Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K

Class IA phosphatidylinositol 3-kinases (PI3K), which generate PIP₃ as a signal for cell growth and proliferation, exist as an intracellular complex of a catalytic subunit bound to a regulatory subunit. We and others have previously reported that heterozygous mutations in PIK3CD encoding the p110δ catalytic PI3K subunit cause a unique disorder termed p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency (PASLI) disease. We report four patients from three families with a similar disease who harbor a recently reported heterozygous splice site mutation in PIK3R1, which encodes the p85α, p55α, and p50α regulatory PI3K subunits. These patients suffer from recurrent sinopulmonary infections and lymphoproliferation, exhibit hyperactive PI3K signaling, and have prominent expansion and skewing of peripheral blood CD8⁺ T cells toward terminally differentiated senescent effector cells with short telomeres. The PIK3R1 splice site mutation causes skipping of an exon, corresponding to loss of amino acid residues 434–475 in the inter-SH2 domain. The mutant p85α protein is expressed at low levels in patient cells and activates PI3K signaling when overexpressed in T cells from healthy subjects due to qualitative and quantitative binding changes in the p85α–p110δ complex and failure of the C-terminal region to properly inhibit p110δ catalytic activity.Primary human immunodeficiency diseases offer insights into genes and pathways critical for host defense and healthy immune homeostasis. We and others have recently described a unique immune disorder featuring recurrent sinopulmonary infections, predisposition to chronic EBV and CMV viremia, lymphoproliferation, and increased lymphoma susceptibility (Angulo et al., 2013; Crank et al., 2014; Kracker et al., 2014; Lucas et al., 2014). Heterozygous gain-of-function mutations in the PIK3CD gene encoding the leukocyte-restricted p110δ catalytic subunit of phosphatidylinositol 3-kinase (PI3K) are responsible for this disorder, which we have termed p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency (PASLI) disease (Lucas et al., 2014). PASLI disease is caused by mutation of at least four different sites in PIK3CD that drive hyperactivation of PI3K signaling in immune cells (Crank et al., 2014; Lucas et al., 2014). Some of the disease-causing amino acid substitutions in p110δ are identical to those occurring in tumor cells at homologous sites in PIK3CA encoding p110α, suggesting a similar molecular mode of action. Indeed, PASLI patients exhibit increased lymphoma risk that is further compounded by immunodeficiency leading to poor control of EBV viral loads (Crank et al., 2014; Kracker et al., 2014). We are now aware of ∼80 PASLI patients worldwide, and the number of patients diagnosed with this disorder is expected to increase. Our previous work clearly established that hyperactivation of the PI3K signaling pathway causes immune dysregulation and raised the question of whether or not mutations in other PI3K genes would cause similar clinical manifestations by augmenting this pathway.The phosphoinositide 3-kinase (PI3K) pathway transduces cell growth and proliferation signals through generation of the PIP₃ second messenger, which is important for recruitment and activation of pleckstrin homology (PH) domain–containing signaling proteins. The class IA PI3K family members include the catalytic p110α, p110β, and p110δ proteins and the regulatory p85α, p55α, p50α, p85β, and p55γ proteins. The complex becomes activated upon recruitment to tyrosine-phosphorylated YXXM motifs with major signaling roles downstream of the insulin receptor, insulin-like growth factor-1 receptor, cytokine receptors, T cell receptor, and others. The class IA PI3Ks exist as a dimer of a catalytic and a regulatory subunit. The major roles of the regulatory subunit are to bind and stabilize p110 (Conley et al., 2012), inhibit p110 kinase activity (Burke et al., 2011), and recruit the PI3K complex to phosphotyrosine where binding of the SH2 domains to phosphotyrosine relieves the inhibitory (but not dimerizing) contacts with the catalytic subunit (Yu et al., 1998). There is debate about the existence and potential roles for free monomeric p85α that is not bound to p110 and its possible function in regulating PI3K activity (Geering et al., 2007b). Evidence against roles for free p85α includes the observation that monomeric p85α is relatively unstable (Brachmann et al., 2005; Zhao et al., 2006) and that p85α and p110 are obligate heterodimers normally present in the cell at 1:1 ratio (Geering et al., 2007a). Whether or not p85α can exist unbound to p110 and whether or not free p85α exerts biological or pathological effects remain open questions.Studies in animal models have revealed a complex relationship between p110 and p85α (Vanhaesebroeck et al., 2005). The total PIK3R1 knockout mouse dies in the perinatal period and shows secondary loss of p110 catalytic protein (Fruman et al., 2000). Mice heterozygous for p85α have normal levels of p110 and show greater insulin-stimulated PI3K activity than WT counterparts but display no overt immunological phenotypes (Ueki et al., 2002, 2003; Vanhaesebroeck et al., 2005). Two inherited human diseases have been associated with mutations in the PIK3R1 gene: (1) SHORT syndrome, a disease of short stature, hyperextensible joints, Rieger anomaly of the eye, teething delay, lipoatrophy, and often insulin resistance, caused by heterozygous PIK3R1 mutations (Chudasama et al., 2013; Dyment et al., 2013; Thauvin-Robinet et al., 2013; Bárcena et al., 2014); and (2) agammaglobulinemia due to absent B cells caused by a homozygous PIK3R1 mutation that leads to loss of p85α with secondary loss of p110 (Conley et al., 2012). Somatic, heterozygous mutations in PIK3R1 have also been found in human glioblastoma (Cancer Genome Atlas Research Network, 2008; Parsons et al., 2008) and colon cancer (Jaiswal et al., 2009). These mutations reduce inhibition of p110 by p85α, leading to hyperactive PI3K signaling and tumorigenesis (Jaiswal et al., 2009; Sun et al., 2010). More recently, somatic PIK3R1 mutations in endometrial carcinoma were discovered that cluster mostly within amino acid residues 434–475 in the inter-SH2 domain of p85α and augment PI3K signaling (Urick et al., 2011). These findings in mice and previously described human diseases shed light on the various physiological roles of PIK3R1 gene products and support the hypothesis that cancer-related PIK3R1 gene mutations could be a driver of PASLI-like disease.We have now discovered heterozygous PIK3R1 splice site mutations in patients with PASLI-like disease characteristics and striking hyperactivation of PI3K signaling in immune cells. An independent report has also recently described similar patients with the same splice site mutation (Deau et al., 2014). Here, we not only describe the clinical findings and gene defect in these patients but also provide biochemical evidence that the mutant p85α protein is expressed in patient cells, associates abnormally with p110δ, and dominantly drives constitutive PI3K signaling due to loss of inhibitory contacts, which results in cellular derangements that contribute to immunodeficiency in this patient population. These findings further provide a possible treatment option for these patients using approved or investigational drugs that target PI3K or its downstream effectors (i.e., mTOR inhibition with rapamycin).     

