Colorectal Cancer Xenograft Model Research Articles

Macrophage-Derived Exosomes Promoted the Development and Stemness of Inflammatory Bowel Disease-Related Colorectal Cancer via nuclear paraspeckle assembly transcript 1-Mediated miRNA-34a-5p/phosphoprotein enriched in astrocytes 15 Axis.

Inflammatory bowel disease (IBD) is closely associated with the development of colorectal cancer (CRC) due to the chronic inflammatory response. Macrophages play critical roles in regulating the microenvironment to facilitate tumor progression. Exosomes are key modulators for the communication between macrophages and tumor cells. The mechanism of macrophage-derived exosomes in IBD-related CRC development remains unclear. The macrophages were isolated using fluorescence activating cell sorter (FACS). The RNA and protein expressions in exosomes and CRC cells were examined by quantitative real-time polymerase chain reaction and western blot assays, respectively. CRC cell development was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, BrdU staining, Transwell assay, and spheroid formation assay. The level of stemness was determined by detecting the proportion of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive CRC cells and the expression of LGR5, CD133, and CD44. Molecular interaction experiments were done using luciferase reporter assay and RNA immunoprecipitation assay. Xenograft tumor model in vivo and immunohistochemistry were used to observe the pathological changes. Macrophage-derived exosomes from IBD-related CRC tissues were enriched with nuclear paraspeckle assembly transcript 1 (NEAT1) and able to promote the progression and stemness of CRC both in vitro and in vivo. The exosomal NEAT1 could sponge miR-34a-5p, leading to the restoration of PEA15 expression in CRC cells and promoting the development of CRC. Inhibition of NEAT1 in exosomes could effectivity inhibit the tumor growth in the CRC xenograft model. These findings provide novel insights into how macrophages affect CRC development and highlight exosomal NEAT1 as a therapeutic target for CRC treatment.

Anti-tumor activity of camptothecin analog conjugate of a RSPO4-based peptibody targeting LGR4/5/6 in preclinical models of colorectal cancer.

Antibody-drug conjugates (ADCs) have emerged as a major modality of targeted cancer therapy, yet no ADC has been approved for colorectal cancer (CRC). LGR4/5/6 (leucine-rich repeat containing, G protein-coupled receptor 4, 5, 6) are three related receptors that are expressed at high levels together or alternately in nearly all cases of CRC. ADCs targeting LGR5 have been shown to have robust anti-tumor potency, but not all CRC cells express LGR5 and LGR5-positive tumor cells may lose LGR5 expression due to cancer cell plasticity. R-spondin 4 (RSPO4) is a natural protein ligand of LGR4/5/6 with high affinity for all three receptors. We fused a mutant form of RSPO4 that retains high affinity binding to LGR4/5/6 to IgG1 Fc to create a peptibody designated R462. Conjugation of R462 with a camptothecin analog (CPT2) at eight drugs per peptibody led to the synthesis of R462-CPT2 that showed highly potent cytotoxic activity in vitro in CRC cell lines expressing any of LG4/5/6. In cell line xenograft and PDX models of CRC, R462-CPT2 demonstrated robust anti-tumor effect. Importantly, R462-CPT2 showed no major adverse effect at therapeutically effective dose levels. These results strongly support the use of RSPO ligand drug-conjugates that target LGR4/5/6 simultaneously for the treatment of CRC.

Establishment and Clinical Significance of the Patient-Derived Xenograft Model of Colorectal Cancer.

