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R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis. INTRODUCTIONR-Ras is a Ras-family GTPase and its amino acid sequence is 55% identical to those of the classical types of Ras (H-, K-, N-Ras, collectively referred to hereafter as "Ras") (Lowe et al., 1987). As is the case with the other Ras-family GTPases, R-Ras is regulated primarily by two classes of protein, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). Reflecting the high sequence similarity among Ras-family GTPases, many GEFs and GAPs for R-Ras catalyze other Ras-family GTPases as well (Ohba et al., 2000). Furthermore, R-Ras is known to interact with many effectors of Ras, such as Raf-1, Ral GEFs, and the p110␣ subunit of phosphoinositide 3-kinase (PI3K) (Rey et al., 1994;Marte et al., 1997). Despite this redundancy between R-Ras and Ras, R-Ras exhibits various properties that are distinct from those of Ras. For example, R-Ras preferentially activates Ral GEFs and PI3K, but it does not activate Raf (Huff et al., 1997;Rodriguez-Viciana et al., 2004). The transforming activity of constitutively active R-Ras is substantially less potent than that of the constitutively active Ras (Cox et al., 1994), although it should be noted that a recent report has suggested the involvement of R-Ras in human gastric cancer (Nishigaki et al., 2005). Meanwhile, R-Ras is known to regulate cell adhesion, cell spreading, and phagocytosis through the activation of integrin (Zhang et al., 1996;Keely et al., 1999; Berrier et al., 2000;Self et al., 2001). R-Ras-null mice have recently been shown to exhibit excessive vascular responses, in spite of the fact that they are otherwise normal (Komatsu and Ruoslahti, 2005). This phenotype seems to reflect higher levels of expression of R-Ras in smooth muscle cells, including blood vessel cells. The results obtained with R-Ras-null mice have also demonstrated that an R-Ras defect can be almost entirely compensated for by other ...

PURPOSE To investigate efficacy and safety of trastuzumab deruxtecan (T-DXd) in human epidermal growth factor receptor 2 (HER2)–low gastric or gastroesophageal junction (GEJ) adenocarcinoma. METHODS Patients with locally advanced or metastatic HER2-low (cohort 1, immunohistochemistry 2+/in situ hybridization–negative; cohort 2, immunohistochemistry 1+) gastric/GEJ adenocarcinoma treated with at least two prior regimens, including fluoropyrimidine and platinum, but anti-HER2 therapy naive, received T-DXd 6.4 mg/kg intravenously once every 3 weeks. The primary end point was confirmed objective response rate by independent central review. RESULTS Among 21 patients enrolled in cohort 1 and 24 enrolled in cohort 2, 19 and 21 patients, respectively, had central HER2 confirmation, received T-DXd, and had measurable tumors at baseline. The confirmed objective response rate was 26.3% (95% CI, 9.1 to 51.2) from five partial responses in cohort 1 and 9.5% (95% CI, 1.2 to 30.4) from two partial responses in cohort 2. Thirteen patients (68.4%) in cohort 1 and 12 (60.0%) in cohort 2 experienced reduced tumor size. The median overall survival was 7.8 months (95% CI, 4.7 to nonevaluable) in cohort 1 and 8.5 months (95% CI, 4.3 to 10.9) in cohort 2; the median progression-free survival was 4.4 months (95% CI, 2.7 to 7.1) and 2.8 months (95% CI, 1.5 to 4.3), respectively. The most common grade ≥ 3 treatment-emergent adverse events in cohorts 1 and 2 were anemia (30.0% and 29.2%), decreased neutrophil count (25.0% and 29.2%), and decreased appetite (20.0% and 20.8%). Drug-related interstitial lung disease/pneumonitis occurred in one patient in each cohort (grade 1 or 2). No drug-related deaths occurred. CONCLUSION This study provides preliminary evidence that T-DXd has clinical activity in patients with heavily pretreated HER2-low gastric/GEJ adenocarcinoma.

