Assessing Reuse of Hypodermic Needles in Mice by means of Digital Imaging, Photomicrography, Bacterial Culture, Analysis of Nest Building, and Animal Vocalization.

Hypodermic needles are sometimes reused in animal research settings to preserve the viability of and to conserve limited quantities of injected material. However, the reuse of needles is strongly discouraged in human medicine to prevent inju- ries and the spread of infectious disease. No official guidelines prohibit needle reuse in veterinary medicine, although the practice may be discouraged. We hypothesized that reused needles would be significantly more blunt than unused needles and that reuse for additional injections would cause more animal stress. To test these ideas, we evaluated mice that were injected subcutaneously in the flank or mammary fat pad to generate cell line xenograft and mouse allograft models. Needles were reused up to 20 times, based on an IACUC-approved protocol. A subset of reused needles was digitally imaged to determine needle dullness based on the area of deformation from the secondary bevel angle; this parameter was not different between new needles and needles that had been reused 20 times. In addition, the number of times a needle was reused was not significantly related to audible mouse vocalization during injection. Finally, nest building scores for mice that were injected with a needle used 0 through 5 times were similar to those of mice injected with a needle had been used 16 through 20 times. Among the 37 reused needles that were tested, 4 were positive for bacterial growth; the only organisms cultured were Staphylococcus spp. Contrary to our hypothesis, reusing needles for subcutaneous injections did not increase animal stress based on analysis of vocalization or nest building.

Comparison of Pyrrolobenzodiazepine Dimer Bis-imine versus Mono-imine: DNA Interstrand Cross-linking, Cytotoxicity, Antibody-Drug Conjugate Efficacy and Toxicity.

Antibody-drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo cross-linking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade.

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

Targeting and pharmacology of an anti-IL13Rα2 antibody and antibody-drug conjugate in a melanoma xenograft model.

IL13Rα2 is a cell surface tumor antigen that is overexpressed in multiple tumor types. Here, we studied biodistribution and targeting potential of an anti-IL13Rα2 antibody (Ab) and anti-tumor activity of anti-IL13Rα2-antibody-drug conjugate (ADC). The anti-IL13Rα2 Ab was labeled with fluorophore AF680 or radioisotope 89Zr for in vivo tracking using fluorescence molecular tomography (FMT) or positron emission tomography (PET) imaging, respectively. Both imaging modalities showed that the tumor was the major uptake site for anti-IL13Rα2-Ab, with peak uptake of 5-8% ID and 10% ID/g as quantified from FMT and PET, respectively. Pharmacological in vivo competition with excess of unlabeled anti-IL13Rα2-Ab significantly reduced the tumor uptake, indicative of antigen-specific tumor accumulation. Further, FMT imaging demonstrated similar biodistribution and pharmacokinetic profiles of an auristatin-conjugated anti-IL13Rα2-ADC as compared to the parental Ab. Finally, the anti-IL13Rα2-ADC exhibited a dose-dependent anti-tumor effect on A375 xenografts, with 90% complete responders at a dose of 3 mg/kg. Taken together, both FMT and PET showed a favorable biodistribution profile for anti-IL13Rα2-Ab/ADC, along with antigen-specific tumor targeting and excellent therapeutic efficacy in the A375 xenograft model. This work shows the great potential of this anti-IL13Rα2-ADC as a targeted anti-cancer agent.

Preclinical Evaluation of 89Zr-Df-IAB22M2C PET as an Imaging Biomarker for the Development of the GUCY2C-CD3 Bispecific PF-07062119 as a T Cell Engaging Therapy.

PURPOSE: A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. PROCEDURES: NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. RESULTS: The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. CONCLUSION: Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

Anti-tumor immunity influences cancer cell reliance upon ATG7.

Macroautophagy (autophagy) is an essential cellular catabolic process required for survival under conditions of starvation. The role of autophagy in cancer is complex, context-dependent and at times contradictory, as it has been shown to inhibit, promote or be dispensable for tumor progression. In this study, we evaluated the contribution of the immune system to the reliance of tumors on autophagy by depleting autophagy-related 7 (ATG7) in murine tumor cells and grafting into immunocompetent versus immunodeficient hosts. Although loss of ATG7 did not affect tumor growth in vitro or in immunodeficient mice, our studies revealed that cancer cell reliance on autophagy was influenced by anti-tumor immune responses, including those mediated by CD8+ T cells. Furthermore, we provide insights into possible mechanisms by which autophagy disruption can enhance anti-tumor immune responses and suggest that autophagy disruption may further benefit patients with immunoreactive tumors.

PF-06804103, A Site-specific Anti-HER2 Antibody-Drug Conjugate for the Treatment of HER2-expressing Breast, Gastric, and Lung Cancers.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Use of translational modeling and simulation for quantitative comparison of PF-06804103, a new generation HER2 ADC, with Trastuzumab-DM1.

