Bearing Mice Research Articles

Photothermal therapy of xenografted tumor by carbon nanoparticles-Fe(II) complex

Due to the unique structure, carbon nanomaterials could convert near-infrared (NIR) light into heat efficiently in tumor ablation using photothermal therapy (PTT). However, none of them has been applied in clinical treatment, because they have not been approved for clinical evaluations and the precise temperature control facility is scarce. In this study, we designed a temperature-responsive controller for PTT and used carbon nanoparticles-Fe(II) complex (CNSI-Fe) as photothermal conversion agent (PTA) for PTT of tumor in vitro and in vivo. CNSI-Fe was an innovative drug under the evaluations in clinical trials. CNSI-Fe showed excellent photothermal conversion ability in water to increase the water temperature by 40 °C within 5 min under irradiation of 808 nm laser at 0.5 W/cm2. The temperature was precisely controlled at 52 °C for both in vitro and in vivo tumor inhibition. CNSI-Fe with NIR irradiation showed higher tumor cell inhibition than CNSI. In tumor bearing mice, CNSI-Fe with NIR irradiation achieved an inhibition rate of 84.7 % and 71.4 % of them were completely cured. Mechanistically, CNSI-Fe under NIR irradiation induced the radical generation, oxidative damage and ferroptosis to kill tumor. In addition, CNSI-Fe showed good biosafety during PTT according to hematological, serum biological and histopathological examinations. These results indicated that the combination of chemotherapy and PTT provided higher antitumor efficiency using CNSI-Fe as PTA.

Preclinical in vitro and in vivo activity of the RAF/MEK clamp avutometinib in combination with FAK inhibition in uterine carcinosarcomas

ObjectivesUterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. MethodsWhole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. ResultsWES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. ConclusionThe combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.

Abstract PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-07.

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-08.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-02.

Abstract PO4-06-11: Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation

Abstract Triple negative breast cancer (TNBC) is characterized by invasiveness and poor survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. In the past 4 years, several targeted therapies for TNBC have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against other biological drivers of TNBC remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in breast cancer. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. PGRN/GP88 expression is elevated in 30% TNBC. The importance of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells was previously established. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days since the start of immunization, several fully human monoclonal antibodies were successfully developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in MDA-MB-231 cells in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited proliferation and migration in a dose-and time-dependent fashion in TNBC cells. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that several fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. In vivo dose response of AG01 in MDAMB-231 tumor bearing mice will be provided. Conclusion: PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization of mouse monoclonal antibodies. The development of fully human anti-progranulin antibodies provides novel targeted therapeutic option for TNBC. This work is supported by a an SBIR grant 5R44 CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Jun Hayashi, Chun Dong. Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-11.

Unacylated Ghrelin Protects Against Muscle Wasting, Mitochondrial Dysfunction, and Neuromuscular Junction Disruption in Tumor Bearing Mice

