Colorectal Cancer Patient-derived Xenograft Research Articles

Therapeutic Responses to Two New SN-38 Derivatives in Colorectal Cancer Patient-Derived Xenografts and Respective 3D In Vitro Cultures.

SN-38, an active metabolite of irinotecan, exhibits toxicity to all proliferating cells, causing dose-limiting and potentially life-threatening side effects. Newly established water-soluble derivatives of SN-38, 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin (BN-MOA) and 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (BN-NMe), exhibit a unique mechanism of spontaneous alkylation of aromatic bases in DNA and show greater in vitro activity on cancer cell lines than SN-38. The aim of this study was to compare the therapeutic responses to irinotecan, BN-MOA and BN-NMe in vivo and in vitro in 3D cultures using colorectal cancer (CRC) patient derived xenografts (PDX). Seven established PDX tissues were subcutaneously grown on the flanks of NSG or NSG-SGM3 mice and tumor diameters were measured with a caliper. Compounds were administrated intraperitoneally at 40 mg/kg every five days. 3D PDX cultures were performed on 96-well LifeGel plates and cell viability was determined with the CellTiter Glo 3D reagent. Treatment with irinotecan significantly delayed or stopped the growth of 5 out of 7 PDXs, with a greater level of inhibition from BN-MOA compared to irinotecan and BN-NMe. In vitro studies exhibited the same trends in SN-38 and BN-NMe but not in BN-MOA. The new SN-38 derivatives, BN-MOA and BN-NMe, showed enhanced therapeutic effects compared to irinotecan in CRC models. BN-MOA demonstrated superior tumor inhibition in vivo, while BN-NMe had similar in vitro activity to SN-38. These findings highlight the potential of BN-MOA for greater antitumor efficacy in vivo, with BN-NMe showing comparable effectiveness to SN-38 in vitro. Future studies should optimize growth models to better predict anticancer drug responses.

Abstract IA003: Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase

Abstract The WRN helicase serves as a key target in the treatment of cancers characterized by microsatellite instability (MSI) because it plays a crucial role in resolving harmful non-canonical DNA structures that arise in cells with defective mismatch repair systems. Despite the critical functions of human DNA and RNA helicases, no drugs targeting these enzymes have been approved, largely due to the difficulties in identifying potent and selective inhibitors. In this study, we present the chemoproteomics-based identification of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This inhibitor specifically interacts with a cysteine residue (C727) within the helicase domain, which undergoes inter-domain movement during the DNA unwinding process. VVD-133214 reacts with the WRN protein in a nucleotide cooperative manner, promoting stable, compact structures that impede the enzyme's dynamic flexibility essential for its helicase activity. Inhibition of WRN by VVD-133214 leads to extensive double-stranded DNA breaks, nuclear enlargement, and ultimately cell death, specifically in MSI-high cells but not in microsatellite stable cells. The inhibitor demonstrated good tolerance in mice and significant tumor reduction in various MSI-high colorectal cancer cell lines and patient-derived xenograft models. Our findings highlight an allosteric strategy to inhibit WRN function that avoids interference from the endogenous ATP cofactor in cancer cells and positions VVD-133214 as a promising therapeutic candidate for patients with MSI-high cancers. Citation Format: Matthew P. Patricelli, Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodilles, Seth M .Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N Williams, Martha K. Pastuszka, John J. Sigler, Piergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Todd M. Kinsella. Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA003.

Patient tissue-derived FGFR4-variant and wild-type colorectal cancer organoid development and anticancer drug sensitivity testing

ObjectivesFGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. MethodsClinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. ResultsWES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. ConclusionsFGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis

BackgroundAberrant alternative splicing (AS) is a pervasive event during colorectal cancer (CRC) development. SF3B3 is a splicing factor component of U2 small nuclear ribonucleoproteins which are crucial for early stages of spliceosome assembly. The role of SF3B3 in CRC remains unknown.MethodsSF3B3 expression in human CRCs was analyzed using publicly available CRC datasets, immunohistochemistry, qRT-PCR, and western blot. RNA-seq, RNA immunoprecipitation, and lipidomics were performed in SF3B3 knockdown or overexpressing CRC cell lines. CRC cell xenografts, patient-derived xenografts, patient-derived organoids, and orthotopic metastasis mouse models were utilized to determine the in vivo role of SF3B3 in CRC progression and metastasis.ResultsSF3B3 was upregulated in CRC samples and associated with poor survival. Inhibition of SF3B3 by RNA silencing suppressed the proliferation and metastasis of CRC cells in vitro and in vivo, characterized by mitochondria injury, increased reactive oxygen species (ROS), and apoptosis. Mechanistically, silencing of SF3B3 increased mTOR exon-skipped splicing, leading to the suppression of lipogenesis via mTOR-SREBF1-FASN signaling. The combination of SF3B3 shRNAs and mTOR inhibitors showed synergistic antitumor activity in patient-derived CRC organoids and xenografts. Importantly, we identified SF3B3 as a critical regulator of mTOR splicing and autophagy in multiple cancers.ConclusionsOur findings revealed that SF3B3 promoted CRC progression and metastasis by regulating mTOR alternative splicing and SREBF1-FASN-mediated lipogenesis, providing strong evidence to support SF3B3 as a druggable target for CRC therapy.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and 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 1900.

Abstract 219: Molecular and therapeutic characterization of a large-scale collection of metastatic colorectal cancer patient-derived xenografts and matched organoids for translational oncology

Abstract Strong evidence supports the use of patient-derived xenografts (PDXs) as faithful models reflecting patient outcomes. Since not all research can support the use of animals, exploiting patient-derived in vitro models such as organoids (also know as tumoroids) is essential. However, current biobanks of metastatic colorectal cancer (mCRC) tumoroids have important limitations, such as small sample size and a lack of systematic in vivo validation with paired xenografts, which decreases the translational value of tumoroid-based pipelines. Here we describe XENTURION, a unique open-science resource of XENografts and Tumoroids for Research In ONcology that encompasses 129 sibling pairs of mCRC PDXs and PDX-derived tumoroids (PDXTs) with accompanying molecular and therapeutic characterization.The vast majority of XENTURION models were analyzed for mutations (targeted next-generation sequencing of 116 relevant CRC genes), gene copy number architecture (DNA shallow sequencing) and global transcriptomics (RNAseq) and benchmarked against large patient datasets (TCGA and MSK-IMPACT). Results showed that the PDX-PDXT collection proved to be representative of the genetic heterogeneity of mCRCs and displayed high genetic and transcriptional similarity between matched pairs. A PDXT-based population trial with the clinically approved anti-EGFR antibody cetuximab was performed in 116 PDXTs, revealing variable sensitivities that were consistent with clinical response biomarkers and with tumor growth changes in 79 matched PDXs. Cetuximab response profiles also recapitulated the outcome of EGFR knock-out by CRISPR-Casp9 technology in 13 representative PDXTs. Adaptive signals upregulated by EGFR blockade both in tumoroids and PDXs were computationally and functionally prioritized. Top candidates were screened in PDXTs and 3 surviving compounds were identified as actionable co-extinction targets with cetuximab in 12 PDXT models. Results were finally validated in PDXs.To our knowledge, this is the first large-scale study in which a systematic comparison of molecular and therapeutic profiles between PDXT-PDX pairs was attempted. As a publicly available resource, XENTURION will offer a knowledge base of disseminatable methods, resources and information to streamline preclinical studies and accelerate new treatments for patients with mCRC. Citation Format: Simonetta M. Leto, Elena Grassi, Marco Avolio, Valentina Vurchio, Francesca Cottino, Martina Ferri, Eugenia R. Zanella, Laura Di Blasio, Alberto Puliafito, Luca Primo, Andrea Bertotti, Livio Trusolino. Molecular and therapeutic characterization of a large-scale collection of metastatic colorectal cancer patient-derived xenografts and matched organoids for translational oncology [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 219.

