Colorectal Cancer Model Research Articles

Bidirectional effects of the tryptophan metabolite indole-3-acetaldehyde on colorectal cancer.

Colorectal cancer (CRC) has a high incidence and mortality. Recent studies have shown that indole derivatives involved in gut microbiota metabolism can impact the tumorigenesis, progression, and metastasis of CRC. To investigate the effect of indole-3-acetaldehyde (IAAD) on CRC. The effect of IAAD was evaluated in a syngeneic mouse model of CRC and CRC cell lines (HCT116 and DLD-1). Cell proliferation was assessed by Ki-67 fluorescence staining and cytotoxicity tests. Cell apoptosis was analysed by flow cytometry after staining with Annexin V-fluorescein isothiocyanate and propidium iodide. Invasiveness was investigated using the transwell assay. Western blotting and real-time fluorescence quantitative polymerase chain reaction were performed to evaluate the expression of epithelial-mesenchymal transition related genes and aryl hydrocarbon receptor (AhR) downstream genes. The PharmMapper, SEA, and SWISS databases were used to screen for potential target proteins of IAAD, and the core proteins were identified through the String database. IAAD reduced tumorigenesis in a syngeneic mouse model. In CRC cell lines HCT116 and DLD1, IAAD exhibited cytotoxicity starting at 24 h of treatment, while it reduced Ki67 expression in the nucleus. The results of flow cytometry showed that IAAD induced apoptosis in HCT116 cells but had no effect on DLD1 cells, which may be related to the activation of AhR. IAAD can also increase the invasiveness and epithelial-mesenchymal transition of HCT116 and DLD1 cells. At low concentrations (< 12.5 μmol/L), IAAD only exhibited cytotoxic effects without promoting cell invasion. In addition, predictions based on online databases, protein-protein interaction analysis, and molecular docking showed that IAAD can bind to matrix metalloproteinase-9 (MMP9), angiotensin converting enzyme (ACE), poly(ADP-ribose) polymerase-1 (PARP1), matrix metalloproteinase-2 (MMP2), and myeloperoxidase (MPO). Indole-3-aldehyde can induce cell apoptosis and inhibit cell proliferation to prevent the occurrence of CRC; however, at high concentrations (≥ 25 μmol/L), it can also promote epithelial-mesenchymal transition and invasion in CRC cells. IAAD activates AhR and directly binds MMP9, ACE, PARP1, MMP2, and MPO, which partly reveals why it has a bidirectional effect.

IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

Phase and Defect Engineering of MoSe2 Nanosheets for Enhanced NIR-II Photothermal Immunotherapy.

Inducing immunogenic cell death (ICD) during photothermal therapy (PTT) has the potential to effectively trigger photothermal immunotherapy (PTI). However, ICD induced by PTT alone is often limited by inefficient PTT, low immunogenicity of tumor cells, and a dysregulated redox microenvironment. Herein, we develop MoSe2 nanosheets with high-percentage metallic 1T phase and rich exposed active Mo centers through phase and defect engineering of MoSe2 as an effective nanoagent for PTI. The metallic 1T phase in MoSe2 nanosheets endows them with strong PTT performance, and the abundant exposed active Mo centers endow them with high activity for glutathione (GSH) depletion. The MoSe2-mediated high-performance PTT synergizing with efficient GSH depletion facilitates the release of tumor-associated antigens to induce robust ICD, thus significantly enhancing checkpoint blockade immunotherapy and activating systemic immune response in mouse models of colorectal cancer and triple-negative metastatic breast cancer.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt -/- ) caused a marked reduction in tumor growth in both syngeneic tumor models and a genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apc min ). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt -/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor cell-intrinsic mechanism to repress antitumor immunity.

Partial hepatectomy accelerates colorectal metastasis by priming an inflammatory premetastatic niche in the liver.

Resection of colorectal liver metastasis is the standard of care for patients with Stage IV CRC. Despite undoubtedly improving the overall survival of patients, pHx for colorectal liver metastasis frequently leads to disease recurrence. The contribution of this procedure to metastatic colorectal cancer at a molecular level is poorly understood. We designed a mouse model of orthograde metastatic colorectal cancer (CRC) to investigate the effect of partial hepatectomy (pHx) on tumor progression. CRC organoids were implanted into the cecal walls of wild type mice, and animals were screened for liver metastasis. At the time of metastasis, 1/3 partial hepatectomy was performed and the tumor burden was assessed longitudinally using MRI. After euthanasia, different tissues were analyzed for immunological and transcriptional changes using FACS, qPCR, RNA sequencing, and immunohistochemistry. Mice that underwent pHx presented significant liver hypertrophy and an increased overall metastatic load compared with SHAM operated mice in MRI. Elevation in the metastatic volume was defined by an increase in de novo liver metastasis without any effect on the growth of each metastasis. Concordantly, the livers of pHx mice were characterized by neutrophil and bacterial infiltration, inflammatory response, extracellular remodeling, and an increased abundance of tight junctions, resulting in the formation of a premetastatic niche, thus facilitating metastatic seeding. Regenerative pathways following pHx accelerate colorectal metastasis to the liver by priming a premetastatic niche.

