Tumor response to a mastermind inhibitor protein.

Background: MDNA11 is an albumin-fused ‘beta-enhanced not-alpha’ IL-2 superkine that preferentially expands and activates immune effector cells over immune suppressive Tregs. MDNA11 effectively inhibited subcutaneously implanted syngeneic tumors with complete tumor regression and a potent memory response. Mice treated with MDNA11 demonstrated increased tumor infiltration of CD8+ T and NK cells and elevated circulating memory and antigen-specific CD8+ T cells. Given these results, we hypothesize that preconditioning of immune cells against cancer cells by MDNA11 prior to tumor resection can prevent metastasis and prolong survival. This study compared the efficacy of MDNA11 in neo-adjuvant and adjuvant settings in an aggressive orthotopic breast cancer model prone to distant metastasis following tumor removal. Method: Female Balb/c mice with 4T1.2 tumors implanted in mammary fat pads were treated with a single dose of MDNA11 prior to or following tumor resection and survival was monitored. Excised tumors are being analyzed for immune cell infiltration. Surviving mice underwent re-challenges by subcutaneous implanting 4T1.2 cells without any additional treatment. Necropsy was performed to identify gross metastasis. Results: All control mice that had tumors resected but received no treatment died by study day 54 with gross metastasis in multiple organs. In the adjuvant group where mice received a single dose of MDNA11 following tumor resection, 3 of 8 (37.5%) survived to study end (study day 134). Of the 5 mice in the adjuvant group that died while on study, 4 had distant metastasis. In contrast, of the 8 mice that were administered a single dose of MDNA11 prior to tumor resection, 7 (87.5%) survived to study day 134 with no observable clinical symptoms. The single mouse that was found dead had no metastasis. Remaining mice in the neo-adjuvant (n = 7) and adjuvant (n = 3) were re-challenged at day 65 by implanting 4T1.2 cells subcutaneously in their flanks. A second re-challenge was performed on study day 98. Mice did not receive any additional treatment and on both re-challenge occasions there was no tumor growth or metastasis whereas control naïve mice that were also implanted with 4T1.2 cancer cells showed robust tumor growth at the implanted site. Conclusion: A single dose of MDNA11 in a neo-adjuvant setting confers immediate and long-term protection from metastatic disease in the 4T1.2 orthotopic breast cancer model. Citation Format: Abdalla Sheikh, Nina Merchant, Aanchal Sharma, Fahar Merchant, Minh D. To. Neo-adjuvant administration of MDNA11, a long-acting IL-2 Superkine, prevents metastasis, protects against tumor rechallenges and provides long-term survival in an orthotopic model of breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-06-28.

Proteinases, including matrix metalloproteinases (MMPs), contribute to cancer progression and other pathologies. Selective MMP expression can be used to distinguish benign from malignant tumors and identify aggressive tumors associated with poor outcome. MMP9, a basement membrane-degrading type-IV collagenase/gelatinase, is associated with tumor invasion and metastasis. In this project, we describe a new class of dendritic nanoparticles, nanodendrons (NDs), with MMP molecular recognition and targeting capabilities. These NDs can be studied as individual dendrons tuned for specific functions such as enhanced imaging or targeted drug treatments. Additionally, the NDs can be coupled to facilitate multifunctional purposes such as in NDs that can self-report drug delivery to tumors. The prototypical system presented here describes NDs that are activated by MMP9: 1) ND-PB, a near infrared imaging beacon; 2) ND-PXL, a therapeutic that delivers paclitaxel (PXL) and 3) NDPB-NDPXL, a bi-functional agent. In vivo studies in two orthotopic models of breast cancer demonstrate efficacy of these NDs to image and treat breast cancer. The proteinase-activated prodrug, NDPXL, delivers PXL to breast cancer through release of the drug in the tumor microenvironment and increases therapeutic efficacy while reducing systemic toxicity (including peripheral neuropathy). The delivery of PXL using the proteolytically activated ND-PXL is effective in inhibiting tumor growth in two orthotopic models of breast cancer (PyVT-R221A and MDA-MB231). Daily treatment of MDA-MB231 tumors with 12.5 mg/kg PXL as either ND-PXL or Abraxane® (Abx), showed similar reduction in tumor growth as compared with vehicle-treated animals. Further investigation of the NDPXL in a fully immunocompetent mouse model (PyVT-R221A) with treatments given on alternate days at a dose of 12.5 mg/kg (ND-PXL or Abx) yielded similar results: an average reduction in tumor growth of 58% and 53% in ND-PXL and Abx cohorts, respectively. Peripheral nerve toxicity, a debilitating, long term side effect of Abx therapy, was assessed in both tumor and non-tumor mice through monitoring behavior indicative of peripheral nerve damage before, during and after administration of each drug. Peripheral neurotoxicity is markedly reduced in the ND-PXL-treated versus Abraxane®-treated mice as evident in a number of behavioral assessments. The development of this novel class of NDs expands upon the current capabilities of modern proteinase-based optical beacons and prodrugs and is a step forward in treatment of both primary and metastatic cancer. [Supported in part by Susan G. Komen for the Cure®] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1947. doi:1538-7445.AM2012-1947

Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts. Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 × 106 + 1.63 × 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 × 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays. Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX. Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Ingrid Rankin, Gregory D. Lyng, Stephano Cairo, Katie Pedrick, Alexandra Kury, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 633.

Ultrasound contrast agent (UCA) based molecular imaging is a rapidly growing field in biomedical research. Ultrasound (US) is a non-radiative imaging modality that is frequently used to investigate various human diseases in the clinic. US molecular imaging is a novel imaging modality that utilizes targeted contrast agents to add molecular information about the imaging target, including vascular biomarkers of cancer. Pre-clinical animal models are commonly used to evaluate and validate targeted contrast agents before extending them for FDA-approval. Key impediments in the preclinical evaluations are the short in vivo half-life of microbubbles (MBs) (less than 30 minutes), limited transducer scanning area, and focusing one target at a time, which all make this strategy low throughput. Moreover, whole-body imaging strategy is not available for US imaging compared to other imaging modalities, such as PET, CT, and MRI. To overcome these imitations, we have developed a multi-mouse US imaging platform using an Automated Breast Volume Scanner (ABVS) system. In addition, we have synthesized a novel breast cancer vascular-specific B7-H3 targeted microbubbles (TMBs) using a microfluidic device. We tested these TMBs in a genetically engineered transgenic mice model [FVB/N-Tg (MMTV-PyMT)634Mul/J] (n=32) that develops breast cancer in the mammary fat pad. The US imaging results of conventional Vevo 2100 system indicated that B7-H3-TMBs could provide target specific contrast signals in the tumor vasculature compared to control MBs. We used this MBs for whole-body imaging using an ABVS system with two mice simultaneously in a stage. We imaged multiple tumors (10 tumors/mouse, 2 mice per scan, n=20 tumors) with a single bolus injection of TMBs per mouse. The results successfully showed tumor specific contrast signals from tumors independent of tumor size. Introduction of this whole-body scanner, and its ability to scan multiple mice simultaneously allows rapid imaging of multiple tumors with 3D quantification of tumor size by administering a single TMB injection enabling without significant contrast loss. Our approach makes this US-MI as high throughput and can be used to test multiple contrast agents for investigating different aspects of biological problems including screening of vascular biomarkers of human diseases. Citation Format: Arutselvan Natarajan, Farbod Tabesh, Dongwoon Hyun, Ramasamy Paulmurugan, Jeremy Dahl. Simultaneous whole-body ultrasound contrast imaging: a novel high throughput preclinical platform for targeted molecular imaging studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB115.

Notch signaling mediates numerous developmental cell fate decisions in organisms ranging from flies to humans, resulting in the generation of multiple cell types from equipotential precursors. In this paper, we present evidence that activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof (Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000) Dev. Biol., 218, 326-340). The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Here we show that Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicated that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreased myocardial gene expression and increased expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling had the opposite effect. Activation or inhibition of Notch also regulated contribution of individual cells to the myocardium. Importantly, expression of Nkx2. 5 and Gata4 remained largely unaffected, indicating that Notch signaling functions downstream of heart field specification. We conclude that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates.

We describe here, a whole-body imageable spontaneous metastatic model of human prostate cancer developed by surgical orthotopic implantation (SOI) and visualized by red fluorescent protein (RFP) expression. Human prostate cancer PC-3 cells were transduced with the pLNCX2-DsRed-2-RFP retroviral vector containing the RFP and neomycin-resistance genes. A stable RFP-expressing PC-3 clone was selected in 800 microg/ml G418 in vitro and injected subcutaneously in nude mice. Stable high-level expression of RFP was maintained in the subcutaneously-growing tumors. To utilize RFP expression for metastasis studies, fragments of the subcutaneously-growing tumor, which were comprised of RFP-expressing cells, were implanted by SOI in the prostate of nude mice. Primary tumor growth, progression, and subsequent lymphatic metastases were visualized in live, intact animals in real time by whole-body RFP fluorescence imaging. In total, 100% of the experimental animals developed lymphatic metastasis, the growth of which was monitored in real time by whole-body imaging. The aggressive lymphatic metastasis in this model reflects one of the major metastatic routes of prostate cancer in human patients. Intravital RFP imaging visualized single cancer cells in the lung and bladder. Open RFP imaging at autopsy visualized extensive primary growth and highly disseminated lymph-node metastases. The long-wavelength emission of RFP enabled high sensitivity and resolution of microscopic tumor growth using appropriate imaging techniques. The model should be useful for the real-time evaluation of novel therapeutics for metastatic prostate cancer.