Abstract 1934: The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts

Abstract Background: Cetuximab and panitumumab are monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), that provide clinical benefit to metastatic colorectal cancer (mCRC) patients with RAS wild-type (RASWT) tumors. Unfortunately, intrinsic or acquired resistance to these therapies limits their clinical effectiveness, necessitating the development of more efficient therapeutic strategies. Onvansertib is an oral, small molecule, selective inhibitor of the PLK1 kinase, currently in clinical development for KRAS-mutant mCRC. The aim of this study was to assess the anti-tumor activity of onvansertib monotherapy and in combination with cetuximab in RASWT CRC patient-derived xenograft (PDX) models. Methods: Twenty RASWT CRC PDXs were selected and engrafted in nude mice. Once tumors reached 200-350-mm3, mice were treated with vehicle, onvansertib (60mg/kg, QD), cetuximab (20mg/kg, BIW) or the combination for 18 days. PDX models were chosen based on their sensitivity to cetuximab, resulting in a selection of 7 cetuximab-sensitive (CetuxS) and 13 cetuximab-resistant (CetuxR) PDXs, including 7 with intrinsic resistance and 6 with acquired-resistance. Tumor volume change from baseline (TVC) was calculated as 100%x(Vt-Vo)/Vo and tumor growth inhibition (TGI) as 100%x(TVCcontrol-TVCtreated)/TVCcontrol. Tumor regression was defined as TVCD18<0 and tumor stasis 0≤TVCD18<100. Results: Cetuximab sensitivity was confirmed in the selected models, cetuximab induced tumor regression in all the CetuxS PDXs, while no or limited activity was observed in the resistant models (median TGI=29%, IQR 16-61). Onvansertib exhibited potent anti-tumor activity in 17 (85%) PDXs, resulting in tumor regression (n=11) or tumor stasis (n=6). Onvansertib TGI at Day 18 was not significantly different in CetuxS PDXs (median TGI 102, IQR 76-103) compared to CetuxR PDXs (median TGI 108, IQR 74-124), supporting that onvansertib anti-tumor activity is independent of the sensitivity/resistance to cetuximab. The combination of onvansertib and cetuximab induced tumor regression in 18 (90%) PDXs. The combination showed significantly improved efficacy compared to individual therapies in some of the models. Conclusions: Onvansertib monotherapy displayed potent anti-tumor activity in RASWT CRC PDX models, independently of their sensitivity to cetuximab. Additionally, onvansertib combined with cetuximab exhibited either comparable or superior anti-tumor activity than the monotherapies. Collectively, this data supports the clinical development of the PLK1 inhibitor onvansertib for RASWT mCRC. Citation Format: Maya Ridinger, Preeti Kanikarla Marie, Fengqin Gao, Zhensheng Liu, Giulia Maddalena, David Menter, Alexey Sorokin, Tod Smeal, Scott Kopetz. The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts [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 1934.

Abstract 2362: Preclinical efficacy and safety of M9140, a novel antibody-drug conjugate (ADC) with topoisomerase 1 (TOP1) inhibitor payload targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-expressing colorectal tumors

Abstract Background: M9140 is the first clinical-stage TOP1 inhibitor payload ADC directed against CEACAM5, which is expressed in colorectal cancer (CRC) and other cancer indications. M9140 is composed of a CEACAM5-specific antibody conjugated to eight, highly stable and hydrophilic ß-glucuronide-exatecan linker-payloads. After linker cleavage, the highly potent and membrane permeable TOP1 inhibitor exatecan is released. Methods: M9140 potency was tested in viability assays using cancer cell lines with different levels of CEACAM5 expression. Potency shift viability assays were conducted using two CRC cell lines, with zosuquidar used as multidrug resistance-1 (MDR-1) inhibitor. The M9140 bystander effect was assessed in co-culture experiments with CEACAM5-negative and -positive cancer cells. Antitumor efficacy and safety were evaluated in patient-derived xenograft (PDX) mouse models and in cynomolgus monkeys, respectively. Results: M9140 demonstrated specific binding to CEACAM5 and selective killing of target-positive cancer cells with potencies between 0.09 nM and 0.62 nM. A potent bystander effect of M9140 was demonstrated in co-culture experiments indicating the potential to treat tumors with heterogeneous target expression. Unlike other ADC payloads such as monomethyl auristatin E (MMAE) and the maytansine derivative DM4, exatecan potency was not affected by MDR-1 inhibition, indicating that this drug efflux resistance mechanism may not be relevant for exatecan. A single treatment with 10 mg/kg M9140 caused strong antitumor effects in all 14 CRC PDX models tested, with tumor stasis in 10 models and tumor regression in four models as the best response. The TOP1 inhibitor-based ADC M9140 caused strong antitumor efficacy with a tumor volume reduction of 74% and 88% as best response in two CRC PDX models where a maytansine-based CEACAM5 ADC was not effective. In cynomolgus monkeys, M9140 showed dose-dependent hematolymphoid and intestinal effects, consistent with exatecan payload, with no indication of any other toxicities such as interstitial lung disease (ILD) or ocular toxicity. Conclusions: M9140 demonstrated high potency, strong antitumor activity, and bystander effect in preclinical models. The side effect profile in monkeys was favorable and in line with the expected toxicity of exatecan. Notably, ILD and ocular toxicity, which are known adverse effects of deruxtecan and maytansine-based ADCs respectively, were absent. A first-in-human study to evaluate the safety, tolerability, pharmacokinetics, and preliminary clinical activity of M9140 in advanced solid tumors is ongoing (NCT05464030). Citation Format: Sabine Raab-Westphal, Felix Hart, Willem Sloot, Min Shan, Nicolas Rasche, Christiane Amendt, Jan Anderl. Preclinical efficacy and safety of M9140, a novel antibody-drug conjugate (ADC) with topoisomerase 1 (TOP1) inhibitor payload targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-expressing colorectal 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 2362.

