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Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase–AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti–IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti–IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone. Mol Cancer Ther; 9(10); 2652–64. ©2010 AACR.

Supplementary Data from Compensatory Insulin Receptor (IR) Activation on Inhibition of Insulin-Like Growth Factor-1 Receptor (IGF-1R): Rationale for Cotargeting IGF-1R and IR in Cancer

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

Supplementary Material from A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor–dependent tumor growth in vivo

Supplementary Figures 1-6 from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Table 1 from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Methods from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Figure Legends from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

The landscape in immuno-oncology (I-O) has undergone profound changes since its early beginnings up through the rapid advances happening today. The current drug development pipeline consists of thousands of potential I-O therapies and therapy combinations, many of which are being evaluated in clinical trials. The efficient and successful development of these assets requires the investment in and utilization of appropriate tools and technologies that can facilitate the rapid transitions from preclinical evaluation through clinical development. These tools include (i) appropriate preclinical models, (ii) biomarkers of pharmacodynamic, predictive and monitoring utility, and (iii) evolving clinical trial designs that allow rapid and efficient evaluation during the development process. This article provides an overview of how novel discoveries and insights into each of these three areas have the potential to further address the clinical management needs for patients with cancer.

Glioblastoma (GBM) is a fast-growing, aggressive type of central nervous system tumor that forms on the supportive tissue of the brain. Primary treatment options for patients with GBMs have not changed much in many years, and still include surgery, radiation and/or chemotherapy treatment with temozolomide (TMZ). GBM is an incurable disease where there is a desperate need for continued development of novel therapeutic options. A number of preclinical models exist to support the early work needed to develop these new drugs. Here we describe three different preclinical models of brain cancer, U87MG-Luc, GL261-Luc and BT142. Each model was evaluated following orthotopic implantation into the brain. As radiation therapy remains a steadfast approach to GBM, we evaluated each model for its response to radiation and tracked disease progression and response to treatment with either bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) along with traditional survival (morbidity/mortality) analysis. U87MG-Luc is a human glioblastoma cell line that was developed from an astrocytoma. We found that orthotopic implant of U87MG-luc results in reliable growth with a tumor doubling time of 4 days and a median survival time of 45 days. We tested this model with radiation therapy (2.5Gy for 5 days on, 2 days off, for 2 cycles) and found a 70% partial response rate as measured by BLI. We also tested this model with TMZ therapy (33.3mg/kg; orally every day for 5 days) and found a significant delay in disease progression, resulting in a 93% increase in life span. GL261-Luc is a murine glioblastoma model that grows quite aggressively in the mouse brain. We find a tumor volume doubling time of 2 days and a median survival time of 14 days. As this model grows in a mouse strain with an intact immune system it allowed us to evaluate the tumor infiltrating immune cell populations. While we found a low number of CD45+ cells infiltrating into GL261-Luc tumors, immune population changes over time will be described. In this model we tested increasing doses of radiation and found that a single dose of 15Gy was curative whereas the tumors were more moderately responsive to a single dose of 7.5Gy (30% tumor regressions). We then combined 7.5Gy with the immune checkpoint inhibitor anti-PD-1 and saw an improved outcome with a 100% response rate. BT142 is a human anaplastic oligoastroytoma that is relatively unique due to its isocitrate dehydrogenase (IDH1/2) mutational status which results in an accumulation of the oncometabolite 2-hydroxygluarate (2-HG) in the tumor. This tumor line grows in the NOD SCID mouse strain and we found a tumor doubling time of 5 days, as measured by MRI. The BT142 model has proven to be sensitive to radiation following a single dose of 10Gy but less responsive when lower dosage levels were administered on a fractionated schedule. Thus, a variety of human and mouse GBM models exist to enable further drug discovery and development efforts for this intractable disease. Citation Format: Erin E. Trachet, Sumithra Urs, Alden Wong, Scott Wise, Maryland Rosenfeld Franklin. Radiation response in preclinical orthotopic models of brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1928.