Emulator-Based Bayesian Calibration of the CISNET Colorectal Cancer Models.

To calibrate Cancer Intervention and Surveillance Modeling Network (CISNET)'s SimCRC, MISCAN-Colon, and CRC-SPIN simulation models of the natural history colorectal cancer (CRC) with an emulator-based Bayesian algorithm and internally validate the model-predicted outcomes to calibration targets. We used Latin hypercube sampling to sample up to 50,000 parameter sets for each CISNET-CRC model and generated the corresponding outputs. We trained multilayer perceptron artificial neural networks (ANNs) as emulators using the input and output samples for each CISNET-CRC model. We selected ANN structures with corresponding hyperparameters (i.e., number of hidden layers, nodes, activation functions, epochs, and optimizer) that minimize the predicted mean square error on the validation sample. We implemented the ANN emulators in a probabilistic programming language and calibrated the input parameters with Hamiltonian Monte Carlo-based algorithms to obtain the joint posterior distributions of the CISNET-CRC models' parameters. We internally validated each calibrated emulator by comparing the model-predicted posterior outputs against the calibration targets. The optimal ANN for SimCRC had 4 hidden layers and 360 hidden nodes, MISCAN-Colon had 4 hidden layers and 114 hidden nodes, and CRC-SPIN had 1 hidden layer and 140 hidden nodes. The total time for training and calibrating the emulators was 7.3, 4.0, and 0.66 h for SimCRC, MISCAN-Colon, and CRC-SPIN, respectively. The mean of the model-predicted outputs fell within the 95% confidence intervals of the calibration targets in 98 of 110 for SimCRC, 65 of 93 for MISCAN, and 31 of 41 targets for CRC-SPIN. Using ANN emulators is a practical solution to reduce the computational burden and complexity for Bayesian calibration of individual-level simulation models used for policy analysis, such as the CISNET CRC models. In this work, we present a step-by-step guide to constructing emulators for calibrating 3 realistic CRC individual-level models using a Bayesian approach. We use artificial neural networks (ANNs) to build emulators that surrogate complex individual-based models to reduce the computational burden in the Bayesian calibration process.ANNs showed good performance in emulating the CISNET-CRC microsimulation models, despite having many input parameters and outputs.Using ANN emulators is a practical solution to reduce the computational burden and complexity for Bayesian calibration of individual-level simulation models used for policy analysis.This work aims to support health decision scientists who want to quantify the uncertainty of calibrated parameters of computationally intensive simulation models under a Bayesian framework.

ATG16L1 in myeloid cells limits colorectal tumor growth in ApcMin/+ mice infected with colibactin-producing Escherichia coli via decreasing inflammasome activation

ABSTRACT Escherichia coli strains producing the genotoxin colibactin, designated as CoPEC (colibactin-producing E. coli), have emerged as an important player in the etiology of colorectal cancer (CRC). Here, we investigated the role of macroautophagy/autophagy in myeloid cells, an important component of the tumor microenvironment, in the tumorigenesis of a susceptible mouse model infected with CoPEC. For that, a preclinical mouse model of CRC, the Apc Min/+ mice, with Atg16l1 deficiency specifically in myeloid cells (Apc Min/+ /Atg16l1[∆MC]) and the corresponding control mice (Apc Min/+ ), were infected with a clinical CoPEC strain 11G5 or its isogenic mutant 11G5∆clbQ that does not produce colibactin. We showed that myeloid cell-specific Atg16l1 deficiency led to an increase in the volume of colonic tumors in Apc Min/+ mice under infection with 11G5, but not with 11G5∆clbQ. This was accompanied by increased colonocyte proliferation, enhanced inflammasome activation and IL1B/IL-1β secretion, increased neutrophil number and decreased total T cell and cytotoxic CD8+ T cell numbers in the colonic mucosa and tumors. In bone marrow-derived macrophages (BMDMs), compared to uninfected and 11G5∆clbQ-infected conditions, 11G5 infection increased inflammasome activation and IL1B secretion, and this was further enhanced by autophagy deficiency. These data indicate that ATG16L1 in myeloid cells was necessary to inhibit colonic tumor growth in CoPEC-infected Apc Min/+ mice via inhibiting colibactin-induced inflammasome activation and modulating immune cell response in the tumor microenvironment. Abbreviation: AOM, azoxymethane; APC, APC regulator of WNT signaling pathway; ATG, autophagy related; Atg16l1[∆MC] mice, mice deficient for Atg16l1 specifically in myeloid cells; CASP1, caspase 1; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; CoPEC, colibactin-producing Escherichia coli; CRC, colorectal cancer; CXCL1/KC, C-X-C motif chemokine ligand 1; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MC, myeloid cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pks, polyketide synthase; qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TME, tumor microenvironment; TNF/TNF-α, tumor necrosis factor.

