Significant Decrease In Tumor Growth Research Articles

Deferasirox’s Anti-Chemoresistance and Anti-Metastatic Effect on Non-Small Cell Lung Carcinoma

Clinically approved iron chelators, originally designed to address iron overload disorders, have emerged as potential anticancer agents. Deferasirox (Def), a tridentate iron chelator, has demonstrated antiproliferative effects in cancer. Background/Objectives: This study aims to elucidate the mechanism of action of Def and its impact on non-small cell lung carcinoma (NSCLC). Methods: NSCLC A549 cells were treated with Def to assess cytotoxicity, the effect on nuclear and mitochondrial pathways, and iron-containing proteins and genes to evaluate anti-metastasis and chemoresistance. A lung carcinoma mouse model was used for in vivo studies. Results: Our findings revealed that Def induced cytotoxicity, effectively chelated intracellular iron, and triggered apoptosis through the increase in phosphatidylserine externalization and caspase 3 activity. Additionally, Def caused G0/G1 cell cycle arrest by downregulating the ribonucleotide reductase catalytic subunit. Furthermore, Def perturbed mitochondrial function by promoting the production of reactive oxygen species and the inhibition of glutathione as a measurement of ferroptosis activation. Def demonstrated inhibitory effects on cell migration in scratch assays, which was supported by the upregulation of n-myc downstream-regulated gene 1 and downregulation of the epidermal growth factor receptor protein. Also, Def downregulated one of the main markers of chemoresistance, the ABCB1 gene. In vivo experiments using a lung carcinoma mouse model showed that Def treatment did not affect the animal’s body weight and showed a significant decrease in tumor growth. Conclusions: This investigation lays the groundwork for unraveling Def action’s molecular targets and mechanisms in lung carcinoma, particularly within iron-related pathways, pointing out its anti-metastasis and anti-chemoresistance effect.

Radiosensitizing effects of heparinized magnetic iron oxide nanoparticles in colon cancer

