Pancreatic Cancer Xenograft Model Research Articles

O-P03 A composite polymeric nanoparticle as a sensitiser for sonodynamic therapy (SDT)-based treatment of pancreatic cancer

Abstract Background Pancreatic cancer remains one of the most recalcitrant forms of cancer with poor prognosis and limited treatment options. SDT is a novel, targeted approach to the treatment of solid tumours. Based on the generation of cytotoxic reactive oxygen species (ROS) following the exposure of a sonosensitiser to ultrasound, the approach is designed to extracorporeally target less accessible lesions. Here we describe the production of a poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), Rose Bengal (RB) and indocyanine green (ICG) containing composite nanoparticles and describe their use in SDT-mediated treatment of pancreatic cancer using both in vitro and in vivo target models. Methods Nanoparticles were prepared using an oil in water emulsion and solvent diffusion-based approach. These were designated RB-ICGNP. In vitro SDT treatment consisted of exposing BxPC3 (human PDAC cells), T110029 (murine PDAC cells) or hPSC (immortalised human pancreatic stellate cells) to RB-ICGNP and subsequently treating with ultrasound for 30 s at a frequency of 1 MHz, a power density of 3.0 W/cm2 (SATP) using a duty cycle of 50% at a pulse repetition frequency of 100 Hz. For in vivo studies, BxPC3 (xenograft) and T110029 (syngeneic) tumours were treated with a power density of 3.5 W/cm2 ultrasound for 3.5 min. Results Conclusions Using in vitro and in vivo (human xenograft and murine syngeneic) models of pancreatic cancer, RB-ICGNP composite nanoparticles may be employed as a sensitiser for SDT-based treatment of pancreatic cancer. Since pancreatic stellate cells were more sensitive to SDT, the latter may have an impact on tumour stroma. Staining of residual tumour tissues from SDT-treated animals for connective tissue (stroma) confirmed the latter. Since tumour stroma presents a significant challenge to treatment of pancreatic cancer and represents a negative prognostic marker, the impact delivered by SDT may be exploited to potentiate alternative therapeutic approaches.

ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody.

Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Abstract P240: Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (<1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials & Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P240.

Exosomal lncRNA UCA1 Derived From Pancreatic Stellate Cells Promotes Gemcitabine Resistance in Pancreatic Cancer via the SOCS3/EZH2 Axis.

ObjectivePancreatic cancer is associated with poor prognosis and dismal survival rates. This study aims to investigate roles of lncRNA UCA1-loaded exosomes secreted by pancreatic stellate cells (PSCs) in Gemcitabine (Gem) resistance of pancreatic cancer under hypoxia, which involves the methylation of SOCS3 and EZH2 recruitment.MethodsThe exosomes were isolated from PSCs and hypoxic PSCs (HPSCs), and co-cultured with pancreatic cancer cells transduced with manipulated lncRNA UCA1, EZH2, and SOCS3. The interaction among lncRNA UCA1, EZH2, and SOCS3 was characterized by RIP and ChIP assays. Next, MTT assay, flow cytometry and TUNEL staining and Transwell assay were used to detect cell viability, apoptosis, invasion, and migration. Gem-resistant pancreatic cancer cell line (GemMIA-R3) was established, which was applied in a mouse xenograft model of pancreatic cancer, with MTT assay to determine Gem sensitivity.ResultsLncRNA UCA1 was highly expressed, while SOCS3 was poorly expressed in pancreatic cancer tissues. Hypoxia induced activation of PSCs and promoted release of exosomes. LncRNA UCA1 delivered by hypoxic PSC-derived exosomes (HPSC-EXO) regulated histone methylation level in SOCS3 gene region through recruitment of EZH2. In vitro and in vivo experimental results confirmed that lncRNA UCA1-loaded HPSC-EXO promoted malignant phenotypes, inhibited apoptosis, and promoted Gem resistance of pancreatic cancer cells as well as tumorigenesis in mice.ConclusionUnder hypoxic conditions, exosomes secreted by hypoxia-induced PSCs deliver lncRNA UCA1 into pancreatic cancer cells, where lncRNA UCA1 recruits EZH2 and regulates histone methylation level in SOCS3 gene region, thereby augmenting pancreatic cancer resistance to Gem.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

