Reduction In Tumor Size Research Articles

Therapeutic potential of LINS01 histamine H3 receptor antagonists as antineoplastic agents for triple negative breast cancer

The aims of this work were to evaluate the expression of histamine H3 receptor (H3R) in triple negative breast cancer (TNBC) samples and to investigate the antitumoral efficacy and safety of the LINS01 series of H3R antagonists, through in silico, in vitro, and in vivo approaches. Antitumor activity of LINS01009, LINS01010, LINS01022, LINS01023 was assayed in vitro in 4T1 and MDA-MB-231 TNBC cells (0.01–100 μM), and in vivo in 4T1 tumors orthotopically established in BALB/c mice (1 or 20 mg/kg). Additionally, H3R expression was assessed in 50 human TNBC samples. We have described a higher H3R mRNA expression in basal-like/TNBC tumors vs. matched normal tissue using TCGA Pan-Cancer Atlas data, and a higher H3R expression in human tumor samples vs. peritumoral tissue evidenced by immunohistochemistry associated with poorer survival. Furthermore, while all the essayed compounds showed antitumoral properties, LINS01022 and LINS01023 exhibited the most potent antiproliferative effects by: i) inducing cell apoptosis and suppressing cell migration in 4T1 and MDA-MB-231 TNBC cells, and ii) inhibiting cell growth in paclitaxel-resistant 4T1 cells (potentiating the paclitaxel antiproliferative effect). Moreover, 20 mg/kg LINS01022 reduced tumor size in 4T1 tumor-bearing mice, exhibiting a safe toxicological profile and potential for druggability estimated by ADME calculations. We conclude that the H3R is involved in the regulation of TNBC progression, offering promising therapeutic potential for the novel LINS01 series of H3R antagonists.

Potential of Nanomedicines as an Alternative for the Treatment of Colorectal Cancer - A Review.

Colorectal cancer is the third most common cancer and the second in cases of cancer-related death. Polytherapy generates many adverse effects, leading the patient to give up. Nanotechnology has been studied in recent years to circumvent limitations. Groups composed of polymeric, lipid, and inorganic nanoparticles are the most purpose. Thus, the objective of this work is to bring information on how nanosystems can improve the chemotherapeutic treatment for colorectal cancer. Therefore, a search in journals such as "LILACS", "SciELO" and "PubMed/Medline" was performed, resulting in 25,000 articles found when applied the search engines "nanoparticle," "colorectal cancer," "malignant neoplasms," and "chemotherapy." After inclusion and exclusion factors, 24 articles remained, which were used as the basis for this integrative review. The results reveal that, regardless of the choice of matrix, nanoparticles showed an increase in bioavailability of the active, increasing the half-life by up to 13 times, modified release, as well as a significant reduction in tumor size, with cell viability up to 20% lower than the free drug tested, in different colorectal cancer cell lines, such as HCT-116, HT-29, and CaCo-2. However, more in vivo and clinical studies need to be performed, regardless of the formulation of its matrix, aiming at a higher rate of safety for patients and stability of the formulations, as well as knowledge of detailed indices of its pharmacokinetics and pharmacodynamics, seeking to avoid further damage to the recipient organism.

Transcranial Agonism of CD40 and ICOS Promotes Meningeal Immunity and Control of Glioblastoma

INTRODUCTION: Glioblastomas generate an immunosuppressive microenvironment that limits antitumor immune responses. METHODS: We used the SB28 model of glioblastoma in C57BL/6J (B6) mice, which is a poorly immunogenic tumor with a lower mutational load than other many glioblastoma cell lines used in rodents. We stereotactically injected 7,000 SB28 cells at a depth of 1 mm into the neocortex of 8- to 10-week-old mice. At day 7, a carboxymethylcellulose gel containing 100 µg of agonistic anti-ICOS and 200 µg anti-CD40 were injected transcranially (i.e., under the scalp) of mice. Control mice received corresponding isotype control antibodies. Meningeal whole mounts were then stained using immunohistochemistry and imaged by confocal microscopy. RESULTS: Transcranial agonism of CD40 and ICOS generated a markedly enhanced and more localized meningeal immune response against SB28 tumors than intravenous agonism. Specifically, transcranial treatment promoted formation of CD4 T cell and B cell clusters in the meninges as well as increased production of class-switched immunoglobulins. These clusters were also associated with reduced tumor size at day 14. CONCLUSIONS: Our results demonstrate that transcranial delivery of antibodies against CD40 and ICOS can drive a local humoral immune response against an immunologically silent SB28 glioblastoma. We postulate that application of these agonistic antibodies in combination with a tumor neoantigen will further enhance tumor control and may offer a possible treatment modality in human glioblastoma patients.

Impact of cytoreductive surgery with and without chemoradiation on the outcome of induced oral squamous cell carcinoma: an experimental study

Objectives The aim of this study is to evaluate the effect of cytoreductive surgery with and without chemoradiation on tumor size, survival rate and systemic status in hamsters with chemically induced oral squamous cell carcinoma (OSCC). Design The study was conducted on eighty-eight male hamsters. OSCC was induced by 7,12-Dimethylbenz[a] anthracene (DMBA) painted on hamsters’ buccal pouch. Hamsters were subsequently divided into four groups, group 1 (n = 22) employed as control group, group 2 (n = 22) with only induced OSCC left without treatment, group 3 (n = 22) underwent surgical removal of 75% of tumor mass, and group 4 (n = 22) underwent surgical procedures combined with chemoradiation. Results Clinical evaluation revealed that cytoreductive surgery combined with chemoradiation was associated with overall tumor size reduction and improvement of survival rate. Histological examination revealed necrosis of tumor tissue and heavy infiltration by chronic inflammatory cells were also observed. Conclusion Chemoradioation therapy after debulking surgery was associated with a decrease of tumor size and improvement in survival rate.