Patient-derived xenograft (PDX) models are widely acknowledged for their ability to reflect the heterogeneity of humancancers and can be used to improve preclinical models. In this study, we evaluatedthe factors affecting the tumor formation rate ofthe PDX colorectal cancer (CRC)model and conducted preliminary drug sensitivity tests. CRC patients who underwent elective surgery at Shaoxing People's Hospital from November 2019 to October 2020 were included. The tumor tissue obtained from surgery was transplanted to the back of NSG mice, and the PDX model was established and subcultured to the F3 generation. Factors that affected tumorigenicity were analyzed and compared histologically. Drug interventions included 5-fluorouracil, oxaliplatin, and propofol. Sixty CRC patients were included in this study, and tumorigenesis was observed in CRC tissue derived from 37 cases (62%). The primary tumor malignancy degree (tumor stage and degree of cell differentiation), preoperative carcinoembryonic antigen level, and tumor location in CRC patients could affect the tumorigenicity of the PDX model. Histopathological analysis of CRC-PDX transplanted tumor tissue was highly consistent with the patient's tumor tissue. All four chemotherapy regimens could inhibit tumor growth and cause tumor tissue damage. Propofol could inhibit diarrhea in mice and protect intestinal mucosa. The CRC-PDX model established in this study can maintain the biological characteristics of primary tumors and can be used as a reference model for the individualized treatment of CRC patients. The degree of malignancy of the primary tumor is the primary factor affecting the tumorigenesis rate of the PDX model.

274 (PB262): Development of Patient-Derived Colorectal Cancer Xenograft and Organoid Models with Acquired Resistance to KRAS G12C Inhibitors

274 (PB262): Development of Patient-Derived Colorectal Cancer Xenograft and Organoid Models with Acquired Resistance to KRAS G12C Inhibitors

Impact of Tumoral β2-Adrenergic Receptor Expression on Chemotherapeutic Response and Prognosis in Patients with Advanced Colorectal Cancer.

The β2-adrenergic receptor (β2-AR) is a therapeutic target for circulatory agonists and exhibits oncogenic activity in several cancers. However, its role in advanced colorectal cancer (CRC) treated using chemotherapy remains unclear. We investigated the potential of β2-AR as a novel chemosensitivity marker and therapeutic target in inoperable CRC. β2-AR expression was evaluated immunohistochemically in 80 advanced or recurrent CRC cases for which untreated resected specimens were available before systemic chemotherapy implementation. We assessed the relationship among β2-AR protein expression, clinicopathological factors, therapeutic response, and prognosis. Furthermore, we evaluated the significance of β2-AR as an in vitro and in vivo therapeutic target using CRC cell lines and a CRC xenograft model treated with the β-blocker, propranolol, and other anticancer agents. High tumoral β2-AR expression was associated with shorter progression-free survival and chemotherapeutic resistance in patients treated with oxaliplatin-based regimens and bevacizumab-based regimens. We found no synergistic effect between propranolol and oxaliplatin. However, combined administration of propranolol and bevacizumab induced significant tumor shrinkage in the CRC xenograft model. β2-AR is a possible biomarker for chemosensitivity and prognosis in advanced CRC. Repositioning existing β-blockers could be beneficial for treating CRC resistant to existing treatment regimens.

Structural insights into molecular-targeting helix–loop–helix peptide against vascular endothelial growth factor-A

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

Novel ST1926 Nanoparticle Drug Formulation Enhances Drug Therapeutic Efficiency in Colorectal Cancer Xenografted Mice.

Cancer is a major public health problem that ranks as the second leading cause of death. Anti-cancer drug development presents with various hurdles faced throughout the process. Nanoparticle (NP) formulations have emerged as a promising strategy for enhancing drug delivery efficiency, improving stability, and reducing drug toxicity. Previous studies have shown that the adamantyl retinoid ST1926 displays potent anti-tumor activities in several types of tumors, particularly in colorectal cancer (CRC). However, phase I clinical trials in cancer patients using ST1926 are halted due to its low bioavailability. In this manuscript, we developed ST1926-NPs using flash nanoprecipitation with polystyrene-b-poly (ethyleneoxide) as an amphiphilic stabilizer and cholesterol as a co-stabilizer. Dynamic light scattering revealed that the resulting ST1926-NPs Contin diameter was 97 nm, with a polydispersity index of 0.206. Using cell viability, cell cycle analysis, and cell death assays, we showed that ST1926-NP exhibited potent anti-tumor activities in human CRC HCT116 cells. In a CRC xenograft model, mice treated with ST1926-NP exhibited significantly lowered tumor volumes compared to controls at low drug concentrations and enhanced the delivery of ST1926 to the tumors. These findings highlight the potential of ST1926-NPs in attenuating CRC tumor growth, facilitating its further development in clinical settings.