In animals, pigment cells are essential for coloration, as seen in feathers, fur, skin and eyes. In mammals, neural crest-derived melanocytes constitute the major population of pigment cells that is located mainly in the skin epidermis, as well as in hair follicles. A minor population of pigment cells originates from the optic cup and forms the retinal pigment epithelium (RPE), a cell monolayer lying between the neural retina and the choroid. In many cell types, the Notch signaling pathway regulates diverse biological processes such as cell fate decision, differentiation, stem cell maintenance or apoptosis. In my thesis project, the role of this pathway was investigated using Notch1, Notch2 and RBP-Jκ conditional knockout mice. Disruption of the Notch pathway by inactivation of Notch1 and Notch2, or RBP-Jκ in the melanocyte lineage using Tyr::Cre transgenic mice resulted in a gene dosage-dependent precocious hair graying, with the most pronounced effect seen in mice lacking both Notch1 and Notch2 in melanocytes. Histologically, this coat color dilution was explained by the progressive elimination of melanocytes and melanocyte stem cells, identified in skin or embryos by X-Gal staining (Dct::LacZ reporter mouse line) and immunofluorescence analyses. As in neural crest-derived melanocytes, members of the Notch signaling pathway are normally expressed in pigment cells of the RPE. The RPE-restricted deletion of RBP-Jκ using Tyrp1::Cre transgenic mice resulted in a microphthalmic eye phenotype and thus indicated a role of the Notch pathway in the development of the RPE. Further analyses demonstrated that misregulation of Notch signaling in RPE induced a reduction in the number of these pigment cells. In addition to these knockout studies, I have generated transgenic mouse lines that express the constitutively activated intracellular part of Notch (NotchIC) in melanocytes (Dct::NotchIC) and RPE cells (Tyrp1::NotchIC). Since these mice rescue the phenotypes observed in the conditional Notch receptor knockouts, they are instrumental to assess the implication of active Notch signaling in pigment cell development and tumorigenesis. Moreover, it might be determined whether constitutive activation of Notch can prevent physiological hair graying and loss of stem cells. In summary, my results underline the importance of fine-tuned Notch signaling for proper hair pigmentation and, more generally, for pigment cell development.

Combined Radiation and Anti-CTLA4 Induces a CD8+ Immune Response in an Orthotopic Mouse Model of Breast Cancer Which is Detectable with CD8 ImmunoPET at an Early Timepoint after Treatment

Intestinal crypts are maintained by long-lived intestinal stem cells (ISCs) which reside near the base of glands. Above the ISC pool, there are short-lived progenitors that can supply lineage-specific differentiated cell types into the villus. Notch and Wnt pathways play a key role for determining the stem/progenitor cell function and their cell fate, and regulating cancer development in the gastrointestine. Although it has been well established that ISCs are a major origin of intestinal cancer, it remains unknown whether intestinal progenitor population can give rise to cancer. We use Mist1-CreERT mice and induce Notch and Wnt activation by mating with LSL-Notch1-IC mice and/or Apc flox mice. We found that a bHLH transcriptional factor Mist1 is expressed in Paneth cells (Lysozyme+CD24high) as well as short-lived secretory progenitors (Lysozyme-CD24low) in the intestine. Mist1+ secretory progenitors are radio- and chemo-resistant, but do not show stem cell interconversion even after intestinal injury or Lgr5 ablation. However, aberrant Notch activation changes Mist1+ cells to Lgr5+ long-lived enterocyte progenitors, with loss of secretory lineage differentiation. Mist1+CD24low progenitor population can form intestinal organoids with Notch activation in vitro. Mist1+ secretory progenitors can give rise to intestinal cancer by simultaneous Notch and Wnt activation by loss of Apc gene, while loss of Apc in Mist1 lineage alone does not develop cancer. In the colon, Mist1 marks colonic secterory progenitors that become to lineage trace and can be a cancer-initiating cell after Notch activation or DSS-induced colonic injury. These results provide the clear evidence of cellular plasticity dependent on Notch signaling in the gut, and suggest short-lived progenitors as another cellular origin of cancer besides ISC pool. Citation Format: Yoku Hayakawa, Kosuke Sakitani, Woosook Kim, Yagnesh Tailor, Karan Nagar, Kazuhiko Koike, Samuel Asfaha, Timothy C. Wang. Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 912.