Background: Cancer cachexia is a complex metabolic syndrome that severely impacts mobility, treatment strategies, and life quality of the patients. However, no treatments are available to mitigate the debilitating consequences of cancer cachexia. Ghrelin is a hormone released from the stomach that increases appetite upon acylation by binding to GHSR1a receptors in the brain. Because of its orexigenic effects and increases in body weight, ghrelin GHSR1a receptor analogs were tested as clinical trials to mitigate muscle wasting and loss of strength in cancer patients in several countries. The analogs (i.e., Anamorelin) increased body weight of the non-small lung cancer patients in European countries and Japan, but failed to increase strength, which is the critical component in cancer cachexia. Mainly due to the lack of improvement in strength and patients’ quality of life, Anamorelin was not approved for clinical use in Europe. While the lack of increases in strength is, at least in part, associated with the adipogenic effects of acylated ghrelin, unacylated ghrelin directly improves satellite cell renewal, muscle growth, and mitochondrial function independent of GHSR1a receptor activation and increases in appetite. Unacylated ghrelin is the predominant population in circulation comprising proximately 90-95% of total ghrelin in humans and rodents. Hypothesis: Unacylated ghrelin projects against muscle wasting, loss of strength, and metabolic dysfunction of tumor bearing mice. Methods and Results: Supplementing the cancer cell conditioned medium (CM) with unacylated ghrelin preserved myotube size. Unacylated ghrelin also diminished the cancer cell CM-induced upregulation of MuRF-1, muscle specific ubiquitin ligase. Interestingly, unacylated ghrelin normalized transcriptional upregulation of denervation markers (i.e., sarcolipin, Runx1, GADD45a) in myotubes treated with CM. To further investigate the therapeutic effects of unacylated ghrelin in vivo, we inoculated lung cancer cell line (LL2, ATCC®) in the flank of 4 months old male mice. Two weeks after the injection prior to development of cachexia, mice were provided with unacylated ghrelin containing drinking water or untreated water as control. Tumor bearing (TB) mice treated with unacylated ghrelin showed 30-40% increases in gastrocnemius and quadriceps weights, while soleus muscle mass was unchanged. In addition to the muscle quantity, unacylated ghrelin increased force generating capacity was also improved by unacylated ghrelin. Notably, unacylated ghrelin increased mitochondrial oxygen consumption rate and protected against neuromuscular junction disruption and denervation in TB control mice. Conclusion: Unacylated ghrelin protects against cancer cachexia by targeting multiple risk factors in skeletal muscle wasting, including protein balance, metabolic alterations, and neuromuscular junction disruption. Our findings show protective effects of unacylated ghrelin against cachexia and neuromuscular impairment in tumor bearing mice, indicating its therapeutic potential in cancer patients. NIA R00 064143 to Ahn B. Pilot funds from the Center for Redox Biology in Medicine, and Cancer Genetics and Metabolism program at the Wake Forest Baptist Comprehensive Cancer Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of PHD inhibitor on tumor microenvironment and tumor immune response

Since the approval of HIF-PH inhibitors, HIF-PH inhibitors have been used clinically, and many studies and clinical case reports have been reported in Japan. A lot of information has been accumulated on clinical usage. However, HIF-PH inhibitors require careful administration for cancer patients due to their action mechanism through upregulating hypoxia-inducible factors (HIFs) level. In cancer cells, HIFs affect tumor progression and contribute to chemo- and radio-resistance. On the other hand, upregulation of HIFs in immune cells is associated with inflammation and suppress tumor progression. However, these controversial effects are not clear in in vivo model. It is needed to reveal whether upregulating HIFs level is beneficial for cancer therapy or not. We have previously reported that HIF-PH inhibitor treatment in tumor bearing mice model led to reconstitute tumor blood vessel and inhibit tumor growth. In addition, these phenomena were caused by tumor infiltrated macrophages and they altered these phenotypes. In this review, we will describe our findings on the mechanism of tumor growth suppression by HIF-PH inhibitors. We also want to mention the risks and benefits of future HIF-PH inhibitors.

Continued use of alcohol accelerates cancer cachexia and impairs ability to induce 4E-BP1 but not p70S6K phosphorylation following muscle contraction

A primary co-morbidity of cancer is cancer cachexia characterized by the wasting of adipose tissue and skeletal muscle. Up to 80% of cancer patients may suffer from this wasting condition leading to reduced quality of life, poor treatment effectiveness and increased morbidity and mortality. The risk of cancer cachexia is enhanced in those with poor lifestyle habits including the habitual consumption of alcohol both prior to a cancer diagnosis and when consumption is continued during disease progression. The use of muscle contraction to improve outcomes in cachexia has been explored in alcohol naïve models with some success, however alcohol at high doses induces anabolic resistance preventing protein synthesis in response to an anabolic stimuli like muscle contraction. Therefore, muscle contraction may not be a suffcient stimuli to overcome cachexia induced catabolism when alcohol has been or is currently being consumed. The purpose of this work was to determine if alcohol induces anabolic resistance in tumor bearing mice or whether it can be used as a therapeutic intervention. It was hypothesized that alcohol would inhibit the stimulation of markers of the anabolic mTORC1 pathway in tumor bearing mice. METHODS: Female BALB/c mice, 12 weeks of age, were randomized into 4 groups (n = 12-14/group): control diet (CON), control diet-cancer (Cancer), Prior EtOH diet-cancer (PE-Cancer), and EtOH diet-cancer (E-cancer). All mice received the isocaloric Lieber DeCarli liquid diet with EtOH mice consuming 20% kcal/daily EtOH for 6 weeks. After 6 weeks, PE-cancer switched to the non-alcohol diet and all cancer groups had C26 carcinoma cells implanted. A single bout of maximal stimulated muscle contraction (10sets of 6 repetitions at 100Hz) was performed ~2 weeks later and muscles were collected 4 hours post contraction. RESULTS: E-cancer lost ~10% body mass, while no other cancer groups exhibited a significant loss in body mass indicating early-stage cachexia. Spleen mass was elevated in all cancer groups relative to control, while quadricep and gastrocnemius muscle weights were reduced in PE-cancer and E-cancer compared to CON. Muscle contraction significantly increased the phosphorylation of S6K1 Thr389 in all of groups except for PE-cancer where a strong trend was still detected (p=0.06). In contrast, phosphorylation of 4EBP1 Ser65 was only increased by muscle contraction in CON, as cancer prevented its stimulation. Therefore, despite accelerating body weight loss, continued alcohol consumption or prior alcohol consumption did not completely impair the anabolic response to maximally stimulated muscle contraction during the early stages of cancer cachexia. However, measurement of rates of degradation and synthesis over time are required to make further conclusions as to its therapeutic potential. Florida Department of Health Grant # 9BC03. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

