Patient-derived Cancer Organoids Research Articles

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo.

Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.

Establishment of patient-derived organoids for guiding personalized therapies in breast cancer patients.

Breast cancer has become the most commonly diagnosed cancer. The intra- and interpatient heterogeneity induced a considerable variation in treatment efficacy. There is an urgent requirement for preclinical models to anticipate the effectiveness of individualized drug responses. Patient-derived organoids (PDOs) can accurately recapitulate the architecture and biological characteristics of the origin tumor, making them a promising model that can overtake many limitations of cell lines and PDXs. However, it is still unclear whether PDOs-based drug testing can benefit breast cancer patients, particularly those with tumor recurrence or treatment resistance. Fresh tumor samples were surgically resected for organoid culture. Primary tumor samples and PDOs were subsequently subjected to H&E staining, immunohistochemical (IHC) analysis, and whole-exome sequencing (WES) to make comparisons. Drug sensitivity tests were performed to evaluate the feasibility of this model for predicting patient drug response in clinical practice. We established 75 patient-derived breast cancer organoid models. The results of H&E staining, IHC, and WES revealed that PDOs inherited the histologic and genetic characteristics of their parental tumor tissues. The PDOs successfully predicted the patient's drug response, and most cases exhibited consistency between PDOs' drug susceptibility test results and the clinical response of the matched patient. We conclude that the breast cancer organoids platform can be a potential preclinical tool used for the selection of effective drugs and guided personalized therapies for patients with advanced breast cancer.

Abstract 2965: GADD45B as a mechanism for enhanced treatment response with the combination of copanlisib and romidepsin in colorectal cancer

Abstract Background: Treatment options for molecular subtypes of colorectal cancer (CRC) are urgently needed, including for PIK3CA mutant CRC. Previous work in our lab demonstrated enhanced activity of copanlisib in combination with romidepsin in PIK3CA mutant CRC models. Here we investigate the induction of GADD45B as a mechanism of the enhanced induction of apoptosis seen with this treatment strategy. Methods: RNA sequencing was done on the CRC 2D cell line, SW48PK (Horizon Discovery, SW48 PIK3CAH1047R/+ mutation) as well as PIK3CA mutant and wild-type patient derived cancer organoids (PDCOs) through the UW Biotechnology Center. All lines were treated with copanlisib, romidepsin, or combination and were compared with untreated controls. GADD45B gene expression was also confirmed on SW48PK, PIK3CA mutant, wild type, and PIK3CA and KRAS mutant PDCOs using RT-qPCR. Results: RNA sequencing determined that there was an increase of GADD45B expression in the SW48PK 2D cell line as well as in PIK3CA mutant and wild-type PDCOs treated with the combination compared to untreated controls (average log2 fold change= 1.83). A significant increase in GADD45B expression was seen in the SW48PK combination treatment group compared to the control group at 6 hours (combination median fold change= 2.7, p= 0.045) and 24 hours (4.1, p< 0.001). For PIK3CA mutant PDCOs treated with the combination, there was significant increase in GADD45B expression at 6 hours (9.45, p= 0.005) and 24 hours (14.7, p= 0.002) compared to untreated control. In wild type PDCOs, there was a significant increase in GADD45B expression in the combination compared to untreated control at 6 hours (32.95, p=0.004) but not after 24 hours (2.6, p= 0.42). For KRAS mutant PDCOs, there was a significant increase in GADD45B expression in combination treated cells at 6 (9.9, p= 0.001) and 24 hours (3.3, p= 0.047), however, this increase was less than PIK3CA mutant PDCOs at 24 hours (3.3 vs 14.7). Conclusions: Copanlisib in combination with romidepsin increases GADD45B expression in PDCOs and SW48PK. Further studies will determine if GADD45B is required for this treatment response. Citation Format: Kennedy Maduscha, Alexa Schmitz, Rebecca DeStefanis, Cheri Pasch, Dustin Deming. GADD45B as a mechanism for enhanced treatment response with the combination of copanlisib and romidepsin in 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 2965.

Abstract 1944: Using patient-derived cancer organoids to determine the effects of the anti-EGFR therapy panitumumab in the presence or absence of KRAS mutation

Abstract Introduction: Tumor heterogeneity is a major cause of resistance to therapies in colorectal cancer (CRC). The anti-EGFR (epidermal growth factor receptor) therapies panitumumab (pani) and cetuximab, are now being investigated for the retreatment of patients when resistant subclones could be more abundant in a previously wild-type cancer. Here we utilize patient-derived cancer organoids (PDCOs) to investigate the potential implications of the abundance of resistant subclones on treatment and duration of response. Methods: Two CRC PDCO lines, one pani sensitive (PS) and one pani resistant (PR), were cultured separately and then mixed to determine how different combinations of mutations altered the time for the overall culture to be resistant to pani. NGS was performed on these cells to determine the mutation profile. The two PDCO lines were cultured together with the following concentrations (v/v) of 100% PS; 20% PR+80% PS; 10% PR+90% PS, 5% PR+95% PS, 1% PR+99% PS, and 100% PR using Matrigel and fed with previously published CRC organoid media. Widefield imaging was performed for diameter change analysis at day 0 and day 4 using ImageJ. Upon a median growth of >20% at baseline/untreated, all mixed populations were treated at increments of 20% physiologic Cmax of pani each week. Once select mixed populations reached 100% Cmax and maintained a median growth of >20% for two consecutive weeks, they were determined to have reached complete resistance to the treatment. Results: DNA sequencing of PDCOs showed PR has mutations associated with pani resistance (KRASG12V, PIK3CAH1047R) and the PS line did not. All the conditions except for 100% PS escalated to ≥40% Cmax within 7 weeks and reached complete resistance to pani by week 22. 100% PS remained at 20% Cmax through week 22. The 100% PR population reached complete resistance to pani the quickest at 100% Cmax at week 8. The conditions with the lowest percentages of PR (5% PR+95% PS and 1% PR+99% PS already started to show resistance above the PS line by 8 weeks and reached complete resistance to pani at week 12. Conclusion: PDCO lines containing low concentrations of resistant clones can quickly lead to resistance to pani treatment, even below the typical level for clinical NGS detection. All KRAS mutant conditions (PR) reached resistance quicker than 100% PS condition. Further studies will evaluate even low concentrations of resistance clones across further CRC PDCO models and will evaluate the mechanism by which these clones are leading to resistance. Citation Format: Jordan N. Stoecker, Shirsa Udgata, Amani A. Gillette, Cheri A. Pasch, Melissa C. Skala, Dustin A. Deming. Using patient-derived cancer organoids to determine the effects of the anti-EGFR therapy panitumumab in the presence or absence of KRAS mutation [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 1944.

