Cancer Patient-derived Xenograft Research Articles

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

Abstract PO2-28-08: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract TNBC is one of the most aggressive subtypes of breast cancer. Combating chemotherapy resistance is critical to improving quality of care and reducing fatality among TNBC patients. In order to understand the mechanisms of chemoresistance in TNBC tumors, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy among tumors, and metastatic potential of certain tumors over others. Thus, this study utilizes spatially resolved transcriptomic (SRT) technology to profile the spatial interactions in the TNBC TME. With the goal of better understanding chemoresistance in TNBC and providing a proof-of-concept for new technology, we have launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Genomics Visium was performed on vehicle, AC-treated residual tumor (21 days post-Tx), and AC-treated regrown tumor (50 days post-Tx when tumors regrew to starting tumor volume). This study is divided into two parts: 1) development of a computational pipeline; 2) spatial colocalization analysis of residual and regrown tumors to understand chemoresistance. PART 1: Due to the lack of specialized tools for processing and sorting xenograft reads from SRT data, we developed the Xenomake pipeline, which combines a xenograft sorting algorithm (Xengsort) and spatial barcode demultiplexing pipeline to assign reads into the host and graft organisms for each spatial spot. RESULTS: Xenomake permits clustering the spatial spots into stroma-rich (enriched for mouse mRNAs), and epithelial-rich (enriched for human mRNAs) regions. We show that Xenomake can find differential cytokine production in the stroma and epithelium. Since PDX data separate the tumor into stroma and epithelium by organism, the pipeline enables fine-tuned downstream analysis such as stromal-stromal and stromal-epithelial interactions. Xenomake is thus generally applicable for SRT involving PDX samples. PART 2: Previously we detailed patterns of tumor regression into a residual tumor state, followed by uncontrolled regrowth in the absence of treatment in multiple PDX models of TNBC. Although using PDX models necessitates immune-compromised mice, several types of stromal populations are present and analyzable in these models. Thus, using organism-assigned reads processed by Xenomake, we computationally inferred the localization pattern of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial cells (ENDO), monocytes, and perivascular-like cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for the ~30 HALLMARK pathways across samples. We correlated the patterns of SPAM with stroma cell-type SLP to survey the stroma-epithelial interactions across samples. RESULTS: Stroma cells are generally distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and with CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Furthermore, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-08.