SP94 engineered erythrocyte membrane enhanced the targeted delivery of biomimetic nanosuspension with IDO immunotherapy and chemotherapy in liver cancer

The efficient eradication of tumor cells remains a critical challenge in the realm of liver cancer therapies. The combination of chemotherapy and immunotherapy holds more significant potential for improving treatment efficacy. However, the effectiveness of immunotherapy is mainly impeded by the high activity of indoleamine 2,3-dioxygenase (IDO), which creates an immunosuppressive tumor microenvironment via robust proliferation of immunosuppressive T regulatory cells (Tregs) and inhibition of cytotoxic T lymphocytes (CTLs). Herein, we developed SR-ATO-ASIV@NS, a liver cancer-targeting peptide (SP94) modified RBCM-coated biomimetic nanosuspension. This innovative formulation incorporates the chemotherapeutic agent arsenic trioxide (ATO) and the immunomodulator astragaloside IV (AS-IV) as IDO inhibitor for the combined chemoimmunotherapy of liver cancer. The SR-ATO-ASIV@NS increased the solubility of both ATO and AS-IV and displayed excellent stability and exhibited high drug-loading efficiency, rendering it suitable for efficient dual drug delivery. Furthermore, this biomimetic nanosuspension efficiently evaded immune response and showed specific uptake in liver cancer cells via SP94 receptor-mediated endocytosis. The remarkable pharmacokinetic profile, characterized by prolonged blood circulation, enhanced the in vivo anti-tumor efficacy in H22 tumor bearing mice through the synergistic action of the dual drugs. On one hand, the released ATO resulted in excessive reactive oxygen species (ROS) generation, which stimulated the induction of apoptosis pathways via Bax/Bcl-2 and caspase mechanism. While on the other hand, AS-IV effectively blocked the IDO activity and hinders the production of Tregs within the tumor microenvironment. Consequently, the reduced Tregs population fosters the proliferation of CTLs, alleviating ITM and intensifying the immunotherapeutic response for the efficient killing of cancer cells. Additionally, the successful inhibition of liver cancer metastasis to the lungs and robust increase in T memory cells further boosted the anti-tumor therapeutic efficacy of SR-ATO-ASIV@NS. In summary, SR-ATO-ASIV@NS represents a novel and scientifically sophisticated strategy in targeted biomimetic nanotherapeutics. This approach not only enhances site-specific drug delivery but also showcases promising potential for advancing the field of combined chemoimmunotherapy for liver cancer.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 μM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Abstract ND07: BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding

Abstract The glycine to cysteine mutation on codon 12 of KRAS (KRASG12C) is found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. This activating mutation pushes the balance of cellular KRAS towards its active, GTP-bound (ON) state that signals downstream and drives cellular proliferation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive GDP-bound (OFF) state, have demonstrated clinical efficacy; however, median duration of response has been &amp;lt; 9 months. Rapid tumor cell adaptation to KRASG12C (OFF)-only inhibitors like sotorasib and adagrasib has been attributed to reactivation of MAPK signaling through increased RTK flux and KRASG12C gene amplification that result in increased KRASG12C (ON) presence and activity. To block rapid KRASG12C (ON)-driven adaptation to KRASG12C (OFF) inhibitors, we have developed a first-in-class, direct, small molecule covalent inhibitor of both KRASG12C (ON) and (OFF) states. BBO-8520 binds in the switch II pocket and covalently modifies both the (ON) and (OFF) forms of KRASG12C independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement by BBO-8520 shows complete modification of both KRASG12C states within 15 minutes, while sotorasib and adagrasib, only modify the (OFF) state. 31P NMR studies demonstrate that BBO-8520 inhibits KRASG12C (ON) by locking the GTP-bound protein in state 1, a conformation where it is unable to bind effectors. The ability to directly bind KRASG12C (ON) leads to potent (~30 nM) inhibition in an effector (Raf1) binding assay where inactive (OFF) inhibitors demonstrate no measurable potency. BBO-8520 displays highly significant binding to KRASG12C in a global cysteine proteome analysis and is 100x more selective for KRASG12C than for WT KRAS and other mutant isoforms, with no measurable activity against N- or H-RAS. Cellular signaling and viability assays show that BBO-8520 has sub-nanomolar potency against KRASG12C mutant cell lines. In effector binding assays, BBO-8520 rapidly and completely blocks the RAS-RAF1 interaction, clearly differentiating it from inactive (OFF) inhibitors that require longer times to allow for cycling to the inactive (OFF) state. Additionally, long-term clonogenic assays that detect the emergence of resistance in the presence of inhibitors show that BBO-8520 is at least 30x more potent than sotorasib and adagrasib at preventing outgrowth. Importantly, the presence of growth factors (e.g., EGF) that push KRASG12C into its (ON) state, and significantly lower the potency of inactive (OFF) inhibitors (&amp;gt;20x), have only minor effects on BBO-8520’s potency. Drug-like pharmacokinetic properties allow BBO-8520 to achieve strong dose- and time-dependent pharmacodynamic effects (&amp;gt;80% inhibition of pERK) following a single, oral dose in KRASG12C mutant tumor bearing mice. In vivo target engagement and pERK inhibition in the MIAPaCa-2 and H358 KRASG12C mutant tumor models resulted in durable tumor regressions at 10 mg/kg. Similarly, daily dosing of 10 mg/kg of BBO-8520 in the KrasG12C-p53 driven GEMM model for 6 weeks resulted in better than 50% lung tumor volume regression. BBO-8520’s potent activity against KRASG12C (ON) presents, for the first time, the potential opportunity to directly target all mutant KRASG12C in cells, including the active form of KRASG12C, enabling efficacy following complete or near-complete target inhibition. Citation Format: Anna E. Maciag, James Stice, Bin Wang, Alok Sharma, Albert Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, Dom Esposito, John Paul Denson, Michela Ranieri, Mary Meynardie, Sadaf Mehdizadeh, Patrick Alexander, Maria Abreu Blanco, David Turner, Rui Xu, Felice C. Lightstone, Kwok Kin Wong, Dhirendra Simanshu, Keshi Wang, Andrew G. Stephen, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND07.