Simple SummaryA feature of pancreatic cancer (PC) is the frequent overexpression of tyrosine kinase membrane receptor HER2 along with its membrane partner the MUC4 oncomucin in the early stages of the pancreatic carcinogenesis. However, therapeutic approaches targeting HER2 in PC are not efficient. MUC4 could indeed represent an alternative therapeutic strategy to target HER2 signaling pathway, but this approach needs to characterize MUC4/HER2 interaction at the molecular level. In this study, we successfully showed the impact of the EGF domains of MUC4 on HER2 binding affinity and demonstrated their “growth factor-like” biological activities in PC cells. Moreover, homology models of the MUC4EGF/HER2 complexes allowed identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results allow a better understanding of the mechanisms involved in the MUC4/HER2 complex formation and may lead to the design of potential MUC4/HER2 inhibitors.The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their “growth factor-like” biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and β-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

Inhibition of JAK2/STAT3 signaling pathway by panaxadiol limits the progression of pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related death with the characteristics of chemoresistance and early metastasis. Panaxadiol, a triterpenoid saponin extracted from the roots of American ginseng, has been proved to display anti-tumor activity in colon cancer. In this study, we found panaxadiol significantly inhibited proliferation, and induced apoptosis in human pancreatic cancer cell lines PANC-1 and Patu8988 in a dose-dependent manner. Furthermore, the expression of apoptosis-related proteins (Bax, Bcl2, Cleaved-caspase3) was detected via western blot and immunofluorescence staining. In addition, panaxadiol was also found to inhibit the migration of pancreatic cancer cells by wound healing and transwell assays. In vivo, the growth of xenograft pancreatic cancer models was also notably suppressed by panaxadiol compared to the control group. Moreover, the down-regulation of JAK2-STAT3 signaling pathway was responsible for the underlying pro-apoptosis mechanism of panaxadiol, and this result was in good agreement with molecular docking analysis between panaxadiol and STAT3. In conclusion, our work comprehensively explored the anti-tumor ability in PANC-1 and Patu8988 cells of panaxadiol and provided a potential choice for the clinical treatment of pancreatic cancer patients.

Possible Role of Cytochrome P450 1B1 in the Mechanism of Gemcitabine Resistance in Pancreatic Cancer.

Patient-derived xenograft models reportedly represent original tumor morphology and gene mutation profiles. In addition, patient-derived xenografts are expected to recapitulate the parental tumor drug responses. In this study, we analyzed the pathways involved in gemcitabine resistance using patient-derived xenograft models of pancreatic cancer. The patient-derived xenograft models were established using samples from patients with pancreatic cancer. The models were treated with gemcitabine to better understand the mechanism of resistance to this anti-cancer drug. We performed comparative gene analysis through the next-generation sequencing of tumor tissues from gemcitabine-treated or non-treated patient-derived xenograft mice and gene set enrichment analysis to analyze mRNA profiling data. Pathway analysis of gemcitabine-treated patient-derived xenografts disclosed the upregulation of multiple gene sets and identified several specific gene pathways that could potentially be related to gemcitabine resistance in pancreatic cancer. Further, we conducted an in vitro analysis to validate these results. The mRNA expression of cytochrome P450 1B1 and cytochrome P450 2A6 was upregulated in a concentration-dependent manner following gemcitabine treatment. Moreover, the sensitivity to gemcitabine increased, and viable cells were decreased by the cytochrome P450 1B1 inhibitor, indicating that the cytochrome P450 1B1 pathway may be related to gemcitabine resistance in pancreatic cancer.

Pancreatic cancer cell-derived microRNA-155-5p-containing extracellular vesicles promote immune evasion by triggering EHF-dependent activation of Akt/NF-κB signaling pathway

Pancreatic cancer (PC)-derived EVs have been extensively investigated due to their promising potential as disease biomarkers for diagnosis, monitoring, and treatment decisionmaking. Herein, we explored the mechanism underlying PC-derived EVs in immune evasion of PC. Initially, microRNA (miR)-155-5p level was quantified by RT-qPCR in tumor tissue samples from PC patients, EVs isolated from PC cell lines and PC cell lines. Then, the interaction between miR-155-5p and EHF was identified using dual-luciferase reporter assay. Ectopic expression and knockdown experiments were conducted in PC cells, PC cells-derived EVs, or mouse xenograft model of PC. Afterwards, cell invasion, proportion of macrophage and immune cell subsets, and expression of NF-κB signaling-related genes were assessed using Transwell assay, flow cytometry, RT-qPCR and western blot analysis, respectively. Accordingly, miR-155-5p was upregulated in clinical tissue samples, Pan02-derived EVs and PC cell lines. miR-155-5p knockdown in PC cells enhanced anti-tumor immunity. PC cell-derived EVs facilitated immunosuppressive microenvironment by promoting T cell depletion. In addition, PC cell-derived EVs transferred miR-155-5p to macrophages and then promoted polarization of macrophages to M2 phenotype. EHF was downregulated in PC and could be targeted by miR-155-5p, which resulted in the activation of the Akt/NF-κB signaling. Our findings revealed a previously unrecognized tumor immune evasion-promoting function of PC-derived EV miR-155-5p in PC development by suppressing EHF and activating NF-κB signaling. This study suggested that the miR-155-5p/EHF/Akt/NF-κB axis can be exploited to prevent cancer immune evasion triggered by therapies.