Radiolabeling and Preclinical Evaluation of Therapeutic Efficacy of 225Ac-ch806 in Glioblastoma and Colorectal Cancer Xenograft Models.

The epidermal growth factor receptor (EGFR) protein is highly expressed in a range of malignancies. Although therapeutic interventions directed toward EGFR have yielded therapeutic responses in cancer patients, side effects are common because of normal-tissue expression of wild-type EGFR. We developed a novel tumor-specific anti-EGFR chimeric antibody ch806 labeled with 225Ac and evaluated its invitro properties and therapeutic efficacy in murine models of glioblastoma and colorectal cancer. Methods: 225Ac-ch806 was prepared using different chelators, yielding [225Ac]Ac-macropa-tzPEG3Sq-ch806 and [225Ac]Ac-DOTA-dhPzPEG4-ch806. Radiochemical yield, purity, apparent specific activity, and serum stability of 225Ac-ch806 were quantified. In vitro cell killing effect was examined. The biodistribution and therapeutic efficacy of 225Ac-ch806 were investigated in mice with U87MG.de2-7 and DiFi tumors. Pharmacodynamic analysis of tumors after therapy was performed, including DNA double-strand break immunofluorescence of γH2AX, as well as immunohistochemistry for proliferation, cell cycle arrest, and apoptosis. Results: [225Ac]Ac-macropa-tzPEG3Sq-ch806 surpassed [225Ac]Ac-DOTA-dhPzPEG4-ch806 in radiochemical yield, purity, apparent specific activity, and serum stability. [225Ac]Ac-macropa-tzPEG3Sq-ch806 was therefore used for both invitro and invivo studies. It displayed a significant, specific, and dose-dependent invitro cell-killing effect in U87MG.de2-7 cells. 225Ac-ch806 also displayed high tumor uptake and minimal uptake in normal tissues. 225Ac-ch806 significantly inhibited tumor growth and prolonged survival in both U87MG.de2-7 and DiFi models. Enhanced γH2AX staining was observed in 225Ac-ch806-treated tumors compared with controls. Reduced Ki-67 expression was evident in all 225Ac-ch806-treated tumors. Increased expression of p21 and cleaved caspase 3 was shown in U87MG.de2-7 and DiFi tumors treated with 225Ac-ch806. Conclusion: In glioblastoma and colorectal tumor models, 225Ac-ch806 significantly inhibited tumor growth via induction of double-strand breaks, thereby constraining cancer cell proliferation while inducing cell cycle arrest and apoptosis. These findings underscore the potential clinical applicability of 225Ac-ch806 as a potential therapy for EGFR-expressing solid tumors.

Redistribution of Defective Mitochondria-mediated Dihydroorotate Dehydrogenase Imparts 5-Fluorouracil Resistance in Colorectal Cancer

Although 5-fluorouracil (5-FU) is the primary chemotherapy treatment for colorectal cancer (CRC), its efficacy is limited by drug resistance. Ferroptosis activation is a promising treatment for 5-FU-resistant cancer cells; however, potential therapeutic targets remain elusive. This study investigated ferroptosis vulnerability and dihydroorotate dehydrogenase (DHODH) activity using stable, 5-FU-resistant CRC cell lines and xenograft models. Ferroptosis was characterized by measuring malondialdehyde levels, assessing lipid metabolism and peroxidation, and using mitochondrial imaging and assays. DHODH function is investigated through gene knockdown experiments, tumor behavior assays, mitochondrial import reactions, intramitochondrial localization, enzymatic activity analyses, and metabolomics assessments. Intracellular lipid accumulation and mitochondrial DHODH deficiency led to lipid peroxidation overload, weakening the defense system of 5-FU-resistant CRC cells against ferroptosis. DHODH, primarily located within the inner mitochondrial membrane, played a crucial role in driving intracellular pyrimidine biosynthesis and was redistributed to the cytosol in 5-FU-resistant CRC cells. Cytosolic DHODH, like its mitochondrial counterpart, exhibited dihydroorotate catalytic activity and participated in pyrimidine biosynthesis. This amplified intracellular pyrimidine pools, thereby impeding the efficacy of 5-FU treatment through molecular competition. These findings contribute to the understanding of 5-FU resistance mechanisms and suggest that ferroptosis and DHODH are promising therapeutic targets for patients with CRC exhibiting resistance to 5-FU.