The combination of radiation treatment and chemotherapy is currently the standard for management of cancer patients. However, safe doses do not often provide effective therapy, then pre-treated patients are forced to repeat treatment with often already increased tumor resistance to drugs and irradiation. One of the solutions we suggest is to improve primary course of radiation treatment via enhancing radiosensitivity of tumors by magnetic-guided iron oxide nanoparticles (magnetite). We obtained spherical heparinized iron oxide nanoparticles (hIONPs, ∼20 nm), characterized it by TEM, Infrared spectroscopy and DLS. Then hIONPs cytotoxicity was assessed for colon cancer cells (XTT assay) and cellular uptake of nanoparticles was analyzed with X-ray fluorescence.Combination of ionizing radiation (IR) and hIONPs in vitro caused an increase of G2/M arrest of cell cycle, mitotic errors and decrease in survival (compared with samples exposed to IR and hIONPs separately). The promising results were shown for magnetic-guided hIONPs in CT26-grafted BALB/C mice: the combination of intravenously administrated hIONPs and IR showed 20,8% T/C ratio (related to non-treated mice), while single radiation had no shown significant decrease in tumor growth (72,4%). Non-guided by magnets hIONPs with IR showed 57,9% of T/C. This indicates that ultra-small size and biocompatible molecule are not the key to successful nano-drug design, in each case, delivery technologies need to be improved when transferred to in vivo model.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract 7465: Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract Immune checkpoint blockade revolutionized cancer treatments by activating anti-tumor immunity. Pancreatic ductal adenocarcinoma (PDA) patients have not benefited from these advances due to an immune suppressive tumor microenvironment. Identifying PDA cell-encoded factors that drive immune evasion is necessary to unlock new immunologically active treatment options in PDA. We have recently demonstrated that the cytokine Leukemia inhibitory factor (LIF) is highly upregulated in PDA compared to the normal healthy pancreas. LIF is known for its immune suppressive qualities, but data on LIF in PDA is currently limited. We generated a conditional Lif flox (L) allele and crossed it into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. LKPC mice (n=29) had longer overall survival (69 days) than control KPC mice (n=29) (59 days), (p<0.05). LKPC mice harbored significantly smaller tumors with an average tumor weight of 0.6g compared to KPC control animals with an average tumor weight of 1.0g (p<0.005). Immunohistochemical evaluation of tumor proliferation measured by Ki-67 positivity in LKPC and KPC tumors at endpoint showed comparable results. Tumor-derived cell lines from KPC and LKPC mice showed no growth differences in vitro nor when reimplanted subcutaneously (sc) in immunodeficient mice. In contrast, we observed a significant decrease in tumor growth and weight in LKPC sc tumors (p<0.05) compared to the KPC sc tumors in syngeneic mice. LKPC sc tumors had significantly fewer neutrophils and more macrophages, particularly MHC II-high macrophages, known for their anti-tumor qualities when compared to KPC sc tumors using flow cytometry. A trend towards more CD8+ T-cells in sc LKPC tumors was also observed. Our genetically engineered mouse model confirmed this finding by immunohistochemical evaluation of tumors at endpoint, which demonstrated a significant increase of CD8+ T-cells in the LKPC animals compared to the KPC controls. In summary, we find that cancer-cell-expressed LIF augments tumor growth and shortens overall survival by changing the immune tumor microenvironment, nominating leukemia inhibitory factor depletion as a promising therapeutic immune target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Alexis J. Combes, Shabana Bano, Lisiena Hysenaj, Jeroen P. Roose, Adam Olshen, Ashley Kiemen, Sarah Umetsu, Man-Tzu Wang, Eric A. Collisson. Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7465.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Abstract 5742: Therapeutic efficacy of CA102N in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer to treat due to its aggressive nature and late-stage diagnosis. Considering the lack of effective therapies, there is an urgent need to develop new and effective therapeutics to improve the prognosis of patients. CA102N is a carrier-mediated novel anticancer product where hyaluronic acid is conjugated to Nimesulide-NH2. CA102N is currently under development for the treatment of solid tumors, including colorectal cancer. CA102N was safe and well tolerated in the first-in-human trial in patients with locally advanced or metastatic solid tumors. In the present study, the in vitro anticancer activity of CA102N was investigated in K-ras mutant PANC-1 pancreatic cancer cells. The antitumor efficacy was also tested in vivo in the PANC-1 mice model. Results demonstrated that CA102N inhibits cell growth, cell migration, angiogenesis, and cell proliferation in both K-ras mutant PANC-1 pancreatic cancer cells or tumors. Administration of CA102N in PANC-1 xenografts resulted in a dose-dependent and statistically significant decrease in tumor growth at 9.8 mg/kg and 14.7 mg/kg Nim equivalents, respectively, compared to the vehicle control group. A significant decrease (80%) in liver metastasis was also noted in the PANC-1 orthotopic mice model at 19.6 mg/kg Nim equivalents dose level. Mechanistically, CA102N was found to suppress the PI3k/Akt/mTOR and Raf/MEK/ERK signaling pathways, in addition to reducing the expressions of the N6-methyladenosine (m6A) reader and writer proteins, OCT3/4 transcription factor and RAS levels in vivo. CA102N also modified the tumor stroma and improved drug permeability in vivo. Based on this compelling data, CA102N shows promise as a potential treatment for pancreatic cancer. Combination therapies and regimens are currently being explored in in vivo models as a strategy for clinical development. Citation Format: Louis Lin, Shuling Tu, Eskouhie Tchaparian. Therapeutic efficacy of CA102N in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5742.

Abstract PR04: USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation

Abstract Functional inactivation of RB1 occurs frequently in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype with poor clinical prognosis when tumors metastasize. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a key epigenetic regulator that is controlled by the RB-E2F pathway and is frequently overexpressed in many types of cancers, including TNBC. Using TCGA and METABRIC databases, we found that UHRF1 overexpression is negatively associated with breast cancer patient survival, including TNBC. Genetic ablation of UHRF1 using CRISPR/Cas9 in TNBC cell lines resulted in a robust decrease in tumorigenicity, including decreased cell clonogenicity (soft agar colony assay), migration (scratch wound healing assay), and invasion (Transwell assay) in vitro and decreased tumor growth and metastasis using orthotopic xenograft in vivo models. Despite strong evidence showing UHRF1 as a promising therapeutic target in several cancers, there is a current lack of UHRF1-specific inhibitors. To overcome this limitation, we explored targeting Ubiquitin-Specific Protease 7 (USP7), a deubiquitinase that prevents UHRF1 degradation, as a therapeutic strategy to trigger UHRF1 degradation. Here, we demonstrate that genetic inactivation or pharmacological inhibition of USP7 decreases TNBC tumor growth and metastasis in a UHRF1-degradation dependent manner. Phenocopying the effects of knocking out UHRF1, genetic ablation of USP7 using CRISPR/Cas9 in TNBC cell lines also resulted in significant decreases in cell clonogenicity, migration, and invasion in vitro. Intriguingly, mice bearing USP7 knockout tumors resulted in a far more robust decrease in tumor growth and absolute absence of metastatic disease. We established that many of the effects driven by USP7 ablation were mediated by the targeted degradation of UHRF1 by comparing vector control cell lines with UHRF1 knockout cells treated with the highly selective USP7 inhibitors, XL177A or FT671. In the absence of UHRF1, USP7 inhibitors showed no effects in cell migration and invasion in the three TNBC cell lines used in this study. Of promise, NSG mice bearing MDA-MB-231 tumors were recruited into vehicle or 50 mg/kg FT671 treatment groups and dosed daily via oral gavage for 20 days. For mice treated with FT671, we observed a significant decrease in tumor growth, with decreased UHRF1 levels in tumors, with minimal systemic toxicities. This study suggests that USP7 is a target with potential clinical relevance in suppressing tumor growth and metastasis in TNBC, and possibly other TP53-null cancers. These are promising results that present a novel opportunity for future clinical interventions for TNBC patients. Citation Format: Ahhyun Kim, Claudia Benavente. USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR04.

