In-Silico Identification of Novel Pharmacological Synergisms: The Trabectedin Case.

The in-silico strategy of identifying novel uses for already existing drugs, known as drug repositioning, has enhanced drug discovery. Previous studies have shown a positive correlation between expression changes induced by the anticancer agent trabectedin and those caused by irinotecan, a topoisomerase I inhibitor. Leveraging the availability of transcriptional datasets, we developed a general in-silico drug-repositioning approach that we applied to investigate novel trabectedin synergisms. We set a workflow allowing the identification of genes selectively modulated by a drug and possible novel drug interactions. To show its effectiveness, we selected trabectedin as a case-study drug. We retrieved eight transcriptional cancer datasets including controls and samples treated with trabectedin or its analog lurbinectedin. We compared gene signature associated with each dataset to the 476,251 signatures from the Connectivity Map database. The most significant connections referred to mitomycin-c, topoisomerase II inhibitors, a PKC inhibitor, a Chk1 inhibitor, an antifungal agent, and an antagonist of the glutamate receptor. Genes coherently modulated by the drugs were involved in cell cycle, PPARalpha, and Rho GTPases pathways. Our in-silico approach for drug synergism identification showed that trabectedin modulates specific pathways that are shared with other drugs, suggesting possible synergisms.

Genomic instability analysis in DNA from Papanicolaou test provides proof-of-principle early diagnosis of high-grade serous ovarian cancer.

Late diagnosis and the lack of screening methods for early detection define high-grade serous ovarian cancer (HGSOC) as the gynecological malignancy with the highest mortality rate. In the work presented here, we investigated a retrospective and multicentric cohort of 250 archival Papanicolaou (Pap) test smears collected during routine gynecological screening. Samples were taken at different time points (from 1 month to 13.5 years before diagnosis) from 113 presymptomatic women who were subsequently diagnosed with HGSOC (pre-HGSOC) and from 77 healthy women. Genome instability was detected through low-pass whole-genome sequencing of DNA derived from Pap test samples in terms of copy number profile abnormality (CPA). CPA values of DNA extracted from Pap test samples from pre-HGSOC women were substantially higher than those in samples from healthy women. Consistently with the longitudinal analysis of clonal pathogenic TP53 mutations, this assay could detect HGSOC presence up to 9 years before diagnosis. This finding confirms the continual shedding of tumor cells from fimbriae toward the endocervical canal, suggesting a new path for the early diagnosis of HGSOC. We integrated the CPA score into the EVA (early ovarian cancer) test, the sensitivity of which was 75% (95% CI, 64.97 to 85.79), the specificity 96% (95% CI, 88.35 to 100.00), and the accuracy 81%. This proof-of-principle study indicates that the early diagnosis of HGSOC is feasible through the analysis of genomic alterations in DNA from endocervical smears.

A pan-cancer landscape of pathogenic somatic copy number variations.

OBJECTIVE: Copy number variations (CNVs) play crucial roles in physiological and pathological processes, including cancer. However, the functional implications of somatic CNVs in tumor progression and evolution remain unclear. This study focuses on identifying CNV alterations with high pathogenic potential that drive and sustain tumorigenesis, distinguishing them from passenger alterations that accumulate during tumor growth. Our goal is to explore the variability of CNVs across different tumor types and infer their impact on tumor cell functions. METHODS: Starting from 7352 copy number profiles across 33 different cancer types, we infer the pathogenicity of each CNV and perform both intra- and inter-tumor analyses to predict the functional impact of different genomic patterns. We evaluate the actionability of genes belonging to altered regions and we correlate the presence of pathogenic regions with genome instability patterns and patients' survival. RESULTS: Our analysis uncovered large heterogeneity among different tumors suggesting in many cases distinct genetic drivers of tumorigenesis. Recurrent genomic alterations frequently coincide with dysfunctional homologous recombination pathways and negative regulation of the immune system. In certain tumors, the number of pathogenic CNVs emerged as a prognostic biomarker, highlighting their significance in cancer progression. CONCLUSION: This study contributes to elucidate the functional impact of pathogenic CNVs in tumor progression and sheds light on their potential as prognostic markers in specific cancer types.

Important functional role of the protein osteopontin in the progression of malignant pleural mesothelioma.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components. Methods: Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model. Results: In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene (Spp1) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo. Conclusion: These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo. These findings have translational potential to improve the therapeutic response of human MPM.

Cholesterol 24-hydroxylase is a novel pharmacological target for anti-ictogenic and disease modification effects in epilepsy.

