An indirect comparison of acalabrutinib with and without obinutuzumab vs zanubrutinib in treatment-naive CLL.

ABSTRACT: The efficacy and safety of acalabrutinib plus obinutuzumab and acalabrutinib monotherapy vs zanubrutinib in patients with treatment-naive chronic lymphocytic leukemia/small lymphocytic lymphoma without del(17p) were compared using an unanchored matching-adjusted indirect comparison. Individual patient-level data from ELEVATE-TN (acalabrutinib plus obinutuzumab, n = 162; acalabrutinib monotherapy, n = 163) were weighted to match published aggregate baseline data from SEQUOIA cohort 1, which excluded patients with del(17p) (zanubrutinib, n = 241), using variables that were prognostic/predictive of investigator-assessed progression-free survival (INV-PFS) in an exploratory Cox regression analysis of ELEVATE-TN. After matching, INV-PFS was longer with acalabrutinib plus obinutuzumab (hazard ratio [HR], 0.41; 95% confidence interval [CI], 0.23-0.74) and comparable with acalabrutinib monotherapy (HR, 0.91; 95% CI, 0.53-1.56) vs zanubrutinib. Acalabrutinib monotherapy had significantly lower odds of any grade hypertension vs zanubrutinib (odds ratio [OR], 0.44; 95% CI, 0.20-0.99), whereas acalabrutinib plus obinutuzumab had significantly higher odds of neutropenia (OR, 2.19; 95% CI, 1.33-3.60) and arthralgia (OR, 2.33; 95% CI, 1.37-3.96) vs zanubrutinib. No other significant differences in safety were observed. In summary, acalabrutinib plus obinutuzumab had longer INV-PFS with increased odds of neutropenia and arthralgia than zanubrutinib, whereas acalabrutinib monotherapy had similar INV-PFS with lower odds of any grade hypertension. These trials were registered at www.ClinicalTrials.gov as #NCT02475681 and #NCT03336333.

Quality-adjusted survival time without symptoms or toxicity of acalabrutinib with or without obinutuzumab in patients with treatment-naive chronic lymphocytic leukemia.

Acalabrutinib monotherapy (A) and acalabrutinib plus obinutuzumab (A + O) demonstrated improved efficacy and safety versus chlorambucil plus obinutuzumab (C + O) among treatment-naive patients with chronic lymphocytic leukaemia (CLL) in the ELEVATE-TN trial. The relative risk-benefit at a median follow-up of 47 months was assessed using Quality-adjusted Time Without Symptoms and Toxicity (Q-TWiST) methodology. Patient data were partitioned into 3 states: time with toxicity (TOX); time without symptoms or toxicity (TWiST); and time after relapse (REL). Mean Q-TWiST was estimated by summing the mean time in each state, multiplied by its respective utility weight. Patients receiving A or A + O experienced significantly longer Q-TWiST versus C + O when toxicity was defined as grade 3-4 adverse events (AEs) (41.79 vs 34.56 months; 42.07 vs 34.56 months) and grade 2-4 AEs (35.07 vs 30.64 months; 34.21 vs 30.64 months). Overall, patients with treatment-naive CLL treated with A or A + O experienced significant gains in Q-TWiST compared with C + O.

The Orai Ca2+ channel inhibitor CM4620 targets both parenchymal and immune cells to reduce inflammation in experimental acute pancreatitis.

KEY POINTS: This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. ABSTRACT: Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

BACKGROUND & AIMS: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. METHODS: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. RESULTS: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. CONCLUSIONS: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

Molecular basis of the CRAC channel.

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.

Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth.

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

A store-operated calcium channel in Drosophila S2 cells.

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating.

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.