Background: Perioperative FLOT is a standard of care for resectable, esophagogastric adenocarcinoma (EGA). This trial evaluates the addition of trastuzumab (tras) and pertuzumab (per) to FLOT for HER2-positive patients (pts).Methods: PETRARCA is a multicenter, randomized, investigator initiated trial planned as a phase II/III study. We report the phase II part of this trial. Pts with HER2+ resectable EGA (! cT2 or cN+) were randomized 1:1 to 4 pre-and post-operative cycles of FLOT (Docetaxel 50 mg/m 2 ; Oxaliplatin 85 mg/m 2 ; Leucovorin 200 mg/m 2 ; 5-FU 2600 mg/m 2 , q2w) (Arm A) or the same regimen with tras 8/6 mg/kg and per 840 mg q3w, followed by 9 cycles tras/per (arm B). Primary endpoint for the phase II part was the rate of pathological complete remission (pCR). Main secondary endpoints were DFS, OS and safety.Results: The trial closed prematurely and did not proceed to phase III. In total, 81 pts were randomized (A, 41; B, 40). Baseline characteristics were balanced (overall, male 79%; median age 60; cT3/T4 86%; cN+ 85%; GEJ 75%). 93% in arm A and 90% in arm B completed pre-OP treatment as planned. More pts had at least one dose modification in arm B (A, 44%; B, 70%). pCR rate was significantly improved with tras/per (A, 12%; B, 35%; p ¼ 0.02). Likewise, the rate of pathological lymph node negativity was higher with tras/per (A, 39%; B, 68%). R0-resection rate (A, 90%; B, 93%) and surgical morbidity (A: 43%; B, 44%) were comparable. Moreover, in-house mortality was equal in both arms (overall 2.5%). Median DFS was 26 months in arm A and not yet reached in arm B (HR 0.58, p ¼ 0.14). After a median follow-up of 22 months median OS was not yet reached. DFS and OS rates [with 95% CI] at 24 months were 54% [38-71%] and 77% [63-90%] in arm A and 70% [55-85%] and 84% [72-96%] in arm B, respectively. More ! grade 3 adverse events were reported with tras/per (75% vs. 85%), especially diarrhea (5% vs. 41%) and leukopenia (13% vs 23%).Conclusions: The addition of tras/per to perioperative FLOT significantly improved pCR and nodal negativity rates in pts with Her2+ resectable EGA at the price of higher rates of diarrhea and leukopenia. Subgroup analyses will be presented.

HER2-targeted anticancer therapies may be associated with cardiovascular adverse events. This study evaluated effects of the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan (T-DXd, DS-8201a) on QT/QTc interval and its pharmacokinetics. Patients with heavily pretreated, metastatic HER2-expressing breast cancer were enrolled at seven study sites in Japan. T-DXd was administered intravenously at 6.4 mg/kg on day 1 of each 21day cycle. Primary end points were baseline-adjusted QTcF interval and pharmacokinetics parameters. Key secondary end points included safety events, serum concentration of T-DXd and DXd at the time of electrocardiographic measurements, and antitumor activity parameters. Among 51 total patients, 47 (92.2%) had HER2-low breast cancer (immunohistochemistry 1+ or 2+ and in situ hybridization-negative/equivocal/missing). Pharmacokinetic parameters after a single dose of T-DXd were consistent with previous studies. After multiple doses, T-DXd showed moderate accumulation (accumulation ratio (cycle 3/cycle 1), 1.35), but DXd showed minimal accumulation (1.09). The upper bound of the 90% confidence interval for mean ΔQTcF interval was < 10 ms at all timepoints, and at mean maximum serum concentration was also < 10 ms. Based on concentration-QT analysis, ΔQTcF increased with increasing concentrations of T-DXd and DXd. No clinically meaningful QTcF prolongation was observed. T-DXd had a manageable safety profile and showed antitumor activity in HER2-low breast cancer. In this study, a T-DXd dose of 6.4 mg/kg, higher than the 5.4-mg/kg dose currently approved for breast cancer, was not associated with clinically relevant QTcF prolongation in heavily pretreated patients with HER2-expressing metastatic breast cancer. This study adds to our understanding of T-DXd for treatment of HER2-low breast cancer.