Telomere dysfunction alters intestinal stem cell dynamics to promote cancer.

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3ß inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.

Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR targeted treatments. Therefore, we designed a protein and genomic based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor tissue microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS) (30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared to ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n=12) vs Non-HomDel (n=37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNASEH2B HomDels in U-LMS was 76%, 93% and 71% respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

Complete loss of TP53 and RB1 is associated with complex genome and low immune infiltrate in pleomorphic rhabdomyosarcoma.

Rhabdomyosarcoma accounts for roughly 1% of adult sarcomas, with pleomorphic rhabdomyosarcoma (PRMS) as the most common subtype. Survival outcomes remain poor for patients with PRMS, and little is known about the molecular drivers of this disease. To better characterize PRMS, we performed a broad array of genomic and immunostaining analyses on 25 patient samples. In terms of gene expression and methylation, PRMS clustered more closely with other complex karyotype sarcomas than with pediatric alveolar and embryonal rhabdomyosarcoma. Immune infiltrate levels in PRMS were among the highest observed in multiple sarcoma types and contrasted with low levels in other rhabdomyosarcoma subtypes. Lower immune infiltrate was associated with complete loss of both TP53 and RB1. This comprehensive characterization of the genetic, epigenetic, and immune landscape of PRMS provides a roadmap for improved prognostications and therapeutic exploration.

The androgen receptor is a therapeutic target in desmoplastic small round cell sarcoma.

Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have direct clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer.

Multi-site desmoplastic small round cell tumors are genetically related and immune-cold.

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive soft tissue sarcoma that is characterized by the EWSR1-WT1 fusion protein. Patients present with hundreds of tumor implants in their abdominal cavity at various sites. To determine the genetic relatedness among these sites, exome and RNA sequencing were performed on 22 DSRCT specimens from 14 patients, four of whom had specimens from various tissue sites. Multi-site tumors from individual DSRCT patients had a shared origin and were highly related. Other than the EWSR1-WT1 fusion, very few secondary cancer gene mutations were shared among the sites. Among these, ARID1A, was recurrently mutated, which corroborates findings by others in DSRCT patients. Knocking out ARID1A in JN-DSRCT cells using CRISPR/CAS9 resulted in significantly lower cell proliferation and increased drug sensitivity. The transcriptome data were integrated using network analysis and drug target database information to identify potential therapeutic opportunities in EWSR1-WT1-associated pathways, such as PI3K and mTOR pathways. Treatment of JN-DSRCT cells with the PI3K inhibitor alpelisib and mTOR inhibitor temsirolimus reduced cell proliferation. In addition, the low mutation burden was associated with an immune-cold state in DSRCT. Together, these data reveal multiple genomic and immune features of DSRCT and suggest therapeutic opportunities in patients.

Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Using single-cell RNA sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAF) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs, which leads to decreased survival. Tumor-promoting FAP+ CAFs (TP-CAF) and tumor-restraining αSMA+ CAFs (TR-CAF) differentially regulate cancer-associated pathways and accumulation of regulatory T cells. Improved efficacy of gemcitabine is observed when IL6 is deleted from αSMA+ CAFs but not from FAP+ CAFs using dual-recombinase genetic PDAC models. Improved gemcitabine efficacy due to lack of IL6 synergizes with anti-PD-1 immunotherapy to significantly improve survival of PDAC mice. Our study identifies functional heterogeneity of CAFs in PDAC progression and their different roles in therapy response. SIGNIFICANCE: PDAC is associated with accumulation of dense stroma consisting of fibroblasts and extracellular matrix that regulate tumor progression. Here, we identify two distinct populations of fibroblasts with opposing roles in the progression and immune landscape of PDAC. Our findings demonstrate that fibroblasts are functionally diverse with therapeutic implications. This article is highlighted in the In This Issue feature, p. 1397.

Prediction of biomarkers and therapeutic combinations for anti-PD-1 immunotherapy using the global gene network association.

