Abstract Integrin beta-6 (ITGb6) is a heterodimeric, cell-surface glycoprotein highly expressed in multiple solid tumor indications, including non-small cell lung cancer, esophageal cancer, head and neck cancers, breast and gastric cancers. Its expression has been associated with pro-tumorigenic activities including proliferation, migration, and invasion. ITGb6 has restricted expression in adult tissues, making it a highly promising target for cancer therapy using antibody drug conjugates (ADCs). STRO-006 is an investigational ADC composed of an ITGb6 targeted human IgG1 antibody conjugated to exatecan, a topoisomerase 1 inhibitor, via a b-glucuronidase-cleavable linker. The anti-ITGb6 antibody was discovered from a Fab ribosome display library using Sutro’s XpressCF+® system. The drug-linker is functionalized with dibenzylcyclooctyne (DBCO) and allows rapid and selective site-specific conjugation to the azide-containing non-natural amino acid p-azidomethylphenylalanine (pAMF) incorporated into the antibody (Ab) sequence using XpressCF+®. This site-specific conjugation is highly efficient, resulting in a well-defined, homogeneous ADC with a drug-antibody ratio (DAR) of 8. The STRO-006 antibody binds specifically to the alpha-v beta-6 heterodimer and has high affinity to human and cynomolgus ITGb6. It demonstrates rapid and efficient internalization, ideal for an ADC mechanism of action. Importantly, the STRO-006 antibody does not compete for LAP binding and therefore does not interfere with TGFb signaling. STRO-006 is optimized for a stable pharmacokinetic profile and exhibits extended half-life of ∼7 days, low clearance of ∼5 mL/d/kg and maintains a stable DAR over the course of 21-days in non-human primates. In preclinical studies, STRO-006 demonstrates potent anti-tumor activity at clinically relevant dose in both PDX and xenograft models of non-small cell lung, head and neck squamous cell and pancreatic carcinomas. STRO-006 demonstrates a favorable safety and pharmacokinetic profile in non-human primates. These results suggest that STRO-006 is a promising candidate for the treatment of multiple carcinomas and supports further clinical investigation. A Phase 1, first-in-human study is planned to assess the safety and activity of STRO-006. Citation Format: Kshama A. Doshi, Stephanie Armstrong, Eunice Kim, Dan Shen, Sihong Zhou, Rhoneil Pena, Robert Yuan, Mark Armanini, Brian Vuillemenot, Xiaofan Li, Guifen Xu, Krishna Bajjuri, Miao Wen, Jeff Hanson, Cuong Tran, Amandeep Gakhal, GARRETT GROSS, Gang Yin, Werner Rubas, Genevive Hernandez, Daniel Calarese, Hans-Peter Gerber, Alice Yam. STRO-006: An Integrin beta-6-targeting ADC demonstrates favorable safety profile and potent antitumor activity in preclinical solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1713.

