Pier-Luigi Lollini

ES2B-C001 represents a new generation of vaccines based on virus-like particles (VLP) that display the full extracellular domain of HER-2 and has now entered clinical development. ES2B-C001 elicited strong anti-HER-2 antibody responses that cured human HER-2 transgenic tumors and metastases in mice. Early vaccine trials may include patients who are still receiving anti-HER-2 monoclonal antibody (MAb) therapy, a clinical scenario that has not typically been addressed in preclinical studies. To evaluate whether the concurrent administration of 4D5 anti-HER-2 MAb affects the immunogenicity or the therapeutic efficacy of ES2B-C001, we administered concurrent treatments to tumor-free or to human HER-2 transgenic mammary carcinoma-bearing mice. In tumor-free mice, 4D5 treatment did not hamper either ES2B-C001 activity, which induced a strong anti-HER-2 IgG production, or T cell responses. In tumor-bearing mice, the therapeutic efficacy of ES2B-C001 was not compromised by 4D5, resulting in 13/20 long-term tumor-free mice with ES2B-C001 alone, versus 15/20 with the combined treatment. ES2B-C001 elicited robust HER-2-specific antibody responses, reaching concentrations in the milligram/mL range and persisting for >6 months post-treatments, regardless of prior 4D5 administration. These findings indicate that co-administration of an anti-HER-2 MAb does not impair the efficacy of ES2B-C001, supporting the feasibility of vaccinating patients undergoing trastuzumab therapy.

GSEAs and qRT-PCR validation reveal a unique transcriptional profile of CDS characterized by enriched expression of a HGMAs/IGF2BPs/IGF2 axis. A, GSEA reveals a significant enrichment of genes upregulated in patients with CDS versus Ewing sarcoma. CDS displayed a signature enriched for genes involved in embryonic stem cell development (Benporath_ES_1), KRAS signaling (Sweet_KRAS_TARGETS_DN), WT1 target genes (Kim_WT1_TARGETS_UP), and chromatin modification (Mikkelsen_NPC_HCP_WITH_H3K27ME3). The enrichment score curve was obtained using GSEA software. In the enrichment plot, the x-axis shows the rank order of genes from the most upregulated to the most downregulated between CDS and Ewing sarcoma samples. The vertical black line indicates the position of the enriched genes (Hit) comprising the gene set. The graph on the bottom shows the ranked list metric (signal-to-noise ratio) for each gene as a function of the rank in the ordered dataset. The heatmaps show the genes that contribute most to the enriched pathway or biological process resulting from the leading edge analysis. B, Validation of the RNA-seq results was performed by qRT-PCR and the results are shown in scatter plots. Each dot represents a tumor sample. Differential expression of genes between CDS and Ewing sarcoma was established by Student t test and correction for multiple comparisons using the Holm-Sidak method: **, P < 0.01; ****, P < 0.0001; ns, nonsignificant. The mean ± SE of relative mRNA expression (2–ΔΔCt) is reported as log2. GAPDH was used as a reference gene. C, Left, CIC-DUX4 silencing in PDX-CDS#4 cells by RT-PCR. Agarose gel electrophoresis image of a 233-base fusion transcript is shown. Right, relative mRNA expression (2−ΔΔCt) of HMGA2, IGF2BP2, IGF2BP3, and IGF2 after cell exposure to siCIC or scrambled (SCR) control siRNAs (40 nmol, 72 hours) by qRT-PCR. The mean ± SE of relative mRNA expression (2−ΔΔCt) is reported as log2. GAPDH was used as a reference gene. *, P < 0.05; **, P < 0.01; one-way ANOVA with respect to the control.