Owing to a lack of response to the anti-PD1 therapy for most cancer patients, we develop a network approach to infer genes, pathways, and potential therapeutic combinations that are associated with tumor response to anti-PD1. Here, our prediction identifies genes and pathways known to be associated with anti-PD1, and is further validated by 6 CRISPR gene sets associated with tumor resistance to cytotoxic T cells and targets of the 36 compounds that have been tested in clinical trials for combination treatments with anti-PD1. Integration of our top prediction and TCGA data identifies hundreds of genes whose expression and genetic alterations that could affect response to anti-PD1 in each TCGA cancer type, and the comparison of these genes across cancer types reveals that the tumor immunoregulation associated with response to anti-PD1 would be tissue-specific. In addition, the integration identifies the gene signature to calculate the MHC I association immunoscore (MIAS) that shows a good correlation with patient response to anti-PD1 for 411 melanoma samples complied from 6 cohorts. Furthermore, mapping drug target data to the top genes in our association prediction identifies inhibitors that could potentially enhance tumor response to anti-PD1, such as inhibitors of the encoded proteins of CDK4, GSK3B, and PTK2.

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.

Multiomics profiling of primary lung cancers and distant metastases reveals immunosuppression as a common characteristic of tumor cells with metastatic plasticity.

BACKGROUND: Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. RESULTS: We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. CONCLUSIONS: Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.

Genomics and the Immune Landscape of Osteosarcoma.

Conventional osteosarcoma (OS) is a high-grade intraosseous malignancy with production of osteoid matrix; however, a deeper dive into the underlying genetics reveals genomic complexity and instability that result in significant tumor heterogeneity. While early karyotyping studies demonstrated aneuploidy with chromosomal complexity and structural rearrangements, further investigations have identified few recurrent genetic alterations with the exception of the tumor suppressors TP53 and RB1. More recent studies utilizing next-generation sequencing (NGS; whole-exome sequencing, WES; and whole-genome sequencing, WGS) reveal a genomic landscape predominantly characterized by somatic copy number alterations rather than point/indel mutations. Despite its genomic complexity, OS has shown variable immune infiltrate and limited immunogenicity. In the current chapter, we review the hallmarks of OS genomics across recent NGS studies and the immune profile of OS including a large institutional cohort of OS patients with recurrent and metastatic disease. Understanding the genomic and immune landscape of OS may provide opportunities for translation in both molecularly targeted therapies and novel immuno-oncology approaches.

Genomic assessment distinguishes intrapulmonary metastases from synchronous primary lung cancers.

BACKGROUND: Multiple synchronous lung tumors (MSLT), particularly within a single lobe, represent a diagnostic and treatment challenge. While histologic assessment was once the only method to possibly distinguish multiple primary lung cancers, there is a growing interest in identifying unique genomic features or mutations to best characterize these processes. METHODS: In order to differentiate multiple primary lung malignancies from intrapulmonary metastases in patients with MSLT, we performed whole exome sequencing (WES) on 10 tumor samples from 4 patients with MSLT. RESULTS: Shared mutations between tumors from the same patient varied from 0-91%. Patient 3 shared no common mutations; however, in Patients 2 and 4, identical mutations were identified among all tumors from each patient, suggesting that the three tumors identified in Patient 3 represent separate primary lung cancers, while those of Patients 1, 2 and 4 signify hematogenous and lymphatic spread. CONCLUSIONS: A high proportion of shared mutations between different lung tumors is likely indicative of intrapulmonary metastatic disease, while tumors with distinct genomic profiles likely represent multiple primary malignancies driven by distinct molecular events. Application of genomic profiling in the clinical setting may prove to be important to precise management of patients with MSLT.

Effect of Wall Thickness on Stress-Strain Response and Buckling Behavior of Hollow-Cylinder Rubber Fenders.

In this study, the effect of wall thickness (15-25 mm) on the stress-strain response of hollow-cylinder rubber fenders were investigated by conducting monotonic compression tests. It was found that a progressive increase in lateral bending deformation was observed during monotonic compression. Simultaneously, the extent of the lateral deflection decreased notably with an increasing wall thickness. From the experimental results, the fact is accepted that buckling occurred in the tested fender due to the fact that the ratio of the height to the wall thickness was higher than four in all of the considered cases. Moreover, an s-shape profile appeared in the stress-strain curves, which became clearer as the wall thickness was reduced from 25 to 15 mm. To assess the performance of fenders objectively, an energy-effectiveness index, C E R , was introduced to quantify the energy absorption capacity of the fender. From the experimental observations, it was inferred that the contact area of the folded inner surface of the fender produced under compression generated an additional reaction force and affected the shape of the stress-strain curve since the measured load consisted of two reaction forces: one caused by the self-contact area, and the other resulted from the compression-bending deformation that occurred in the side wall of the fender. To examine this assertion, a finite element analysis (FEA) was conducted and confirmed the effect of the reaction force on the sensitivity of the s-shape characteristic of the stress-strain curve. Finally, a polynomial regression was conducted and the calculated results based on the fourth-degree stress polynomial function correlated very well with the measured stress-strain curves.