Tissue Factor (TF) is a type I transmembrane protein that is aberrantly expressed in multiple solid tumor indications including cervical, head and neck, non-small cell lung, and pancreatic cancers. Its expression is associated with poor prognosis and has been linked to pro-tumorigenic activities such as metastasis, inflammation, and angiogenesis. Under normal physiological conditions, TF plays an important role in blood clotting and, as such, is broadly expressed in many normal tissues, typically in the subendothelium. Upon endothelial damage, TF can combine with clotting factors in circulation to initiate a coagulation cascade, thus constituting a "hemostatic envelope". TF-targeted vedotin ADCs are approved in metastatic cervical cancer and in clinical testing across a range of solid tumor indications. However, toxicities such as peripheral neuropathy, epistasis, and ocular toxicities have been dose-limiting. We have developed STRO-004, a novel TF-targeted ADC with a DAR8 β-glucoronidase-exatecan linker-payload, with enhanced potency and safety in preclinical models. STRO-004 is a TF-targeted ADC, engineered for a wider therapeutic index. The ADC is constructed on an antibody that has been designed to avoid interference in the blood coagulation pathways, thus reducing the potential risk for bleeding. The anti-TF antibody is conjugated to exatecan, a topoisomerase 1 inhibitor, via a β-glucoronidase-cleavable linker (SC3417) at a drug-to-antibody ratio (DAR) of 8. The drug-linker is site-specifically conjugated to the non-natural amino acid p-azidomethylphenylalanine (pAMF) incorporated at optimized sites within the antibody (Ab) sequence using Xpress CF+TM. In preclinical xenograft models, STRO-004 demonstrates potent, dose-dependent activity from 0.25-7.5 mg/kg, including in models with low and heterogeneous levels of TF expression. STRO-004 was evaluated in patient-derived xenograft (PDx) models of cancers with prevalent TF expression and demonstrated 50% overall response rate and 70% disease control rate across all models after single dose treatment. Exploratory toxicology studies show that STRO-004 has a favorable safety profile in cynomolgous monkeys up to 50 mg/kg, the highest dose tested. In circulation, STRO-004 demonstrated extended half-life, low clearance, and stable drug-linkage. Consistent with this finding, only low levels of free exatecan could be detected. Based on these promising preclinical observations, STRO-004 is advancing to IND-enabling studies for the treatment of TF expressing malignancies. Andrew McGeehan, Helena Kiefel, Brian Vuillemenot, Guifen Xu, Sihong Zhou, Grace Lee, Mark Armanini, Xiaofan Li, Krishna Bajjuri, Miao Wen, Cuong Tran, Jeff Hanson, Daniel Calarese, Amandeep Gakhal, Hanspeter Gerber, Werner Rubas, Gang Yin, Alice Yam. Preclinical activity and safety of STRO-004, a novel ADC targeting tissue factor for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1572.

Supplementary Figure 3. LSN2478185 reduces the loss of skeletal muscle mass under conditions of caloric restriction.

Background Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a cell-surface, oncofetal protein whose expression is correlated with oncogenic properties such as enhanced proliferation, survival, and chemoresistance. Despite its prevalence across many cancer indications, ROR1 has restricted expression in adult tissues, making it an ideal target for targeted therapies. Methods We have designed a novel ROR1-targeted ADC, STRO-003, which is composed of an anti-ROR1 human IgG1 antibody (SP11285) conjugated to an exatecan warhead via a stable, β-glucuronide linker (SC3417) with a drug-to-antibody ratio (DAR) of 8. SP11285 was discovered using Fab ribosome display as a selective and high affinity ROR1 antibody that exhibits favorable internalization activity upon cell binding, consistent with an ADC mechanism of action. The exatecan payload is a potent topoisomerase I inhibitor of the camptothecin class and has anti-proliferative activity against a variety of cancer cell lines in vitro. The SC3417 linker-payload releases exatecan upon glucuronidase linker cleavage and is site-specifically conjugated using strain promoted alkyne-azide cycloaddition (SPAAC) to form a stable, homogeneous ADC. Results The anti-Ror1 antibody SP11285 binds with high affinity to the ROR1 immunoglobulin domain and is cross-reactive to both rodent and monkey ROR1. Unlike other camptothecin analogs, exatecan is resistant to overexpression of Pgp, and therefore has reduced risk of multi-drug resistance. Exatecan also induces immunogenic cell death, thereby providing an avenue for engaging the immune system and enriching the anti-tumor response. STRO-003 has exhibited potent and specific activity in xenograft models for TNBC and lung cancer, and importantly, elicited significant anti-tumor suppression in a panel of NSCLC PDx models, including those with low ROR1 expression. In nonclinical safety studies conducted in cynomolgus monkeys, STRO-003 was tolerated up to 45 mg/kg without signs of hematological toxicity or tissue-specific lesions. By comparison, a similar exatecan-ADC with a cathepsin-sensitive linker was more poorly tolerated and elicited signs of inflammation in the lung. Conclusions Our data suggests that STRO-003 is a promising clinical candidate for solid tumor indications and we have initiated IND-enabling studies. Ethics Approval All in vivo procedures were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Sutro Biopharma or commissioned contract research organization (CRO).