A Histone Deacetylase Inhibitor, Panobinostat, Enhances Chimeric Antigen Receptor T-cell Antitumor Effect Against Pancreatic Cancer.

In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model. We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano). The combination treatment enabled significant suppression of Her2+ pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice. We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.

Nanoprobe-Based Magnetic Resonance Imaging of Hypoxia Predicts Responses to Radiotherapy, Immunotherapy, and Sensitizing Treatments in Pancreatic Tumors.

Accurate diagnosis of tumors and predicting the therapeutic responses are highly demanded in the clinic to improve the treatment efficacy and survival rates. Since hypoxia develops in the progression of tumors and inversely correlates with prognosis and promotes resistance to radiotherapies and immunotherapies, it is a potential marker for therapeutic prediction. Therefore, effective discrimination of tumor hypoxia for predicting therapeutic outcomes is critical. Here, a magnetic resonance imaging (MRI)-based diagnosis strategy using contrast-amplifying nanoprobes that sense tumor acidosis and real-time observation of hypoxic conditions in tumors has been developed, aiming at accurate detection of pancreatic tumors and prediction of therapeutic effects. Our approach selectively probed xenograft, allograft, and transgenic spontaneous models of intractable pancreatic cancer, which lacks standardized predictive markers to identify patients who benefit most from treatments, and effectively discriminated the intratumoral hypoxia levels. By stratification of pancreatic tumors based on quantitative MR imaging of hypoxia, it enabled prediction of the responses to radiotherapy and immune checkpoint inhibitors. Moreover, the nanoprobe-based MRI could monitor hypoxia reduction by tumor normalization treatments, which permits visualizing pancreatic tumors that will respond to immune checkpoint blockade therapy, enhancing the response rate. The results demonstrate the potential of our strategy for accurate tumor diagnosis, patient stratification, and effective therapy.

A calcium phosphate drug carrier loading with 5-fluorouracil achieving a synergistic effect for pancreatic cancer therapy

Calcium based biomaterials were widely used for drug delivery application due to their biodegradability, biocompatibility, and high drug loading capacity. Herein, amino-capped polyamidoamine (PAMAM) dendrimer was applied as a macromolecular template to form amino-modified calcium phosphate hollow sphere (CaPO-NH2). After loading with 5-fluorouracil (5Fu), this system performed synergistic cancer chemotherapy. In this study, the 5Fu/CaPO-NH2 particles could be efficiently uptaken by cancer cells, and then decompose into Ca2+ and release 5Fu drug in the cytoplasm; therefore calcium overload and reactive oxygen species (ROS) accumulation were found in PSN1 cells that could induce cell membrane damage and elicit cell apoptosis through a series of biochemical reactions including endoplasmic reticulum stress, lipid peroxidation and mitochondrial apoptosis. In the PSN1 pancreatic cancer xenograft model, the 5Fu/CaPO-NH2 system performed high tumor inhibition via chemotherapy and calcium overload induced apoptosis. Comparingly, the normal cells and organs were insensitive to this synergistic therapy, which indicated the well biocompatibility of delivery system. Thus, this study provided a promising CaPO-NH2 drug delivery platform for enhanced 5Fu chemotherapeutic effect.