Immuno-genomic landscape of osteosarcoma.

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.

Stress-Strain Response of Cylindrical Rubber Fender under Monotonic and Cyclic Compression.

The study was devoted to the observation and modeling the mechanical behaviors of a hybrid SBR/NR (Styrene-Butadiene/Natural Rubber) hybrid vulcanized rubber fender under monotonic/cyclic compression. In experimental observations of the monotonic compression tests, it was found that lateral deformation occurred on the tested fender and was more significant with increasing the extent of the compressive strain. The relationship between the transmission stress S c and the compressive strain e c was nonlinear and the absorbed strain-energy-density was increased monotonically with the increment of the compressive strain. Among all cyclic compression tests with strain controlled, the reductions in both the stress range and the absorbed strain-energy-density up to the ten-thousandth cycle were found and then both of the cyclic properties remain approximately constant in the following compression cycles. Two new properties, the softening factor and the energy reduction factor, were introduced to quantify the effect of the strain range on the extent of the reduction in stress range and that on the absorbed strain-energy-density, respectively. It was found that both of the calculated values of the new properties increase with the increment of strain range. In mathematical modeling of the relationship between the transmission stress and the compressive strain, a new approach based on energy-polynomial-function E s ( e c ) was presented and was successfully used to simulate the monotonic curve and the stable hysteresis loop curves of the tested rubber fender in compression. Essentially, the energy-polynomial-function E s ( e c ) was obtained by performing a polynomial regression on a large amount of ( e c , E s ) data. Moreover, the least-square approach was applied to determine the corresponding regression coefficients in E s ( e c ) . Clearly, the stress-polynomial-function in modeling the S c - e c curve could be obtained from the differentiation of the energy-polynomial-function with respect to the compressive strain. In addition, to provide an adequate estimation of the mechanical properties of the cylindrical rubber fender under compression, the named cyclic stress-strain curve and cyclic energy-strain curve were developed and also modeled in this study.

Author Correction: Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer.

In the version of this article originally published, information regarding several funding sources was omitted from the Acknowledgements section. The following sentences should have been included: "This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program, the UT Lung SPORE 5 P50 CA07090, and the MD Anderson Cancer Center Support Grant P30CA01667. V.P is supported by R01CA155196-01A1 from the National Cancer Institute." Also, reference 18 was incorrect. The original reference was: Kim, E. S. et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 1, 44-53 (2011). It should have been: Papadimitrakopoulou, V. et al. The BATTLE-2 study: a biomarker-integrated targeted therapy study in previously treated patients with advanced non-small-cell lung cancer. J Clin. Oncol. 34, 3638-3647 (2016). The errors have been corrected in the HTML and PDF versions of this article.

FusionPathway: Prediction of pathways and therapeutic targets associated with gene fusions in cancer.

Numerous gene fusions have been uncovered across multiple cancer types. Although the ability to target several of these fusions has led to the development of some successful anti-cancer drugs, most of them are not druggable. Understanding the molecular pathways of a fusion is important in determining its function in oncogenesis and in developing therapeutic strategies for patients harboring the fusion. However, the molecular pathways have been elucidated for only a few fusions, in part because of the labor-intensive nature of the required functional assays. Therefore, we developed a domain-based network approach to infer the pathways of a fusion. Molecular interactions of a fusion are first predicted by using its protein domain composition, and its associated pathways are then inferred from these molecular interactions. We demonstrated the capabilities of this approach by primarily applying it to the well-studied BCR-ABL1 fusion. The approach was also applied to two undruggable fusions in sarcoma, EWS-FL1 and FUS-DDIT3. We successfully identified known genes and pathways associated with these fusions and satisfactorily validated these predictions using several benchmark sets. The predictions of EWS-FL1 and FUS-DDIT3 also correlate with results of high-throughput drug screening. To our best knowledge, this is the first approach for inferring pathways of fusions.