Abstract A059: Targeting pancreatic cancer metabolic dependencies through glutamine antagonism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. A factor that contributes to the poor prognosis of the disease is the complex tumor microenvironment (TME). The PDAC TME is composed of excessive fibrosis and desmoplasia, which creates a harsh environment resulting in hypoxia and altered nutrient availability. To promote survival and proliferation in this environment, PDAC cells can reprogram glutamine (Gln) metabolism. Previous studies have demonstrated that PDAC cells use Gln to support proliferation and redox balance. However, earlier attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. We hypothesized that a Gln analogue, such as 6-Diazo-5-oxo-L-norleucine (DON), could broadly target Gln metabolism in PDAC and prevent rapid adaptation. Indeed, DON treatment led to a significant decrease in PDAC proliferation and a profound reduction of various metabolites involved in central carbon and nucleotide metabolism, suggesting that DON creates a metabolic crisis. In addition, we observed a significant decrease in tumor growth in various in vivo models (syngeneic, immunodeficient and PDXs) using DRP-104 (sirpiglenastat), a novel pro-drug version of DON that was designed to circumvent DON associated GI toxicity and allow the therapeutic exploration of broad Gln antagonism. Mechanistically, we found that ERK signaling is increased as a compensatory mechanism through the increased activity of receptor tyrosine kinases (RTKs). Combinatorial treatment of DRP-104 and Trametinib (MEK1/2) inhibitor led to a significant increase in survival in a syngeneic model PDAC. Taken together, these pre-clinical results suggest that broadly targeting Gln metabolism could provide a new therapeutic avenue for PDAC and that the combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome. Citation Format: Joel Encarnacion-Rosado, Alec C. Kimmelman, Robert Wild. Targeting pancreatic cancer metabolic dependencies through glutamine antagonism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A059.

RCAd-LTH-shPD-L1, a double-gene recombinant oncolytic adenovirus with enhanced antitumor immunity, increases lymphocyte infiltration and reshapes the tumor microenvironment

BackgroundWith the successful development of modern immunotherapy, immune checkpoint inhibitors (ICIs) are currently considered potential therapeutic options for patients with cancer. However, the therapeutic potential of ICIs in human cancer...

Qpctl Inhibition, By Targeting the Inflammatory Tumor Microenvironment, Constitutes a Novel Therapeutic Approach for Diffuse Large B-Cell Lymphoma

