Sharon Barr

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

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.

Loss of homotypic cell adhesion by epithelial-mesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition.

Overexpression and enhanced activation of the epidermal growth factor receptor (EGFR) is frequently observed in human carcinomas. Inhibitors of EGFR signaling have shown clinical utility; however, understanding response at the molecular level is important to define patient subsets most likely to benefit, as well as to support the rational design of drug combinations. Pancreatic and colorectal tumor cell lines insensitive to EGFR inhibition were those that had lost or mutated the epithelial junction constituents E-cadherin and γ-catenin, had lost homotypic adhesion, and often gained proteins associated with an epithelial to mesenchymal–like transition, such as vimentin, zeb1, or snail. In matched pairs of colorectal tumor cells, the epithelial lines showed an average 7-fold greater sensitivity than mesenchymal-like lines. In human pancreatic and colorectal tumor tissues, gain of mesenchymal characteristics and loss of epithelial characteristics correlated with advancing tumor stage. These data indicate an especially sensitive patient subset as well as a rationale for the combination of EGFR antagonists with agents that affect the epithelial to mesenchymal–like transition process as a mechanism to enhance sensitivity for more advanced mesenchymal-like tumors. [Mol Cancer Ther 2007;6(2):532–41]

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

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.

Critical roles for mTORC2- and rapamycin-insensitive mTORC1-complexes in growth and survival of BCR-ABL-expressing leukemic cells

mTOR-generated signals play critical roles in growth of leukemic cells by controlling mRNA translation of genes that promote mitogenic responses. Despite extensive work on the functional relevance of rapamycin-sensitive mTORC1 complexes, much less is known on the roles of rapamycin-insensitive (RI) complexes, including mTORC2 and RI-mTORC1, in BCR-ABL-leukemogenesis. We provide evidence for the presence of mTORC2 complexes in BCR-ABL-transformed cells and identify phosphorylation of 4E-BP1 on Thr37/46 and Ser65 as RI-mTORC1 signals in primary chronic myelogenous leukemia (CML) cells. Our studies establish that a unique dual mTORC2/mTORC1 inhibitor, OSI-027, induces potent suppressive effects on primitive leukemic progenitors from CML patients and generates antileukemic responses in cells expressing the T315I-BCR-ABL mutation, which is refractory to all BCR-ABL kinase inhibitors currently in clinical use. Induction of apoptosis by OSI-027 appears to negatively correlate with induction of autophagy in some types of BCR-ABL transformed cells, as shown by the induction of autophagy during OSI-027-treatment and the potentiation of apoptosis by concomitant inhibition of such autophagy. Altogether, our studies establish critical roles for mTORC2 and RI-mTORC1 complexes in survival and growth of BCR-ABL cells and suggest that dual therapeutic targeting of such complexes may provide an approach to overcome leukemic cell resistance in CML and Ph+ ALL.In recent years, there have been substantial research advances on the mechanisms by which BCR-ABL transforms hematopoietic cells and promotes leukemic cell growth and survival. Among the diverse signaling cascades activated by BCR-ABL, the mTOR pathway plays a critical role in mRNA translation of genes that promote leukemogenesis and mitogenic responses. We have recently shown that dual targeting of mTORC1 and mTORC2 complexes using a catalytic mTOR inhibitor, OSI-027, results in generation of potent antileukemic effects against BCR-ABL transformed cells. Such effects were also seen in cells expressing the T315I mutation, which is resistant to all currently approved BCR-ABL kinase inhibitors. Our studies also demonstrate that such dual catalytic inhibition of mTORC2 and mTORC1 complexes in BCR-ABL-expressing K562 cells results in induction of autophagy, and that inhibition of the autophagic process using chloroquine promotes apoptosis of these cells. Altogether, our studies suggest that autophagy may be a limiting factor for the induction of apoptosis during dual mTORC2-mTORC1 targeting, in at least some types of BCR-ABL-expressing cells and have raised the potential of combinations of catalytic inhibitors of mTOR with autophagy inhibitors for the treatment of refractory Ph(+) leukemias.

Dual mTORC2/mTORC1 targeting results in potent suppressive effects on acute myeloid leukemia (AML) progenitors

Purpose: To determine whether mTORC2 and rapamycin-insensitive (RI)-mTORC1 complexes are present in acute myeloid leukemia (AML) cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors. Experimental Design: Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell-proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor-colony formation. Results: mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. RI-mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027 blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro. Conclusions: Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin. Clin Cancer Res; 17(13); 4378–88. ©2011 AACR.

Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors

The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist.

Abstract 2403: Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway.

Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets. Methods: Expression of >140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC 50 s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1. Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients. Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O9Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [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 2403. doi:10.1158/1538-7445.AM2013-2403

Abstract A2: Selective inhibition of mTORC1/mTORC2 sensitizes prostate cancer cells to the effects of AR or IGF-1R/IR antagonists, providing an approach to overcome intrinsic mechanisms of resistance

Prostate cancer is a complex disease primarily characterized by dependence on androgen receptor (AR) signaling. Androgen deprivation therapy is efficacious; however, prostate tumors which initially respond to castration or androgen antagonists eventually progress. These castration-resistant tumors may develop hypersensitivity to low levels of androgens and AR overexpression or may adapt to rely on alternate signaling pathways such as the PI3K/mTOR axis. Deregulation of the PI3K/mTOR axis is a feature of prostate cancer, as evidenced by the fact that 40% of primary and 70% of metastatic prostate tumors exhibit loss of the tumor suppressor PTEN. Recent publications have shown that crosstalk between the AR and PTEN/PI3K/mTOR pathways plays a role in prostate cancer development and progression (1,2). Building upon these data, we evaluated the effects of ASP7486 (OSI-027), a selective inhibitor of mTORC1/mTORC2 as monotherapy and combined with bicalutamide, an androgen receptor antagonist. ASP7486 inhibited proliferation across a panel of prostate cancer cell lines, including those refractory to bicalutamide, supporting the hypothesis that tumors which have acquired androgen independence may rely upon mTOR signaling for survival. ASP7486 but not rapamycin, an allosteric mTORC1 inhibitor, induced apoptosis, implying that mTORC2 signaling is an important regulator of survival in prostate cancer. ASP7486, but not rapamycin, significantly upregulated AR expression which may serve to sensitize cells to the effect of an antiandrogen while limiting sensitivity to mTOR inhibition as a monotherapy. Consistent with this, the combination of ASP7486 and bicalutamide synergistically inhibited proliferation in vitro. In prostate tumors which have acquired resistance to androgen antagonists, activation of alternate RTKs may provide a survival mechanism in the absence of functional AR. PI3K pathway alterations and increased expression of IFG-1R are observed in castrate-resistant tumors (3,4). Treatment of prostate cancer cells with ASP7486 leads to increased phosphorylation of multiple RTKs, including IGF-1R and IR. We reasoned that ASP7486-mediated activation of IGF-1R and IR would sensitize these cells to the effects of OSI-906, a selective IGF-1R/IR inhibitor. The combination of ASP7486 and OSI-906, a selective inhibitor of IGF-1R/IR, synergistically inhibited proliferation and induced apoptosis. The combination of the two drugs provided greater inhibition of key signaling effectors than either monotherapy. These effects are not limited to prostate cancer. We have observed ASP7486-induced upregulation of RTK expression and phosphorylation in multiple tumor types, and the combination of ASP7486 and OSI-906 synergistically inhibited proliferation in the majority of cell lines tested. Together these data demonstrate that combinations of ASP7486 with targeted inhibitors can attenuate crosstalk between signaling networks and provide synergistic efficacy in vitro. The importance of AR and IGF-1R/PI3K/mTOR signaling in prostate cancer provides a rationale for targeting the disease and its intrinsic mechanisms of resistance with inhibitors of these pathways or in combination.