Hyperoside Inhibits RNF8-mediated Nuclear Translocation of β-catenin to Repress PD-L1 Expression and Prostate Cancer.

Hyperoside is a flavonol glycoside isolated from Hypericum perforatum L. that has inhibitory effects on cancer cells; however, its effects on prostate cancer (PCa) remain unclear. Therefore, we studied the anti-PCa effects of hyperoside and its underlying mechanisms in vitro and in vivo. This study aimed to explore the mechanism of hyperoside in anti-PCa. 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT), transwell, and flow cytometry assays were used to detect PCa cell growth, invasion, and cell apoptosis. Immunoblot analysis, immunofluorescence, immunoprecipitation, and quantitative real-time PCR (qRT-PCR) were used to analyze the antitumor mechanism of hyperoside. Hyperoside inhibited PCa cell growth, invasion, and cell cycle and induced cell apoptosis. Furthermore, RING finger protein 8 (RNF8), an E3 ligase that assembles K63 polyubiquitination chains, was predicted to be a direct target of hyperoside and was downregulated by hyperoside. Downregulation of RNF8 by hyperoside impeded the nuclear translocation of β-catenin and disrupted the Wnt/β-catenin pathway, which reduced the expression of the target genes c-myc, cyclin D1, and programmed death ligand 1 (PD-L1). Decreased PD-L1 levels contributed to induced immunity in Jurkat cells in vitro. Finally, in vivo studies demonstrated that hyperoside significantly reduced tumor size, inhibited PD-L1 and RNF8 expression, and induced apoptosis in tumor tissues of a subcutaneous mouse model. Hyperoside exerts its anti-PCa effect by reducing RNF8 protein, inhibiting nuclear translocation of β-catenin, and disrupting the Wnt/β-catenin pathway, in turn reducing the expression of PD-L1 and improving Jurkat cell immunity.

Safety, Efficacy, and Biological Data of T-Cell-Enabling Oncolytic Adenovirus TILT-123 in Advanced Solid Cancers from the TUNIMO Monotherapy Phase I Trial.

TILT-123 (igrelimogene litadenorepvec) is an oncolytic adenovirus armed with TNFa and IL2, designed to induce T-cell infiltration and cytotoxicity in solid tumors. TUNIMO (NCT04695327) was a single-arm, multicenter phase I dose-escalation trial designed to assess the safety of TILT-123 in advanced solid cancers refractory to standard therapy. Patients received intravenous and intratumoral TILT-123. The primary endpoint was safety by adverse events (AE), laboratory values, vital signs, and electrocardiograms. Secondary endpoints included tumor response, pharmacokinetics, and predictive biomarkers. Twenty patients were enrolled, with a median age of 58 years. Most prevalent cancer types included sarcomas (35%), melanomas (15%) and ovarian cancers (15%). No dose-limiting toxicities were observed. The most frequent treatment-related AEs included fever (16.7%), chills (13.0%), and fatigue (9.3%). Ten patients were evaluable for response on day 78 with RECIST 1.1, iRECIST or PET-based evaluation. The disease control rate by PET was 6/10 (60% of evaluable patients) and 2/10 by RECIST 1.1 and iRECIST(20%of evaluable patients). Tumor size reductions occurred in both injected and non-injected lesions. TILT-123 was detected in injected and non-injected tumors, and virus was observed in blood after intravenous and intratumoral injections. Treatment resulted in reduction of lymphocytes in blood, with concurrent lymphocyte increases in tumors, findings compatible with trafficking. TILT-123 was safe and able to produce antitumor effects in local and distant lesions in heavily pre-treated patients. Good tolerability of TILT-123 facilitates combination studies, several of which are ongoing (NCT04217473, NCT05271318, NCT05222932, and NCT06125197). See related commentary by Silva-Pilipich and Smerdou, p. 3649.

Allosteric SHP2 inhibition enhances regorafenib's effectiveness in colorectal cancer treatment

Colorectal cancer (CRC) is the third most common cancer globally. Regorafenib, a multi-target kinase inhibitor, has been approved for treating metastatic colorectal cancer patients who have undergone at least two prior standard anti-cancer therapies. However, regorafenib efficacy as a single agent remains suboptimal. A promising target at the crossroads of multiple signaling pathways is the Src homology 2 domain-containing protein tyrosine phosphatase (SHP2). However, a combination approach using SHP2 inhibitors (SHP099) and anti-angiogenic drugs (Regorafenib) has not been reported in current research. In this study, we conducted in vitro experiments combining SHP099 and regorafenib and established an MC-38 colon cancer allograft mouse model. Our results revealed that co-treatment with SHP099 and regorafenib significantly inhibited cell viability and altered the biological characteristics of tumor cells compared with treatment alone in vitro. Furthermore, the combination strategy demonstrated superior therapeutic efficacy compared to monotherapy with either drug. This was evidenced by reduced tumor size, decreased proliferation, increased apoptosis, normalized tumor microvasculature, and improved antitumor immune response in vivo. These findings suggest that the combination of an SHP2 inhibitor and regorafenib is a promising therapeutic approach for patients with colorectal cancer.

