Patient-derived Xenograft Research Articles

Advancing Clinical Response Against Glioblastoma: Evaluating SHP1705 CRY2 Activator Efficacy in Preclinical Models and Safety in Phase I Trials.

It has been reported that circadian clock components, Brain and Muscle ARNT-Like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK), are uniquely essential for glioblastoma (GBM) stem cell (GSC) biology and survival. Consequently, we developed a novel Cryptochrome (CRY) activator SHP1705, which inhibits BMAL1-CLOCK transcriptional activity. We analyzed buffy coats isolated from Phase 1 clinical trial subjects' blood to assess any changes to circadian, housekeeping, and blood transcriptome-based biomarkers following SHP1705 treatment. We utilized GlioVis to determine which circadian genes are differentially expressed in non-tumor versus GBM tissues. We employed in vitro and in vivo methods to test the efficacy of SHP1705 against patient-derived GSCs and xenografts in comparison to earlier CRY activator scaffolds. Additionally, we applied a novel-REV-ERB agonist SR29065, which inhibits BMAL1 transcription, to determine whether targeting both negative limbs of the circadian transcription-translation feedback loop (TTFL) would yield synergistic effects against various GBM cells. SHP1705 is safe and well-tolerated in Phase I clinical trials. SHP1705 has increased selectivity for the CRY2 isoform and potency against GSC viability compared to previously published CRY activators. SHP1705 prolonged survival in mice bearing GBM tumors established with GSCs. When combined with the novel REV-ERB agonist SR29065, SHP1705 displayed synergy against multiple GSC lines and differentiated GSCs (DGCs). These demonstrate the efficacy of SHP1705 against GSCs, which pose for GBM patient outcomes. They highlight the potential of novel circadian clock compounds in targeting GBM as single agents or in combination with each other or current standard-of-care. SHP1705 is a novel CRY2 activator that has shown success in Phase 1 safety trialsSHP1705 has a significantly improved efficacy against GSCs and GBM PDX tumorsNovel REV-ERB agonist SR29065 and SHP1705 display synergistic effects against GSCs. CRY2 is decreased in GBM tissues compared to CRY1 suggesting that promoting CRY2 activity will be an efficacious GBM treatment paradigm. SHP1705, a CRY2 activator that has shown success in Phase 1 safety trials, has significantly improved preclinical efficacy. Novel REV-ERB agonist SR29065 displays synergistic effects against diverse GBM cells.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

Expression and Clinical Significance of Nuclear Phosphoglucomutase-1 in Hepatocellular Carcinoma.

This study aimed to evaluate the predictive values of phosphoglucomutase-1 (PGM1) expression for prognosis in patients with hepatocellular carcinoma (HCC). PGM1 expression was assessed by immunohistochemistry in tissue microarrays. The relationship of PGM1 expression level with pathologic parameters and prognosis values was respectively analyzed by χ2 test and Cox regression. The accuracy of independent risk factors in predicting prognosis was calculated by receiver operating characteristic curve. HCC patient-derived xenograft models were performed to evaluate the nuclear PGM1 antitumor effect. The results showed that PGM1 expression was low in HCC tissues. Nuclear PGM1 was an independent prognostic factor for overall survival and time to recurrence. Cox regression showed that nuclear PGM1, serum α-fetoprotein, liver cirrhosis, and TNM staging stage were independent risk predictors for HCC. Receiver operating characteristic curve demonstrated that combination of independent predictors had better prognostic value than TNM staging alone. Moreover, patient-derived xenograft models showed antitumor effect of nuclear PGM1. We found that low expression of nuclear PGM1 was detected in HCC tissues and associated with poor prognostic. Nuclear PGM1 was an independent prognostic factor in patients with HCC. Furthermore, nuclear PGM1 combining other independent risk factors showed a better prognostic value. Nuclear PGM1 was a useful prognostic biomarker for patients with HCC.

