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Background: Prolactin receptor (PRLR) is an attractive antibody therapeutic target with expression across a broad population of breast cancers. Antibody efficacy, however, may be limited to subtypes with either PRLR overexpression and/or those where estradiol no longer functions as a mitogen and are, therefore, reliant on PRLR signaling for growth. In contrast a potent PRLR antibody-drug conjugate (ADC) may provide improved therapeutic outcomes extending beyond either PRLR overexpressing or estradiol-insensitive breast cancer populations., Methods: We derived a novel ADC targeting PRLR, ABBV-176, that delivers a pyrrolobenzodiazepine (PBD) dimer cytotoxin, an emerging class of warheads with enhanced potency and broader anticancer activity than the clinically validated auristatin or maytansine derivatives. This agent was tested in vitro and in vivo cell lines and patient derived xenograft models., Results: In both in vitro and in vivo assays, ABBV-176 exhibits potent cytotoxicity against multiple cell line and patient-derived xenograft breast tumor models, including triple negative and low PRLR expressing models insensitive to monomethyl auristatin (MMAE) based PRLR ADCs. ABBV-176, which cross links DNA and causes DNA breaks by virtue of its PBD warhead, also demonstrates enhanced anti-tumor activity in several breast cancer models when combined with a poly-ADP ribose polymerase (PARP) inhibitor, a potentiator of DNA damage., Conclusions: Collectively the efficacy and safety profile of ABBV-176 suggest it may be an effective therapy across a broad range of breast cancers and other cancer types where PRLR is expressed with the potential to combine with other therapeutics including PARP inhibitors.

Rationale: TRPM2 (transient receptor potential melastatin-2) expressed in endothelial cells (ECs) is a cation channel mediating Ca 2+ entry in response to intracellular generation of adenosine diphosphoribose-the TRPM2 ligand., Objective: Because polymorphonuclear neutrophils (PMN) interaction with ECs generates reactive oxygen species, we addressed the possible role of TRPM2 expressed in ECs in the mechanism of transendothelial migration of PMNs., Methods and Results: We observed defective PMN transmigration in response to lipopolysaccharide challenge in adult mice in which the EC expressed TRPM2 is conditionally deleted ( Trpm2 iΔEC ). PMN interaction with ECs induced the entry of Ca 2+ in ECs via the EC-expressed TRPM2. Prevention of generation of adenosine diphosphoribose in ECs significantly reduced Ca 2+ entry in response to PMN activation of TRPM2 in ECs. PMNs isolated from gp91phox -/- mice significantly reduced Ca 2+ entry in ECs via TRPM2 as compared with wild-type PMNs and failed to induce PMN transmigration. Overexpression of the adenosine diphosphoribose insensitive TRPM2 mutant channel (C1008→A) in ECs suppressed the Ca 2+ entry response. Further, the forced expression of TRPM2 mutant channel (C1008→A) or silencing of poly ADP-ribose polymerase in ECs of mice prevented PMN transmigration., Conclusions: Thus, endotoxin-induced transmigration of PMNs was secondary to TRPM2-activated Ca 2+ signaling and VE-cadherin phosphorylation resulting in the disassembly of adherens junctions and opening of the paracellular pathways. These results suggest blocking TRPM2 activation in ECs is a potentially important means of therapeutically modifying PMN-mediated vascular inflammation., (© 2017 American Heart Association, Inc.)

Objective: Transient receptor potential melastatin-2 (TRPM2) channel is a nonselective cation channel that mediates influx of Ca(2+) and Na(+) with relative permeability of PCa:PNa ≈0.6 in response to cellular oxidative stress. As angiogenesis and ischemic neovascularization are both significantly dependent on oxidant signaling, here we investigated the possible role of vascular endothelial growth factor (VEGF)-induced reactive oxygen species production in activating TRPM2-dependent Ca(2+) signaling and in the mechanism of angiogenesis and ischemic neovascularization., Approach and Results: We observed that VEGF stimulation rapidly induced the association of TRPM2 and cellular Src kinase with vascular endothelial-cadherin forming a signalplex at vascular endothelial-cadherin junctions in endothelial cells. Using endothelial cells isolated from TRPM2(-/-) mice or after small interfering RNA depletion of TRPM2, we demonstrated that TRPM2-activated Ca(2+) signaling was required for cellular Src kinase-induced phosphorylation of vascular endothelial-cadherin at Y658 and Y731, the crucial sites involved in vascular endothelial-cadherin internalization in response to VEGF. VEGF-induced reactive oxygen species generation activated TRPM2-induced Ca(2+) entry, whereas the reactive oxygen species-insensitive TRPM2 mutant (C1008→A) showed impaired Ca(2+) entry. Endothelial cells depleted of TRPM2 also displayed significantly perturbed migratory phenotype and impaired activation of cellular Src in response to VEGF. TRPM2(-/-) mice reconstituted with wild-type myeloid cells demonstrated aberrant angiogenesis and neovascularization in the hindlimb ischemia model as compared with wild-type mice., Conclusions: VEGF-induced angiogenesis and postischemic neovascularization in mice required reactive oxygen species generation in endothelial cells and resultant TRPM2 activation. Thus, our findings provide novel insight into the role of TRPM2 in mechanism of angiogenesis and ischemic neovascularization., (© 2015 American Heart Association, Inc.)