Abstract 2118: SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models

Abstract Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the cell surface of most of the colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and of more than 2/3 of gastric cancer (GC) and non-small cell lung cancer (NSCLC) while normal tissue expression is limited. The high prevalence of CEACAM5 expression prompted us to develop a novel CEACAM5 topoisomerase I inhibitor (Topo1i) antibody drug conjugate (ADC) with a DAR of 8, SGN-CEACAM5C/SAR445953. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The Topo1i payload was optimized for potency and enhanced bystander activity. SGN-CEACAM5C/SAR445953 is rapidly internalized and demonstrates in vitro cytotoxicity with EC50 values in the sub-nM range while it induces no toxicity on CEACAM5-negative cells. The potent anti-tumor activity is mediated by direct cytotoxicity on CEACAM5-expressing tumor cells and by a strong bystander effect due to the diffusion of the payload to the neighboring CEACAM5-negative tumor cells. Accordingly, the ADC incubated in a co-culture of CEACAM5-positive and -negative cells at a ratio of 1/1 induces a ~50% growth inhibition of CEACAM5-negative cells. Interestingly, with incubated ADC, only 1% of CEACAM5-positive cells in co-culture are sufficient to provide 11% growth inhibition of CEACAM5-negative cells. In vivo, SGN-CEACAM5C/SAR445953 is stable in circulation in SCID mice with a t1/2 close to 15 days. In vivo efficacy at 1, 3 and 10 mg/kg (single administration) was evaluated in panels of 4 CRC, 3 PDAC, 4 NSCLC and 3 GC patient-derived xenograft (PDX) models. At 10 mg/kg, the ADC elicits antitumor regression in 14/14 models. At 3 mg/kg, tumor regression occurs in 12/14 models. This potent, specific and dose dependent anti-tumor activity was further confirmed in Single Mouse Trials (SMT) of 20 CRC PDX models, 31 lung cancer PDX models and 19 gastric cancer PDX models. SMT consists in use of one animal per PDX model per treatment arm and for which the evaluation of efficacy is based on RECIST (Response Evaluation Criteria In Solid Tumors) criteria used in clinic. In CRC, gastric and lung cancer SMT, disease control rates are 95%, 84% and 87%, respectively with overall response rates of 55%, 68% and 71% including 15%, 10% and 26% of complete responses, respectively. The high anti-tumor activity across panels of PDX models of several CEACAM5 positive indications supports further evaluation of SGN-CEACAM5C/SAR445953 in patients with CRC, PDAC, GC and lung cancers (NCT06131840). Citation Format: Yves Baudat, Celine Nicolazzi, Johann Petur Sigurjonsson, Celine Amara, Astrid Clarke, Ryan lyski, Dave Meyer, Valeria Fantin, Marielle Chiron, Stephanie Decary. SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor 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 2118.

Structure-Activity Relationship of FL118 Platform Position 7 Versus Position 9-Derived Compounds and Their Mechanism of Action and Antitumor Activity.

