The PI3K-mTOR Pathway in Mammals: From Therapeutics to Fundamentals

Cells from multicellular organisms are under the control of extracellular signals to ensure the activation of certain cellular processes only in specific contexts. These extracellular signals act through intracellular signaling components that share some features with unicellular organisms such as yeast, but with additional layers of complexity and regulation. In metazoans, activation of the PI3K-mTOR signaling network is a shared response to engagement of diverse types of extracellular signals. Depending on the cell type and stimulus, activation of this pathway can promote different cellular fates including cell growth, proliferation, survival, migration and differentiation. Dysregulation of these processes is highly implicated in a variety of diseases such as cancer, diabetes, and immune disorders. Indeed, either decreased or hyperactivation of the PI3K-mTOR signaling pathway is observed in many human diseases making this network a crucial target for therapy. In this dissertation, I present a series of preclinical studies showing that indeed, pharmacological or genetic perturbation of the PI3K-mTOR pathway has an impact in the context of cancer and immunity.Since the identification of phosphoinositide 3-kinase (PI3K) enzyme activity in transformed cells nearly three decades ago, there has been significant advancement in the field leading to the development of a variety of novel chemical tools for therapeutic intervention. In chapter 2, I focus on the published promising finding that cancer cells with mutations in one of the most frequently mutated PI3K isoforms, p110α (encoded by PIK3CA), can be selectively targeted with a novel p110α-selective chemical inhibitor while preserving adaptive immune function. As it is becoming increasingly clear that an intact adaptive immune system is critical for tumor regression, these findings raise confidence that selective p110α inhibitors in cancer therapy will not be as immunosuppressive as global PI3K inhibitors currently in clinical trials.Chapter 3 focuses on the surprising immunomodulatory effects of targeting the mammalian target of rapamycin (mTOR) downstream of PI3K with second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs). Unlike the long-known immunosuppressant rapamycin that targets mTOR through an allosteric mechanism, we observed immunoenhancing effects of TOR-KIs specifically in the context of B cell antibody class-switch recombination (CSR). I will present published work where I genetically validated that the mechanism of action of TOR-KIs on CSR is through inhibition of the mTOR complex 2 (mTORC2) signaling axis. These findings have strong implications for utilizing currently available inhibitors of the PI3K-mTOR pathway beyond cancer therapy and in modulating immunity.Chapter 4 returns to the evolutionarily conserved fundamental role of the PI3K-mTOR pathway, which is to regulate cellular growth (mass increase). A working concept has been proposed and experimentally supported in that cells from multicellular organisms can uncouple cell growth from other cellular processes such as cell proliferation through engagement of distinct mTORC1 effectors. However, given the unique characteristics of primary lymphocytes in that they require a very long phase of growth prior to rapid divisions to produce a large number of identical cells, I hypothesized that a common intracellular effector may coordinate both cell growth and proliferation in this cell type. This work has led to the identification of the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) downstream of mTORC1 as the critical effector in coordinating the two processes during lymphocyte activation. These results are surprising as the 4E-BPs specifically regulate only cell proliferation in other mammalian cell types. The findings highlight the amazing specificity of this pathway in coordinating cell growth to cell proliferation in a cell-type specific manner.Despite the fundamental aspect of this research, this work has provided some major advances in the clinical context as well. The FDA-approved immunosuppressant rapamycin has long been known to have selective potency against lymphocytes although its target mTOR is ubiquitously expressed. In work presented in Chapter 4, I show that rapamycin acts upon the 4E-BPs specifically in primary lymphocytes but not other cell types, providing an explanation for its selective effects on these cells. Implications of this work beyond immunity will be discussed in Chapter 5.In terms of basic cell biology, a fascinating aspect of this research is that during metazoan evolution, distinct cell types have adopted specialized mechanisms to utilize the PI3K-mTOR pathway for fundamental cellular processes such as growth and proliferation, in order to serve each of their roles in a multicellular system. These fundamental studies are critical as loss of proper PI3K-mTOR regulation in each cell type can ultimately lead to diseases such as cancer where cells have lost the properties to adapt to multicellularity. Lastly, I believe that my findings support and provide more evidence towards a paradigm-shifting concept that protein synthesis can no longer be regarded as a housekeeping function that automatically pumps out proteins at the end of gene regulation. Rather, the findings strongly argue that protein synthesis itself can be regulated and perhaps is the most important step of gene regulation given that proteins are the most important effectors that mediate cellular functions. This paradigm will be discussed and extended in the context of understanding adaptive immunity. Recent progress in understanding lymphocyte activation has mainly focused on concepts of transcriptional regulation and metabolic reprogramming. Protein synthesis in lymphocyte activation has been studied vigorously in the late 70s. The finding that cap-dependent translation is a critical step in lymphocyte activation not only rejuvenates the idea that regulation of protein synthesis plays a critical role in immunity and suggests the possibility of regulation of select transcripts that may have therapeutic potential

Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function.

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function

Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function.

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function

Abstract IA16: Mechanisms of resistance to mTOR inhibitors in leukemia and lymphoma

Abstract Targeting mTOR signaling is a promising approach for treating blood cancers. We reported that mTOR kinase inhibitors, including PP242 and MLN0128, synergize with ABL tyrosine kinase inhibitors (TKIs) to cause cell cycle arrest and death in acute leukemia cells driven by BCR-ABL. mTOR kinase inhibitors are more effective than rapamycin in these models and have minimal effects on normal hematopoietic cells and immune responses at anti-leukemic doses. Ongoing studies indicate that mTOR kinase inhibitors are immunosuppressive at slightly higher concentrations. Moreover, the compounds are generally not cytotoxic as single agents in leukemia or lymphoma models. These findings emphasize the need for rational combinations to unleash the therapeutic potential of mTOR kinase inhibitors. To this end, we have tested various classes of anti-cancer agents guided by gene expression and proteomic data. Our data reveal synergies between mTOR kinase inhibitors with histone deacetylase inhibitors in B-ALL, and with BCL2 antagonists in DLBCL. Unexpectedly, mTOR kinase inhibitors protect B-ALL cells from methotrexate and 6-mercaptopurine, chemotherapeutic agents used in the treatment of B-ALL patients. ABL TKIs can also protect B-ALL cells from methotrexate by inhibiting downstream mTOR signaling. Together these studies identify potential applications and limitations of mTOR-targeted therapy in blood cancers. Citation Format: Thanh-Trang Vo, Jong-Hoon Scott Lee, Lomon So, Brandon Beagle, Matthew R. Janes, David A. Fruman. Mechanisms of resistance to mTOR inhibitors in leukemia and lymphoma. [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 IA16

Resistance to mTOR Kinase Inhibitors in Lymphoma Cells Lacking 4EBP1

<div><p>Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive “active-site” mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.</p></div

mTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition

The mammalian target of rapamycin (mTOR) is a kinase that functions in two distinct complexes, mTORC1 and mTORC2. In peripheral B cells, complete deletion of mTOR suppresses germinal center B-cell responses, including class switching and somatic hypermutation. The allosteric mTORC1 inhibitor rapamycin blocks proliferation and differentiation, but lower doses can promote protective IgM responses. To elucidate the complexity of mTOR signaling in B cells further, we used ATP-competitive mTOR kinase inhibitors (TOR-KIs), which inhibit both mTORC1 and mTORC2. Although TOR-KIs are in clinical development for cancer, their effects on mature lymphocytes are largely unknown. We show that high concentrations of TOR-KIs suppress B-cell proliferation and differentiation, yet lower concentrations that preserve proliferation increase the fraction of B cells undergoing class switching in vitro. Transient treatment of mice with the TOR-KI compound AZD8055 increased titers of class-switched high-affinity antibodies to a hapten–protein conjugate. Mechanistic investigation identified opposing roles for mTORC1 and mTORC2 in B-cell differentiation and showed that TOR-KIs enhance class switching in a manner dependent on forkhead box, subgroup O (FoxO) transcription factors. These observations emphasize the distinct actions of TOR-KIs compared with rapamycin and suggest that TOR-KIs might be useful to enhance production of class-switched antibodies following vaccination