Abstract LB106: The EGF domains of MUC4 oncomucin interact with ErbB2 and mediate tumorigenic activity of cancer cells represent new potential therapeutic targets

Abstract The MUC4 mucin and its membrane partner ErbB2 form an oncogenic complex at the surface of epithelial cancer cells that promotes tumor progression. They directly physically interact via an extracellular region of MUC4 encompassing three EGF domains opening the route to therapeutic targeting of ErbB2 over-expressing cancers in which MUC4-ErbB2 is present. However, before small inhibitory molecules may be designed, it is mandatory (i) to decipher at the molecular level which amino acid regions are involved in the interaction and (ii) show that they mediate cancer cell tumorigenicity. By using in vitro (2D cell culture, Micro Scale Thermophoresis, PLA assay, Co-IP, GST pull down, Molecular Dynamics simulations) and in vivo (xenograft model of pancreatic cancer) approaches, we show that the EGF1 domain of MUC4 is central both in the interaction with ErbB2 and in the activation of cell proliferation and migration and tumor growth via activation of key oncogenic pathways (Src, Ras, p70S6K/AKT/mTOR, β-catenin, FAK). Finally, we have identified hotspots of interaction paving the way to the design of therapeutic inhibitory molecules targeting the EGF domains of MUC4 which may represent a new alternative strategy to overcome ErbB2 therapeutic failure in cancer. Citation Format: Nicolas STOUP, Maxime LIBERELLE, Céline SCHULZ, Romain VASSEUR, Romain MAGNEZ, Xavier THURU, Patricia MELNYK, Nicolas RENAULT, Nicolas JONCKHEERE, Nicolas LEBEGUE, Isabelle VAN SEUNINGEN. The EGF domains of MUC4 oncomucin interact with ErbB2 and mediate tumorigenic activity of cancer cells represent new potential therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB106.

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Abstract 957: Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2

Abstract HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The HER2/HER3 complex is most active in the presence of heregulin (HRG) and upregulation of HRG is one of the key resistance mechanisms after TKI treatment. Zenocutuzumab is an HER2/HER3 bispecific antibody that specifically and potently blocks HRG-driven signaling of the heterodimeric complex. Zenocutuzumab has demonstrated clinical activity in patients with advanced cancer where the HRG-driven HER2/HER3 pathway is active [Ref].Zenocutuzumab was discovered through a large unbiased screen with HER2×HER3 Biclonics® (bAbs). Zenocutuzumab specifically blocks HRG-induced signaling of HER2:HER3 but not of HER2:HER4, EGFR:HER2 or EGFR:HER3 heterodimers. Zenocutuzumab demonstrates an in vitro potency superior to other high affinity anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of HRG, thereby overcoming one of the resistance mechanisms of current HER2 therapies. The potent activity of Zenocutuzumab is based in-part on its binding to domain I of HER2. Docking of Zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm. As a result, Zenocutuzumab displaces HRG binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HRG-mediated HER2/HER3 signaling. In binding experiments on cells expressing different levels of HER2 and HER3, HER2:HER3 ratios point to the anti-HER2 Fab binding arm being the main driver of Zenocutuzumab avidity. In an in vivo orthotopic breast cancer xenograft model that used Micro-PET imaging, the tumor penetration of a variant of Zenocutuzumab is HER2-dependent despite the high subnanomolar affinity of the HER3 Fab arm for its receptor. Gamma-counter quantification of radioactivity confirmed that antibody levels in the tumors with the HER2xHER3 bAb were 2.5-fold higher than for the parental HER3 mAb. Zenocutuzumab reduced tumor growth in an in vivo orthotopic pancreatic cancer xenograft model that expresses HRG. The brain is one of the organs with high HRG concentrations to which many tumors metastasize. Mice bearing intracranial HER2-amplified breast cancer cells presented at the end of the treatment period 100% survival when treated with MCLA-128, in contrast to 38% survival when treated with HER2 antibody drug conjugate T-DM1.Conclusion: Zenocutuzumab uses a Dock & Block™ mechanism for potent and specific tumor targeting and simultaneous inhibition of HRG-driven proliferation of HER2-amplified tumors, even at supramaximal concentrations of HRG. Citation Format: Cecile Geuijen, David Maussang-Detaille, Tristan Gallenne, Linda Hendriks, Arjen Kramer, Jan Gerlach, Mark Throsby, John de Kruif. Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 957.

Abstract 1271: In vitro and in vivo characterization of BI 1823911 - a novel KRASG12C selective small molecule inhibitor

