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Activated phosphoinositide 3-kinase delta syndrome (APDS) is a recently described form of inborn error of immunity (IEI) caused by heterozygous mutations in PIK3CD or PIK3R1 genes, respectively, encoding leukocyte-restricted catalytic p110δ subunit and the ubiquitously expressed regulatory p85 α subunit of the phosphoinositide 3-kinase δ (PI3Kδ). The first described patients with respiratory infections, hypogammaglobulinemia with normal to elevated IgM serum levels, lymphopenia, and lymphoproliferation. Since the original description, it is becoming evident that the onset of disease may be somewhat variable over time, both in terms of age at presentation and in terms of clinical and immunological complications. In many cases, patients are referred to various specialists such as hematologists, rheumatologists, gastroenterologists, and others, before an immunological evaluation is performed, leading to delay in diagnosis, which negatively affects their prognosis. The significant heterogeneity in the clinical and immunological features affecting APDS patients requires awareness among clinicians since good results with p110δ inhibitors have been reported, certainly ameliorating these patients’ quality of life and prognosis.

B cells express four PI3K isoforms (p110α, p110β, p110γ, and p110δ) but have a unique dependence on p110δ for survival. The design of isoform-selective inhibitors is possible, and pharmacologic inhibition of p110δ is toxic to neoplastic CLL cells for both cell-intrinsic and cell-extrinsic reasons. Idelalisib is a first-in-class p110δ inhibitor that exhibits efficacy for the treatment of relapsed CLL irrespective of adverse prognostic features. Toxicities of presumed autoimmune origin (hepatitis, enteritis/colitis, and pneumonitis), opportunistic infections, and bacterial infections were more pronounced in treatment naïve patients and when the drug was given in combination with chemotherapy, limiting further development. Duvelisib is a p110γ/δ inhibitor with a similar efficacy and safety profile to idelalisib. Recent data indicate that umbralisib, a p110δ/CK-1ε dual inhibitor, is safe and effective when administered to patients with CLL in either the treatment naïve or relapsed/refractory setting. Future studies are needed to establish where umbralisib should be incorporated into the treatment of CLL, what drug classes are most effective when combined with umbralisib and why umbralisib’s safety profile is superior. Whether other PI3K inhibitors in development can similarly retain efficacy while mitigating toxicity remains to be determined; most are exploring intermittent dosing schedules for that purpose.

Highlights • SHC014748M was proved to be more selective for PI3Kδ inhibition relative to other class i PI3K enzymes. • SHC014748M showed in vitro activity in most of 23 B lymphoma cell lines and primary CLL cells and also inhibited phosphorylation of AKT, targets downstream of PI3Kδ. • In vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. • SHC014748M seemed to be a novel promising compound in the treatment of B cell lymphomas and CLL., PI3Kδ (phosphatidylinositol 3-kinase-δ), one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ for patients with indolent non-Hodgkin lymphomas. Here in this paper, we comprehensively evaluated the in vitro and in vivo antitumor activity of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation. Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary chronic lymphocytic leukemia (CLL) cells. Scid mice were subcutaneously inoculated with the SU-DHL-6 cell line. SHC014748M was more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines and primary CLL cells. SHC014748M also inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in both lymphoma cells and primary CLL cells. In vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 in patient serum decreased sharply after SHC014748M treatment. According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL.

Phosphoinositide 3-kinase catalytic subunit δ isoform (P110δ) is mainly expressed in white blood cells. It is involved in T and B lymphocyte differentiation, maturation and the neutrophil chemotaxis process. Apolipoprotein E (ApoE) is an arginine-rich alkaline protein, which is present in plasma chylomicron, low-density lipoprotein and very low-density lipoprotein. The present study aimed to determine the effects of P110δ deletion on myocarditis in ApoE−/− mice. A mouse model of ApoE and P110δ double deletion was initially constructed; hematoxylin and eosin (H&E) staining was performed to detect the histological alterations in the mouse myocardium. Systolic and diastolic alterations, and alterations in the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were examined by electrocardiogram. Blood cell of ApoE and P110δ double mice was used to detect changes in white blood cells and monocytes. Western blotting was used to detect the expression levels of apoptosis-associated proteins, whereas flow cytometry was used to detect the percentage of apoptosis. Morphological alterations in myocardial cells were observed under a microscope. The results of polymerase chain reaction demonstrated that double deletion mice were successfully constructed. H&E staining revealed that cells in the ApoE−/− mice were spindle-shaped; however, the nuclei were smaller in the double deletion mice. There was no change in cardiac contraction in normal mice; however, in double deletion mice, the systolic and diastolic contractions were markedly reduced. LVFS and LVEF were decreased compared with in the control group. Blood cell analysis indicated that the content of white blood cells and monocytes in the experimental group was significantly higher than that in the control group. Western blotting demonstrated that the expression levels of apoptotic proteins in double deletion mice were significantly higher compared with in the control group. Flow cytometry revealed that the apoptotic ratio was increased in double deletion mice compared with in the control group (42 vs. 21%). These findings suggested that deletion of P110δ may induce monocyte peritoneal infiltration and increase apoptosis, thus promoting the development of myocarditis.