Structurally, FL118 is a camptothecin analogue and possesses exceptional antitumor efficacy against human cancer through a novel mechanism of action (MOA). In this report, we have synthesized and characterized 24 FL118 Position 7-substituted and 24 FL118 Position 9-substituted derivatives. The top compounds were further characterized for their MOA in colorectal cancer (CRC) models using CRC patient-derived xenograft (PDX) models and pancreatic cancer PDX models to evaluate their antitumor activities. Four FL118 Position 7-substituted derivatives showed significantly better antitumor efficacy than the FL118 Position 9-substituted derivatives. The four identified compounds also appeared to have better antitumor activity than their parental platform FL118. Interestingly, RNA-Seq analyses indicated that three of the four compounds exerted antitumor effects via an MOA similar to FL118, which provided an intriguing opportunity for follow-up studies. Extended in vivo studies revealed that FL77-6 (7-(4-ethylphenyl)-FL118), FL77-9 (7-(4-methoxylphenyl)-FL118), and FL77-24 (7-(3, 5-dimethoxyphenyl)-FL118) exhibit potential for further development toward clinical trials.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Abstract B147: Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models

Abstract Background: Resistance to topo-I inhibitors is of significant importance in treating patients with metastatic CRC (mCRC). Selinexor and eltanexor are selective inhibitors of nuclear export, by binding to exportin 1 (XPO1, the primary nucleic export receptor, and is overexpressed in CRC). Eltanexor is an investigational XPO1 inhibitor, with similar mechanism and potency but improved tolerability in animal models. XPO1 inhibition results in nuclear localization of tumor suppressor proteins, oncoprotein mRNAs, and topoisomerases, and decreases DNA damage repair (DDR) response. Irinotecan is a topo-I inhibitor which induces DNA damage and is commonly used in mCRC. We hypothesized that XPO1 inhibition may improve response to topo-I inhibitor in mCRC by impairing the DDR response. Methods: The synergism of XPO1 inhibition (selinexor or eltanexor) with SN38 was assessed in CRC cell lines by CellTiter-Glo 2.0 and quantitated using Bliss Additivity Model using Synergy Finder 2.0. Each condition was plated either concurrent (both drugs for 72 hours) or sequential (SN38 for 24 hours then removed, followed by XPO1 inhibitor for 48 hours). Nude mice were implanted with one microsatellite stable CRC PDX model in the flanks followed by treatment with vehicle, XPO1 inhibitor, irinotecan (15 mg/kg intraperitoneal weekly), or combination. Tumor growth was measured by specific growth rate (SGR), within an IACUC-approved protocol. Tumor tissue was collected at day 7 for molecular analysis. Tissues were lysed and protein was run on 4%-12% SDS-PAGE Gel. p21 Waf/Cip and P-Histone H2A.X were used to determine DNA damage by western blot. P-Histone H2AX (pH2AX) was evaluated by immunofluorescence using primary conjugated P-Histone H2A.X antibody and DAPI for nuclear counter stain. Slides were imaged using Olympus iX83 microscope and analysis was performed using Olympus CellSens software. Results: In CRC cell lines, concurrent combination eltanexor and SN38 did not synergistically decrease viability but increased pH2AX compared to SN38 alone (~30-fold). However, sequential SN38 followed by eltanexor synergistically decreased viability at multiple dose levels; evaluation of pH2AX is ongoing. In PDX model CRC254, eltanexor (10 mg/kg and reduced to 7.5 mg/kg on day 8 to improve tolerability, orally 5 days per week) with irinotecan decreased SGR compared to irinotecan alone (SGR=0.007 vs 0.026, respectively, p=0.0003) and compared to eltanexor alone (SGR=0.029, p <0.0001). Combination treatment increased pH2AX and increased p21 expression compared to either single-agent treatment. Similar SGR results were observed with selinexor (5 mg/kg orally twice weekly). Conclusions: XPO1 inhibition and topo-I inhibition decreased CRC tumor growth in PDX models, presumably by increasing DNA double strand breaks. Cell line data suggests sequential dosing of topo-I inhibition followed by XPO1 inhibition may be effective. This work supports continued development of this combination in mCRC. Citation Format: Robert W Lentz, Adrian Dominguez, Stacey Bagby, Cameron Binns, Alexis Leal, Sunnie Kim, Sarah Lindsey Davis, Christopher H Lieu, Wells A Messersmith, Todd M Pitts. Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B147.