Abstract KRASG12C mutations are predominantly found in non-small cell lung cancer (NSCLC, 13%), in colorectal cancer (CRC, 3%), and with a lower prevalence in pancreatic ductal adenocarcinoma (PDAC, 1%). The amino acid exchange at position 12 from glycine to cysteine renders RAS insensitive to GAP-catalyzed hydrolysis but not to intrinsic hydrolysis and consequently, KRASG12C is still dependent on GEF stimulation to achieve full activation. The active GTP-loaded form of KRASG12C is favored and leads to activation of downstream signaling and proliferation. A number of recent publications has shown that targeting this mutant form of KRAS, using several covalent KRASG12C inhibitors binding to the inactive GDP-KRASG12C form, leads to anti-proliferative effects and induction of apoptosis in KRASG12C mutant cancer cell lines, CDX and PDX models. Early clinical data for AMG 510 and MRTX849 revealed a response rate of 35-45% in NSCLC and of 7-17% in CRC patients. Here, we show that BI 1823911 has potent anti-proliferative activity in a panel of KRASG12C mutant cancer cell lines with higher or similar potency compared to these two most advanced compounds in clinical development. In a panel of KRASG12C NSCLC cell lines, treatment with BI 1823911 results in downregulation of MAPK pathway-responsive genes, such as DUSP6 and CCND1, and the extent of pathway modulation correlates with sensitivity. Likewise, we observe strong and sustained inactivation of the MAPK pathway at the protein level using p-ERK as a pharmacodynamic (PD) biomarker. A MIA PaCa-2 cell line-derived pancreatic cancer xenograft model was selected for extensive PK/PD/efficacy analyses in vivo. Briefly, BI 1823911 tested at 60 mg/kg showed similar anti-tumor activity compared to both competitor compounds dosed at clinically relevant exposures. Results of the ongoing in-depth PK/PD/efficacy analysis will be shared. Furthermore, BI 1823911 was tested with a daily oral dose of 60 mg/kg in a panel of NSCLC or CRC CDX or PDX mouse models and showed comparable efficacy to AMG 510 and MRTX849, respectively. Preclinical and clinical data suggest that monotherapy with a KRASG12C inhibitor will not be sufficient to achieve durable response. Combination therapy of a KRASG12C inhibitor may therefore lead to enhanced anti-tumor efficacy and may address adaptive resistance mechanisms. Therefore, we selected a panel of KRASG12C mutant cancer cell lines and tested a large set of compounds in combination with BI 1823911 to identify synergistic anti-proliferative activity. Among other MAPK and PI3K pathway inhibitors, a SOS1::KRAS inhibitor was confirmed as promising combination partner. We will show results from in vitro and in vivo combination studies in NSCLC and CRC tumor models that show deep and durable responses upon combination of BI 1823911 with SOS1::KRAS inhibitor BI 1701963 providing a strong rationale for clinical investigation of this combination. Citation Format: Fabio Savarese, Andreas Gollner, Dorothea Rudolph, Jesse Lipp, Johannes Popow, Marco H. Hofmann, Heribert Arnhof, Jörg Rinnenthal, Francesca Trapani, Michael Gmachl, Daniel Gerlach, Joachim Broeker, Peter Ettmayer, Andreas Mantoulidis, Jason Phan, Christian A. Smethurst, Matthias Treu, Alex G. Waterson, Hengyu Lu, Annette Machado, Joseph Daniele, Stephan W. Fesik, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek, Darryl B. McConnell, Mark Petronczki, Norbert Kraut, Irene C. Waizenegger. In vitro and in vivo characterization of BI 1823911 - a novel KRASG12C selective small molecule inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1271.

A Liposomal Gemcitabine, FF-10832, Improves Plasma Stability, Tumor Targeting, and Antitumor Efficacy of Gemcitabine in Pancreatic Cancer Xenograft Models

PurposeThe clinical application of gemcitabine (GEM) is limited by its pharmacokinetic properties. The aim of this study was to characterize the stability in circulating plasma, tumor targeting, and payload release of liposome-encapsulated GEM, FF-10832.MethodsAntitumor activity was assessed in xenograft mouse models of human pancreatic cancer. The pharmacokinetics of GEM and its active metabolite dFdCTP were also evaluated.ResultsIn mice with Capan-1 tumors, the dose-normalized areas under the curve (AUCs) after FF-10832 administration in plasma and tumor were 672 and 1047 times higher, respectively, than after using unencapsulated GEM. The tumor-to-bone marrow AUC ratio of dFdCTP was approximately eight times higher after FF-10832 administration than after GEM administration. These results indicated that liposomal encapsulation produced long-term stability in circulating plasma and tumor-selective targeting of GEM. In mice with Capan-1, SUIT-2, and BxPC-3 tumors, FF-10832 had better antitumor activity and tolerability than GEM. Internalization of FF-10832 in tumor-associated macrophages (TAMs) was revealed by flow cytometry and confocal laser scanning microscopy, and GEM was efficiently released from isolated macrophages of mice treated with FF-10832. These results suggest that TAMs are one of the potential reservoirs of GEM in tumors.ConclusionThis study found that FF-10832 had favorable pharmacokinetic properties. The liposomal formulation was more effective and tolerable than unencapsulated GEM in mouse xenograft tumor models. Hence, FF-10832 is a promising candidate for the treatment of pancreatic cancer.