Prolactinoma Presenting with Secondary Amenorrhoea in a Young Girl

Prolactinoma is the most common secreting pituitary adenoma. It has different clinical presentation in men and women which varies with age. Young women present with menstrual problems, infertility and galactorrhoea, whilst older women and men may present with loss of libido, visual disturbances and in late cases, ith osteoporosis. In approaching a young woman with secondary amenorrhoea, other cuses such as pregnancy, functional hypothalamic amenorrhea, pituitary adenoma, Polycystic Ovarian Syndrome (PCOS) or primary ovarian insufficiency should be ruled out. Prolactinoma can be confirmed in the presence of high serum prolactin level and adenoma on pituitary gland magnetic resonance imaging (MRI). Dopaminergic agonist, cabergoline is efficacious in restoring gonadal and sexual functions by normalising prolactin levels. We presented a case report onan 18-year-old girl who presented with secondary amenorrhoea for six months. On further evaluation she was noted to have galactorrhoea with substantially high prolactin level of 466.9 ng/mL (normal range: 4.0- 23) and the MRI showed a pituitary tumour measuring 1.2 x 1.1 x 1.4 cm. A diagnosis of macroprolactinoma was made and she was managed with cabergoline with gradual dose increment. At 6 months of treatment, her menstrual cycle and serum prolactin had normalised. A repeat MRI pituitary one year later showed reduction in tumour size.

Abstract 3327: L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors

Abstract Introduction: Immunotherapy is a promising therapeutic approach for a wide range of cancers. However, some tumors called "cold tumors" lack infiltrating T-cells and are not responsive to immune checkpoint inhibitors (ICIs), leading to worse clinical outcomes. This study demonstrates that L-pampo, a novel TLR2/3 agonist, promotes an anti-tumor immune response in cold tumors. Also, combining L-pampo with ICIs can improve the anti-tumor efficacy more safely than standard-of-care (SoC) chemotherapies. Method: We utilized syngeneic tumor models using CT26 colon cancer and 4T1 triple-negative breast cancer (TNBC). L-pampo was administered alone or in combination with ICIs such as anti-PD-1 (aPD-1) and anti-CTLA-4 (aCTLA-4) antibodies to tumor-bearing mice (n=8/group). Tumor size was measured every three days, and the survival rate was monitored until day 60. On day 21 or 27 after transplantation, we analyzed the tumor weight and tumor-infiltrating immune cells. Furthermore, we assessed the respective anti-tumor efficacies and safety of L-pampo and SoC chemotherapies (FOLFOX and paclitaxel). Result: In the CT26 colon cancer model, L-pampo effectively reduced the tumor size compared to the vehicle group (83.7%). The combination of L-pampo and ICI reduced tumor size by 86.1% (with aCTLA4) and 90.5% (with aPD-1), while the ICI alone did not effectively inhibit tumor growth (aPD-1; 17.3%, aCTLA-4; 23.6%). Notably, the triple concurrent treatment with L-pampo, aPD-1 and aCTLA-4 reduced tumor size by 96.6% and induced completed tumor regression in 1 out of 8. In the 4T1 TNBC model, L-pampo reduced tumor size by 64.2% compared to the vehicle treatment, and combined treatment with L-pampo and a-PD-1 suppressed tumor growth compared to aPD-1 alone (9.6% vs. 61.5%). Additionally, L-pampo improved median overall survival in each tumor model (CT26; 25.5 vs. 35.5, 4T1; 29.5 vs. 53.5 days). To compare the anti-tumor efficacy of L-pampo and conventional cytotoxic chemotherapy, we treated tumor-bearing mice with FOLFOX or paclitaxel as SoC regimen, respectively. Compared with each chemotherapy, L-pampo revealed comparable antitumor efficacies (Tumor growth inhibition CT26; 75.6% vs. 70.6%, 4T1; 65.5% vs. 58.8%). Interestingly, L-pampo inhibited tumor growth without significant adverse effects. In contrast, conventional chemotherapies induced acute hematological side effects such as leukopenia/neutropenia/thrombocytopenia and rapid weight loss. Furthermore, L-pampo increased the active cytotoxic T-cells infiltration through antigen-presenting cells stimulation within the tumors. Conclusion: This study suggests that L-pampo, a novel TLR2/3 agonist, can be a potent anti-tumor agent, improving the anti-tumor efficacy of ICIs in cold tumors with enhanced anti-tumor immunity and minimal toxicities than conventional chemotherapies. Citation Format: Guentae Kim, Yoonki Heo, Hye Jung Kim, Sejung Park, Byung Cheol Ahn, Won Suk Lee, Chan Kim, Hong Jae Chon, Eunyoung Chun, Jung Sun Yum. L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors [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 3327.