Abstract 7600: Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer related mortality in the United States, largely due to ineffective systemic therapy. While novel KRAS target strategies are in development, few agents have been investigated to target the transcriptional machinery for global inhibition of expression. Recent small molecule inhibitors targeting transcription (i.e., CDK7) have shown preliminary evidence of disease control; however, combination therapies have lagged, likely due to uncertainty in clinical toxicity. Text mining analysis suggests combination BET and CDK9 inhibition may be promising against PDAC. Here, we present preclinical evidence of synergistic activity for the combination of bromodomain and extraterminal (BET) inhibitor (ZEN3694) and CDK9 inhibitor (VIP152) using patient derived cancer organoids (PCOs). Methods: Dose response curves were generated for ZEN3694 and VIP152 to assess their single agent activity against PCOs using a low volume format. ZEN3694 was treated continuously while VIP152 was removed after 24h to mimic pharmacokinetics with assay endpoint at 144h. Synergy assays were designed using a combination dose titration grid. Viability was determined in all assays using 3D CellTiter Glo (CTG, 50% v/v). Synergy scores were produced using SynergyFinder 3.0 including Zero Interaction Potency (ZIP), Bliss Independence (Bliss), Highest Single Agent (HSA), and Loewe Additivity (Loewe). Western blot analysis was performed against global markers of active RNA polymerase II (phosphorylation at serine 2, 5, 7) and antiapoptotic protein, BCL xL, and MCL 1. Results: ZEN3694 was observed to have a single agent IC50 in the single uM range across 3 independent PDAC PCO lines (PDAC1 3.7uM, PDAC2 1.1uM, PDAC3 2.9uM). VIP152 showed significant potency in the same 3 PDAC PCO lines (PDAC1 140.2nM, PDAC2 30.5nM, PDAC3 43.4nM). The combination of ZEN3694 and VIP152 was found to have increased therapeutic activity with double digit synergy scores across all reference models: PDAC1 (Loewe 17.3, HSA 19.8, ZIP 20.2, and Bliss 17.2) and PDAC2 (Loewe 34.7, HSA 28.3, ZIP 19.7, and Bliss 19.6). Western blots of protein collected from organoids treated for 48h showed on target activity, including decrease in the phosphorylation of Rbp1 (Ser 2, Ser 5, and Ser 7) compared to both control and single agents and decreased expression in antiapoptotic proteins (MCL 1 and BCL xL). Conclusion: Here, we show the activity of dual transcription targeting with BET and CDK9 inhibition in patient derived pancreatic cancer organoid models. The combination of ZEN3694 and VIP152 has in vitro synergy in PDAC PCO models with on target activity. Ongoing work is validating this combination in animal models to assess toxicity, in vivo activity, and RNA expression profiling to understand transcriptional pathway dependency and therapeutic resistance. Citation Format: Lucas J. Koeppel, Austin Stram, Robert J. Millikin, Ellie Riedl, Md Shahadat Hossan, Ethan Samuel Lin, Ron Stewart, Jeremy D. Kratz. Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of 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 7600.

Abstract 4242: Preserving lung cancer patient mutations in 3D organoid models: Advancing cancer research through innovative culture systems

Abstract Lung cancer is one of the most prevalent cancers worldwide. There are currently many anticancer drugs available, but they have numerous issues in terms of treatment, such as drug resistance and recurrence. Culturing systems that can replace a patient's cancer tissue are crucial, and the existing 2D culturing systems have many limitations. Patient-derived cancer organoid culture is the most advanced culture system for understanding mechanism of cancers. Using a 3D organoid culture system, we have successfully established a culture system for lung cancer using biopsy/surgical specimens and malignant effusions, including a long-term culture system. There are two critical indicators to determine the success of 3D organoid culture. One is whether the organoids retain their cancer-specific mutations from patient’s lung cancer tissue, and the second is whether the organoids grow well and can be maintained in long-term culture for more than 20-30 passages. We typically assess gene mutations in EGFR, ALK, ROS, and P53 when evaluating lung cancer organoids. We confirm these mutations when we establish organoids. and currently we have established a total of 127 patient-derived lung cancer organoids. Among the 127 organoids, we completed NGS analysis on 108 of them and validated that 86.5% exhibit similarity when compared to lung cancer tissues. Additionally, we achieved a 100% retention of mutations in the long-term culturing of these organoids. Our studies indicate that patient-derived lung cancer organoids represent a meaningful organoid system for cancer tissue replacement and treatment response in the context of precision cancer medicine. Citation Format: Ji-yeon Park, Seo Rin Gu, Jiyeon Lee, Na-Gyeong Lee, Dojin Choi, Sang Eun Park, Ji Min Kim, Jinguen Rheey. Preserving lung cancer patient mutations in 3D organoid models: Advancing cancer research through innovative culture systems [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 4242.