Phosphoinositide-3-kinase δ (PI3Kδ) is found in immune cells and is part of the PI3K/AKT/mTOR/S6K signalling pathway essential to cell survival, growth and differentiation. Hyperactivation of PI3Kδ enzyme results in Activated PI3-kinase delta syndrome (APDS). This childhood onset, autosomal dominant, combined immunodeficiency, is caused by heterozygous gain of function (GOF) mutations in PIK3CD (encodes PI3Kδ catalytic subunit p110δ), mutations in PIK3R1 (encodes PI3Kδ regulatory subunit p85α) or LOF mutations in PTEN (terminates PI3Kδ signalling) leading to APDS1, APDS2 and APDS-Like (APDS-L), respectively. APDS was initially described in 2013 and over 285 cases have now been reported. Prompt diagnosis of APDS is beneficial as targeted pharmacological therapies such as sirolimus and potentially PI3Kδ inhibitors can be administered. In this review, we provide an update on the clinical and laboratory features of this primary immunodeficiency. We discuss the common manifestations such as sinopulmonary infections, bronchiectasis, lymphoproliferation, susceptibility to herpesvirus, malignancy, as well as more rare non-immune features such as short stature and neurodevelopmental abnormalities. Laboratory characteristics, such as antibody deficiency and B cell and T cell, phenotypes are also summarised.

The mouse lung telocyte cell line (TCSV40) recently established provides further opportunities to learn TC biology and functions. The present study aims at investigating regulatory roles of phosphoinositide 3-kinase (PI3K) isoforms in TC proliferation and movement and in TGFβ1-induced sensitivity and response of lung TCs to PI3K inhibitors. Network and molecular interactions of genes coding PI3K family or TGFβ family proteins in mouse primary TCs were defined. Mouse lung TCSV40 proliferation, apoptosis, cell cycle, and dynamical bio-behaviors were measured with or without TGFβ1 stimulation or PI3K catalytic isoform protein (PI3K/mTOR, PI3Kα/δ/β, PI3K p110δ, or pan-PI3K) inhibitions. The present study showed the difference of network characteristics and interactions of genes coding PI3K isoform proteins or TGFβ family proteins in primary lung telocytes from mouse lungs compared to those of other cells residing in the lung. TGFβ1 had diverse effects on TC proliferation with altered TC number in G2 or S phase, independent upon the administered dose of TGFβ1. PI3Kα/δ/β, PI3K/mTOR, and PI3K p110δ were involved in TC proliferation, of which PI3Kα/δ/β was more sensitive. The effects of pan-PI3K inhibitor indicate that more PI3K isoforms were stimulated by the administering of external TGFβ1 and contributed to TGFβ1-induced TC proliferation. PI3K p110δ upregulated TC proliferation and movement dynamically without TGFβ1, and downregulated TC proliferation with TGFβ1 stimulation, but not TC movement. PI3Kα/δ/β and PI3K/mTOR were more active in TGFβ1-induced S phase accumulation and had similar dynamic effects to PI3K p110δ. Gene expression of PI3K isoforms in TCs was upregulated after TGFβ1 stimulation. The expression of PIK3CA coding p110-α or PIK3CG coding p110-γ were up- or downregulated in TCs without TGFβ1, respectively, when PI3K/mTOR, PI3Kα/δ/β, PI3K p110δ, or pan-PI3K were inhibited. TGFβ1 upregulated the expression of PIK3CA and PIK3CB, while downregulated the expression of PIK3CD and PIK3CG. Our data imply that TGFβ1 plays divergent roles in the expression of PI3K isoform genes in lung TCs and can alter the sensitivity and response of lung TCs to PI3K inhibitors.

Fcγ receptor (FcγR) signalling in monocyte derived macrophages from chronic lymphocytic leukaemia (CLL) patients is poorly understood. This signalling pathway is the key determinant of the ability of the macrophages to respond to therapeutic antibodies in current clinical use for CLL. Muted FcγR signalling activity accompanies disease progression and results in resistance to therapeutic antibodies. The molecular mechanisms controlling FcγR signalling and resistance are unknown. Here, we demonstrate that the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is essential for CLL-derived macrophages to respond to therapeutic antibodies. Inhibition of p110δ in the macrophages reduces FcγR-mediated antibody immune responses. Surprisingly, our studies indicated that FcγR downstream signalling is independent of SYK and BTK activity. Thus, we show that FcγR antibody responses occur via a previously unidentified p110δ-dependent pathway, which is independent of the previously described SYK/BTK activation pathway. These data provide novel insights into the effectors of antibody responses. Our data also provide mechanistic insights into therapy resistance in CLL.

Alzheimer's disease (AD) is associated with the cleavage of the amyloid precursor protein (APP) to produce the toxic amyloid-β (Aβ) peptide. Accumulation of Aβ, together with the concomitant inflammatory response, ultimately leads to neuronal death and cognitive decline. Despite AD progression being underpinned by both neuronal and immunological components, therapeutic strategies based on dual targeting of these systems remains unexplored. Here, we report that inactivation of the p110δ isoform of phosphoinositide 3-kinase (PI3K) reduces anterograde axonal trafficking of APP in hippocampal neurons and dampens secretion of the inflammatory cytokine tumor necrosis factor-alpha by microglial cells in the familial AD APPswe/PS1ΔE9(APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δD910A) had reduced Aβ peptides levels and plaques in the brain and an abrogated inflammatory response compared with APP/PS1 littermates. Mechanistic investigations reveal that PI3Kδ inhibition decreases the axonal transport of APP by eliciting the formation of highly elongated tubular-shaped APP-containing carriers, reducing the levels of secreted Aβ peptide. Importantly, APP/PS1/δD910Amice exhibited no spatial learning or memory deficits. Our data highlight inhibition of PI3Kδ as a new approach to protect against AD pathology due to its dual action of dampening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing.SIGNIFICANCE STATEMENTDuring Alzheimer's disease (AD), the accumulation of the toxic amyloid-β (Aβ) peptide in plaques is associated with a chronic excessive inflammatory response. Uncovering new drug targets that simultaneously reduce both Aβ plaque load and neuroinflammation holds therapeutic promise. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of phosphoinositide 3-kinase (PI3K) is involved in anterograde trafficking of the amyloid precursor protein in neurons and in the secretion of tumor necrosis factor-alpha from microglial cells. Genetic inactivation of PI3Kδ reduces Aβ plaque deposition and abrogates the inflammatory response, resulting in a complete rescue of the life span and spatial memory performance. We conclude that inhibiting PI3Kδ represents a novel therapeutic approach to ameliorate AD pathology by dampening plaque accumulation and microglial-dependent neuroinflammation.