Synthesis and Evaluation of a Novel c-Met-Targeting Cyclic Peptide as a Potential Diagnostic Agent for Colorectal Cancer.

The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase linked to the proliferation, survival, invasion, and metastasis of several types of cancers, including colorectal cancer (CRC), particularly when aberrantly activated. Our study strategically designs peptides derived from interactions between c-Met and the antibody Onartuzumab. By utilizing a cyclic strategy, we achieved significantly enhanced peptide stability and affinity. Our in vitro assessments confirmed that the cyclic peptide HYNIC-cycOn exhibited a higher affinity (KD = 83.5 nM) and greater specificity compared with its linear counterpart. Through in vivo experiments, [99mTc]Tc-HYNIC-cycOn displayed exceptional tumor-targeting capabilities and minimal absorption in nontumor cells, as confirmed by single-photon emission computed tomography. Notably, the ratios of tumor to muscle and tumor to intestine, 1 h postinjection, were 4.78 ± 0.86 and 3.24 ± 0.47, respectively. Comparable ratios were observed in orthotopic CRC models, recording 4.94 ± 0.32 and 3.88 ± 0.41, respectively. In summary, [99mTc]Tc-HYNIC-cycOn shows substantial promise as a candidate for clinical applications. We show that [99mTc]Tc-HYNIC-cycOn can effectively target and visualize c-Met-expressing tumors in vivo, providing a promising approach for enhancing diagnostic accuracy when detecting c-Met in CRC.

Inactivation of KDM6A promotes the progression of colorectal cancer by enhancing the glycolysis

KDM6A (lysine demethylase 6A) has been reported to undergo inactivating mutations in colorectal cancer, but its function in the progression of colorectal cancer has not been evaluated using animal models of colorectal cancer. In this study, we found that knocking out KDM6A expression in mouse intestinal epithelium increased the length of villus and crypt, promoting the development of AOM (azoxymethane)/DSS (dextran sulfate sodium salt)-induced colorectal cancer. On the other hand, knocking down KDM6A expression promoted the growth of colorectal cancer cells. In molecular mechanism studies, we found that KDM6A interacts with HIF-1α; knocking down KDM6A promotes the binding of HIF-1α to the LDHA promoter, thereby promoting LDHA expression and lactate production, enhancing glycolysis. Knocking down LDHA reversed the malignant phenotype caused by KDM6A expression loss. In summary, this study using animal models revealed that KDM6A loss promotes the progression of colorectal cancer through reprogramming the metabolism of the colorectal cancer cells, suggesting that restoring the function of KDM6A is likely to be one of the strategies for colorectal cancer treatment.

Prognostic Analysis Combining Histopathological Features and Clinical Information to Predict Colorectal Cancer Survival from Whole-Slide Images.

Colorectal cancer (CRC) is a malignant tumor within the digestive tract with both a high incidence rate and mortality. Early detection and intervention could improve patient clinical outcomes and survival. This study computationally investigates a set of prognostic tissue and cell features from diagnostic tissue slides. With the combination of clinical prognostic variables, the pathological image features could predict the prognosis in CRC patients. Our CRC prognosis prediction pipeline sequentially consisted of three modules: (1) A MultiTissue Net to delineate outlines of different tissue types within the WSI of CRC for further ROI selection by pathologists. (2) Development of three-level quantitative image metrics related to tissue compositions, cell shape, and hidden features from a deep network. (3) Fusion of multi-level features to build a prognostic CRC model for predicting survival for CRC. Experimental results suggest that each group of features has a particular relationship with the prognosis of patients in the independent test set. In the fusion features combination experiment, the accuracy rate of predicting patients' prognosis and survival status is 81.52%, and the AUC value is 0.77. This paper constructs a model that can predict the postoperative survival of patients by using image features and clinical information. Some features were found to be associated with the prognosis and survival of patients.

Constructing a prognostic model for colorectal cancer with synchronous liver metastases after preoperative chemotherapy: a study based on SEER and an external validation cohort.