Efficacy of combined treatment with trabectedin and NVP-BEZ235 against CDS tumor growth and metastasis and schematic representation of the CDS-specific HMGA2/IGF2BPs/IGF2/IGF1R/AKT-mTOR pathway. A, Inhibition of PDX-CDS #4-C tumor growth after treatments with trabectedin and/or NVP-BEZ235. Significant reduction in tumor growth was observed after single treatments (*, P < 0.05, Student t test) but the inhibition increased after combination of the two drugs (significance was at least ***, P < 0.001, Student t test starting from day 23) or to single treatments (*, P < 0.05). Points, tumor diameter means (calculated as geometric mean in mm); bars, SE. Drugs were administered as indicated in the Supplementary Material and Methods. B, Tumor-free survival curves of mice treated with trabectedin and/or NVP-BEZ235. Kaplan–Meier curves and Mantel–Cox tests (compared with untreated mice) are shown: *, P < 0.05; **, P < 0.01. C, Inhibition of PDX-CDS#4-C experimental metastases to the lungs, liver, and other sites (mainly interscapular adipose tissue and lymph nodes) after treatments with trabectedin and/or NVP-BEZ235, starting from 7 days after intravenous cell injection. All of the treated groups developed a significantly lower number of metastases than untreated mice by the nonparametric Mann–Whitney test (P < 0.01; n = 7 for untreated control group and trabectedin; n = 5 for NVP-BEZ235; n = 6 for the combination Trabectedin + NVP-BEZ235). In the lung, the combined treatment led to a significant lower number of metastases compared with trabectedin (*, P < 0.05), or with NVP-BEZ235 (**, P < 0.01) as single agents. Bars, metastasis number means and SE. Incidence of mice with metastasis to the different sites (mice with metastasis/total number of mice per group) is reported as percentage. D, In the nucleus, high expression of HMGA2 favors the transcription of IGF2BP2 and IGF2BP3. In the cytoplasm, IGF2BP2 and IGF2BP3 directly bind to and stabilize IGF2 and IGF1R mRNAs, which subsequently activate IGF1R signaling. The repression of MAPK signaling by the CIC-DUX4 fusion protein renders CDSs mainly dependent on the AKT/mTOR pathway. Trabectedin can impair HMGA2 activity by preventing its binding to promoters, thus inhibiting the transcription of its targets IGF2BP2 and IGF2BP3 and decreasing IGF2/IGF1R signaling. NVP-BEZ235 is a dual PI3K/mTOR inhibitor used in phase II clinical trials. The combination of trabectedin with NVP-BEZ235 synergistically inhibits tumor growth.

CDS PDX models recapitulated the phenotypic characteristics and transcriptional profiles of the primary tumor. A, Histologic and IHC features of CDS patient samples and corresponding PDXs at different in vivo passages (P1, first passage; P3, third passage). Sections were stained with hematoxylin-eosin (H&E) or with antibodies against the antigens ETV4 and CD99. Bar, 100 μm. B and C, Spearman correlation analysis among the gene expression profiles of CDS tumors, their corresponding PDX models (P3), and the PDX-derived cell lines are shown based on the signatures of 3,179 (B) and 71 (C) DEGs. Correlation coefficients are shown.

Drug sensitivity in CDS and Ewing sarcoma PDX-derived cell lines.

The HMGA2/IGF2BP2–3/IGF2 network is upregulated and sustains AKT pathway activity in CDS PDX-derived cell lines versus Ewing sarcoma PDX-derived cell lines. A, qRT-PCR (top) and Western blot (bottom) analysis of HMGA2, IGF2BP2, IGF2BP3, and IGF2 mRNA and protein in CDS PDX-derived cell lines versus Ewing sarcoma PDX-derived cell lines. Three independent biological replicates were performed for qRT-PCR and Western blotting. One representative immunoblot is shown. For qRT-PCR, the data represent the mean ± SE. **, P < 0.01; ****, P < 0.0001, Student t test comparing the CDS and Ewing sarcoma groups. GAPDH was used as a reference gene. B, Immunoblots showing the protein expression of IGF1R and the downstream mediators AKT and MAPK under basal conditions. Two independent experiments were performed. One representative immunoblot is shown. GAPDH was employed for normalization purposes. C, Immunoblots showing the expression of AKT and MAPK after serum starvation with or without subsequent stimulation with IGF2. Two independent experiments were performed. One representative immunoblot is shown. GAPDH was employed for normalization purposes.