Abstract 2086: Effect of XPO1 inhibition on colorectal cancer tumorigenesis

Abstract Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the U.S. A subset of individuals faces a notably higher likelihood of developing CRC within their lifetime. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing CRC tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. The overexpression of XPO1 has been identified across several cancers, including CRC. XPO1 interacts with a diverse spectrum of more than 1000 proteins. The overexpression of XPO1 leads to the excessive removal of tumor suppressors from the nucleus, rendering these proteins functionally inert. To counteract the overexpression of XPO1 in tumors, a novel class of drugs known as Selective Inhibitors of Nuclear Export (SINE) has been developed. Among these, Eltanexor (KPT-8602) has emerged as a promising therapeutic agent, demonstrating fewer side effects compared to its precursors, and is currently under evaluation in Phase 1/2 clinical trials. The treatment effects of SINE compounds on CRC remain largely unknown. We have assessed Eltanexor's ability to limit CRC tumorigenesis using a genetic CRC model in Apcmin/+ mice. Our findings indicate that Eltanexor substantially reduces tumor burden by approximately 3-fold and induces a significant reduction of tumors exceeding 1mm in size. Furthermore, a xenograft experiment using the human CRC cell line, HCT116, reveals nearly a 3-fold reduction in tumor size upon Eltanexor treatment. In both CRC models, Eltanexor exhibits noteworthy tolerability. Additionally, XPO1 protein levels are consistently diminished in both models, aligning with our in vitro observations. To further ascertain Eltanexor's specificity for CRC tumors versus normal tissue, we conducted drug sensitivity assays using organoids derived from Apcmin/+ tumors and wild-type mice small intestine tissue. We found that Apcmin/+ tumoroids have heightened sensitivity to Eltanexor-treatments, when compared to wild-type organoids. In an endeavor to elucidate Eltanexor's potent in vivo tumorigenesis inhibitory effects, we conducted multiple assays which revealed a reduction in COX-2 RNA and protein expression levels in Eltanexor-treated cells and reduced COX-2 protein in the tumors of Eltanexor-treated Apcmin/+ mice. In CRC, COX-2 is implicated in CRC development and is currently the target of multiple CRC chemopreventive agents. Our observations reveal that Eltanexor-treated CRC cells exhibit reduced COX-2 gene expression through both inhibiting COX-2’s promoter and its mRNA post-transcriptional stability. Collectively, our findings underscore XPO1 as a potent target for inhibiting CRC tumorigenesis. Future studies will aim to further assess how Eltanexor is impacting COX-2’s promoter, while additionally testing Eltanexor’s tumorigenesis-inhibiting potential in a colitis-induced CRC mouse model. Citation Format: Andrew E. Evans, Dan A. Dixon. Effect of XPO1 inhibition on colorectal cancer tumorigenesis [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 2086.

Abstract 3307: c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC

Abstract The interaction between E3 ligases and chaperones is crucial for proteostasis, regulating more than 10% of total proteasomes and over 30% of human E3 ligases (Kevei, 2017). Chaperones also can recognize their substrates and direct them to degradation by the ubiquitin-proteasome system (UPS). Developing a targeted protein degradation (TPD) technology that utilizes these advantageous attributes of chaperones, termed chaperone-mediated protein degradation (CMPD) technology, would broaden the range of disease-causing target proteins and would efficiently degrade them by recruiting various intracellular E3 ligases through interaction with chaperones, compare to other TPD approaches. The c-mesenchymal-epithelial transition (c-MET) is recognized as primary cause of drug-resistance to EGFR inhibitors in non-small cell lung cancer (NSCLC). Herein, to degrade the c-MET, we synthesized bifunctional small molecule compound inducing c-MET and chaperones in proximity, termed CMPD-MET degrader, and evaluate it in MET-mutant NSCLC cell lines and mouse xenograft models. The efficacy and potency of CMPD-MET degrader were assessed using H596 and H1437 cells in vitro. Cell proliferation was measured using MTT assay and c-Met degradation assay was conducted, along with the examination of proteasome-dependent degradation via western blot. An in vivo H596 xenograft model gauged impact of CMPD-MET degrader via I.P. administration. In NSCLC with MET Exon 14 skipping mutation cell line H596 and H1437, treatment a OZD-MET degrader resulted inhibition of cell proliferation with IC50< 5 µM and subsequent proteasome-dependent c-MET degradation with DC50 ranging from 4-6 µM for 24h. The degradation of c-MET levels significantly induced by OZD-MET degrader, especially, the c-Met was degraded by 70.4% and 51.7% at 5 µM for 24h in H596 and H1437 cells, respectively. Also, OZ-MET degrader effectively suppressed c-MET mediated signaling and showed cleavage of PARP-1 in a dose-dependent manner. OZD-MET degrader induced selective degradation of c-MET showing at least more than about 5-fold selectivity over other chaperones-regulated client proteins.In the H596 xenograft model, OZD-MET degrader was administered in 5 dosage groups (minimum dose is 2.5 mg/kg) resulted in dramatic reduction in tumor size compared to the vehicle-treated group. Remarkably, administration of OZD-MET degrader revealed more than 90% degradation of c-Met in tumor tissues compared to the vehicle groups via western blots and fluorescence image. In contrast, c-MET inhibitor treated group showed no effect on degradation of c-MET in tumor tissues. In summary, we developed a novel c-Met targeting chaperone-mediated protein degrader, showed highly potent and selective proteasome-dependent c-MET degradation and notable inhibition of tumor size in xenograft models. These results provide a strong foundation for further evaluation in clinical trials. Citation Format: Han Jung Chae, Jisun Kim, SunHye Lee, Ho Yoo, Hyun-Kyoung Kim, Young-Jin Yu. c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC [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 3307.

