Recurrent AKT mutations in human cancers: functional consequences and effects on drug sensitivity.

Precision oncology trials based on tumor gene sequencing depend on robust knowledge about the phenotypic consequences of the genetic variants identified in patients' tumors. Mutations in AKT1-3 occur in 3-5% of human cancers. Although a single hotspot mutation, E17K, is the most common, well characterized activating mutations account for a minority of Akt variants that have been identified in large tumor sequencing studies to date. In order to determine the potential clinical relevance of both common and rare Akt mutations, we expressed a set of over twenty recurrent Akt mutants in three different cell lines and evaluated activation of Akt pathway signaling and effects on growth. We determined their relative sensitivity to allosteric and ATP-competitive Akt inhibitors in clinical development. Most Akt mutants did not activate pathway signaling compared to wild type Akt and did not affect growth properties. In addition, the most common activating Akt mutations, including Akt1 E17K, L52R, and Q79K conferred neither sensitivity nor resistance to Akt inhibitors. Equivocal evidence was found that Akt1 D323H and Akt2 W80C mutants are relatively resistant to the allosteric Akt inhibitor MK-2206, but not an ATP-competitive inhibitor. Our results suggest that the vast majority of rare Akt variants are passenger mutations with no effect on drug sensitivity. The hypothesis that activating Akt mutations predict for Akt inhibitor sensitivity remains to be tested clinically, but is not yet supported by our preclinical data.

Engineering targeted chromosomal amplifications in human breast epithelial cells.

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system.

PURPOSE: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown. EXPERIMENTAL DESIGN: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background. RESULTS: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206. CONCLUSIONS: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies.

Functional analysis of non-hotspot AKT1 mutants found in human breast cancers identifies novel driver mutations: implications for personalized medicine.

The phosphatidylinositol 3-kinase (PI3-kinase)-Akt-mTOR pathway is mutated at high frequency in human breast cancer, and this pathway is the focus of active drug discovery and clinical investigation. Trials of personalized cancer therapy seek to leverage knowledge of cancer gene mutations by using mutations to guide the choice of targeted therapies. At the same time, cancer genome sequencing studies are identifying low frequency variants of unknown significance in known cancer genes, as well as genes of unknown function. We have performed functional analysis of six non-hotspot AKT1 pleckstrin homology domain mutants identified in recent large-scale breast cancer sequencing studies. Three of these mutants cause constitutive activation of Akt1 in the absence of growth factors, leading to phosphorylation of downstream target proteins. Like the hotspot E17K mutation, these mutants confer constitutive membrane localization of Akt1. Finally, the same three mutants showed oncogenic activity in a cellular transformation assay. The other three mutants were inactive in all assays. These findings validate novel driver mutations in AKT1, and extend the number and type of mutations that activate the PI3-kinase pathway in human breast cancers.

Fine tuning the immune response through B7-H3 and B7-H4.

SUMMARY: B7-H3 and B7-H4 belong to a new class of immune regulatory molecules, which primarily execute their functions in peripheral tissues to fine tune immune responses in target organs. In normal circumstances, while the mRNA for both molecules is broadly distributed, tight control at the post-transcriptional level is imposed. Under a pathogenic environment, such as inflammation and cancer, the control is often aberrant. Upon engaging their receptors, these molecules regulate the immune response in positive or negative ways depending on the expression and type of cells bearing the receptors. Thus, manipulation of the expression of these molecules and/or their receptors may represent a realistic opportunity to fine tune immune responses and to design new immunotherapeutic approaches.

Augmentation of anti-tumor responses of adoptively transferred CD8+T cells in the lymphopenic setting by HSV amplicon transduction.

Treatment of cancer with cytotoxic agents may induce lymphopenia. Adoptively transferred T cells have been reported to display enhanced anti-tumor efficacy in the lymphopenic setting. We reasoned that the anti-tumor effects of adoptively transferred cells in the lymphopenic host could be further augmented through local provision of an innate stimulus in the tumor bed. Utilizing a model in which mice were irradiated to induce lymphopenia, with limited shielding to allow tumor growth, we demonstrate that "triple" therapy consisting of radiation-induced lymphopenia, adoptive transfer of naïve CD8+ T cells, and intra-tumoral HSV amplicon injection resulted in reduced tumor growth compared to the combination of any two of the aforementioned interventions. To gain insight into the mechanism underlying this effect we studied the effects of HSV amplicon transduction into tumors on cytokine expression and on anti-tumor specific T cells. HSV amplicon transduction specifically induced several cytokine mRNAs including IFN-gamma, and IP-10. Adoptively transferred transgenic OT-1 T cells directed against Ovalbumin were more effective against Ovalbumin-expressing tumors when combined with intra-tumoral HSV amplicon injections in the lymphopenic host. Following intra-tumoral HSV-amplicon injections, anti-tumor T cells secreted higher levels of interferon-gamma in response to in-vitro re-stimulation with tumor cells, implying that HSV amplicon injection provided a strong signal for T cell activation. Combining adoptive transfer of naïve T cells in the lymphopenic setting with local T cell stimulation may facilitate expansion and activation of anti-tumor T cell populations in vivo, resulting in enhanced anti-tumor responses without the need to resort to prolonged in vitro T cell culture and/or manipulation.

Adoptively transferred tumor-specific T cells stimulated ex vivo using herpes simplex virus amplicons encoding 4-1BBL persist in the host and show antitumor activity in vivo.

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.