The inositol lipid phosphatases PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from systemic lupus erythematosus and type 1 diabetes patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye toward eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low dosages of the p110δ inhibitor idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely induced B cell–targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low-dosage p110δ inhibitor therapy blocks disease progression in this model of type 1 diabetes. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

Activated phosphoinositide 3-kinase delta syndrome (APDS) is a novel primary immunodeficiency (PID) caused by heterozygous gain of function mutations in PI3Kδ catalytic p110δ (PIK3CD) or regulatory p85α (PIK3R1) subunits leading to APDS1 and APDS2, respectively. Patients with APDS present a spectrum of clinical manifestations, particularly recurrent respiratory infections and lymphoproliferation. We searched PubMed, Web of Science, and Scopus databases for APDS patients and screened for eligibility criteria. A total of 243 APDS patients were identified from 55 articles. For all patients, demographic, clinical, immunologic, and molecular data were collected. Overall, 179 APDS1 and 64 APDS2 patients were identified. The most common clinical manifestations were respiratory tract infections (pneumonia (43.6%), otitis media (28.8%), and sinusitis (25.9%)), lymphoproliferation (70.4%), autoimmunity (28%), enteropathy (26.7%), failure to thrive (20.6%), and malignancy (12.8%). The predominant immunologic phenotype was hyper-IgM syndrome (48.1%). Immunologic profiling showed decreased B cells in 74.8% and CD4+ T cells in 64.8% of APDS patients. The c.3061 G>A (p. E1021K) mutation in APDS1 with 85% frequency and c.1425+1 G> (A, C, T) (p.434–475del) mutation in APDS2 with 79% frequency were hotspot mutations. The majority of APDS patients were placed on long-term immunoglobulin replacement therapy. Immunosuppressive agents such as rituximab, tacrolimus, rapamycin, and leniolisib were also administered for autoimmunity and inflammatory complications. In addition, hematopoietic stem cell transplantation (HSCT) was used in 12.8% of patients. APDS has heterogynous clinical manifestations. It should be suspected in patients with history of recurrent respiratory infections, lymphoproliferation, and raised IgM levels. Moreover, HSCT should be considered in patients with severe and complicated clinical manifestations with no or insufficient response to the conventional therapies., This work was supported by vice chancellor for research, Alborz University of Medical Sciences, under Grant No. IR.ABZUMS.REC.1397.051.

In chronic lymphocytic leukemia (CLL), signaling through several prosurvival B cell surface receptors activates the PI3K signaling pathway. Idelalisib is a highly selective PI3K (PI3Kδ) isoform-specific inhibitor effective in relapsed/refractory CLL and follicular lymphoma. However, severe autoimmune adverse effects in association with the use of idelalisib in the treatment of CLL, particularly as a first-line therapy, gave indications that idelalisib may preferentially target the suppressive function of regulatory T cells (Tregs). On this background, we examined the effect of idelalisib on the function of human Tregs ex vivo with respect to proliferation, TCR signaling, phenotype, and suppressive function. Our results show that human Tregs are highly susceptible to PI3Kδ inactivation using idelalisib compared with CD4+ and CD8+ effector T cells (Teffs) as evident from effects on anti-CD3/CD28/CD2–induced proliferation (order of susceptibility [IC50]: Treg [.5 μM] > CD4+ Teff [2.0 μM] > CD8+ Teff [6.5 μM]) and acting at the level of AKT and NF-κB phosphorylation. Moreover, idelalisib treatment of Tregs altered their phenotype and reduced their suppressive function against CD4+ and CD8+ Teffs. Phenotyping Tregs from CLL patients treated with idelalisib supported our in vitro findings. Collectively, our data show that human Tregs are more dependent on PI3Kδ-mediated signaling compared with CD4+ and CD8+ Teffs. This Treg-preferential effect could explain why idelalisib produces adverse autoimmune effects by breaking Treg-mediated tolerance. However, balancing effects on Treg sensitivity versus CD8+ Teff insensitivity to idelalisib could still potentially be exploited to enhance inherent antitumor immune responses in patients.

This study reports on a novel activating p110δ mutation causing adult-onset hypogammaglobulinemia with lymphopenia without the classical presentation of atypical Activated phosphoinositide 3-kinase δ syndrome (ADPS-1), underlining thus the heterogeneous clinical and immunological presentation of p110δ mutated individuals and offers additional data on the role of p110δ in early and late B cell development in humans.

Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8+ T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8+ T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8+ T cell fate.

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

The presence of regulatory T cells (Tregs) in skin is important in controlling inflammatory responses in this peripheral tissue. Uninflamed skin contains a population of relatively immotile Tregs often located in clusters around hair follicles. Inflammation induces a significant increase both in the abundance of Tregs within the dermis, and in the proportion of Tregs that are highly migratory. The molecular mechanisms underpinning Treg migration in the dermis are unclear. In this study we used multiphoton intravital microscopy to examine the role of RGD-binding integrins and signalling through phosphoinositide 3-kinase P110δ (PI3K p110δ) in intradermal Treg migration in resting and inflamed skin. We found that inflammation induced Treg migration was dependent on RGD-binding integrins in a context-dependent manner. αv integrin was important for Treg migration 24 hours after induction of inflammation, but contributed to Treg retention at 48 hours, while β1 integrin played a role in Treg retention at the later time point but not during the peak of inflammation. In contrast, inhibition of signalling through PI3K p110δ reduced Treg migration throughout the entire inflammatory response, and also in the absence of inflammation. Together these observations demonstrate that the molecular mechanisms controlling intradermal Treg migration vary markedly according to the phase of the inflammatory response.

