Jamie L. Jordan

Predicting Drug Responsiveness in Human Cancers Using Genetically Engineered Mice

Purpose: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system. Experimental Design: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53−/− OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data. Results: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer. Conclusions: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer. Clin Cancer Res; 19(17); 4889–99. ©2013 AACR.

Effects of tumor microenvironment heterogeneity on nanoparticle disposition and efficacy in breast cancer tumor models.

Purpose: Tumor cells are surrounded by a complex microenvironment. The purpose of our study was to evaluate the role of heterogeneity of the tumor microenvironment in the variability of nanoparticle (NP) delivery and efficacy. Experimental Designs: C3(1)-T-Antigen genetically engineered mouse model (C3-TAg) and T11/TP53Null orthotopic syngeneic murine transplant model (T11) representing human breast tumor subtypes basal-like and claudin-low, respectively, were evaluated. For the pharmacokinetic studies, non-liposomal doxorubicin (NL-doxo) or polyethylene glycol tagged (PEGylated) liposomal doxorubicin (PLD) was administered at 6 mg/kg i.v. x1. Area under the concentration versus time curve (AUC) of doxorubicin was calculated. Macrophages, collagen, and the amount of vasculature were assessed by IHC. Chemokines and cytokines were measured by multiplex immunochemistry. NL-doxo or PLD was administered at 6 mg/kg i.v. weekly x6 in efficacy studies. Analyses of intermediary tumor response and overall survival were performed. Results: Plasma AUC of NL-doxo and PLD encapsulated and released doxorubicin was similar between two models. However, tumor sum total AUC of PLD was 2-fold greater in C3-TAg compared with T11 (P < 0.05). T11 tumors showed significantly higher expression of CC chemokine ligand (CCL) 2 and VEGF-a, greater vascular quantity, and decreased expression of VEGF-c compared with C3-TAg (P < 0.05). PLD was more efficacious compared with NL-doxo in both models. Conclusion: The tumor microenvironment and/or tumor cell features of breast cancer affected NP tumor delivery and efficacy, but not the small-molecule drug. Our findings reveal the role of the tumor microenvironment in variability of NP delivery and therapeutic outcomes. Clin Cancer Res; 20(23); 6083–95. ©2014 AACR.

Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with CDK4/6 sensitive tumors

Inhibition of the p16INK4a/cyclin D/CDK4/6/RB pathway is an effective therapeutic strategy for the treatment of estrogen receptor positive (ER+) breast cancer. Although efficacious, current treatment regimens require a dosing holiday due to severe neutropenia potentially leading to an increased risk of infections, as well as tumor regrowth and emergence of drug resistance. Therefore, a next generation CDK4/6 inhibitor that can inhibit proliferation of CDK4/6-dependent tumors while minimizing neutropenia could reduce both the need for treatment holidays and the risk of inducing drug resistance. Here, we describe the preclinical characterization and development of G1T38; a novel, potent, selective, and orally bioavailable CDK4/6 inhibitor. In vitro, G1T38 decreased RB1 (RB) phosphorylation, caused a precise G1 arrest, and inhibited cell proliferation in a variety of CDK4/6-dependent tumorigenic cell lines including breast, melanoma, leukemia, and lymphoma cells. In vivo, G1T38 treatment led to equivalent or improved tumor efficacy compared to the first-in-class CDK4/6 inhibitor, palbociclib, in an ER+ breast cancer xenograft model. Furthermore, G1T38 accumulated in mouse xenograft tumors but not plasma, resulting in less inhibition of mouse myeloid progenitors than after palbociclib treatment. In larger mammals, this difference in pharmacokinetics allowed for 28 day continuous dosing of G1T38 in beagle dogs without producing severe neutropenia. These data demonstrate G1T38 has unique pharmacokinetic and pharmacodynamic properties, which result in high efficacy against CDK4/6 dependent tumors while minimizing the undesirable on-target bone marrow activity, thus potentially allowing G1T38 to be used as a continuous, daily oral antineoplastic agent.

