Enhancing chimeric antigen receptor T cell therapy by modulating the p53 signaling network with Δ133p53α.

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.

The landscape of etiological patterns of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in Thailand.

Thailand is among countries with the highest global incidence and mortality rates of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). While viral hepatitis and liver fluke infections have been associated with HCC and iCCA, respectively, other environmental risk factors, overall risk factor commonality and combinatorial roles, and effects on survival have not been systematically examined. We conducted a TIGER-LC consortium-based population study covering all high-incidence areas of both malignancies across Thailand: 837 HCC, 1474 iCCA, and 1112 controls (2011-2019) were comprehensively queried on lifelong environmental exposures, lifestyle, and medical history. Multivariate logistic regression and Cox proportional hazards analyses were used to evaluate risk factors and associated survival patterns. Our models identified shared risk factors between HCC and iCCA, such as viral hepatitis infection, liver fluke infection, and diabetes, including novel and shared associations of agricultural pesticide exposure (OR range of 1.50; 95% CI: 1.06-2.11 to 2.91; 95% CI: 1.82-4.63) along with vulnerable sources of drinking water. Most patients had multiple risk factors, magnifying their risk considerably. Patients with lower risk levels had better survival in both HCC (HR 0.78; 95% CI: 0.64-0.96) and iCCA (HR 0.84; 95% CI: 0.70-0.99). Risk factor co-exposures and their common associations with HCC and iCCA in Thailand emphasize the importance for future prevention and control measures, especially in its large agricultural sector. The observed mortality patterns suggest ways to stratify patients for anticipated survivorship and develop plans to support medical care of longer-term survivors, including behavioral changes to reduce exposures.

p53 mutations and inflammation-associated cancer are linked through TNF signaling.

In this issue, Di Minin et al. (2014) link mutant p53 and chronic inflammation to tumorigenic progression via TNF signaling. Mutp53 interacts with the tumor suppressor DAB2IP in the cytoplasm, and induces a TNF-dependent transcriptional profile via NF-kB and JNK.

Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis.

BACKGROUND: Engineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3' end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results. RESULTS: We collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients. By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported. We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients. CONCLUSIONS: Our findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells.

BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated ß-galactosidase (SA-ß-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

tsRNA signatures in cancer.

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called "ts-101" and "ts-53," respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.

The Werner syndrome RECQ helicase targets G4 DNA in human cells to modulate transcription.

The Werner syndrome (WS) is a prototypic adult Mendelian progeroid syndrome in which signs of premature aging are associated with genomic instability and an elevated risk of cancer. The WRN RECQ helicase protein binds and unwinds G-quadruplex (G4) DNA substrates in vitro, and we identified significant enrichment in G4 sequence motifs at the transcription start site and 5' ends of first introns (false discovery rate < 0.001) of genes down-regulated in WS patient fibroblasts. This finding provides strong evidence that WRN binds G4 DNA structures at many chromosomal sites to modulate gene expression. WRN appears to bind a distinct subpopulation of G4 motifs in human cells, when compared with the related Bloom syndrome RECQ helicase protein. Functional annotation of the genes and miRNAs altered in WS provided new insight into WS disease pathogenesis. WS patient fibroblasts displayed altered expression of multiple, mechanistically distinct, senescence-associated gene expression programs, with altered expression of disease-associated miRNAs, and dysregulation of canonical pathways that regulate cell signaling, genome stability and tumorigenesis. WS fibroblasts also displayed a highly statistically significant and distinct gene expression signature, with coordinate overexpression of nearly all of the cytoplasmic tRNA synthetases and associated ARS-interacting multifunctional protein genes. The 'non-canonical' functions of many of these upregulated tRNA charging proteins may together promote WS disease pathogenesis. Our results identify the human WRN RECQ protein as a G4 helicase that modulates gene expression in G4-dependent fashion at many chromosomal sites and provide several new and unexpected mechanistic insights into WS disease pathogenesis.

Are Colon and Rectal Cancer Two Different Tumor Entities? A Proposal to Abandon the Term Colorectal Cancer.

Colon cancer (CC) and rectal cancer (RC) are synonymously called colorectal cancer (CRC). Based on our experience in basic and clinical research as well as routine work in the field, the term CRC should be abandoned. We analyzed the available data from the literature and results from our multicenter Research Group Oncology of Gastrointestinal Tumors termed FOGT to confirm or reject this hypothesis. Anatomically, the risk of developing RC is four times higher than CC, while physical activity helps to prevent CC but not RC. Obvious differences exist in molecular carcinogenesis, pathology, surgical topography and procedures, and multimodal treatment. Therefore, we conclude that CC is not the same as RC. The term "CRC" should no longer be used as a single entity in basic and clinical research as well as other areas of classification.

Menstrual and reproductive factors and lung cancer risk: A pooled analysis from the international lung cancer consortium.