IKAROS, encoded by the IKZF1 gene, is a DNA-binding protein that functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL). Recent studies have identified IKAROS&rsquo, s novel function in the epigenetic regulation of gene expression in T-ALL and uncovered many genes that are likely to be directly regulated by IKAROS. Here, we report the transcriptional regulation of two genes, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE), by IKAROS in T-ALL. PIK3CD encodes the protein p110&delta, subunit of phosphoinositide 3-kinase (PI3K). The PI3K/AKT pathway is frequently dysregulated in cancers, including T-ALL. IKAROS binds to the promoter regions of PIK3CD and PIKFYVE and reduces their transcription in primary T-ALL. Functional analysis demonstrates that IKAROS functions as a transcriptional repressor of both PIK3CD and PIKFYVE. Protein kinase CK2 (CK2) is a pro-oncogenic kinase that is overexpressed in T-ALL. CK2 phosphorylates IKAROS, impairs IKAROS&rsquo, s DNA-binding ability, and functions as a repressor of PIK3CD and PIKFYVE. CK2 inhibition results in increased IKAROS binding to the promoters of PIK3CD and PIKFYVE and the transcriptional repression of both these genes. Overall, the presented data demonstrate for the first time that in T-ALL, CK2 hyperactivity contributes to PI3K signaling pathway upregulation, at least in part, through impaired IKAROS transcriptional regulation of PIK3CD and PIKFYVE. Targeting CK2 restores IKAROS&rsquo, s regulatory effects on the PI3K oncogenic signaling pathway.

Background Duvelisib, an FDA-approved oral phosphoinositide 3-kinase (PI3K)-δ,γ inhibitor, targets tumor cells of B/T cell malignancies, but may modulate non-malignant immune cells in the tumor microenvironment (TME) of many cancers. PI3K–δ and PI3K–γ downmodulate immunosuppressive Tregs and myeloid cells in solid tumors.1, 2, 3 We used single-cell RNA analysis of PIK3CD and PIK3CG to explore resistance mechanisms to checkpoint inhibitors (CPI). Methods Single-cell melanoma (SKCM) RNAseq datasets: GSE120575;4 CD45+ cells from 48 CPI responders and non-responder tumors, and GSE115978;5 33 treatment-naive and CPI-progressing (resistant) tumors. Cancer cells and CD45+ TME subpopulations, specified by gene expression signatures and tSNE plots, had PI3K gene expressions profiled. Differential gene expression (DE) was gated in MAST/Seurat. Fishers test Odds Ratio (OR) was calculated for ‘high’ expression. Results PIK3CD expression is higher in SKCM than most cancers (10.8 median RSEM log 2).7 By single-cell analysis, PIK3CD (> 0.3 log2 TPM) occurs in 68.2% of cancer cells, with PIK3CB, PIK3CA, and PIK3CG expressed in 32.3%, 12.0%, and 7.2% respectively. PIK3CD-high cancer cells (>4 log2 TPM) have a 711-gene DE gene signature mostly related to immune processes. A higher proportion of cancer cells in CPI resistant tumors express PIK3CD, than untreated tumors (OR 2.02, 95% CI 1.65–2.48, p=3.04 × 10–12), as do PIK3CD+PIK3CG-expressing cancer cells (OR 2.14, 95% CI 1.47–3.13, p=4.2 × 10-5). Additionally, in PI3K–δ or PI3K–γ high melanoma cell lines duvelisib inhibited proliferation, p-AKT and c-myc.7 PIK3CD and PIK3CG are prominently expressed in many SKCM CD45+ TME cells (84.5% and 31.7% CD45+ respectively). PIK3CD (>0.3 log2 TPM) occurs in a high fraction of T (85.7%), CD8+ T (86.3%), CD4+ T (86.9%), B (78.5%), macrophages (88%), and NK (85%). PIK3CG is highest in B, dendritic, cycling lymphocytes and plasma cells. Strikingly, a significantly higher proportion of PIK3CD+ cells occur in resistant tumors compared to untreated for all CD45+ cells, (OR 1.64, 95% CI 1.40–1.94, p=4.79 × 10-10), CD8+ T (OR 2.15, 95% CI 1.61–2.86, p=6.5 × 10-8), and an exhausted C8+ T subpopulation (OR 3.17, 95% CI 1.89–5.37, p=2.95 × 10-6). PIK3CD+PIK3CG-expressing CD45+ cells are significantly increased in CPI-resistant tumors (OR 1.22, 95% CI 1.07–1.39, p=0.002). Conclusions These findings support a mechanism where CPI therapies may contribute to modulation of PI3Kδ expression in cancer cells and the immune TME. The PI3K-δ,γ inhibitor duvelisib is being investigated in combination with CPI and evaluated in the context of CPI resistance in clinical trials: pembrolizumab (HNSC, NCT04193293), and nivolumab (Richter’s Syndrome, NCT03892044). References Ali K, Soond DR, Pineiro R, Hagemann T, Pearce W, Lim EL, Bouabe H, Scudamore CL, Hancox T, Maecker H, Friedman L, Turner M, Okkenhaug K, Vanhaesebroeck B. Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer Nature 2014; 510(7505):407–411. Kaneda MM, Messer KS, Ralainirina N, Li H, Leem CJ, Gorjestani S, Woo G, Nguyen AV, Figueiredo CC, Foubert P, Schmid MC, Pink M, Winkler DG, Rausch M, Palombella VJ, Kutok J, McGovern K, Frazer KA, Wu X, Karin M, Sasik R, Cohen EE, Varner JA. PI3Kγ is a molecular switch that controls immune suppression. Nature 2016; 539(7629):437–442. De Henau O, Rausch M, Winkler D, Campesato LF, Liu C, Cymerman DH, Budhu S, Ghosh A, Pink M, Tchaicha J, Douglas M, Tibbitts T, Sharma S, Proctor J, Kosmider N, White K, Stern H, Soglia J, Adams J, Palombella VJ, McGovern K, Kutok JL, Wolchok JD, Merghoub T. Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells. Nature 2016; 539(7629):443–447. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, Freeman SS, Reuben A, Hoover PJ, Villani AC, Ivanova E, Portell A, Lizotte PH, Aref AR, Eliane JP, Hammond MR, Vitzthum H, Blackmon SM, Li B, Gopalakrishnan V, Reddy SM, Cooper ZA, Paweletz CP, Barbie DA, Stemmer-Rachamimov A, Flaherty KT, Wargo JA, Boland GM, Sullivan RJ, Getz G, Hacohen N. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018; 175: 998–1013. Jerby-Arnon L, Shah P, Cuoco MS, Rodman C, Su MJ, Melms JC, Leeson R, Kanodia A, Mei S, Lin JR, Wang S, Rabasha B, Liu D, Zhang G, Margolais C, Ashenberg O, Ott PA, Buchbinder EI, Haq R, Hodi FS, Boland GM, Sullivan RJ, Frederick DT, Miao B, Moll T, Flaherty KT, Herlyn M, Jenkins RW, Thummalapalli R, Kowalczyk MS, Canadas I, Schilling B, Cartwright ANR, Luoma AM, Malu S2, Hwu P, Bernatchez C, Forget MA, Barbie DA, Shalek AK, Tirosh I, Sorger PK, Wucherpfennig K, Van Allen EM, Schadendorf D, Johnson BE, Rotem A, Rozenblatt-Rosen O, Garraway LA, Yoon CH, Izar B, Regev A. A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade. Cell 2018; 175: 984–997. Firebrowse Gene Expression Viewerhttp://firebrowse.org/viewGene.html. Coma S, Weaver DT, Pachter JA. [Poster #663] The dual PI3K-δ/PI3K-γ inhibitor duvelisib inhibits signaling and proliferation of solid tumor cells expressing PI3K-δ and/or PI3K-γ. AACR. 2020.

Activated phosphoinositide 3-kinase delta syndrome 1 (APDS-1) is a recently described inborn error of immunity caused by monoallelic gain-of-function mutations in the PIK3CD gene. We reviewed for the first time medical records and laboratory data of eight Italian APDS-1 patients. Recurrent sinopulmonary infections were the most common clinical feature at onset of disease. Seven patients presented lymphoproliferative disease, at onset or during follow-up, one of which resembled hemophagocytic lymphohistiocytosis (HLH). Genetic analysis of the PIK3CD gene revealed three novel mutations: functional testing confirmed their activating nature. In the remaining patients, the previously reported variants p.E1021K (n = 4) and p.E525A (n = 1) were identified. Six patients were started on immunoglobulin replacement treatment (IgRT). One patient successfully underwent hematopoietic stem cell transplantation (HSCT), with good chimerism and no GVHD at 21 months post-HSCT. APDS-1 is a combined immune deficiency with a wide variety of clinical manifestations and a complex immunological presentation. Besides IgRT, specific therapies targeting the PI3K&delta, pathway will most likely become a valid aid for the amelioration of patients&rsquo, clinical management and their quality of life.

Blood monocytes mediate the hematogenous dissemination of human cytomegalovirus (HCMV) in the host. However, monocytes have a short 48-hour (h) lifespan and are not permissive for viral replication. We previously established that HCMV infection drives differentiation of monocytes into long-lived macrophages to mediate viral dissemination, though the mechanism was unclear. Here, we found that HCMV infection promoted monocyte polarization into distinct macrophages by inducing select M1 and M2 differentiation markers and that Akt played a central role in driving differentiation. Akt&rsquo, s upstream positive regulators, PI3K and SHIP1, facilitated the expression of the M1/M2 differentiation markers with p110&delta, being the predominant PI3K isoform inducing differentiation. Downstream of Akt, M1/M2 differentiation was mediated by caspase 3, whose activity was tightly regulated by Akt in a temporal manner. Overall, this study highlights that HCMV employs the PI3K/SHIP1/Akt pathway to regulate caspase 3 activity and drive monocyte differentiation into unique macrophages, which is critical for viral dissemination.

The catalytic subunit p110δ of phosphoinositide 3-kinase (PI3K) encoded by PIK3CD has been implicated in some human solid tumors. However, its roles in colorectal cancer (CRC) remain largely unknown. Here we found that PIK3CD was overexpressed in colon cancer tissues and CRC cell lines and was an independent predictor for overall survival (OS) of patients with colon cancer. The ectopic overexpression of PIK3CD significantly promoted CRC cell growth, migration and invasion in vitro and tumor growth in vivo. In contrast, inhibition of PIK3CD by specific small-interfering RNA or idelalisib dramatically suppressed CRC cell growth, migration and invasion in vitro and tumor growth in vivo. Moreover, PIK3CD overexpression increased AKT activity, nuclear translocation of β-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity and decreased glycogen synthase kinase 3β (GSK-3β) activity, whereas PIK3CD inhibition exhibited the opposite effects. Furthermore, PIK3CD-mediated cell growth, migration and invasion were reversed by blockade of AKT signaling or depletion of β-catenin. In addition, PIK3CD expression in colon cancer tissues positively correlated with β-catenin abnormal expression, which was an independent predictor for OS of colon cancer patients. Taken together, our findings demonstrate that PIK3CD is an independent prognostic factor in CRC and that PIK3CD induces CRC cell growth, migration and invasion by activating AKT/GSK-3β/β-catenin signaling, suggesting that PIK3CD might be a novel prognostic biomarker and a potential therapeutic target for CRC.

Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δ(D910A/D910A)), we show that fetuses carried by p110δ(D910A/D910A) females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δ(D910A/D910A) females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.

Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3′-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δ(D910A/D910A)) in the Eμ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δ(D910A/D910A) mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δ(D910A/D910A) mice exhibited compromised CD4(+) and CD8(+) T cell response, and depletion of CD4(+) or CD8(+) T cells restored leukemia. Interestingly, p110δ(D910A/D910A) mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δ(D910A/D910A) mice with p110δ(WT/WT) Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition.

Oncotarget 9(51), 29565-29573 (2018). doi:10.18632/oncotarget.25619, Published by Impact Journals LLC, [S.l.]

Metformin exerts direct anti-tumor effects by activating AMP-activated protein kinase (AMPK), a major sensor of cellular metabolism in cancer cells. This, in turn, inhibits pro-survival mTOR signaling. Metformin has also been shown to disrupt complex 1 of the mitochondrial electron transport chain. Here, we explored the lymphoma specific anti-tumor effects of metformin using Daudi (Burkitt), SUDHL-4 (germinal center diffuse large B-cell lymphoma; GC DLBCL), Jeko-1 (Mantle-cell lymphoma; MCL) and KPUM-UH1 (double hit DLBCL) cell lines. We demonstrated that metformin as a single agent, especially at high concentrations produced significant reductions in viability and proliferation only in Daudi and SUDHL-4 cell lines with associated alterations in mitochondrial oxidative and glycolytic metabolism. As bcl-2 proteins, cyclin dependent kinases (CDK) and phosphoinositol-3- kinase (PI3K) also influence mitochondrial physiology and metabolism with clear relevance to the pathogenesis of lymphoma, we investigated the potentiating effects of metformin when combined with novel agents Venetoclax (bcl-2 inhibitor), BAY-1143572 (CDK9 inhibitor) and Idelalisib (p110δ- PI3K inhibitor). Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM of metformin resulted in 1.4 fold and 8.8 fold decreases, respectively, in IC-50 values of Venetoclax. By contrast, 3-fold and 10 fold reduction in IC-50 values of BAY-1143572 in Daudi and Jeko-1 cells respectively was seen in the presence of 10 mM of metformin. No change in IC-50 value for Idelalisib was observed across cell lines. These data suggest that although metformin is not a potent single agent, targeting cancer metabolism with similar but more effective drugs in novel combination with either bcl-2 or CDK9 inhibitors warrants further exploration.

Background Glioblastoma (GBM) is difficult to treat. Phosphoinositide 3-kinase (PI3K) is an attractive therapeutic target for GBM; however, targeting this pathway to effectively treat GBM is not successful because the roles of PI3K isoforms remain to be defined. The aim of this study is to determine whether PIK3CB/p110β, but not other PI3K isoforms, is a biomarker for GBM recurrence and important for cell survival. Methods Gene expression and clinical relevance of PI3K genes in GBM patients were analyzed using online databases. Expression/activity of PI3K isoforms was determined using immunoblotting. PI3K genes were inhibited using short hairpin RNAs or isoform-selective inhibitors. Cell viability/growth was assessed by the MTS assay and trypan blue exclusion assay. Apoptosis was monitored using the caspase activity assay. Mouse GBM xenograft models were used to gauge drug efficacy. Results PIK3CB/p110β was the only PI3K catalytic isoform that significantly correlated with high incidence rate, risk, and poor survival of recurrent GBM. PIK3CA/p110α, PIK3CB/p110β, and PIK3CD/p110δ were differentially expressed in GBM cell lines and primary tumor cells derived from patient specimens, whereas PIK3CG/p110γ was barely detected. PIK3CB/p110β protein levels presented a stronger association with the activities of PI3K signaling than other PI3K isoforms. Blocking p110β deactivated PI3K signaling, whereas inhibition of other PI3K isoforms had no effect. Specific inhibitors of PIK3CB/p110β, but not other PI3K isoforms, remarkably suppressed viability and growth of GBM cells and xenograft tumors in mice, with minimal cytotoxic effects on astrocytes. Conclusions PIK3CB/p110β is a biomarker for GBM recurrence and selectively important for GBM cell survival.

Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor–associated protein CD79B. CD79B-mutant PCNSLs showed enrichment of mammalian target of rapamycin (mTOR)-related gene sets and increased staining with PI3K/mTOR activation markers. Inhibition of the PI3K isoforms p110α/p110δ or mTOR synergized with ibrutinib to induce cell death in CD79B-mutant PCNSL cells. Significance: Ibrutinib has substantial activity in patients with relapsed or refractory B-cell lymphoma of the CNS. Response rates in PCNSL were considerably higher than reported for diffuse large B-cell lymphoma outside the CNS, suggesting a divergent molecular pathogenesis. Combined inhibition of BTK and PI3K/mTOR may augment the ibrutinib response in CD79B-mutant human PCNSLs. Cancer Discov; 7(9); 1018–29. ©2017 AACR. See related commentary by Lakshmanan and Byrd, p. 940. This article is highlighted in the In This Issue feature, p. 920

The PI3Kδ isoform (PIK3CD), also known as P110δ, is predominately expressed in leukocytes and has been implicated as a potential target in the treatment of hematological malignancies. In this report, we detailed the pharmacologic properties of puquitinib, a novel, orally available PI3Kδ inhibitor. Puquitinib, which binds to the ATP-binding pocket of PI3Kδ, was highly selective and potent for PI3Kδ relative to other PI3K isoforms and a panel of protein kinases, exhibiting low-nanomolar biochemical and cellular inhibitory potencies. Additional cellular profiling demonstrated that puquitinib inhibited proliferation, induced G1 -phase cell-cycle arrest and apoptosis in acute myeloid leukemia (AML) cell lines, through downregulation of PI3K signaling. In in vivo AML xenografts, puquitinib alone showed stronger efficacy than the well-known p110δ inhibitor, CAL-101, in association with a reduction in AKT and ERK phosphorylation in tumor tissues, without causing noticeable toxicity. Furthermore, the combination of puquitinib with cytotoxic drugs, especially daunorubicin, yielded significantly stronger antitumor efficacy compared with each agent alone. Thus, puquitinib is a promising agent with pharmacologic properties that are favorable for the treatment of AML.