IL2 Inducible T-cell Kinase, a Novel Therapeutic Target in Melanoma.

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation. Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined. Results: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0–G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Ptennull/BrafV600E) melanomas. Conclusions: We conclude that ITK, formerly considered an immune cell–specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanoma model supports this possibility. Clin Cancer Res; 21(9); 2167–76. ©2015 AACR.

Abstract 2649: Inhibition of mTOR, but not PI3K, is required for the anti-tumor efficacy in breast cancer of dual PI3K/mTOR inhibitors given in combination with MEK inhibitors

Background: The mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are deregulated in many cancers including breast cancer. Additionally, studies have shown that significant crosstalk exists between these pathways; often making single pathway inhibition ineffective. While, combined PI3K/mTOR and MEK inhibition appears highly active in pre-clinical models, the combination has been reported to be toxic in human use. In an effort to produce a more targeted regimen, we examined whether both PI3K and mTOR inhibition are required for pre-clinical anti-tumor efficacy in combination with a MEK inhibitor. Methods: In vivo studies assessed overall survival and best tumor response upon administration of dual PI3K/mTOR (BEZ235 and GSK806) versus allosteric mTOR inhibition (Everolimus) in combination with MEK inhibition (AZD244 and GSK212) in genetically engineered murine models (GEMM) of breast cancer [T11 and C(3)Tag]. Activation and response of the PI3K/mTOR and MEK/ERK pathways to dual PI3K/mTOR, allosteric mTOR inhibition, and MEK inhibition were assessed in vitro in murine cell lines derived from the above GEM models. Results: Single agent regimens of MEK, allosteric mTOR, or dual PI3K/mTOR inhibition were minimally effective in C(3)Tag or T11 tumors in vivo. As previously reported, combined PI3K/mTOR and MEK inhibition showed potent pre-clinical activity in both the T11 and C3Tag models, prolonging overall survival relative to both vehicle or single-agent treated mice. Surprisingly, combinations of MEK inhibitors with allosteric mTOR inhibitors were as active as the MEK/PI3K/mTOR combination. In vitro studies in cell lines derived from these tumor models showed that mTORC1 inhibition, but not PI3K inhibition, resulted in compensatory activation of MEK. Conclusions: Combined targeting of the mTOR and MEK pathways results in improved response and survival in faithful GEM models of breast cancer. The combination of dual PI3K/mTOR catalytic inhibitors with MEK inhibition does not afford benefit over combined allosteric mTOR and MEK inhibition Citation Format: Aleisha M. Smith, Jessie Xiong, Lucas Hunter, Jamie Jordan, Kelly Clark, David B. Darr, Sharpless Norman, Charles M. Perou, William Y. Kim. Inhibition of mTOR, but not PI3K, is required for the anti-tumor efficacy in breast cancer of dual PI3K/mTOR inhibitors given in combination with MEK inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2649. doi:10.1158/1538-7445.AM2015-2649