Abstract 220: Thresholding response assessment in biliary tract cancer organoids to inform sensitivity to chemotherapy across molecular subtypes

Abstract Background: Cholangiocarcinoma (CCA) remains a highly morbid cancer for which accurate models. Patient-derived cancer organoid (PCOs) have been generated in CCA, however their utility in clinical prediction has not been validated. Z’ factoring often fails in assessing CCA organoid response. Prior analysis for organoid response has shown an effect size by Glass’s Delta (GΔ) of ≥1.25 in growth inhibition is predictive of clinical response. Here, we present subclonal organoid response across a diverse set of CCA models in response to therapy by growth and organoid level viability response. Methods: PCOs were expanded from the NCI’s Patient-Derived Model Repository (PDMR), tissue biopsy, and rapid autopsy. Organoid response was tracked from growth using Z-stacked high content imaging (Cytation5) with endpoint at 144h and stained using established viability markers, Caspase-3/7 (C3/7) and ToPro3. Treatment groups included media control, positive control of cycloheximide 200uM continuous, gemcitabine (gem) 10uM 24h, cisplatin (cis) 5uM 48h, combination gem+cis, and FGFR2 inhibitor futibatinib (futi) 300nM continuous. Results: Across models, PCO growth directly correlated with Z’ factor thresholding (R= 0.868). NCI PDMR organoids, CK11523 and CK10519 are IDH1mutant (mt), and both showed superior response to gem when compared to cis by growth (gem GΔ 1.18 and 0.44 v. cis GΔ 0.11 and 0.23) and C3/7 (gem GΔ 3.25 and 2.34 v. cis GΔ 1.46 and 0.33). BRCA1mt CCA was found to achieve modest growth inhibition with gem and gem+cis (GΔ 1.09 and 1.17) and yet significant increase in C3/7 (gem GΔ 1.68 and gem+cis GΔ 1.36) and ToPro3 (gem GΔ 1.65 and gem+cis GΔ 1.82). Despite this result, the patient developed early clinical recurrence after neoadjuvant gem+cis chemotherapy (<3 months). FGFR2-HPGDS fusion CCA achieved no meaningful difference in growth response with gem+cis (GΔ 0.94) or futi (GΔ 0.36). These findings were consistent with the clinical outcome of primary chemotherapy resistance and progression on futi. However, we observed increase in C3/7 positivity with both gem+cis (GΔ 2.11) and futi (GΔ 1.37). Conclusions: Primary resistance to both targeted and chemotherapy remains a formidable barrier to therapeutic development in CCA. While growth inhibition remains a validated marker of clinical response, further work is needed to understand thresholds for C3/7 and ToPro3 across physiologic drug dosing. This is the first report of modeling the novel FGFR2-HPGDS fusion CCA in organoids. Ongoing work is evaluating therapeutic sensitivity across prospective specimens of CCA in support of establishing thresholding for response to apoptosis (C3/7) and necrosis (ToPro3). Citation Format: Eleanor Riedl, Austin Stram, Md Shahadat Hossan, Ethan Samuel Lin, Jamie Warner, Luke Koeppel, Jeremy D. Kratz. Thresholding response assessment in biliary tract cancer organoids to inform sensitivity to chemotherapy across molecular subtypes [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 220.

Abstract 4249: Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing

Abstract Purpose: Ovarian cancer is the most lethal gynecologic cancer. Majority of ovarian cancers belong to the subgroup of high-grade serous ovarian cancer (HGSC). Its standard treatment is surgery and chemotherapy with platinum and taxane-based compounds, for which almost all patients become resistant. We have established a panel of patient derived 3-dimensional (3D) HGSC long-term tumor organoid cultures for personalized disease modeling, drug screening and evaluation of the efficiency of potential new treatments to be used in preclinical research. Methods: Tumor tissues or ascites from > 30 HGSC tumors were set up as long-term 3D organoid cultures (PDOs). RNA and whole genome sequencing was performed from the original tumors and from PDOs. Pluripotency and proliferation were assessed via high-throughput immunofluorescent detection of PAX8 and Ki67. Resistance towards standard treatment and response to additional selected drugs and drug combinations were evaluated in cell death and proliferation assays. Imaging and image-based quantifications were performed under 3D setup. PDOs were set up for in vivo drug testing in immunodeficient NOD/Shi-scid/IL-2Rgnull (NOG) mice. Summary: A set of HGSC PDOs of different longitudinal, prospective, and multiregional samples has been established representing the three evolutionary states of HGSC: evolving, maintaining and adaptive state. They are ready to be used as rational and effective models for studying drug interventions. Supportively, results of preclinical studies with PI3K inhibitor Alpelisib matched the published patient responses. Tumor evolutionary analysis based on data of longitudinal, prospective, and multiregional collected samples suggest further targetable pathways that can mediate chemotherapy resistance and pinpoint potential targets for drug intervention. Conclusion: Inhibition of these pathways with simultaneous targeting of other cellular functions, such as lysosomal-mediated cell death, may be highly effective alternative treatment option. 3D imaging and efficient image analysis are necessary tools to understand tumor organoid biology and drug responses to assist development and pre-clinical evaluation of new treatments. This study is a part of the DECIDER project (https://www.deciderproject.eu/, NCT04846933). Citation Format: Tuula Kallunki, Anna R. Lauridsen, Aikaterini Skorda, Alexandra Lahtinen, Marie L. Bay, Benita Rasmussen, Kaisa Huhtinen, Taru Muranen, Jaana Oikkonen, Johanna Hynninen, Sampsa Hautaniemi. Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing [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 4249.