Abstract LB439: Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics

Abstract Background. Intra-tumoral (IT) injections of therapeutics hold promise for the treatment of solid tumors. However, drugs administered directly in the tumor have short IT residence times potentially limiting their exposure and therefore efficacy. We are developing a platform of novel hydrogel microsphere (MS) conjugates that enables long-acting tumor residence after a single injection of the conjugate. We demonstrate this approach not only solves the problem of short acting intra-tumoral therapy, but also allows for the safe administration of two therapies with overlapping toxicities such as SN-38 and a PARP inhibitor (PARPi). Methods. Hydrogel microsphere (MS) conjugates were prepared and characterized. Using MS conjugates labeled with rhodamine and fluorescein, we established methods to directly measure the IT concentration and half-life of the released drug after IT administration into CT26 tumor bearing mice. We next measured the tissue pharmacokinetics and efficacy of MSs attached to SN-38, a potent topoisomerase 1 inhibitor and immunomodulator, using NSG mice bearing 22Rv1 ATM-/- xenografts. Finally, we measured the anti-tumor efficacy of MS~SN-38 in combination with an oral PARPi, talazoparib, in 22Rv1 ATM-/- xenografts. Results. We successfully established methods to quantify the concentration of released drug and MS bound drug from tumor biopsies using fluorescent surrogates. When applied to MS~SN-38 conjugates, locally injected tissues showed high levels of SN-38 with a long half-life of about one week. In contrast, the systemic half-life of free SN-38 is ~30 minutes. IT MS~SN-38 was ~10-fold more efficacious as an anti- tumor agent than systemic SN-38 from subcutaneous administration of MS~SN-38. Finally, we show high anti-tumor synergy from the SN-38 localized in a tumor by IT MS~SN-38 in combination with systemic administration of the PARPi talazoparib. Conclusions. This microsphere technology allows for sustained delivery of high local concentrations of drugs administered intratumorally. Here, IT MS~SN-38 resulted in an intra-tumoral half-life of ~1 week and marked anti-tumor efficacy in 22Rv1 ATM-/- xenografts. We also demonstrated that long-acting IT injections enables safe use of drug combinations with well-described overlapping toxicities such as SN-38 and PARPi. The microsphere technology may offer an alternative strategy for localizing and sustaining drugs in tumors and may mitigate toxicities from combination therapy where two agents have a similar toxicological profile. Other combinations, including small molecules and immuno-oncology agents, are being explored. Citation Format: John A. Hangasky, Jeff Henise, Deborah Charych, Christopher W. Carreras, Gary Ashley, Daniel V. Santi. Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB439.

Abstract LB281: A preclinical therapeutic study of minibody 177Lu-DOTAGA-IAB56 targeting αVβ6 for the treatment of pancreatic cancer

Abstract Pancreatic cancer represents 3% of all cancers and 7% of all cancer deaths in the USA, with a majority of cases diagnosed late and with limited treatment efficacy. Radiopharmaceutical Therapy (RPT) using antibodies (Abs) is emerging as a new approach for the treatment of solid cancers. Due to the long circulation time in blood, full-length antibodies bear concerns for hematological exposure to radioactivity. Antibody fragments with lower molecular weight like minibodies (Mb, 80 kDa) show rapid blood clearance, thus reducing off-target exposure, while maintaining the high target affinity typical of immunoglobulins. Mbs consist of two scFv antigen-binding domains fused to the CH3 domain of human IgG. Integrin αVβ6 is mainly absent in healthy adult tissues but upregulated in a variety of carcinomas, including pancreatic cancer. In this preclinical proof of concept study, we evaluated a Mb targeting human αVβ6 (IAB56) as an RPT agent for the treatment of pancreatic cancer. To this end we first evaluated the β-emitter Mb 177Lu-DOTAGA-IAB56 in a dosimetry study in mice and extrapolated the organ exposures to humans. Then we tested its therapeutic efficacy in female Nu/J mice bearing the subcutaneous ductal adenocarcinoma xenograft CFPAC-1. The IAB56 Mb was conjugated with DOTAGA and radiolabeled with 177Lu. Doses of 1.48 MBq/animal were administered intravenously (iv) in non-tumor bearing mice and biodistributions collected at 0.5, 3, 6, 24, 48, 120 and 192 hours postinjection (p.i.) to quantify time course uptake at major organs. Dosimetry data extrapolated from these biodistributions showed that exposure in humans at clearance organs (kidneys and liver), had an average dose of 3.1 and 5.0 Gy which is 8 times and 6 times below the published dosimetry thresholds of 23 Gy and 32 Gy respectively. Bone marrow and the lungs, two additional radiation sensitive sites, show doses of 0.04 and 0.4 Gy, 50.0 times and 67.5 times below the tox thresholds (2 Gy for bone marrow and 27 Gy for the lungs). The spleen had an average dose of 2.2 Gy, which is 18.2 times below the threshold of 40 Gy. Preclinical RPT studies were conducted in CFPAC-1 xenografts. A pancreatic carcinoma cell-line reported to have intermediate-to-low expression level of human αVβ6 (4-fold lower than BxPC3 pancreatic adenocarcinoma). When the tumor size reached 100-150 mm3 mice were randomized into three groups: vehicle, single dose of 18.5 MBq, or two doses 11.1 MBq and 8.1 MBq 7 days apart of 177Lu radiolabeled DOTAGA-IAB56. A rapid tumor growth inhibition response for up to 36 days was observed in both RPT groups which evoked a significant improvement in survival compared to control. Overall, our data indicate the anti-αvβ6 Mb, IAB56 is efficacious for the treatment of pancreatic cancer supporting the use of this agent as a RPT approach for solid tumors. Citation Format: Leticia M. De Souza Cordeiro, Kelley C. Atkinson, Fang Jia, Argin Aivazian, Gareth E. Smith, Ian Wilson, Alessandro Mascioni. A preclinical therapeutic study of minibody 177Lu-DOTAGA-IAB56 targeting αVβ6 for the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB281.

Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade

BackgroundTigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that...

Focused ultrasound-mediated blood–brain barrier opening is safe and feasible with moderately hypofractionated radiotherapy for brainstem diffuse midline glioma

BackgroundDiffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic strategies have been unsuccessful and radiotherapy (RT) remains the standard-of-care. A central impediment to treatment is the blood–brain barrier (BBB), which precludes drug delivery to the central nervous system (CNS). Focused ultrasound (FUS) with microbubbles can transiently and non-invasively disrupt the BBB to enhance drug delivery. This study aimed to determine the feasibility of brainstem FUS in combination with clinical doses of RT. We hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT.MethodsTo establish a safety timeline, we administered FUS to the brainstem of non-tumor bearing mice concurrent with or adjuvant to RT; our findings were validated in a syngeneic brainstem murine model of DMG receiving repeated sonication concurrent with RT. The brainstems of male B6 (Cg)-Tyrc-2J/J albino mice were intracranially injected with mouse DMG cells (PDGFB+, H3.3K27M, p53−/−). A clinical RT dose of 39 Gy in 13 fractions (39 Gy/13fx) was delivered using the Small Animal Radiation Research Platform (SARRP) or XRAD-320 irradiator. FUS was administered via a 0.5 MHz transducer, with BBBO and tumor volume monitored by magnetic resonance imaging (MRI).ResultsFUS-mediated BBBO did not affect cardiorespiratory rate, motor function, or tissue integrity in non-tumor bearing mice receiving RT. Tumor-bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy/13fx offered local control, though disease progression occurred 3–4 weeks post-RT.ConclusionRepeated FUS-mediated BBBO is safe and feasible concurrent with RT. In our syngeneic DMG murine model, progression occurs, serving as an ideal model for future combination testing with RT and FUS-mediated drug delivery.

Abstract 6816: Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase

Abstract Background: Cancer cachexia is a devastating syndrome characterized by progressive muscle wasting and hyperinflammation. The underlying mechanisms remain poorly understood with no treatment options available. Cachexia is driven by systemic inflammation, pro-inflammatory cytokines, and apoptotic cell death (debris). The resolution of inflammation as an active biochemical process is regulated by novel pro-resolving lipid mediators. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are lipid mediators stimulating inflammation resolution. EETs are rapidly metabolized by the enzyme soluble epoxide hydrolase (sEH). sEH is a critical cause of inflammation and a biomarker in several chronic inflammatory diseases. sEH inhibitors promote tissue regeneration and counter-regulate pro-inflammatory cytokines. We hypothesized that pharmacological inhibition of the sEH may prevent cachexia. Methods: We investigated murine cancer cachexia models using genetically engineered pancreatic (KPC) and prostate (TRAMP C1) tumor cell lines, and “TRAMP” mice (transgenic adenocarcinoma of the mouse prostate). We performed LC-MS/MS-based profiling of bioactive lipids in plasma from KPC and colon cancer (CT26)-induced cachectic mice. Results: KPC cells injected intraperitoneally induced 19.7% and 54.1% reduction in muscle weight in the tibialis anterior and soleus, respectively, in immunocompetent C57BL/6 mice compared to non-tumor bearing (NTB) mice. LC-MS/MS revealed KPC and CT26 cachexia induced a pro-inflammatory eicosanoid-driven cytokine storm. The human sEH inhibitor EC5026 delayed the onset of cachexia, leading to sustained survival over 250 days post-injection in the KPC model (n=15 mice/group). sEH expression was increased in the gastrocnemius of KPC mice by day 22 post-tumor cell injection vs. NTB. EC5026 counter-regulated sEH expression compared to vehicle-treated. In the TRAMP model, 5/5 of mice treated with EC5026 survived 230 days post-treatment compared to no survival of vehicle-treated mice (n=5). The transplanted KPC and TRAMP showed reduced CD4+ and CD8+ T lymphocytes and B lymphocytes in the tibialis anterior and gastrocnemius vs. NTB after flow cytometry analysis. EC5026 stimulated a general increase in CD3+ T lymphocytes and counter-regulated an eicosanoid-driven cytokine storm as shown by cytokine assays. EC5026 treatment increased tibialis anterior and gastrocnemius muscle weights and body weights compared to vehicle-treated KPC and TRAMP mice. Conclusions: sEH inhibition may be a novel therapeutic approach to cachexia by targeting the immune response via stimulation of resolution of inflammation without toxicity or immunosuppression. Since sEH inhibitors have proven safe and effective in clinical trials for controlling multiple inflammatory diseases, this study provides a basis for the rapid clinical translation of sEH inhibitors to prevent/reverse cachexia in cancer patients. Citation Format: Rachel L. Bayer, Sarina Virani, Michael Gillespie, Jacqueline Capuano, Keira Smith, Katherine Quinlivan, Kimberly Vasquez, Jun Yang, Haixia Yang, Nicholas Mitsiades, Bruce D. Hammock, Dipak Panigrahy. Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase [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 6816.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models [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 538.