The combination of preoperative chemotherapy and surgical treatment has been shown to significantly enhance the prognosis of colorectal cancer with liver metastases (CRLM) patients. Nevertheless, as a result of variations in clinicopathological parameters, the prognosis of this particular group of patients differs considerably. This study aimed to develop and evaluate Cox proportional risk regression model and competing risk regression model using two patient cohorts. The goal was to provide a more precise and personalized prognostic evaluation system. We collected information on individuals who had a pathological diagnosis of colorectal cancer between 2000 and 2019 from the Surveillance, Epidemiology, and End Results (SEER) Database. We obtained data from patients who underwent pathological diagnosis of colorectal cancer and got comprehensive therapy at the hospital between January 1, 2010, and June 1, 2022. The SEER data collected after screening according to the inclusion and exclusion criteria were separated into two cohorts: a training cohort (training cohort) and an internal validation cohort (internal validation cohort), using a random 1:1 split. Subgroup Kaplan-Meier (K-M) survival analyses were conducted on each of the three groups. The data that received following screening from the hospital were designated as the external validation cohort. The subsequent variables were chosen for additional examination: age, gender, marital status, race, tumor site, pretreatment carcinoembryonic antigen level, tumor size, T stage, N stage, pathological grade, number of tumor deposits, perineural invasion, number of regional lymph nodes examined, and number of positive regional lymph nodes. The primary endpoint was median overall survival (mOS). In the training cohort, we conducted univariate Cox regression analysis and utilized a stepwise regression approach, employing the Akaike information criterion (AIC) to select variables and create Cox proportional risk regression models. We evaluated the accuracy of the model using calibration curve, receiver operating characteristic curve (ROC), and area under curve (AUC). The effectiveness of the models was assessed using decision curve analysis (DCA). To evaluate the non-cancer-related outcomes, we analyzed variables that had significant impacts using subgroup cumulative incidence function (CIF) and Gray's test. These analyses were used to create competing risk regression models. Nomograms of the two models were constructed separately and prognostic predictions were made for the same patients in SEER database. This study comprised a total of 735 individuals. The mOS of the training cohort, internal validation cohort, and QDU cohort was 55.00months (95%CI 46.97-63.03), 48.00months (95%CI 40.65-55.35), and 68.00months (95%CI 54.91-81.08), respectively. The multivariate Cox regression analysis revealed that age, N stage, presence of perineural infiltration, number of tumor deposits and number of positive regional lymph nodes were identified as independent prognostic risk variables (p < 0.05). In comparison to the conventional TNM staging model, the Cox proportional risk regression model exhibited a higher C-index. After controlling for competing risk events, age, N stage, presence of perineural infiltration, number of tumor deposits, number of regional lymph nodes examined, and number of positive regional lymph nodes were independent predictors of the risk of cancer-specific mortality (p < 0.05). We have developed a prognostic model to predict the survival of patients with synchronous CRLM who undergo preoperative chemotherapy and surgery. This model has been tested internally and externally, confirming its accuracy and reliability.

Notch-Targeted Therapeutic in Colorectal Cancer by Notch1 Attenuation Via Tumor Microenvironment-Responsive Cascade DNA Delivery.

The Notch signaling is a key molecular pathway that regulates cell fate and development. Aberrant Notch signaling can lead to carcinogenesis and progression of malignant tumors. However, current therapies targeting Notch pathway lack specificity and induce high toxicity. In this report, a tumor microenvironment-responsive and injectable hydrogel is designed to load plasmid DNA complexes as a cascade gene delivery system to achieve precise Notch-targeted gene therapy of colorectal cancer (CRC). The hydrogels are prepared through cross-linking between phenylboric acid groups containing poly(oligo(ethylene glycol)methacrylate) (POEGMA) and epigallocatechin gallate (EGCG), used to load the complexes between plasmid DNA encoding short hairpin RNAs of Notch1 (shNotch1) and fluorinated polyamidoamine (PAMAM-F) (PAMAM-F/shNotch1). In response to low pH and H2O2 in tumor microenvironment, the hydrogel can be dissociated and release the complexes for precise delivery of shNotch1 into tumor cells and inhibit Notch1 activity to suppress malignant biological behaviors of CRC. In the subcutaneous tumor model of CRC, PAMAM-F/shNotch1-loaded hydrogels can accurately attenuate Notch1 activity and significantly inhibit tumor growth without affecting Notch signal in adjacent normal tissues. Therefore, this therapeutic system can precisely inhibit Notch1 signal in CRC with high responsiveness and low toxicity, providing a promising Notch-targeted gene therapeutic for human malignancy.