Abstract 674: Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway

Abstract Aberrant migration and proliferation of cancer cells are the hallmark of metastatic tumors. Protein Regulator of Cytokinesis 1 (PRC1) is a scaffold protein that regulates metabolic pathways of cancer cell proliferation, survival, and metastasis. BKT300 is a small molecule (MW 399.33, C22H25NO6), that selectively inhibits the migration and survival of hematological and solid cancer cells. BKT300 inhibits, at high affinity, both actin and microtubule formation and organization leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis through the caspase 3 pathway. These effects are selective to cancer cells and were not observed on normal cells. BKT300 was found to arrest PRC1 in a phosphorylated form at position T481, down regulates the expression of CDC25C and up regulate p21. Suppression of PRC1 using SiRNA ablation inhibited BKT300 induced apoptosis on several tumor cell lines and inhibited the effect of BKT300 on G2M cell cycle arrest. Furthermore, using Biacore SPR, microscale thermophoresis (MST), and ELISA assays we were able to demonstrate the direct binding of BKT300 to PRC1. Beyond its single agent activity, BKT300 was shown to synergize with standard of care (SoC) treatments, including anti PD-1 immunotherapy, BCL-2 inhibitor (Ventoclax), topoisomerase I inhibitor (Irinotecan) topoisomerase II inhibitor (Doxorubicin), anti-metabolite Fluorouracil (5-FU) and alkylating agent (Temozolomide). BKT300 has a remarkable safety profile when administered at high doses to mice rats, minipigs and non-human primates. Treatment with BKT300 showed almost no effects on hematopoiesis and biochemistry in doses far higher than the affective ones. The in-vivo activity of BKT300 was demonstrated in multiple Xenograft, Syngeneic and PDX models in mice. In-vivo treatment with BKT300 resulted in preventive (small tumors) as well as therapeutic (large tumors) activity in triple negative breast cancer (TNBC) xenograft, MDA-MB-453 and MDA-MB-468 models leading to over then 70%-90% reduction in tumor size following one to two treatments cycles. Similar results were obtained using PDX models derived from patients with Colon, Ovarian and Pancreatic cancer. BKT300 is a novel, first in class, targeted anti-cancer treatment that inhibits the activity of the scaffold protein PRC1. BKT300 is expected to provide a new treatment option to, the high unmet medical need of, advanced difficult to treat cancers, specifically ones that overexpress PRC1 and CDC25C. Citation Format: Amnon Peled, Michal Abraham, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon. Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway [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 674.

Abstract 2508: EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies

Abstract Background: Glioblastoma (GBM) is characterized as the most aggressive and lethal primary brain tumor. One of the hallmarks of GBM tumor growth and progression lies within the proangiogenic tumor microenvironment. Current anti-angiogenic drugs such as the anti-VEGF antibody take effect by inducing tumor cell starvation, leading to a functional normalization of the tumor vasculature. However, the survival benefits of anti-angiogenic drugs have been very limited. To this end, we hypothesized that the tetraspan Epithelial Membrane Protein-2 (EMP2) may help mediate resistance. Objective: We hypothesize that Epithelial Membrane Protein-2 may serve as a targetable biomarker for anti-VEGF resistance. We further explored the effectiveness of combination therapy in SB28 and GL261 GBM mouse models using anti-VEGF drug, Aflibercept, with a novel anti-EMP2 monoclonal antibody (mAb). Results: Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF2a using standard immunohistochemistry. Patients had banked tumor tissue both prior to and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. Similarly, HIF-2a showed increased expression post-bevacizumab treatment and showed expression in both tumor and stromal cells. Limited animal models exist for anti-VEGF therapy for GBM. To establish syngeneic models, 10 week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. To next investigate if this model could recapitulate the response obtained patients tumors, formalin fixed paraffin embedded tissue was analyzed for both EMP2 and HIF-2a expression. Both markers were significantly upregulated in Aflibercept treated samples compared to the control.To next investigate if combination treatment could produce a significant reduction in tumor load, animals were treated with a combination treatment with Aflibercept and anti-EMP2 mAb. The combination therapy significantly reduced tumor volume and weight in comparison to control-treated tumors. Combination treatment in SB28 tumors resulted in a two fold reduction in tumor load and 50% reduction in tumor weight. Moreover, tumors showed reduced Ki67 expression. Similarly, treated GL261 tumors produced a 2.3 fold reduction in tumor size and ~2.7 fold reduction in tumor weight. Our results suggest that EMP2 can be a promising new target therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients. Citation Format: Brian Aguirre, Mahlet Mekonnen, Riddhi Mehta, Meera Suresh, Anubhav Chandla, Karam Han, Isaac Yang, Madhuri Wadehra. EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies [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 2508.