The tolerability of single daily gavage doses of 0.5% or 2.0% (wt/vol) sodium lauryl sulfate (SLS) in 11- to 12-week-old male CD-1 mice was evaluated in a study of 3 months in duration. Live-phase, gross necropsy, and histopathologic parameters were evaluated. Mortality of 14% occurred in mice administered formulations containing SLS. Clinical observations in mice administered SLS included abnormal respiration (audible, irregular, and/or labored), swollen abdomen, rough haircoat, hunched appearance, and hypoactivity. Necropsy findings in mice administered SLS consisted of enlarged intestines containing abnormal contents with gas. There were no instances of mechanical gavage–related injury. Histologic evaluation of the respiratory tract revealed injury to the nasal passages and nasopharynx, including, but not limited to, inflammation, exudate, apoptosis/necrosis of epithelium, and atrophy of epithelium or olfactory nerves. Collectively, the data indicated that under the experimental conditions of our 3-month study in male CD-1 mice, once-daily gavage administration of vehicle formulations containing SLS at 0.5% or 2.0% resulted in nasal injury and 14% mortality supportive of gastroesophageal reflux. Sponsors utilizing formulations containing SLS in toxicity studies in CD-1 mice should exclude gastroesophageal reflux as a confounding factor in studies with morbidity or mortality associated with respiratory distress or evidence of aerophagia.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Abstract There is growing evidence that tumor-targeted cytotoxins can also enhance anti-tumor immunity by inducing immunogenic cell death (ICD) in tumor cells and promoting recruitment of immune effector cells. We sought to investigate the immune stimulating potential of STRO-002, an antibody drug conjugate (ADC) composed of an anti-Folate receptor alpha (FolRα) antibody conjugated to a tubulin-targeting hemiasterlin warhead via a cleavable linker. FolRα is a single chain glycosylphosphatidylinositol-anchored membrane receptor glycoprotein with minimal expression in normal tissues. Its overexpression in several cancer indications has been described, including in ovarian, endometrial, non-small cell lung carcinoma (NSCLC), and triple negative breast cancer (TNBC), thus making it an ideal ADC target. We have previously demonstrated potent in vitro and in vivo activity of STRO-002 in several FolRα expressing models. Here we show that the hemiasterlin warhead, SC209, and STRO-002 ADC induced ICD in vitro as evidenced by presentation of cell-surface calreticulin and release of HMGB1 and ATP. As a result of ICD, STRO-002 treated FolRα positive cancer cells induced antigen-dependent monocyte activation, as well as, increased phagocytic activity in PBMCs co-cultured with tumor cells. To determine if these immunogenic properties could improve therapeutic efficacy, we evaluated STRO-002 in combination with the immune checkpoint inhibitor Avelumab (anti-PD-L1) in a mouse syngeneic MC38 model engineered to express human FolRα (MC38-hFolRα). Results showed that STRO-002 and Avelumab alone inhibited tumor growth and could induce complete responses (e.g. no palpable tumors) at low frequency (< 15%), while co-administration of STRO-002 and Avelumab significantly enhanced efficacy leading to complete response in the majority of animals. Furthermore, when animals that initially achieved complete response were re-challenged with MC38-hFolRα cells, they showed durable anti-tumor immunity, indicating formation of immunological memory. Immunohistochemical analysis conducted seven days after treatment revealed a significant increase in tumor-infiltrating cytotoxic CD8+ T cells in animals treated with combination of STRO-002 + Avelumab versus either monotherapy. Cumulatively, these results suggest that STRO-002 synergizes with Avelumab to enhance anti-tumor response by inducing ICD in tumor cells, which in turn, promote T cell recruitment. These data support the rationale for combining STRO-002 with immune checkpoint inhibitors to potentially enhance their clinical efficacy. Citation Format: Millicent Embry, Sihong Zhou, Christine Cheng, Janice Yu, Cristina L. Abrahams, Xioafan Li, Jeff Hanson, Cuong Tran, Gang Yin, Shamim Ahmad, Krishna Bajjuri, Venita DeAlmeida, Mark Lupher, Trevor Hallam. STRO-002, an anti-FolRαADC, demonstrates immune-modulating properties and potentiates PD-L1 blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2250.