Therapies for epilepsy mainly provide symptomatic control of seizures since most of the available drugs do not target disease mechanisms. Moreover, about one-third of patients fail to achieve seizure control. To address the clinical need for disease-modifying therapies, research should focus on targets which permit interventions finely balanced between optimal efficacy and safety. One potential candidate is the brain-specific enzyme cholesterol 24-hydroxylase. This enzyme converts cholesterol to 24S-hydroxycholesterol, a metabolite which among its biological roles modulates neuronal functions relevant for hyperexcitability underlying seizures. To study the role of cholesterol 24-hydroxylase in epileptogenesis, we administered soticlestat (TAK-935/OV935), a potent and selective brain-penetrant inhibitor of the enzyme, during the early disease phase in a mouse model of acquired epilepsy using a clinically relevant dose. During soticlestat treatment, the onset of epilepsy was delayed and the number of ensuing seizures was decreased by about 3-fold compared to vehicle-treated mice, as assessed by EEG monitoring. Notably, the therapeutic effect was maintained 6.5 weeks after drug wash-out when seizure number was reduced by about 4-fold and their duration by 2-fold. Soticlestat-treated mice showed neuroprotection of hippocampal CA1 neurons and hilar mossy cells as assessed by post-mortem brain histology. High throughput RNA-sequencing of hippocampal neurons and glia in mice treated with soticlestat during epileptogenesis showed that inhibition of cholesterol 24-hydroxylase did not directly affect the epileptogenic transcriptional network, but rather modulated a non-overlapping set of genes that might oppose the pathogenic mechanisms of the disease. In human temporal lobe epileptic foci, we determined that cholesterol 24-hydroxylase expression trends higher in neurons, similarly to epileptic mice, while the enzyme is ectopically induced in astrocytes compared to control specimens. Soticlestat reduced significantly the number of spontaneous seizures in chronic epileptic mice when was administered during established epilepsy. Data show that cholesterol 24-hydroxylase contributes to spontaneous seizures and is involved in disease progression, thus it represents a novel target for chronic seizures inhibition and disease-modification therapy in epilepsy.

Targeted Mutational Analysis of Circulating Tumor DNA to Decipher Temporal Heterogeneity of High-Grade Serous Ovarian Cancer.

We have previously demonstrated that longitudinal untargeted analysis of plasma samples withdrawn from patients with high-grade serous ovarian cancer (HGS-EOC) can intercept the presence of molecular recurrence (TRm) earlier than the diagnosis of clinical recurrence (TRc). This finding opens a clinical important temporal window to acquire through plasma sample analysis a real-time picture of those emerging molecular lesions that will drive and sustain the growth of relapsed disease and ultimately will confer resistance. In this proof of principle study, the same genomic libraries obtained at the diagnosis (T0), TRm and TRc were further analyzed by targeted resequencing approach to sequence the coding region of a panel of 65 genes to provide longitudinal analysis of clonal evolution as a novel strategy to support clinical decisions for the second-line treatment. Experiments were performed on plasma and tumor tissues withdrawn on a selection of previously analyzed cohorts of cases (i.e., 33 matched primary and synchronous lesions and 43 plasma samples from 18 patients). At T0, the median concordance of mutations shared by each tumor tissue biopsy and its matched plasma sample was 2.27%. This finding confirms the limit of a single tumor biopsy to be representative of the entire disease, while plasma analysis can recapitulate most of the main molecular lesions of the disease. A comparable scenario was observed during longitudinal analysis, where, with the exception of the TP53 gene and germline mutations in BRCA1/2 genes, no other gene shared the same locus specific gene mutation across T0, TRm and TRc time points. This high level of temporal heterogeneity has important implications for planning second-line treatment. For example, in three out of 13 cases, plasma ctDNA analysis at TRm or TRc reported acquired novel variants in the TP53BP1 gene not present at T0. In particular, patient 21564, potentially eligible for PARP-inhibitor (PARPi) treatment at the time of diagnosis (BRCA1 c.5182delA mutation), would unlikely respond to these drugs in second-line therapy due to the presence of eight distinct TP53BP1 variants in plasma samples collected TRc. This study demonstrates that liquid biopsy provides a real-time molecular picture to intercept those actionable genetic vulnerabilities or drug resistance mechanisms that could be used to plan a more rational second-line treatment.

Tumor treating fields affect mesothelioma cell proliferation by exerting histotype-dependent cell cycle checkpoint activations and transcriptional modulations.

Although clinical antitumor activity of Tumor Treating Fields (TTFields) has been reported in malignant pleural mesothelioma (MPM) patients, the mechanisms behind the different selectivity displayed by the various MPM histotypes to this physical therapy has not been elucidated yet. Taking advantage of the development of well characterized human MPM cell lines derived from pleural effusion and/or lavages of patients' thoracic cavity, we investigated the biological effects of TTFields against these cells, representative of epithelioid, biphasic, and sarcomatoid histotypes. Growth inhibition and cell cycle perturbations caused by TTFields were investigated side by side with RNA-Seq analyses at different exposure times to identify pathways involved in cell response to treatment. We observed significant differences of response to TTFields among the cell lines. Cell cycle analysis revealed that the most sensitive cells (epithelioid CD473) were blocked in G2M phase followed by formation of polyploid cells. The least sensitive cells (sarcomatoid CD60) were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptosis was present in all samples, but while epithelioid cell death was already observed during the first 24 h of treatment, sarcomatoid cells needed longer times before they engaged apoptotic pathways. RNA-Seq experiments demonstrated that TTFields induced a transcriptional response already detectable at early time points (8 h). The number of differentially expressed genes was higher in CD473 than in CD60 cells, involving several pathways, such as those pertinent to cell cycle checkpoints, DNA repair, and histone modifications. Our data provide further support to the notion that the antitumor effects of TTFields are not simply related to a non-specific reaction to a physical stimulus, but are dependent on the biological background of the cells and the particular sensitivity to TTFields observed in epithelioid MPM cells is associated with a higher transcriptional activity than that observed in sarcomatoid models.

Epithelioid Pleural Mesothelioma Is Characterized by Tertiary Lymphoid Structures in Long Survivors: Results from the MATCH Study.

Pleural mesothelioma (PM) is an aggressive tumor with few therapeutic options. Although patients with epithelioid PM (ePM) survive longer than non-epithelioid PM (non-ePM), heterogeneity of tumor response in ePM is observed. The role of the tumor immune microenvironment (TIME) in the development and progression of PM is currently considered a promising biomarker. A few studies have used high-throughput technologies correlated with TIME evaluation and morphologic and clinical data. This study aimed to identify different morphological, immunohistochemical, and transcriptional profiles that could potentially predict the outcome. A retrospective multicenter cohort of 129 chemonaive PM patients was recruited. Tissue slides were reviewed by dedicated pathologists for histotype classification and immunophenotype of tumor-infiltrating lymphocytes (TILs) and lymphoid aggregates or tertiary lymphoid structures (TLS). ePM (n = 99) survivors were further classified into long (>36 months) or short (<12 months) survivors. RNAseq was performed on a subset of 69 samples. Distinct transcriptional profiling in long and short ePM survivors was found. An inflammatory background with a higher number of B lymphocytes and a prevalence of TLS formations were detected in long compared to short ePM survivors. These results suggest that B cell infiltration could be important in modulating disease aggressiveness, opening a pathway for novel immunotherapeutic approaches.

Regional and temporal heterogeneity of epithelial ovarian cancer tumor biopsies: implications for therapeutic strategies.

Stage III/IV epithelial ovarian cancer (EOC) is a systemic disease. The clonal relationship among different tumor lesions at diagnosis (spatial heterogeneity) and how tumor clonal architecture evolves over time (temporal heterogeneity) have not yet been defined. Such knowledge would help to develop new target-based strategies, as biomarkers which can adjudge the success of therapeutic intervention should be independent of spatial and temporal heterogeneity. The work described in this paper addresses spatial and temporal heterogeneity in a cohort of 71 tumor biopsies using targeted NGS technology. These samples were taken from twelve high grade serous (HGS) and seven non HSG-EOC, both at the time of primary surgery when the tumor was naïve to chemotherapy and after chemotherapy. Matched tumor lesions growing in the ovary or at other anatomical sites show very different mutational landscapes with branched tumor evolution. Mutations in ATM, ATR,TGFB3,VCAM1 and COL3A1 genes were shared across all lesions. BRCA1 and BRCA2 genes were frequently mutated in synchronous lesions of non HGS-EOC. Relapsed disease seems to originate from resistant clones originally present at the time of primary surgery rather than from resistance acquired de novo during platinum based therapy. Overall the work suggests that EOC continues to evolve. More detailed mapping of genetic lesions is necessary to improve therapeutic strategies.

Mechanisms of responsiveness to and resistance against trabectedin in murine models of human myxoid liposarcoma.