Abstract PO1-04-14: PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts

Abstract Paclitaxel is a first-line chemotherapy for hormone receptor positive (HR+) metastatic breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Response rate to paclitaxel ranges between 20-40%, and most patients progress due to intrinsic or acquired resistance, leaving them with limited treatment choices. Therapeutic strategies to overcome paclitaxel resistance and extend its clinical benefit are urgently needed. Paclitaxel-resistant tumors have increased expression of polo-like kinase 1 (PLK1), a serine-threonine-protein kinase that regulates mitosis and cell cycle progression. PLK1 has also been shown to mediate resistance to CDK4/6 inhibitor palbociclib in HR+ breast cancer. Onvansertib is an orally bioavailable, highly potent, and selective PLK1 inhibitor in clinical development. Onvansertib exhibited synergy in vitro and showed potent anti-tumor activity in vivo in combination with paclitaxel in ovarian cancer and triple negative breast cancer (TNBC) preclinical models. A phase 1b/2 clinical trial is ongoing to evaluate the safety and efficacy of onvansertib plus paclitaxel in advanced TNBC (NCT05383196). Here we investigated whether the potential of onvansertib and paclitaxel combination could be extended to HR+ breast cancer. Co-treatment with onvansertib and paclitaxel synergistically inhibited the viability of HR+ breast cancer cell lines (MCF-7, T-47D, EFM-19, CAMA-1, ZR-75-1). Compared to the single agents, the combination induced increased apoptosis. The combination was further tested in vivo in patient-derived xenograft (PDX) models. To this end, six HR+ breast cancer PDXs with intrinsic or acquired resistance to palbociclib were established from primary breast tumors (n=1) or metastatic bone biopsies (n=5). The PDX tumors were engrafted subcutaneously in nude mice and the effect of onvansertib (45 mg/kg, oral, 5 days a week) and paclitaxel (15-25mg/kg, IP, once a week) was investigated as monotherapy and in combination. The combination of onvansertib and paclitaxel was tolerated with minimal toxicity and showed enhanced anti-tumor activity compared to either agent alone in all 6 PDX models. Paclitaxel and onvansertib monotherapies had little to no anti-tumor activity in 4 out of 6 PDX models (HBCx124palboR25, HBCx-139 palboR5, HBCx-202 and HBCx-131). Importantly, the combination of onvansertib and paclitaxel induced strong anti-tumor activity in these 4 models. Tumor regression was observed in the 2 PDX models with acquired resistance to palbociclib, HBCx-124palboR25 and HBCx-139palboR5, with 62% (5/8 mice) and 54% (6/11 mice) complete response rates, respectively. The combination showed tumor stasis in HBCx-131 and induced tumor regression in HBCx-202, established from patient resistant to palbociclib. In 2 of the 6 PDXs, paclitaxel exhibited anti-tumor activity, inducing tumor regression in HBCx-137paboR26 and tumor stasis in HBCx-86. In comparison, the combination of onvansertib and paclitaxel had greater activity, inducing tumor regression in both models with a higher rate of complete response than the monotherapy. Complete response rates for paclitaxel versus the combination were 62% and 100% in the HBCx-137palboR26 model and 0% and 67% in the HBCx-86 model. Notably, the antitumor activity of the onvansertib and paclitaxel combination was very durable, showing a robust delay in tumor relapse after stopping therapy. Together, our data strongly support that combining paclitaxel with the PLK1 inhibitor onvansertib extends its benefit and overcomes paclitaxel resistance, and represents a promising therapeutic strategy for HR+ breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Citation Format: Maya Ridinger, Pierre Painsec, Sreeja Sreekumar, Dalia Gonzalez, Davis Klein, Laura Sourd, Elodie Montaudon, Paul Cottu, Tod Smeal, Elisabetta Marangoni. PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-04-14.

Advancements in the Understanding of Small-Cell Neuroendocrine Cervical Cancer: Where We Stand and What Lies Ahead

Small-cell neuroendocrine cervical carcinoma (SCNCC) is a rare yet aggressive gynecological malignancy associated with dismal clinical outcomes. Its rarity has led to a limited number of retrospective studies and an absence of prospective research, posing significant challenges for evidence-based treatment approaches. As a result, most gynecologic oncology centers have limited experience with this tumor, emphasizing the urgent need for a comprehensive review and summary. This article systematically reviews the pathogenesis, immunohistochemical and molecular characteristics, prognostic factors, and clinical management of gynecologic SCNCC. We specifically focused on reviewing the distinct genomic characteristics of SCNCC identified via next-generation sequencing technologies, including loss of heterozygosity (LOH), somatic mutations, structural variations (SVs), and microRNA alterations. The identification of these actionable genomic events offers promise for discovering new molecular targets for drug development and enhancing therapeutic outcomes. Additionally, we delve deeper into key clinical challenges, such as determining the optimal treatment modality between chemoradiation and surgery for International Federation of Gynecology and Obstetrics (FIGO) stage I phase patients within a precision stratification framework, as well as the role of targeted therapy within the homologous recombination (HR) pathway, immune checkpoint inhibitors (ICIs), and prophylactic cranial irradiation (PCI) in the management of SCNCC. Finally, we anticipate the utilization of multiple SCNCC models, including cancer tissue-originated spheroid (CTOS) lines and patient-derived xenografts (PDXs), to decipher driver events and develop individualized therapeutic strategies for clinical application.

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.

Mutant RAS-driven secretome causes skeletal muscle defects in breast cancer.

Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple negative breast cancer (TNBC), Estrogen Receptor-positive/Progesterone Receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in ~37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Since RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects.