Abstract 935: Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents

Abstract Background: EC is the most commonly diagnosed gynecologic cancer in the US and has both rising incidence and mortality. Uterine serous cancer (USC) and uterine carcinosarcoma (UCS) are high grade ECs characterized by TP53 mutation and molecular alterations suggesting high replication stress (RS) and cell cycle dysregulation. These molecular alterations could engender sensitivity to RS-targeting strategies such as WEE1 inhibition. In a single arm phase 2 of 35 patients, the WEE1 inhibitor adavosertib demonstrated clinical activity in recurrent USC, with an overall response rate of 29.4%. To support the development of predictive biomarkers of sensitivity to RS targeting agents, we report on the establishment of co-clinical EC PDOs from pre- and on-treatment biopsies from patients with USC or UCS enrolled to a translational trial of adavosertib. Methods: Patients with USC or UCS consented for trial participation underwent pre- and on-treatment biopsies while receiving adavosertib monotherapy. Biopsies were dissociated and passaged as 3D organoid cultures in Matrigel in an optimized media. Established PDOs were seeded in 3D microfluidic devices, treated with adavosertib in a dose-dependent manner and assessed for viability after 6 days of treatment by dual labeling live/dead imaging. IC50 values were calculated using log(inhibitor) vs response - variable slope (four parameters) model in GraphPad Prism. To assess the effect of adavosertib on replication fork speed, DNA fiber assays were conducted with PDOs treated with adavosertib for 72 hours. Finally, FFPE blocks were created from PDOs for assessment of DNA damage repair biomarkers by IHC. Results: 19 biopsies were obtained from 11 participating patients. 6 biopsies did not yield sufficient tumor cells for PDO generation. From the remaining 13 samples, 5 PDOs were successfully established (3 USC and 2 UCS), with a higher success rate when 3 biopsy cores were received (3 of 4; 75%) compared to when 2 biopsy cores were received (2 of 9; 22 %). Dual labeling live/dead imaging of PDOs treated with adavosertib resulted in IC50 values ranging from 113nM to 856nM. DNA fiber assays revealed consistent decrease in fork speed with adavosertib exposure, consistent with the hypothesized effect of adavosertib in increasing RS in these cells. A dose dependent effect on replication fork speed was observed, with greater decreases in fork speed with increasing concentrations of adavosertib. Due to the limited number of samples and the number of PDOs generated from on-treatment as opposed to pre-treatment biopsies, clinical correlation between PDO adavosertib sensitivity and clinical activity was not formally assessed. IHC analysis of potential DNA damage biomarkers is ongoing. Conclusions: We describe the successful establishment of co-clinical EC PDOs for functional evaluation of sensitivity to RS targeting agents. Citation Format: Elena Ivanova, Aisha L. Saldanha, Shrabasti Roychoudhury, Bose Kochupurakkal, Ari P. Zlota, Clare E. Padrick, Ha V. Vo, Eli K. Greenberg, Hannah Sawyer, Carina Feeney, Courtney H. Qi, Swati Narayan, Jennifer D. Curtis, Nabihah Tayob, Marisa R. Nucci, Panagiotis Konstantinopoulos, Dipanjan Chowdhury, Geoffrey Shapiro, Cloud P. Paweletz, Ursula A. Matulonis, Joyce F. Liu. Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents [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 935.

Abstract 6901: Paired colorectal and pancreatic cancer patient-derived tumor and organoids biobank

Abstract Patient-derived xenograft (PDX) models constitute a crucial tool in precision cancer medicine owing to their high accuracy in predicting the response to therapeutic drugs among patients in clinical settings. Our center has established a biobank that collects more than 80 colorectal cancer and 25 pancreatic cancer PDX tissues, along with essential information about patients’ tumors and PDX models. However, cost-intensive and time-consuming limit the usage of PDX model in high throughput screening during drug development. To overcome this hurdle, we are generating a PDX-match paired organoid (PDXO) biobank by using emerging three-dimensional culture techniques. Before large-scaled expansion, the concordance between PDX tissues and PDXOs of partial specimens was evaluated firstly using short-tandem repeat (STR) analysis, immunohistochemistry (IHC) and H&E staining. Data of STR analysis performed by using AmpFLSTR Identifiler PLUS PCR Amplification Kit demonstrated that PDX tissues and PDXOs have identical DNA fingerprinting. IHC and H&E staining revealed PDX tissues and PDXOs have highly similar organ-specific markers expression (e.g., Cytokeratin 20, Caudal Type Homeobox 2, and Pan Cytokeratin) and histological morphology. Even after passing to at least five generations, passed PDXOs maintained high fidelity to their original state, as confirmed by whole-exome sequencing (WES) and STR analysis across passages. The WES analysis for passage 0 (P0) and passage 5 (P5) of PDXOs from different PDX tissues revealed there is more than 95% intersection between P0 and P5 PDXOs with the same missense mutation gene. The results of STR analysis showed that P0 and P5 PDXOs have identical STR profile. To evaluate the capability of PDXOs in clinical response prediction, several effective and ineffective drugs (e.g., oxapliplatin, 5-FU, gemcitabine) were tested. The results of drug testing indicated that PDXOs can be used to accurately predict the response to therapeutic drugs as PDX model but more efficient than PDX model. Therefore, PDXO biobank can serve as a valuable resource for high throughput in vitro drug screening prior in vivo efficacy confirmation using PDX models. Citation Format: Juiling Wang, Wen-Hui Ma, Yun-Chi Lo, Hsiao-Chun Hsieh, Li-Ju Hsiao, Yu-Pin Bau, Nai-Chieh Yang, Shaw-Jenq Tsai, Yan-Shen Shan, Tzu-Yu Chen, Yu-Ling Chen, Pei-Ju Chiang, Tzu-Ling Chen, Hsiu-Ping Chang, Hsian-Jean Chin. Paired colorectal and pancreatic cancer patient-derived tumor and organoids biobank [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 6901.