The EGF domains of MUC4 oncomucin interact with ErbB2 and mediate tumorigenic activity of cancer cells represent new potential therapeutic targets

The MUC4 mucin and its membrane partner ErbB2 form an oncogenic complex at the surface of epithelial cancer cells that promotes tumor progression. They directly physically interact via an extracellular region of MUC4 encompassing three EGF domains opening the route to therapeutic targeting of ErbB2 over-expressing cancers in which MUC4-ErbB2 is present. However, before small inhibitory molecules may be designed, it is mandatory (i) to decipher at the molecular level which amino acid regions are involved in the interaction and (ii) show that they mediate cancer cell tumorigenicity. By using in vitro (2D cell culture, Micro Scale Thermophoresis, PLA assay, Co-IP, GST pull down, Molecular Dynamics simulations) and in vivo (xenograft model of pancreatic cancer) approaches, we show that the EGF1 domain of MUC4 is central both in the interaction with ErbB2 and in the activation of cell proliferation and migration and tumor growth via activation of key oncogenic pathways (Src, Ras, p70S6K/AKT/mTOR, beta-catenin, FAK). Finally, we have identified hotspots of interaction paving the way to the design of therapeutic inhibitory molecules targeting the EGF domains of MUC4 which may represent a new alternative strategy to overcome ErbB2 therapeutic failure in cancer.

Cancer Nanomedicine Developed From Total Human Serum: A Novel Approach for Making Personalized Nanomedicine

Aim: To develop a method formaking total serum nanoparticles (TSN) loaded with cytotoxic chemodrugs for cancer therapy. Materials & methods: TSN loaded with paclitaxel (PTX) or piperlongumine (PL) were prepared using high-pressure homogenization and tested for immunogenicity in healthy animals and antitumor properties in pancreatic cancer xenograft models. Results: TSN-PL nanoparticles of average size 104nm and encapsulation efficiency approximately50% showed enhanced dose-dependent cytotoxicity compared with TSN-PTX or clinically used combination of gemcitabineand nano-PTX in two pancreatic cell lines. Significant antitumor efficacy was also established in the pancreatic xenograft model. Conclusion: We developed a unique method of converting total blood serum into chemo drug-loaded nanoparticles and demonstrated its efficacy in vitro and in vivo.

Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer

PurposeRadioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines. MethodsThe human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H2DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion. ResultsThe acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines. ConclusionsOur data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype.

Inhibition of bone morphogenetic protein receptor 2 suppresses pancreatic ductal adenocarcinoma growth by regulating GRB2/PI3K/AKT axis.

Bone morphogenetic protein receptor 2 (BMPR2) is an important transmembrane serine/threonine kinase that involves oncogenic processes in multiple cancers. However, the role of BMPR2 and its regulatory mechanism in pancreatic ductal adenocarcinoma (PDAC) remain unknown. We performed a tissue array to explore the expression of BMPR2 in PDAC tissues. The Cell Counting Kit-8 (CCK-8) and colony formation assays were used to measure PDAC cells' proliferation. Proteomics and mass spectrometry technology was applied to analyze the BMPR2-regulating proteins. Flow cytometry was used to analyze the cell cycle distribution of PDAC cells. Orthotopic pancreatic cancer (PC) and patient-derived xenograft (PDX) models were used for in vivo experiments. This study revealed the over-expression of BMPR2 in PDAC tissues and its proliferation-promoting role in PDAC cells. By carrying out protein mass spectrometry technique as well as bioinformatics analysis, we identified that BMPR2 regulated the growth factor receptor-bound protein 2/phosphatidylinositol 3-kinase/protein kinase B (GRB2/PI3K/AKT) signaling pathway, and further in vitro experiments showed that inhibition of BMPR2 resulted in suppressing proliferation and G2/M arrest by inhibiting the GRB2/PI3K/AKT signaling pathway in PDAC cells. The inhibition of BMPR2 by LDN193189 showed similar results in PDAC cells, orthotopic PC, and PDX models, which revealed that inhibition of BMPR2 significantly suppressed tumor growth by suppressing the GRB2/PI3K/AKT axis. Inhibition of BMPR2 suppresses PDAC growth by regulating the GRB2/PI3K/AKT axis and is a promising PDAC treatment strategy.