Abstract 4761: Defining the adaptive kinome response to BRAF and MEK inhibition in melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Early clinical studies have shown that concurrent administration of BRAF and MEK inhibitors, dabrafenib and trametinib, is more active in patients with BRAFV600E/K melanoma than either single agent alone. Even though the combination of BRAF/MEK inhibitors is initially highly effective in treating melanoma in a subset of patients, progression to resistance ultimately occurs–commonly via reactivation of the BRAF/MEK/ERK pathway. Therefore, therapeutic strategies to counter primary resistance and prevent the emergence of secondary resistance are needed. Following pharmacological or progressive genetic perturbations, dynamic and system-wide adaptive changes (“reprogramming”) in the expression and activity of multiple kinases (collectively termed the kinome) clearly occur in tumor cells. Rich networks of serine/threonine and tyrosine kinases, many of which are understudied, are integrated into the signaling systems controlled by oncogenes known to drive melanoma. Thus, a comprehensive understanding of the kinome at baseline, during the adaptive response to kinase inhibitor treatment, and in the context of acquired resistance is critical. We use Multiplexed Inhibitor Beads (MIBs), mixtures of covalently immobilized, linker-adapted kinase inhibitors coupled with mass spectrometry (MS), to assess changes in activation of kinases in untreated samples, during acute inhibition of BRAF and/or MEK and in the context of acquired resistance. Our enhanced MIB/MS technology allows us to study over 75-80% of the expressed kinome (and numerous lipid and metabolic kinases) simultaneously using lysates from cell lines, genetically engineered mouse model (GEMM) tumors, patient-derived xenografts or human melanoma. We have identified several kinases (e.g. PDGFRβ, DDR1, MAP3K1) that are activated upon acute treatment with dabrafenib, trametinib, or dabrafenib/trametinib as well as in drug-resistant human melanoma cells and BRAFV600E/PTEN-/- GEMM-derived tumors and cell lines. Thus, studies using MIB/MS to measure dynamic kinome responses have identified kinases that have not been previously shown to be involved in the adaptive response to BRAF and/or MEK inhibition. We are currently targeting kinases involved in adaptive kiome reprogramming, pharmacologically or by RNAi, to determine whether their activation leads to vulnerability. This work will lead to the identification of novel and rationally predicted therapies for patients with advanced melanoma. Citation Format: Steven P. Angus, Timothy J. Stuhlmiller, Rachel Reuther, Trang T. Pham, Deborah A. Granger, David B. Darr, Jamie L. Jordan, Stergios J. Moschos, Gary L. Johnson, Norman E. Sharpless. Defining the adaptive kinome response to BRAF and MEK inhibition in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4761. doi:10.1158/1538-7445.AM2014-4761

Abstract 5024: mDX400, the murine analog against the anti-PD1 antibody MK-347 is active in immunocompetent, autochthonous murine models of melanoma and breast cancer

Although effective therapies exist for BRAF-mutant metastatic melanomas (MM) and ER+/PR+/HER2+ breast cancers, fewer options are available for the more aggressive triple negative breast cancers (TNBC) and Ras-mutant MM. Immune infiltration is frequently observed in patient subsets with MM or TNBC. An anti-tumor host immune response may be restrained by the expression of immune checkpoint proteins, such as the programmed death 1 (PD1) protein. We evaluated mDX400, a murine version of the anti-PD-1 antibody MK-3475 that is currently in human clinical trials, in genetically engineered murine models (GEMMs) of melanoma and breast cancer (BC). PD1 antibody was used alone and in combination. Models studied were for basal-like, TNBC (C3TAg), Claudin-Low breast cancer p53null, luminal BC (MMTV-Her2/Neu), RAS-driven melanoma (Tyr-H-Ras and null for Ink4a/Arf (TRIA)) and BRaf-mutant, Pten-deficient melanoma (BRaf/Pten). Mice were housed, treated, and evaluated in the UNC Lineberger Comprehensive Cancer Center Mouse Phase I Unit (MP1U). mDX400 and isotype antibody (10mg/kg IP qw) were supplied by Merck & Co., Inc. Murine cohorts were assessed weekly and therapeutic intervention began once tumors reached 40-64mm3. TNBC models were treated with Carboplatin (C, 50mg/kg IP qwk) in combination with mDX400. mDX400 was administered as a single agent in Melanoma GEMMs. Endpoints were maximal response at ≥21 days and median overall survival (OS). In the C3TAg BC model, mDX400 did not exhibit single-agent activity, but substantial synergy was observed in combination with C [partial response (PR) or CR in 12 of 15 vs. 0 of 19 in non-treated animals, Fisher9s exact p=0.006 or vs. C alone 5 of 13 animals, p=0.05] and prolonged OS (41 vs. 28 days in NT, log-rank p=0.006 or C only 28 days, p=0.006) in the C3TAg model. Treatment with single-agent mDX400 induced a marked response in the RAS-driven TRIA melanoma model that is resistant to multiple systemic treatments. mDX400-treated TRIA mice enjoyed an almost tripling of OS [median 56 vs. 21 days, p=0.006]. BRaf/Pten melanomas did not respond to mDX400. Likewise mDX400 plus C provided no benefit to the p53null or MMTV-Her2/Neu BC GEMMs vs. C alone. Our data show differential response of PD-1 antibody therapies to various melanoma and BC models, the latter in combination with C. The responsiveness of a RAS-driven melanoma model to mDX400 is consistent with earlier reports about the clinical benefit of patients with NRAS-mutant melanoma to Ipilimumab, and the lack of PD-L1 expression and immune infiltrates of PTEN-deficient melanomas. At the meeting we will report expression of PD-L1 and other immune checkpoint proteins by cancer cells. The basis whereby carboplatin potentiates the activity of anti-PD1 in TNBC is an area of ongoing study. Citation Format: David Darr, Kelly S. Clark, Joseph H. Phillips, Elaine Pinheiro, Venkataraman Sriram, Jessie Xiong, Jamie Jordan, Norman E. Sharpless, Charles Perou, Stergios Moschos. mDX400, the murine analog against the anti-PD1 antibody MK-347 is active in immunocompetent, autochthonous murine models of melanoma and breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2014-5024

Abstract 3354: Relationship between tumor-associated macrophages (TAMs), tumor delivery, and efficacy of PEGylated liposomal doxorubicin (PLD) and non-liposomal doxorubicin (NL-doxo) in genetically engineered mouse models (GEMMs) of breast cancer (BC).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Solid tumors consist of both malignant cells and non-malignant stromal cells. Stromal cells typically include vascular, mesenchymal, and pro-inflammatory cells. Macrophages are the most abundant pro-inflammatory cell type in solid tumors and are termed TAMs. TAMs are derived from monocytes (mononuclear phagocyte system, MPS) and are associated with tumor progression, angiogenesis, and metastasis. The MPS are responsible for the clearance and distribution of nanoparticle (NP) anticaner agents such as PLD. However, the role of MPS in the tumor delivery of NP have not been evaluated. In this study, we evaluated the relationship between TAM-related tumor microenvironment factors, tumor delivery, and efficacy of PLD and NL-doxo in GEMMs of BC. Methods: Basal-like (C3(1)T-antigen) and claudin-low (T11/TP53-/-) GEMMs of human BC subtypes were evaluated. PLD or NL-doxo was administered at 6 mg/kg IV x 1 via a tail vein. For each GEMM, mice (n=3) were sacrificed prior to dose, and from 5 min to 96 h post dose. Plasma encapsulated and released doxo and sum total doxo conc in tumor was measured by HPLC. Area under the plasma and tumor conc versus time curves (AUC) were calculated. TAM infiltration was visualized and measured via immunohistochemistry (IHC) for F4/80. Endothelial cells in tumors were detected by CD31 IHC. Density of TAMs and endothelial cells in tumors were measured by the Aperio analysis algorithms. In efficacy study, PLD or NL-doxo at 6 mg/kg was administered every week for 6 weeks (n=20 per model). Tumor growth and overall survival were monitored. Results: Mean ± SD of plasma AUC of PLD encapsulated doxo in T11 and C3tag were1,449 ± 57 and 1,610 ± 111 (μg·h/ml), respectively. Mean ± SD of tumor sum total AUC of PLD in T11 and C3tag were 210 ± 26 and 480 ± 71 (μg·h/ml), respectively. Mean ± SD of tumor AUC of NL-doxo in T11 and C3tag were 61 ± 12 and 57 ± 10 (μg·h/ml), respectively. Mean ± SD baseline % positive cells of TAM in T11 and C3tag were 113 ± 45 and 110 ± 50, respectively. Mean ± SD baseline microvessel density in T11 and C3tag were 8.8 ± 2.5 and 6.1 ± 1.8 (number of vessels x10ˆ5 per unit area), respectively. C3tag demonstrated greater antitumor response to PLD compared to T11. Conclusions: There was a substantial difference in tumor exposure of PLD, but not NL-doxo, in the two GEMMs. These findings suggest that TM factors in BC may affect the delivery of NP agents, but not small molecules (SM). However, the similar TAM and microvessel baseline density in GEMMs suggest alternative tumor factors specifically affecting the delivery of NP agents. Further studies are warranted to elucidate the mechanisms underlying the delivery of NP agents in these and other tumors which may explain different tumor phenotypes and therapeutic outcomes related to treatment with NP and SM agents. Citation Format: Gina Song, David B. Darr, Charlene M. Santos, Taylor F. White, Jamie L. Jordan, Mimi Kim, Bentley R. Midkiff, Nana N. Feinberg, Ryan Miller, Arlin B. Rogers, Andrew C. Dudley, Charles M. Perou, William C. Zamboni. Relationship between tumor-associated macrophages (TAMs), tumor delivery, and efficacy of PEGylated liposomal doxorubicin (PLD) and non-liposomal doxorubicin (NL-doxo) in genetically engineered mouse models (GEMMs) of breast cancer (BC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2013-3354