Abstract 228: Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients

Abstract Glioblastoma (GBM) is the most common and incurable adult primary brain tumour. Treatment resistance and relapse is the primary cause of death in GBM. Tumour heterogeneity is one of the significant determinants of treatment failure. Patient-derived cancer organoids have been shown to maintain genetic and phenotypic tumour features and recapitulate intratumoral heterogeneity. Here, we report the development and molecular characterisation of patient-derived GBM organoid (PGO) models to study treatment response to existing and new treatment combinations. GBM biopsies were dissociated, suspended in Cultrex, and cultured in a brain stem cell medium. PGOs were characterised by whole-exome (WES) single-cell karyotype (scKaryoSeq) and RNA sequencing. The IC-50 and viability of PGO upon temozolomide (TMZ) and small molecule inhibitors were determined. Gene expression profiling was performed before and after TMZ treatment. Single-cell spatial heterogeneity was determined using Multiplex Immunostaining of PGO and matched patient samples. PGOs were established with a success rate of 80% (n=31). WES analysis between PGOs from the same patient at multiple time points (1-3 months) showed high maintenance of Single Nucleotide Variants (92.3%-97.7%). In PGOs key somatic oncogene variants and copy number variations were retained compared to the primary tumour (58%-90%). scKaryoseq (N=7) of different clinical GBM subtypes showed a high degree of genetic heterogeneity, with some PGO having up to 10 subclones. Single-cell phenotypic landscaping of patient biopsies showed mixed cellular states maintained after long-term culture in the matched PGO. PGOs showed a differential response to TMZ, corresponding with MGMT methylation and patient mutation status. RNAseq of PGO revealed TMZ-responsive genes, including upregulation of the prognostic JUN kinase pathway. Combination treatment of a JUN kinase inhibitor and TMZ showed a synergistic effect. In addition, PGOs were screened for their sensitivity to clinically approved brain-penetrable small molecule inhibitors targeting key GBM driver pathways. Combining small molecular inhibitors of MEK/PI-3/CDK4/EGFR kinase strongly reduced cell viability and activation of cell death pathways. phosphoKinome screens detected compensatory pathway activation upon kinase inhibition (MEK/CDK4 inhibitor) in several PGO. Data will show novel kinase interaction pathways that increase drug sensitivity in PGO. These data show that PGOs can be used as a stable phenotypic and genetic representative of the patient's tumour and have the potential to function as patient avatars for adaptive treatment selection and actionable drug target discovery. Citation Format: Marc A. Vooijs, Linde Hoosemans, Jolanda Piepers, Maikel Verduin, Maxime Vanmechelen, Mike Van Heumen, Frederik De Smet, Ann Hoeben. Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients [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 228.

Abstract 1952: Co-alterations in PIK3CA and ARID1A lead to enhanced sensitivity to PI3K inhibition

Abstract Background: There is increasing interest in developing precision medicine strategies for cancers with PIK3CA mutations. Co-alterations in PIK3CA and ARID1A (AD) occur in ~1% of all cancers. Previously, patients with co-alterations in PIK3CA and AD have shown both an improved objective response rate and improved progression free survival following copanlisib (cop) treatment compared to cancers with PIK3CA mutations and wild-type AD. Here we evaluate potential mechanisms for this enhanced clinical benefit. Methods: AD was knocked out in SW48 and SW48PIK3CA-H1047R (SW48PK) human 2D colorectal cancer cell lines using CRISPR (SW48AD; SW48PKAD). Knockouts were confirmed using PCR genotyping, amplicon sequencing, and western blot (WB). A cell proliferation assay (WST-1) was performed at increasing doses of cop for 48hrs to determine cell viability. WBs were performed at 6hrs and 24hrs after cop treatment to determine levels of phosphorylation of AKT, S6, and 4EBP1 and levels of apoptosis-related proteins (MCL-1, BCL-xL, BCL-2, PARP). Athymic nude mice were flank injected with SW48PK and SW48PKAD and treated with cop (10mg/kg in PEG400/acidified water (0.1N HCl, pH 3.5; 20/80, v/v) or vehicle for 25 days or until moribund. Results: WST-1 assay showed highest sensitivity to cop in SW48PKAD cells compared to SW48, SW48PK and SW48AD. At 10nM (p=0.01), there was a significant difference in cell viability between SW48PK and SW48PKAD. No significant difference was seen in the phosphorylation (p) of AKT, S6, or 4EBP1 between SW48PK and SW48PKAD. Expression of MCL-1 protein decreased in SW48PKAD compared to SW48PK. No difference was seen in levels of BCL-xL and BCL-2. On treatment with cop, there was a greater reduction in pS6 in SW48PKAD at 24 hours compared to SW48PK, but no significant difference in pAKT and p4EBP1. There was a gradual increase in MCL-1, BCL-xL, and cleaved PARP in SW48PKAD overtime following cop treatment compared to SW48PK. In vivo, untreated SW48PKAD tumors grew more quickly than the SW48PK tumors (8-fold increase in tumor size in SW48PKAD compared to 5-fold increase in SW48PK by day 25, p=0.06). SW48PKAD mice had significant tumor reduction (p=0.002) on cop treatment by day 25; however, SW48PK mice did not. Conclusions: ARID1A loss along with PIK3CA mutation leads to greater PI3K inhibition response most likely due to enhanced induction of apoptosis. Further confirmation of this enhanced sensitivity is required with better in vitro models such as patient-derived cancer organoids. Citation Format: Shirsa Udgata, Jordan N. Stoecker, Anna L. Lippert, Cheri A. Pasch, Dustin A. Deming. Co-alterations in PIK3CA and ARID1A lead to enhanced sensitivity to PI3K inhibition [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 1952.