Radiotherapy is a highly utilized clinical treatment modality. More than 50% of all cancer patients receive some type of radiation therapy during the course of their illness. In mouse models, radiation treatment has been shown to increase the level of tumor antigen presentation and the variety of peptides available for cross-presentation. Current work in the field focuses on using radiation as a tool to bridge the gap from tumor equilibrium to tumor elimination, which could improve the response rate of immuno-oncology agents. 4T1 is a murine breast cancer model known to have a large percentage of myeloid derived suppressor cells (MDSC) making the model resistant to many immunotherapies and is considered an immunologically cold tumor. We hypothesized that treatment with focal radiation (RT, Xstrahl) would sensitize 4T1 tumors to anti-CTLA-4 treatment. To determine an appropriate dose of RT, mice with 4T1-Lu2 tumors were treated with a single, focal dose of 5, 10 or 20Gy RT. No response was seen at 5Gy, 10Gy resulted in 10 days growth delay, and 20Gy resulted in 16 days growth. In subsequent work, mice were placed into groups and treated with either isotype control (10mg/kg, MPC-11), anti-CTLA-4 (10mg/kg, 9D9), 10Gy RT, or the combination. Mice were monitored over time for changes in primary tumor volume by caliper measurements and for metastatic disease by bioluminescence imaging (BLI) of the thoracic region. Satellite groups were included for immunohistochemistry and the evaluation of changes in tumor infiltrating lymphocytes. Median tumor growth delay with anti-CTLA-4, RT, or the combination, was 2.4, 3.5 and 9.4 days, respectively. Time to progression, to 1,200mm3 tumors, was increased by 25% in the combination group but not significantly in the monotherapy groups. While all mice showed evidence of metastatic disease, mice in the combination group displayed a lower level of BLI signal on day 30 when compared to other groups. To examine the effects on immune cell infiltration, 11 subsets were profiled by flow cytometry. B cells and regulatory T cells were significantly reduced (>95% and 90%, respectively) in the combination group when compared to an untreated group. B cell reductions that exceeded 90% were also observed in some animals within the isotype control group. Examination of the CD8+ T cell phenotype demonstrated that combination therapy, but not monotherapies, increased expression of both CD69 and PD-1 on CD8+ T cells suggesting an enhancement of anti-tumor cytotoxicity in these cells. Interestingly, we found that the combination selectively reduced the levels of phosphorylated STAT3 in MDSC subsets. Collectively, this intimates that anti-CTLA-4 and RT selectively disrupt STAT3 signaling in MDSCs and combine to enhance CD8+ T cell activity, which may play a role in the 4T1 tumor growth delay observed. Citation Format: Maryland Rosenfeld Franklin, David Draper, Sumithra Urs, Scott Wise. Preclinical use of focal radiation and immune checkpoint blockade to improve therapeutic response in an immunologically cold tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 535.