An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

Exploration of novel isoxazole-fused quinone derivatives as anti-colorectal cancer agents through inhibiting STAT3 and elevating ROS level

Colorectal cancer (CRC) is trending to be a major health problem throughout the world. Therapeutics with dual modes of action have shown latent capacity to create ideal anti-tumor activity. Signal transducer and activator of transcription 3 (STAT3) has been proved to be a potential target for the development of anti-colon cancer drug. In addition, modulation of tumor redox homeostasis through deploying exogenous reactive oxygen species (ROS)-enhancing agents has been widely applied as anti-tumor strategy. Thus, simultaneously targeting STAT3 and modulation ROS balance would offer a fresh avenue to combat CRC. In this work, we designed and synthesized a novel series of isoxazole-fused quinones, which were evaluated for their preliminary anti-proliferative activity against HCT116 cells. Among these quinones, compound 41 exerted excellent in vitro anti-tumor effect against HCT116 cell line with an IC50 value of 10.18 ± 0.4 nM. Compound 41 was proved to bind to STAT3 by using Bio-Layer Interferometry (BLI) assay, and can significantly inhibit phosphorylation of STAT3. It also elevated ROS of HCT116 cells by acting as a substrate of NQO1. Mitochondrial dysfunction, apoptosis, and cell cycle arrest, which was caused by compound 41, might be partially due to the inhibition of STAT3 phosphorylation and ROS production induced by 41. Moreover, it exhibited ideal anti-tumor activity in human colorectal cancer xenograft model and good safety profiles in vivo. Overall, this study provided a novel quinone derivative 41 with excellent anti-tumor activity by inhibiting STAT3 and elevating ROS level, and gave insights into designing novel anti-tumor therapeutics by simultaneously modulation of STAT3 and ROS.

Preclinical evaluation of AGTR1-Targeting molecular probe for colorectal cancer imaging in orthotopic and liver metastasis mouse models

Despite advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable for patients with distant liver metastasis. Fluorescence molecular imaging with specific probes is increasingly used to guide CRC surgical resection in real-time and treatment planning. Here, we demonstrate the targeted imaging capacity of an MPA-PEG4-N3-Ang II probe labeled with near-infrared (NIR) fluorescent dye targeting the angiotensin II (Ang II) type 1 receptor (AGTR1) that is significantly upregulated in CRC. MPA-PEG4-N3-Ang II was highly selective and specific to in vitro tumor cells and in vivo tumors in a mouse CRC xenograft model. The favorable ex vivo imaging and in vivo biodistribution of MPA-PEG4-N3-Ang II afforded tumor-specific accumulation with low background and >10 contrast tumor-to-colorectal values in multiple subcutaneous CRC models at 8 h following injection. Biodistribution analysis confirmed the probe's high uptake in HT29 and HCT116 orthotopic and liver metastatic models of CRC with signal-to-noise ratio (SNR) values of tumor-to-colorectal and -liver fluorescence of 5.8 ± 0.6, 5.3 ± 0.7, and 2.7 ± 0.5, 2.6 ± 0.5, respectively, enabling high-contrast intraoperative tumor visualization for surgical navigation. Given its rapid tumor targeting, precise tumor boundary delineation, durable tumor retention and docking study, MPA-PEG4-N3-Ang II is a promising high-contrast imaging agent for the clinical detection of CRC.

The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

Abstract LB199: Condensate modulator sequesters beta-catenin into depot condensates and demonstrates competitive anti-tumor activity in animal models ofcolorectal cancer