Abstract 6649: Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with few therapeutic options. Tumor Treating Fields (TTFields) are low-intensity electric fields that disrupt cellular processes critical for cancer cell division and tumor progression and are approved for the treatment of glioblastoma and pleural mesothelioma. A phase 3 clinical trial is in progress to test the efficacy and safety of TTFields concomitant with standard-of-care gemcitabine and nab-paclitaxel (GnP) as front-line treatment of locally advanced PDAC. TTFields have been shown to induce immunogenic cell death (ICD) in cancer cells and elicit a systemic anti-cancer immune response. Accordingly, the benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in patients with non-small cell lung carcinoma. This preclinical study investigated TTFields-induced immune responses in PDAC. Methods: An orthotopic PDAC tumor model was established by inoculating 4 × 105 Panc02 pancreatic cancer cells with stable luciferase expression into the pancreas tail of immune-competent C57BL/6 mice. The mice were randomized into four groups following bioluminescence detection of tumor volume: control, TTFields (150 kHz, 10 days), GnP (50 mg/kg for each drug, i.p. injected once a week for a total of two administrations), and GnP + TTFields. At the end of treatment, tumor volume was measured using caliper, and blood was collected for spectral flow cytometry-based immunophenotyping and ELISA testing for the ICD marker HMGB-1. In vitro, Panc02 cells were treated for 72h with TTFields (150 kHz, 1.62 V/cm RMS) and examined by flow cytometry for apoptosis and ATP secretion (using quinacrine staining) and surface exposure of calreticulin. Results: TTFields markedly reduced the tumor size in Panc02 tumor-bearing mice compared to control mice. Analysis of peripheral blood effector (CD62L-CD44+) and central (CD62L+CD44+) memory T cells demonstrated the induction of circulating effector-memory cytotoxic CD8+ T cells. Additionally, serum HMGB-1 concentrations were significantly elevated in mice treated with TTFields compared to control mice, implying the involvement of ICD in the response to TTFields. In vitro, TTFields application induced apoptosis of Panc02 cells and increased ICD, as indicated by increased surface exposure of calreticulin and ATP depletion. When TTFields were used in Panc02 tumor bearing mice concomitantly with GnP, a substantial reduction in tumor size was detected, and a complete tumor regression was observed in a number of mice. Conclusions: Our results suggest that TTFields may enhance the effects of standard-of-care chemotherapy for the treatment of PDAC. Furthermore, TTFields were shown to induce ICD and activate an antitumor immune response in PDAC, supporting future studies evaluating the efficacy of TTFields together with ICI in PDAC. Citation Format: Tal Kan, Tharwat Haj Khalil, Yiftah Barsheshet, Tali Voloshin, Avital Vorontsov, Boris Brant, Lilach Koren, Roni Blatt, Shay Cahal, Efrat Zemer-Tov, Rom Paz, Anat Klein-Goldberg, Catherine Tempel Brami, Sara Jacobovitch, Alexandra Volodin, Bella Koltun, Cfir David, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model [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 6649.

Abstract 6318: Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities

Abstract Purpose: Driven by a relentless unmet need with unsatisfied cancer therapy, we aimed to develop chimeric antigen receptor T-cell (CAR-T) therapy for targeting advanced gastric cancer (AGC) expressing PD-L1. We constructed a 2nd generation CAR lentiviral vector expressing a unique anti-PD-L1 scFv with moderate affinity (VaxCAR001) in order to compare scFvs derived from Avelumab or Atezolizumab. This project focused on the efficacy and safety of PD-L1 CAR-Ts, which are affected by their binding affinity and dissociation with the target protein, PD-L1 during the treatment period. Methods and Results: The findings of this study revealed significant differences among PD-L1-targeting CAR-T cells with different affinities and dissociations. VaxCAR001 cells, showing intermediate affinity, demonstrated a higher CD8 ratio (up to 78% compared to 58% in Avelumab-derived and 45% in Atezolizumab-derived CAR-T), reduced expression of inhibitory markers including Tim-3, LAG-3, and TIGIT, (8%, 39% and 13% in VaxCAR001 vs 35%, 55% and 42 % in Avelumab, 19%, 51% and 34% in Atezolizumab scFv-derived CAR-T, respectively) and more sustained cytotoxicity. Notably, it maintained strong cytotoxicity even one month after production, while the Avelumab or Atezolizumab ScFv-derived CAR-T cells did not. In multicellular spheroid 3D co-culture with CAR T cells, VaxCAR001 exhibited superior infiltration into the core of spheroid and induced higher CAR-T-mediated killing for 3D tumor spheroids as indicated by LDH assay (68%, 800% increased than Avelumab or Atezolizumab scFv-derived CAR-T). Moreover, in an MSI-high AGC mouse model, treatment with VaxCAR001 cells resulted in a significant reduction in tumor size and prolonged survival until the end of the study period. On the other hand, treatment with the Avelumab or Atezolizumab scFv-derived CAR-T cells led to high mortality, with most mice succumbing within three weeks after CAR-T cell injection, suggesting an on-target off-tumor effect. Conclusion: This study demonstrates that VaxCAR001 cells bearing intermediate affinity and high dissociation rate for target PD-L1 showed the superior killing of PD-L expressing tumor cells in vitro and in vivo and manifested better safety profiles, as compared to CAR-T cells bearing scFv derived from commercial anti-PD-L1 targeting monoclonal antibodies such as avelumab and atezolizumab. VaxCAR001 revealed extended cytotoxicity, enhanced infiltration into tumor spheroids, and greater tumor size reduction in in vivo models. Importantly, it also demonstrated superior safety profiles, as evidenced by reduced expression of inhibitory markers and fewer off-tumor adverse effects. These findings would accelerate the clinical stage developments as an effective and safer treatment strategy against AGC. Citation Format: Minwoo Baek, Jae-In Yu, Eun-Ji Choi, Yeojin Lim, Jiwon Yang, Se-Ryeong Choi, Yoonjoo Choi, Mihwa Kim, Joon Haeng Rhee, Je-Jung Lee. Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities [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 6318.

