Dieter Weichenhan's research while affiliated with German Cancer Research Center and other places

Cancer is a heterogeneous disease caused by genetic alterations. Computational analysis of cancer genomes led to the expansion of the catalog of driver mutations. While individual high-impact mutations have been discovered also in gene promoters, frequency-based approaches have only characterized a few novel candidates. To investigate the promoter mutation paucity in cancer, we developed the REMIND-Cancer workflow to predict activating promoter mutations in silico, irrespective of their recurrence frequency, and applied it to the PCAWG dataset. We positively validated 7 candidates by luciferase assay including mutations within the promoters of ANKRD53 and MYB. Our analysis indicates that particular mutational signatures and necessary co-alterations constrain the creation and positive selection of functional promoter mutations. We conclude that activating promoter mutations are more frequent in the PCAWG dataset than previously observed, which has potential implications for personalized oncology.

AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.

The epiCMIT (epigenetically determined Cumulative MIToses) mitotic clock traces B-cell mitotic history via DNA methylation changes in heterochromatin and H3K27me3-containing chromatin. While high scores correlated with poor outcomes in CLL and MCL, its prognostic significance in SMZL remains unknown. Derived from 142 SMZL cases using DNA methylation microarrays, epiCMIT values were correlated with genomic, transcriptomic, and clinical data. EpiCMIT as a continuous variable was significantly higher in females (p=0.02), patients with IGHV1-2*04 allele usage (p<0001), intermediate IGHV somatic hypermutation load (97-99.9% identity, p=0.04), elevated mutational burden (25 vs. 17 mut/Mb, p=0.001), driver gene mutations [KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01), KMT2D (p<0.001)], and del(7q) (p=0.01). Negative correlation between epiCMIT and telomere length (r=-0.29 p<0.001) supported the association between cumulated proliferation and telomere attrition. While univariate analysis highlighted epiCMIT as robust predictor of shorter treatment-free survival (TFS), multivariate analysis confirmed epiCMIT as an independent marker for shorter TFS. In summary, our matched multi-omic datasets facilitate the clinico-biological characterization of SMZL and introduces epiCMIT as a strong prognostic marker, identifying high-risk patients and predicting reduced treatment-free survival, hence providing a new tool for risk-adapted patient management.

Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

The epiCMIT (epigenetically-determined Cumulative MIToses) mitotic clock (Durran-Ferrer, 2020) tracks DNA hypo- and hyper-methylation changes in heterochromatin and H3K27me3-containing chromatin, respectively, providing a comprehensive record of B cell mitotic history (pre- and post-malignant transformation). Higher epiCMIT levels correlate with poor survival in CLL and MCL, suggesting that a greater mitotic history predicts future proliferative capacity. However, no studies have investigated epiCMIT in splenic marginal zone lymphoma (SMZL). We studied 142 SMZL cases, a rare lymphoid malignancy with specific genetic characteristics [del(7q), KLF2and NOTCH2 mutations, biased use of IGHV1-2*04]. After calculating epiCMIT scores from 450/850K Illumina arrays we compared these data with gene mutations from targeted resequencing and WGS, copy number variations, telomere length, RNA-Seq, miRNA-Seq data, and clinical outcome data. By defining epiCMIT-hyper [mean: 0.6, range: 0.29-0.91] and -hypo [mean: 0.71, range: 0.39-0.90] values in our cohort, we demonstrate a strong positive correlation between hyper- and hypo- scores, as previously observed in MCL and CLL (Durran-Ferrer, 2020). The highest score obtained for epiCMIT-hyper or -hypo per sample was then selected as the epiCMIT score per patient [mean: 0.72, range: 0.39-0.91]. We identified significant correlations between epiCMIT and key clinico-biological characteristics, including a significant negative correlation between epiCMIT and telomere length (R 2=0.67 p<0.001) supporting cell proliferation and concomitant telomere attrition. High epiCMIT scores were associated with female gender (p=0.02), IGHV1-2*04 alleles (p<0.001) with intermediate levels of IGHV somatic hypermutation (SHM) load (97-99.9%, p=0.04), driver somatic gene mutations, including those in KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01) and KMT2D (p<0.001) and deletion of 7q (p=0.01) ( Fig1). Using previously defined classification systems (Arribas, 2015 and Bonfiglio, 2022), we show enrichment of high-risk subgroups [Arribas- high global methylation subgroup (High-M, p<0.001) and Bonfiglio- NFKB, NOTCH and KLF2 genetic subgroup (NNK, p=0.01)] in cases with high epiCMIT. In a sub-cohort of 42 SMZL cases, with a spectrum of epiCMIT values and WGS/ (mi)RNA-Seq data available, we demonstrated 1) a significantly higher genome-wide somatic mutational burden in patients with high epiCMIT-total scores (25 v 17mut/Mb, p=0.001), and 2), correlations between epiCMIT and specific gene/miRNA expression associated with cellular survival and proliferation ( CARD11 [R 2=0.6, p<0.01], MAP2K1 [R 2=0.61, p<0.01], miRNA-155 [R 2=0.58, p=0.018]), apoptosis ( BCOR [R 2=0.61, p<0.01]), and epigenetic regulation ( EZH2 [R 2=0.58, p<0.01]). We then conducted univariate Cox Proportional Hazards analysis to investigate the prognostic significance of clinical and molecular features on time to first treatment (TTFT; most prevalent treatments: splenectomy, n=51; rituximab, n=20) and overall survival (OS). The epiCMIT score and epiCMIT-hyper values showed the highest hazard ratios (HR) for TTFT (HR=26.6, p=0.001) and OS (HR=9.45, p=0.043) respectively ( Fig2). Other factors associated with shorter TTFT included TP53 mutation, 3q gain, IGHV1-2*04 usage, High-M, short telomeres and female gender. Shorter OS was significantly (p<0.05) linked to mutations in key genes (including TP53 and NOTCH2), genomic complexity and del(7q). To avoid redundancy and collinearity, only the most significant epiCMIT metric was included in a multivariate Cox Proportional Hazards model (backwards selection, 94 patients/63 events), and epiCMIT-total emerged as an independent marker for shorter TTFT (HR=44.4, p=0.004), together with gain of chromosome 3q (HR=2.6, p=0.002). Kaplan-Meier analysis further confirmed the importance of epiCMIT-total, with higher scores (>median: 0.73) associated with significantly shorter TTFT (median 23 vs 3.5 months) and higher mortality (p=0.004). To conclude, we demonstrate the potential clinical utility of epiCMIT score in SMZL patients, identifying that SMZL patients with high epiCMIT harbour specific genetic characteristics, and exhibit reduced treatment-free survival. EpiCMIT could be valuable in clinical practice, helping to identify those patients destined to progress and require closer monitoring.

Background Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. Dysregulation of the epigenetic machinery is a significant contributor to disease development. Some AML patients benefit from treatment with hypomethylating agents (HMAs), but no predictive biomarkers for therapy response exist. Here, we investigated whether unbiased genome-wide assessment of pre-treatment DNA-methylation profiles in AML bone marrow blasts can help to identify patients who will achieve a remission after an azacytidine-containing induction regimen. Results A total of n = 155 patients with newly diagnosed AML treated in the AMLSG 12-09 trial were randomly assigned to a screening and a refinement and validation cohort. The cohorts were divided according to azacytidine-containing induction regimens and response status. Methylation status was assessed for 664,227 500-bp-regions using methyl-CpG immunoprecipitation-seq, resulting in 1755 differentially methylated regions (DMRs). Top regions were distilled and included genes such as WNT10A and GATA3. 80% of regions identified as a hit were represented on HumanMethlyation 450k Bead Chips. Quantitative methylation analysis confirmed 90% of these regions (36 of 40 DMRs). A classifier was trained using penalized logistic regression and fivefold cross validation containing 17 CpGs. Validation based on mass spectra generated by MALDI-TOF failed (AUC 0.59). However, discriminative ability was maintained by adding neighboring CpGs. A recomposed classifier with 12 CpGs resulted in an AUC of 0.77. When evaluated in the non-azacytidine containing group, the AUC was 0.76. Conclusions Our analysis evaluated the value of a whole genome methyl-CpG screening assay for the identification of informative methylation changes. We also compared the informative content and discriminatory power of regions and single CpGs for predicting response to therapy. The relevance of the identified DMRs is supported by their association with key regulatory processes of oncogenic transformation and support the idea of relevant DMRs being enriched at distinct loci rather than evenly distribution across the genome. Predictive response to therapy could be established but lacked specificity for treatment with azacytidine. Our results suggest that a predictive epigenotype carries its methylation information at a complex, genome-wide level, that is confined to regions, rather than to single CpGs. With increasing application of combinatorial regimens, response prediction may become even more complicated.

Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners ( MNX1 and ETV6 ) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6 . The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key points Expression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) MNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1 :: ETV6 oncofusion gene.