Abstract Constitutive activation of beta-catenin is a well-known driver of malignancy. However, traditional drug discovery approaches have proven challenging and largely unsuccessful in identifying therapeutic agents to modulate the function of beta-catenin. Biomolecular condensates have recently been demonstrated to play key roles in regulating most cellular processes and biological pathways by compartmentalizing biomolecules in membranelles organelles, thus lending novel opportunities to drug previously “undruggable” targets. Here, we leverage condensate biology to discover small molecules that reverse the hyperactive function of beta-catenin in colorectal cancer by entrapping it into depot condensates. We developed a high-throughput phenotypic assay that identifies beta-catenin condensate-modifying compounds (c-mods). Through condensate screening and functional secondary assays, we have identified c-mods that sequester beta-catenin into depots, induce selective cancer cell killing, and reverse oncogenic beta-catenin specific gene expression programs. C-mods identified in our screen are effective in genetically diverse colorectal cancers and a range of Wnt-associated cancers, providing the opportunity to treat a broad patient population. Furthermore, oral dosing of lead c-mod demonstrates competitive tumor growth inhibition as a single agent in xenograft and PDX models of colorectal cancer. Taken together, these results highlight the promise of condensate biology in drugging previously intractable high-value targets in oncology and developing novel treatments for patients suffering from diseases of high unmet need. Citation Format: Costa Salojin, Douglas Baumann, Adam Talbot, Kip West, Thomas Durand-Reville, Isaac Klein, Ann Boija. Condensate modulator sequesters beta-catenin into depot condensates and demonstrates competitive anti-tumor activity in animal models ofcolorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB199.

Abstract 3326: PMR-116, a second generation RNA Polymerase I inhibitor

Abstract Introduction: Inhibition of RNA Polymerase I, the enzyme responsible for the synthesis of ribosomal RNAs, the nucleic scaffold of ribosomes, has been proven as a novel therapeutic target as most cancer cells are "addicted" to ribosome biogenesis. Methods: In collaboration with Pimera Inc, we developed and tested the efficacy of a second generation RNA polymerase I inhibitor, PMR-116, in various pre-clinical cancer models. On-target activity of PMR-116 was confirmed in peripheral blood cells isolated from patients with solid tumors enrolled in a phase I trial of PMR-116. Results: PMR-116 potently inhibits Pol I transcription with ~200 fold higher selectivity towards Pol I compared to Pol II transcription. PMR-116 inhibits rRNA synthesis by stalling the Pol I complex at the rDNA promoter preventing promoter escape and consequently elongation. PMR-116 anti-cancer activity was evaluated against a panel of over 100 cancer cell lines representative of 20 major types of solid and haematological malignancies. This new Pol I inhibitor exhibited broad anti-proliferative and cytotoxic activity with an IC50 ranging from 32-4500nM and a median IC50 of 300nM. In contrast, cells derived from normal tissues were significantly less sensitive (IC50 ranging from 6-33μM). In vivo, PMR-116 significantly improved survival and reduce tumor burden in several preclinical models such as Vk*MYC transgenic model of indolent multiple myeloma, CT26 xenograft model of colorectal cancer, MMTV-PyMT transgenic model of metastatic breast cancer, mixed-lineage leukemia (MLL)-eleven nineteen leukemia (ENL)+Nras acute myeloid leukemia (+/-p53 mutation) and patient-derived xenografts of prostate cancer and AML. In a phase I trial of PMR-116, on-target activity of PMR-116 was observed in cells derived from the peripheral blood. Conclusion: PMR-116 significantly reduced cancer growth in a wide range of tumor models, including GEMM, syngeneic and PDX models. PMR-116 is undergoing clinical testing in phase I in solid tumors with the objective to determine maximum tolerated dose and identify a phase II dose. Citation Format: Luc Furic, Nadine Hein, Rita Ferreira, Konstantin Panov, Alisee Huglo, Richard Rebello, Eric Kusnadi, Denis Drygin, Mustapha Haddach, Ross Hannan. PMR-116, a second generation RNA Polymerase I 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 3326.

Abstract 4551: A novel MET antibody-drug conjugate based combination therapy to overcome colorectal cancer plasticity and drug resistance