Myxoid liposarcoma (MLPS) is a rare soft-tissue sarcoma characterised by the expression of FUS-DDIT3 chimera. Trabectedin has shown significant clinical anti-tumour activity against MLPS. To characterise the molecular mechanism of trabectedin sensitivity and of resistance against it, we integrated genomic and transcriptomic data from treated mice bearing ML017 or ML017/ET, two patient-derived MLPS xenograft models, sensitive to and resistant against trabectedin, respectively. Longitudinal RNA-Seq analysis of ML017 showed that trabectedin acts mainly as a transcriptional regulator: 15 days after the third dose trabectedin modulates the transcription of 4883 genes involved in processes that sustain adipocyte differentiation. No such differences were observed in ML017/ET. Genomic analysis showed that prolonged treatment causes losses in 4p15.2, 4p16.3 and 17q21.3 cytobands leading to acquired-resistance against the drug. The results dissect the complex mechanism of action of trabectedin and provide the basis for novel combinatorial approaches for the treatment of MLPS that could overcome drug-resistance.

Tumor Immune Microenvironment and Genetic Alterations in Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and fatal disease of the pleural lining. Up to 80% of the MPM cases are linked to asbestos exposure. Even though its use has been banned in the industrialized countries, the cases continue to increase. MPM is a lethal cancer, with very little survival improvements in the last years, mirroring very limited therapeutic advances. Platinum-based chemotherapy in combination with pemetrexed and surgery are the standard of care, but prognosis is still unacceptably poor with median overall survival of approximately 12 months. The genomic landscape of MPM has been widely characterized showing a low mutational burden and the impairment of tumor suppressor genes. Among them, BAP1 and BLM are present as a germline inactivation in a small subset of patients and increases predisposition to tumorigenesis. Other studies have demonstrated a high frequency of mutations in DNA repair genes. Many therapy approaches targeting these alterations have emerged and are under evaluation in the clinic. High-throughput technologies have allowed the detection of more complex molecular events, like chromotripsis and revealed different transcriptional programs for each histological subtype. Transcriptional analysis has also paved the way to the study of tumor-infiltrating cells, thus shedding lights on the crosstalk between tumor cells and the microenvironment. The tumor microenvironment of MPM is indeed crucial for the pathogenesis and outcome of this disease; it is characterized by an inflammatory response to asbestos exposure, involving a variety of chemokines and suppressive immune cells such as M2-like macrophages and regulatory T cells. Another important feature of MPM is the dysregulation of microRNA expression, being frequently linked to cancer development and drug resistance. This review will give a detailed overview of all the above mentioned features of MPM in order to improve the understanding of this disease and the development of new therapeutic strategies.

DNA Damage Response and Immune Defense.

DNA damage is the cause of numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. The DNA damage response (DDR), in turn, coordinates DNA damage checkpoint activation and promotes the removal of DNA lesions. In recent years, several studies have shown how the DDR and the immune system are tightly connected, revealing an important crosstalk between the two of them. This interesting interplay has opened up new perspectives in clinical studies for immunological diseases as well as for cancer treatment. In this review, we provide an overview, from cellular to molecular pathways, on how DDR and the immune system communicate and share the crucial commitment of maintaining the genomic fitness.

Is DNA repair a potential target for effective therapies against malignant mesothelioma?

Malignant pleural mesothelioma (MPM) is a rare malignancy mainly caused by asbestos exposure. Germinal and acquired mutations in genes of DNA repair pathways, in particular of homologous recombination repair, are frequent in MPM. Here we overview the available experimental data suggesting that an impaired DNA repair system affects MPM pathogenesis by leaving lesions through the genome unresolved. DNA repair defects represent a vulnerability of MPM, and it seems plausible to propose that leveraging these deficiencies could have therapeutic potential for patients with MPM, for whom there is an urgent need of more effective therapies.

Correction: Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combination of PPARγ Agonist Pioglitazone and Trabectedin Induce Adipocyte Differentiation to Overcome Trabectedin Resistance in Myxoid Liposarcomas.

PURPOSE: This study was aimed at investigating whether the PPARγ agonist pioglitazone-given in combination with trabectedin-is able to reactivate adipocytic differentiation in myxoid liposarcoma (MLS) patient-derived xenografts, overcoming resistance to trabectedin. EXPERIMENTAL DESIGN: The antitumor and biological effects of trabectedin, pioglitazone, and the combination of the two drugs were investigated in nude mice bearing well-characterized MLS xenografts representative of innate or acquired resistance against trabectedin. Pioglitazone and trabectedin were given by daily oral and weekly i.v. administrations, respectively. Molecular studies were performed by using microarrays approach, real-time PCR, and Western blotting. RESULTS: We found that the resistance of MLS against trabectedin is associated with the lack of activation of adipogenesis. The PPARγ agonist pioglitazone reactivated adipogenesis, assessed by histologic and gene pathway analyses. Pioglitazone was well tolerated and did not increase the toxicity of trabectedin. The ability of pioglitazone to reactivate adipocytic differentiation was observed by morphologic examination, and it is consistent with the increased expression of genes such as ADIPOQ implicated in the adipogenesis process. The determination of adiponectin by Western blotting constitutes a good and reliable biomarker related to MLS adipocytic differentiation. CONCLUSIONS: The finding that the combination of pioglitazone and trabectedin induces terminal adipocytic differentiation of some MLSs with the complete pathologic response and cure of tumor-bearing mice provides a strong rationale to test the combination of trabectedin and pioglitazone in patients with MLS.

Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

BACKGROUND: Myxoid liposarcoma is a histological subtype of liposarcoma particularly sensitive to trabectedin. In clinical use this drug does not cause cumulative toxicity, allowing prolonged treatment, generally until disease progression. No other effective therapies are available for trabectedin-resistant patients. METHODS: Through repeated in vivo treatment in athymic nude mice, we have obtained a patient-derived xenograft with acquired resistance to trabectedin. RESULTS: At basal level, the morphology of the resistant and sensitive models did not differ, in keeping with the finding that the transcriptional profiles of the resistant and sensitive tumours were very similar. After trabectedin treatment adipogenesis was induced in the parental xenograft but not in the resistant one, as assessed by pathological and molecular analysis. A defective transcription-coupled-nucleotide excision repair in the resistant tumour due to mutation of the UVSSA gene may be implicated in the mechanism of resistance. CONCLUSIONS: This is the first in vivo model of myxoid liposarcoma with acquired resistance to trabectedin. Although further studies are necessary to characterise the resistance mechanisms, this is a useful tool for studying new therapeutic strategies to overcome trabectedin resistance in patients.

Multisite analysis of high-grade serous epithelial ovarian cancers identifies genomic regions of focal and recurrent copy number alteration in 3q26.2 and 8q24.3.

High-grade serous epithelial ovarian cancer (HGS-EOC) is a systemic disease, with marked intra and interpatient tumor heterogeneity. The issue of spatial and temporal heterogeneity has long been overlooked, hampering the possibility to identify those genomic alterations that persist, before and after therapy, in the genome of all tumor cells across the different anatomical districts. This knowledge is the first step to clarify those molecular determinants that characterize the tumor biology of HGS-EOC and their route toward malignancy. In our study, -omics data were generated from 79 snap frozen matched tumor biopsies, withdrawn before and after chemotherapy from 24 HGS-EOC patients, gathered together from independent cohorts. The landscape of somatic copy number alterations depicts a more homogenous and stable genomic portrait than the single nucleotide variant profile. Genomic identification of significant targets in cancer analysis identified two focal and minimal common regions (FMCRs) of amplification in the cytoband 3q26.2 (region α, 193 kb long) and 8q24.3 (region ß, 495 kb long). Analysis in two external databases confirmed regions α and ß are features of HGS-EOC. The MECOM gene is located in region α, and 15 genes are in region ß. No functional data are yet available for the genes in the ß region. In conclusion, we have identified for the first time two FMCRs of amplification in HGS-EOC, opening up a potential biological role in its etiopathogenesis.

Targeted gene sequencing of Lynch syndrome-related and sporadic endometrial carcinomas.

About one-third of endometrial carcinomas (ECs), mainly of endometrioid histology, harbor the mismatch repair (MMR) defects and microsatellite instability (MSI). Among these, ECs arising in women with Lynch syndrome (LS) account for a large proportion. To date, no somatic genetic analyses have been published comparing LS-ECs with sporadic ECs. In this work, we examined the mutational profiles of a well-characterized series of sporadic and LS-related ECs, performing exonic targeted sequencing of 16 genes mainly involved in MSI ECs. Next-generation sequencing analysis was performed in 35 ECs on the MiSeq platform (Illumina, San Diego, CA), and the mutational profile was analyzed integrating molecular and immunohistochemical data. PTEN, ARID1A, and ARID2 were the most frequently mutated genes regardless of MSI status or family history. MSI ECs showed a higher mutational load than MMR-proficient cases, exhibiting an MMR-deficient mutational signature. Among MSI tumors, LS-related and sporadic ECs exhibited similar mutational profiles, with MSH2 as the most commonly mutated gene. KRAS mutations seemed to be more common in sporadic MSI ECs than in LS-related ECs even if further studies are needed to confirm this finding. MMR-deficient ECs carried a higher mutational load and an excess of C>T transitions compared with MMR-proficient ECs, suggesting that the use of a small gene panel may be adequate to highlight significant differences between these 2 groups. An integrated analysis of genetic and epigenetic features of LS-related and sporadic ECs provides useful insights into disease biology and diagnostic classification of these tumors.