Abstract 4239: Pancreatic cancer organoids from the Human Cancer Models Initiative biobank reflect disease genotypes, capture patient heterogeneity, and are amenable to therapeutic screening

Abstract Pancreatic cancer is the fourth leading cause of cancer-related death in the United States (~3% of all cancer cases) and has one of the lowest 5-year relative survival rates. There is a lack of clinically representative, easily available, validated, in vitro pancreatic cancer models that reflect the genomic and phenotypic diversity of the disease. The Human Cancer Models Initiative (HCMI) is an international collaborative project devoted to the development and distribution of approximately 1,000 novel human primary tissue-derived tumor models supported with clinical and molecular annotation. These models were manufactured, characterized, and validated at a not-for-profit biobank and made available for academic and commercial use in basic research, compound screening, and drug development. Available pancreatic cancer organoids (PCOs) from the HCMI biobank (n=46 unique models, n=41 unique donors) were acquired and subjected to a targeted next-generation sequencing hybridization capture, pan-cancer panel of 663 genes. Data were compared to publicly available clinical and genotyping results from the patient’s tumor (n=42) that the model was derived from as well as cancer datasets such as The Cancer Genome Atlas (TCGA). A subset of models was cultured following standard procedures and tested for response to chemotherapeutics (cisplatin, nab-paclitaxel), KRAS-targeting inhibitors (MRTX1133, KR-pep-2D), or PARP inhibitors (PJ34, olaparib) at three time points. A luminescent ATP viability assay was used to assess response. PCOs from the HCMI were predominantly derived from primary tumors (75%), had an adenocarcinoma ductal histological type, and were from female patients (56%) with an average age of 65 years at diagnosis. Models had mutations in key disease-relevant driver genes including KRAS, TP53, SMAD4, and/or CDKN2A at frequencies similar to the TGCA cohort. Sequencing revealed mutations in genes less commonly associated with pancreatic cancer, including BRCA, APC, BRAF, TGFBR2, and ARID1A, albeit at lower frequencies. Most models had mutations in KRAS including G12D, G12V, G12C, and G12R variants. Significant but not complete concordance was seen in comparison with the mutation status of the patient’s respective tumor, indicating potential genotypic drift had occurred. Response to acute treatment varied by model, genotype, timepoint, and drug. Chemotherapeutics like Cisplatin were broadly cytotoxic while MRTX1133 exhibited greater toxicity in models with KRAS G12D variants as compared with KRAS wild type. Patient-derived cancer organoids from the HCMI recapitulate genotypes seen in patient populations and respond to therapeutic treatment in vitro. Citation Format: Stephen Friend, Rachel Demerath, Matthew Graziano, James Clinton. Pancreatic cancer organoids from the Human Cancer Models Initiative biobank reflect disease genotypes, capture patient heterogeneity, and are amenable to therapeutic screening [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 4239.

Abstract 4243: Revolutionizing cancer research, new drug screening approach with vascularized cancer organoid platform mimicking patient tumor microenvironment

Abstract Patient-derived organoids (PDOs) are improving our understanding of cancer heterogeneity and personalized medicine. In particular, patients' intrinsic and acquired treatment resistance is being evaluated with PDOs, which has become an essential factor in developing new anticancer drugs and designing successful clinical trials. However, there are obstacles to cancer heterogeneity and patient-specific cancer therapy due to lack of understanding of tumoral diversity resulting from the tumor microenvironment (TME). Complex direct and indirect interactions between various types of TME cells and the resulting tumoral diversity are challenges that must be addressed for customized cancer therapy. Standard cancer therapy targets tumor cells without considering possible damage on the tumor microenvironment that could impair therapy response. Therefore, the construction and analysis of a platform for co-culturing cancer organoids with other cell types that make up the TME over a long period of time is necessary. We generated and performed characterization of blood vessel organoid (BVO). The BVO expressed endothelial marker CD31 and pericyte marker PDGFRβ. We attempted to co-culture with patient-derived lung cancer organoid (LCO) on Matrigel by dissociating the constructed BVO, and it was confirmed that BVO and LCO were interconnected after 10 days. This new platform was treated with Gefitinib and Poziotinib, and after 5 days of drug treatment, the IC50 value increased compared to LCO-only culture. Additionally, flow cytometry analysis confirmed that co-culturing with BVO during drug treatment resulted in a lower percentage of dead LCO cells compared to LCO-only culture. Image-based analysis also verified that co-culturing with blood vessels enhances the viability of LCOs when treated with drugs. Taken together, the co-culture of cancer organoids and BVOs present a platform that closely mimics the in vivo TME compared to the existing cancer organoid culture method. This innovative platform demonstrates higher drug resistance than existing approaches, offering a novel method for drug screening. Citation Format: Mingyun Lee, Yeo-Joon Yoon, Yong-Soo Lee, Jinguen Rheey. Revolutionizing cancer research, new drug screening approach with vascularized cancer organoid platform mimicking patient tumor microenvironment [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 4243.

Abstract 3972: Personalized immune-organoid co-culture system for modeling the anti-tumor immune response in breast cancer patients

