Ionizing radiation is a potent carcinogen, inducing cancer through DNA damage. The signatures of mutations arising in human tissues following in vivo exposure to ionizing radiation have not been documented. Here, we searched for signatures of ionizing radiation in 12 radiation-associated second malignancies of different tumour types. Two signatures of somatic mutation characterize ionizing radiation exposure irrespective of tumour type. Compared with 319 radiation-naive tumours, radiation-associated tumours carry a median extra 201 deletions genome-wide, sized 1–100 base pairs often with microhomology at the junction. Unlike deletions of radiation-naive tumours, these show no variation in density across the genome or correlation with sequence context, replication timing or chromatin structure. Furthermore, we observe a significant increase in balanced inversions in radiation-associated tumours. Both small deletions and inversions generate driver mutations. Thus, ionizing radiation generates distinctive mutational signatures that explain its carcinogenic potential.

SummaryHistone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

It is never too late to stop drinking, even with the most severe degrees of cirrhosis on biopsy. Early drinking status is the most important factor determining long-term survival in alcohol-related cirrhosis.

Background and AimAlcohol-related liver disease (ALD) remains a leading cause of liver-related morbidity and mortality. Age, fibrosis stage, MELD score and continued alcohol consumption predict outcome in everyday clinical practice. In previous studies increased hepatocyte nuclear area and hepatocyte expression of p21, both markers of senescence, were associated with increased fibrosis stage and a poor outcome in non-alcohol-related fatty liver disease, while increased hepatocyte nuclear area was related to liver dysfunction in ALD cirrhosis. This study, therefore, investigated the pattern of hepatocyte cell cycle phase distribution and hepatocyte p21 expression in relation to outcome in ALD.MethodsLiver sections from two cohorts were studied. The first comprised 42 patients across the full spectrum of ALD. The second cohort comprised 77 patients with ALD cirrhosis. Immunohistochemistry assessed hepatocyte expression of cell cycle phase markers and p21. Regenerating liver (n=12) and “normal” liver sections (n=5) served as positive and negative controls, respectively.ResultsIn the first cohort there was little cell cycle progression beyond G1/S phase and increased hepatocyte p21 expression (p<0.0001), which correlated independently with fibrosis stage (p=0.005) and an adverse liver-related outcome (p=0.03). In the second cohort, both hepatocyte p21 expression (p<0.001) and MELD score (p=0.006) were associated independently with an adverse liver-related outcome; this association was stronger with hepatocyte p21 expression (AUROC 0.74; p=0.0002) than with MELD score (AUROC 0.59; p=0.13). Further, hepatocyte p21 expression co-localised with increased hepatic stellate cell activation.ConclusionsThe findings are consistent with impaired cell cycle progression beyond the G1/S phase in ALD. The striking independent associations between increased hepatocyte p21 expression and both fibrosis stage and an adverse liver-related outcome in both cohorts suggests hepatocyte senescence plays an important role in ALD. Measuring hepatocyte p21 expression is simple and cheap and in this series was a useful measure of long-term prognosis in ALD.

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.iscerning the source of spontaneous germline mutations is fundamental to understanding the causes of many diseases, including monogenic developmental disorders (1) and complex conditions such as autism (2, 3) and schizophrenia (4). Recent whole genome sequencing studies of parent-child trios show that most mutations (such as nucleotide substitutions) originate from the paternal germline and increase in frequency with the father's age (5, 6), an issue of particular significance given the demographic shift to delayed reproduction in many populations (7). The deduction that the testes of older men harbor a greater burden of mutations, compared with younger men, is consistent with indirect measures of genetic decline, ranging from high indices of arrested germ cell divisions to complete involution of the seminiferous tubules (7-9). Surprisingly, however, it has not previously been possible to trace specific mutations back to their origins within individual germ cells (spermatogonia) of human testes.One mechanism proposed to contribute to the age-related increase in male mutations is selfish spermatogonial selection, a process equivalent to neoplasia but occurring in the unique context of the germ cell (10). In this process, specific point mutations that confer gain-of-function to components of the growth factor receptor-RAS signaling pathway occur rarely in spermatogonial stem cells of the adult testis but show a steep increase in prevalence with age, attributed to clonal expansion of mutant spermatogonia over time (11)(12)(13)(14)(15)(16). Fertilization of the egg by a mutant sperm leads to serious congenital disorders in the next generation, characterized by multiple malformations and, in some cases, a predisposition to malignancy. These disorders include Apert, Crouzon, and (16, 26). Based on the unexpectedly high birth prevalence of several of the associated congenital d...

Immunotherapy treatments with anti-PD-1 boost recovery in less than 30% of treated cancer patients, indicating the complexity of the tumor microenvironment. Expression of HLA-E is linked to poor clinical outcomes in mice and human patients. However, the contributions to immune evasion of HLA-E, a ligand for the inhibitory CD94/NKG2A receptor, when expressed on tumors, compared with adjacent tissue and peripheral blood mononuclear cells, remains unclear. In this study, we report that epithelial-derived cancer cells, tumor macrophages, and CD141 þ conventional dendritic cells (cDC) contributed to HLA-E enrichment in carcinomas. Different cancer types showed a similar pattern of enrichment. Enrichment correlated to NKG2A upregulation on CD8 þ tumor-infiltrating T lymphocytes (TIL) but not on CD4 þ TILs. CD94/NKG2A is exclusively expressed on PD-1 high TILs while lacking intratumoral CD103 expression. We also found that the presence of CD94/NKG2A on human tumor-specific T cells impairs IL2 receptor-dependent proliferation, which affects IFNg-mediated responses and antitumor cytotoxicity. These functionalities recover following antibody-mediated blockade in vitro and ex vivo. Our results suggest that enriched HLA-E:CD94/NKG2A inhibitory interaction can impair survival of PD-1 high TILs in the tumor microenvironment.

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition. Cancer Res; 74(20); 5866-77. Ó2014 AACR.

Enrichment of CD103 þ tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103 þ cytotoxic CD8 þ T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103 þ CTLs by assessing T-cell receptor (TCR)-matched CD103 þ and CD103cancer-specific CTL immunity in vitro and its immunophenotype ex vivo. Interestingly, we found that differentiated CD103 þ cancer-specific CTLs expressed the active form of TGFb1 to continually self-regulate CD103 expression, without relying on external TGFb1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103 þ CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103 þ cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.