P4-03-03: Therapeutic Sensitivities of Mouse Models of Human Breast Cancer.

Genetically engineered mouse (GEM) models have provided a wealth of information regarding the genetic causes of cancer, but their utility for preclinical drug evaluation has not been well examined. Here we have used three mammary tumor GEM models that represent three human breast cancer subtypes and have evaluated their sensitivities to chemotherapy and to three biologically targeted agents. We have selected three mouse models that resemble human breast cancer subtypes based upon common gene expression profiles; Basal-like tumors are represented by the C3(1)-T-antigen (C3-TAg) model, Luminal B tumors are represented by the MMTV-Neu model, and the P53 null transplant T11 line represents the newly described Claudin-low breast tumor subtype. On each of these models we have tested the therapeutic efficacy of: four chemotherapeutics (doxorubicin, carboplatin, paclitaxel, and cyclophosphamide), two chemotherapy combinations (carboplatin/paclitaxel and doxorubicin/cyclophosphamide), and three biologically targeted agents (erlotinib, lapatinib, and ABT-888, alone and combined with selected chemotherapies). The results from individual models were as follows: The MMTV-Neu tumors were sensitive to the single-agent chemotherapeutics carboplatin and cyclophosphamide, and cyclophosphamide dramatically increased overall survival of the MMTV-Neu mice. The targeted agents lapatinib and erlotinib were extremely effective; lapatinib produced a near complete regression in every MMTV-Neu mouse tested and both compounds lead to greatly increased survival times. In the Claudin-low T11 line, the tumors were very sensitive to cyclophosphamide. Alone and in combination with doxorubicin, cyclophosphamide was the only chemotherapeutic able to successfully cause tumor regression in this model. None of the biological inhibitors were effective as single agents in these mice, nor were they effective in combination with chemotherapeutics other than cyclophosphamide In the C3-TAg basal-like model, carboplatin alone and in combination with other drugs caused volume reduction in some of the tumors. Erlotinib was able to cause volume reductions in a third of the treated C3-Tag tumors, which revealed a heterogeneity of response within this GEM strain. None of the single agent treatments significantly increased overall survival in these mice. Those combination treatments that were effective showed a range of responses from tumor regression to slowed progression. Finally, we closely examined the heterogeneous responses of the C3-Tag tumors to carboplatin/paclitaxel and performed expression profiling of sensitive and resistant tumors. We identified a gene signature from these treated mouse tumors that was able to predict pathological complete response of human patients receiving multiagent taxane-containing neoadjuvant chemotherapy regimens. These results show that genomically selected GEM models can recapitulate findings seen in human tumors (like lapatinib responsiveness in HER2+ tumors and carboplatin sensitivity in basal-like tumors) and that GEM models can potentially be used to develop biomarkers and to test new drug combinations prior to their being tested in humans. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-03.