We have previously described that diffuse large B-cell lymphomas (DLBCL) harboring an inflammatory and immunosuppressive microenvironment (i.e., IN-LME) carry independent poor prognosis while proof-of-principle experiments modifying the composition of the LME were sufficient to induce lymphoma regression in mice models ( Kotlov, Cancer Discovery 2021). However, identification of pharmaceutical approaches that by modifying the LME will induce an anti-lymphoma effect while being suitable for short-term human translation remains challenging. The IN-LME is characterized by the presence of pro-inflammatory and immunosuppressive chemokines and cytokines that frequently undergo post-translational modifications. QPCTL (glutaminyl-peptide cyclotransferase-like) is an intracellular enzyme that mediates a N-terminal pyroglutamylation of CD47 and several chemokines, including CCL2 and CCL7, ultimately resulting in their sustained expression and/or secretion. SC-2882 is a first-in-class specific QPCTL inhibitor that induces secondary proteolytic degradation of the monocyte chemoattractants CCL2 and CCL7 ( da Silva, Nature Immunology 2022) and inactivation of the “do-not-eat-me” signal CD47 ( LogtenbergNature Medicine 2019). In a dataset of 470 DLBCL patients we found that the expression of QPCTL correlates negatively with overall survival and progression free survival (0.019 and <0.001, respectively). To determine whether QPCTL could be a potential target in DLBCL, we investigated its expression along with its targets CCL2, CCL7 and CD47 in additional 216 DLBCL cases categorized by LME. We found that while QPCTL and CD47 are expressed at similar levels in all DLBCL cases regardless their COO class and LME category, CCL2 was highly expressed in DLBCL harboring a IN-LME (p <0.05 vs. other LME categories), that also correlate with higher presence of tumor-associated macrophages (TAMs) and exhausted CD8+PD1 HIGH T cells (p <0.01 and p<0.001, vs. other LME categories, respectively). CCL7 was not expressed at detectable levels in majority of LMEs in DLBCL. In preclinical models, murine and human DLBCL cell lines expressed baseline QPCTL and CD47 while CCL2 and CCL7 expression (qPCR) and secretion (ELISA) increased at higher levels upon culturing these cells for 24 h embedded in splenic extracellular matrix 3D cultures. Exposure of a panel of 3 murine and 5 human DLBCL cells to increasing concentrations of SC-2882 (vs. vehicle) for up to 72 h and up to a 10 micromolar dose had no noticeable decrease in cell viability or proliferation. However, SC-2882 (vs. vehicle) at nanomolar concentrations increased anti-Cd20-dependent lymphoma cell phagocytosis by in vitro differentiated macrophages (from splenic monocytes in mice and THP1 cells in humans) in 3D co-cultures (p < 0.05, for all conditions). To further test the anti-lymphoma effect of SC-2882 we used a murine GCB-DLBCL model by implanting GEMM-derived aggressive Ezh2 mutant (EZH2 Y646equivalent) + BCL2 amplified lymphoma cells in the spleen of immunocompetent C57BL/mice (n=14). After implantation, mice were randomized to receive SC-2882 by oral gavage (n=7) or vehicle (n=14) for 14 days. Tumor burden was followed by luciferase imaging (Caliper), and spleens were analyzed at sacrifice by multiparametric imaging, flow-cytometry, and spatial transcriptomics. Oral SC-2882 (vs. vehicle) induced a significant decrease in tumor growth (p=0.0079, Figure 1) without evidence of systemic toxicity by body weight, blood cell count and chemistry panel. Analysis of the LME in remnant splenic tumors showed in SC-2882 (vs. vehicle) treated mice a significant decrease in lymphoma cells and Ki67+ cells (p<0.001) as well as F480+ TAMs (p<0.01) while there was an increase of CD3+ T cell infiltration (p<0.001). We also found a decrease in Cd4+Foxp3+ Tregs and increase in Cd8+Gzb+ cells in SC-2882 treated mice by flow-cytometry. These changes mirrored the findings in the spatial transcriptomics. Additional analysis of LME cell subpopulations by SC-RNA-seq is ongoing. Overall, these data indicate that QPCTL is a potential target in DLBCL, and that QPCTL inhibition exhibits potent anti-lymphoma effects primarily by targeting the IN-LME.

Tumor Regression with 5-Aminolevulinic Acid (5-ALA)-Mediated Radiodynamic Therapy (RDT) Using Different Megavoltage Energies

5-aminolevulinic acid (5-ALA) accumulates in target tumor cells, where it is metabolized to a photosensitizer - protoporphyrin IX (PpIX). Cherenkov light induced by high-energy photon beams effectively activates the PpIX due to the Soret band. The activated photosensitizers lead to cellular toxicities to kill malignant tumor cells by converting surrounding tissue oxygens into singlet oxygens. Radiodynamic therapy (RDT) utilizes cellular damage caused by both radiation dose and activated photosensitizer. The emission of Cherenkov light was observed higher with higher energy irradiation in phantom and ex-vivo tissues. Therefore, using an in-vivo mouse model, this study aimed to investigate the efficacy and energy dependency of RDT combined with 5-ALA and different ranges of megavoltage photon irradiation. In order to investigate individual and synergistic effects of 5-ALA administration and radiation treatment, the tumors (n = 344) were randomized into eight groups: control (untreated), 5-ALA only, 6-, 15-, and 45-MV conventional radiation treatment (RT) only, 6-, 15-, and 45-MV RDT. 4 Gy in a single fraction was delivered to the tumors using three different energy photons for RT only and RDT groups. 5-ALA was systemically injected into 5-ALA only and RDT groups at 100 mg/kg by tail-vein 4 hours before irradiation for endogenous PpIX accumulation in the tumor. Tumor growth was measured using a 1.5 T MR scanner on the day of treatment (prior to the treatment), 3, 7, and 14 days post-treatment. Two-way repeated ANOVA with Bonferroni correction was used to compare each treatment group to determine the statistically significant difference in tumor growth. A total of 45 MV RDT resulted in the most significant decrease in tumor growth by 58.8 ± 3.4 %, 58.0 ± 3.0 %, and 55.0 ± 3.0 % compared to 5-ALA, 45 MV RT, and control group on 7 days post-treatment (P<0.001), respectively. Moreover, a synergistic effect of 45 MV RDT causes a 47.1-54.1 % additional decrease in tumor growth toward the effective treatment outcome. Compared to the different energies, 45 MV RDT resulted in a 52.2 ± 3.1 % and 19.7 ± 7.2 % decrease in tumor growth compared to 6 MV RDT and 15 MV RDT on 7 days post-treatment, respectively (P<0.001), whereas the conventional RT was not. Radiodynamic therapy using 5-ALA administration and 45MV photon beam irradiation resulted in the most significant tumor growth control. A photon energy dependency was observed in radiodynamic therapy. 45MV photon beams showed greater in activating PpIX, improving the synergistic effects of radiation dose and activated photosensitizer than the other energies. The preliminary results provide a foundation for new innovative treatment strategies that have the potential to improve cancer treatment.

