cells and LU99 cells (MTAPnull). The in vitro and in vivo pharmacokinetic properties were assessed with corresponding assay methods. Results: ATG-042-198 demonstrated potent and selectively inhibitory effect on HCT116 MTAP ko cells’ proliferation (IC50=44nM) and SDMA expression (IC50=0.146nM), but very weak effect on HCT116 MTAP wt cells’ proliferation (IC50=4550nM) and SDMA expression (IC50>500nM). Consistently, ATG-042-198 showed excellent anti-proliferation activities on multiple endogenous MTAPnull cell lines (A549, IC50=2nM; LU99, IC50=6nM; U87MG, IC50=11nM; NALM-6, IC50=37nM; MIA PaCa-2, IC50=43nM). ATG-042-198 demonstrated high permeability (Caco2 assay, ER=2), good metabolic stability (Human liver microsomes assay, T1/2>145min), and low risk of drug interaction (CYP inhibition assay, 3A4/2C19/2D6, all IC50>10μM). In vivo PK study showed that ATG-042-198 was well absorbed, with a dose-dependent increase in plasma distribution and high oral bioavailability (F=108%). Plasma free drug concentrations were continuously higher than the IC50 of cell proliferation inhibition within 24 hours after 30mpk administration. In three CDX models (HCT116 MTAP wt, HCT116 MTAP ko and LU99), ATG-042-198 at a dose of 30 mg/kg QD potently and selectively inhibited tumor growth (TGI=-1%, 89% and 106%) without inducing body weight loss. Conclusions: In summary, ATG-042-198 is an oral MTAP null-selective PRMT5 inhibitor with potent efficacy against MTAPnull tumor. It also demonstrated good tolerability and preclinical PK profiles. Citation Format: Ya Kong, Ming Zhang, Lulu Jiang, Ruiting Xu, Bin Jiang, Guoqiang Dai, Yulong He, Huiling Liu, Jay Mei, Bing Hou, Bo Shan. Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor [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 4592.

Abstract 4591: PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors

Abstract Background: Methylthioadenosine phosphorylase (MTAP) gene deletion is highly prevalent across many cancer types such as brain, pancreatic and lung cancer. Accumulated MTA in MTAP loss cells competes with S-adenosylmethionine (SAM, the methyl donor) and leads to partial inhibition on protein arginine methyltransferase 5 (PRMT5), resulting in the sensitivity of these cells to PRMT5 inhibition. On this basis, MTA cooperative PRMT5 inhibitors (such as MRTX1719 and TNG908) have been designed to stabilize the binding of PRMT5 to MTA, but not SAM, resulting in further improvement of selectivity window. The clinical data showed that MRTX1719 has the capability to inhibit MTAP-loss tumor growth while avoid hematology toxicity observed with first generation PRMT5 inhibitors. Methods: A pair of HCT116 isogenic cell lines (MTAP-/- and MTAP+/+) were used to determine the effects of PH020-2 on cell proliferation and intracellular symmetric dimethylarginine (SDMA) content. Human CD34+ hematopoietic stem cells (HSCs) were collected to evaluate hematological toxicity. Intravenous injection of PH020-2 in rats were performed to assess the brain-penetration capability. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with tumor harboring MTAP gene deletion. The in vitro and in vivo pharmacokinetic (PK) and safety properties were assessed with corresponding assay methods. Results: PH020-2 dramatically decreased SDMA content and inhibited proliferation in HCT116 MTAP-/- cells (IC50, SDMA, 0.31 nM; proliferation, 5.9 nM), but had very weak effect on HCT116 WT cells (IC50, SDMA, 296.2 nM; proliferation, 1381 nM), suggesting an excellent selectivity (proliferation, 253×). In comparison, IC50 of MRTX1719 and TNG908 on MTAP-/- cell viability was 8.38 and 52.75 nM, respectively, generating a selectivity of 118× and 28×, respectively. PH020-2 mildly inhibited CD34+ HSC (IC50=807.1 nM), compared to the test on MRTX1719 (IC50=481.9 nM). The tests on a panel of cancer cells confirmed activity and selectivity of PH020-2. PH020-2 possessed excellent PK profiles in all species with absolute bioavailability of 98% in dogs. At the same time, PH020-2 showed outstanding brain penetration in rats (Kp, 0.67). In three CDX models (H838, H2170, Hup-T4), PH020-2 at a dose of 25 mg/kg BID potently inhibited tumor growth, and tumor regression was observed at higher doses in all tested models. Methyltransferase screening (44 targets) showed that PH020-2 potently inhibits PRMT5, but not others. Safety panel assays covering 47 targets demonstrated excellent safety profiles of PH020-2. All other in vitro and in vivo tests showed favorable results and supported for preclinical development. Conclusion: The data suggest that PH020-2 is an MTA-cooperative PRMT5 inhibitor with excellent brain-penetration that selectively targets MTAP-deleted tumors. Citation Format: Feng Gao, Bin Liu, Jing Wang, Liandong Jing, Yongyong Wu, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4591.