DB-1310, an ADC comprised of a novel anti-HER3 antibody conjugated to a DNA topoisomerase I inhibitor, is highly effective for the treatment of HER3-positive solid tumors

BackgroundHER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Multiple HER3-targeting antibodies and antibody–drug conjugates (ADCs) were developed for the solid tumor treatment, however none of HER3-targeting agent has been approved for tumor therapy yet. We developed DB-1310, a HER3 ADC composed of a novel humanized anti-HER3 monoclonal antibody covalently linked to a proprietary DNA topoisomerase I inhibitor payload (P1021), and evaluate the efficacy and safety of DB-1310 in preclinical models.MethodsThe binding of DB-1310 to Her3 and other HER families were measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast, lung, prostate and colon cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3 + breast, lung, colon and prostate cancer xenograft models. The safety profile was also measured in cynomolgus monkey.ResultsDB-1310 binds HER3 via a novel epitope with high affinity and internalization capacity. In vitro, DB-1310 exhibited cytotoxicity in numerous HER3 + breast, lung, prostate and colon cancer cell lines. In vivo studies in HER3 + HCC1569 breast cancer, NCI-H441 lung cancer and Colo205 colon cancer xenograft models showed DB-1310 to have dose-dependent tumoricidal activity. Tumor suppression was also observed in HER3 + non-small cell lung cancer (NSCLC) and prostate cancer patient-derived xenograft (PDX) models. Moreover, DB-1310 showed stronger tumor growth-inhibitory activity than patritumab deruxtecan (HER3-DXd), which is another HER3 ADC in clinical development at the same dose. The tumor-suppressive activity of DB-1310 synergized with that of EGFR tyrosine kinase inhibitor, osimertinib, and exerted efficacy also in osimertinib-resistant PDX model. The preclinical assessment of safety in cynomolgus monkeys further revealed DB-1310 to have a good safety profile with a highest non severely toxic dose (HNSTD) of 45 mg/kg.ConclusionsThese finding demonstrated that DB-1310 exerted potent antitumor activities against HER3 + tumors in in vitro and in vivo models, and showed acceptable safety profiles in nonclinical species. Therefore, DB-1310 may be effective for the clinical treatment of HER3 + solid tumors.

Abstract LB406: Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer

Abstract Folate receptor alpha (FRα) is a cell surface GPI-anchored protein overexpressed in several solid tumors with highest prevalence in ovarian cancer and lung adenocarcinoma but restricted expression in normal tissues. AZD5335, an FRα-targeting antibody conjugated to AZ’s proprietary topoisomerase 1 inhibitor (TOP1i) payload, AZ14170132, is currently being investigated in the FONTANA Phase 1/2 clinical trial (NCT05797168). Since homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRRm+) are associated with sensitivity to DNA damaging agents, we evaluated the efficacy of AZD5335 in ovarian cancer patient-derived xenograft (PDX) models assessed for HRRm, as described1. Sixteen ovarian cancer PDX models were each treated with a single dose of AZD5335 (5 mg/kg IV). Of the HRRm- cohort, 8/11 (73%) cohort responded to treatment with a tumor regression >30% from baseline. In the HRRm+ cohort 4/5 (80%) responded to AZD5335. PARP1 can act as a positive regulator of genomic stability by counteracting TOP1-induced DNA damage (Malanga and Althaus, 2004) that led to the hypothesis that the efficacy of TOP1i drugs can be enhanced by combination with PARP inhibitors. To evaluate for potential synergy of AZD5335 with the PARP1 inhibitor, saruparib (AZD5305): Three FRα-expressing cancer cell lines, KB, IGROV-1 and OVCAR-3, were treated with AZD5335 +/- saruparib, revealing evidence of synergistic cytotoxicity. These results encouraged us to investigate this combination in pre-clinical HRRm+ in vivo xenograft models (OVCAR-3 and OV2022F). In OVCAR-3 xenograft models, single agent activity with saruparib (1 mg/kg PO QD x28) was 37% tumor growth inhibition (TGI) and with AZD5335 (0.625 mg/kg IV x1) was 50% TGI while combined administration at the same doses improved TGI to 86%. Similar combinatorial activity was seen in OV2022F with TGI 61%, 54%, and 97% for saruparib, AZD5335, and saruparib +AZD5335, respectively. In conclusion, AZD5335 exhibits a similar degree of single agent antitumor activity in both HRRm+ and HRRm- preclinical models, and the efficacy can be potentiated by combining with the PARP inhibitor, saruparib. 1. Clarke et al. 2022, NEJM Evid 2022;1(9) DOI: 10.1056/EVIDoa22000432. Malanga and Althaus 2004, JBC 2004; 279(7) DOI: 10.1074/jbc.C3004372003. Citation Format: Marco Gymnopoulos, Ravinder Tammali, Ana De Almeida, Jixin Wang, Claire Myers, Mark Albertella, Paula G. Fraenkel, Puja Sapra. Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB406.

