Brian C. Grabiner

Response and Acquired Resistance to Everolimus in Anaplastic Thyroid Cancer

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.

A Diverse Array of Cancer-Associated MTOR Mutations Are Hyperactivating and Can Predict Rapamycin Sensitivity

Genes encoding components of the PI3K-AKT-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene encoding the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor DEPTOR. mTOR complex 1 (mTORC1) signaling in cells expressing various activating mutations remains sensitive to pharmacologic mTOR inhibition, but is partially resistant to nutrient deprivation. Finally, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency.

CARMA3 is Crucial for EGFR-Induced Activation of NF-κB and Tumor Progression

EGF activates NF-κB, and constitutively activated NF-κB contributes to EGFR mutation-associated tumorigenesis, but it remains unclear precisely how EGFR signaling leads to NF-κB activation. Here we report that CARMA3, a caspase recruitment domain (CARD)-containing scaffold molecule, is required for EGF-induced NF-κB activation. CARMA3 deficiency impaired the activation of the IKK complex following EGF stimulation, resulting in a defect of EGF-induced IκBα phosphorylation and NF-κB activation. We found that CARMA3 and Bcl10 contributed to several characteristics of EGFR-associated malignancy, including proliferation, survival, migration, and invasion. Most importantly, CARMA3 contributed to tumor growth in vivo. Our findings elucidate a crucial link between EGFR-proximal signaling components and the downstream IKK complex, and they suggest a new therapeutic target for treatment of EGFR-driven cancers.

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

Summary During antibody affinity maturation, germinal center (GC) B cells cycle between affinity‐driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen‐dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell‐cycle progression itself and must be regulated dynamically during cyclic re‐entry to ensure efficient affinity‐based selection. Graphical Abstract Figure. No Caption available. HighlightsPositive selection of GC B cells triggers mTORC1 activation prior to clonal expansionmTORC1 is required for biomass accumulation but dispensable for cell‐cycle progressionDeregulated mTORC1 promotes DZ phenotype and leads to loss of GC competitivenessRapamycin‐resistant mouse strain is used for cell‐intrinsic mTORC1 inhibition &NA; During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst.

Abstract PR04: mTOR mutations in cancer

Genes in the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer but relatively few mutations have been observed in the MTOR gene itself. By mining publicly available cancer sequencing data, we recently reported that MTOR is commonly mutated in cancer, with more than 35 previously uncharacterized pathway activating mutations. Most were recurrent, localized within distinct clusters in the C-terminal half of the protein, and induced activation of mTOR Complex 1 (mTORC1), mTORC2, or both. The mutations did not prevent mTOR inhibition by rapamycin or ATP-competitive inhibitors, although several conferred resistance to nutrient withdrawal. Importantly, cancer cell lines with activating mTOR mutations are especially sensitive to rapamycin both in vitro and in vivo, indicating pathway dependency. In a companion report, we also demonstrated that the tumor of a bladder cancer patient with exquisite sensitivity to a rapamycin analog (rapalog) harbored two activating mTOR mutations, likely driving mTOR pathway dependency. However, after 14 months of rapalog response, the patient displayed progressive disease, indicating that the tumor had acquired resistance to the targeted therapy. Here, we demonstrate two mechanisms by which cancers may display resistance to rapamycin through mTOR mutation. In the first example, we acquired a tumor biopsy from an anaplastic thyroid carcinoma patient before they were enrolled on a rapalog clinical trial. The patient displayed a durable 18 month response to the rapalog before progressing, at which time we acquired a second biopsy. Whole exome sequencing of the biopsies revealed the presence of a loss-of-function mutation in the mTORC1 negative regulator Tuberin (TSC2), likely driving pathway addiction and thus sensitivity to the rapalog. Sequencing of the post-resistance biopsy revealed that the tumor had acquired a mutation in the rapamycin-binding domain of mTOR. Cells made to exogenously express this mutation displayed potent resistance to rapamycin-mediated inhibition of cell proliferation and phosphorylation of the mTORC1 substrate S6K1. Importantly, the cells remained sensitive to an ATP-competitive mTOR inhibitor because the two classes of mTOR inhibitors rely upon completely different mechanisms of action. Thus, we show that as patients acquire resistance to rapalogs, they may still be effectively treated with ATP-competitive mTOR inhibitors. The rapalog-resistant mutation described above is an example of an acquired mutation, although due to the genetic instability of cancer cells, a tumor may harbor pre-existing rapamycin-resistant mutations. To determine the prevalence of such mutations, we again turned to publicly available cancer sequencing data, identifying approximately 30 mutations in the rapamycin-binding domain of mTOR. The majority these mutations did not alter mTOR pathway activation and remained rapamycin sensitive. Importantly, one breast cancer sample harbored a rapamycin-resistant mTOR mutation, although it remained sensitive to an ATP-competitive mTOR inhibitor. This rare event is highly relevant given that rapalogs are currently approved for the treatment of breast cancer. Had this patient been treated with a rapalog, they would have only experienced the side-effects and none of the benefits of treatment. Collectively, we demonstrate that mTOR mutations are common in cancer, many of which confer pathway activation and addiction. Additionally, both acquired and pre-existing mTOR mutations can confer resistance towards rapamycin treatment, although this resistance can be overcome with ATP-competitive inhibitors. This work has important ramifications for the treatment of cancer patients with mTOR inhibitors, by identifying both those patients likely to respond or display resistance to such treatments. This abstract is also being presented as Poster A44. Citation Format: Brian Grabiner, Nikhil Wagle, Eliezer Van Allen, Levi Garraway, Jochen Lorch, David Sabatini. mTOR mutations in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR04.