Abstract 5921: PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors

Abstract Background: High microsatellite instability (MSI-H) is highly prevalent across many cancer types including endometrium, gastric, ovary and colorectal cancer. It is well known that there is a clear synthetic lethality (SL) interaction between MSI-H and Werner syndrome protein (WRN) inhibition. WRN is a member of RecQ helicase family and involved in the maintenance of genome integrity. Different from other RecQ family members, WRN has two activity centers responsible for helicase and exonuclease activity, respectively. Further studies confirmed that an inhibition on helicase activity, but not exonuclease activity, is responsible for SL interaction with MSI-H. Also, Recent clinical observations supported that though loss-of-function of WRN could cause an early-aged disease Werner syndrome, selective loss of helicase but not exonuclease leads to only mild to moderate symptoms, suggesting a selective inhibitor of helicase activity has the potential to induce the SL interaction with MSI-H and keep patients safe. Methods: Biochemical assays and cell-based assays were performed to evaluate in vitro activity and selectivity of PH027-1. The in vivo anti-tumor efficacy was tested in two cell-derived xenograft (CDX) mouse models with tumors harboring MSI-H. The in vivo pharmacokinetics (PK) properties were evaluated in mice, rats and dogs, and other in vitro and in vivo PK and safety properties were assessed with corresponding assay methods. Results: Biochemical assays showed that PH027-1 potently inhibited helicase activity of WRN (IC50, 0.8 nM), but had no effect on other RecQ family members even at the highest tested concentration of 10 μM (IC50, all≥10 μM for BLM, RecQ4 and RecQ5). At the same time, PH027-1 did not affect the exonuclease activity of WRN at all tested concentrations. In cell-based assays, PH027-1 potently inhibited cell viability of MSI-H cancer cells SW48 and HCT116 at tens of nM, but had no effect on MSS cells (HT29 cancer cells and CD34+ hematopoietic stem cells) even at 10 μM. The tests were further expanded to 18 cancer cell lines covering uterus, gastric, ovary and colorectal cancer. Nine of twelve MSI-H cell lines were very sensitive to WRN inhibition, and all six MSS cell lines were totally resistant to the treatment. PH027-1 possessed excellent PK profiles as shown by in vivo PK studies, with absolute bioavailability of 58%~102% in mice, rats and dogs. In two CDX models (SW48 and HCT116), oral administration of PH027-1 dose-dependently inhibited tumor growth, and a nearly complete inhibition was observed at a dose of 20 mg/kg QD. Tumor regression was clearly shown at higher doses. All other in vitro and in vivo PK and safety tests showed favorable results and supported for preclinical development of PH027-1. Conclusion: As a highly selective and potent inhibitor on helicase activity of WRN, PH027-1 has the potential to become a novel and safe approach for treatment of MSI-H tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Jing Wang, Yongyong Wu, Jun Wan, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5921.