Abstract 4463: OSI-027, a dual mTORC1/mTORC2 inhibitor, induces autophagy in cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Inhibitors of mTORC1 have proven clinical activity in renal cell carcinoma (RCC), indicating that mTOR is an important signaling hub in this tumor type. mTOR exists in two functionally distinct protein complexes: mTORC1 and mTORC2. mTORC1 regulates multiple cellular functions including protein translation and tumor metabolism While this complex binds rapamycin some aspects of mTORC1 function are rapamycin insensitive. mTORC2 controls distinct cellular functions including survival and is not directly inhibited by rapalogs. In order to evaluate the relative contribution of mTORC1 and mTORC2 to the survival of RCC cells, we compared the effects of rapamycin, an allosteric inhibitor of mTORC1, to OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2. In RCC cell lines, OSI-027 provided greater maximal inhibition of proliferation than rapamycin, with reduction of cell viability to less than baseline in 5 of 12 cell lines, however caspase 3/7-dependent apoptosis was observed only in one cell line. We identified a subset of RCC cell lines which were sensitive to OSI-027 but not rapamycin, and another set which was relatively sensitive to both. Comparison of gene expression profiles of these groups identified MAP1LC3, encoding LC3, a critical effector of autophagy, as significantly upregulated in cell lines which were preferentially sensitive to OSI-027. We therefore sought to further define the role of autophagy in response to OSI-027 treatment. Autophagy is a catabolic process by which cells consume proteins and organelles to promote survival under adverse conditions, and which can result in cell death. Both mTORC1 and mTORC2 have been shown to negatively regulate autophagy, and inhibition of mTOR has been shown to induce formation of autophagic vesicles. Treatment in vitro with OSI-027 resulted in the conversion of LC3 to the lipidated form LC3-II as well as accumulation of LC3 in autophagosomes, robust indicators of autophagy. Increased autophagosome content occurred in a dose-dependent manner and is inversely proportional to viable cell number although induction of apoptosis was not observed, implying that in these cells OSI-027-induced autophagy leads to complete growth arrest or apoptosis-independent cell death. OSI-027-mediated induction of autophagy occurred in the majority of RCC cell lines tested, whereas little or no autophagy resulted from rapamycin treatment. In xenograft tumors sensitive to OSI-027 but not rapamycin, we analyzed induction of LC3-II and apoptosis in tumor lysates. Consistent with our observations in vitro, we observed that treatment with OSI-027 but not rapamycin resulted in induction of autophagy in vivo, while neither compound induced apoptosis. Together these data indicate that autophagy may play a key role in mediating OSI-027 but not rapamycin efficacy and supports further exploration of the utility of dual mTORC1/mTORC2 inhibitors for the treatment of renal cell carcinoma. 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 4463. doi:10.1158/1538-7445.AM2011-4463

Abstract 1632: Co-targeting mTOR and IGF-1R/IR results in synergistic activity against a broad array of tumor cell lines, independent of KRAS mutation status

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The IGF-1 receptor (IGF-1R) and insulin receptor (IR) are known to couple via IRS-1 to the PI3K signaling axis. Upon activation, these receptor tyrosine kinases stimulate PI3K, initiating a cascade of ser/thr kinases, including mTOR. mTOR is associated with two complexes: mTORC1, rapamycin sensitive, and mTORC2, which does not bind rapamycin. Since mTOR functions downstream of IGF-1R and IR, it is thought that tumor cells sensitive to IGF-1R inhibition would also respond to mTORC1/mTORC2 inhibition. To test this hypothesis we evaluated the effects of OSI-906, a selective small molecule dual kinase inhibitor of IGF-1R and IR and OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2, alone and in combination in cell proliferation assays using a broad array of tumor cell lines. Across multiple tumor types, cells with activating KRAS mutations were typically less sensitive to OSI-027 than those expressing wild-type KRAS. In contrast, mutant KRAS did not reduce sensitivity to OSI-906. Cell lines with activating mutations in PIK3CA or loss of PTEN were sensitive to OSI-027 but the presence of a concurrent mutation in KRAS decreased their sensitivity to OSI-027. In KRAS mutant cells, OSI-906 was more effective than OSI-027 at reducing phospho-AKT levels. Conversely, in tumor cell lines with wild-type KRAS cells, OSI-027 more effectively reduced phospho-AKT levels compared to OSI-906. Similar effects were observed in a pair of isogenic cell lines engineered to express either wild-type or mutant KRAS. The observation that tumor cell lines exhibit differential sensitivity to inhibitors of IGF-1R/IR or mTORC1/mTORC2 supports the concept that IGF-1R and IR can activate multiple pathways and are not limited to signaling through the mTOR axis. Also, mTOR is a central signaling hub which integrates signaling from multiple inputs, not only the IGF-1R/IR cascade. The combination of OSI-906 and OSI-027 synergistically inhibited the proliferation of cancer cell lines, including those expressing mutant KRAS. In some cases, the combination also resulted in synergistic induction of apoptosis. These data provide support for further exploration of the potential utility of combining OSI-906 (dual IGF-1R/IR inhibitor) with OSI-027 (dual mTORC1/mTORC2 inhibitor) for the treatment of human cancers, including those with activating mutations in KRAS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1632.