To advance immuno-oncology drug development, there is a need for well-characterized syngeneic tumor models. In addition to subcutaneous modeling, orthotopic models are equally important for preclinical development as they are posited to have improved clinical translatability compared to subcutaneous implantation. To this end, we have generated both subcutaneous and systemic A20 murine B cell lymphoma models. Specific to orthotopic modeling, a luciferase-enabled A20 model (A20-luc) was created to monitor tumor progression by bioluminescence imaging. Systemic parental A20 or A20-luc models show slight differences in median survival (34.5 days and 39 days, respectively) but similar take rates and clinical manifestations, including abdominal distension and metastatic foci in the liver. These data suggest that parental A20 and A20-luc behave similarly in systemic disease. To understand how checkpoint blockade and costimulatory agonists act on A20, anti-mPD-1, anti-mPD-L1, anti-mCD137, and anti-mGITR were evaluated for efficacy against both established subcutaneous and systemic A20 models. Our data indicate that neither anti-mPD-1 nor anti-mGITR demonstrate antitumor activity in either model. Anti-mPD-L1 is inactive in systemic disease but shows some efficacy on A20 subcutaneous tumors with delayed growth observed in 40% of mice. Contrastingly, the costimulatory agonist anti-mCD137 elicits a stronger response, with 50% of established subcutaneous A20 tumors having delayed growth including two complete responses and one tumor free survivor. Similarly, in systemic A20 disease, 50% of animals treated with anti-mCD137 exhibited a profound decrease in luciferase signal. While modestly responsive, both A20 and A20-luc show promise for use in combination strategies. To determine whether this modest activity is due to an infiltration of immunosuppressive cells into the tumor, the composition of immune cells in established subcutaneous A20 tumors was evaluated. Despite modest activity, 50% of total CD45+ infiltrate were T cells and NK cells, 7% were MDSCs and macrophages, and the remaining were dendritic in nature. As high T cell infiltrate is more common in tumors responsive to immunotherapy, these infiltrated T cells are hypothesized to lack signals required for activation or are reactive to non-tumor-associated antigen. Similar assays with metastatic foci resulting from systemic disease are planned. Both subcutaneous and orthotopic A20 models exhibit favorable characteristics for evaluation of investigational agents. Response to immunotherapy is minimal to moderate in both models but are generally aligned. The high infiltrate of T cells in the subcutaneous model have potential for exploitation of this model in development of agents targeting T cells. Taken together, our results indicate that A20 is a favorable syngeneic tumor model for both subcutaneous and orthotopic applications. Citation Format: Sheri R. Barnes, Sumithra Urs, David Draper, Scott Wise, Maryland Rosenfeld Franklin. Systemic and subcutaneous modeling of A20 murine B cell lymphoma in mice: A comparative assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3719.

The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor β (aTGFβ), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFβ caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFβ induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated, antibody-producing plasma cells in the bone marrow. Genetic mutations within the myeloma cells and their interaction with various cytokines and growth factors contribute to the invasiveness of the disease and enhanced drug resistance. Patients are generally asymptomatic until very late-state disease. However, once disease is present, localization in the bones, especially the spine, is common. There are few preclinical models that recapitulate human disease. In this work, we have evaluated the human MM.1S model and the murine 5TGM-1 model. MM.1S was derived from a 42-year-old African American woman and has been documented to express CD25, CD38, CD52, and CD59. It also expresses the glucocorticoid receptor and is dexamethasone sensitive. To more effectively monitor in vivo disease progression, we transfected the MM.1S line with luciferase (MM.1S-pMMP-LucNeo). With bioluminescence imaging (BLI) we track and monitor disseminated disease progression over time and find reproducible in vivo growth in SCID beige mice. The median tumor volume doubling time is ~2 days and median time to morbidity/mortality endpoint is 35-45 days. MM.1S-pMMP-LucNeo was also evaluated in NSG mice which show more aggressive disease onset (staging 7 days post implant vs. 15 days in SCID mice). In NSG mice the tumor volume doubling time is ~1.8 days and median time to endpoint is ~21 days, roughly half the overall survival time observed in the SCID mouse. In all mouse strains we have investigated, the animals are relatively asymptomatic throughout the lifetime of the study and only begin to present symptoms at late-stage disease, similar to how the disease manifests itself in humans. Common, late-stage clinical signs in the mice include lethargy and paralysis. BLI on these animals shows localization of the signal in the spine, long bones, and mandible. As in the clinic, bortezomib (Velcade®) has limited effect on disease progression in this model. Expression of CD138 is a hallmark of plasma cells and multiple myeloma cells. In the NSG study we determined, by flow cytometry, that >85% of the CD45- gate in the bone marrow was CD138+ 21 days post-implant, suggesting significant engraftment of the tumor cells within the bone marrow. 5TGM-1 is a syngeneic model generated from the C57BL/KaLwRij mouse which is predisposed to develop several monoclonal B-cell proliferative disorders, including a low percentage (0.5%) of the mice developing multiple myeloma. From this strain, several cell clones were isolated and called the 5TMM series, which included the 5T33MM clone. 5TGM-1 is a sub-clone of 5T33MM cells and we have used 5TGM-1-luc to monitor disease progression and localization in vivo. The 5TGM-1-luc model is aggressive and BLI has shown localization of disease in the long bones. We have compared this model in both the syngeneic C57BL/KaLwRij strain and the immune-deficient NIH III (triple deficient) strain. Disease progression behaves similarly in both strains of mice, with significant tumor cells localizing in the spine and the long bones. The model is sensitive to traditional chemotherapies like doxorubicin and cyclophosphamide, but bortezomib (Velcade) only produces modest activity and carfilzomib has shown little activity at the dose and schedule tested. A greater understanding of the preclinical behavior of these models, including response to standard of care and molecular profiling, will provide critical insight into designing more clinically relevant preclinical experiments. Citation Format: Erin Trachet, Maryland Rosenfeld Franklin. Preclinical models of multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 36.

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR– insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR–directed Akt signaling, where all affect feedback loops converging at the level of IRS-1. [Cancer Res 2008;68(20):8322–32]

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis.

T cell-derived cytokines are important in the development of an effective immune response, but when dysregulated they can promote disease. Here we identify a four-helix bundle cytokine we have called interleukin 31 (IL-31), which is preferentially produced by T helper type 2 cells. IL-31 signals through a receptor composed of IL-31 receptor A and oncostatin M receptor. Expression of IL-31 receptor A and oncostatin M receptor mRNA was induced in activated monocytes, whereas epithelial cells expressed both mRNAs constitutively. Transgenic mice overexpressing IL-31 developed severe pruritus, alopecia and skin lesions. Furthermore, IL-31 receptor expression was increased in diseased tissues derived from an animal model of airway hypersensitivity. These data indicate that IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.

The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono- and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. To this end, using the CT26 murine syngeneic colorectal cancer model we have developed a 10 color flow cytometry antibody panel that focuses on the identification of tumor-infiltrating immune cell subsets derived from myeloid lineage precursors utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer with autosampler. The panel includes a combination of antibodies against CD45, CD3, CD19, CD49b, CD335, CD11b, CD11c, Ly-6G, Ly-6C, F4/80, and CD115. By excluding cells of lymphoid lineage, we show that this panel facilitates analysis of myeloid derived cells including natural killer (NK) cells, macrophages, neutrophils, dendritic cells (DCs), and monocytic or granulocytic myeloid-derived suppressor cell (mMDSCs and gMDSCs) subsets in tumor and peripheral blood. In addition, this combination of antibodies allows for a more complete analysis of MDSCs which can differentially express several disease-relevant myeloid specific markers including Ly-6G, Ly-6C, F4/80, CD11c, and CD115. In the tumor, the Ly-6G-high population demonstrates differential expression of Ly-6C, 21% Ly-6C-high (granulocytes) and 77% Ly-6C-low (mMDSCs). The majority of the granulocytic population was identified as gMDSCs and the remainder as neutrophils based on CD115 expression. Macrophages constitute 27% of Ly-6G-low cells. In blood, 98% of the Ly-6G-high population was also Ly-6C-high, and this population is predominantly neutrophils. No macrophages (Ly6G-low and F4/80+) were identified in the peripheral blood. These data confirm the expected distribution of myeloid lineages in the tissue types investigated. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of additional potentially responsive immune compartments will facilitate identification and development of potential combination therapies otherwise overlooked by looking primarily at T-cells. This panel allows for a significant expansion of our ability to provide a complex description of the myeloid subset. Citation Format: Matt Thayer, David Draper, Daniel Saims, Maryland Rosenfeld-Franklin, Scott C. Wise. In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [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 3242.

Immunotherapy has led to major treatment breakthroughs for a number of cancers including non-small cell lung cancer (NSCLC). Although initial responses to immune checkpoint inhibitors are promising, a significant percentage of patients do not respond or rapidly acquire resistance. Although the mechanisms underlying intrinsic and acquired resistance remain largely unexplained; the expression of programmed cell death-ligand 1 (PD-L1), lack of tumor cell capacity to effectively present neoantigens, and presence of immunosuppressive cellular subsets have been implicated as potential mechanisms. Histone deacetylase (HDAC) inhibitors have emerged as a class of agents that may combat checkpoint inhibitor resistance by reversing immune evasion and eliciting an anti-tumor activity through a multi-faceted immuno-stimulatory mechanism of action. Mocetinostat is a spectrum-selective Class I/IV HDAC inhibitor specifically targeting HDAC-1, -2, -3 and -11. The present studies were designed to explore mocetinostat's effect as an immune-enhancer and ultimately, to evaluate its potential to be used in combination with immune checkpoint inhibitors (e.g., PD-1/PD-L1 antagonists). Specifically, we assessed mocetinostat's effect on the expression of various immunomodulatory factors by tumor cells as well as its effect on immune cell sub-populations in the tumor microenvironment in vivo. Mocetinostat elicited a concentration-dependent increase in PD-L1 mRNA expression which translated into increased PD-L1 surface protein expression in a panel of NSCLC cell lines. In addition, mocetinostat elicited a concentration-dependent increase in expression of MHC-class I related polypeptide-related sequence A (MIC-A) and MIC-B, and cluster of differentiation 86 (CD86). Furthermore, mocetinostat induced expression of several human leukocyte antigen (HLA) gene complex family members including HLA-A, -B, -DRA, and -DPA among others. To determine the effect of mocetinostat on systemic and tumor immune cell subpopulations we treated CT26 tumor-bearing mice. Mocetinostat increased splenic CD4-positive T effector cells and tumor mature cytolytic CD8-postive T cells and at the same time decreased tumor FoxP3-positive T regulatory cells and CD11b/Gr1-positive myeloid-derived suppressor cells (MDSC). These data provide evidence that mocetinostat modulates key immune regulators both in tumor cells as well as in relevant immune cell types in the tumor microenvironment and provides strong rationale for combination with immune checkpoint inhibitors. Citation Format: David Briere, Niranjan Sudhakar, Lars Engstrom, Jill Hallin, Ruth Tang, Harrah Chiang, Maryland Rosenfeld-Franklin, Peter Olson, James Christensen. The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations. [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 4021.

Background: The IGF-1 receptor (IGF-1R) has been implicated in the promotion of tumorigenesis, metastasis and resistance to cancer therapies. Therefore, this receptor has become a major focus for the development of anticancer agents. Results: Our lead optimization efforts that blended structure-based design and empirical medicinal chemistry led to the discovery of OSI-906, a novel small-molecule dual IGF-1R/insulin receptor (IR) kinase inhibitor. OSI-906 potently and selectively inhibits autophosphorylation of both human IGF-1R and IR, displays in vitro antiproliferative effects in a variety of tumor cell lines and shows robust in vivo anti-tumor efficacy in an IGF-1R-driven xenograft model when administered orally once daily. Conclusion: OSI-906 is a novel, potent, selective and orally bioavailable dual IGF-1R/IR kinase inhibitor with favorable preclinical drug-like properties, which has demonstrated in vivo efficacy in tumor models and is currently in clinical testing.

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase–AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti–IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti–IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone. Mol Cancer Ther; 9(10); 2652–64. ©2010 AACR.

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

A series of novel, potent quinolinyl-derived imidazo[1,5-a]pyrazine IGF-IR (IGF-1R) inhibitors--most notably, cis-3-(3-azetidin-1-ylmethylcyclobutyl)-1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (AQIP)--is described. Synthetic details, structure-activity relationships, and in vitro biological activity are reported for the series. Key in vitro and in vivo biological results for AQIP are reported, including: inhibition of ligand-stimulated autophosphorylation of IGF-IR and downstream pathways in 3T3/huIGFIR cells; inhibition of proliferation and induction of DNA fragmentation in human tumor cell lines; a pharmacokinetic profile suitable for once-per-day oral dosing; antitumor activity in a 3T3/huIGFIR xenograft model; and effects on insulin and glucose levels.