Abstract 2050: Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC

Abstract SLITRK6 is a type I transmembrane member of the SLITRK family of proteins and is highly expressed in certain epithelial tumors including urothelial, lung, breast cancer and glioblastoma, with minimal-to-low expression in normal tissues. Recent studies with a vedotin-based SLITRK6-directed ADC, AGS15E, have offered clinical proof-of-concept of harnessing this target with the ADC modality for addressing high unmet need at least in bladder cancer. PRO1106 is comprised of a proprietary humanized SLITRK6-targeting IgG1 (dubbed as “mAb”) and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1106 and the parent mAb exhibited strong binding (with low nanomolar affinity) to recombinant human or cynomolgus monkey SLITRK6 on bio-layer interferometry. No binding was observed with human SLITRK1 or SLITRK4 or in additional negative-binding assays. Potent cellular binding (with sub-nanomolar EC50) was also observed for the parent mAb and ADC with bladder, head and neck, and brain cancer cell lines as well as SLITRK6-overexpressing 293F cells. Target-binding was superior to sirtratumab (the parent antibody in AGS15E) and the binding epitope appeared to be distinct from that of sirtratumab in cross-block studies. PRO1106 displayed rapid internalization and elicited robust cytotoxicity (IC50 in the sub-nanomolar to low nanomolar range) in SLITRK6-expressing tumor cells but not target-negative cells. PRO1106 produced strong tumor growth inhibition in multiple cell-derived xenograft (CDX) models in mice representing bladder cancer and esophageal squamous cell carcinoma. PRO1106 showed a stable PK with much higher exposure compared to an in-house synthesized analog of AGS15E in rats. In an exploratory PK study in cynomolgus monkeys, PRO1106 at 5 mg/kg exhibited extended PK with minimal release of exatecan in circulation and with no apparent toxicity observed. In summary, PRO1106 is an ADC built on a clinically validated linker-drug directed at a clinically validated target, with promising data in preclinical pharmacology studies. As SLITRK6 expression in skin is negligible, PRO1106 may offer an exciting new option for patients with bladder cancer where the current standard-of-care ADC therapy (enfortumab vedotin) confers significant skin-related toxicity burden as well as for patients with additional cancer types. Citation Format: Wanwan Shen, Haotian Lu, Yang Xiao, Xuan Qiu, Haidong Liu, Lei Wang, Zhu Chen. Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC [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 2050.

Abstract 7590: Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair

Abstract Colorectal Cancer (CRC) is the third most common cause of cancer mortality in the US. A major problem in CRC management is relapse and progression to metastatic disease leading to poor overall survival. The tumor suppressor p53 is mutated in most CRCs and often drives metastasis and therapy resistance. Current therapeutic options targeting p53 mutant cancers show poor efficacy and frequently exhibit high toxicity. To address this clinical problem, we developed a novel therapeutic strategy for selective targeting of p53 mutant cancers. The strategy combines a thymidine analogue (e.g., trifluorthymidine/TFT, a component of TAS102) and poly (ADP-ribose) polymerase inhibitor (PARPi). We observed that TAS102 does not block DNA replication, but rather prompts post-replicative base-excision DNA repair (BER), and PARPi increases double-strand DNA breaks (DSBs) in p53 mutant cancer cells. Normal p53 wild type (p53wt) cells are arrested in the G1 phase and repair DNA. Thus, the TAS102-PARPi combination selectively targets p53-mutant cancer cells, resulting in the accumulation of DSBs and cell death. This novel strategy was examined in p53wt and p53-mutant tumor cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. The drug combination was significantly more effective in inhibiting p53 mutant tumor growth and in prolonging survival compared to monotherapies. This strategy is currently being tested in a first-in-human dose-escalation Phase I study (NCT04511039) in patients with advanced CRC. Further investigation revealed that the TAS102-PARPi combination induced DNA damage response (DDR) and the G2 checkpoint, leading to G2 arrest in p53 mutant cancer cells. We observed that TAS102-PARPi induced phosphorylation of CDC2 (also known as CDK1) but did not increase phosphorylation of histone H3 (a marker of mitosis), therefore indicating activation of the G2 checkpoint. Furthermore, the roles of ATM and ATR kinases in the DDR and G2 responses were clarified using highly specific kinase inhibitors. The analysis of the DDR/G2 signaling response in isogenic p53wt and mutant CRC cell lines showed that inhibition of WEE1 kinase can selectively abrogate the G2 checkpoint in p53-mutant cells, resulting in marked DNA damage and cell death. Importantly, the blockade of WEE1 strongly synergized with the TAS102-PARPi combination, further enhancing its potency and efficacy against p53-mutant cancer cells. In summary, our research provides a foundation for a novel therapeutic strategy for p53 mutant cancers that may provide a paradigm shift in the treatment of these deadly cancers, increasing efficacy while minimizing toxic adverse effects. Citation Format: Mohammed M. Alruwaili, Natsumi Naranjo, Priyanka Rajan, Kyeong Beom Jo, Dae-Kyum Kim, Thomas Melendy, Robert Straubinger, Christos Fountzilas, Andrei Bakin. Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair [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 7590.