Too much of a good thing: immunodeficiency due to hyperactive PI3K signaling

Primary immune deficiency diseases arise due to heritable defects that often involve signaling molecules required for immune cell function. Typically, these genetic defects cause loss of gene function, resulting in primary immune deficiencies such as severe combined immune deficiency (SCID) and X-linked agammaglobulinemia (XLA); however, gain-of-function mutations may also promote immune deficiency. In this issue of the JCI, Deau et al. establish that gain-of-function mutations in PIK3R1, which encodes the p85α regulatory subunit of class IA PI3Ks, lead to immunodeficiency. These observations are consistent with previous reports that hyperactivating mutations in PIK3CD, which encodes the p110δ catalytic subunit, are capable of promoting immune deficiency. Mutations that reduce PI3K activity also result in defective lymphocyte development and function; therefore, these findings support the notion that too little or too much PI3K activity leads to immunodeficiency.     

Therapeutic implications of activating noncanonical PIK3CA mutations in head and neck squamous cell carcinoma

Alpelisib selectively inhibits the p110α catalytic subunit of PI3Kα and is approved for treatment of breast cancers harboring canonical PIK3CA mutations. In head and neck squamous cell carcinoma (HNSCC), 63% of PIK3CA mutations occur at canonical hotspots. The oncogenic role of the remaining 37% of PIK3CA noncanonical mutations is incompletely understood. We report a patient with HNSCC with a noncanonical PIK3CA mutation (Q75E) who exhibited a durable (12 months) response to alpelisib in a phase II clinical trial. Characterization of all 32 noncanonical PIK3CA mutations found in HNSCC using several functional and phenotypic assays revealed that the majority (69%) were activating, including Q75E. The oncogenic impact of these mutations was validated in 4 cellular models, demonstrating that their activity was lineage independent. Further, alpelisib exhibited antitumor effects in a xenograft derived from a patient with HNSCC containing an activating noncanonical PIK3CA mutation. Structural analyses revealed plausible mechanisms for the functional phenotypes of the majority of the noncanonical PIK3CA mutations. Collectively, these findings highlight the importance of characterizing the function of noncanonical PIK3CA mutations and suggest that patients with HNSCC whose tumors harbor activating noncanonical PIK3CA mutations may benefit from treatment with PI3Kα inhibitors.     

PI3-kinase mutation linked to insulin and growth factor resistance in vivo

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is central to the action of insulin and many growth factors. Heterozygous mutations in the gene encoding the p85α regulatory subunit of PI3K (PIK3R1) have been identified in patients with SHORT syndrome — a disorder characterized by short stature, partial lipodystrophy, and insulin resistance. Here, we evaluated whether SHORT syndrome–associated PIK3R1 mutations account for the pathophysiology that underlies the abnormalities by generating knockin mice that are heterozygous for the Pik3r1^Arg649Trp mutation, which is homologous to the mutation found in the majority of affected individuals. Similar to the patients, mutant mice exhibited a reduction in body weight and length, partial lipodystrophy, and systemic insulin resistance. These derangements were associated with a reduced capacity of insulin and other growth factors to activate PI3K in liver, muscle, and fat; marked insulin resistance in liver and fat of mutation-harboring animals; and insulin resistance in vitro in cells derived from these mice. In addition, mutant mice displayed defective insulin secretion and GLP-1 action on islets in vivo and in vitro. These data demonstrate the ability of this heterozygous mutation to alter PI3K activity in vivo and the central role of PI3K in insulin/growth factor action, adipocyte function, and glucose metabolism.     

Activated PI3Kδ signals compromise plasma cell survival via limiting autophagy and increasing ER stress

While phosphatidylinositide 3-kinase delta (PI3Kδ) plays a critical role in humoral immunity, the requirement for PI3Kδ signaling in plasma cells remains poorly understood. Here, we used a conditional mouse model of activated PI3Kδ syndrome (APDS), to interrogate the function of PI3Kδ in plasma cell biology. Mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) in B cells generated increased numbers of memory B cells and mounted an enhanced secondary response but exhibited a rapid decay of antibody levels over time. Consistent with these findings, aPIK3CD expression markedly impaired plasma cell generation, and expression of aPIK3CD intrinsically in plasma cells was sufficient to diminish humoral responses. Mechanistically, aPIK3CD disrupted ER proteostasis and autophagy, which led to increased plasma cell death. Notably, this defect was driven primarily by elevated mTORC1 signaling and modulated by treatment with PI3Kδ-specific inhibitors. Our findings establish an essential role for PI3Kδ in plasma cell homeostasis and suggest that modulating PI3Kδ activity may be useful for promoting and/or thwarting specific immune responses.     

Elaborating piperazinyl-furopyrimidine based scaffolds as phosphoinositol-3-kinase enzyme alpha (PI3Kα) inhibitors to combat pancreatic cancer

Phosphoinositol-3-kinase enzyme (PI3K) plays a crucial role in driving oncogenic growth in various mammalian cells, particularly pancreatic cells. In the current study a series of novel furo[2,3-d]pyrimidine based-compounds were designed and synthesized as potential PI3K-α inhibitors. In accordance to the structure–activity relationship (SAR) studies of known PI3K-α inhibitors, different linkers including amide, urea and ether were attached to a piperazinyl furo[2,3-d]pyrimidine core. The synthesized compounds that revealed moderate PI3K-α inhibitory activity were tested for their anti-proliferative activities against pancreatic carcinoma on the PANC-1 cell line. Compounds 7b and 8a showed the highest anti-proliferative activity with IC₅₀ values of 4.5 μM and 6 μM, respectively and relatively, the best in vitro PI3K inhibition ability within the newly synthesized compounds. Additionally, all the newly synthesized final compounds were tested on 60 human cancer cell lines. A docking study was carried out on the PI3K-α active site showing a comparable binding mode to that of FDA approved PI3K-α inhibitors. These newly discovered lipid kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agents.

Phosphoinositol-3-kinase alpha (PI3K-α) enzyme inhibition to combat pancreatic cancer.     

Hematopoiesis and RAS-driven myeloid leukemia differentially require PI3K isoform p110α

The genes encoding RAS family members are frequently mutated in juvenile myelomonocytic leukemia (JMML) and acute myeloid leukemia (AML). RAS proteins are difficult to target pharmacologically; therefore, targeting the downstream PI3K and RAF/MEK/ERK pathways represents a promising approach to treat RAS-addicted tumors. The p110α isoform of PI3K (encoded by Pik3ca) is an essential effector of oncogenic KRAS in murine lung tumors, but it is unknown whether p110α contributes to leukemia. To specifically examine the role of p110α in murine hematopoiesis and in leukemia, we conditionally deleted p110α in HSCs using the Cre-loxP system. Postnatal deletion of p110α resulted in mild anemia without affecting HSC self-renewal; however, deletion of p110α in mice with KRAS^G12D-associated JMML markedly delayed their death. Furthermore, the p110α-selective inhibitor BYL719 inhibited growth factor–independent KRAS^G12D BM colony formation and sensitized cells to a low dose of the MEK inhibitor MEK162. Furthermore, combined inhibition of p110α and MEK effectively reduced proliferation of RAS-mutated AML cell lines and disease in an AML murine xenograft model. Together, our data indicate that RAS-mutated myeloid leukemias are dependent on the PI3K isoform p110α, and combined pharmacologic inhibition of p110α and MEK could be an effective therapeutic strategy for JMML and AML.     