Trabectedin impairs the HMGA2/IGF2BP2–3/IGF network and inhibits AKT pathway activity in combination with NVP-BEZ235 treatment. A and B, qRT-PCR analysis of HMGA2/IGF2BP2–3 mRNA expression (A) and Western blot analysis of HMGA2/IGF2BP2–3/IGF2/IGF1R protein expression (B) in PDX-CDS#4-C and PDX-CDS#3-C cells treated with 3 nmol/L trabectedin for the indicated time. At least three independent biological replicates were performed. One representative immunoblot is shown. For qRT-PCR, the data represent the mean ± SE. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, one-way ANOVA. Each dot represents an independent experiment. GAPDH was used as a reference gene. C, Western blot analysis showing activation of IGF1R and the downstream mediator AKT in PDX-CDS#4-C and PDX-CDS#3-C cells treated with 3 nmol/L trabectedin for 24 hours with or without a subsequent 24 hours treatment with different doses of NVP-BEZ235. Three independent biological replicates were performed. One representative immunoblot is shown. GAPDH was employed for normalization purposes.

Introduction: Resistance to anti-HER2 therapies and incomplete response remain considerable challenges in managing HER2-positive tumors. Furthermore, some healthy organs, like the heart, express low levels of HER2, entailing a risk of toxic side effects, such as cardiotoxicity. Thus, the development of new anti-HER2 agents, to improve therapy outcomes, is still ongoing and requires preclinical evaluations of their side effects. Nonhuman primates are crucial in toxicology due to their high genetic similarity to humans. In line with the 3Rs principles, their use should be minimized by prioritizing the development of more predictive alternative methods. However, most in vitro assays (e.g., ELISA) only show the binding of anti-HER2 agents to cynomolgus HER2, without revealing the functional activities, such as growth inhibition. Methods: We obtained cell lines expressing cynomolgus ( Macaca fascicularis ) HER2 (cyHER2), to evaluate the functional inhibitory activity of anti-human HER2 therapeutic agents on endogenous cynomolgus HER2 in three-dimensional (3D) growth condition in vitro . Results: Our model, based on NIH 3T3 cells, became sensitive to the monoclonal antibody trastuzumab and to the selective HER2 tyrosine kinase inhibitor tucatinib. Discussion: The results suggest that this model could be a promising tool for preclinical functional cross-reactivity tests of anti-HER2 therapies before in vivo studies.

Chondrosarcoma (CS), the second most common malignant bone tumor after osteosarcoma, accounts for 20–30% of all malignant bone tumors. It mainly affects adults, middle-aged, and elderly people. The CS family includes various entities displaying peculiar biological, genetic, and epigenetic characteristics and clinical behaviors. Conventional CS is the most common subtype. High-grade, dedifferentiated, and mesenchymal CS, as well as unresectable and metastatic CS, exhibit poor prognoses due to their intrinsic resistance to radiotherapy and chemotherapy, underscoring the urgent need for novel therapeutic strategies. CS research is dealing with several challenges. Experimental studies can rely on animal and patient-derived models, but the paucity of representative near-patient preclinical models has hampered predictive drug screening research. This review describes the main clinical and molecular features of CS subtypes, discussing recent data on the genetic alterations and molecular mechanisms involved in CS pathogenesis and progression. The review provides an overview of the current in vitro and in vivo CS models, discusses their advantages and limitations, and highlights the recent efforts in the development of new targeted therapies against CS dependencies, including IDH1/2 mutations, NAD⁺ dependency, and SIRT1-HIF-2α axis, or exploring DR5 targeting, antiangiogenic therapies, epigenetic drugs, and immunological approaches. All such strategies, in combination with advanced preclinical modeling and personalized multi-omic profiling, hold promise for improving the survival of patients with advanced CS.