Integrative Molecular Analyses of the MD Anderson Prostate Cancer Patient-derived Xenograft (MDA PCa PDX) Series.

Develop and deploy a robust discovery platform that encompasses heterogeneity, clinical annotation, and molecular characterization and overcomes the limited availability of prostate cancer models. This initiative builds on the rich MD Anderson (MDA) prostate cancer (PCa) patient-derived xenograft (PDX) resource to complement existing publicly available databases by addressing gaps in clinically annotated models reflecting the heterogeneity of potentially lethal and lethal prostate cancer. We performed whole-genome, targeted, and RNA sequencing in representative samples of the same tumor from 44 PDXs derived from 38 patients linked to donor tumor metadata and corresponding organoids. The cohort includes models derived from different morphologic groups, disease states, and involved organ sites (including circulating tumor cells), as well as paired samples representing heterogeneity or stages before and after therapy. The cohort recapitulates clinically reported alterations in prostate cancer genes, providing a data resource for clinical and molecular interrogation of suitable experimental models. Paired samples displayed conserved molecular alteration profiles, suggesting the relevance of other regulatory mechanisms (e.g., epigenomic) influenced by the microenvironment and/or treatment. Transcriptomically, models were grouped on the basis of morphologic classification. DNA damage response-associated mechanisms emerged as differentially regulated between adenocarcinoma and neuroendocrine prostate cancer in a cross-interrogation of PDX/patient datasets. We addressed the gap in clinically relevant prostate cancer models through comprehensive molecular characterization of MDA PCa PDXs, providing a discovery platform that integrates with patient data and benchmarked to therapeutically relevant consensus clinical groupings. This unique resource supports robust hypothesis generation and testing from basic, translational, and clinical perspectives.

Sialyl-Tn serves as a potential therapeutic target for ovarian cancer

BackgroundOvarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer.MethodsWe aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples.ResultsOur in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis.ConclusionsOur preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft 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 538.

Abstract 3876: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract Combating chemotherapy resistance in TNBC is critical to improving quality of care and reducing fatality. To understand chemoresistance, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy and metastatic potential. Thus, this study utilizes spatially resolved transcriptomics (SRT) to profile the spatial interactions in TNBC TME. With the goal of understanding chemoresistance and providing a proof-of-concept for new technology, we launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Visium was performed on Veh, AC-residual tumor (21 d post-Tx), and AC-regrown tumor (50 d post-Tx when tumors regrew). This study is divided into two parts. 1. Due to the lack of specialized tools for processing xenograft reads from SRT, we developed the Xenomake pipeline, which combines xenograft sorting and spatial barcode demultiplexing to assign reads into the host and graft organisms per spatial spot. It permits clustering the spots into stroma-rich (mouse), and epithelial-rich (human) regions. We show that it can find differential cytokines in the stroma (S) and epithelium (E), and finding SS, EE, and SE interactions. 2. Although using PDX necessitates immune-compromised mice, several types of stromal populations are present and analyzable. Thus, using Xenomake, we inferred the localization of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial (ENDO), monocytes, and perivascular cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for HALLMARK pathways. We correlated SPAM with stroma cell type SLP to survey stroma-epithelial interactions. Stroma cells are distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Further, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [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 3876.

