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Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

Purpose:Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer type, lacking effective therapies and associated with a dismal prognosis. Palbociclib is a selective CDK4/6 inhibitor, which has been shown to suppress cell proliferation in many experimental cancer models. Recently, we demonstrated that pan-mTOR inhibitors, such as MLN0128, effectively induce apoptosis, although have limited efficacy in restraining proliferation of ICC cells. Here, we tested the hypothesis that palbociclib, due to its antproliferative properties in many cancer types, might synergize with MLN0128 to impair ICC growth.Experimental Design:Human ICC cell lines and the AKT/YapS127A ICC mouse model were used to test the therapeutic efficacy of palbociclib and MLN0128, either alone or in combination.Results:Administration of palbociclib suppressed in vitro ICC cell growth by inhibiting cell-cycle progression. Concomitant administration of palbociclib and MLN0128 led to a pronounced, synergistic growth constraint of ICC cell lines. Furthermore, while treatment with palbociclib or MLN0128 alone resulted in tumor growth reduction in AKT/YapS127A mice, a remarkable tumor regression was achieved when the two drugs were administered simultaneously. Mechanistically, palbociclib was found to potentiate MLN0128 mTOR inhibition activity, whereas MLN0128 prevented the upregulation of cyclin D1 induced by palbociclib treatment.Conclusions:Our study indicates the synergistic activity of palbociclib and MLN0128 in inhibiting ICC cell proliferation. Thus, combination of CDK4/6 and mTOR inhibitors might represent a novel, promising, and effective therapeutic approach against human ICC.See related commentary by Malumbres, p. 6

Supplementary Figure 1. Activation of the CDK4/6 axis in human intrahepatic cholangiocarcinoma (ICC) specimens. Supplementary Figure 2. Levels of Rb1, CDK4, CDK6, E2F1, and Cyclin E1 mRNA in human intrahepatic cholangiocarcinoma (ICC) from The Cancer Genome Atlas (TCGA) and National Cancer Institute (NCI) datasets. Supplementary Figure 3: Effect of Palbociclib on ICC cell growth. Supplementary Figure 4: Effect of Palbociclib on the levels of putative target proteins in human ICC cell lines. Supplementary Figure 5. Inhibition of CyclinD1 (CCND1) expression enhances tumor cell growth suppressor capacity of Palbobiclib. Supplementary Figure 6. Correlation analysis of Retinoblastoma (Rb) expression and the growth inhibitory effects of Palbociclib. Supplementary Figure 7. Palbociclib inhibits proliferation of ICC cell lines in colony formation assays. Supplementary Figure 8. Retinoblastoma-dependent cell growth inhibition effect of long-term Palbociclib treatment in ICC cells. Supplementary Figure 9. Effect of combined Palbociclib/MLN0128 treatment on cell proliferation. Supplementary Figure 10. Synergistic effect of Palbociclib/MLN0128 combination in ICC cell lines. Supplementary Figure 11. Effect of combined Palbociclib/MLN0128 administration on cell cycle of ICC cell lines. Supplementary Figure 12. Effect of Palbociclib/MLN0128 on putative target proteins in intrahepatic cholangiocarcinoma (ICC) cell lines. Supplementary Figure 13. Overview of the apoptosis rate in ICC lesions from AKT/YapS127A mice subjected to the various treatment regimens. Supplementary Figure 14. Effect of Palbociclib/MLN0128 treatment on putative target proteins in AKT/YapS127A mice. Supplementary Table 1: Clinicopathological features of intrahepatic cholangiocarcinoma (ICC) patients Supplementary Table 2: Western blotting antibody information

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10–20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.