Pan cancer characterization of genes whose expression has been associated with LINE-1 antisense promoter activity

BackgroundLong interspersed nuclear element-1 (LINE-1 or L1) comprises 17% of the human genome. As the only autonomous and active retrotransposons, L1 may take part in cancer initiation and progression in some ways. The studies of L1 in cancer mainly focus on the impact of L1 insertion into the new genome locus. The L1 5´ untranslated region (UTR) also contains antisense promoter (ASP) activity, generating L1-gene chimeric transcripts to a neighbor exon. Some of these ASP-associated genes have been reported to be overexpressed in cancer and promote cancer cell growth. However, little is known about overall expression patterns and the roles of L1 ASP-associated genes in human cancers.ResultsL1 ASP-associated genes were frequently dysregulated in cancer and associated with the cell cycle, the PI3K/AKT pathway, and the GTPase signaling pathway. The expression of L1 ASP-associated genes was correlated with tumor patient prognosis. Hub L1 ASP-associated genes CENPU and MCM2 showed a correlation with immune infiltration, clinical T stage, and cancer stemness in pan-cancer. Knockdown of L1 ASP-associated gene LINC00491 resulted in a significant decrease in tumor growth and migration ability.ConclusionsThe expression of L1 ASP-associated genes is significantly dysregulated at the pan-cancer level, which is closely related to the tumor microenvironment, progression, and patient prognosis. Hub genes CENPU and MCM2 are expected to be new tumor diagnostic markers and therapeutic targets.

HGG-19. CLINICAL RESPONSE TO THE PDGFRA/KIT INHIBITOR AVAPRITINIB IN PEDIATRIC AND YOUNG ADULT HIGH-GRADE GLIOMA PATIENTS WITH H3K27M OR PDGFRA GENOMIC ALTERATIONS

Abstract PDGFRA is commonly altered in pediatric and young adult high-grade gliomas (pHGGs) including histone 3 lysine 27-mutated diffuse midline gliomas (H3K27M DMG), a fatal disease with no current options for cure. We performed comprehensive genomic and transcriptomic analyses of n=259 pediatric high-grade glioma cases which revealed PDGFRA genomic alterations in ~15% of patients. H3K27M DMGs had significantly higher PDGFRA expression compared to H3 wild-type tumors regardless of genomic alteration. Tumors with PDGFRA gene amplification demonstrated significantly elevated PDGFRA expression in both H3K27M DMGs and H3 wild-type pHGGs relative to tumors with wild-type or point mutated PDGFRA. We tested a range of PDGFRA inhibitors against a panel of patient derived pediatric H3K27M DMG, pHGG, and adult HGG. Amongst the inhibitors tested, avapritinib, a potent small molecule inhibitor with relatively selective activity against both wild-type and mutant PDGFRA showed potent toxicity against a wide array of pediatric and adult cell lines. This molecule also demonstrated supra-micromolar blood brain barrier penetration in pre-clinical in vivo models, and demonstrated significant decrease in tumor growth and improved survival in orthotopic mouse xenograft models. Finally, building on this preclinical activity, we report the first clinical experience using avapritinib in eight pediatric and young adult patients with high-grade glioma (H3K27M DMG and/or PDGFRA altered). Avapritinib usage showed no significant acute toxicities within this patient cohort. Most importantly, our preliminary data reveal radiographic response evaluated by RAPNO criteria in 50% of patients, a striking outcome rarely seen in this patient population. In summary, we report that avapritinib, a selective, CNS penetrant small molecule inhibitor of PDGFRA has potent activity in preclinical models and produced promising clinical responses with good tolerability in pediatric and young adult patients with high-grade glioma, suggesting a promising role for avapritinib therapy in pediatric high grade glioma.