Abstract 6663: Innovative CPMV immunotherapy: A canine model for poor-prognosis breast cancer treatment

Abstract Cowpea Mosaic Virus (CPMV) is a plant comovirus non-infectious to humans or animals. In mice models, intratumor immunotherapy (ITI) with CPMV devoid of nucleic acids (empty CPMV, eCPMV) and full CPMV results in a highly potent, systemic, antitumor immune response, with full CPMV being much more effective. Dogs with spontaneous mammary (breast) cancer (MC) serve as a valuable model for studying breast cancer (BC), bridging the gap between studies in rodents and human clinical trials. Canine MC develops in the presence of an intact immune system and has a unique advantage of shorter endpoint periods compared to women BC clinical trials. New immunotherapeutic approaches are necessary for poor-prognosis BC, such as triple-negative BC, metastatic BC, and the very aggressive and lethal inflammatory BC (IBC). Previous studies by our group using eCPMV ITI in dogs with MC revealed a substantial reduction in tumor size in both injected and non-injected tumors (abscopal effect), extended survival, and improved quality of life without any adverse effects or toxicity. In this clinical trial, we employed full CPMV nanoparticles ITI in dogs with poor-prognosis (inoperable) MC, comparing them with untreated controls. Regular tumor volume assessments and clinical evaluations have been conducted. Histopathology and immunohistochemistry of immune cell populations were performed to study tumor microenvironment (TME) modifications. Thus far, 5 dogs have been treated: four cases of inflammatory mammary cancer (IMC) and one case of metastatic MC. After at least two weekly CPMV ITI sessions, a significant reduction in tumor volumes was observed. In IMC cases, marked clinical improvement of typical signs (warmth, edema, erythema, and cutaneous pain) was observed in 3 out of 4 cases, and tumor reduction was so remarkable in one dog that surgery was recommended and successfully carried out. In IMC CPMV ITI cases, the survival of 2 dogs exceeded historical IMC records; the dog with metastatic MC maintains stable disease after one year, which is also superior to controls. TME studies showed that eCPMV immunotherapy significantly increased intratumor inflammatory infiltrates, neutrophils, T lymphocytes, and macrophages. Additionally, Treg cells (FOXP3+)/T lymphocytes (CD3+) ratio was significantly reduced. Our results demonstrate that immunotherapy with eCPMV induces a potent immunostimulatory effect on dogs with poor-prognosis MC by recruiting and activating neutrophils as the main drivers of the inflammatory response. This process reduces the immunosuppressive action of regulatory T lymphocytes and produces a breakdown of tumor immune suppression. These findings underscore the therapeutic potential of CPMV nanoparticles ITI in the management of canine IMC and metastatic MC. From a translational point of view, this study lays the groundwork for advancing the treatment of human breast cancer. Citation Format: Guillermo Valdivia, Dolores Pérez-Alenza, Lucía Barreno, Ángela Alonso-Diez, Jessica Fernanda Affonso de Oliveira, María Suárez-Redondo, Steven F. Fiering, Nicole F. Steinmetz, Laura Peña. Innovative CPMV immunotherapy: A canine model for poor-prognosis breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6663.

Abstract 4225: Analysis of the landscape of cancer-associated neurons (CANs) in diverse cancers through retrograde tracing and single cell molecular profiling

Abstract Hyperinnervation emerges as a common hallmark in the tumor microenvironment (TME) of most cancers, with clinical studies establishing a direct link between increased innervation and poor prognosis. Despite the acknowledged pivotal role of the TME, previous molecular profiling excluded tumor-infiltrating neurons due to the unique anatomical characteristics of peripheral neurons. While their axons extend into tumor masses, their DNA- and mRNA-containing cell bodies reside in adjacent ganglia, rendering them absent from virtually all large data sets analyzing tumor material. To fill this critical gap of the role of neurons for cancer growth, therapy resistance and metastasis, we developed a multi-layered approach to unravel the molecular basis of the neural influence in a.o. pancreatic (PDAC), colon cancer and melanoma including their metastasis. First, we unveiled a complex neural network within tumors and metastases through tissue clearing and 3D imaging of patient-derived xenografts, GEMMs, and human samples. This approach was complemented by the introduction of 'Trace-n-Seq,' a novel methodology integrating retrograde axonal tracing, FACS analysis, and single-cell RNA sequencing of neurons innervating both healthy organs and tumors. Expanding this technique unveiled the molecular profiles of over 3000 individual sympathetic, parasympathetic, and sensory neurons infiltrating various tumors, metastases, and pancreatitis, which were compared to neurons innervating healthy organs. Thereby we identified novel neuronal subtypes and observed tumor-induced neuronal reprogramming. Our single-cell data, when integrated with scRNA sequencing of PDAC, unveiled comprehensive neuronal interactions. An interactome analysis identified key genes in tumor/stroma-nerve signaling. To address the functional consequence of hyperinnervation of PDAC tumors, we show data demonstrating that surgical or pharmacological denervation of tumors caused a reduction in tumor size, concomitant with diminished stromal and increased immune compartments. Our data also suggest that the anti-cancer activity of paclitaxel -not oxaliplatin- was partially dependent on its ability to directly target CANs. In fact, in a neoadjuvant setting, PDAC patients treated with Gemcitabine/nab-paclitaxel, but not FOLFIRINOX, displayed a reduction in neuronal structures, underscoring the clinical significance of these observations. In summary, our comprehensive approach has elucidated the role of neurons in PDAC, melanoma, pancreatitis, and metastasis, leveraging innovative tools for studying the molecular programs of CANs at the single-cell level. These insights not only unveil novel mechanisms orchestrated by neuronal activity but also pave the way for imminent clinical trials, aiming to combat PDAC by targeting cancer-associated neurons. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Daniel Azorin, Nicolas Dross, Albrecht Stenzinger, Sebastian Schölch, Frank Winkler, Martin Sprick, Andreas Trumpp. Analysis of the landscape of cancer-associated neurons (CANs) in diverse cancers through retrograde tracing and single cell molecular profiling [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 4225.

