A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in ß-thalassemic mice.

ABSTRACT: Iron plays a major role in the deterioration of ß-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 ß-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 ß-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 ß-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 ß-thalassemia.

INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/ß-catenin signaling in breast cancer.

INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 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)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3ß lysosomal degradation and activation of Wnt/ß-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)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/ß-catenin therapies.

Gßγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.

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 Gßγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gßγ 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 Gßγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gßγ 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 Gßγ 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 Gßγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

Inositol polyphosphate 4-phosphatase II (INPP4B) is associated with chemoresistance and poor outcome in AML.

Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P3 3-phosphatase (PTEN), PtdIns(3,4,5)P3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4- and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function.

INPP4B is highly expressed in prostate intermediate cells and its loss of expression in prostate carcinoma predicts for recurrence and poor long term survival.

BACKGROUND: Phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in prostate carcinoma due to the loss of tumor suppressor PTEN, which leads to increased Akt activity. Expression of INPP4B, another negative regulator of the PI3K/Akt pathway, is also reduced in prostate carcinoma. However, uncertainty exists regarding the association of INPP4B expression and biochemical and clinical relapse of prostate carcinoma. METHODS: INPP4B expression in benign prostate acini was analyzed by co-immunofluorescence with cytokeratins (CK) 5, 8, 19, androgen receptor (AR), c-MET, chromogranin A and Ki67. INPP4B expression in prostate carcinoma was analyzed in two independent cohorts (n = 406). The association of INPP4B with biochemical and clinical prostate carcinoma relapse was assessed by Kaplan-Meier and Cox proportional hazards modeling. RESULTS: INPP4B was expressed in luminal epithelium within benign ducts, and was highly expressed in CK5+/CK8+/CK19+/AR-/c-MET+/Ki67- intermediate cells in proliferative inflammatory atrophic acini. Overall, INPP4B expression was reduced in prostate carcinoma compared to benign epithelium. Absent/low INPP4B expression was associated with reduced biochemical relapse-free survival (P = 0.01) and increased risk of clinical relapse (P = 0.01). Absence of INPP4B expression was an independent predictor of clinical relapse free survival (P = 0.004) when modeled with Gleason score (P = 0.027) and pathologic stage (P = 0.07). CONCLUSIONS: INPP4B is highly expressed in intermediate cells within proliferative inflammatory atrophic ducts, and expression is reduced in prostate carcinoma. Absence of INPP4B expression is associated with poor outcome following radical prostatectomy, and represents an independent prognostic marker of prostate carcinoma clinical recurrence.

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells.

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.