XRT-02. SINGLE-CELL RNA SEQUENCING IDENTIFIES FUNCTIONAL MACROPHAGE SUBSETS THAT ARE ENRICHED IN RESPONSE TO DIFFERENTIAL PHAGOCYTOSIS INDUCTION AGAINST GLIOMA

Abstract Pediatric diffuse midline gliomas (DMGs), are fatal brain tumors of childhood arising in the ventral pons. Currently, radiation therapy (RT) is the mainstay treatment for DIPG. However, RT is not a curative treatment and provides only temporary relief for most patients. Recent advances in immunotherapy have yielded some fantastic opportunities to effectively treat patients with high-grade pediatric brain tumors. In this study, we demonstrate that fractionated RT (4Gy X 3) induces immunogenic cell death and activates multiple damage-associated molecular patterns (DAMPs) on DMG/DIPG cells. Furthermore, combining 4Gy X 3 with anti-CD47 therapy enhances the in vitro phagocytosis of DIPG/DMG cells by peripheral blood mononuclear cell-derived macrophages. Next, using single-cell RNA sequencing (scRNA-seq), we investigated the transcriptomic profile of macrophages that were co-cultured with irradiated or non-irradiated DMG cells in the presence of anti-CD47 monoclonal antibodies for 24 hours. Our findings identified eleven distinct macrophage clusters displaying different gene expression patterns in all the treatment conditions. However, PBS treatment led to a marked increase in macrophages expressing antigen-presentation genes (HLA-DR and HLA-DQB1). 4Gy X 3 treatment led to the enrichment of macrophages expressing genes related to the oxidative phosphorylation pathway, whereas anti-CD47 treatment led to the enrichment of macrophages expressing genes related to interferon-gamma response and ERK-signaling pathway. Lastly, mice intracranially transplanted with DMG/DIPG that received 4Gy X 3 and anti-CD47 therapy showed a significant decrease in tumor growth and an increase in survival rate than those receiving either monotherapy alone. We are currently performing scRNA-seq validation studies on tumors obtained from orthotopic and syngeneic models of DMG. In summary, our results highlight the functional heterogeneity of macrophages and suggest that combining fractionated RT with anti-CD47 therapy has distinct anti-tumor effects and can be used as a therapeutic approach for treating DIPG patients.

Abstract LB018: Therapeutic efficacy of inhibition of specific sirtuins against melanoma in BrafV600E/PtenNULL and PDX models

Abstract Melanoma is a highly immunogenic malignancy and one of the deadliest skin cancers due to its ability to metastasize and its resistance to existing therapies. Current treatment regimens include immunotherapies as well as targeted therapies (e.g. BRAF and MEK inhibitors). However, these immuno- or targeted therapies have been associated with acquired resistance and, therefore, failure of therapy. Therefore, development of novel mechanistically-targeted therapies is of paramount importance for an efficient management of this neoplasm. Sirtuins, a seven-member family of class III histone deacetylases, have been implicated in the development/progression of multiple cancers, including melanoma. Previous studies from our laboratory and elsewhere have shown the pro-proliferative functions of sirtuins SIRT1 and SIRT3 in melanoma. We have also shown that the small molecule inhibitor 4’-bromoresveratrol (4’-BR; co-inhibitor of SIRT1 and SIRT3) has antiproliferative effects in melanoma cell lines as well as in a BrafV600E/PtenNULL mouse model of melanoma. This study was designed to further expand on our previous findings and to determine the therapeutic significance of SIRT1 and SIRT3 in melanoma. To determine the effect of specific inhibition of SIRT3 on melanoma progression, we treated BrafV600E/PtenNULL mice with 100 μg small interfering RNA (siRNA) against SIRT3 (siSIRT3) or its non-targeting control (siNTC) twice weekly (intraperitoneal injections for 5 weeks). We found a trend of decreased tumor volumes in the siSIRT3-treated mice, though not significant. A similar trend was seen in the final tumor weight of these mice. In the next series of experiments, we used two human melanoma patient-derived xenografts (PDXes) to compare the effect of 4’-BR or siSIRT3 on melanoma tumor growth. Similar to our published study in the BrafV600E/PtenNULL mice, we found that 4’-BR treatment (50 mg/kg, via intraperitoneal injection, twice weekly for 5 weeks) resulted in a significant decrease in tumor growth when compared to vehicle control mice. Additionally, the final tumor weight was significantly decreased in the 4’-BR group. However, the siSIRT3 treatments resulted in a decrease but non-significant decrease in melanoma tumor growth or final weights, supporting our observations in BrafV600E/PtenNULL mice. Considering the lung is a common site for metastasis in melanoma, metastatic nodules in the lungs were quantified in one of our PDX models. We observed a decreased number in both the siSIRT3 and 4’-BR groups vs their respective controls. Overall, our data suggests that targeting SIRT3 alone may not be sufficient for melanoma treatment, and that the concomitant inhibition of SIRT1 and SIRT3 (and potential other sirtuins with pro-proliferative functions in melanocytic cells) may be better for melanoma management. Indeed, additional research is needed to validate our findings in relevant models. Citation Format: Karla Anaya Aldrete, Gabriella R. Zaemisch, Mary A. Ndiaye, Gagan Chhabra, Nihal Ahmad. Therapeutic efficacy of inhibition of specific sirtuins against melanoma in BrafV600E/PtenNULL and PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB018.