Abstract 1724: Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Understanding genetic mechanisms of sensitivity and resistance to targeted anticancer therapies may improve patient selection and rational treatment designs. One approach to increase this understanding involves the study of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder on a study of the allosteric mTOR inhibitor everolimus in thyroid cancer: a 57-yr-old woman with refractory metastatic anaplastic thyroid carcinoma (ATC), a highly aggressive neoplasm with no adequate therapies and a median survival of 5 months. After beginning treatment with everolimus, the patient experienced a near complete response that lasted for 18 months, followed by progressive disease, which was then re-biopsied. To date, mechanisms of clinical resistance to mTOR inhibition have not been described. We performed whole exome sequencing (WES) of both pre-treatment and drug resistant tumor tissue to look for the underlying mechanisms of exquisite sensitivity and acquired resistance to everolimus. WES of the pre-treatment tumor revealed a somatic nonsense mutation in TSC2, a tumor suppressor gene whose inactivation is known to activate the mTOR pathway and result in sensitivity to mTOR inhibition in some cancers. WES of the drug resistant tumor additionally revealed a mutation in mTOR (mTOR-F2108L) not detected in the pre-treatment tumor. Structural modeling demonstrated that this mutation occurs in the FKBP12-rapamycin binding domain of mTOR and is predicted to prevent binding of the drug to the protein. Overexpressing mTOR-F2108L in HEK-293T cells resulted in significant resistance to rapamycin compared to cells expressing wild type (wt) mTOR. In cells expressing the mutant mTOR, rapamycin did not decrease phosphorylation of S6K1, a downstream target of mTOR, compared with cells expressing wt mTOR. Notably, cells expressing mTOR-F2108L remained sensitive to the direct TOR inhibitor torin, suggesting a therapeutic approach to overcome resistance in this patient. In summary, we add ATC to the growing list of cancers found to be exquisitely sensitive to everolimus when activating mTOR pathway mutations are present. Moreover, we present the first reported, to our knowledge, mechanism of acquired resistance to everolimus identified in patients. The fact that this occurs via a binding domain mutation that blocks allosteric mTOR inhibition suggests that followup therapy with direct TOR inhibitors may still have benefit in some patients who develop resistance to everolimus. The use of precision medicine approaches in ATC to screen for alterations in the mTOR pathway may help identify subsets of patients who would benefit from targeted therapies directed against mTOR. Moreover, the use of serial biopsies to profile patients who develop resistance to everolimus could dictate optimal followup treatment in ATC and other cancers. Citation Format: Nikhil Wagle, Brian C. Grabiner, Eliezer M. Van Allen, Ali Amin-Mansour, Scott C. Carter, Nathanael Gray, Justine A. Barletta, Scott J. Swanson, Daniel Ruan, David J. Kwiatkowski, Glenn J. Hanna, Robert I. Haddad, David Sabatini, Pasi A. Janne, Levi A. Garraway, Jochen H. Lorch. Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma. [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 1724. doi:10.1158/1538-7445.AM2014-1724

Abstract A163: A diverse array of mutations in MTOR are hyperactivating and correlate with rapamycin sensitivity.

Components of the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer but relatively few mutations have been observed in the MTOR gene itself, which encodes the mTOR kinase. We used cancer genome sequencing data to generate a catalogue of over 450 MTOR point mutations found in cancer and from these identified a set of over 25 that cause mTOR pathway hyperactivation in cells. Mutations occur in multiple cancer types and cluster in several distinct regions in the C-terminal half of mTOR. The distribution and diversity of hyperactivating mutations is distinct from those found in kinases such as AKT1 or BRAF but similar to the pattern seen in PIK3CA. The mutations do not affect the sub-cellular localization of mTOR or its capacity to nucleate the mTORC1 and mTORC2 complexes, but the mutants do bind less DEPTOR, an mTOR inhibitor. In kinetic analyses, several of the most frequently recurrent mutations demonstrate increased K-cat over wild-type mTOR, suggesting that these mutations alter the mTOR kinase itself. Lastly, cancer cell lines with naturally arising mTOR mutations that fall within the clusters exhibited hypersensitivity to mTOR inhibitors. Thus, a diverse set of mutations in mTOR can activate the mTOR pathway and may predict which patients are most likely to respond to rapamycin-based therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A163. Citation Format: Brian Grabiner, David Sabatini. A diverse array of mutations in MTOR are hyperactivating and correlate with rapamycin sensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A163.