Many clinical features of lung cancer are different in women and men. Sex steroid hormones exert effects in nonreproductive organs, such as the lungs. The association between menstrual and childbearing factors and the risk of lung cancer among women is still debated. We performed a pooled analysis of eight studies contributing to the International Lung Cancer Consortium (4,386 cases and 4,177 controls). Pooled associations between menstrual or reproductive factors and lung cancer were estimated using multivariable unconditional logistic regression. Subgroup analyses were done for menopause status, smoking habits and histology. We found no strong support for an association of age at menarche and at menopause with lung cancer, but peri/postmenopausal women were at higher risk compared to premenopausal (OR 1.47, 95% CI 1.11-1.93). Premenopausal women showed increased risks associated with parity (OR 1.74, 95% CI 1.03-2.93) and number of children (OR 2.88, 95% CI 1.21-6.93 for more than 3 children; p for trend 0.01) and decreased with breastfeeding (OR 0.54, 95% CI 0.30-0.98). In contrast, peri/postmenopausal subjects had ORs around unity for the same exposures. No major effect modification was exerted by smoking status or cancer histology. Menstrual and reproductive factors may play a role in the genesis of lung cancer, yet the mechanisms are unclear, and smoking remains the most important modifiable risk factor. More investigations in large well-designed studies are needed to confirm these findings and to clarify the underlying mechanisms of gender differences in lung cancer risk.

Alcohol and lung cancer risk among never smokers: A pooled analysis from the international lung cancer consortium and the SYNERGY study.

It is not clear whether alcohol consumption is associated with lung cancer risk. The relationship is likely confounded by smoking, complicating the interpretation of previous studies. We examined the association of alcohol consumption and lung cancer risk in a large pooled international sample, minimizing potential confounding of tobacco consumption by restricting analyses to never smokers. Our study included 22 case-control and cohort studies with a total of 2548 never-smoking lung cancer patients and 9362 never-smoking controls from North America, Europe and Asia within the International Lung Cancer Consortium (ILCCO) and SYNERGY Consortium. Alcohol consumption was categorized into amounts consumed (grams per day) and also modelled as a continuous variable using restricted cubic splines for potential non-linearity. Analyses by histologic sub-type were included. Associations by type of alcohol consumed (wine, beer and liquor) were also investigated. Alcohol consumption was inversely associated with lung cancer risk with evidence most strongly supporting lower risk for light and moderate drinkers relative to non-drinkers (>0-4.9 g per day: OR = 0.80, 95% CI = 0.70-0.90; 5-9.9 g per day: OR = 0.82, 95% CI = 0.69-0.99; 10-19.9 g per day: OR = 0.79, 95% CI = 0.65-0.96). Inverse associations were found for consumption of wine and liquor, but not beer. The results indicate that alcohol consumption is inversely associated with lung cancer risk, particularly among subjects with low to moderate consumption levels, and among wine and liquor drinkers, but not beer drinkers. Although our results should have no relevant bias from the confounding effect of smoking we cannot preclude that confounding by other factors contributed to the observed associations. Confounding in relation to the non-drinker reference category may be of particular importance.

Genome-wide scan of 29,141 African Americans finds no evidence of directional selection since admixture.

The extent of recent selection in admixed populations is currently an unresolved question. We scanned the genomes of 29,141 African Americans and failed to find any genome-wide-significant deviations in local ancestry, indicating no evidence of selection influencing ancestry after admixture. A recent analysis of data from 1,890 African Americans reported that there was evidence of selection in African Americans after their ancestors left Africa, both before and after admixture. Selection after admixture was reported on the basis of deviations in local ancestry, and selection before admixture was reported on the basis of allele-frequency differences between African Americans and African populations. The local-ancestry deviations reported by the previous study did not replicate in our very large sample, and we show that such deviations were expected purely by chance, given the number of hypotheses tested. We further show that the previous study's conclusion of selection in African Americans before admixture is also subject to doubt. This is because the FST statistics they used were inflated and because true signals of unusual allele-frequency differences between African Americans and African populations would be best explained by selection that occurred in Africa prior to migration to the Americas.

Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers.

BACKGROUND & AIMS: A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. METHODS: We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn's disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. RESULTS: Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. CONCLUSIONS: Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.

The landscape of recombination in African Americans.

Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10(-245)). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.

Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs.

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.

Inflammation-mediated genetic and epigenetic alterations drive cancer development in the neighboring epithelium upon stromal abrogation of TGF-ß signaling.

Deletion of tumor suppressor genes in stromal fibroblasts induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. However, the underlying mechanisms remain to be elucidated. Here we report that deletion of the gene encoding TGFß receptor 2 (Tgfbr2) in the stromal fibroblasts (Tgfbr2(fspKO)) induces inflammation and significant DNA damage in the neighboring epithelia of the forestomach. This results in loss or down-regulation of cyclin-dependent kinase inhibitors p15, p16, and p21, which contribute to the development of invasive squamous cell carcinoma (SCC). Anti-inflammation treatment restored p21 expression, delayed tumorigenesis, and increased survival of Tgfbr2(fspKO) mice. Our data demonstrate for the first time that inflammation is a critical player in the epigenetic silencing of p21 in tumor progression. Examination of human esophageal SCC showed a down-regulation of TGFß receptor 2 (TßRII) in the stromal fibroblasts, as well as increased inflammation, DNA damage, and loss or decreased p15/p16 expression. Our study suggests anti-inflammation may be a new therapeutic option in treating human SCCs with down-regulation of TßRII in the stroma.