Erlotinib is a tyrosine kinase inhibitor of EGFR approved for use in non-small cell lung cancer (NSCLC) and pancreatic cancer in combination with Gemcitabine. Within the context of NSCLC it has been observed that patients harboring an activating mutation within the kinase domain of EGFR demonstrate a high response to treatment. However, their disease often progresses within 1 year. OSI-906 targets IGF-1R and IR receptor tyrosine kinases and has shown combination efficacy with erlotinib in preclinical models of NSCLC with wild type EGFR. Here we report preclinical data suggesting that the combination of erlotinib and OSI-906 is more efficacious in NSCLC models expressing an EGFR mutation than either single agent. A panel of NSCLC cells lines with confirmed mutations in the EGFR kinase domain was screened for erlotinib and OSI-906 single agent drug sensitivities. All mutant EGFR models exhibited sensitivity to EGFR inhibition by erlotinib (IC50 7–33 nM) while none showed sensitivity to OSI-906 (IC50 7gt;10 uM). In vitro, the combination of erlotinib and OSI-906 resulted in synergistic inhibition of cell proliferation and induction of apoptosis in PC3 (JCP-1) cells. Mechanistically we found the combination had enhanced inhibition of signaling through the PI3K/AKT pathway compared to treatment with either single agent. We also observed that while OSI-906 showed no induction of cell death, erlotinib treatment caused almost complete cell death. However after 9 days of erlotinib treatment small colonies of viable cells still remained. Removal of drug allowed the cells to grow and expand. When cells were treated with both erlotinib and OSI-906, complete cell death occurred without any evidence of cell regrowth following removal of both drugs. In order to evaluate this combination effect in vivo the EGFR mutant human tumor xenograft models NCI-H1650 and PC-14 were employed. Both lines are highly sensitive to erlotinib single agent treatment but show no anti-tumor activity with OSI-906 single agent treatment. The combination of 100 mg/kg erlotinib and 10 mg/kg OSI-906 demonstrated enhanced tumor growth delay when compared to either single agent dosed at MTD. However, as a first step towards understanding preclinical mechanism of this interaction we investigated the combination of 25 mg/kg of erlotinib with 30 mg/kg of OSI-906 and identified potential in vivo synergy. These studies with 25 mg/kg of erlotinib and 30 mg/kg of OSI-906 showed initial tumor regressions, enhanced tumor inhibition and substantially prolonged tumor growth delay when compared to either single agent. Drug-drug interaction PK studies suggest that OSI-906 is not acting to enhance erlotinib exposure. This data provides preclinical proof-of-concept for the use of OSI-906 in combination with erlotinib to obtain greater anti-tumor activity in the mutant EGFR setting. 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 LB-388. doi:10.1158/1538-7445.AM2011-LB-388