Abstract A097: HBI-2438, HUYABIO selective KRASG12C inhibitor with BBB penetration, inhibited tumor growth in a metastatic brain model as single agent and also displayed synergy in combination with HBI-2376 (HUYABIO SHP2 inhibitor) in a CRC PDX model

Abstract Recent advances in targeting KRASG12C mutation and approval of sotorasib and adagrasib provided new treatment avenues for cancer patients. Additionally, about 10% of NSCLC (non-small cell lung cancer) patients carrying KRASG12C mutation are diagnosed with brain metastasis. Nearly 50% of NSCLC patients with recurrent disease also have metastatic lesions in the brain. This further signifies the therapeutic value of KRASG12C inhibitors with BBB (blood brain barrier) penetration properties. Early studies showed HBI-2438, HUYABIO selective and potent KRASG12C inhibitor, was detectable and quantifiable in CSF when administered orally. Here we tested efficacy of HBI-2438 in an intra-carotid brain metastatic model and compared its efficacy vs. AMG510 and MRTX849 in similar dose and intervals. Bioluminescent imaging and quantification showed major tumor regression in tumors treated with HBI-2438 and MRTX849 and to a lesser extent in AMG510 treated tumors, all at 30 mg/kg. Consistent with preclinical data, HBI-2438 monotherapy showed early clinical efficacy signal in Cohort 1 (150 mg) and Cohort 2 (300 mg). HBI-2438 was well tolerated and did not show any SAEs or DLTs in these early cohorts. SHP2 signaling is one of the main components of the RAS/MAPK pathway and is acting upstream of KRAS. Therefore, and has been shown recently, targeting both KRAS and SHP2 in combination therapy may enhance the response to either agent as monotherapy. Here we tested combination of HBI-2438 and HBI-2376 (HUYABIO selective SHP2 inhibitor) in a KRASG12C CRC (colorectal cancer) PDX (patient derived xenograft) model that has demonstrated partial resistance to treatment with KRASG12C inhibitors. As expected, tumor bearing mice treated with HBI-2438 or MRTX849 monotherapy showed partial tumor growth inhibition in this model. However, combination treatment, HBI-2438 (10 or 30 mpk) plus HBI-2376 (5 mpk), showed significant inhibition in tumor growth. Additionally, the combination therapy at a higher dose (HBI-2438 at 100 mpk plus HBI-2376 at 5 mpk) achieved complete tumor regression vs. MRTX849+TNO155 demonstrating synergy between the two compounds in this model. These data further demonstrated greater inhibition of KRAS/MAPK pathway leading to a better tumor growth inhibition and justifies testing combination of HUYABIO KRAS G12Ci plus SHP2i in the clinic. Therefore, we believe HBI-2438 is a well differentiated KRAS G12C inhibitor vs. the competitors due to i) BBB penetration ii) superiority in combination therapy with SHP2 inhibitor and iii) efficacy signal in early cohorts in the clinic. Citation Format: Farbod Shojaei, Jill M Ricono, Che Fang, John Ning, Gloria Lee, Mireille Gillings. HBI-2438, HUYABIO selective KRASG12C inhibitor with BBB penetration, inhibited tumor growth in a metastatic brain model as single agent and also displayed synergy in combination with HBI-2376 (HUYABIO SHP2 inhibitor) in a CRC PDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A097.

Fisetin induces the upregulation of AKAP12 mRNA and anti-angiogenesis in a patient-derived organoid xenograft model