The effect of selective and non-selective cyclo-oxygenase inhibition on tumour growth and metastasis in an orthotopic model of breast cancer was investigated. 4T1 mammary adenocarcinoma cells were injected into the mammary fat pad of female BALB/c mice. When tumours reached a mean tumour diameter of 8.4±0.4 mm, mice were randomised into three groups (n=6 per group) and received daily intraperitoneal injections of the selective cyclo-oxygenase-2 inhibitor, SC-236, the non selective cyclo-oxygenase inhibitor, Indomethacin, or drug vehicle. Tumour diameter was recorded on alternate days. From 8 days after initiation of treatment, tumour diameter in animals treated with either SC-236 or indomethacin was significantly reduced relative to controls. Both primary tumour weight and the number of lung metastases were significantly reduced in the SC-236 and indomethacin treated mice. Microvessel density was reduced and tumor cell apoptosis increased in the primary tumour of mice treated with either the selective or non-selective cyclo-oxygenase inhibitor. In vitro, cyclo-oxygenase inhibition decreased vascular endothelial growth factor production and increased apoptosis of tumour cells. Our results suggest that cyclo-oxygenase inhibitors will be of value in the treatment of both primary and metastatic breast cancer.British Journal of Cancer (2002) 87, 231–237. doi:10.1038/sj.bjc.6600462www.bjcancer.com© 2002 Cancer Research UK

Highlights from Recent Cancer Literature

The purpose of this study was to compare the expression and function of NOTCH1 in chronic lymphocytic leukemia (CLL) patients harboring a wild-type (WT) or mutated NOTCH1 gene. NOTCH1 mRNA and surface protein expression levels were independent of the NOTCH1 gene mutational status, consistent with the requirement for NOTCH1 signaling in this leukemia. However, compared with NOTCH1-WT CLL, mutated cases displayed biochemical and transcriptional evidence of an intense activation of the NOTCH1 pathway. In vivo, expression and activation of NOTCH1 was highest in CLL cells from the lymph nodes as confirmed by immunohistochemistry. In vitro, the NOTCH1 pathway was rapidly downregulated, suggesting that signaling relies upon micro-environmental interactions even in NOTCH1-mutated cases. Accordingly, co-culture of Jagged1(+) (the NOTCH1 ligand) nurse-like cells with autologous CLL cells sustained NOTCH1 activity over time and mediated CLL survival and resistance against pro-apoptotic stimuli, both abrogated when NOTCH1 signaling was pharmacologically switched off. Together, these results show that NOTCH1 mutations have stabilizing effects on the NOTCH1 pathway in CLL. Furthermore, micro-environmental interactions appear critical in activating the NOTCH1 pathway both in WT and mutated patients. Finally, NOTCH1 signals may create conditions that favor drug resistance, thus making NOTCH1 a potential molecular target in CLL.