Abstract 6913: A preclinical platform of prostate cancer PDX and derived cellular models as a tool for pharmacological screening and functional studies

Abstract Prostate Cancer (PCa) is the most frequently diagnosed cancer in men and remains one of the leading causes of cancer death worldwide. Despite undeniable progress in understanding the biology and genetics of this highly heterogenous and complex disease, the development of effective therapies is hampered by the lack of relevant experimental models recapitulating its diversity. To tackle this issue, over the last years, we have generated and characterized at the molecular and pharmacology level a collection of prostate cancer patient-derived xenografts (PDX) that accurately reproduce PCa histological and molecular heterogeneity. Although the development of these PCa PDX has been challenging, we are now able to offer a preclinical platform of 11 fully characterized PCa PDX models and in vitro cellular counterparts for preclinical evaluation of novel treatment modalities. Our platform consists of a collection of 4 androgen receptor high (ARPC), 2 androgen receptor low (ARLPC), 1 amphicrine prostate carcinoma (AMPC) and 4 castration-resistant neuroendocrine prostate cancer (CRPC-NE) PDX models and 6 cellular models derived from these PDX (PDXDC). Most of these PDX models were obtained by transplantation of post-surgery tumor specimens either by grafting of tumor fragments in the interscapular region of nude mice or in the subrenal capsule of NOD scid gamma mice. One model is a CTC-derived eXplant established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis injected into the fat pad of NOD scid gamma mice. PDXDC were obtained from dissociated PDX tumors cultured under various media and matrix conditions. They were characterized by comparison with the parental PDX by Short Tandem Repeat (STR) profiling before performing a master bank. All models (PDX and PDXDC) were characterized at molecular level by whole exome and RNA sequencing. PDX were characterized pharmacologically for response to standards of care, physical castration and response to the androgen receptor inhibitor enzalutamide. In vitro drug sensitivity of PDXDC was compared with their parental PDX in vivo drug response. This unique PDX panel reflects the molecular heterogeneity of prostate cancer and reproduces the molecular and drug response profile of human tumors. Associated to in vitro cell derivatives, our collection provides a powerful preclinical platform for translational research to identify mechanisms underlying acquired resistance to current therapies and develop novel treatment strategies against PCa and CRPC. Citation Format: Emilie Indersie, Léa Sinayen, Ludovic Bigot, Katell Mevel, Aurore Gorse, Marie Tavernier, Enora Le Ven, Luc Friboulet, Françoise Farace, Ester Morgado, Laurent Pouyet, Yohann Loriot, François Romagné, Olivier Déas. A preclinical platform of prostate cancer PDX and derived cellular models as a tool for pharmacological screening and functional studies [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 6913.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical 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 6579.

Abstract 907: ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction

Abstract Precision oncology hinges on accurate prediction of patient-specific treatment response from tumor molecular inputs. While deep learning (DL) models achieve state-of-the-art response prediction in cell lines, existing methods do not readily translate to the clinic where training data is limited. Here, we have developed and validated ScreenDL, a novel DL framework designed explicitly for use in clinical precision oncology applications. The underlying architecture of ScreenDL consists of two fully connected branches dedicated to learning rich embeddings of a drug’s chemical structure and a tumor’s transcriptomic profile respectively. These tumor omic and drug molecular embeddings are fed into a shared response prediction subnetwork which outputs predicted ln(IC50) values. The ScreenDL training schema incorporates three phases, each designed to improve clinical response prediction: (1) an initial pretraining phase leverages large-scale cell line pharmacogenomic datasets to extract patterns/relationships linking tumor omics and drug response; (2) subsequent transfer learning integrates pharmacogenomic data from patient-derived models of cancer, adapting the pretrained model to a more patient-relevant context; (3) patient-level omics and drug screening data (e.g., from matched patient-derived organoid (PDO) models) is integrated through patient-specific fine-tuning, generating a patient-specific response prediction model. Importantly, biomaterial from surgical tumor resection is often available for organoid establishment and patient-specific drug screening in practical clinical scenarios. To assess the utility of our ScreenDL framework for clinical response prediction, we applied ScreenDL to predict treatment response in 50 advanced/metastatic triple negative breast cancer (TNBC) patient-derived xenograft models (PDX). We leveraged tumor omics profiles for each PDX and drug screening data from matched PDX-derived organoids (PDxOs), mirroring the combination of patient-level tumor omic characterization and functional drug screening in matched PDOs, a protocol currently being piloted in Functional Precision Oncology trials at the University of Utah Huntsman Cancer Institute. After cell line pretraining, ScreenDL achieves a modest median Pearson correlation per drug of 0.15 relative to 0.03 for the leading compared model. However, transfer learning and subsequent PDX-specific fine-tuning significantly improve performance, producing median Pearson correlations per drug of 0.39 and 0.51, respectively. These results demonstrate the ability of our ScreenDL framework to dramatically improve response prediction in clinically relevant domains, bringing DL-based precision treatment selection closer to clinical application. Citation Format: Casey Sederman, Tonya Di Sera, Yi Qiao, Xiaomeng Huang, Bryan E. Welm, Alana L. Welm, Gabor Marth. ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction [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 907.

