Abstract INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P 2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA -mutant ER + breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA -mutant ER + breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P 2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER + breast cancer via PI(3,4)P 2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.
Iron plays a major role in the deterioration of β-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in β-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in β-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the β-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of β-thalassemia.
Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.
Abstract Acute myeloid leukaemia (AML) is an aggressive blood cancer that is usually fatal within weeks without effective therapy. Treatment currently involves standard chemotherapy agents such as cytarabine (Ara-C) and anthracyclines but these drugs fail to elicit complete clinical responses in 20-30% of cases. An understanding of the mechanisms in AML mediating resistance to chemotherapy may uncover new oncoproteins amenable to medicinal targeting. Activation of the phosphoinositide 3-kinase (PI3-K)/AKT pathway is prevalent in AML and linked to chemotherapy failure and poor outcomes. Homeostatic regulation of PI3K activity is orchestrated by a triad of lipid phosphatases, categorized functionally as 3-, 4-, or 5-phosphoinositide phosphatases (PIPs). We screened for pathological expression of 3-, 4-, or 5-PIPs in AML using a quantitative multiplexed MassArray platform (Sequenom®) and found marked overexpression of inositol polyphosphate 4-phosphatase II (INPP4B) in a subset of primary AMLs compared to normal bone marrow. Immunohistochemical staining of 120 primary AML bone marrow samples showed INPP4B was overexpressed (>20% blasts positive) in 16% of cases. INPP4B overexpression was associated with an inferior complete response rate to intensive chemotherapy (33%), compared to AMLs low (5-20%; CR 66%) or negative (<5%; CR 93%) for INPP4B. Ectopic overexpression of INPP4B in MV4;11 and HEL AML cells conferred resistance to in vitro cell death (flow-based viability and colony forming capacity) when exposed to standard agents used to treat AML, including cytarabine, daunorubicin and etoposide. High levels of INPP4B in AML also limited the cytotoxic efficacy of drugs such as cytarabine in vivo using human xenograft models of AML. Although INPP4B phosphatase function was proven to be catalytically active in primary AML, expression of a phosphatase inactive mutant (INPP4B C842A) did not abrogate chemoresistance. Conversely, siRNA-mediated knockdown of endogenous INPP4B expression in KG1 and OCI-AML3 AML increased leukemic sensitivity to cytarabine. These findings infer 1) the presence of a novel phosphatase-independent function for this protein and 2) a previously unsuspected role for INPP4B overexpression in mediating chemoresistance and poor clinical outcome in a subset of patients with AML. Citation Format: Sewa Rijal, Nhu-Y Nugyen, Tse-Chieh Teh, Natalie K. Rynkiewicz, Nik Cummings, Lisa Ooms, Sharon Avery, Julie Mcmanus, Mark A. Guthridge, Catriona Mclean, Christina A. Mitchell*, Andrew Wei*. Elucidating a novel role for inositol polyphosphate 4-phosphatase type II (INPP4B) in mediating chemoresistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5283. doi:10.1158/1538-7445.AM2014-5283
The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.
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