Notch and Wnt/β-catenin pathways play essential roles in intestinal development, homeostasis and neoplasia. Citrobacter rodentium (CR) induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. In the current study, we investigated functional cross-talk between the Notch and Wnt pathways and how they regulate colonic crypt hyperplasia and/or colitis in response to CR infection. Infection of NIH:Swiss outbred mice with CR induced gross thickening and rigidity of the distal colon and crypt hyperplasia which peaked by day 12, persisted until day 27 before declining by day 34. Nuclear staining for Notch target Hes-1 increased at days 6-27 before declining at day 34. Acute (5 days) treatment with Notch blocker dibenzazepine (DBZ; @10μmol/Kg) beginning on the 1st day of CR infection followed by euthanasia at day 6 blocked both Notch and Wnt signaling and induced goblet cells hyperplasia; acute DBZ treatment beginning on the 7th day of CR infection followed by euthanasia at day 12 inhibited Notch signaling without blocking the components of the Wnt pathway: both β-catenin and its downstream target Jagged-1, a Notch ligand, increased significantly at day 12 leading to crypt hyperplasia. Chronic (10 days) DBZ treatment beginning on the 1st day of CR infection followed by euthanasia at day 12 blocked both Notch and Wnt signaling, increased epithelial permeability and induced colitis. To directly establish that the disruption of mucus gel layer overlying the gut epithelium by DBZ may be the cause of colitis in CR infected animals, we utilized mice lacking Core 3 beta1,3-N-acetylglucosaminyltransferase, an enzyme important in synthesis of Core 3-derived O-glycans, the primary component of the intestinal mucins. The Core-3−/− mice are susceptible to experimental triggers of colitis. Infection of Core-3−/− mice with CR destroyed the intestinal barrier and induced colitis which was exacerbated following DBZ treatment. The changes accrued were not due to loss of colonic stemness as DBZ did not alter putative stem cell marker DCAMKL-1 expression in either strain. Dietary bael extract (4%) and curcumin (4%) restored the Notch/Wnt cross-talk and promoted crypt regeneration/hyperplasia in CR-infected/DBZ-treated SW mice. Thus, 1. TMCH exhibits active Notch and Wnt signaling. 2. Blocking Notch signaling early induces goblet cells hyperplasia but no colitis. 3. Acute but late stage blocking of Notch signaling does not abrogate Wnt/β-catenin-induced crypt hyperplasia. 4. Chronic defect in Notch signaling also blocks Wnt pathway and induces colitis in response to CR infection due to loss of barrier function and lack of crypt regeneration despite lack of alterations in colonic stemness. Thus, both Wnt and Notch cross-talk and epithelial barrier regulate hyperplasia and/or colitis in response to CR infection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2833. doi:10.1158/1538-7445.AM2011-2833

Background: The combination of 18mg Lenvatinib and 5mg Everolimus per day is used in the clinic for advanced renal cancer patients. However, due to the severe side effects of Lenvatinib, the dose is often reduced to 12mg. Archexin, a fully phosphorothioated 20-mer antisense oligonucleotide, inhibits AKT-1 mRNA translation and inhibits tumor growth. Combination of Archexin with reduced-dosed Lenvatinib and Everolimus may reduce adverse reactions while enhancing therapeutic efficacy. Methods: First, Lenvatinib and Everolimus monotherapies were evaluated for antitumor efficacy in an orthotopic Renca-luciferase syngeneic model. 45 mice were randomly divided into 9 groups (5 in each group), vehicle control, Lenvatinib (5, 10, 20, and 30 mg/kg) and Everolimus (0.2, 0.5, 1, 2, and 4 mg/kg) via daily oral administration for 20 days. Next, we investigated the anti-tumor effect of the two- and three-drug combination. 35 mice were randomly divided into 7 groups (5 in each group), including vehicle control, Lenvatinib/Everolimus combinations (5+0.2, 10+0.2 and 10+0.5 mg/kg) and Archexin/Lenvatinib/Everolimus combinations (60+5+0.2, 60+10+0.2 and 60+10+0.5 mg/kg) via tail vein injection (vehicle/Archexin, Q3D*6 times) or oral administration (Lenvatinib and Everolimus, QD*18 days). Body weight was monitored daily. Tumor burden was measured by bioluminescence through whole-body fluorescence images, performed twice a week. Results: The results showed that Everolimus inhibited tumor growth in a dose-dependent manner. Partial response was observed in mice treated with varying doses of Lenvatinib(TGIs: 57.94%,76.65% and 60.22%), and no significant dose-effect correlation was found. The dose of Lenvatinib and Everolimus that partially inhibited the orthotopic tumor growth had also been identified and selected for subsequent study investigating drug combinations. The drugs combination study showed that three drug combinations (Archexin/Lenvatinib/Everolimus) had superior efficacy compared to the Lenvatinib and Everolimus combination. The fluorescence signal intensities (Lg10) of the three-drug combination were significantly lower than that of the two-drug combination group with P value <0.05. No severe adverse effects were observed in the three drug combinations group. Conclusion: Three drugs combination of Archexin, Lenvatinib and Everolimus, demonstrated superior anti-tumor efficacy compared to the two-drug combination currently used in the clinic. This finding suggests the three-drug combination warrant investigation in the clinical trial in the patients with advanced renal cancer. Citation Format: Fengyang Xie, Jia Wei, Lan Deng, Chao Wang, Ben Zhao, Robert J. Lee, Yuxin Angela Men. Evaluation of an AKT-1 antisense oligonucleotide in combination with Lenvatinib and Everolimus in Renca-luciferase syngeneic orthotopic murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 701.

Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy, and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively, and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo. NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity.