Sabine Siegemund

Balancing pro- and anti-inflammatory TLR4 signaling

Limiting immune responses is critical for protecting the host from harm. The p110δ isoform of the kinase PI(3)K acts as a balance between pro- and anti-inflammatory TLR4 signaling in dendritic cells.

IP3 3-kinase B controls hematopoietic stem cell homeostasis and prevents lethal hematopoietic failure in mice.

Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures lifelong hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify Inositol-trisphosphate 3-kinase B (Itpkb) as an essential regulator of HSC homeostasis. Young Itpkb(-/-) mice accumulated phenotypic HSC, which were less quiescent and proliferated more than wild-type (WT) controls. Itpkb(-/-) HSC downregulated quiescence and stemness associated, but upregulated activation, oxidative metabolism, protein synthesis, and lineage associated messenger RNAs. Although they had normal-to-elevated viability and no significant homing defects, Itpkb(-/-) HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb(-/-) mice lost hematopoietic stem and progenitor cells and died with severe anemia. WT HSC normally repopulated Itpkb(-/-) hosts, indicating an HSC-intrinsic Itpkb requirement. Itpkb(-/-) HSC showed reduced colony-forming activity and increased stem-cell-factor activation of the phosphoinositide-3-kinase (PI3K) effectors Akt/mammalian/mechanistic target of rapamycin (mTOR). This was reversed by treatment with the Itpkb product and PI3K/Akt antagonist IP4. Transcriptome changes and biochemistry support mTOR hyperactivity in Itpkb(-/-) HSC. Treatment with the mTOR-inhibitor rapamycin reversed the excessive mTOR signaling and hyperproliferation of Itpkb(-/-) HSC without rescuing colony forming activity. Thus, we propose that Itpkb ensures HSC quiescence and function through limiting cytokine-induced PI3K/mTOR signaling and other mechanisms.

Inositol tetrakisphosphate limits NK cell effector functions by controlling PI3K signaling

Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell-based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP(3) generation by PI3K and generation of diacylglycerol and IP(3) by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP(3) metabolite inositol (1,3,4,5)tetrakisphosphate (IP(4)) in NK cells. IP(4) promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP(4) limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP(3) effector-kinase Akt. This identifies IP(4) as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP(4) is a broadly important signaling paradigm.

Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent

β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement. DOI: http://dx.doi.org/10.7554/eLife.10786.001

Abstract LB-020: IP3 3-kinase B suppresses B-cell lymphoma by antagonizing PI3K/mTOR in B cells

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Accounting for ∼4% of all cancers in the US, Non-Hodgkin Lymphoma (NHL) is the most prevalent blood cancer. Diffuse large B-cell Lymphoma (DLBCL) is the most common and aggressive type of NHL. Here, we identify inositol-trisphosphate (IP3) 3-kinase B (Itpkb) as a novel tumor suppressor whose deficiency triggers DLBCL in mice. We found that aging Itpkb-/- mice die prematurely with anemia and splenomegaly. 15% of these mice showed multiorgan-infiltration with neoplastic germinal-center phenotype B cells reminiscent of DLBCL. Itpkb-/- B cell lymphomas are mono-or oligoclonal, transplantable, and constitutively hyperactivate the oncogenic phosphoinositide 3-kinase (PI3K) signaling pathway. Growth of Itpkb-/- B cell lymphoma xenografts in immunodeficient mice is sensitive to treatment with PI3K or mTOR inhibitors. In humans, subsets of DLBCL patients carry Itpkb missense mutations, deletions or copy number reductions. Additionally, Itpkb expression is altered in several other hematopoietic and non-hematopoietic human cancers. We propose that Itpkb suppresses tumors by producing inositol-tetrakisphosphate (IP4), a soluble analog of the PI3K product phosphatidylinositol-trisphosphate (PIP3). We and others have previously shown that IP4 antagonism with PIP3 for Akt effector kinase binding prevents excessive Akt/mTORC1 signaling in hematopoietic cells. Our new results suggest that this mechanism prevents oncogenic PI3K signaling in the B cell lineage and possibly in other cell types. Thus, Itpkb might be a novel biomarker for tumor aggressiveness or prognosis, and Itpkb activating drugs or IP4 might have therapeutic potential. This work was supported by NIH grants AI070845 and GM100785 to KS, The Leukemia & Lymphoma Society Scholar Award 1440-11 to KS, the AAI Careers in Immunology Fellowship Program to SR, DFG Fellowship SI 1547/1-1 to SS, and an ARC fellowship to CC. Citation Format: Karsten Sauer, Claire Conche, Hyun-Yong Jin, Kelly Bethel, Stephanie Rigaud, Luise Westernberg, Sabine Siegemund, Changchun Xiao. IP3 3-kinase B suppresses B-cell lymphoma by antagonizing PI3K/mTOR in B cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-020.