A modeling and simulation approach was used for quantitative comparison of a new generation HER2 antibody drug conjugate (ADC, PF-06804103) with trastuzumab-DM1 (T-DM1). To compare preclinical efficacy, the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of PF-06804103 and T-DM1 was determined across a range of mouse tumor xenograft models, using a tumor growth inhibition model. The tumor static concentration was assigned as the minimal efficacious concentration. PF-06804103 was concluded to be more potent than T-DM1 across cell lines studied. TSCs ranged from 1.0 to 9.8 µg/mL (n = 7) for PF-06804103 and from 4.7 to 29 µg/mL (n = 5) for T-DM1. Two experimental models which were resistant to T-DM1, responded to PF-06804103 treatment. A mechanism-based target mediated drug disposition (TMDD) model was used to predict the human PK of PF-06804103. This model was constructed and validated based on T-DM1 which has non-linear PK at doses administered in the clinic, driven by binding to shed HER2. Non-linear PK is predicted for PF-06804103 in the clinic and is dependent upon circulating HER2 extracellular domain (ECD) concentrations. The models were translated to human and suggested greater efficacy for PF-06804103 compared to T-DM1. In conclusion, a fit-for-purpose translational PK/PD strategy for ADCs is presented and used to compare a new generation HER2 ADC with T-DM1.

A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers.

PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Comparison of the molecular and cellular phenotypes of common mouse syngeneic models with human tumors.

BACKGROUND: The clinical success of immune checkpoint inhibitors demonstrates that reactivation of the human immune system delivers durable responses for some patients and represents an exciting approach for cancer treatment. An important class of preclinical in vivo models for immuno-oncology is immunocompetent mice bearing mouse syngeneic tumors. To facilitate translation of preclinical studies into human, we characterized the genomic, transcriptomic, and protein expression of a panel of ten commonly used mouse tumor cell lines grown in vitro culture as well as in vivo tumors. RESULTS: Our studies identified a number of genetic and cellular phenotypic differences that distinguish commonly used mouse syngeneic models in our study from human cancers. Only a fraction of the somatic single nucleotide variants (SNVs) in these common mouse cell lines directly match SNVs in human actionable cancer genes. Some models derived from epithelial tumors have a more mesenchymal phenotype with relatively low T-lymphocyte infiltration compared to the corresponding human cancers. CT26, a colon tumor model, had the highest immunogenicity and was the model most responsive to CTLA4 inhibitor treatment, by contrast to the relatively low immunogenicity and response rate to checkpoint inhibitor therapies in human colon cancers. CONCLUSIONS: The relative immunogenicity of these ten syngeneic tumors does not resemble typical human tumors derived from the same tissue of origin. By characterizing the mouse syngeneic models and comparing with their human tumor counterparts, this study contributes to a framework that may help investigators select the model most relevant to study a particular immune-oncology mechanism, and may rationalize some of the challenges associated with translating preclinical findings to clinical studies.

Cystine-glutamate antiporter xCT deficiency suppresses tumor growth while preserving antitumor immunity.

T cell-invigorating cancer immunotherapies have near-curative potential. However, their clinical benefit is currently limited, as only a fraction of patients respond, suggesting that these regimens may benefit from combination with tumor-targeting treatments. As oncogenic progression is accompanied by alterations in metabolic pathways, tumors often become heavily reliant on antioxidant machinery and may be susceptible to increases in oxidative stress. The cystine-glutamate antiporter xCT is frequently overexpressed in cancer and fuels the production of the antioxidant glutathione; thus, tumors prone to redox stress may be selectively vulnerable to xCT disruption. However, systemic inhibition of xCT may compromise antitumor immunity, as xCT is implicated in supporting antigen-induced T cell proliferation. Therefore, we utilized immune-competent murine tumor models to investigate whether cancer cell expression of xCT was required for tumor growth in vivo and if deletion of host xCT impacted antitumor immune responses. Deletion of xCT in tumor cells led to defective cystine uptake, accumulation of reactive oxygen species, and impaired tumor growth, supporting a cancer cell-autonomous role for xCT. In contrast, we observed that, although T cell proliferation in culture was exquisitely dependent on xCT expression, xCT was dispensable for T cell proliferation in vivo and for the generation of primary and memory immune responses to tumors. These findings prompted the combination of tumor cell xCT deletion with the immunotherapeutic agent anti-CTLA-4, which dramatically increased the frequency and durability of antitumor responses. Together, these results identify a metabolic vulnerability specific to tumors and demonstrate that xCT disruption can expand the efficacy of anticancer immunotherapies.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1).

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1-resistant population was generated. T-DM1-resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)-positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243-53. ©2017 AACR.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Advances in patient-derived tumor xenografts: from target identification to predicting clinical response rates in oncology.

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology. Therefore, improvement in the predictive values of preclinical efficacy models for clinical outcome holds great promise to reduce the clinical attrition rates of experimental compounds. Recent reports suggest that pharmacology studies conducted with patient derived xenograft (PDX) tumors are more predictive for clinical outcome compared to conventional, cell line derived xenograft (CDX) models, in particular when therapeutic compounds were tested at clinically relevant doses (CRDs). Moreover, the study of the most malignant cell types within tumors, the tumor initiating cells (TICs), relies on the availability of preclinical models that mimic the lineage hierarchy of cells within tumors. PDX models were shown to more closely recapitulate the heterogeneity of patient tumors and maintain the molecular, genetic, and histological complexity of human tumors during early stages of sequential passaging in mice, rendering them ideal tools to study the responses of TICs, tumor- and stromal cells to therapeutic intervention. In this commentary, we review the progress made in the development of PDX models in key areas of oncology research, including target identification and validation, tumor indication search and the development of a biomarker hypothesis that can be tested in the clinic to identify patients that will benefit most from therapeutic intervention.

The interaction of PKN3 with RhoC promotes malignant growth.

PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3-RhoC complex represents an attractive therapeutic target in late-stage malignancies.

2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor agents selectively active in securin(-/-) cells.

A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure-activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.

Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression.

We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.

Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.