Abstract 3847: Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian cancer

Abstract Background: Ovarian carcinomas are known to have a high degree of immune suppression, which may be related, in part, to high levels of vascular endothelial growth factor-A (VEGF-A). Bevacizumab (anti-VEGF antibody) has been shown to increase infiltration of T-cells in the tumor microenvironment (TME). We have also identified EGFL6 as an important angiogenic protein. Here, we examined the biological effects of blocking EGFL6 and VEGF-A in ovarian cancer models. Methods: We studied the in vitro (endothelial tube formation and migration assays) and in vivo (ID8KO-Trp53;Brca2 and SKOV3-ip1 models) biological effects of EGFL6 and VEGF-A blockade. Tumor-infiltrating immune cells were studied by flow cytometry and immunohistochemistry staining. Results: Blocking EGFL6 and VEGF-A simultaneously with monoclonal antibodies in RF24 endothelial cells decreased tube formation and migration significantly, in comparison to VEGF-A blockade alone (p&amp;lt;0.001). EGFL6 monotherapy in ID8KO-Trp53;Brca2 tumor bearing syngeneic mice decreased tumor burden (~2.5 fold, p&amp;lt;0.0001) and increased the infiltration of IFNγ+ (~2.75 fold, p&amp;lt;0.0001) and GzmB+ (~1.75 fold, p&amp;lt;0.01) CD8+ T-cells and IFNγ+ (~2 fold, p&amp;lt;0.0001) and GzmB+ (~2 fold, p&amp;lt;0.01) CD4+ T-cells while decreasing PD-L1+ cells (~1.8 fold, p=0.01). Dual blockade of EGFL6 and VEGF-A in SKOV3-ip1 tumor bearing nude mice significantly decreased tumor burden (~3.72 fold, p&amp;lt;0.01) and angiogenesis (~2.36 fold, p&amp;lt;0.05) compared to controls and single-agent blockade. Similar blockade in ID8KO-Trp53;Brca2 tumor bearing mice significantly decreased tumor burden (~4.2 fold, p=0.0004), improved CD8+ (~3.32 fold, p=0.0004) and CD4+ (~2.81 fold, p&amp;lt;0.0001) T-cell infiltration, NK cell infiltration (~2.3 fold, p=0.0005) and decreased myeloid-derived suppressor cells (MDSCs) (~1.92 fold, p=0.0278) compared to EGFL6 and VEGF-A blockade alone. Conclusions: Our results demonstrate that simultaneously targeting the angiogenesis drivers EGFL6 and VEGF-A in ovarian cancer significantly decreased tumor burden in vivo and increased the infiltration of T lymphocytes compared to single-agent blockade. AACR Category: Tumor Microenvironment and Immune Oncology Key words: ovarian cancer, immune suppression, angiogenesis, VEGF-A, EGFL6 Citation Format: Sujanitha Umamaheswaran, Lingegowda S. Mangala, Robiya Joseph, Nicholas B. Jennings, Mark S. Kim, Yunfei Wen, Emine Bayraktar, Ningyan Zhang, Zhiqiang An, Anil K. Sood. Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian 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 3847.