Abstract Despite therapeutic advancements, colorectal cancer (CRC) remains the second deadliest malignancy in the United States. Challenges encountered in CRC treatment may be attributed to cancer stem cells (CSC), an immortal tumor cell population. CSCs potentiate tumor relapse by exploiting its infinite replicative potential and inherent drug resistance. Moreover, CSCs exhibit plasticity, allowing them to transition between differentiated and undifferentiated states in response to environmental cues to evade therapy and drive metastatic progression. MET is a receptor tyrosine kinase frequently upregulated in CRCs. We previously demonstrated that treatment with chemotherapies or antibody-drug conjugates (ADCs) targeting the CSC marker LGR5 led to the loss of LGR5 expression with concomitant MET-STAT3 pathway activation in therapy-resistant CRC cells. Further, we showed that LGR5 couples to the scaffold protein IQGAP1, which correlates with poor prognosis in several cancer types. Interestingly, our new data revealed that LGR5 knockdown enhances IQGAP1 interaction with MET and STAT3 via co-immunoprecipitation assays, suggesting a role for IQGAP1 in mediating MET-STAT3 activation and plasticity of LGR5+ CRC cells. To overcome therapy-induced plasticity, we generated MET-targeting ADCs conjugated to DNA-crosslinking payloads. First, we cloned and produced MET monoclonal antibodies (mAbs) and evaluated them for specificity and binding in a panel of CRC cell lines expressing different MET protein levels. MET mAbs demonstrated high selectivity for MET-expressing CRC cells and internalized to lysosomes, which is necessary for ADC payload release. To generate MET ADCs, we employed a site-specific conjugation methodology to attach cleavable peptide linker-drug moieties to the highest affinity MET mAb. MET ADCs were evaluated for cancer cell-killing efficacy in vitro in parallel with MET mAb, non-targeting control mAb (cmAb), and control ADC (cADC). Our MET ADC displayed high potency and efficacy in MET-expressing CRC cells, whereas MET mAb, cmAb, and cADC had minimal effects. MET ADCs were then tested in combination with chemotherapies or other targeted therapies. The combination of MET ADCs with 5-fluorouracil showed a synergistic effect in vitro. Future work involves investigating the safety and efficacy of MET ADCs alone and combined with chemotherapies in xenograft models of CRC. These findings present a mechanism underpinning CRC plasticity and the rationale for a novel treatment modality to potentially overcome CRC resistance and relapse. Citation Format: Shraddha Subramanian, Tressie A. Posey, Joan Jacob, Adela Aldana, Kendra S. Carmon. A novel MET antibody-drug conjugate based combination therapy to overcome colorectal cancer plasticity and drug resistance [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 4551.

Abstract 7221: A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model

Abstract Anti VEGF A antibody Bevacizumab combined with standard chemotherapy has improved outcomes for CRC patients. However, a subset of CRC patients acquires resistance to anti-VEGF treatment and underlying mechanism is poorly understood. Our recent study reported that circadian clock transcription factor BMAL1 drives transcription of VEGFA conferring resistance to bevacizumab treatment. Our rational is to stabilize negative regulator of circadian clock CRY protein to inhibit BMAL1 activity in a negative feedback loop. In this study, we evaluated whether CRY stabilizers SHP656 and SHP1705 in combination with bevacizumab can synergistically enhance anti-tumor effect in CRC mouse models. Methods: In vitro tube formation assay was performed with HUVECs cells to investigate the effect of CRY stabilizer on tube formation. For in vivo experiments, 106 HCT116 human CRC cells were injected subcutaneously into the right flank of 8-week-old nude mice. Mice (5mice/group) were randomized into: A. vehicle (2% carboxymethylcellulose PO 5 days/week; control IgG IP, 2 days/week); B. bevacizumab (5mg/kg IP, twice/week); C. 10mg/kg SHP656; D. 30mg/kg SHP656; E. 30mg/kg SHP1705; F. 10mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP1705 + bevacizumab. SHP 656 & 1705 was given PO; 5days/week. Tumor volume was measured three times a week. The survival curve was plotted using Kaplan and Meier method and assessed for statistical significance with log-rank test. Results: The in vitro tube formation assay showed significant inhibition of tube formation in HUVEC cells treated with SHP1705 plus bevacizumab compared to treated with bevacizumab alone. In in vivo study, the tumors treated with 30mg/kg SHP1705 + bevacizumab significantly suppressed tumor growth compared to bevacizumab monotherapy (one way-ANOVA, P <0.05). 30mg/kg SHP1705 + bevacizumab significantly improved survival of the mice (Log-rank test, P <0.05). No significant survival benefit was observed with bevacizumab alone. 3/5 mice treated with SHP1705 + bevacizumab showed decreased tumor volume and achieved complete regression for more than 4 months without additional treatment. In combination with bevacizumab, higher dose of SHP656 (30mg/kg) delayed tumor growth and extended survival compared with bevacizumab monotherapy (though did not reach statistical significance) whereas the lower dose of 10mg/kg did not show anti-tumor effect, demonstrating a dose dependent response. Conclusion: Our study shows that CRY stabilizers in combination with bevacizumab significantly enhances anti-tumor efficacy in CRC mouse models. Although mechanism of action of SHP compounds on angiogenesis pathway still needs further investigation, these findings may support development of novel treatment strategies modulating circadian clock to overcome bevacizumab resistance in CRC patients. Citation Format: Jae Ho Lo, Shivani Soni, Goar Smbatyan, Lesly Torres-Gonzalez, Pooja Mittal, Yan Yang, Francesca Battaglin, Priscilla Chan, Yuanzhong Pan, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Indrakant K. Singh, Evanthia T. Roussos Torres, Shannon M. Mumenthaler, Steve A. Kay, Heinz-Josef Lenz. A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model [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 7221.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [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 6487.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR < 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p <0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal 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 3241.