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [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 1356.

Abstract 3147: DXC025, a novel anti-MUC1/EGFR bispecific antibody-tubulysin conjugate with a function linker, exhibits potent anti-tumor efficacy

Abstract Antibody-drug conjugates (ADCs) have become a promised drug class in cancer therapy. Armed with three main components-an antibody, a linker molecule, and a cytotoxic agent (“payload”), plus sites of the antibody conjugation and the distribution of tumor-associate antigens, nowadays ADCs have proved the unique ability of the precision of targeted therapy, a great leap forward from traditional chemotherapeutic approaches that cause widespread effects without specificity. Both epidermal growth factor receptor (EGFR) and mucin 1 (MUC1) are tumor-associated antigens (TAA) that are co-expressed in many solid tumors, such as, colon cancer, non-small cell lung cancer (NSCLC), esophageal cancer, epidermal cancer, and pancreatic cancer. In particular, MUC1, a glycoprotein essential for the formation of the epithelial mucous barrier, is hypoglycosylated and dimerizes with EGFR, a potent oncoprotein, in transformed cells. For our anti-Muc1 ADC clinical program, for treatment of colon and pancreatic cancers, it was found out that the highly glycosylated transmembrane Muc1 had much more turning over rates on the luminal surface of epithelial cells. Here, we first designed and generated bispecific Fab/scFv antibodies targeting both MUC1 and EGFR, using the knob-into-hole technology. The addition of the affinity- and internalization-optimized anti-EGFR arm to the other single Muc1 arm was aimed to broaden tumor selectivity and to reduce on-target toxicity, for the treatment of gastrointestinal tumors in comparison to individual full IgG1-ADC, either EGFR or Muc1-ADC. Indeed, many of generated anti- MUC1/EGFR bispecific antibodies (BsAbs) showed strong binding affinity in MUC1modetateEGFRmodetate cells, in addition to both MUC1 and EGFR highly expressed cells. Internalization assays demonstrated that the BsAbs were endocytosed in tumor cells co-expressing EGFR and MUC1 more efficiently than monoclonal antibodies targeting MUC1. The selected BsAbs were subsequently conjugated with tubulysin B analogs with varieties of Tubulysin B analog payloads and function peptidyl spacer/linkers to generate bispecific ADC (DXC025) candidates. DXC025 candidates exhibited very potent cytotoxicity in vitro against epidermal cancer, lung adenocarcinoma, and pancreatic cancer cell lines with IC50 of single or tens digital pM. In in vivo cell-derived xenograft (CDX) models, DXC025 candidates demonstrated much superior efficacies of tumor growth inhibition in comparison with parental (monoclonal), either Muc1- or EGFR- ADC for the treatment of gastrointestinal tumors. These results indicated that the MUC1/EGFR-ADC (DXC025) would be a promising ADC candidate for targeted treatment of either Muc1- or EGFR-expressing gastrointestinal tumors. Citation Format: Xingyan Jiang, Junxiang Jia, Huihui Guo, Xiangfei Kong, Yongfang Xu, Sishi Ye, Yong Du, Zhicang Ye, Qikuang Cen, Lingli Zhang, Yongxiang Chen, Gaituo Chen, Lu Bai, Yunxia Zheng, Wei Zheng, Jun Zheng, Juan Wang, Wenjun Li, Yuanyuan Huang, Linyao Zhao, Yifang Xu, Mengmeng Liu, Binbin Chen, Meng Dai, Miaomiao Chen, Zhixiang Guo, Qingliang Yang, Robert Y. Zhao. DXC025, a novel anti-MUC1/EGFR bispecific antibody-tubulysin conjugate with a function linker, exhibits potent anti-tumor efficacy [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 3147.