Abstract 4257: A novel 3D co-culture platform for patient-specific immune-oncology treatment

Abstract Lung cancer is the leading cause of global cancer-related mortality resulting in ~1.8 million deaths. annually. Over the past decade, the contribution of tumor microenvironment (TME) in lung cancer progression has been highly appreciated and provided significant improvements of survival outcomes for patients. That includes the complexity of the tumor immune microenvironment (TIME) playing a critical role in cancer progression. However, drug resistance usually arises and there is an urgent need for novel immune cell-based therapies. 3D tumor cell structures that recapitulate the architecture and functionality of the original tumor have revolutionized the understanding of disease mechanisms and testing potential therapeutics. However, there are certain drawbacks that need to be overcome. For example, these models mainly consist of only the epithelial compartment and lack signals from the microenvironment. Recent advances with engineering co-culture systems with immune cells at Champions Oncology help to emulate cell-cell and immune cell infiltration. We have successfully established a biobank of TumorGraft3D (CTG3Ds) derived from non-small cell lung cancer (NSCLC) patient-derived xenografts as well as their autologous and allogeneic immune cells (TILs, PBMCs) to establish a proprietary co-culture platform to investigate disease mechanisms and test potential therapeutics. In brief, surgical resections from patients were used to generate patient-derived xenografts organoids, and matched immune cells were expanded using standard rapid expansion protocol. The combination of a comprehensive staining system and high-content imaging as an endpoint allowed for 3D confocal analysis and deep evaluation of cancer-immune cellular interaction at the single cell level. Our pipeline of image analysis not only differentiates cancer-immune cell spatial distribution and cellular death but also allows for the evaluation of tumor infiltration and drug cytotoxicity. The characterization of the platform showed proliferation of the cancer cells and functional immune profiling by flow cytometry. The molecular profile of the organoids composing the platform showed a tight overlap with the original tumor highlighting the clinical relevance of the model. In addition, this novel co-culture platform has been tested with several NSCLC models to test the efficacy of various therapeutics such as immune-checkpoint inhibitors (ICIs) in reproducible, cost-effective, and patient-specific manner. In summary, Champion’s novel co-culture platform allows screening of novel immune targeting agents to determine impacts tumor cytotoxicity. Furthermore, our lung cancer tumorGraft3D platform serves as a powerful tool to facilitate the practice of precision medicine by investigating a variety of therapeutic modalities, alone or in combination, to identify patient-specific treatment strategies. Citation Format: Taylor Light, Samaneh Kamali, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. A novel 3D co-culture platform for patient-specific immune-oncology treatment [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 4257.