Cost effectiveness of colorectal cancer screening in the Dominican Republic.

e23033 Background: Colorectal cancer (CRC) incidence in the Dominican Republic (DR) is increasing, and CRC now ranks as the third-most prevalent cancer in the DR. There are no national CRC screening guidelines in the DR, and CRC screening is not routinely performed. We evaluated the impact of nationwide CRC screening by comparing the cost-effectiveness of four CRC screening strategies to the current practice of no screening. Methods: We developed a Markov model comparing four CRC screening strategies to the current practice of no screening, or natural history (NH): colonoscopy every ten years (Colo); sigmoidoscopy every five years (Sig); fecal immunochemical test biennially (FIT); and fecal occult blood test biennially (FOBT). Model inputs for screening arms were derived from international randomized controlled trials whenever possible. Data from The Rosa Emilia Sánchez Pérez de Tavares National Cancer Institute in the DR was used to estimate baseline CRC incidence, stage distribution and mortality. Screening was implemented from age 45 to 75. We assumed a 60.6% adherence rate for each screening arm. The model was cross validated by comparing the estimated lifetime impact of each screening arm on CRC incidence and mortality to estimates derived from the MIcrosimulation SCreening Analysis (MISCAN) Colorectal Cancer Model in the US. The primary outcome was the incremental cost-effectiveness ratio (ICER) of each screening strategy. Secondary outcomes included life expectancy, total cost, CRC incidence and CRC-related mortality. The willingness to pay threshold (WTP) was set to the 2022 DR gross domestic product per person ($10,111.20/life year gained). Results: In the NH strategy, 2.3% of the population developed CRC, and 1.2% died from CRC, with a lifetime cost of 203.47 per person. Compared to NH, FIT prevented 27% of CRCs and 37% of CRC deaths, with a lifetime cost of 244.76 per person. Colo was more effective than FIT, preventing 33% and 43% of CRC cases and deaths, respectively, compared to NH. However, the ICER for Colo exceeded the WTP threshold. One-way sensitivity analyses showed that the model is most sensitive to test performance characteristics and test costs. Conclusions: Among the evaluated CRC screening strategies, FIT is the cost-effective option, averting a significant number of CRC cases and deaths, at an incremental cost that is well within the WTP threshold for the DR. Inclusion of local data on the effectiveness and acceptability of the tested CRC screening modalities would improve this model, and strengthen the credibility of our findings. [Table: see text]

An overview of chemically induced rodent models for sporadic colorectal cancer: Histopathological and translational perspectives.

Globally, colorectal cancer (CRC) is one of the most frequently diagnosed human gastrointestinal neoplasia and the second leading cause of cancer-related death in both men and women. Despite considerable efforts currently devoted to the study of the biology and treatment of CRC, patient prognosis and survival are still poor. Sporadic CRC is a complex multistep disease and usually emerges in the setting of lifestyle and dietary changes mainly observed in industrialized countries with high human development index (HDI) (westernized style). The molecular pathogenesis of sporadic CRC presents genetic heterogeneity with APC, RAS, PIK3CA, TGFBR, SMAD4, and TP53 mutations usually detected during the progression of this malignancy. The establishment of sporadic CRC models has become essential for both basic and translational research to improve our understanding of the pathophysiology, unravel new molecular drivers, and preventive/therapeutic improvement of this malignancy. Chemically induced rodent models of sporadic CRC recapitulate most key morphological and genetic/epigenetic events observed during the promotion and progression of this malignancy, establishing effective diagnostic and prevention strategies to be translated into clinical practice. The present review gathers the main features of the state-of-the-art evidence on chemically induced rodent models, widely applied for translational modelling of sporadic CRC with a specific focus on histopathology and prevention perspectives. Our narrative review reinforces the persistent value of these bioassays and encourages the use of multimodel strategies for further investigations.

A phase 1, first-in-human, open-label study evaluating the safety, tolerability, pharmacokinetics, and efficacy of TT125-802 in patients with advanced solid tumors.