Abstract Recent studies have reported the effectiveness of immune checkpoint blockade (ICB) therapy in breast cancer patients. However, only a subset of patients benefits from ICB therapy due to various immunosuppressive mechanisms. The development of personalized predictive models for the anti-tumor immune response is essential to broaden the indication of ICB therapy. Here, we suggest a novel model platform using a co-culture system with patient tumor-derived organoids and matched immune cells to predict the immune response of individual patients. We successfully established fifty-nine cases of patient-derived breast cancer organoids with matched peripheral blood mononuclear cells (PBMCs) (subtype: Luminal A, 18.6%; Luminal B, 28.8%; Triple-negative breast cancer, 35.6%; HER2-positive, 17.0%). Three rapidly proliferating organoids (one Luminal B and two TNBC organoids) were selected to develop an immune-organoid co-culture (IOC) system. To mimic the anti-tumor immune response, cytotoxic immune cells were expanded from PBMCs by modified cytokine-induced killer (CIK) cell expansion protocol utilizing IL-2/IL-15. The CIK cells were co-cultured with patient-derived tumor organoids for killing assay to enable the prediction of ICB responsiveness in vitro. The CIK cells were expanded in the IOC system by stimulation of PBMC with IL-2/IL-15 in the presence of matched tumor organoids for 11 to 14 days. The CIK cells exhibited tumor organoid-specific killing (measured by Caspase-3, 15.22% ± 8.58, mean ± SEM) on organoid co-culture and showed induction of CD107a and CD137 expression in CD8+ T cells and NK cells with the PD-1 blocking antibody. The CIK-mediated tumor organoid deaths and immune cell activation were reduced by HLA class I blocking antibody (W6/32) treatment, confirming HLA-TCR mediated immune response and killing of tumor organoids in the IOC system (-14.85% in organoid-specific lysis and -7.46% in CD107a expression). In the live cell imaging of the IOC system, the interaction between T cells and tumor organoid cells and subsequent induction of Caspase 3 was noted. Our IOC system could directly measure the immunogenic tumor organoid killing by immune cells on ICB treatment in individual breast cancer patients. Moreover, the analysis of immune receptor profiles may reveal immune modulating mechanisms in each patient. We expect the IOC system to serve as a useful platform for personalized immunotherapy and exploration of combinatory therapy to enhance the anti-tumor efficacy of ICB in breast cancer patients. Citation Format: Cheol-Hwa Hong, Won-Ji Ryu, Shinyoung Park, Tae Yeong Kim, Yumi Hwang, Hyunju Han, Seongyeon Jo, Taeyong Kweon, Gun Min Kim, Min Hwan Kim, Hyung Seok Park, Joo Hyuk Sohn. Personalized immune-organoid co-culture system for modeling the anti-tumor immune response in breast cancer patients [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 3972.

Abstract 4644: Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or a PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and a USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alexandre Andre B. A. Da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David Martigneti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Golbahar Sadatrezai, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey Shapiro, Kalindi Parmar, Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid 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 4644.

Abstract 6737: Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors

Abstract Introduction: Immunogenic neoantigens derived from somatic tumor mutations are essential to initiate and sustain robust anti-tumor immune responses. Frameshift insertion/deletions (fs-indels) are a key source of immunogenic neoantigens. However, fs-indels often result in the introduction of premature termination codons (PTCs), leading to mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. In human malignancies, NMD activity is increased in response to the heightened burden of somatic fs-indel mutations and effectively prevents the generation of highly immunogenic tumor-specific neoantigens, thereby promoting immune evasion. Approach: Here, we pharmacologically targeted SMG1, a core component of the NMD pathway, for the first time in patient tumor and normal samples as well as in a panel of human cancer cell lines and patient-derived organoids, to prevent the degradation of highly immunogenic frame-shifted transcripts. We analyzed the changes in transcriptome, proteome and immunopeptidome following SMG1 inhibition (SMG1i). We then made use of tumor-T cell co-cultures and Patient-Derived Tumor Fragments (PDTFs) to assess the effects of SMG1i on anti-tumor immunogenicity. Ex vivo immunological responses were measured by high-dimensional flow cytometry, cytometric bead array and single cell RNA and TCR sequencing. Results: Our data demonstrates a significant increase in T cell activation, expansion of tumor-reactive intratumoural CD8+ lymphocytes and cytokine secretion following SMG1 inhibition in ex vivo PTDFs (n=15) but not in matched normal-adjacent tissue (n=10). Mechanistically, SMG1 inhibition increased the abundance of frame-shifted mRNA transcripts, and aberrant HLA-presented peptides. Co-culture of tumor cells and patient-derived organoids with CD8+ T cells upon SMG1 inhibition induced potent MHC class I antigen-dependent T cell activation and tumor cell killing. Conclusion: Our findings, in a clinically relevant platform, highlight SMG1 inhibition as a novel cancer immunotherapy approach to promote neoantigen production and to induce anti-tumor immunogenicity irrespective of the tumor type or TMB status. Citation Format: Roberto Vendramin, Hongchang Fu, Yue Zhao, Shanila Fernandez-Patel, Danwen Qian, Lorena Ligammari, Gordon Beattie, Ronen Levy, Polina Greenberg, Osnat Bartok, Krupa Thakkar, Jun Murai, Despoina Karagianni, Chris Sng, Mansi Shah, Felipe Galvez-Cancino, Krijn Djikstra, Sergio Quezada, Yardena Samuels, TRACERx consortium, James Reading, Charles Swanton, Kevin Litchfield. Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient 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 6737.

Abstract 1750: Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration

Abstract Breast cancer's complex transcriptional landscape requires a deep understanding of sample and cell diversity to identify effective treatments. In this study, we amalgamate single-cell RNA sequencing data from breast cancer patient-derived xenografts (PDX), organoids, cell lines, patient tumors and reduction mammoplasties resulting in a comprehensive dataset of 117 samples with 506,719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 enriched (HER2E), and triple-negative breast cancer (TNBC) subtypes. We aimed to delineate similarities and distinctions across model systems and patient samples while also exploring stratification of therapeutic drug efficacy based on subtype proportions within tumors. Mammary tumor PDXs, organoids, and established cell lines exhibited higher proliferation and lower heterogeneity observed via UMAP dimensionality reduction compared to most patient tumors or normal breast epithelium. TNBCs had elevated proliferative and pro-metastatic signatures compared to HR+ and HER2E samples. Interestingly, compared to matched PDX tumors, organoids from these same models were found to exhibit stark differences in gene expression, including upregulation of metabolically active aldo-keto reductase family genes, highlighting differences in the model systems with implications for pre-clinical drug testing. Single-cell tumor subtyping analyses with scSubtype and TNBCtype methods found that therapeutically treated samples had shifts in the proportions of cell-wise subtype annotations when compared to matched untreated samples. Similarly, patient lymph node metastasis when compared with matched primary tumors were significantly linked to decreases in Basal-like and HER2-enriched cell-wise annotations in untreated ER+ samples. In vitro assessment of anti-cancer compounds on PDX cells showed significant correlation of subtype proportion with cell viability following treatment with targeted therapeutic agents. This subtyping methodology offers a powerful tool to monitor the evolving gene expression landscape within samples and predict responses to therapeutic agents. We present here a dynamic approach to cell-wise sample annotation and a substantial multi-model dataset for use facilitating informed decision-making in preclinical research and therapeutic development. Citation Format: Julia E. Altman, Carson J. Walker, Emily K. Zboril, Nicole S. Hairr, Rachel K. Myrick, David C. Boyd, Jennifer E. Koblenski, Madhavi Puchalapalli, Bin Hu, Mikhail G. Dozmorov, Xi Chen, Yunshun Chen, Charles M. Perou, Brian D. Lehmann, Jane E. Visvader, Amy L. Olex, J. Chuck Harrell. Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration [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 1750.

