Epigenetic Impact on a Cancer Associated Signature of Alternative Splicing

• Applicant: Professorin Dr. Margarete Odenthal
• Subject Area: General Genetics and Functional Genome Biology ; Pathology ; Cell Biology
• Term: from 2020 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433117128

Chromatin remodelling due to histone modification is a hallmark of cancer. The epigenetic mediator LSD1, catalyzing the demethylation of histone 3 (H3) lysines (K), namely H3K4 and H3K9, is highly overexpressed in a wide variety of cancer types, including non-small cell lung cancer (NSCLC). Current comprehensive screening of LSD1 interaction partners in different cancer cells revealed a cooperative binding to main alternative splicing factors. Most notably, primary findings show that in response to LSD1 inhibition the splicing pattern was markedly changed in lung cancer cells. Based on this data, we suggest a main function of LSD1 in the shift from a homeostatic to an oncogenic signature of alternative splicing, triggering cancer initiation and progression. In the present project, we will address the new function of the epigenetic factor LSD1, not only in transcriptional regulation, but also affecting the splicing process resulting in a divergent splicing profile. We will study the mechanistic link of LSD1 to the splicing machinery on NSCLC cell types (PC9 and A549), carrying the NSCLC relevant KRAS and EGFR mutations. These NSCLC representative cancer cell systems will be used to inhibit LSD1 function by means of the LSD1 inhibitor HCI2509. Furthermore, LSD1 mutants, either affecting the catalytic or the protein interaction site, were generated in NSCLC PC9 and A549 cells by CRISPR/Cas based homologous recombination. In NSCLC cells, the altered pattern of splice isoforms, in response to drug or mutation mediated LSD1 inhibition, will be investigated by single molecule real time (SMRT) sequencing which highly benefits from long read sequencing. Since splicing depends on the transcription rate, which in turn is affected by the histone modification, the LSD1 targeted methylation mark is suggested to influence also splicing. In addition, the methylated histone signature might serve as capture structures for the splicing machinery. To analyse the histone methylation pattern in exon intron junctions, chromatin immunoprecipitation followed by whole genome analysis (ChIP-Seq) will be performed. This data will be used to compare the genome-wide histone H3K4 and H3K9 methylation signature with the identified profiles of LSD1 dependent splice isoforms. Furthermore, the mechanistic link of putative direct or indirect LSD1 interaction and oncogenic alternative splicing will be identified by RNA seq of LSD1 RNA-pull down populations from LSD1 CRIPR/Cas modified NSCLC cell types and corresponding controls. In summary, the aspired comprehensive sequencing approaches will provide novel insights in the mechanistic linkage of LSD1 to the splicing machinery.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Maria Anokhina, Ph.D.