TPS3171 Background: TT125-802 is a selective, potent, orally bioavailable small molecule inhibitor of the bromodomain of CBP and p300, two highly homologues lysine acetyltransferases. CBP/p300 act as transcriptional co-activators with multiple domains and functions, including well-documented roles in cell cycle regulation and several critical tumorigenic pathways (1-3). TT125-802 has shown mono-therapy efficacy in preclinical models of castration-resistant prostate cancer, KRAS-mutated colorectal and non-small cell lung cancer (4,5). Furthermore, TT125-802 is capable to interfere with transcriptional resistance mechanisms limiting targeted therapies (6) which will be explored clinically upon determination of a safe, well-tolerated, and biologically active single-agent dose. Methods: This study aims to establish the safety, tolerability, and pharmacokinetic profile of TT125-802. The maximum tolerated dose/recommended dose for expansion (MTD/RDE) of repeat daily dosing for TT125-802 monotherapy will be determined in subjects with advanced solid tumors in a sequential escalating cohorts design, (1 cycle = 21 days) using the Bayesian Logistics Regression Model (BLRM) with sentinel patients (2-day window for first patient enrolled in each cohort). Cohorts will consist of 3 to 6 subjects who will be followed over a dose-limiting toxicity (DLT) period of at least 21 days. TT125-802 will be administered orally once daily; additional schedules may be evaluated. Dose escalation is planned to follow two-fold increments. It may be adapted upon dose level revision and guided by pharmacokinetic analysis. Additional subjects (up to 6) may be enrolled in at least two monotherapy cohorts deemed safe and tolerable to optimize the recommended phase 2 dose (RP2D). Up to a total of 20 additional subjects may be enrolled. Translational/pharmacodynamic endpoints: Exploratory endpoints will provide proof-of-principle and early biomarker data for CBP/p300 inhibition. These analyses will include: 1. Single-cell gene expression evaluation of peripheral blood mononuclear cells (PBMCs) at multiple time points, 2. anagen hair bulb bulk RNAseq analysis, and 3. paired tumor biopsies will be analyzed with single-cell RNA sequencing. Together, these investigations are expected to support optimal dose determination. Additionally, circulating tumor DNA might be evaluated to define molecular responses. Outlook: The recommended phase 2 dose will be used further to explore the efficacy of TT125-802 as monotherapy. TT125-802 will be evaluated with targeted therapies (for example, KRASG12C/D, BRAF, EGFR, etc., inhibitors) to exploit the blocking of transcriptional escape mechanisms driven by CBP/p300. 1. Iyer, 2007. 2. Dutta, 2016. 3. Attar, 2017. 4. AACR2023 posters #6268. 5. AACR poster #3907. 6. AACR2023 poster #3907. Clinical trial information: 2022-500849-24-00 .

Comparing invitro cytotoxic drug sensitivity in colon and pancreatic cancer using 2D and 3D cell models: Contrasting viability and growth inhibition in clinically relevant dose and repeated drug cycles.

In vitro drug screening that is more translatable to the invivo tumor environment can reduce both time and cost of cancer drug development. Here we address some of the shortcomings in screening and show how treatment with 5-fluorouracil (5-FU) in 2D and 3D culture models of colorectal cancer (CRC) and pancreatic ductal adenocarcinomas (PDAC) give different responses regarding growth inhibition. The sensitivity of the cell lines at clinically relevant 5-FU concentrations was monitored over 4 days of treatment in both 2D and 3D cultures for CRC (SW948 and HCT116) and PDAC (Panc-1 and MIA-Pa-Ca-2) cell lines. The 3D cultures were maintained beyond this point to enable a second treatment cycle at Day 14, following the timeline of a standard clinical 5-FU regimen. Evaluation after one cycle did not reveal significant growth inhibition in any of the CRC or PDAC 2D models. By the end of the second cycle of treatment the CRC spheroids reached 50% inhibition at clinically achievable concentrations in the 3D model, but not in the 2D model. The PDAC models were not sensitive to clinical doses even after two cycles. High content viability metrics point to even lower response in the resistant PDAC models. This study reveals the limitations of testing drugs in 2D cancer models and short exposure in 3D models, and the importance of using appropriate growth inhibition analysis. We found that screening with longer exposure and several cycles of treatment in 3D models suggests a more reliable way to assess drug sensitivity.

Efficacy of fadraciclib (CYC065), a novel dual CDK2/9 inhibitor, on patient-derived models of colorectal cancer.

3596 Background: Colorectal cancer (CRC) is the third most common type of cancer among adults in the United States. Given the heterogeneous nature of the disease and the limited number of treatments available, there is an unmet need to identify new therapeutic vulnerabilities for advanced CRC. One promising approach is targeting of cyclin dependent kinases (CDKs). In our current study, we sought to understand the potential efficacy of fadraciclib (CYC065), a novel dual CDK 2/9 inhibitor, as a novel treatment for metastatic CRC. Methods: Eighteen CRC patient-derived organoids (PDOs) were generated from patients undergoing biopsy or resection for their primary or metastatic CRC under an IRB approved protocol at Duke University. IHC and H&amp;E staining were first performed on the established PDOs to confirm the diagnosis of CRC. PDOs were treated with standard of care chemotherapy (oxaliplatin, irinotecan (SN38) and 5-Flurouracil), CDK4/6 inhibitor palbociclib, and fadraciclib (CYC065). All drugs were used in doses ranging from 6.4nM to 100µM, and drug sensitivities were determined using Cell-Titer Glo (CTG) 72 hours after treatment. Subsequently, three matching PDXs were generated for in vivo validation and treated with fadraciclib (CYC065) via oral gavage at a dose of 25 mg/kg BID five days a week for two weeks. Target validation for CDK2/9 inhibition was performed via western blotting, cell cycle arrest was determined via flow cytometry, and killing by anaphase catastrophe was determined by immunofluorescence staining. Results: CRC PDOs were more sensitive to fadraciclib (CYC065) than chemotherapy and palbociclib, with IC50 values lower than chemotherapy or palbociclib (2.65±3.92µM for fadraciclib (CYC065) vs. 29.92±41.98µM for chemotherapy (P&lt;0.05) or palbociclib (12.33±9.87µM)). Subsequently, we validated our findings in vivo using 3 matched PDX showing significant tumor growth inhibition (TGI) with fadraciclib (CYC065) compared to control (p&lt;0.05) without serious adverse effects. We showed that fadraciclib (CYC065) induces G2/M cell cycle arrest, resulting in anaphase catastrophe as evident by the presence of multipolar mitosis in fadraciclib (CYC065)-treated cells. CDK2/9 inhibition leads to downregulation of c-Myc levels and upregulation of apoptosis induction as a second mechanism of action for this drug. Conclusions: Our data using both in vitro and in vivo patient-derived models support further exploration of fadraciclib (CYC065) has a potential therapy for advanced CRC. CDK2/9 inhibition impacts multiple critical pathways involved in transcription, mitosis and apoptosis. Further studies will be needed to reveal molecular biomarkers of sensitivity and resistance to this novel agent.