Abstract 4568: Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic cancer

Abstract Background: Pancreatic cancer (PaCa) is a highly aggressive malignancy with a very low five-year survival rate secondary to late diagnosis, limited treatment options and resistance to existing therapies. While the emergence of mutant-selective KRASG12D (G12D) inhibitors brings promise for treating PaCa patients carrying these mutations, their effectiveness as single agents is expected to be limited as evident from the clinical efficacy of KRASG12C inhibitors. Experimental Design: First, multiple G12D-mutated PaCa cell lines were treated with a G12D inhibitor (MRTX1133) and subjected to immunoblot analysis for the expression of KRAS effectors and BCL-2 family proteins. Next, a proteolysis-targeting chimera (PROTAC)-based platelet-sparing BCL-xL degrader (DT2216) and/or an mTOR inhibitor (everolimus) were evaluated in combination with MRTX1133 for viability, clonogenicity and apoptosis. Subsequently, mechanistic investigations were carried out using immunoblotting, senescence-associated β-galactosidase staining and qPCR. Finally, therapeutic efficacy and safety of the combinations were evaluated in cell-derived and patient-derived xenograft models. Results: MRTX1133 treatment creates apoptotic vulnerabilities by inducing aberrations in BCL-2 family proteins, with elevated BIM levels and decreased NOXA levels, in G12D-mutated PaCa cells. Furthermore, DT2216 and everolimus were shown to release BIM and stabilize NOXA, respectively, and their combination with MRTX1133 synergistically killed G12D-mutated PaCa cells in vitro and caused significant tumor growth inhibition or regression compared to single agents in vivo. Additionally, MRTX1133 treatment led to induction of senescence in a fraction of PaCa cells. These senescent cells were found to be dependent on BCL-xL and MCL-1 for their survival and were effectively killed with a combination of DT2216 and everolimus through BCL-xL degradation and NOXA-mediated MCL-1 inhibition, respectively. Conclusions: Our findings suggest that the single agent efficacy of MRTX1133 is limited due to apoptosis inhibition and complimentary senescence induction, and therefore its combination with DT2216 and/or everolimus is synergistic, which can potentially overcome resistance, by enhanced apoptosis and clearance of senescent cells. Citation Format: Sajid Khan, Lin Cao, Vignesh Vudhata, Yang Yang, Peiyi Zhang, Guangrong Zheng, Jose G. Trevino, Daohong Zhou. Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4568.

Abstract 5733: Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs

Abstract Topoisomerase 1 (TOP1) inhibitors are a class of cytotoxic small molecules that have demonstrated effectiveness as therapeutic agents in cancer. However, the broad clinical application has been hindered by poor pharmacokinetic profiles and off-target toxicities. Antibody-drug conjugates (ADCs) enable the targeted delivery of cytotoxic payloads by specifically targeting cell-surface antigens expressed on cancers reducing systemic exposure and toxicity. TOP1-ADCs have received FDA approval for HER2- (trastuzumab-deruxtecan) and Trop2- (sacituzumab-govitecan) expressing solid tumors, demonstrating the promise for this drug class to improve the lives of patients. We have developed a novel drug-linker using a potent TOP1 inhibitor with desirable ADME properties, and a hydrophilic glycoside linker as a platform technology enabling ADCs with favorable physicochemical and biological properties. The TOP1 inhibitor payload was optimized for potency, with reduced P-glycoprotein efflux and enhanced permeability. The hydrophilicity of the linker facilitates the development of highly loaded ADCs (8 drugs/mAb) with high plasma stability, low aggregation, and pharmacokinetics similar to parental antibody. The resultant ADCs are potent and immunologically specific with activity in multidrug-resistant tumor models and bystander activity in heterogenous antigen models. Human cancer cell derived xenograft models demonstrated potent and specific ADC anti-tumor activity with several targets and indications. Improved activity was observed compared to corresponding govitecan- and deruxtecan-based ADCs. ADCs prepared from this drug-linker technology were well tolerated in rats after repeat administration of 60 mg/kg every 4 days for 4 doses. The excellent anti-tumor activity in several models and favorable safety profile demonstrates this platform TOP1 drug-linker technology has the potential for development as ADCs against many cancer targets. Citation Format: Ryan Lyski, Lauren Bou, Johann Sigurjonsson, David Meyer, Calvin Neace, David Ortiz, Katie Snead, Kaleb Smith, Steven Jin, Nicole Stevens, Julia Cochran, Kaveh Alizadeh, Narayana Yeddula, Erica McKinney, Adam Peterson, Lindsay Dotloe, Jessica Simmons, Christopher Carosino, Kim Emmerton, Nancy Everds, Scott Jeffrey, Peter Senter. Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs [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 5733.