Retracted Article: Salvianolic acid B inhibits inflammatory response and cell apoptosis via the PI3K/Akt signaling pathway in IL-1β-induced osteoarthritis chondrocytes

Osteoarthritis (OA) is the most common joint disease among late middle-aged or elderly people. The pathological process of OA mainly involves the degeneration of cartilage tissue and deficiency of joint function. Salvianolic acid B (Sal B) is the main active ingredient of Salvia miltiorrhiza Bge, which possesses anti-inflammatory, anti apoptotic and other pharmacological activities. In this study, primary chondrocytes were cultured to investigate the effects of Sal B on the inflammatory response and apoptosis of OA induced by IL-1β, and to explore the possible mechanism. First, we determined the cytotoxicity of Sal B; the results showed that the cell activity of chondrocytes was not influenced by Sal B when the concentration was below 150 μM. Moreover, Sal B (40 and 80 μM) suppressed the expression of iNOS in OA chondrocytes induced by IL-1β, and restrained the secretion of NO, IL-6, IL-17 and TNF-α in chondrocytes obviously. Sal B (40, 80 μM) significantly alleviated the inhibitory effect of cell activity stimulated by IL-1β and up-regulated the expression of Col II and reduced the expression of Col X. Besides, Sal B down-regulated the expression level of Bax and promoted the expression of Bcl-2, showed a significant effect on promoting proliferation and inhibiting cell apoptosis. In addition, we found that IL-1β significantly reduced the ratio of p-PI3K/PI3K, p-Akt/Akt induced the nuclear translocation of AKT and inhibited the activation of the PI3K/Akt signaling pathway. Finally, the PI3K inhibitor, LY-294002, was added in IL-1β-induced chondrocytes. The results suggest that Sal B ameliorates IL-1β induced inflammation and suppresses apoptosis in OA by activating the PI3K/Akt signaling pathway. Our study reveals the mechanism of Sal B acts on OA and may provide a basis for the treatment of OA with Sal B.

Osteoarthritis (OA) is the most common joint disease among late middle-aged or elderly people.     

Agammaglobulinemia and absent B lineage cells in a patient lacking the p85α subunit of PI3K

Whole exome sequencing was used to determine the causative gene in patients with B cell defects of unknown etiology. A homozygous premature stop codon in exon 6 of PIK3R1 was identified in a young woman with colitis and absent B cells. The mutation results in the absence of p85α but normal expression of the p50α and p55α regulatory subunits of PI3K. Bone marrow aspirates from the patient showed <0.1% CD19(+) B cells with normal percentages of TdT(+)VpreB(+)CD19(-) B cell precursors. This developmental block is earlier than that seen in patients with defects in the B cell receptor signaling pathway or in a strain of engineered mice with a similar defect in p85α. The number and function of the patient's T cells were normal. However, Western blot showed markedly decreased p110δ, as well as absent p85α, in patient T cells, neutrophils, and dendritic cells. The patient had normal growth and development and normal fasting glucose and insulin. Mice with p85α deficiency have insulin hypersensitivity, defective platelet function, and abnormal mast cell development. In contrast, the absence of p85α in the patient results in an early and severe defect in B cell development but minimal findings in other organ systems.     

Thymic development beyond β-selection requires phosphatidylinositol 3-kinase activation by CXCR4