CD74 is a type II transmembrane glycoprotein that functions as an MHC class II chaperone and displays diverse roles in immune responses. Recently, anti‐CD74 immunotherapy has shown promise as an effective treatment strategy for lymphoid neoplasms in preclinical models. Using a human anti‐CD74 antibody (SP7219), we defined the expression of CD74 protein in both normal and over 790 neoplastic hematolymphoid tissue samples. We found that CD74 is expressed broadly in normal B‐cell compartments including primary and secondary lymphoid follicles and in the thymic medulla. The vast majority of lymphomas expressed CD74, including Hodgkin lymphomas (98%), B‐cell lymphomas (96%), extranodal NK/T‐cell lymphomas (88%), mature T‐cell lymphomas (80%), and plasma cell myeloma (75%). Our findings confirm and expand previous observations regarding the expression of CD74 and suggest that CD74 expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and plasma cell malignancies.

Antiangiogenesis-based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor-positive hematopoietic cells could be impacted by VEGF pathway-targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell-containing Lin-cKit(+)Sca1(+) (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function.

Abstract Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.

AimsDevelopment of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method.Methods and resultsExcellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P<0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R-2>0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21.ConclusionsThe anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

Beverly L. Falcon, Julie Stewart, Scharri Ezell, Jeff Hanson, John Wijsman, Xiang Ye, Eric Westin, Greg Donoho, Kelly Credille and Mark T. Uhlik 1 Eli Lilly and Company, Department of Cancer Angiogenesis, Lilly Corporate Center, Indianapolis, IN 46285, USA 2 Eli Lilly and Company, Department of Translational Science, Lilly Corporate Center, Indianapolis, IN 46285, USA 3 Eli Lilly and Company, Department of IT, Lilly Corporate Center, Indianapolis, IN 46285, USA 4 Eli Lilly and Company, Department of Oncology, Lilly Corporate Center, Indianapolis, IN 46285, USA 5 Eli Lilly and Company, Department of Investigational Pathology, Lilly Corporate Center, Indianapolis, IN 46285, USA

Targeting multiple hallmarks of cancer with drug combinations may provide unique opportunities for cancer therapeutics; however, phenotypic quantification is necessary to understand in vivo mechanisms of action of each drug alone or in combination. Immunohistochemistry (IHC) can quantify phenotypic changes, but traditional methods are not amenable for high-throughput drug discovery. In this article, we describe a high-content method to quantify changes in tumor angiogenesis, vascular normalization, hypoxia, tumor cell proliferation, and apoptosis using IHC. This method to quantify tumor model phenotypes can be useful for cancer drug discovery by increasing the understanding of: (i) tumor models used in efficacy studies, (ii) changes occurring during the growth of the tumor, and (iii) novel mechanisms of actions of cancer therapeutics.

Abstract The Notch pathway is a highly conserved signaling system that plays an important role in development and tissue homeostasis. While Notch mutations are well characterized and implicated in hematological malignancies such as T-cell acute lymphoblastic leukemia, in solid tumors ligand or receptor over-expression may lead to enhanced/sustained Notch function, triggering increased tumor cell proliferation/survival, angiogenesis and tumor formation. In order to delineate an oncogenic role of activated Notch in tumors of epithelial origin, we carried out a series of in-vitro and in-vivo studies. We demonstrated that the activated Notch1 receptor (a - secretase-dependent Notch1 E with the transmembrane domain and a-secretase-independent constitutively activated Notch1 intracellular fragment) can transform normal rat cells, RK3E. These transformed cells formed colonies in soft agar, confirming their anchorage-independent growth potential, and when implanted subcutaneously, formed tumors in athymic nude mice. Inhibition of Notch signaling through a small molecule inhibitor of -secretase, a key regulator of Notch processing, may provide an attractive targeted cancer therapeutic strategy. We have identified and characterized a novel small molecule that is an exquisitely potent inhibitor of Notch signaling in tumor cell lines and endothelial cells with an IC50 ranging from 0.005 nM to 20 nM. The Notch inhibitor meets all pharmacokinetic criteria in pre-clinical species. In a xenograft tumor model, the novel compound inhibited Notch cleavage in a dose-dependent manner at 6 hours after a single oral dose. This inhibition of Notch cleavage resulted in the induction of apoptosis (as measured by activated caspase-3 levels) that was statistically significant at 24 hours after a single oral dose of 3 mg/kg. Analysis of tumors from animals treated with the Notch inhibitor revealed inhibition of angiogenesis through formation of leaky vasculature which may also contribute to observed anti-tumor activity. Furthermore, Notch inhibition produced tumor regression in the Notch-dependent tumor models. Anti-tumor activity was also observed in several human xenograft tumors of epithelial origin. To mitigate mucoid gasteroentropathy due to Notch inhibition, PK/PD data were incorporated in devising dosing strategies that identified an optimal intermittent dosing schedule without negatively impacting efficacy. Furthermore, the mucoid gastroentropathy was also mitigated by the prophylactic administration of dexamethasone without negatively impacting Notch inhibitor mediated efficacy. In summary, we have characterized an orally bio-available small molecule Notch inhibitor that may provide therapeutic benefit to cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B188.

