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BackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.MethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines,in vitroandvivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.ResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.ConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

Summary Background Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. Methods We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675–1·3 mg/m2 every 3 weeks; olaparib 100–300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. Findings Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2–6; range 1–17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5–23). Considering all dose levels, the most common grade 3–4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6–27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. Interpretation Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. Funding Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies., Some colorectal cancer patients respond well to treatment with anti-EGFR antibodies, however response is almost invariably followed by acquired resistance. Here, the authors show that patients with shorter responses acquire RAS mutations, while those relapsing later preferentially develop mutations in the extracellular domain of EGFR.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Selective tumor targeting is expected to enhance drug delivery and to decrease toxicity, resulting in an improved therapeutic index. We have recently identified the HSYWLRS peptide sequence as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current therapies. Here we validated the specific binding of HSYWLRS to neuroblastoma cell suspensions obtained either from cell lines, animal models, or Schwannian-stroma poor, stage IV neuroblastoma patients. Binding of the biotinylated peptide and of HSYWLRS-functionalized fluorescent quantum dots or liposomal nanoparticles was dose-dependent and inhibited by an excess of free peptide. In animal models obtained by the orthotopic implant of either MYCN-amplified or MYCN single copy human neuroblastoma cell lines, treatment with HSYWLRS-targeted, doxorubicin-loaded Stealth Liposomes increased tumor vascular permeability and perfusion, enhancing tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy, as evaluated by bioluminescence imaging and micro-PET, leading to (i) delay of tumor growth paralleled by decreased tumor glucose consumption, and (ii) abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span. Our findings are functional to the design of targeted nanocarriers with potentiated therapeutic efficacy towards the clinical translation.

Neuronal pentraxins (NPTX) and their corresponding receptors (NPTXR) have been studied as synapse-associated proteins in the nervous system, but their role in cancer is largely unknown. By applying a multidisciplinary, high-throughput proteomic approach, we have recently identified a peptide ligand motif for targeted drug delivery to neuroblastoma. Here, we report the sequence similarity between this peptide and a conserved portion of the pentraxin domain that is involved in the homo- and hetero-oligomerization of NPTX2 and NPTXR. We show that, in comparison with normal tissues, NPTX2 and NPTXR are overexpressed in vivo in mouse models, as well as in human Schwannian stroma-poor, stage IV neuroblastoma. Both proteins are concentrated in the vicinity of tumor blood vessels, with NPTXR also present on neuroblastic tumor cells. In vivo targeting of NPTX2 and NPTXR with the selected peptide or with specific antibodies reduces tumor burden in orthotopic mouse models of human neuroblastoma. In vitro interference with this ligand/receptor system inhibits the organization of neuroblastoma cells in tumor-like masses in close contact with vascular cells, as well as their adhesion to normal microenvironment-derived cells, suggesting a role in the cross-talk between tumor and normal cells in the early steps of neuroblastoma development. Finally, we show that NPTX2 is a marker of poor prognosis for neuroblastoma patients. Cancer Res; 75(20); 4265–71. ©2015 AACR.

Introduction. The identification of peptide ligands specific for solid tumors is expected to provide targeting moieties to improve delivery and to decrease toxicity of chemotherapy. We have recently identified the peptide HSYWLRS as a specific ligand for neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. Experimental procedures. The capability of peptide HSYWLRS to recognize NB cells was evaluated by coupling Qdot fluorescent nanoparticles with HSYWLRS or its scrambled version (SCR). NB cell association and internalization of HSYWLRS-targeted liposomes were tested by FACS and confocal microscopy studies. We further evaluated a potential role of this peptide in perturbing tumor-stroma interactions and tumor growth. NB cell lines stably transfected with eGFP were mixed with endothelial cells in the presence of either SCR or HSYWLRS peptides. Cell morphology and reciprocal cellular interactions were evaluated by optical and fluorescence microscopy. We finally performed therapeutic experiments with mice orthotopically injected with luc-trasfected NB cells and treated with HSYWLRS-targeted, doxorubicin-loaded liposomes (HSYWLRS-SL[DXR]). Anti-tumor efficacy was evaluated by BLI imaging. In vivo imaging was also performed by injecting mice with a bolus of fluorodeoxyglucose during a list mode acquisition lasting one hour using a dedicated micro-PET system. After framing rate optimization, tumor glucose consumption was measured using Patlak graphical approach and normalizing the slope of regression line for serum glucose level. Results. FACS analysis showed that HSYWLRS-Qdot and SCR-Qdot bound NB cells in a dose-dependent manner, however with different intensity, being HSYWLRS-Qdot the more potent. The binding of HSYWLRS-Qdot was efficiently inhibited by an excess of HSYWLRS, but not by control SCR peptide. In contrast, the binding of SCR-Qdot was not inhibited neither by an excess of SCR nor by HSYWLRS peptide, suggesting that the binding of SCR-Qdot is not specific. Again, the specific peptide-driven binding of HSYWLRS-SL to NB cells was inhibited by an excess of HSYWLRS peptide. In all cases, HSYWLRS specifically altered in vitro the interactions of NB cells with endothelium. Similarly, this peptide statistically decreased tumor take and growth when co-injected with tumor cells in the adrenal gland of nude mice. Preliminary in vivo results obtained by BLI and micro-PET devises indicated that HSYWLRS-SL[DXR] decrease tumor growth through a reduction of tumor glucose consumption, leading to an enhanced life span in treated mice. Conclusion. Our findings demonstrate that HSYWLRS peptide recognizes NB cells and is functional in the design of nanocarriers with therapeutic efficacy paving the way to its clinical development. Citation Format: Alice Bartolini, Monica Loi, Daniela Di Paolo, Laura Emionite, Angelina Sacchi, Flavio Curnis, Gianluca Bottoni, Michela Massollo, Cristina Gagliani, Silvia Bruno, Alessandro Gori, Renato Longhi, Michele Cilli, Carlo Tacchetti, Angelo Corti, Gianmario Sambuceti, Mirco Ponzoni, Serena Marchiò, Fabio Pastorino. Characterization and anti-tumor functionality of a neuroblastoma-specific peptide, either free or conjugated to nanocarriers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2014-1778

Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.

Disseminated neuroblastoma (NB) is refractory to most current therapeutic regimens. The therapeutic index of anticancer drugs is increased by liposome encapsulation and further improvements is obtained by coupling tumor-targeting ligands to the surface of the lipidic envelop. Phage display technology is a powerful tool in discovering novel ligands specific to receptors on the surface of tumor epithelial and endothelial cells. Therapeutic targeting to tumor blood vessels combines blood vessel destruction with the expected anti-tumor activities of the drug, resulting in increased efficacy and reduced toxicity. To find NB-specific targeting moieties, we established a protocol for the isolation of heterogeneous cell populations by tissue fractionation of primary tumors and metastases from two models of human NB (with tumor cells injected either intravenously, to mimic minimal residual disease, or orthotopically in the adrenal gland of mice, to reflect the growth of advanced NB in children with large adrenal gland tumors and small metastatic lesions) and from stage IV, stroma poor, NB-derived specimens immediately after surgical removal. Cells extracted from corresponding healthy organs from mice and patients were used both in a negative pre-selection step and as a negative control for specific phage enrichment. The NB cell suspensions were subjected to multi-step screenings with the phage-displayed peptide library CX7C (where C = cysteine and X = any aminoacid). We globally isolated 135 NB-binding peptides. Of these, 31 were selected for binding to the primary tumor mass, 16 to the metastatic mass, 63 to tumor endothelial cells, and 25 to endothelial cells of metastases. Several proteins presenting sequence homology with the discovered peptides have been identified by BLAST analysis and were evaluated for their expression in NB tumors, derived from both mouse xenogratfs and patient specimens. Specifically, 5 novel phage display derived-peptides showed specific binding on NB specimens and homing to tumor cells and tumor vasculature, 10 minutes and 24 hours after injection through the tail vein of NB-bearing mice. We are testing the new molecular, tumor- and vasculature-specific peptides for generating novel tumor-specific liposomal therapies against NB. The availability of novel ligands binding to additional tumor-associated antigens and to targets on both endothelial and perivascular tumor cells will allow to design more sophisticated liposomal targeted anticancer strategies that exhibit high levels of selective toxicity for the cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2011-3625

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2–/– interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.

A cross-kingdom tale of drug resistance Physicians who treat bacterial infections and those who treat cancer often face a common challenge: the development of drug resistance. It is well known that when bacteria are exposed to antibiotics, they temporarily increase their mutation rate, thus increasing the chance that a descendant antibiotic-resistant cell will arise. Russo et al. now provide evidence that cancer cells exploit a similar mechanism to ensure their survival after drug exposure (see the Perspective by Gerlinger). They found that human colorectal cancer cells treated with certain targeted therapies display a transient up-regulation of errorprone DNA polymerases and a reduction in their ability to repair DNA damage. Thus, like bacteria, cancer cells can adapt to therapeutic pressure by enhancing their mutability. Science , this issue p. 1473 ; see also p. 1458

Background Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. Patients and methods We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying aCAD-ALK translocation during treatment with an ALK inhibitor. Results Using a custom next generation sequencing panel we identified the genomicCAD-ALK rearrangement and aTP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of theCAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient’s clinical course. Detection of theCAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identifiedALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. Conclusion We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB.By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.