Abstract 3651: The role of caspase-1 in basal-like breast cancer and the tumor microenvironment

Abstract Introduction: Breast cancer is the most common malignancy in women world-wide. Basal-like breast cancer (BLBC) is an aggressive subtype with poor prognosis for which there are no known targeted therapies. Understanding what drives complex cell-cell interactions within the tumor microenvironment (TME) is critical to developing new strategies. BLBC has previously been shown to have high expression of the inflammatory cytokine IL1β, which then promotes the recruitment of pro-tumoral tumor-associated macrophages (TAMs) to the TME. Here we report that BLBC is uniquely capable of IL1β secretion due to elevated expression of caspase-1, a key component of the inflammasome required for IL1β maturation. Methods: Using publicly available gene expression data sets, the association between caspase-1, breast cancer subtype and estrogen receptor (ER) co-expression was examined. These associations were further assessed in vitro by exposing luminal (T47D/MCF7) or BLBC (MDA-MB231) cell lines to siER knockdown or ER overexpression. Using both a CRISPR/Cas9 generated caspase-1 knockout (KO) BLBC mouse line as well as pharmacological inhibition with the caspase-1 inhibitor VX-765, the effect of caspase-1 on tumor growth was studied in syngeneic immunocompetent mice. Immune infiltrates were assessed via flow cytometry of the excised murine tumors as well as immunohistochemistry. To prove these results were IL1β specific, caspase-1 KO tumor allografts containing self-cleaving IL1β were used for comparison. Finally, to determine potential synergy between caspase-1 and immune checkpoint inhibition, wild type and caspase-1 KO allografts were simultaneously treated with anti-PD1 immunotherapy. Tumor growth and immune infiltrates were analyzed between the treatment groups as stated above. Results: Caspase-1 was found to be associated with the basal-like subtype and have an inverse relationship with ER expression. In vitro, inhibition of ER increased caspase-1 expression, whereas ER overexpression decreased caspase-1. Caspase-1 KO or pharmacological inhibition resulted in a significant decrease in tumor growth and TAM infiltration, and these findings were rescued back to wild type levels with the addition of a self-cleaving IL1β. Finally, caspase-1 inhibition reversed resistance to anti-PD1 immunotherapy in murine allografts, resulting in significant deceleration of tumor growth when used in combination. Summary: The lack of ER in BLBC promotes caspase-1 expression, allowing IL1β maturation, macrophage recruitment and tumor progression. Genetic or pharmacologic inhibition of caspase-1 inhibits TAM recruitment and reverses resistance to immune checkpoint inhibition in BLBC. Conclusions: Our data provides new insights into the biology of BLBC and identifies the combination of caspase-1/IL1β inhibition and immunotherapy as a novel therapeutic strategy to combat this disease. Citation Format: Wanda Marini, Weiyue Zheng, Kiichi Murakami, Pamela S. Ohashi, Michael Reedijk. The role of caspase-1 in basal-like breast cancer and the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3651.

Abstract 6397: Eribulin acts as an immune adjuvant to enhance the antitumor efficacy of STING agonists in triple-negative breast cancer models

Abstract Triple-negative breast cancer (TNBC) is a heterogenous subtype of breast cancer lacking effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment. Strategies to improve the response of TNBC patients to immunotherapy include the upregulation of the cGAS-STING innate immune sensing pathway and STING agonists are in clinical development. We previously showed that eribulin, a microtubule destabilizer used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. In separate studies, we demonstrated that eribulin also significantly enhances type I interferon expression induced by STING agonists through a second TBK1-dependent mechanism downstream of STING activation. Both mechanisms of eribulin-mediated activation of interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizer paclitaxel. Our current studies demonstrate the in vivo immunological effects of the microtubule destabilizer eribulin with the STING agonist ADU-S100 that lead to a significant decrease in tumor growth in the spontaneous MMTV-PyVT mammary tumor model upon combination treatment. The immunostimulatory effects of eribulin with or without concurrent administration of a STING agonist were determined in the spleen, tumor draining lymph nodes, and tumors with distinct effects observed in the activation of innate and adaptive responses. We further demonstrate the immunological and antitumor efficacy of combinations of eribulin and ADU-S100 in the syngeneic 4T1 model that is resistant to the antimitotic effects of eribulin as a single agent. Even in the absence of direct antimitotic effects on the tumor, eribulin was sufficient to promote activation of CD4+CD25− effector T-cells in the spleen and lymph nodes specifically in tumored animals but not untumored controls. Additionally, eribulin was able to promote a similar increase in the activation of CD4+ effector T-cells even in untumored animals when administered in combination with a STING agonist. Together, these data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC that could inform on their more rational use in the clinic as single agents or as chemotherapeutic backbones in combination with targeted therapy, including immunotherapeutics. More specifically, our data demonstrate that eribulin can act as an immune adjuvant to promote STING signaling and T-cell activation in multiple in vivo TNBC models in addition to its well established anti-mitotic effects to enhance antitumor efficacy. Citation Format: Leila Takahashi-Ruiz, Charles S. Fermaintt, Nancy Wilkinson, Peter Y. Chan, Susan L. Mooberry, April L. Risinger. Eribulin acts as an immune adjuvant to enhance the antitumor efficacy of STING agonists in triple-negative breast cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6397.