Updated results from ERAS-007 plus encorafenib and cetuximab (EC) in patients (pts) with EC-naïve metastatic BRAF V600E colorectal cancer (CRC) in the phase 1b/2 HERKULES-3 study.

3517 Background: The RAS/MAPK pathway (including BRAF) is dysregulated in a broad range of cancers including CRC, resulting in downstream activation of ERK1/2. Patients (pts) with metastatic CRC with BRAF V600E mutation (BRAF V600E mCRC) have dramatically poorer survival rates than those with non-BRAF V600E mutated CRC, highlighting the need for novel therapies. The combination of a BRAF plus EGFR inhibitor is approved for the treatment of pts with BRAF V600E mCRC; however, objective response occurs in only 20% of pts. ERAS-007 is a novel, potent, and orally bioavailable inhibitor of ERK. ERAS-007 alone or in combination with encorafenib and cetuximab (EC) showed promising in vitro and in vivo activity in BRAF V600E CRC models, supporting investigation of the potential clinical benefit of ERAS-007 + EC in pts with BRAF V600E mCRC. Methods: HERKULES-3 is a Phase 1b/2 study to assess the safety, tolerability, pharmacokinetics (PK), and preliminary clinical activity of ERAS-007 combinations targeting the MAPK pathway in pts with advanced GI cancers. Within this study, we are currently evaluating the safety, tolerability, PK, and preliminary clinical activity of the combination of ERAS-007 + EC in pts with EC-naïve BRAF V600E CRC. Results: As of 30 November 2023, 19 (EC-naïve and EC-pretreated) pts have been dosed with ERAS-007 100 mg twice daily-once a week (BID-QW) in combination with EC (300 mg oral daily + 500 mg/m2 intravenous infusion every 2 weeks). The treatment-emergent AEs (TEAEs) occurring in ≥25% of pts were dermatitis acneiform (52.6%); nausea and fatigue (47.4% each); diarrhea (42.1%); constipation, headache, and hematuria (31.6% each); and vomiting, anemia, and infusion-related reaction (26.3% each). There were no Grade 4 TEAEs. One pt suffered a Grade 5 unrelated event of respiratory failure. There were no Grade 3 treatment-related AEs that occurred in more than 1 pt. There were no ERAS-007-related AEs that led to discontinuation of ERAS-007. Of the 19 pts, 11 were EC naïve and 10 of 11 were efficacy evaluable. In the EC naïve efficacy evaluable pts, the ORR was 40% with 3 of the 4 responses confirmed. The maximum duration of response is &gt; 15 months; updated data will be presented. Conclusions: ERAS-007 100 mg BID-QW in combination with EC was safe and well tolerated with preliminary evidence of promising clinical activity. These results support continued evaluation of this combination in EC naïve pts with BRAF V600E CRC. Clinical trial information: NCT05039177 .