The EGFR kinase inhibitor erlotinib is approved as a maintenance therapy in 1st line NSCLC as well as for treatment of 2nd/3rd line NSCLC and in combination with Gemcitabine for pancreatic cancer. It has been observed that the most pronounced responses to EGFR tyrosine kinase inhibitors (TKI's) were observed in patients whose tumors expressed a mutated form of the EGFR kinase. These mutations mapped to the kinase domain of the receptor and functionally have been shown to render tumors and cells lines onco-addicted to EGFR signaling. Although patients expressing a mutated EGFR show a dramatic initial response to EGFR TKI's, ∼50% of these patients will progress while on therapy after 1-2 years. The mechanisms that underlie this acquired resistance to EGFR TKI therapy have been intensively studied and include but are not limited to the presence of a second mutation, T790M, or increased HGF-MET signaling. Previously, the role of epithelial to mesenchymal transition (EMT) in resistance to EGFR kinase inhibitors has been described in the context of wild type EGFR. EMT. We were therefore interested in understanding whether EMT could play a role in resistance to EGFR TKIs in the context of an EGFR mutation. Here we show that a panel of NSCLC cell lines, expressing mutant EGFR, can undergo an EMT in response to TGFβ treatment. The cell lines take on a scattered and spindle-like morphology and also down regulate the expression of E-cadherin and up regulate expression of vimentin, classic protein markers of an EMT. Importantly we show that after undergoing EMT, the EGFR mutant line HCC827 has reduced sensitivity to erlotnib treatment which is regained after reversal of the EMT. To further explore whether EMT could play a role in acquired resistance to erlotinib, we generated in vitro cell line models that were resistant to EGFR inhibition through continued culturing in the presence of erlotinib over a 6 month period. Resistant clones generated from parental HCC4006 cells acquired a more scattered and spindle-like morphology consistent with an EMT. These clones had down-regulated E-cadherin and ErbB3 expression and upregulated vimentin, fibronectin and Zeb1 expression and also showed a gene expression pattern consistent with having undergone an EMT. In addition, the resistant H4006 clones were more migratory and invasive than their parental counterpart. Finally we show that the resistant clones are enriched for stem cell markers and have enhanced signaling through the Src family kinases and the JAK-STAT pathway suggesting a mechanistic rationale for their reduced sensitivity to EGFR inhibitors. Taken together. these data indicate that NSCLC cell lines that express a mutant version of EGFR are able to undergo an EMT which can influence the efficacy of EGFR TKIs, suggesting that this may be an additional mechanism underlying the acquired resistance of NSCLC patients to EGFR therapy in the clinic. 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 3370. doi:10.1158/1538-7445.AM2011-3370

The Type 1 insulin-like growth factor receptor (IGF-1R) is a well established mediator of tumor cell proliferation and survival. IGF-1R is required for cellular transformation by a number of oncogenes including Ras, and expression of IGF-1R can promote tumor formation in vivo. Blockade of IGF-1R signaling by either genetic or pharmacological methods results in inhibition of tumor cell proliferation. This understanding has spurred the evaluation of a number of IGF-1R inhibitors in the clinic, including both neutralizing antibodies and small molecule kinase inhibitors. IGF-1R is structurally and functionally related to the insulin receptor (IR). Although IR is appreciated for its classical role in glucose metabolism, IR can also regulate cellular proliferation. Furthermore, there is evidence for compensatory crosstalk between IGF-1R and IR. In embryonic development IR signaling can compensate for loss of IGF-1R to maintain normal embryonic weight. A growing body of data indicates that tumor cells may also exploit IR signaling for proliferation and survival. Tumor cells frequently co-express both IGF-1R and IR, and increased expression of both IGF ligands and insulin are associated with increased risk of cancer. Elevated expression of the IR(A) fetal variant, which is potently activated by both insulin and IGF-2, is observed in select human tumors. Although the tumorigenicity for IGF-1R is well described, the potential for IR as a tumor maintenance gene in vivo has thus far not been established. Herein, we sought to address the tumorigenic potential for IGF-1R compared with IR(A). We used a mouse mammary tumor model driven by an inducible human HER2 oncogene under doxycyclin-directed expression, where repression of HER2 expression upon doxycyclin withdrawal was followed by introduction of genes encoding either IGF-1R or IR(A) in combination with IGF2. We find that either IGF-1R or IR(A), in combination with the ligand IGF2, can complement tumor growth. The growth of both IGF-1R and IR(A) direct complementation (DC) tumor models could be inhibited by the dual IGF-1R/IR small molecule inhibitor OSI-906. Cell lines derived from the IGF-1R and IR(A) DC tumors show that expression of either receptor individually can maintain signaling through the IRS-AKT signaling pathway. Collectively, these observations show that either IGF-1R or IR(A) have tumorigenic potential, and indicate that dual targeting of IGF-1R and IR may be required for optimal activity in tumors where both receptors are present and activated. 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 1631. doi:10.1158/1538-7445.AM2011-1631