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection.T lymphocytes develop in the thymus from a multistep differentiation program characterized by the sequential VDJ rearrangement of the tcrb and tcra genes in combination with stringent quality control checkpoints. The most immature T lymphocytes are CD4 and CD8 double-negative (DN), and this population can be further subdivided based upon their expression of CD44 and CD25 (Godfrey et al., 1993). At the CD25⁺CD44^lo DN3 stage, thymocytes undergo the β-selection checkpoint. DN3 cells are tested for the successful expression of a TCRβ polypeptide in the context of the invariant pTα subunit and CD3, which together form the preTCR (Yamasaki and Saito, 2007). DN3 cells that have successfully rearranged TCRβ undergo proliferative expansion, further differentiation, and allelic exclusion of the TCRβ locus. This occurs as DN3 cells lose expression of CD25 to become DN4. Subsequently, thymocytes express CD8 and CD4, which together define the double-positive (DP) population, where tcra rearrangement occurs and the mature TCR is expressed. The completion of β-selection is contingent upon Notch signaling, which is necessary for survival, metabolic fitness, and proliferation after TCRβ rearrangement (Ciofani and Zuñiga-Pflucker, 2005; Maillard et al., 2006). Together, Notch and preTCR signaling are thought to constitute the minimal requirements for continued differentiation beyond β-selection; however, our understanding of how the signaling events downstream of these receptors are integrated is limited.The class I phosphatidylinositol 3-kinase (PI3K) family of enzymes mediates the phosphorylation of phosphatidylinositol-4,5-trisphosphate (PIP₂) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP₃). This lipid binds the pleckstrin homology domains of effector molecules, which in turn regulate cellular processes including survival, proliferation, metabolism, differentiation, and movement (Fruman and Bismuth, 2009). The class I PI3Ks are comprised of two subgroups designated class IA and IB. The class IA subgroup, whose members are activated by tyrosine kinase–associated receptors, consists of three ∼110-kD catalytic subunits termed α, β, and δ that pair with regulatory subunits p50α, 55α, 85α, p85β, and p55γ. The class IB subgroup, activated by G protein–coupled receptors, consists of p110γ that interacts with p101 or p84 regulatory subunits.Functional redundancy between PI3K isoforms for T cell development is revealed in mice lacking both p110γ and p110δ in the germline. These mice have small thymi characterized by a marked reduction in DP thymocyte numbers (Webb et al., 2005; Swat et al., 2006; Ji et al., 2007), reflecting defective survival of DP thymocytes (Webb et al., 2005; Swat et al., 2006). The conditional deletion in DN T cells of phosphatase and tensin homologue, which opposes PI3K activity by converting PIP₃ to PIP₂, rescues T cell development in mice with defective IL-7 or preTCR signaling (Hagenbeek et al., 2004; Shiroki et al., 2007). Despite this, it remains unclear how P110γ and P110δ contribute to the earlier stages of T cell development and to which activating receptors these PI3K isoforms are coupled (Webb et al., 2005; Swat et al., 2006). Genetic and biochemical studies have revealed important roles for the PI3K-dependent protein kinases PDK1 and Akt in T cell development (Hinton et al., 2004; Fayard et al., 2007; Juntilla et al., 2007; Mao et al., 2007). DN4 cells of these mutant mice were small and failed to up-regulate nutrient receptors despite having normal expression of intracellular TCRβ, whereas expression of constitutively active Akt resulted in the rescue of the DN3 developmental block in Rag2-deficient mice (Mao et al., 2007).Although the preTCR utilizes protein tyrosine kinases for signaling, a direct link between the preTCR and PI3K activation has yet to be shown. Similarly, the PI3K effector molecules PDK1 and AKT have been reported to be required for Notch-dependent metabolic changes in DN3 cells (Ciofani and Zuñiga-Pflucker, 2005; Kelly et al., 2007). However, the mechanistic basis for the interaction between Notch and PI3K remains to be clarified. Further, the implication of class IB PI3K in early T cell development raises the possibility that a GPCR may also be involved in this process, but such a receptor has not been identified.Among GPCRs, chemokine receptors are important in directing thymocytes through the cortex and medulla during their development, thereby guiding thymocytes to the appropriate microenvironment for their specific developmental stage (Petrie and Zuñiga-Pflucker, 2007). However, it is not known if chemokines can directly contribute to the induction of thymocyte proliferation and differentiation. CXCL12, and its receptor CXCR4, was first identified as a growth factor for pre B cells (Nagasawa et al., 1994) and using mutant mice it was subsequently shown that CXCR4 is critical for B cell and myeloid hematopoiesis (Ma et al., 1998; Nagasawa et al., 1996; Tachibana et al., 1998; Zou et al., 1998). In the thymus, CXCR4 is highly expressed on thymocytes from the DN2 to the DP stage (Ara et al., 2003; Plotkin et al., 2003), but its role during T cell development is controversial. Both CXCR4 and CXCL12 are required for embryo viability, and some studies examining T cell lymphopoiesis at fetal stages of development have concluded that T cell development is normal in the absence of CXCR4 (Ma et al., 1998; Zou et al., 1998) Conversely, competitive chimera studies using donor fetal liver cells showed a reduced reconstitution of CXCR4-deficient thymocytes compared with WT (Ara et al., 2003). In addition, cre-mediated CXCR4 deletion in DN1 thymocytes resulted in complete developmental arrest of T cells (Plotkin et al., 2003) and human CD34⁺ cells seeded into mouse fetal thymic organ cultures treated with neutralizing antibodies to CXCL12 or CXCR4 showed impaired differentiation and reduced numbers of recovered T cells (Hernández-López et al., 2002).We have revealed the role of P110γ and P110δ in T cell development using a combination of genetic and pharmacological approaches. Stimulation of different PI3K mutant DN4 cells through the preTCR showed that p110δ is essential for the initiation of PI3K signaling from this receptor, but we found no role for p110γ. Experiments with chimeric mice and in vitro cell differentiation cultures demonstrated the combined deletion of p110δ and the class IB regulatory subunit p101 recapitulate the phenotype of p110γ^−/−δ^−/− mice, suggesting a requirement for G-protein coupled receptor (GPCR)–dependent PI3K activation during T cell development. Further evidence for this was shown using small molecule inhibitors of CXCR4 to impede the proliferation and differentiation of DN3 thymocytes in vitro. Moreover, we found that stimulation of DN3 thymocytes with the chemokine CXCL12 elicits a PI3K-mediated signaling response which is predominantly p110γ-dependent, but also involves p110δ. Analysis of CXCR4-deficient embryos confirmed that signaling through CXCR4 is critical for normal thymic T cell development. Using these insights, we tested the requirement for CXCR4 signaling by culturing DN3 cells with Notch ligand in the presence or absence of CXCL12. Only in the presence of CXCL12 could DN3 cells differentiate to become DP. These insights permitted the differentiation and expansion of DN3 in the absence of supporting stromal cells and that the additional inclusion of a GSK3 inhibitor allowed PI3K-mediated accessory cell–free proliferation and differentiation to a similar extent as that seen upon co-culture of DN3 thymocytes with stromal cells.     

Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.     

Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma

Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTEN^Null melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase–dependent and –independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAF^V600E-induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway–targeted inhibitors. These experiments revealed that mutationally activated PIK3CA^H1047R cooperates with BRAF^V600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAF^V600E/PTEN^Null melanomas in vivo and in tissue culture. Combined inhibition of BRAF^V600E and PI3K had more potent effects on the regression of established BRAF^V600E/PTEN^Null melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAF^V600E/PIK3CA^H1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAF^V600E/PTEN^Null melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAF^V600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway–targeted inhibition of PI3K and BRAF^V600E in the therapeutic management of BRAF^V600E/PTEN^Null melanomas.     

Inhibition of the p110α isoform of PI 3-kinase stimulates nonfunctional tumor angiogenesis

Understanding the direct, tumor cell–intrinsic effects of PI 3-kinase (PI3K) has been a key focus of research to date. Here, we report that cancer cell–extrinsic PI3K activity, mediated by the p110α isoform of PI3K, contributes in an unexpected way to tumor angiogenesis. In syngeneic mouse models, inactivation of stromal p110α led to increased vascular density, reduced vessel size, and altered pericyte coverage. This increased vascularity lacked functionality, correlating with enhanced tumor hypoxia and necrosis, and reduced tumor growth. The role of p110α in tumor angiogenesis is multifactorial, and includes regulation of proliferation and DLL4 expression in endothelial cells. p110α in the tumor stroma is thus a regulator of vessel formation, with p110α inactivation giving rise to nonfunctional angiogenesis, which can stunt tumor growth. This type of vascular aberration differs from vascular endothelial growth factor–centered antiangiogenesis therapies, which mainly lead to vascular pruning. Inhibition of p110α may thus offer a new antiangiogenic therapeutic opportunity in cancer.Constitutive activation of the PI3K pathway is very common in cancer, and PI3K inhibitors are currently progressing through oncology trials (Rodon et al., 2013). At present, the role of PI3K in the tumor microenvironment, such as in cancer-associated fibroblasts, endothelial cells (ECs), mural cells, and immune cells, is largely unexplored. With the exception of white blood cells, where p110γ and p110δ both play important roles (including in cancer; p110γ; Schmid et al., 2011), the ubiquitously expressed p110α is likely to be a critical PI3K isoform in nonleukocyte stromal cell types, based on the notion that p110α plays a nonredundant key role in vascular development (Lelievre et al., 2005; Graupera et al., 2008) and fibroblast proliferation (Foukas et al., 2006; Zhao et al., 2006). However, the role of p110α in the tumor stroma is unknown. To assess the importance of the p110α PI3K axis in the cancer stromal compartment, we manipulated this pathway in syngeneic mouse cancer models.     

Amino acid sequences characterization and anti-inflammatory potency evaluation of Portulaca oleracea L. oligopeptides in macrophages

The Portulaca oleracea L. oligopeptides are seldom explored because they are often present in a complex matrix. In the current study, eleven novel Portulaca oleracea L. oligopeptides (POPs) were isolated and their mino acid sequence identified. Further, the anti-inflammatory potency was explored in lipopolysaccharide (LPS)-induced RAW264.7 cells. Results showed that POP-1∼[EHGEYE] possessed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways. This may be an important molecular mechanism of POPs in anti-inflammatory damage.

POP-1 performed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways.     