Abstract 3122: Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6

Abstract Claudin-6 is a member of the claudin family and consists of four transmembrane helices, two extracellular loops, and an amino- and carboxyl-terminal tail with a PDZ-binding motif in the cytoplasm. It is involved in the formation of tight junctions and is expressed in developing human epithelial structures during early-to-mid gestation while expression in iadult tissues is mostly absent. Claudin-6 expression is upregulated in ovarian cancer, testicular cancer, endometrial cancer, non-small cell lung cancer (NSCLC) and gastric cancer. Here, the generation and preclinical characterization of a novel Claudin-6 specific antibody-drug conjugate (ADC) is described, composed of a humanized IgG1 directed against human Claudin-6 site-specifically conjugated a novel payload containing a valine-alanine cleavable linker and a camptothecin warhead with a drug to antibody ratio of 6. FcγR-mediated effector function of the ADC was abrogated via mutations in the Fc portion of the antibody. The purpose of this study was to evaluate the expression of Claudin-6 in different tumour indications and characterise the in vitro and in vivo anti-tumour activity in human cancer cell lines and patient-derived xenograft (PDX) models. In addition, the tolerability of the ADC was evaluated in a repeat-dose toxicity study cynomolgus monkeys. Cell binding studies using Fabs showed high affinity binding to Claudin-6, while affinity for Claudin-9 was 16-fold lower with no binding to Claudin-3 and Claudin-4. Cross-reactivity with cynomolgus monkey and mouse Claudin-6 was also demonstrated. Fcgamma receptor binding studies confirmed the strongly reduced binding of the antibody to FcγI, FcγI,IIa, FcγIIb and FcγIIa, consistent with the introduction of mutations in the Fc domain of the antibody. In vitro, the Claudin-6 ADC showed potent and specific cytotoxicity against various Claudin-6-expressing solid cancer cell lines, including OVCAR3, PA-1, OV-90, FU-97, BeWo, NTERA-2 and NUGC3 in comparison to a non-binding control ADC conjugated to the same payload, and it also showed potent bystander killing. In vivo, a single dose intravenous (IV) of the Claudin-6 ADC demonstrated potent anti-tumour activity in multiple solid tumour xenograft models, including OVCAR3 and PA-1. Furthermore, administration of the Claudin-6 ADC to cynomolgus monkeys was well tolerated up to 40 mg/kg IV when given on Day 1 and 22. Cell membrane expression of Claudin-6 was confirmed by immunohistochemistry (IHC) in ovarian cancer (70%), testicular cancer (98%), endometrial cancer (30%), NSCLC (28%) and gastric cancer (24%), confirming expression of Claudin-6 in indications with high unmet need. In summary, this novel Claudin-6 ADC demonstrated potent, specific anti-tumour activity in Claudin-6 expressing cancer-derived models and was well tolerated in cynomolgus monkey, supporting future clinical development of this ADC. Citation Format: Nicola Tsang, Nicolas Veillard, Elizabeth Horsley, Karin Havenith, Narinder Janghra, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Paul W. Hogg, Francesca Zammarchi, Lolke de Haan, Patrick H. van Berkel. Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6 [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 3122.

Abstract 1862: ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications

Abstract Background: Folate Receptor alpha (FRα) is a validated cancer target that is prevalently expressed in multiple cancers with high unmet need, including ovarian cancer and other gynecological cancers, NSCLC, endometrial cancer and TNBC. Due to FRα’s favorable expression profile, multiple antibody-drug conjugates (ADCs) are being explored in this setting. Here we present the preclinical characterization of a new anti-FRα ADC, ZW191. ZW191 is a bystander active antibody drug conjugate (ADC) comprised of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor (TOPO1i) payload, ZD06519, via a maleimidocaproyl (MC) anchor and a glycyl glycyl phenylalanyl glycyl (GGFG)-aminomethyl (AM) cleavable linker at a drug-to-antibody ratio (DAR) of 8. ZW191 is projected to undergo IND submission in 2024. Materials and Methods: The novel antibody and drug-linker components of ZW191 were generated, characterized, and optimally integrated. The apparent binding affinity and cellular internalization of the ZW191 antibody were determined in FRα-expressing cells. Tumor spheroid cancer cell cultures were utilized to determine ZW191’s degree of tissue penetration and its cytotoxicity. The bystander activity of ZW191 was assessed using antigen positive and negative co-culture experiments. The anti-tumor activity of ZW191 was evaluated in a panel of ovarian cancer, NSCLC, endometrial cancer and TNBC patient-derived xenografts (PDX) spanning a range of FRα expression. ZW191 was evaluated in toxicology and pharmacokinetic (PK) studies performed in non-human primates (NHP). Results: The antibody component of ZW191 features a favorable binding profile with strong binding to FRα, and drives superior tumor spheroid penetration, cellular internalization, and payload delivery compared to FRα targeted antibodies used in multiple other ADCs. ZW191 demonstrates potent activity in FRα expressing cell cultures and effective bystander activity. In a panel of PDX models representing a range of FRα expression, ZW191 demonstrates compelling anti-tumor activity across ovarian and endometrial cancers, NSCLC and TNBC. ZW191 demonstrates an encouraging tolerability profile in NHP, with favorable PK. The promising efficacy, tolerability, and PK support the potential of ZW191 as a novel therapeutic agent that may help address unmet need in patients with high and low FRα expressing cancers. Citation Format: Sam Lawn, Andrea Hernandez Rojas, Raffaele Colombo, Jodi Wong, Kaylee Wu, Vincent Fung, Manuel Lasalle, Mark E. Petersen, Lemlem Degefie, Araba Sagoe-Wagner, Dunja Urosev, Luying Yang, Ambroise Wu, Catrina Kim, Amos Chua, Kurt Stahl, Geoffrey C. Winters, John C. Fann, Jamie Rich, Stuart Barnscher. ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications [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 1862.