Colorectal cancer (CRC) is associated with high incidence and mortality rates. Targeted therapies for CRC cause various adverse effects, necessitating the development of novel approaches to control CRC progression. In this milieu, we investigated the anti-CRC effects of fisetin, a natural plant flavonoid. Cytotoxicity was performed in CRC patient-derived organoids (30 T and 33 T). Fisetin-induced tumor growth was evaluated in a CRC patient-derived organoid xenograft (PDOX) model. RNA sequencing, immunohistochemistry, and western blotting were performed subsequently. Fisetin significantly decreased organoid viability in a dose-dependent manner. In the PDOX model, fisetin significantly delayed tumor growth, showing a decrease in Ki-67 expression and the induction of apoptosis. In tumor tissues, four genes were identified as differentially expressed between the control and fisetin-treated groups. Among these, A-kinase anchoring protein 12 (AKAP12) level was significantly increased by fisetin treatment (fold change > 2, p < 0.05). Notably, fisetin significantly inhibited vascular endothelial growth factor (VEGF) and epithelial cell adhesion molecule (EpCAM) via upregulation of AKAP12. Our results demonstrate the upregulation of AKAP12 mRNA and inhibition of angiogenesis by fisetin as a therapeutic strategy against CRC.

Lasting response by vertical inhibition with cetuximab andtrametinib in KRAS-mutated colorectal cancer patient-derived xenografts.

Although approximately half of all metastatic colorectal cancers (mCRCs) harbour mutations in KRAS or NRAS, hardly any progress has been made regarding targeted treatment for this group over the last few years. Here, we investigated the efficacy of vertical inhibition of the RAS-pathway by targeting epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase kinase (MEK) in patient-derived xenograft (PDX) tumours with primary KRAS mutation. In total, 19 different PDX models comprising 127 tumours were tested. Responses were evaluated according to baseline tumour volume changes and graded as partial response (PR; ≤ - 30%), stable disease (SD; between -30% and +20%) or progressive disease (PD; ≥ + 20%). Vertical inhibition with trametinib and cetuximab induced SD or PR in 74% of analysed models, compared to 24% by monotherapy with trametinib. In cases of PR by vertical inhibition (47%), responses were lasting (as long as day 137), with a low incidence of secondary resistance (SR). Molecular analyses revealed that primary and SR was driven by transcriptional reprogramming activating the RAS pathway in a substantial fraction of tumours. Together, these preclinical data strongly support the translation of this combination therapy into clinical trials for CRC patients.

Chromatin accessibility dynamics in colorectal cancer liver metastasis: Uncovering the liver tropism at single cell resolution

Tumor metastasis causes over 90% of cancer related death and no currently available therapies target it. However, there is limited understanding regarding the epigenetic regulation of genes during this complex process. Here by integrating single-cell ATAC-seq (scATAC-seq), single-cell RNA-seq (scRNA-seq), microarray, bulk RNA-seq, immunohistochemistry (IHC) staining, as well as proteomics datasets from paired primary and liver metastatic colorectal cancer (CRC) patient-derived xenograft (PDX) model and patients, we discovered that liver metastatic CRC cells lose their colon-specific chromatin accessible sites yet gain liver-specific ones. Importantly, we observed elevated accessibility of HNF4A, a liver-specific transcription factor, in liver metastatic CRC cells. Subsequently, we performed clustering analysis of liver metastatic CRC cells together with cells involved in liver development, revealing significant heterogeneity among the liver metastatic CRC cells. Over 50% of the liver metastatic CRC cells exhibited characteristics similar to those of erythroid progenitors and hepatocytes, showing increased expression of genes involved in oxidative phosphorylation and glycolysis. Moreover, our discovery further revealed that the MHC and IFN response genes in these cells exhibit moderate epigenetic activity, which is significantly associated with the low objective response rates in checkpoint blockade immunotherapy. Our findings uncovered the critical roles of HNF4A and the cell populations within liver metastatic CRC cells might serve as crucial therapeutic targets for addressing liver metastasis and improving the immunotherapy response in patients with CRC.

An amino acid transporter subunit as an antibody–drug conjugate target in colorectal cancer