Abstract 6590: Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells

Abstract Microsatellite-unstable (MSI) cancers depend on the WRN helicase to resolve replication stress from expanded (TA)-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours and WRN inhibitors are being developed. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI synthetic lethality, validating the helicase domain as the primary target and guiding inhibitor discovery. Fragment-based screening led to the discovery of potent and highly selective WRN helicase covalent inhibitors from the same chemical series. These compounds strikingly and selectively suppressed MSI model growth in vitro and in vivo by mimicking genetic WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage response. We characterized WRN inhibition across cancer cell lines and patient-derived tumour organoids, enabling biomarker assessment of TA-repeat expansions and MMR status to refine patient selection, with compound efficacy correlating with TA-repeat expansions in MSI cells. Efficacy was also confirmed in patient-derived organoids and PDX models refractory to immunotherapy. The discovery of potent and selective covalent WRN inhibitors provides proof-of-concept for targeting MSI cancers and provides tools to unlock the molecular dissection of the WRN biology and MSI synthetic lethality. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Sara F. Vieira, Yang Lee, Ray Shenje, Shriram Bhosle, Samantha Walker, Kieron May, Carmen Herranz-Ors, Cansu Dicer, Freddy Gibson, Ruby Banerjee, Thilo Werner, Josh Cottom, Yang Peng, Nanhua Deng, Phil Landis, Thomas Angel, Jacob Bush, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Howard Lightfoot, Francesca Zappacosta, Jonathan Pettinger, Syd Barthorpe, Chris Eberl, Brian Jones, Jess Schneck, Dennis Murphy, Josh Taygerly, Mike DeMartino, Matthew Coelho, Jon Houseley, Benjamin Schwartz, Mathew J. Garnett. Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells [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 6590.

Abstract 3994: N glycosylation inhibition hinders immunosuppressive tumor microenvironment cells improving CAR T cell efficacy

Abstract Adoptive transfer of CAR T cells demonstrated impressive results against B-cell malignancies, but still limited efficacy against solid tumors. In this context, multiple challenges need to be overcome, including poor tumor recognition and strong immunosuppression within the tumor microenvironment (TME). Our Unit has recently reported that pharmacological inhibition of N-glycan synthesis in cancer cells increases CAR T cell efficacy by improving tumor recognition and preventing T cell exhaustion. In this project, we investigated the role of N-glycosylation blockade on TME cells in the context of colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC)-derived liver metastases, exploiting CEA-specific CAR T cell therapy. To understand the effect of N-glycosylation blockade on TME cells (both M2-like macrophages, M2-M and Hepatic stellate cells, HepSCs), we analyzed the phenotypic and transcriptional profile and we performed in vitro functional assays, such as tripartite co-cultures, suppressive assays and released-cytokines analysis. In vitro studies revealed that N-glycosylation inhibition abolishes the ability of both TME cells to restrain T cell proliferation and increases the elimination of cancer cell lines and patient-derived tumor organoids (PDOs from CRC-liver metastases). Interestingly, these effects were associated with profound phenotypic and transcriptional changes in M2-M and HepSCs. In particular, the treatment was able to inhibit M2-polarization in terms of surface markers expression, IL-10 secretion and gene expression profile, and was shown to hinder the activation of HepSCs. Moreover, to evaluate the effect of N-glycosylation inhibition on TME cells in vivo, we exploited immunodeficient mice reconstituted with a human immune system (huSGM3), engrafted intra-liver with tumor cells and treated with CEA CAR T cells. Interestingly, in this in vivo model, the presence of human immune cells supports CAR T cell responses and helps recreate an immune TME more representative of the human disease. Importantly, using these mice we observed that N-glycosylation inhibition improves CEA CAR T cells fitness, enhancing their antitumor activity in terms of survival. This effect is associated with a modulation of human immune TME populations and in particular with the downregulation of immunosuppressive genes in tumor-infiltrating human immune cells (from scRNAseq and Nanostring analysis). Overall, these data suggest that blocking N-glycosylation can help overcome multiple barriers that currently limit CAR T cell efficacy in solid tumors, acting not only on tumor cells, but also on immunosuppressive tumor microenvironment cells. Citation Format: Camilla Sirini, Beatrice Greco, Chiara Balestrieri, Barbara Camisa, Laura Falcone, Rita El Khoury, Oronzina Botrugno, Giulio Giovannoni, Giovanni Tonon, Emanuela Brunetto, Maria Giulia Cangi, Federica Pedica, Alice Bergamini, Fabio Ciceri, Chiara Bonini, Monica Casucci. N glycosylation inhibition hinders immunosuppressive tumor microenvironment cells improving CAR T cell efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3994.