Abstract 5586: Hijacking mutant E3 ligase for targeted protein degradation: Discovery of cancer-specific PROTACs for the treatment of FBXW7 R465C driven cancers

Abstract Many cancers carry LOF mutations of tumor suppressor genes and drug discovery against these cancers are challenging. Fbxw7 is a major tumor suppressor E3 ligase and is frequently mutated (LOF) in cancer. Fbxw7 R465C mutation correlates with worse survival and resistance to both chemo- and targeted therapies. Blueray have discovered Fbxw7 R465C-selective ligands and developed PROTAC molecules selectively targeting cancers carrying Fbxw7 R465C mutation. The Fbxw7 R465C-based PROTACs show superior degradation activity for several target proteins and inhibit proliferation of cancer cells carrying Fbxw7 R465C mutations. These PROTACs show tumor growth inhibition in Fbxw7 R465C mutant cell derived xenograft models with clear PK/PD correlations. Development of bi-functional molecules such as PROTACs bring new options to target cancers with LOF mutations. Citation Format: Hailong Zhang. Hijacking mutant E3 ligase for targeted protein degradation: Discovery of cancer-specific PROTACs for the treatment of FBXW7 R465C driven 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 5586.

Abstract 2833: Validating cancer biomarker expression on mouse cell-derived xenograft models of human cancer

Abstract Background and Objective: The target antigen expression level is critical for the potency of immune therapies like ADC, mRNA-LNP, and CAR-T. Selecting an appropriate cancer model remains a challenge to the pre-clinical drug testing. HD Biosciences offers over 1000 cell lines and 150 CDX mouse models of human cancers. Furthermore, we have validated the expression of three therapeutic targets Nectin-4, Trop2, and PD-L1 in various CDX tumor tissues. This poster will highlight some recently established models as well as the antigen validation result. Our study provides a reference that guides the model selection of efficacy studies targeting Nectin-4, Trop2, or PD-L1. In addition, we offer a platform to validate the expression of target antigens in CDX mouse models covering numerous cancer types. Method and Result: Biomarker surface expression of Nectin-4, Trop2, and PD-L1 on xenograft tumors was validated by IHC staining and FACS analysis. We highlight these models with high expression of each marker. These models are available to support further efficacy studies of anti-cancer drugs targeting the marker. We also analyzed the biomarker surface expression in vitro using cell pellets via FACS analysis to compare with the in vivo expression level. Hence, the biomarker gene expression level was confirmed by FACS. Interestingly, the result indicates biomarker expression in mouse tumor tissues is often inconsistent with in vitro cell expression. For example, the A375 CDX model on humanized immunodeficient mice is commonly used for anti-PD-1/PD-L1 drug efficacy tests. However, the surface expression of PD-L1 is only detected in the A375 tumor tissues and is undetectable in the A375 cell pellet. This finding could explain the inconsistency of in vitro and in vivo drug test results. The in vivo CDX model-based biomarker would better predict the drug’s efficacy, as the model better mimics the clinical situation of tumor burden in patients. Conclusion: In vitro gene expression and protein expression of cell surface biomarkers may not be relevant to the in vivo tumor surface expression level, resulting in inconsistent efficacy data in vitro and in vivo. Thus, validating targeted biomarkers in vivo is crucial for choosing the best cancer tumor model with high target expression for drug efficacy validation. HD Biosciences provides validated 150 CDX models expressing Nectin-4, Trop2, and PD-L1 and have in vivo capability for target validation and drug efficacy study. Citation Format: Minqi Huang, Alyssa E. Wiggins, D Marroquin, Mengling Liu, Luke Tsai. Validating cancer biomarker expression on mouse cell-derived xenograft models of human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2833.