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patient's B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.

The expression of different toll-like receptors (TLRs) on tumor cells has been associated with disease aggressiveness, treatment resistance, and poor prognosis. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is considered critical for cancer cell survival and proliferation. Thus, we investigated the effect of TLR-stimulated PI3K activation on the epithelial-to-mesenchymal transition (EMT) of primary (Caov-3) and metastatic (SK‑OV‑3) epithelial ovarian cancer cell lines in this study. TLR engagement with various ligands promoted the expression of class IA PI3K (p110α, p110β, and p110δ) and increased the expression of mesenchymal markers (N-cadherin, Slug, Vimentin, Snail, α-SMA, and TCF) in SK‑OV‑3 cells. The migratory activity and secretion of EMT-related cytokines of SK‑OV‑3 were significantly higher compared to those of Caov-3 after activation with TLR agonist. Although the invasive capacity and production of EMT-related cytokines of LPS-stimulated SK‑OV‑3 cells were significantly suppressed by all pharmacological inhibitors of the p110 isoform, the Syk/Src-dependent p110β isoform prominently attenuated migration activity. In contrast, the production of IL-10 and galectin-1 was mainly affected by the p110δ isoform. Gene silencing of TLR4 and galectin-1 with siRNA decreased the expression of matrix metalloproteinase-2 (MMP2) and MMP9 and reduced mesenchymal markers in LPS-treated SK‑OV‑3 cells. This study demonstrated that TLR-mediated PI3K activation modulated the invasion and metastasis of ovarian cancer through the production of galectin-1, suggesting that inhibition of the p110 isoform is a promising therapeutic approach against metastatic ovarian cancer.

Although new targeted therapies, such as ibrutinib and idelalisib, have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies, or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors, possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We discovered a single molecule, MDVN1003 (1-(5-amino-2,3-dihydro-1H-inden-2-yl)-3-(8-fluoro-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), that inhibits Bruton's tyrosine kinase and phosphatidylinositol-3-kinase δ, two proteins regulated by the B cell receptor that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2, two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together, these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

// Simona Ultimo 1, * , Carolina Simioni 1, * , Alberto M. Martelli 2 , Giorgio Zauli 1 , Camilla Evangelisti 3 , Claudio Celeghini 4 , James A. McCubrey 5 , Giorgia Marisi 6 , Paola Ulivi 6 , Silvano Capitani 1, 7 , Luca M. Neri 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 3 Institute of Molecular Genetics, Rizzoli Orthopedic Institute, National Research Council, Bologna, Italy 4 Department of Life Sciences, University of Trieste, Trieste, Italy 5 Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA 6 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e Cura dei Tumori (IRST) IRCCS, Meldola, Italy 7 LTTA Center, University of Ferrara, Ferrara, Italy * These authors contributed equally to this work Correspondence to: Luca M. Neri, email: luca.neri@unife.it Silvano Capitani, email: silvano.capitani@unife.it Keywords: PI3K isoforms, Bcr-Abl, Ph + B-acute lymphoblastic leukemia, autophagy, tyrosine kinase inhibitors Received: December 08, 2016 Accepted: February 12, 2017 Published: February 20, 2017 ABSTRACT B-acute lymphoblastic leukemia (B-ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. Philadelphia chromosome-positive (Ph + ) B-ALL is a subtype that expresses the Bcr-Abl fusion protein which represents a negative prognostic factor. Constitutive activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) network is a common feature of B-ALL, influencing cell growth and survival. In the present study, we aimed to investigate the efficacy of PI3K isoform inhibition in B-ALL cell lines harboring the Bcr-Abl fusion protein. We studied the effects of anti Bcr-Abl drugs Imatinib, Nilotinib and GZD824 associated with PI3K isoform inhibitors. We used a panel of six compounds which specifically target PI3K isoforms including the pan-PI3K inhibitor ZSTK474, p110α BYL719 inhibitor and the dual p110γ/p110δ inhibitor IPI145. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTS assays, apoptosis and autophagy by flow cytometry and Western blot, as well as the phosphorylation status of the pathway. ZSTK474, BYL719 and IPI145 administered in combination with imatinib, nilotinib and GZD824 for 48 h, decreased cell viability, induced apoptosis and autophagy in a marked synergistic manner. These findings suggest that selected PI3K isoform inhibitors used in combination with anti Bcr-Abl drugs may be an attractive novel therapeutic intervention in Ph + B-ALL.

Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in either the leukocyte-restricted p110δ catalytic (PIK3CD) subunit or the ubiquitously expressed p85α regulatory (PIK3R1) subunit of class IA phosphoinositide 3-kinases (PI3Ks). There are two classes of APDS: APDS1 that arises from p110δ mutations that are analogous to oncogenic mutations found in the broadly expressed p110α subunit and APDS2 that occurs from a splice mutation resulting in p85α with a central deletion (Δ434–475). As p85 regulatory subunits associate with and inhibit all class IA catalytic subunits, APDS2 mutations are expected to similarly activate p110α, β, and δ, yet APDS2 largely phenocopies APDS1 without dramatic effects outside the immune system. We have examined the molecular mechanism of activation of both classes of APDS mutations using a combination of biochemical assays and hydrogen–deuterium exchange mass spectrometry. Intriguingly, we find that an APDS2 mutation in p85α leads to substantial basal activation of p110δ (>300-fold) and disrupts inhibitory interactions from the nSH2, iSH2, and cSH2 domains of p85, whereas p110α is only minimally basally activated (∼2-fold) when associated with mutated p85α. APDS1 mutations in p110δ (N334K, E525K, E1021K) mimic the activation mechanisms previously discovered for oncogenic mutations in p110α. All APDS mutations were potently inhibited by the Food and Drug Administration-approved p110δ inhibitor idelalisib. Our results define the molecular basis of how PIK3CD and PIK3R1 mutations result in APDS and reveal a potential path to treatment for all APDS patients.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality among immunocompromised and immunonaive individuals. HCMV-induced signaling initiated during viral entry stimulates a rapid noncanonical activation of Akt to drive the differentiation of short-lived monocytes into long-lived macrophages, which is essential for viral dissemination and persistence. We found that HCMV glycoproteins gB and gH directly bind and activate cellular epidermal growth factor receptor (EGFR) and integrin β1, respectively, to reshape canonical Akt signaling within monocytes. The remodeling of the Akt signaling network was due to the recruitment of nontraditional Akt activators to either the gB- or gH-generated receptor signaling complexes. Phosphoinositide 3-kinase (PI3K) comprised of the p110β catalytic subunit was recruited to the gB/EGFR complex despite p110δ being the primary PI3K isoform found within monocytes. Concomitantly, SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) was recruited to the gH/integrin β1 complex, which is critical to aberrant Akt activation, as SHIP1 diverts PI3K signaling toward a noncanonical pathway. Although integrin β1 was required for SHIP1 recruitment, gB-activated EGFR mediated SHIP1 activation, underscoring the importance of the interplay between gB- and gH-mediated signaling to the unique activation of Akt during HCMV infection. Indeed, SHIP1 activation mediated the increased expression of Mcl-1 and HSP27, two Akt-dependent antiapoptotic proteins specifically upregulated during HCMV infection but not during growth factor treatment. Overall, our data indicate that HCMV glycoproteins gB and gH work in concert to initiate an HCMV-specific signalosome responsible for the atypical activation of Akt required for infected monocyte survival and ultimately viral persistence. IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic throughout the world regardless of socioeconomic conditions and geographic locations with a seroprevalence reaching up to 100% in some developing countries. Although asymptomatic in healthy individuals, HCMV can cause severe multiorgan disease in immunocompromised or immunonaive patients. HCMV disease is a direct consequence of monocyte-mediated systematic spread of the virus following infection. Because monocytes are short-lived cells, HCMV must subvert the natural short life-span of these blood cells by inducing a distinct activation of Akt, a serine/theonine protein kinase. In this work, we demonstrate that HCMV glycoproteins gB and gH work in tandem to reroute classical host cellular receptor signaling to aberrantly activate Akt and drive survival of infected monocytes. Deciphering how HCMV modulates the cellular pathway to induce monocyte survival is important to develop a new class of anti-HCMV drugs that could target and prevent spread of the virus by eliminating infected monocytes.

In patients, gain-of-function (GOF) mutations in PIK3CD break tolerance, causing highly penetrant secretion of autoreactive IgM. Mouse models reveal that Pik3cd GOF subverts the response to self-antigen, preventing the induction of anergy and instead stimulating plasmablast and GC formation., Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity., Graphical Abstract

22q13 deletion syndrome is a genetic disorder caused by the deletion or disruption of the segment of the long arm of chromosome 22. The characteristic clinical features of this syndrome include delayed expressive speech, autistic behavior and hypotonia, and clinically severe complications associated with autoimmunity are rarely reported. We herein report a girl with 22q13 deletion syndrome complicated with multiple inflammatory and autoimmune diseases during early childhood. We performed whole-exome sequencing to identify the genes responsible for her autoimmune diseases and identified the de novo variant p.R512W in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) gene. We suspected it to be the disease-causing variant at the conserved residue in PIK(3)C p110δ. Alternatively, haplo-insufficiency of SHANK3 or other genes by 22q13 deletion and the PIK3CD variant might have synergistically contributed to the onset of the distinctive clinical manifestations in this patient.

International audience; Infection of T cells by Epstein-Barr virus (EBV) causes chronic active EBV infection (CAEBV) characterized by T cell lymphoproliferative disorders (T-LPD) of unclear etiology. Here, we identified two homozygous biallelic loss-of-function mutations in PIK3CD and TNFRSF9 in a patient who developed a fatal CAEBV. The mutation in TNFRSF9 gene coding CD137/4-1BB, a costimulatory molecule expressed by antigen-specific activated T cells, resulted in a complete loss of CD137 expression and impaired T cell expansion toward CD137 ligand-expressing cells. Isolated as observed in one sibling, CD137 deficiency resulted in persistent EBV-infected T cells but without clinical manifestations. The mutation in PIK3CD gene that encodes the catalytic subunit p110δ of the PI3K significantly reduced its kinase activity. Deficient T cells for PIK3CD exhibited reduced AKT signaling, while calcium flux, RAS-MAPK activation, and proliferation were increased, suggestive of an imbalance between the PLCγ1 and PI3K pathways. These skewed signals in T cells may sustain accumulation of EBV-infected T cells, a process controlled by the CD137-CD137L pathway, highlighting its critical role in immunity to EBV.