Phase III randomized study of taselisib or placebo with fulvestrant in estrogen receptor-positive, PIK3CA-mutant, HER2-negative, advanced breast cancer: the SANDPIPER trial.

BACKGROUND: The phase III SANDPIPER study assessed taselisib (GDC-0032), a potent, selective PI3K inhibitor, plus fulvestrant in estrogen receptor-positive, HER2-negative, PIK3CA-mutant locally advanced or metastatic breast cancer. PATIENTS AND METHODS: Postmenopausal women with disease recurrence/progression during/after an aromatase inhibitor were randomized 2 : 1 to receive taselisib (4 mg; taselisib arm) or placebo (placebo arm) plus fulvestrant (500 mg). Stratification factors were visceral disease, endocrine sensitivity, and geographic region. Patients with PIK3CA-mutant tumors (central cobas® PIK3CA Mutation Test) were randomized separately from those without detectable mutations. The primary endpoint was investigator-assessed progression-free survival (INV-PFS) in patients with PIK3CA-mutant tumors. Secondary endpoints included objective response rate, overall survival, clinical benefit rate, duration of objective response, PFS by blinded independent central review (BICR-PFS), safety, and time to deterioration in health-related quality of life. RESULTS: The PIK3CA-mutant intention-to-treat population comprised 516 patients (placebo arm: n = 176; taselisib arm: n = 340). INV-PFS was significantly improved in the taselisib {7.4 months [95% confidence interval (CI), 7.26-9.07]} versus placebo arm (5.4 months [95% CI, 3.68-7.29]) (stratified hazard ratio [HR] 0.70; 95% CI, 0.56-0.89; P = 0.0037) and confirmed by BICR-PFS (HR 0.66). Secondary endpoints, including objective response rate, clinical benefit rate, and duration of objective response, showed consistent improvements in the taselisib arm. Safety was assessed in all randomized patients who received at least one dose of taselisib/placebo or fulvestrant regardless of PIK3CA-mutation status (n = 629). Serious adverse events were lower in the placebo versus taselisib arm (8.9% versus 32.0%). There were more discontinuations (placebo arm: 2.3%; taselisib arm: 16.8%) and dose reductions (placebo arm: 2.3%; taselisib arm: 36.5%) in the taselisib arm. CONCLUSION: SANDPIPER met its primary endpoint; however, the combination of taselisib plus fulvestrant has no clinical utility given its safety profile and modest clinical benefit.

A novel Rab9 effector required for endosome-to-TGN transport.

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p-Emp47p hybrid protein was mislocalized to the cell surface in the alpha-COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.

Vesicle traffic: get your coat!

The budding of transport vesicles from the Golgi complex is initiated by activation of the small GTPase ARF; the discovery of enzymes that can convert soluble ARF-GDP to the active, membrane-associated form ARF-GTP will shed light on the mechanism and regulation of the formation of transport vesicles.

Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution.

We report here the identification and characterization of a human 76kDa membrane protein that is found predominantly in endosomes. This protein is related to the Saccharomyces cerevisiae EMP70 gene product, a precursor protein whose 24kDa cleavage product (p24a) was found in yeast endosome-enriched membrane fractions (Singer-Krüger et al., 1993. J. Biol. Chem. 268, 14376-14386). Northern blot analysis indicated that p76 mRNA is highly expressed in human pancreas but could be detected in all tissues examined. p76 is highly conserved throughout evolution, as related proteins have also been detected in Caenorhabditis elegans and Arabidopsis thaliana. This family of proteins has a relatively divergent, hydrophilic N-terminal domain and a well-conserved, highly hydrophobic C-terminal domain which contains nine potential membrane-spanning domains. Transiently expressed, myc-tagged human p76 appears to be localized to endosomes by virtue of its apparent colocalization with transferrin receptors and some mannose 6-phosphate receptors. Furthermore, p76 adopts a type-I topology within the membrane, with its hydrophilic N-terminus facing the lumen of cytoplasmic membranes. The structural features of p76 suggest that it may function as a channel or small molecule transporter in intracellular compartments throughout phylogeny. 1998 Elsevier Science B.V.

Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast.

The YPT7 gene encodes the Saccharomyces cerevisiae homolog of mammalian rab7 protein. Data obtained from studies on a delta ypt7 mutant suggested that Ypt7p is involved in the endocytic pathway in yeast (Wichmann et al., Cell 71, 1131-1142, 1992). We report here that endocytosed pheromone alpha-factor accumulates in late endosomes in delta ypt7 cells, indicating that Ypt7p is involved in the regulation of transport steps from late endosomes to the vacuole. We also show that alpha-factor can be degraded in a PEP4-dependent manner in a prevacuolar/endosomal compartment in delta ypt7 cells, providing independent evidence that the pathways of vacuole biogenesis and endocytosis in yeast may intersect in the endosomal membrane system.

Calcium-independent calmodulin requirement for endocytosis in yeast.

We have recently shown that actin and fimbrin are required for the internalization step of endocytosis in yeast. Using a yeast strain with a temperature-sensitive allele of CMD1, encoding calmodulin, we demonstrate that this protein is also required for this process. Calmodulin mutants that have lost their high-affinity calcium binding sites are, however, able to carry out endocytosis normally. A mutation in Myo2p, an unconventional myosin that is a possible target of calmodulin, did not inhibit endocytosis. The function of calmodulin in endocytosis seems to be specific among membrane trafficking events, because the calmodulin mutants are not defective for biogenesis of soluble vacuolar hydrolases nor invertase secretion. Calmodulin does not seem to play a major role in the post-internalization steps of the endocytic pathway in yeast.

Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast.

GPI-anchored proteins are attached to the membrane via a glycosylphosphatidylinositol-(GPI) anchor whose carbohydrate core is conserved in all eukaryotes. Apart from membrane attachment, the precise role of the GPI-anchor is not known, but it has been proposed to play a role in protein sorting. We have investigated the transport of the yeast GPI-anchored protein Gas1p. We identified two mutant strains involved in very different cellular processes that are blocked selectively in the transport of GPI-anchored proteins before arrival to the Golgi. The end8-1/lcb1-100 mutant is defective in ceramide synthesis. In vitro data suggest a requirement for ceramides after the exit from the ER. We therefore propose that ceramides might function in the fusion of a GPI-containing vesicle with the Golgi, but we cannot exclude a role in the ER. The second mutant that blocks the transport of GPI-anchored proteins to the Golgi is ret1-1, a mutant in the alpha-subunit of coatomer. In both mutants, GPI-anchor attachment is normal and in ret1-1 cells, the GPI-anchors are remodeled with ceramide to the same extent as in wild-type cells.

The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.