Splicing and epigenomic analyses in high-risk CLL for the development of novel therapeutic strategies.
Final Report Abstract
Chronic lymphocytic leukemia is one of the most common leukemias in the Western world and presents with a wide variety in disease outcome. While some patients have an indolent disease for several years, others require immediate treatment. Therefore, early patient stratification is of clinical importance. Here, we evaluated a previously published prognostic epigenetic marker set consisting of the DNA methylation levels of five CpGs in a German CLL cohort and confirmed it’s prognostic value. We evaluated the methylation levels in 70 CLL patients presenting with an either indolent disease (i.e. more than 5 years without treatment requirement) or progressive disease (i.e. relapse after therapy) and observed a specificity of 80% and a sensitivity of 83%, which confirms the prognostic value of this marker set. The splicing factor 3b Subunit 1, SF3B1, is one of the most frequently mutated genes in CLL. To better understand the functional consequences, we evaluated the impact of SF3B1 mutations on gene expression and splicing in cell lines and primary CLL material. We evaluated the RNA expression and splicing alterations mediated by SF3B1 mutations in CLL patients and in two isogenic cell line pairs (K562, Nalm6) either wild type or carrying a heterozygous mutation in SF3B1 and identified commonly deregulated genes and splicing alterations, among which was MAP3K7. In SF3B1 mutated cells, a cryptic 3’ splice site in MAP3K7 was used resulting in a premature stop codon which most likely results in nonsense mediated decay. Moreover, SF3B1 mutated samples display an inclusion of a cryptic exon in BRD9, which predicts a premature stop codon and nonsense mediated decay. Using an methylated immunoprecipitation followed by sequencing (MeDIP-Seq) approach to interrogate the whole-genome DNA methylation profile we detected several differentially methylated regions, the majority being hypomethylated in the SF3B1 mutated samples. These differential methylation sites are not due to the developmental stage of the initiating CLL cell, but rather seem to dependent upon the mutational status of the cells. The differentially methylated regions did not overlap with alternative splicing sites, underscoring different mechanisms for SF3B1mut effects. Furthermore, the analysis of histone modifications (H3K36me3, H3K4me3) and POL2 (POL2, phosphorylated at serine 2 (POL2S2P) and serine 5 (POL2S5P), C-terminal domain) distribution on chromatin by ChIP-Seq analyses pointed to a defect in transcription, with an increase of the activating H3K4me3 mark at the promoter and a decrease of POL2, POL2S2P, POL2S5P at the promoter site in SF3B1 mutated cells. Thus, we thought that SF3B1 binding to RNA might be altered and performed iCLIP analyses. Interestingly, these analyses did not reveal any gross alterations in RNA binding when comparing SF3B1 mutated and wild type cells indicating that an altered protein-protein interaction might cause the alternative splicing events. Indeed, by performing chromatin immunoprecipitations followed by mass spectrometry analyses we discovered that the p.K700E, the most common mutation in SF3B1, results in an altered interactome, in particular on chromatin organization, DNA damage response, nuclear transport and translation. In a small scale screen in an isogenic cell line pair either with wild type or mutated SF3B1, we identified three compounds displaying increased sensitivity in SF3B1 mutated cells. We are right now in the progress to extend these findings to a preclinical mouse model. At this point we encountered necessary deviations from the original project plan which resulted in a delay of the animal experiments. First of all the import of the Sf3b1K700E/wt animals into our animal facility by embryo transfer took longer than envisaged and was then, shut down by corona regulations. Moreover, initially, we planned to use Cd19Cre/wt/Sf3b1K700E/wt/Atmfl/fl animals for our experiments. However, during the project time it was shown that the incidence of developing a CLL-like disease within these animals is only 9% at an age of 18 months. Therefore, we now use Cd19Cre/wt/Sf3b1K700E/wt/TCL1tg/wt animals, which develop a CLL-like disease due to the presence of the TCL1 transgene. In the meantime we focused on the data analyses and interpretation of large-scale analyses to functionally delineate the mechanism of SF3B1mut. All data is made publicly available by deposition in public databases.
Publications
- Epigenomic profiling of non-small cell lung cancer xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance. Genome Med. 2018 Jul 20;10(1):55
Grasse S, Lienhard M, Frese S, Kerick M, Steinbach A, Grimm C, Hussong M, Rolff J, Becker M, Dreher F, Schirmer U, Boerno S, Ramisch A, Leschber G, Timmermann B, Grohé C, Lüders H, Vingron M, Fichtner I, Klein S, Odenthal M, Büttner R, Lehrach H, Sültmann H, Herwig R, Schweiger MR
(See online at https://doi.org/10.1186/s13073-018-0562-1) - Combined Targeted Resequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for Poly(ADP-Ribose) Polymerase 1 Inhibitor Sensitivity Testing. J Mol Diagn. 2019 Mar;21(2):198-213
Grimm C, Fischer A, Farrelly AM, Kalachand R, Castiglione R, Wasserburger E, Hussong M, Schultheis AM, Altmüller J, Thiele H, Reinhardt HC, Hauschulz K, Hennessy BT, Herwig R, Lienhard M, Buettner R, Schweiger MR
(See online at https://doi.org/10.1016/j.jmoldx.2018.10.007) - Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients. Int J Mol Sci. 2021 Aug 28;22(17):9337
Pacholewska A, Grimm C, Herling CD, Lienhard M, Königs A, Timmermann B, Altmüller J, Mücke O, Reinhardt HC, Plass C, Herwig R, Hallek M, Schweiger MR
(See online at https://doi.org/10.3390/ijms22179337) - Pericentromeric Satellite III transcripts induce etoposide resistance. Cell Death Dis. 2021 May 24;12(6):530
Kanne J, Hussong M, Isensee J, Muñoz-López Á, Wolffgramm J, Heß F, Grimm C, Bessonov S, Meder L, Wang J, Reinhardt HC, Odenthal M, Hucho T, Büttner R, Summerer D, Schweiger MR
(See online at https://doi.org/10.1038/s41419-021-03810-9)