BackgroundAdvanced colorectal cancer (CRC) is difficult to treat. For that reason, the development of novel therapeutics is necessary. Here we describe a potentially actionable plasma membrane target, the amino acid transporter protein subunit CD98hc.MethodsWestern blot and immunohistochemical analyses of CD98hc protein expression were carried out on paired normal and tumoral tissues from patients with CRC. Immunofluorescence and western studies were used to characterize the action of a DM1-based CD98hc-directed antibody–drug conjugate (ADC). MTT and Annexin V studies were performed to evaluate the effect of the anti-CD98hc-ADC on cell proliferation and apoptosis. CRISPR/Cas9 and shRNA were used to explore the specificity of the ADC. In vitro analyses of the antitumoral activity of the anti-CD98hc-ADC on 3D patient-derived normal as well as tumoral organoids were also carried out. Xenografted CRC cells and a PDX were used to analyze the antitumoral properties of the anti-CD98hc-ADC.ResultsGenomic as well proteomic analyses of paired normal and tumoral samples showed that CD98hc expression was significantly higher in tumoral tissues as compared to levels of CD98hc present in the normal colonic tissue. In human CRC cell lines, an ADC that recognized the CD98hc ectodomain, reached the lysosomes and exerted potent antitumoral activity. The specificity of the CD98hc-directed ADC was demonstrated using CRC cells in which CD98hc was decreased by shRNA or deleted using CRISPR/Cas9. Studies in patient-derived organoids verified the antitumoral action of the anti-CD98hc-ADC, which largely spared normal tissue-derived colon organoids. In vivo studies using xenografted CRC cells or patient-derived xenografts confirmed the antitumoral activity of the anti-CD98hc-ADC.ConclusionsThe studies herewith reported indicate that CD98hc may represent a novel ADC target that, upon well-designed clinical trials, could be used to increase the therapeutic armamentarium against CRC.

Multi-label transcriptional classification of colorectal cancer reflects tumor cell population heterogeneity

BackgroundTranscriptional classification has been used to stratify colorectal cancer (CRC) into molecular subtypes with distinct biological and clinical features. However, it is not clear whether such subtypes represent discrete, mutually exclusive entities or molecular/phenotypic states with potential overlap. Therefore, we focused on the CRC Intrinsic Subtype (CRIS) classifier and evaluated whether assigning multiple CRIS subtypes to the same sample provides additional clinically and biologically relevant information.MethodsA multi-label version of the CRIS classifier (multiCRIS) was applied to newly generated RNA-seq profiles from 606 CRC patient-derived xenografts (PDXs), together with human CRC bulk and single-cell RNA-seq datasets. Biological and clinical associations of single- and multi-label CRIS were compared. Finally, a machine learning-based multi-label CRIS predictor (ML2CRIS) was developed for single-sample classification.ResultsSurprisingly, about half of the CRC cases could be significantly assigned to more than one CRIS subtype. Single-cell RNA-seq analysis revealed that multiple CRIS membership can be a consequence of the concomitant presence of cells of different CRIS class or, less frequently, of cells with hybrid phenotype. Multi-label assignments were found to improve prediction of CRC prognosis and response to treatment. Finally, the ML2CRIS classifier was validated for retaining the same biological and clinical associations also in the context of single-sample classification.ConclusionsThese results show that CRIS subtypes retain their biological and clinical features even when concomitantly assigned to the same CRC sample. This approach could be potentially extended to other cancer types and classification systems.

Delivery of Engineered Primary Tumor-Derived Exosomes Effectively Suppressed the Colorectal Cancer Chemoresistance and Liver Metastasis.

Liver metastasis is one of the major causes of colorectal cancer (CRC)-related morbidity and mortality. Delivering small interfering RNAs (siRNAs) or noncoding RNAs has been reported as a promising method to target liver metastasis and chemoresistance in CRC. Here, we report a noncoding RNA delivery system using exosomes derived from primary patient cells. Coiled-coil domain-containing protein 80 (CCDC80) was strongly associated with CRC liver metastasis and chemoresistance, a finding validated by bioinformatic analysis and clinical specimens. Silencing CCDC80 significantly increased sensitivity to chemotherapy agents in OXA-resistant cell lines and a mouse model. The primary cell-derived exosome delivery system was designed to simultaneously deliver siRNAs targeting CCDC80 and increase chemotherapy sensitivity in the distant CRC liver metastasis mouse models and patient-derived xenograft mouse models. We further validated the antitumor effect in an ex vivo model of chemoresistant CRC organoids and a patient-derived organoid xenograft model. Tumor-bearing mice treated with the siRNA-delivering exosomes and hepatectomy showed ideal overall survival. Our results provide a therapeutic target and represent a possible therapeutic alternative for patients with CRC and distant metastasis and in cases of chemoresistance.