Notoginsenoside R1 prevents H9c2 cardiomyocytes apoptosis against hypoxia/reoxygenation via the ERs/PI3K/Akt pathway

Notoginsenoside R₁ (NGR₁) is separate from Panax notoginsenosides (PNS), and plays a role similar to phytoestrogen in preventing and treating cardiovascular diseases. However, the protective mechanism of NGR₁ in the myocardial ischemia/reperfusion injury via the estrogen receptor (ER) pathway remains unclear, which hinder its application. This study aimed to study the preventive mechanisms of NGR₁ in the apoptosis of H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R). NGR₁ did not affect the expression of ERα and ERβ proteins in normal H9c2 cardiomyocytes. However, NGR₁ could upregulate the ERα and G protein-coupled receptor 30 (GPR30) proteins in H9c2 cardiomyocytes after H/R without affecting ERβ levels. Moreover, it significantly affected the expression levels of PI3K and its downstream apoptosis proteins such as Bcl-2 Associated X Protein (Bax), B cell lymphoma/lewkmia-2 (Bcl-2), caspase-3, and so forth. Whereas, after adding the PI3K protein antagonist, the modulatory expression levels of PI3K and its downstream apoptosis proteins were remarkably abolished. After adding ERα and GPR30 antagonists, NGR₁ had no significant effect on the expression of PI3K and its downstream Akt protein in the model group. The data of flow cytometry showed that after adding the ERα, GPR30 and PI3K antagonists, the apoptotic rate of cardiomyocytes had no significant changes compared with the model group. This study demonstrated that NGR₁ protected H9c2 cardiomyocytes from the injury after H/R by affecting ERα and GPR30 to regulate the expression levels of PI3K and its downstream apoptosis proteins.

Notoginsenoside R₁ (NGR₁) is separate from Panax notoginsenosides (PNS), and plays a role similar to phytoestrogen in preventing and treating cardiovascular diseases.     

Gαq-containing G proteins regulate B cell selection and survival and are required to prevent B cell–dependent autoimmunity

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G_αq subunit of trimeric G proteins (Gnaq^−/− mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq^−/− B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq^−/− chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq^−/− B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.Autoreactive B cells significantly contribute to the morbidity and mortality associated with many autoimmune diseases (Manjarrez-Orduño et al., 2009). B cell tolerance is normally controlled at several checkpoints in the BM and periphery (Goodnow, 2007). In the periphery, BCR- and BAFFR-dependent signals are required for the differentiation of immature transitional B cells into mature B cells and the continued maintenance of mature B cells (Cancro, 2009). B cells that express BCRs with intermediate affinity for autoantigens are less competitive than nonautoreactive B cells for access to the survival niches in the spleen and are eliminated at the transitional T1 stage of development (Lesley et al., 2004; Thien et al., 2004). However, in the presence of excess BAFF (also known as BLyS), autoreactive B cells can pass the T1 checkpoint and enter the mature B cell pool (Thien et al., 2004). Thus, the appropriate survival and selection of B cells in the periphery appears to be dependent on a dynamic integration of BAFFR and BCR signals.Engagement of either the BCR or BAFFR alone is insufficient to maintain mature B cell survival in the periphery (Cancro, 2009), as BCR signaling is required to sustain NF-κB–dependent BAFFR signaling (Stadanlick et al., 2008). In addition to the NF-κB–dependent cross talk between BAFFR and the BCR, both receptors can also activate PI3K (Fruman and Bismuth, 2009) and its downstream target Akt (Pogue et al., 2000; Patke et al., 2006), a serine threonine kinase which functions as a prosurvival factor in many cell types (Manning and Cantley, 2007). One recent study showed that BCR-dependent survival of mature B cells is highly dependent on PI3K (Srinivasan et al., 2009), and another study showed that activation of the PI3K pathway can rescue normally anergic autoreactive B cells (Browne et al., 2009). The PI3K–Akt signaling pathway is also engaged by activation of seven transmembrane-spanning G protein–coupled receptors (GPCRs; Yanamadala et al., 2009). GPCRs associate with heterotrimeric G proteins in their GDP-bound state (Wettschureck et al., 2004). Upon ligand binding to the GPCR, GDP is exchanged for GTP, which causes G protein release and the disassociation of the GTP-bound α subunit and the βγ dimer. Signal transduction is mediated by both the GTP-bound α subunit and the βγ dimer, but specialization and diversification of the response is often mediated by the GTP-bound α subunits (Wettschureck et al., 2004). There are 16 α subunits that fall into four classes, G_αi, G_αs, G_αq/11, and G_12/13, based on their downstream signaling targets. PI3K can be activated by the βγ dimers released from G_αi-coupled receptors (Wettschureck et al., 2004). In contrast, G_αq, a member of the G_αq/11 family, normally inhibits PI3K activation and prevents activation of Akt (Harris et al., 2006). In cardiomyocytes, Akt activation and cell survival is enhanced when the amount of active GTP-bound G_αq is low (Howes et al., 2006). However, when the amount of active G_αq is increased in cardiomyocytes, Akt activity is inhibited (Ballou et al., 2003) and survival of the cells upon stimulation is reduced (Howes et al., 2003). Examination of cardiomyocytes from transgenic mice expressing G_αq in the cardiomyocytes indicated that the level of cardiomyocyte apoptosis correlated directly with the amount of active G_αq expressed in the cells (Adams et al., 1998). Likewise, increased expression levels of G_αq are associated with changes in cardiomyocyte survival and in the development of cardiac disease in patients (Liggett et al., 2007; Frey et al., 2008). Together, these data suggest that one major function of G_αq is to suppress the PI3K/Akt signaling axis and cell survival. Surprisingly, despite that fact G_αq is expressed ubiquitously in B cells and myeloid cells (Wilkie et al., 1991), nothing is known regarding the requirement for G_αq-containing G proteins in regulating Akt activity or hematopoietic cell survival.In this paper, we show that G_αq regulates peripheral B cell tolerance by suppressing the survival and selection of autoreactive B cells. In the absence of G_αq, B cells constitutively express higher levels of activated Akt and preferentially survive BCR-induced cell death signals and BAFF withdrawal in vitro and in vivo. Most importantly, G_αq-deficient mice rapidly develop an autoreactive B cell repertoire and systemic autoimmunity. Together, these data show that G_αq-containing G proteins, working in concert with the BCR and BAFFR signaling networks, regulate B cell development and peripheral tolerance induction. In addition, the data provide the first example of G protein–dependent suppression of B cell–mediated autoimmunity.     