FABP4-mediated lipid metabolism promotes TNBC progression and breast cancer stem cell activity

Metabolic remodeling is a pivotal feature of cancer, with cancer stem cells frequently showcasing distinctive metabolic behaviors. Nonetheless, understanding the metabolic intricacies of triple-negative breast cancer (TNBC) and breast cancer stem cells (BCSCs) has remained elusive. In this study, we meticulously characterized the metabolic profiles of TNBC and BCSCs and delved into their potential implications for TNBC treatment. Our findings illuminated the robust lipid metabolism activity within TNBC tumors, especially in BCSCs. Furthermore, we discovered that Fabp4, through its mediation of fatty acid uptake, plays a crucial role in regulating TNBC lipid metabolism. Knocking down Fabp4 or inhibiting its activity significantly suppressed TNBC tumor progression in both the MMTV-Wnt1 spontaneous TNBC model and the TNBC patient-derived xenograft model. Mechanistically, Fabp4's influence on TNBC tumor progression was linked to its regulation of mitochondrial stability, the CPT1-mediated fatty acid oxidation process, and ROS production. Notably, in a high-fat diet model, Fabp4 deficiency proved to be a substantial inhibitor of obesity-accelerated TNBC progression. Collectively, these findings shed light on the unique metabolic patterns of TNBC and BCSCs, underscore the biological significance of Fabp4-mediated fatty acid metabolism in governing TNBC progression, and offer a solid theoretical foundation for considering metabolic interventions in breast cancer treatment. SignificanceTriple-negative breast cancer progression and breast cancer stem cell activity can be restricted by targeting a critical regulator of lipid responses, FABP4.

Inhibition of hTERT/telomerase/telomere mediates therapeutic efficacy of osimertinib in EGFR mutant lung cancer.

The inevitable acquired resistance to osimertinib (AZD9291), an FDA-approved third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating or T790M resistant mutations, limits its long-term clinical benefit. Telomere maintenance via telomerase reactivation is linked to uncontrolled cell growth and is a cancer hallmark and an attractive cancer therapeutic target. Our effort toward understanding the action mechanisms, including resistance mechanisms, of osimertinib has led to the identification of a novel and critical role in maintaining c-Myc-dependent downregulation of hTERT, a catalytic subunit of telomerase, and subsequent inhibition of telomerase/telomere and induction of telomere dysfunction in mediating therapeutic efficacy of osimertinib. Consequently, osimertinib combined with the telomere inhibitor, 6-Thio-dG, which is currently tested in a phase II trial, effectively inhibited the growth of osimertinib-resistant tumors, regressed EGFRm NSCLC patient-derived xenografts, and delayed the emergence of acquired resistance to osimertinib, warranting clinical validation of this strategy to manage osimertinib acquired resistance.

LncRNA-HMG incites colorectal cancer cells to chemoresistance via repressing p53-mediated ferroptosis

Upon chemotherapy, excessive reactive oxygen species (ROS) often lead to the production of massive lipid peroxides in cancer cells and induce cell death, namely ferroptosis. The elimination of ROS is pivotal for tumor cells to escape from ferroptosis and acquire drug resistance. Nevertheless, the precise functions of long non-coding RNAs (lncRNAs) in ROS metabolism and tumor drug-resistance remain elusive. In this study, we identify LncRNA-HMG as a chemoresistance-related lncRNA in colorectal cancer (CRC) by high-throughput screening. Abnormally high expression of LncRNA-HMG predicts poorer prognosis in CRC patients. Concurrently, we found that LncRNA-HMG protects CRC cells from ferroptosis upon chemotherapy, thus enhancing drug resistance of CRC cells. LncRNA-HMG binds to p53 and facilitates MDM2-mediated degradation of p53. Decreased p53 induces upregulation of SLC7A11 and VKORC1L1, which contribute to increase the supply of reducing agents and eliminate excessive ROS. Consequently, CRC cells escape from ferroptosis and acquire chemoresistance. Importantly, inhibition of LncRNA-HMG by anti-sense oligo (ASO) dramatically sensitizes CRC cells to chemotherapy in patient-derived xenograft (PDX) model. LncRNA-HMG is also a transcriptional target of β-catenin/TCF and activated Wnt signals trigger the marked upregulation of LncRNA-HMG. Collectively, these findings demonstrate that LncRNA-HMG promotes CRC chemoresistance and might be a prognostic or therapeutic target for CRC.

Highly Efficient Synergistic Chemotherapy and Magnetic Resonance Imaging for Targeted Ovarian Cancer Therapy Using Hyaluronic Acid-Coated Coordination Polymer Nanoparticles.

The diagnosis and treatment of ovarian cancer (OC) are still a grand challenge, more than 70% of patients are diagnosed at an advanced stage with a dismal prognosis. Magnetic resonance imaging (MRI) has shown superior results to other examinations in preoperative assessment, while cisplatin-based chemotherapy is the first-line treatment for OC. However, few previous studies have brought together the two rapidly expanding fields. Here a technique is presented using cisplatin prodrug (Pt-COOH), Fe3+, and natural polyphenols (Gossypol) to construct the nanoparticles (HA@PFG NPs) that have a stable structure, controllable drug release behavior, and high drug loading capacity. The acidic pH values in tumor sites facilitate the release of Fe3+, Pt-COOH, and Gossypol from HA@PFG NPs. Pt-COOH with GSH consumption and cisplatin-basedchemotherapy plus Gossypol with pro-apoptotic effects displays asynergisticeffect forkillingtumorcells. Furthermore, the release of Fe3+ at the tumor sites promotes ferroptosis and enables MRI imaging of OC. Inthe patient-derived tumor xenograft (PDX)model, HA@PFG NPs alleviate the tumor activity. RNA sequencing analysis reveals that HA@PFG NPs ameliorate OC symptoms mainly through IL-6 signal pathways. This work combines MRI imaging with cisplatin-based chemotherapy, which holds great promise for OC diagnosis and synergistic therapy.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

GDF-15 predicts epithelioid hemangioendothelioma aggressiveness and is down-regulated by sirolimus through ATF4/ATF5 suppression.

Epithelioid hemangioendothelioma (EHE) poses a therapeutic challenge due to limited efficacy of conventional chemotherapy in advanced cases, necessitating exploration of new treatment avenues and identification of novel aggressiveness biomarkers. This study aimed at i) utilizing an EHE patient-derived xenograft (PDX) model and its associated cell line to assess the efficacy of sirolimus and ii) analyzing two distinct patient cohorts to pinpoint circulating biomarkers of EHE aggressiveness. A PDX model and corresponding cell line were established from an advanced EHE patient, demonstrating consistency with the original tumor in terms of histomorphology, WWTR1::CAMTA1 fusion presence, and genomic and transcriptomic profiles. Two independent patient series were employed to investigate the association between Growth/Differentiation Factor 15 (GDF-15) serum levels and EHE aggressiveness. ELISA analyses on EHE cell culture medium and blood from EHE-carrying mice revealed the release of GDF-15 by EHE cells. Sirolimus exhibited markedly higher anti-tumor activity compared to doxorubicin, concurrently reducing GDF-15 expression/release both in vivo and in vitro. This reduction was attributed to the drug-induced inhibition of phosphorylation/activation of 4E-BP1 and subsequent downregulation of the GDF-15 transcription factors ATF4 and ATF5. Blood sample analyses from two independent patient series showed a significant correlation between GDF-15 and EHE aggressiveness. This study identifies GDF-15 as a novel biomarker of EHE aggressiveness and underscores the superior efficacy of sirolimus compared to doxorubicin in our experimental models. The observed inhibition of GDF-15 release by sirolimus suggests its potential as a biomarker for monitoring the drug's activity in patients.

Abstract A067: Identifying and targeting FOLFIRINOX resistance mechanisms to improve outcomes in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 12%. More than 80% of PDAC patients cannot undergo potentially curative surgical resection due to the presentation of advanced, metastatic disease, and are instead offered systemic chemotherapy regimens to which resistance is rapidly acquired. FOLFIRINOX chemotherapy offers an 11.1-month median survival compared to 6.8 months with Gemcitabine. Rapid onset of resistance to FOLFIRINOX, coupled with the lack of alternative targeted therapies, ultimately results in patients succumbing to the disease. My project aims to identify mechanisms by which PDAC cells acquire resistance to FOLFIRINOX treatment and investigate interventions aimed at enhancing and/or reinstating FOLFIRINOX sensitivity. Our lab has propagated distinct patient derived xenografts (PDXs) from the Avner Australian Pancreatic Cancer Matrix Atlas (APMA) and I have exposed them to 11 - 15 rounds of FOLFIRINOX (or vehicle control) in vivo to reflect the clinical management of PDAC. Additionally, intrapancreatic orthotopic tumors derived from cells from the KPC mouse model (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) have received 5 - 6 rounds of FOLFIRINOX (or vehicle control). These tumors will undergo quantitative data-independent acquisition mass spectrometry proteomic analysis and RNA sequencing transcriptomic analysis (RNAseq) to reveal resistance pathways and/or proteins operating in the chronically treated tumors and the surrounding pancreatic tumor microenvironment. Additionally, we have developed and cultured matched treatment naïve and FOLFIRINOX-treated patient-derived cell lines (PDCLs) and mouse cell lines from the PDX and KPC-derived tumors. These lines will allow us to validate future targets via gene knockdown and/or pharmacological intervention. 3D assays, such as our organotypic matrix models, will be performed to assess the metastatic potential of cancer cells and their response to FOLFIRINOX in vitro. Furthermore, subcutaneous and orthotopic (intrapancreatic) models coupled with intravital (in vivo) imaging and biosensor technology will allow us to assess targeting in live tumors and metastatic sites in real time. It is the hope that this research will lead to the identification of proteins or protein pathways which can be manipulated in order to re-sensitize tumors to FOLFIRINOX chemotherapy. Citation Format: Victoria Tyma, Ying-Fei Liew, Alice Tran, Anna Howell, Shona Ritchie, Avner Australian Pancreatic Cancer Matrix Atlas, Australian Pancreatic Genome Initiative, Thomas Cox, Marina Pajic, David Herrmann, Brooke Pereira, Paul Timpson, Katie Gordon. Identifying and targeting FOLFIRINOX resistance mechanisms to improve outcomes in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A067.

Abstract B004: Targeting Metabolic Vulnerabilities in Pancreatic Cancer: The Synergistic Anti- Tumor Effects of a Ketogenic Diet and IDH1 Inhibition

Abstract Background: A ketogenic diet, characterized by high fat (90% kCal) and low carbohydrates (5% kCal), induces ketosis. Previous research suggests that a ketogenic diet shifts cancer cell metabolism from glycolysis to the tricarboxylic acid (TCA) cycle in the mitochondria. While the overall impact on cancer growth remains uncertain, most studies suggest an anti-cancer effect. We hypothesize that understanding the molecular and biochemical adaptations of pancreatic cancer cells to a ketogenic diet will reveal metabolic vulnerabilities and lead to rational therapeutic strategies that complement the diet. Methods: Mice were fed a ketogenic diet, with control mice on a normal diet. Pancreatic cancer xenografts were monitored for growth and subjected to biochemical analyses. Tumor metabolites were measured using LC/GC-MS, and oxidative stress was assessed by NADP+/NADPH, NAD+/NADH levels, and lipid peroxidation assays. Enzyme expression was determined by Western blot. In vitro, mitochondrial function was analyzed using the Seahorse FX Analyzer, and cell growth was measured by PicoGreen DNA quantitation. Results: A ketogenic diet slowed pancreatic cancer growth in multiple in vivo models, including MIA-PaCa2, KPC, and patient-derived xenografts. TCA metabolites increased in xenografts exposed to the ketogenic diet, with a significant upregulation of TCA cycle-associated enzymes. Specifically, the expression of BDH1 (a ketolytic enzyme), IDH1 (supports antioxidant defense and mitochondrial function), and IDH2 (mitochondrial homolog of IDH1) were elevated. Conversely, the glycolytic enzyme G3P was reduced. Additionally, a ketogenic diet induced oxidative stress in pancreatic cancer in vivo. Combining a ketogenic diet with an IDH1 inhibitor impaired antioxidant defense and mitochondrial function, resulting in significantly reduced tumor proliferation compared to either treatment alone. In vitro studies indicated that low glucose and fatty acids primarily drive the reduced proliferation of pancreatic cancer cells on a ketogenic diet, while exogenous ketone bodies are utilized for energy under glucose deprivation, generally promoting tumor growth. Conclusion: A ketogenic diet exerts a strong anti-tumor effect in multiple pancreatic cancer mouse models, likely due to low glucose and increased fatty acid load. The metabolic shift towards an oxidative phosphorylation (OXPHOS) phenotype makes pancreatic cancer cells particularly vulnerable to antioxidant and mitochondrial inhibitors. Citation Format: Omid Hajihassani, Mehrdad Zarei, Soubhi Tahan, Peter Gallagher, William Beegan, John Speers, James Choi, Aya Murray, Alexander Loftus, Helen Cheng, Anusha Mudigonda, Karen Ji, Jonathan Hue, Hallie Graor, Jordan Winter. Targeting Metabolic Vulnerabilities in Pancreatic Cancer: The Synergistic Anti- Tumor Effects of a Ketogenic Diet and IDH1 Inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B004.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C027.

Abstract A027: Enhancing pancreatic cancer chemotherapy through photochemical internalisation

Abstract Introduction Pancreatic cancer is the deadliest common cancer. Its poor prognosis is associated with its complex biology and lack of early detection strategies. Although much progress has been made in finding more effective therapeutic strategies, pancreatic tumours remain difficult to treat. A major reason why chemotherapy is relatively ineffective against this type of cancer is inefficient delivery of drugs to their target sites combined with multidrug resistance. Our aim is to use the technique called Photochemical Internalisation (PCI), a light-triggered intracellular drug delivery method which combines low dose Photodynamic Therapy (PDT) with chemotherapy, to induce efficient cytosolic delivery of therapeutic compounds to their specific subcellular targets. Methodology Treatment outcomes using saporin (a type I ribosome-inactivating protein) and gemcitabine as monotherapies, or in combination with a light-activated photosensitizer (the porphyrin TPPS2a), were thoroughly analysed in both established pancreatic cancer cell lines and patient-derived xenograft (PDX) models. The efficacy of these treatments was assessed in both 2D and 3D tumour models through various cell viability assays, including MTT, crystal violet staining, and neutral red staining. Additionally, molecular techniques such as western blotting, protein arrays, and flow cytometry were employed to determine the effects on cell viability and to elucidate changes in signalling pathways leading to the therapeutic response. Results In this study, we observed minimal cytotoxicity induced by saporin, gemcitabine or TPPS2a + light monotherapies. However, PCI synergistically enhanced saporin and gemcitabine cytotoxicity (p&amp;lt;0.001) using very low concentrations in all tumour models. Moreover, we determined the mechanism of cell death triggered by these protocols. PCI induced endosomal disruption following light excitation and apoptosis leading to caspases 3/7 activation, in a time dependent manner. Conclusions Overall, our findings demonstrate the potential of PCI to enhance the efficacy of cancer chemotherapy for pancreatic cancer using lower doses than in monotherapy and open a new window for future translational studies. By using low doses of light therapy, we have found a promising avenue for future studies that could eventually lead to better treatments for this challenging disease. Citation Format: Maria Rosado, Ismahan Mahamed, Andres Garcia-Sampedro, Sandy MacRoberts, Pål Selbo, Stephen Pereira, Pilar Acedo. Enhancing pancreatic cancer chemotherapy through photochemical internalisation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A027.

Abstract C011: Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression

Abstract Pancreatic ductal adenocarcinoma (PDAC) belongs to the tumors with worst prognosis due to the lack of effective treatment. It was previously demonstrated that combined elimination of EGFR and RAF1, two main mediators of the KRAS signaling, induces complete regression of a limited subset of small tumors in a genetically engineered mouse model (Blasco et al., 2019). In the current study, we show that resistance of PDACs with big sizes to the elimination of RAF1 and EGFR is mediated by STAT3. Indeed, combined genetic ablation of EGFR, RAF1 and STAT3 both in vitro and in vivo, results in complete and irreversible regression of more than 40 independent mouse PDAC tumors driven by Kras/Trp53 mutations. Importantly, all PDACs were characterized by increased initial tumor size and highly heterogeneous molecular profile similar to that of Basal human tumors. Moreover, we have validated the efficacy of this novel therapeutic strategy using pharmacological agents, such as EGFR inhibitors approved for clinical use and the STAT3 PROTAC degrader SD-36. Finally, inhibition of RAF1, EGFR and STAT3 expression effectively blocked in vitro and in vivo tumor progression in ten independent patient-derived organoid (PDO) and patient-derived xenograft (PDX) cultures carrying KRAS and TP53 mutations. These results open the door to the development of novel targeted therapies for PDAC patients. Citation Format: Vasiliki Liaki, Mariano Barbacid, Carmen Guerra. Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C011.

Abstract B033: MUC16 - Potential Target for Antibody-based Immunotherapy in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most devastating and lethal cancer. PDAC patients with the current standard of care produce dismal overall survival benefits with high toxicities. Thus, there is an unmet clinical need to develop targeted therapeutics against PDAC. Approximately 50 to 70% of PDAC patients overexpress MUC16, which is associated with disease progression, metastasis, drug resistance, and poor patient survival. We demonstrated that MUC16 enhances PDAC tumor growth via binding and activating epidermal growth factor (EGF) receptor family members and its downstream target PI3K/AKT signaling pathways. Here, we show the efficacy of humanized anti-MUC16 antibody (huAR9.6) against MUC16-positive PDAC. Methods: We tested the in vitro efficacy of humanized anti-MUC16 antibody (huAR9.6) against MUC16+ PDAC cells, patient-derived xenografts (PDXs), and patient-derived organoids (PDOs). We evaluated the in vivo therapeutic efficacy of the huAR9.6 antibody in MUC16+ PDAC cells implanted in subcutaneous and orthotopic tumor models. Fucosylated and afucosylated huAR9.6 induced direct tumor-killing activity was evaluated by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with MUC16 positive PDAC cells. Results: Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16+ PDAC cells and patient-derived organoids (PDOs). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC, PDOs, and PDX-derived cells. HuAR9.6 treatment induces ADCC and CDC against MUC16+ cells. Removing fucose residue on the N-glycans of the huAR9.6-IgG substantially increased huAR9.6’s ADCC activity. More importantly, huAR9.6 treatment caused PDAC regression in subcutaneous and orthotopic tumor models. Conclusion: The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs (detected in ∼ 50-70% of patients). Citation Format: Cory L Brooks, Prakash Radhakrishnan. MUC16 - Potential Target for Antibody-based Immunotherapy in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B033.

Abstract C025: A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer

Abstract BACKGROUND: The absence of symptoms often leads to late diagnosis of pancreatic ductal adenocarcinoma (PDAC) limiting treatment options. Tumor cell proliferation and invasion/metastasis are usually driven by KRAS mutations and activation of Wnt/β-catenin pathway components. The recent approval of KRASG12C-specific inhibitors for lung cancer has validated RAS as a druggable target but has had limited application for PDAC given the rarity of this codon in PDAC. In addition, no Wnt/β-catenin inhibitors have been FDA approved. Thus, drugs which inhibit these important targets are urgently needed for PDAC and other cancers. Here, we describe treatment results with ADT-030, a novel dual pan-RAS/β-catenin inhibitor in murine models of PDAC and lung cancer. METHODS: In vitro assays of growth inhibition, colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were performed to assess potency and selectivity of ADT-030. Murine 2838c3-Luc KRASG12D PDAC cells were implanted orthotopically into the pancreas of C57BL/6J mice and cells from two PDAC patient-derived xenograft (PDX) models harboring KRASG12D or KRASG12C mutations were implanted subcutaneously (SC) in NSG mice to evaluate the antitumor activity of ADT-030. ADT-030 was administered once daily by gavage 5x/week for 4 weeks. RESULTS: ADT-030 potently inhibited the growth of PDAC cells in vitro harboring the most prevalent KRASG12D mutation as well as those with KRASG12C. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using propidium iodide staining revealed that ADT-030 arrested cells in the G2/M phase. Other experiments showed that ADT-030 simultaneously suppressed both MAPK/AKT signaling and β-catenin transcriptional by inhibiting PDE10 and activating cGMP/PKG signaling. ADT-030 caused tumor regression in SC PDAC PDX mouse tumor models harboring either KRASG12D or KRASG12C mutations without discernable toxicity. Inhibition of orthotopic PDAC tumor growth by ADT-030 was dose-dependent, causing up to 80% reduction in tumor volume following a 4-week treatment period. In addition, ADT-030 significantly extended survival and yielded a high percentage of cures after stopping treatment in a murine model of KRAS- mutant lung cancer. CONCLUSION: ADT-030, novel dual pan-RAS/β-catenin inhibitor, showed strong and durable antitumor activity in clinically relevant and aggressive murine models of PDAC and lung cancer. ADT-030 is a promising development candidate for treatment of PDAC and other RAS-driven cancers, particularly given the complex KRAS mutation landscape of many cancers. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C025.

Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma

BackgroundMacrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies.MethodsIn this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT.ResultsMacrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME.ConclusionsIn summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

Overcoming Osimertinib Resistance with AKT Inhibition in EGFRm-Driven Non-Small Cell Lung Cancer with PIK3CA/PTEN Alterations.