Ferroportin depletes iron needed for cell cycle progression in head and neck squamous cell carcinoma.

Ferroportin (FPN), the only identified eukaryotic iron efflux channel, plays an important role in iron homeostasis and is downregulated in many cancers. To determine if iron related pathways are important for Head and Neck Squamous Cell Carcinoma (HNSCC) progression and proliferation, we utilize a model of FPN over-expression to simulate iron depletion and probe associated molecular pathways. The state of iron related proteins and ferroptosis sensitivity was assessed in a panel of metastatic HNSCC cell lines. Stable, inducible expression of FPN was confirmed in the metastatic HNSCC lines HN12 and JHU-022 as well as the non-transformed normal oral keratinocyte (NOK) cell line and the effect of FPN mediated iron depletion was assessed in these cell lines. HNSCC cells are sensitive to iron chelation and ferroptosis, but the non-transformed NOK cell line is not. We found that FPN expression inhibits HNSCC cell proliferation and colony formation but NOK cells are unaffected. Inhibition of cell proliferation is rescued by the addition of hepcidin. Decreases in proliferation are due to the disruption of iron homeostasis via loss of labile iron caused by elevated FPN levels. This in turn protects HNSCC cells from ferroptotic cell death. Expression of FPN induces DNA damage, activates p21, and reduces levels of cyclin proteins thereby inhibiting cell cycle progression of HNSCC cells, arresting cells in the S-phase. Induction of FPN severely inhibits Edu incorporation and increased β-galactosidase activity, indicating cells have entered senescence. Finally, in an oral orthotopic mouse xenograft model, FPN induction yields a significant decrease in tumor growth. Our results indicate that iron plays a role in HNSCC cell proliferation and growth and is important for cell cycle progression. Iron based interventional strategies such as ferroptosis or iron chelation may have potential therapeutic benefits in advanced HNSCC.

TMET-02. THERAPEUTIC EFFICACY OF A NOVEL BRAIN PENETRANT SCD INHIBITOR ON BREAST CANCER BRAIN METASTASES

Abstract Brain metastases are a significant therapeutic challenge and are associated with high morbidity and mortality. Recent evidence suggests that brain metastases have increased fatty acid synthesis compared to extracranial tumors. The enzyme stearoyl-CoA desaturase (SCD), which converts saturated long-chain fatty acids into monounsaturated fatty acids, is often upregulated in breast cancer brain metastasis (BCBM) and is considered a promising therapeutic target. However, the lack of brain penetrant SCD inhibitors limited their use in patients. This study aimed to investigate the therapeutic efficacy of a clinical-stage brain penetrant inhibitor of SCD to treat BCBM. We found that pharmacologic inhibition of SCD in multiple breast cancer cell lines induced significant cytotoxicity, mainly by apoptosis. Gas chromatography/mass spectrometry confirmed that the fatty acid desaturation index, which measures SCD activity, was significantly lower after treatment. SCD inhibition triggered endoplasmic reticulum (ER) stress and increased DNA damage due to a higher production of reactive oxygen species. Further, SCD inhibition decreased DNA damage repair by inhibiting homology-directed repair in vitro and in a mouse model of breast cancer. Notably, inhibition of SCD led to a significant decrease in tumor growth in a subcutaneous mouse model of breast cancer and a significant increase in overall survival in a BCBM mouse model. Cytotoxicity was enhanced when this SCD inhibitor was combined with either a DNA damage-inducing agent (temozolomide or radiation) or with a PARP inhibitor (Niraparib). Finally, we observed that combination therapy of SCD inhibition and Niraparib significantly increased overall survival in a BCBM mouse model. Our results suggest that inhibition of SCD alone or in combination with a PARP inhibitor is a promising therapeutic strategy to treat BCBM.