Abstract 7268: The SOS1 inhibitor MRTX0902 demonstrates activity across cancer models with mutations in proximal components of the RAS-MAPK pathway

Abstract Genetic alterations in the RAS-MAPK pathway are one of the most frequent causes of human cancers. While targeted therapies are approved for patients harboring mutations in KRAS G12C in advanced or metastatic non-small cell lung cancer, there remains a need for an effective therapy for cancers characterized by additional RAS-MAPK pathway mutations across cancer types. Previous studies have shown that genetic or pharmacological inhibition of Son of Sevenless homolog 1 (SOS1) demonstrates antiproliferative activity in cancer cells addicted to KRAS signaling, including cell lines with oncogenic mutations in KRAS, SOS1, PTPN11, or EGFR or a loss-of-function mutation in NF1. These genetic alterations each function to increase SOS1-mediated GTP-loading of KRAS and increase signaling flux through the MAPK pathway. Inhibition of SOS1 by MRTX0902 is anticipated to shift the tumor cell equilibrium of KRAS towards the inactive GDP-bound KRAS, leading to MAPK pathway inhibition. MRTX0902 is a selective inhibitor of SOS1 under evaluation in clinical trials for the treatment of advanced, unresectable, or metastatic cancers with mutations in the KRAS-MAPK pathway. In the present studies, we have shown that MRTX0902 demonstrates single-agent anti-proliferative activity and modulates ERK1/2 phosphorylation (pERK) across a panel of human tumor cell lines harboring oncogenic mutations in SOS1, PTPN11, BRAF (class III) and NF1. Furthermore, oral administration of MRTX0902 as a monotherapy in preclinical human tumor cell-derived xenograft models harboring these oncogenic mutations results in significant tumor growth inhibition. Additionally, combination with the RAF/MEK clamp avutometinib demonstrated greater antitumor activity versus either monotherapy treatment. These data support the clinical utility of MRTX0902 as a single agent or combination partner of avutometinib for the treatment of human cancers harboring oncogenic mutations implicated in dysregulation of RAS-dependent signaling including SOS1, PTPN11, NF1, and BRAF (class III). Citation Format: Niranjan Sudhakar, Larry Yan, Fadia Qiryaqos, Jade Laguer, David Briere, Allan Hebbert, Andrew Calinisan, Silvia Coma, Jonathan Pachter, James G. Christensen, Peter Olson, Shilpi Khare. The SOS1 inhibitor MRTX0902 demonstrates activity across cancer models with mutations in proximal components of the RAS-MAPK pathway [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 7268.

Abstract 2167: Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression

Abstract Colorectal cancer (CRC) affects a significant number of individuals each year, ranking as a leading cause of cancer-related deaths in the U.S. Bile acids (BAs), natural compounds crucial for digesting dietary fats, not only influence the gut microbiota but are also involved in gut health through their interaction with the Farnesoid X Receptor (FXR). Recent findings have highlighted certain microbiota-generated BAs like 3-oxo-lithocholic acid (LCA) and IsoLCA, modulate host immunity, hinder the expansion of intestinal pathogens, and potentially anti-aging. Despite the advancements in identifying the gut microbes behind these BAs, their precise impact on intestinal cells (IECs) and their role in disease progression are largely unexplored. Our pilot investigation unveiled 3oxoLCA as an FXR agonist, restoring FXR signaling in both cancer cells and APCmin/+ mice (a classic CRC mice model). As a result, 3-oxo-LCA inhibits the growth of both human and mouse CRC cells, such as MC38, CT26 and HCT116. Additionally, 3-oxo-LCA also restrained the intestinal stem cells (ISCs)’ proliferation in organoids from both wild-type and APCmin/+ mice, and patient-derived CRC organoids (PDCOs). Remarkably, treatment with 3-oxo-LCA decreased BAs levels, improved gut barrier function, reduced tumor load, and inhibited tumor progression in APCmin/+ mice. Furthermore, 3-oxo-LCA significantly suppressed tumor growth in cancer cell-derived xenograft model mice. Crucially, the 3-oxo-LCA-FXR interaction transcriptionally regulated key apoptotic genes, encouraging cancer cell death. These discoveries highlight the therapeutic promise of incorporating 3-oxo-LCA into strategies for treating CRC. Citation Format: Fei Sun, Xingchen Dong, Ming Qi, Ting Fu. Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression [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 2167.

Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024.