Rationale: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supranormal Ca2+ influx required for initiating of apoptosis are not understood., Objective: Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short variant (TRPM2-S) in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis., Methods and Results: We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma, and this interaction functioned to suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-α activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The protein kinase C-α-activated phosphoswitch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase., Conclusions: Here, we describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species-induced protein kinase C-α activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.

Oxidative [Au1]stress, through the production of oxygen metabolites such as hydrogen peroxide[Au2] (H(2)O(2)), increases vascular endothelial permeability and plays a crucial role in several lung diseases. The transient receptor potential (melastatin) 2 (TRPM2) is an oxidant-sensitive, nonselective cation channel that is widely expressed in mammalian tissues, including the vascular endothelium. We have demonstrated the involvement of TRPM2 in mediating oxidant-induced calcium entry and endothelial hyperpermeability in cultured pulmonary artery endothelial cells. Here, we provide evidence that neutrophil activation-dependent increase in endothelial permeability and neutrophil extravasation requires TRPM2 in cultured endothelial cells. In addition, protein kinase Calpha (PKCalpha) that rapidly colocalizes with the short (nonconducting) TRPM2 isoform after exposure to hydrogen peroxide positively regulates calcium entry through the functional TRPM2 channel. Thus, increase in lung microvessel permeability and neutrophil sequestration depends on the activation of endothelial TRPM2 by neutrophilic oxidants and on PKCalpha regulation of TRPM2 channel activity. Manipulating TRPM2 function in the endothelium may represent a novel strategy aimed to prevent oxidative stress-related vascular dysfunction.

The transient receptor potential (melastatin) 2 (TRPM2), is an oxidant-activated non-selective cation channel that is widely expressed in mammalian tissues including the vascular endothelium. Oxidative stress, through the generation of oxygen metabolites including H(2)O(2), stimulates intracellular ADP-ribose formation which, in turn, opens TRPM2 channels. These channels act as an endogenous redox sensor for mediating oxidative stress/ROS-induced Ca(2+) entry and the subsequent specific Ca(2+)-dependent cellular reactions such as endothelial hyperpermeability and apoptosis. This review summarizes recent findings on the mechanism by which oxidants induce TRPM2 activation, the role of these channels in the signalling vascular endothelial dysfunctions, and the modulation of oxidant-induced TRPM2 activation by PKCalpha and phospho-tyrosine phosphates L1.

Oxidative stress through the production of oxygen metabolites such as hydrogen peroxide (H2O2) increases vascular endothelial permeability. H2O2 stimulates ADP-ribose formation, which in turn opens transient receptor potential melastatin (TRPM)2 channels. Here, in endothelial cells, we demonstrate transcript and protein expression of TRPM2, a Ca2+-permeable, nonselective cation channel. We further show the importance of TRPM2 expression in signaling of increased endothelial permeability by oxidative stress. Exposure of endothelial cell monolayers to sublytic concentrations of H2O2 induced a cationic current measured by patch-clamp recording and Ca2+ entry detected by intracellular fura-2 fluorescence. H2O2 in a concentration-dependent manner also decreased trans-monolayer transendothelial electrical resistance for 3 hours (with maximal effect seen at 300 micromol/L H2O2), indicating opening of interendothelial junctions. The cationic current, Ca2+ entry, and transendothelial electrical resistance decrease elicited by H2O2 were inhibited by siRNA depleting TRPM2 or antibody blocking of TRPM2. H2O2 responses were attenuated by overexpression of the dominant-negative splice variant of TRPM2 or inhibition of ADP-ribose formation. Overexpression of the full-length TRPM2 enhanced H2O2-mediated Ca2+ entry, cationic current, and the transendothelial electrical resistance decrease. Thus, TRPM2 mediates H2O2-induced increase in endothelial permeability through the activation of Ca2+ entry via TRPM2. TRPM2 represents a novel therapeutic target directed against oxidant-induced endothelial barrier disruption.