The success of molecular research and its applications in both the clinical and basic research arenas is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. This tissue bank was set up to bank fresh surgically obtained human tissue using a Clinical Annotated Tissue Database (CATD) in order to capture the associated patient clinical data and demographics using a one way patient encryption scheme to protect patient identification. In this study, we determined that high quality of tissue samples is imperative for both genomic and proteomic molecular research. This paper also contains a brief compilation of the literature involved in the patient ethics, patient informed consent, patient de-identification, tissue collection, processing, and storage as well as basic molecular research generated from the tissue bank using good clinical practices. The current applicable rules, regulations, and guidelines for handling human tissues are briefly discussed. More than 6,610 cancer patients have been consented (97% of those that were contacted by the consenter) and 16,800 tissue specimens have been banked from these patients in 9 years. All samples collected in the bank were QC’d by a pathologist. Approximately 1,550 tissue samples have been requested for use in basic, clinical, and/or biomarker cancer research studies. Each tissue aliquot removed from the bank for a research study were evaluated by a second H&E, if the samples passed the QC, they were submitted for genomic and proteomic molecular analysis/study. Approximately 75% of samples evaluated were of high histologic quality and used for research studies. Since 2003, we changed the patient informed consent to allow the tissue bank to gather more patient clinical follow-up information. Ninety two percent of the patients (1,865 patients) signed the new informed consent form and agreed to be re-contacted for follow-up information on their disease state. In addition, eighty five percent of patients (1,584) agreed to be re-contacted to provide a biological fluid sample to be used for biomarker research.

Aberrant changes in post-translational histone modification have been shown to occur in cancer cells. Although histone modifications have been recently reported to be associated with risk of prostate cancer recurrence, global histone modification in tumor progression remains elusive. In this study, using immunohistochemistry (IHC), we characterized patterns of global histone modifications in 136 clinical specimens from different stages of human tumors of breast and colon. These antibodies included: histone H2AX, H3, H4 and 6 modification-specific antibodies (H2AX modified: S139-phos; H3 modified: S10-phos, K9-Ace, K18-Ace, K4 modified: K12-Ace, K16-Ace). A decision tree paradigm was used for statistical analysis in evaluating the multiple change of histone modification. In the breast carcinoma cases, only the histone H3, H3-K18-Ace, and H4-K16-Ace were found to be associated with high grade tumor. There were no changes in the other histone markers between normal breast tissue, localized adenocarcinoma, invasive adenocarcinoma and metastatic carcinoma and various histological grades of breast tumors. Tumors of the colon were split into left and right sides for statistical analysis due to different genetic pathways. In tumors originating from the left colon, H3 and H4-K12-Ace were highly associated with high grade and stage tumors, while only H3 was associated with high grade stage tumors originating from the right colon. There were no changes in the other Histone markers between normal colonic tissue, localized adenocarcinoma, invasive adenocarcinoma and metastatic carcinoma, and various histological grades of colon tumors. In conclusion, H3 was observed in high grade and stage tumors of the breast and colon. H4-K12-Ace antibody was seen in high grade and stage tumors originating from the left colon. H3-K18-Ace and H4-K16-Ace were associated with high grade and stage tumors of the breast.