Patient-derived Xenograft Research Articles

GDF-15 Predicts Epithelioid Hemangioendothelioma Aggressiveness and Is Downregulated by Sirolimus through ATF4/ATF5 Suppression.

Epithelioid hemangioendothelioma (EHE), an ultra-rare sarcoma, poses therapeutic challenges because of limited efficacy of conventional chemotherapy in advanced cases, necessitating exploration of new treatment avenues and identification of novel aggressive biomarkers. This study aimed at (i) utilizing a patient-derived xenograft model of EHE 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 patient-derived xenograft model and corresponding cell line were established from a patient with advanced EHE, 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 antitumor activity compared with 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 with 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<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 exhibits a strong correlation with DNA hypermethylation, suggesting that this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E colorectal cancer 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 colorectal cancer 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 inhibition with 5-azacitidine treatment additively improved efficacies in BRAFV600E colorectal cancer cells. In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E colorectal cancer, and simultaneous blockade of DNA methyltransferase and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated colorectal cancer.

Dronedarone inhibits the proliferation of esophageal squamous cell carcinoma through the CDK4/CDK6-RB1 axis in vitro and in vivo.

Treatment options for patients with esophageal squamous cell carcinoma (ESCC) often result in poor prognosis and declining health-related quality of life. Screening FDA-approved drugs for cancer chemoprevention is a promising and cost-efficient strategy. Here, we found that dronedarone, an antiarrhythmic drug, could inhibit the proliferation of ESCC cells. Moreover, we conducted phosphorylomics analysis to investigate the mechanism of dronedarone-treated ESCC cells. Through computational docking models and pull-down assays, we demonstrated that dronedarone could directly bind to CDK4 and CDK6 kinases. We also proved that dronedarone effectively inhibited ESCC proliferation by targeting CDK4/CDK6 and blocking the G0/G1 phase through RB1 phosphorylation inhibition by in vitro kinase assays and cell cycle assays. Subsequently, we found that knocking out CDK4 and CDK6 decreased the susceptibility of ESCC cells to dronedarone. Furthermore, dronedarone suppressed the growth of ESCC in patient-derived tumor xenograft models in vivo. Thus, our study demonstrated that dronedarone could be repurposed as a CDK4/6 inhibitor for ESCC chemoprevention.

C-Myc alone is enough to reprogram fibroblasts into functional macrophages

BackgroundMacrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases.MethodsThe composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac.ResultsHere we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models.ConclusionsOur findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

Macroautophagy/autophagy promotes resistance to KRASG12D-targeted therapy through glutathione synthesis

KRASG12D mutation-driven pancreatic ductal adenocarcinoma (PDAC) represents a major challenge in medicine due to late diagnosis and treatment resistance. Here, we report that macroautophagy (hereafter autophagy), a cellular degradation and recycling process, contributes to acquired resistance against novel KRASG12D-targeted therapy. The KRASG12D protein inhibitor MRTX1133 induces autophagy in KRASG12D-mutated PDAC cells by blocking MTOR activity, and increased autophagic flux prevents apoptosis. Mechanistically, autophagy facilitates the generation of glutamic acid, cysteine, and glycine for glutathione synthesis. Increased glutathione levels reduce reactive oxygen species production, which impedes CYCS translocation from mitochondria to the cytosol, ultimately preventing the formation of the APAF1 apoptosome. Consequently, genetic interventions (utilizing ATG5 or BECN1 knockout) or pharmacological inhibition of autophagy (with chloroquine, bafilomycin A1, or spautin-1) enhance the anticancer activity of MRTX1133 in vitro and in various animal models (subcutaneous, patient-derived xenograft, and orthotopic). Moreover, the release of histones by apoptotic cells triggers an adaptive immune response when combining an autophagy inhibitor with MRTX1133 in immunocompetent mice. These findings establish a new strategy to overcome KRASG12D-targeted therapy resistance by inhibiting autophagy-dependent glutathione synthesis.

Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy

BackgroundPediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient...

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

SNHG14 promotes triple-negative breast cancer cell proliferation, invasion, and chemoresistance by regulating the ERK/MAPK signaling pathway.

The functional role and molecular mechanisms of small-nucleolar RNA host gene 14 (SNHG14) in triple-negative breast cancer (TNBC) progression remain unclear. The expression levels of SNHG14 in breast cancer samples and cell lines were determined using real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion abilities were detected using MTS and transwell assays. By RNA sequencing, differentially expressed genes were identified between the SNHG14 siRNA and the negative control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to predict the targets and pathways regulated by SNHG14. pRAF, pMEK, and pERK expression were measured by western blot. The xenograft model was constructed to access the biological function of SNHG14 in vivo. A minimal patient-derived xenograft model was established to evaluate the sensitivity to chemotherapy drugs. Our data indicated that SNHG14 expression was increased in TNBC tissues and cell lines. SNHG14 knockdown attenuated the proliferation, migration, and invasion abilities of TNBC cells both in vivo and in vitro. High SNHG14 expression was associated with lymph node metastasis and a high Ki67 index. The targets of SNHG14 were mainly enriched in the MAPK signaling pathway. pRAF, pMEK, and pERK expression were downregulated after being transfected with SNHG14 siRNA. Compared with the negative control group, the expression of CACNA1I, DUSP8, FGF17, FGFR4, FOS, PDGFRB, and DDIT3 was increased, and the expression of MKNK1 was decreased in the SNHG14 siRNA group. Minimal patient-derived xenograft model demonstrated that knockdown of SNHG14 enhanced the sensitivity to Docetaxel in vivo. Compared with the DMSO group, the proliferation of Docetaxel-resistant MDA-MB-231 cells was decreased in Dabrafenib, PD184352, and FR180204 treatment groups. SNHG14 knockdown inhibits TNBC progression by regulating the ERK/MAPK signaling pathway, which provides evidence for SNHG14 as a potential target for TNBC therapy.

Cancer Patient-Derived Cell-Based Models: Applications and Challenges in Functional Precision Medicine.

The field of oncology has witnessed remarkable progress in personalized cancer therapy. Functional precision medicine has emerged as a promising avenue for achieving superior treatment outcomes by integrating omics profiling and sensitivity testing of patient-derived cancer cells. This review paper provides an in-depth analysis of the evolution of cancer-directed drugs, resistance mechanisms, and the role of functional precision medicine platforms in revolutionizing individualized treatment strategies. Using two-dimensional (2D) and three-dimensional (3D) cell cultures, patient-derived xenograft (PDX) models, and advanced functional assays has significantly improved our understanding of tumor behavior and drug response. This progress will lead to identifying more effective treatments for more patients. Considering the limited eligibility of patients based on a genome-targeted approach for receiving targeted therapy, functional precision medicine provides unprecedented opportunities for customizing medical interventions according to individual patient traits and individual drug responses. This review delineates the current landscape, explores limitations, and presents future perspectives to inspire ongoing advancements in functional precision medicine for personalized cancer therapy.

Analyzing the distribution of progression-free survival for combination therapies: A study of model-based translational predictive methods in oncology

Progression-free survival (PFS) is an important clinical metric in oncology and is typically illustrated and evaluated using a survival function. The survival function is often estimated post-hoc using the Kaplan-Meier estimator but more sophisticated techniques, such as population modeling using the nonlinear mixed-effects framework, also exist and are used for predictions. However, depending on the choice of population model PFS will follow different distributions both quantitatively and qualitatively. Hence the choice of model will also affect the predictions of the survival curves.In this paper, we analyze the distribution of PFS for a frequently used tumor growth inhibition model with and without drug-resistance and highlight the translational implications of this. Moreover, we explore and compare how the PFS distribution for combination therapy differs under the hypotheses of additive and independent-drug action.Furthermore, we calibrate the model to preclinical data and use a previously calibrated clinical model to show that our analytical conclusions are applicable to real-world setting. Finally, we demonstrate that independent-drug action can effectively describe the tumor dynamics of patient-derived xenografts (PDXs) given certain drug combinations.