Abstract 4596: Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497

Abstract It remains largely challenging to treat cancer patients with chromosome 9p21-deletion which occurs in approximately 15% of human cancers. Recently, specific targeting of protein arginine methyltransferase 5 (PRMT5), a key member of the type-II PRMT family and a master epigenetic modulator of arginine methylome essential for cancer progression, has emerged as a promising therapy in various hematological and solid tumors with MTAP deletion serving as a precision biomarker. To optimize the potential of targeting PRMT5, we developed CTS3497, a potent, brain-penetrable, orally bioavailable MTA-cooperative PRMT5 inhibitor, using our EpigenPLUSTM platform. The overall excellent drug-like properties of CTS3497 support fast track IND application, and it is anticipated to enter Phase 1 trials in 2024. CTS3497 demonstrated potent PRMT5 inhibitory activity in cellular pharmacodynamics (PD) assays, evidenced by the suppression of symmetric dimethylarginine (SDMA). Additionally, it exhibited profound cell growth inhibition with a low single-digit nM IC50 and 171-fold high selectivity for MTAPnull over isogenic MTAPwt cell lines. Moreover, CTS3497 displayed strong inhibitory effects on cell growth across diverse panels of MTAPnull cancer cell lines and patient-derived xenograft organoids (PDXOs) spanning various lineages. In vivo, oral administration of CTS3497 resulted in significant tumor regression in multiple MTAP-deleted xenograft models, notably MTAPnull brain tumors. The observed anti-tumor activity correlated with PRMT5 modulation, as indicated by reduced SDMA levels. Impressively, sustained tumor growth inhibition led to the complete tumor regression, with no regrowth even after discontinuation of CTS3497 treatment. The brain penetration property of CTS3497 underscores its therapeutic potential for both primary brain tumors, and brain metastases arising from specific cancers like lung cancer. At the molecular level, PRMT5 inhibition by CTS3497 induced substantial and distinctive RNA splicing defects and differentially expressed gene signatures, impacting key cellular activities downstream of arginine and epigenetic reprogramming in MTAPnull cancer cells. Furthermore, a synergistic anti-tumor effect was observed in multiple MTAP-deleted tumors when combining PRMT5 inhibition with CTS2190, an investigational type-I PRMT inhibitor currently in phase I/II clinical trials. In summary, CTS3497, a PRMT5 inhibitor selectively targeting MTAPnull tumor cells, demonstrated robust and durable anti-tumor responses both in vitro and in vivo across diverse lineages of MTAP-deleted tumors. Its superior ADME properties and favorable safety profiles observed in preclinical studies mark a breakthrough in the next generation epigenetic therapy. CTS3497 emerges as a promising therapeutic option for patients with MTAP-deleted malignancies. Citation Format: Yilin Liu, Long Wang, Hui Shi, Yuanyuan Liu, Qiugeng Ouyang, Xingnian Fu, Meng Wang, Jiannan Guo, Yiqin Wang, Youzhen Wang, Guoliang Xu, Yuan Mi, Haiping Wu. Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497 [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 4596.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast 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 1986.