RAS interaction with PI3K p110α is required for tumor-induced angiogenesis

Direct interaction of RAS with the PI3K p110α subunit mediates RAS-driven tumor development: however, it is not clear how p110α/RAS-dependant signaling mediates interactions between tumors and host tissues. Here, using a murine tumor cell transfer model, we demonstrated that disruption of the interaction between RAS and p110α within host tissue reduced tumor growth and tumor-induced angiogenesis, leading to improved survival of tumor-bearing mice, even when this interaction was intact in the transferred tumor. Furthermore, functional interaction of RAS with p110α in host tissue was required for efficient establishment and growth of metastatic tumors. Inhibition of RAS and p110α interaction prevented proper VEGF-A and FGF-2 signaling, which are required for efficient angiogenesis. Additionally, disruption of the RAS and p110α interaction altered the nature of tumor-associated macrophages, inducing expression of markers typical for macrophage populations with reduced tumor-promoting capacity. Together, these results indicate that a functional RAS interaction with PI3K p110α in host tissue is required for the establishment of a growth-permissive environment for the tumor, particularly for tumor-induced angiogenesis. Targeting the interaction of RAS with PI3K has the potential to impair tumor formation by altering the tumor-host relationship, in addition to previously described tumor cell–autonomous effects.     

PIK3CA and KRAS mutations predict for response to everolimus therapy: now that’s RAD001

Targeted cancer therapeutics can be effective when patients are preselected to maximize the chance of response. Increasingly, molecular markers such as oncogenic DNA mutations are being exploited to help guide patient preselection. These DNA lesions can predict for either a positive or negative response to a given drug. Finding such predictive biomarkers is an ongoing challenge. New work by Di Nicolantonio and colleagues in this issue of the JCI demonstrates that PI3K catalytic α subunit (PIK3CA) mutations can sensitize cancer cells to the mammalian target of rapamycin (mTOR) inhibitor everolimus. In addition, they show that the concurrent presence of PIK3CA mutations and mutations in either KRAS or BRAF predict for resistance to this drug. These data suggest that mTOR inhibitors currently in use will be ineffective against cancers that have a mutation in either KRAS or BRAF despite having PI3K/AKT/mTOR pathway activation. In the past few decades, developers of new anticancer therapies have moved away from cytotoxic drugs that simply target the proliferative hallmark of all cancer cells. Currently, targeted therapies dominate cancer drug development with the aim of blocking the growth and spread of cancer by interfering with specific molecules involved in the progression of a given tumor. One of the most successful targeted anticancer therapies developed is the kinase inhibitor imatinib, which targets the product of the BCR-ABL oncogene that drives disease in all patients with chronic myeloid leukemia (CML) (1). However, for most targeted therapies, only a subset of the patients predicted to respond do so. For example, EGFR-directed therapies were thought to inhibit the growth of non–small-cell lung cancers with EGFR overexpression, but only those cancers with certain activating EGFR mutations respond to these small molecule inhibitors (2, 3). It has therefore become critically important to develop predictive biomarkers for patients who are likely to respond to a given therapy and, equally important, for those who will not. As an example, testing for KRAS mutations has become mandatory for colorectal cancer patients receiving EGFR-directed therapies because the presence of KRAS mutations predicts for resistance to this class of drugs (4). In this issue of the JCI, Di Nicolantonio and colleagues have now uncovered mutations that seem to predict for response to the anticancer drug everolimus, which targets mammalian target of rapamycin (mTOR) (5).