Joy Gary

B Cell–Specific Deficiencies in mTOR Limit Humoral Immune Responses

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP–specific or anti-Pn14–specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A–specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell–specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun‐exposed sites. Most occur in the oral cavity, with a subset having intra‐epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ‐line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c‐kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma

UNLABELLED Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. SIGNIFICANCE Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.

Molecular Pathways: Increased Susceptibility to Infection Is a Complication of mTOR Inhibitor Use in Cancer Therapy

As one of the earliest examples of “chemical biology,” the Mechanistic Target of Rapamycin (mTOR) protein and its chemical inhibitors have been extensively studied across a spectrum of physiologic and pathologic processes at the molecular, organismal, and patient population levels. There are several FDA-approved mTOR inhibitors (sirolimus, everolimus, and temsirolimus) with indications for cancer treatment and for prevention of solid organ rejection. Dozens of mTOR inhibitors are currently being evaluated in hundreds of ongoing clinical trials across a spectrum of diseases, including numerous cancer indications, autoimmune diseases, and a number of congenital disorders. As many of the approved and investigational indications for mTOR inhibitors require long-term treatment, the magnitude and incidence of particular side effects differ from those observed in shorter-term treatments. Here, we focus on the increased risk of infections in patients being treated with mTOR inhibitors. While increased infection rates might be expected from a class of drugs approved as posttransplant immunosuppressants, we review reports from clinical, mechanistic, and genetically engineered mouse model studies detailing a much more nuanced view of mTOR inhibitor drug action and target biology. Clin Cancer Res; 22(2); 277–83. ©2015 AACR.

Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient–derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo. Mol Cancer Ther; 16(9); 2008–21. ©2017 AACR.

cFOS-SOX9 Axis Reprograms Bone Marrow-Derived Mesenchymal Stem Cells into Chondroblastic Osteosarcoma

Summary Bone marrow-derived mesenchymal stem cells (BMSCs) are proposed as the cells of origin of several subtypes of osteosarcoma (OS). However, signals that direct BMSCs to form different subtypes of OS are unclear. Here we show that the default tumor type from spontaneously transformed p53 knockout (p53_KO) BMSCs is osteoblastic OS. The development of this default tumor type caused by p53 loss can be overridden by various oncogenic signals: RAS reprograms p53_KO BMSCs into undifferentiated sarcoma, AKT enhances osteoblastic OS, while cFOS promotes chondroblastic OS formation. We focus on studying the mechanism of cFOS-induced chondroblastic OS formation. Integrated genome-wide studies reveal a regulatory mechanism whereby cFOS binds to the promoter of a key chondroblastic transcription factor, Sox9, and induces its transcription in BMSCs. Importantly, SOX9 mediates cFOS-induced cartilage formation in chondroblastic OS. In summary, oncogenes determine tumor types derived from BMSCs, and the cFOS-SOX9 axis is critical for chondroblastic OS formation.

Abstract 2833: Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins

The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice. Citation Format: Shuling Zhang, Joy M. Gary, John K. Simmons, Jinfei Xu, Benjamin J. Gamache, Ke Zhang, Nicholas Watson, Alexander L. Kovalchuk, Aleksandra M. Michalowski, Jin-Qiu Chen, Michelle A. Herrmann, Tuddow Thaiwong, Matti Kiupel, Wendy Dubois, Joseph R. Testa, Beverly A. Mock. Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins. [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 2833.

Abstract 2309: Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL

The PI3K/AKT/mTOR pathway is frequently hyperactivated in T- cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with constitutively-active AKT specific to T-lymphocytes (Lck-MyrAkt2) were crossed with mice that have genetically-reduced mTOR expression (mTOR knock-down, KD). Lck-MyrAkt2 mice with mTOR KD had altered thymic T-lymphocyte development; these mice also had delayed thymic pre-T cell lymphoblastic leukemia/lymphoma (pre-T LBL) progression and increased survival relative to wild type (WT) mTOR/ Lck-MyrAkt2 mice (average survival of 24 versus 14 weeks, respectively). Delayed pre-T LBL formation was also observed when WT mTOR/Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex. Transcriptional profiling of thymic lymphomas from the WT vs KD mTOR/ Lck-MyrAkt2 mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T- cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. Tumor cells from WT mTOR/Lck-MyrAkt2 mice were more sensitive to CDK4/6 inhibitors than tumor cells from KD mTOR/Lck-MyrAkt2 mice. Combining an mTOR inhibitor (rapamycin) with a CDK4/6 inhibitor (PD-0332991) synergistically inhibited the viability and decreased downstream signaling in both mouse lymphoma cells and human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Combining a dual mTORC inhibitor (PP242) with a CDK4/6 inhibitor decreased downstream signaling to an even greater extent in human T-ALL cell lines. Our results suggest that an mTORi/CDKi combination may be beneficial in the treatment of human T-ALL. Citation Format: Joy M. Gary, Jinfei Xu, John Simmons, Shuling Zhang, Benjamin Gamache, Ke Zhang, Alexander Kovalchuk, Aleksandra Michalowski, Jin-Qiu Chen, Michelle Herrmann, Wendy Dubois, Joseph Testa, Beverly A. Mock. Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL. [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 2309. doi:10.1158/1538-7445.AM2015-2309

Abstract 1555: Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance.

The AKT/PI3K/mTOR signaling pathway is hyperactivated in cancers, including both solid tumors and hematologic malignancies. This hyperactivation is often due to an overexpression or mutation in PI3K, which is upstream of AKT, or a down regulation of PTEN, an inhibitor of the PI3K pathway. mTOR is a central downstream target of this pathway and is a critical modulator of cell cycle, size, proliferation and metabolism. Resistance to mTOR inhibition has been reported in many tumors as well as in certain cell lines, resulting in decreased efficacy of cancer therapy involving this class of inhibitor. In these studies, mice (Mtortm1.1Lgm) with constitutive reduction in mTOR protein level were crossed to mice (Lck-MyrAkt2) that spontaneously develop thymic lymphomas due to constitutive AKT activation to simulate mTOR inhibitor treatment of thymic lymphoma development. The Lck-MyrAkt2 mice spontaneously develop thymic lymphomas at 10-20 weeks of age, and frequently harbor a 14;15 translocation that leads to the apposition of the TCRalpha and Myc genes. Tumor formation was significantly delayed in the mTOR knockdown x Lck-MyrAkt2 cohort, and the mice had a median survival of 181 days compared to 88 days in mice with normal mTOR expression. A similar t(14;15) translocation frequency was observed in thymic lymphomas of mice from both groups. To evaluate potential mTOR resistance mechanisms, gene expression profiling (GEP) of tumors arising from both groups of mice was performed. Molecular and functional enrichment analysis of the genes differentially expressed in the tumors arising despite mTOR inhibition was performed. Increased expression of an oncogenic transcription factor was observed, and is the subject of on-going investigation. Citation Format: Joy Gary, Jinfei Xu, John Simmons, Alexander Kovalchuk, Wendy Dubois, Joseph Testa, Beverly Mock. Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance. [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 1555. doi:10.1158/1538-7445.AM2013-1555