Abstract 1658: CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis

Abstract Activating mutations in BRAF at residue V600 (typically V600E) lead to dysregulation of the MAPK pathway and occur in approximately 8% of all human cancers including 60% of melanoma, up to 12% of colorectal cancer (CRC), and 4% of NSCLC. Currently approved BRAF inhibitors (BRAFi) are selective for BRAF V600X mutant proteins and are typically used in combination with MEK inhibitors (MEKi) in melanoma and NSCLC, or anti-EGFR antibodies such as cetuximab in CRC. However, their activity is limited by primary or acquired resistance often mediated by RAF dimer-inducing mechanisms. Furthermore, progression of BRAF V600X melanoma after BRAFi/MEKi treatment frequently involves brain metastasis, and currently approved BRAFi have relatively poor brain penetration. CFT1946 is a potent, orally bioavailable, cereblon-based BiDACTM degrader that selectively degrades BRAF V600X mutant protein and is currently under investigation in a Phase I clinical trial. CFT1946 has the potential to overcome limitations of clinically approved BRAFi, as degradation of BRAF V600X should abrogate RAF dimer-driven resistance and paradoxical activation. Indeed, we have previously demonstrated that CFT1946 is efficacious in an A375 BRAF V600E/NRAS Q61K xenograft model of BRAFi resistant melanoma. Here we substantially expand our preclinical characterization of CFT1946 across multiple models of BRAF V600X-driven cancers including BRAF V600X-driven CRC and NSCLC, additional BRAFi-resistant melanoma models, and a brain metastatic melanoma model. Single agent CFT1946 outperformed the SOC encorafenib + cetuximab combination in a panel of BRAF-V600X CRC xenograft models. Pathway analysis in BRAF V600X CRC models revealed that CFT1946 suppresses EGFR-mediated MAPK pathway reactivation, a mechanism known to diminish the impact of approved BRAF inhibitors in this indication. CFT1946 also demonstrated regression in a BRAF-V600X NSCLC PDX model where the SOC dabrafenib + trametinib showed only modest tumor growth inhibition. Consistent with our previous results in the A375 melanoma model, single agent CFT1946 showed superior activity versus the SOC dabrafenib + trametinib combination in all additional melanoma models tested, with complete regression achieved using a CFT1946 + trametinib combination in a PDX model bearing a BRAF-V600E kinase duplication that showed minimal response to dabrafenib + trametinib. Finally, using an A375 intracranial model, treatment with CFT1946 gave robust, dose dependent efficacy and survival advantage over encorafenib. The promising activity of CFT1946 in a broad range of BRAF V600X preclinical models supports its ongoing clinical investigation in BRAF V600 mutant solid tumors (NCT05668585). Citation Format: Bridget Kreger, Jacob R. Stephenson, Mathew E. Sowa, Samantha A. Perino, Laura L. Poling, Eunju Hurh, Michael J. Thomenius, Yanke Liang, Stewart L. Fisher, Roy M. Pollock. CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis [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 1658.