Abstract Background Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. Results We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared “murine MRD genes” profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. Conclusions Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies. Graphical Abstract
The Krüppel-like factor (KLF) family consists of transcription factors that can activate or repress different genes implicated in processes such as differentiation, development, and cell cycle progression. Moreover, several of these proteins have been implicated in glucose homeostasis, making them candidate genes for involvement in type 2 diabetes (T2D).Variants of nine KLF genes were genotyped in T2D cases and controls and analysed in a two-stage study. The first case-control set included 365 T2D patients with a strong family history of T2D and 363 normoglycemic individuals and the second set, 750 T2D patients and 741 normoglycemic individuals, all of French origin. The SNPs of six KLF genes were genotyped by Taqman SNP Genotyping Assays. The other three KLF genes (KLF2, -15 and -16) were screened and the identified frequent variants of these genes were analysed in the case-control studies.Three of the 28 SNPs showed a trend to be associated with T2D in our first case-control set (P < 0.10). These SNPs, located in the KLF2, KLF4 and KLF5 gene were then analysed in our second replication set, but analysis of this set and the combined analysis of the three variants in all 2,219 individuals did not show an association with T2D in this French population. As the KLF2, -15 and -16 variants were representative for the genetic variability in these genes, we conclude they do not contribute to genetic susceptibility for T2D.It is unlikely that variants in different members of the KLF gene family play a major role in T2D in the French population.
Abstract Context: The transcription factor Krüppel-like zinc finger 11 (KLF11) has been suggested to contribute to genetic risk of type 2 diabetes (T2D). Our previous results showed that four KLF11 variants, in strong linkage disequilibrium (LD block including +185 A>G/Gln62Arg and −1659 G>C) were associated with T2D in a north European case-control study. Here we further analyzed these variants for T2D association in a general Danish population and assess their possible effect on gene function. Methods: We genotyped Gln62Arg variant, representative for the LD block, in 5864 subjects of the INTER99 study to assess association to T2D and glucose metabolism-related quantitative traits. We studied effects of LD-block variants on KLF11 function and in particular, the effect of −1659G>C on transcriptional regulation of KLF11 using EMSA, chromatin immunoprecipitation, gene reporter assays, and small interfering RNA transfection. Results: We could not confirm T2D association of the KLF11 LD block, however, in glucose-tolerant subjects; it was significantly associated with higher fasting serum insulin and C-peptide levels and increased homeostasis model assessment insulin resistance indexes (P = 0.00004, P = 0.006, and P = 0.00002, respectively). In addition, binding of signal transducer and activator of transcription (STAT)-3 to the wild-type (−1659G>C) allele stimulated gene transcription, whereas STAT3 did not bind onto the mutant allele. Conclusions: We showed that KLF11 may interfere with glucose homeostasis in a Danish general population and that STAT3-mediated up-regulation of KLF11 transcription was impaired by the −1659G>C variant. Overall, KLF11 variants may have a deleterious effect on insulin sensitivity, although that may not be sufficient to lead to T2D.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is the main and the deadliest form of pancreatic cancer. This is a major problem of public health since it will become the second leading cause of death by cancer in the next few years, mainly due to the lack of efficient therapies. Transient Receptor Potential Cation Channel Subfamily M Member 7 (TRPM7) protein, a cation channel fused with a serine/threonine kinase domain is overexpressed in PDAC and associated with a low survival. In this work, we aim to study the role of kinase domain on pancreatic cell fates by using a model of kinase domain deletion by CRISPR-Cas9. PANC-1 and MIA PaCa-2 PDAC cell lines were used and kinase domain was deleted by CRISPR-Cas9 strategy. Kinase domain deletion (ΔK) was validated by RT-qPCR and western-blots. The effect of kinase domain deletion on channel function was studied by patch-clamp and Mn2+-quenching. The cell phenotype was studied by MTT and cell migration/invasion assays. Finally, the role of kinase domain was studied in vivo in xenografted mice. Here we show that TRPM7 kinase domain is required to maintain a mesenchymal phenotype in PDAC cells. We also demonstrated that TRPM7 and PAK1 interact in the same protein complexes. Moreover, TRPM7 kinase domain is required for carcinogenesis and cancer cell dissemination in vivo. Intriguingly, the role of TRPM7 kinase is cell specific and may depend on the KRAS oncogene mutation status. In conclusion, TRPM7 kinase domain is required to maintain a mesenchymal and aggressive phenotype in PDAC cells, and it could be a promising target against PDAC.
Individual genotyping of single n u cleotide polymorphisms (SNPs) remain s expensive, especially for linkage disequili b rium mapping strategies involving high throughput SNP genotyping. On one hand , current methods may suit scientific and la b oratory needs in regard to accuracy, repr o ducibility/robustness, and large-scale appl i cation. On the other hand, a cheaper an d less time-consuming alternative to indivi d ual genotyping is the use of SNP allele fr e quencies determined in DNA pools. W e have developed an accurate and repr o ducible protocol for allele frequency dete r mination using Pyrosequencin g ™technol o gy in large genomic DNA pools (37 4 individuals). The measured correlation (R 2 ) in large DNA pools was 0.980. In the co n text of disease-associated SNPs studies, w e compared the allele frequencies betwee n the disease (e.g., type 2 diabetes and obes i ty) and control groups detected by either i n dividual genotyping or Pyrosequencin g o f DNA pools. In large pools, the variation b e tween the two methods was 1.5 ± 0.9%. I t may be concluded that the allele frequenc y determination protocol could reliably detec t over 4% differences between populations . The method is economical in regard t o amounts of DNA, PCR, and primer exte n sion reagents required. Furthermore, it a l lows the rapid determination of allele fr e quency differences in case/control group s for association studies and susceptibilit y gene discovery in complex diseases .
Krüppel-like transcription factors (KLFs) have elicited significant attention because of their regulation of essential biochemical pathways and, more recently, because of their fundamental role in the mechanisms of human diseases. Neonatal diabetes mellitus is a monogenic disorder with primary alterations in insulin secretion. We here describe a key biochemical mechanism that underlies neonatal diabetes mellitus insulin biosynthesis impairment, namely a homozygous mutation within the insulin gene (INS) promoter, c.-331C>G, which affects a novel KLF-binding site. The combination of careful expression profiling, electromobility shift assays, reporter experiments, and chromatin immunoprecipitation demonstrates that, among 16 different KLF proteins tested, KLF11 is the most reliable activator of this site. Congruently, the c.-331C>G INS mutation fails to bind KLF11, thus inhibiting activation by this transcription factor. Klf11(-/-) mice recapitulate the disruption in insulin production and blood levels observed in patients. Thus, these data demonstrate an important role for KLF11 in the regulation of INS transcription via the novel c.-331 KLF site. Lastly, our screening data raised the possibility that other members of the KLF family may also regulate this promoter under distinct, yet unidentified, cellular contexts. Collectively, this study underscores a key role for KLF proteins in biochemical mechanisms of human diseases, in particular, early infancy onset diabetes mellitus.
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