Osimertinib is an EGFR tyrosine kinase inhibitor indicated for the treatment of EGFR-mutated (EGFRm)-driven lung adenocarcinomas. Osimertinib significantly improves progression-free survival in first-line-treated patients with EGFRm advanced non-small cell lung cancer (NSCLC). Despite the durable disease control, the majority of patients receiving osimertinib eventually develop disease progression. ctDNA profiling analysis of on-progression plasma samples from patients treated with osimertinib in both first- (phase III, FLAURA trial) and second-line trials (phase III, AURA3 trial) revealed a high prevalence of PIK3CA/AKT/PTEN alterations. In vitro and in vivo evidence using CRISPR-engineered NSCLC cell lines and patient-derived xenograft (PDX) models supports a functional role for PIK3CA and PTEN mutations in the development of osimertinib resistance. These alterations are functionally relevant as EGFRm NSCLC cells with engineered PIK3CA/AKT/PTEN alterations develop resistance to osimertinib and can be resensitized by treatment with the combination of osimertinib and the AKT inhibitor capivasertib. Moreover, xenograft and PDX in vivo models with PIK3CA/AKT/PTEN alterations display limited sensitivity to osimertinib relative to models without alterations, and in these double-mutant models, capivasertib and osimertinib combination elicits an improved antitumor effect versus osimertinib alone. Together, this approach offers a potential treatment strategy for patients with EGFRm-driven NSCLC who have a suboptimal response or develop resistance to osimertinib through PIK3CA/AKT/PTEN alterations. See related commentary by Vokes et al., p. 3968.

NVL-655 Is a Selective and Brain-Penetrant Inhibitor of Diverse ALK-Mutant Oncoproteins, Including Lorlatinib-Resistant Compound Mutations.

Three generations of tyrosine kinase inhibitors (TKI) have been approved for anaplastic lymphoma kinase (ALK) fusion-positive non-small cell lung cancer. However, none address the combined need for broad resistance coverage, brain activity, and avoidance of clinically dose-limiting TRK inhibition. NVL-655 is a rationally designed TKI with >50-fold selectivity for ALK over 96% of the kinome tested. In vitro, NVL-655 inhibits diverse ALK fusions, activating alterations, and resistance mutations, showing ≥100-fold improved potency against ALKG1202R single and compound mutations over approved ALK TKIs. In vivo, it induces regression across 12 tumor models, including intracranial and patient-derived xenografts. NVL-655 inhibits ALK over TRK with 22-fold to >874-fold selectivity. These preclinical findings are supported by three case studies from an ongoing first-in-human phase I/II trial of NVL-655 which demonstrate preliminary proof-of-concept clinical activity in heavily pretreated patients with ALK fusion-positive non-small cell lung cancer, including in patients with brain metastases and single or compound ALK resistance mutations. Significance: By combining broad activity against single and compound ALK resistance mutations, brain penetrance, and selectivity, NVL-655 addresses key limitations of currently approved ALK inhibitors and has the potential to represent a distinct advancement as a fourth-generation inhibitor for patients with ALK-driven cancers.

Epigenome reprogramming through H3K27 and H3K4 trimethylation as a resistance mechanism to DNA methylation inhibition in BRAFV600E-mutated colorectal cancer.

BRAFV600E-mutated colorectal cancer (CRC) exhibits a strong correlation with DNA hypermethylation suggesting this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E CRC in vivo. We randomized and treated mice implanted with patient-derived tumor xenografts harboring BRAFV600E mutation with control, 5-azacitidine, vemurafenib (BRAF inhibitor), or the combination. Comprehensive epigenomic profiling was conducted on control and 5-azacitidine-treated tumor samples, including DNA methylation, histone modifications, chromatin accessibility, and gene expression. Combinations of epigenetic agents were explored in preclinical BRAFV600E CRC models. A profound reduction of DNA methylation levels upon 5-azacitidine treatment was confirmed, however, transcriptional repression was not relieved. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon 5-azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around these hypomethylated regions suggesting the involvement of polycomb repressive complex (PRC) activity around the genome with loss of DNA methylation, therefore maintaining the repression of key tumor suppressor genes. Combined inhibition of PRC activity through EZH2 inhibitor with 5-azacitidine treatment additively improved efficacies in BRAFV600E CRC cells. In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E CRC, and simultaneous blockade of DNMT and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated CRC.