Abstract 478: Targeted combination nano-drug delivery system to enhance anti-cancer efficacy and reduce side effects

Abstract Background: Colorectal cancer (CRC) is a global health concern, with significant morbidity and mortality. Oxaliplatin (OXL) is a common treatment for CRC but its effectiveness is hampered by side effects, including gastrointestinal and neurotoxic effects. APX3330 exerts anti-angiogenic and neuroprotective effects. It is proposed that the combined use of OXL and APX3330-loaded nanoparticles (NPs) would improve drug delivery to cancer cells with enhanced therapeutic efficacy while reducing side effects of OXL. Methods: We developed dual-loaded NPs containing OXL and APX3330 linked to a cancer targeting monoclonal antibody (NPs+mAb). We characterized the physicochemical properties of the NPs and evaluated their cytotoxicity and drug release using CRC cell lines. In vivo, we assessed their therapeutic efficiency and side effects using an orthotopic CRC mouse model. Results: NPs exhibited an average size of 229±26 nm with a zeta potential of -24.1±2.1 mV. The drug loading and encapsulation efficiency were 3.3±0.2% and 71.9%, respectively. Morphological analysis via scanning electron microscopy confirmed NP structure. In vitro drug release studies showed a slower release profile from NPs compared to free OXL, with a cumulative release of 34% (w/w) after 48 hours. Notably, cytotoxicity evaluation on CT-26 cells revealed a significant reduction in the IC50 for NPs compared to free APX3330 and OXL. In vivo studies demonstrated a remarkable 31% reduction in tumor growth rate in animals treated with NPs+mAb compared to those receiving free forms of OXL and APX3330. Body weight for animals treated with NPs+mAb remained similar to the healthy group, indicating minimal systemic toxicity. Moreover, the length of the colon in healthy organs remained steady compared to animals treated with OXL, which experienced a reduction in colon length. Animals treated with the free form of OXL exhibited diarrhoea, while the stool water content for animals treated with NPs+mAb remained at a similar level as the healthy group, suggesting reduced gastrointestinal side effects. Conclusions: This study demonstrates the potential of NPs with improved retention and encapsulation efficiency for effective drug delivery. Our research validates our novel method for creating NPs with mAb conjugation, enhancing cytotoxicity against CRC cell lines. In conclusion, the designed NPs+mAb formulation improves OXL treatment efficacy, providing targeted delivery of anti-cancer drugs to the tumor site and reducing tumor size while minimizing side effects. Citation Format: Niloufar Rashidi, Majid Davidson, Vasso Apostolopoulos, Mark R. Kelley, Kulmira Nurgali. Targeted combination nano-drug delivery system to enhance anti-cancer efficacy and reduce side effects [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 478.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p<0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p<0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q<0.05). IPA results showed significant (p<0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p<0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p<0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [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 6832.

Abstract 7117: Convection enhanced delivery of a novel ATR inhibitor synergizes with systemic lomustine for improved treatment of glioblastoma

Abstract Developing effective strategies for treating glioblastoma (GBM), the most common and aggressive form of brain cancer, has been a long-standing challenge. Despite rigorous treatment regimens including surgical resection with concomitant radiotherapy and chemotherapy, the prognosis for this disease remains poor, largely due to tumor recurrence, resistance to conventional therapeutics, pharmacokinetic and brain delivery challenges, and dose-limiting toxicities of existing therapies. The DNA damage response and DNA repair mechanisms such as the ataxia telangiectasia and Rad3-related (ATR) pathway are key mediators of therapeutic responses, and recent studies have shown that targeting DNA repair proteins alongside standard-of-care options is a promising anti-tumor strategy. Here, we employ a combination approach using a next-generation macrocyclic ATR inhibitor, ATRIN-333, to sensitize GBM tumors to alkylators. We establish the potency of ATRIN-333 and report a robust synergistic interaction between it and the alkylator lomustine in GBM cell-based assays and in a murine flank tumor model. This combinatorial treatment was then applied to a mouse intracranial tumor model, where we utilized a convection enhanced delivery (CED) system combined with nanoparticle technology for direct intracranial administration of ATRIN-333. ATRIN-333 was successfully encapsulated into a novel polymeric nanoparticle formulation using poly(ethylene glycol)-co-poly(ethylene brassylate-co-dioxanone) (PEG-EB-co-DO). CED of both free and encapsulated ATRIN-333 potently sensitized intracranial GBM tumors to low-dose (5 mg/kg) systemically-delivered lomustine, reducing tumor size and conferring a substantial survival benefit compared to cohorts treated with lomustine or the ATR inhibitor alone. The combination approach was well tolerated by the mice, with no hematological toxicity observed as assessed by bone marrow analysis and blood cell counts. Our results demonstrate that ATR inhibitor/lomustine combination therapy, used in conjunction with a CED platform, is a powerful avenue for GBM treatment and a viable option for potential clinical implementation. Note: AJ and TL contributed equally to this work. Citation Format: Alexander Josowitz, Teresa Lee, Ranjini K. Sundaram, Jinal Pothupitiya, Eric J. Brown, Amrita Sule, Jason Beckta, Yongheng Wang, Joseph Vacca, Oren Gilad, Ranjit S. Bindra, W Mark Saltzman. Convection enhanced delivery of a novel ATR inhibitor synergizes with systemic lomustine for improved treatment of glioblastoma [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 7117.