Combining the potency of a neoantigen-expressing DNA vector, an anti-CTLA-4 antibody, and an attenuated poxvirus in a personalized immunization platform: Preclinical studies of ODI-2001.

e14674 Background: Immune checkpoint inhibitors (ICI) have transformed cancer treatment. Their impressive effectiveness is crucially dependent on pre-existing neoepitope-specific immune responses. ODI-2001 aims at increasing the immune response against tumors by combining a DNA expressing multiple neoepitopes of the tumor, a MVA vaccinia virus as physiologic adjuvant and DNA carrier, and an anti-CTLA4 antibody as amplifier of the immune response. Methods: ODI-2001 was administered by repeated subcutaneous injections after tumor cells implantation in mice melanoma model B16F10 and colorectal cancer model CT26, and both before and after implantation in breast cancer model 4T1. In B16F10 model combination of ODI-2001 with anti-PD1 was assessed also. Neoepitopes were chosen from literature in B16F10 and CT26 models. In 4T1 model they were predicted using a dedicated neoepitope prediction algorithm (ImmunoEngine, myNEO, Gent, Belgium). Tumor volume and mice survival were assessed over time. Tumors were also analysed by immunofluorescence for immune cell infiltration. Each experiment received approval from the ethics committee for animals. Results: A significant improvement in survival was observed in mice treated with ODI-2001 in B16F10, and CT26 syngeneic mice models. In B16F10 the combination of ODI-2001 with anti-PD1 antibody demonstrated synergistic activity and a significant improvement in survival, while anti-PD1 alone or combined with anti-CTLA-4 failed to show any therapeutic effect (ODI-2001 + anti-PD1 vs. anti-CTLA-4 + anti-PD1 p&lt;0.01). ODI-2001 treatment was associated with tumor rejections in the three animal models. In 4T1 model the ImmunoEngine epitope prediction algorithm demonstrated an improved efficacy as compared to a set of neoepitopes chosen from the literature. In B16F10, following immunization with ODI-2001, an increased CD8 immune cell infiltration of the tumor was documented. ODI-2001 was well tolerated by the treated animals. Conclusions: These data demonstrate a significant antitumoral activity of ODI-2001 as a neoantigen immunization platform across different preclinical cancer models, both in monotherapy and combined with anti-PD1. ODI-2001 represents a promising option to induce de novo cancer-specific immune responses, potentially increasing the proportion of patients responding to immunotherapy treatments. Based on this preclinical program a phase I with ODI-2001 in patients with advanced colorectal carcinoma is currently under preparation.

Effect of TT-816, a novel immune response modifier targeting cannabinoid CB2 receptor, on antitumor immunity and cancer growth.

e14595 Background: The endocannabinoid system is widely expressed in the human body, including the innate and adaptive immune system, where endocannabinoids, Δ9-tetrahydrocannabinol and synthetic ligands regulate immune suppression. The effects of endocannabinoids on immune regulation are primarily mediated by G-protein coupled cannabinoid CB2 receptors (CB2R) via several mechanisms, including immune cell development, migration, proliferation and T and NK cell effector functions. The upregulated expression of CB2R and elevated levels of endocannabinoids have been observed in a variety of tumor microenvironments and are associated with the aggressiveness of cancer. Methods: NK cell function was determined by co-culturing TT-816 pretreated NK cells with K562 cancer cells for 24 hours. The mixed lymphocyte reaction assay was conducted by co-culturing human CD4+ T cells with monocyte-derived dendritic cells. Cell viability was measured by FACS and IFN-g by MSD. The in vivo efficacy of TT-816 was determined in the murine syngeneic B16F10 melanoma and MC38 colorectal cancer models. Tumor-bearing C57BL/6 mice were randomized 6 days after inoculation and treated with vehicle (0.5% CMC, QD) or with TT-816 at 1, 4 or 10 mg/kg (QD) orally, or anti-PD1 (RMP1 14) at 5 mg/kg (Q3D), intraperitoneally. Tumor size and body weight were determined every 2 to 3 days. Data represent mean±SEM. Results: TT-816 concentration-dependently enhanced the functions of NK cells, dendritic cells and T cells. It increased NK cell killing of the human cancer cells and IFN-g production, significantly stimulated the expression of CD86, HLA-DR, IL-12 and TNF-a in monocyte-derived dendritic cells, and enhanced CD4+ T cell proliferation and IFN-g production in a mixed lymphocyte reaction assay. In B16F10 melanoma and MC38 colorectal cancer models, TT-816 dose-dependently inhibited the tumor growth. Furthermore, combination of TT-816 with anti-PD1 synergistically inhibited both tumor growth. In B16F10 model, treatment with TT 816 (4 mg/kg, QD) alone significantly inhibited the tumor growth by 45%. By contrast, anti-PD1 had little effect (TGI 5%). However, the combination of TT-816 with anti-PD1 resulted in a TGI of 72%. In the MC38 model, treatment with TT-816 (4 mg/kg, QD) and anti-PD1 alone, achieved TGI of 50% and 36%, respectively. When combined, TGI was increased to 88%. Conclusions: TT-816 is a novel, oral small molecule immune response modifier that selectively blocks CB2R on immune cells and cancer cells. Preclinical data have demonstrated that it stimulates antitumor immune response and inhibits cancer growth in both hot and cold tumor models. TT-816 is currently undergoing phase 1 clinical trials for the treatment of a broad range of solid tumors.