Investigating the Role of ZNF384 Rearrangements in Acute Leukemia

"Chromosomal rearrangements involving ZNF384 are the defining lesion in 5% of pediatric and adult B-cell acute lymphoblastic leukemia and tumors are characterized by aberrant myeloid marker expression. Additionally, ZNF384 rearrangements are the defining lesion in nearly half of pediatric B/myeloid mixed phenotype acute leukemia. These fusions juxtapose full-length ZNF384 to the N terminal portion of a diverse range of partners, most often, transcription factors or epigenetic modifiers. It has been shown that ZNF384-rearranged tumors have a distinct gene expression profile that is consistent between disease groups and N terminal partners. Genomic analyses of patient tumors has shown that ZNF384 fusions arise in hematopoietic stem cells and that expression of the fusion, but not the concomitant genetic alterations, results in lineage aberrancy, however, the mechanistic role of ZNF384 rearrangements has not been formally studied. The goal of this project was to investigate the role of ZNF384 rearrangements, together with concomitant genetic alterations, in leukemogenesis, with an emphasis on characterizing the role of cell-of-origin and lineage of the resulting leukemias. Additionally, I aimed to explore the mechanism that leads to a distinct gene expression profile and immunophenotype. Using viral overexpression and newly developed genetically engineered mouse models I have shown the effect of ZNF384 fusion expression at multiple stages of hematopoietic development in mouse and human systems. These experiments revealed the hematopoietic skewing toward immature, myeloid differentiation caused by expression of ZNF384 fusions. While expression of ZNF384 fusion oncoproteins in either mouse or human hematopoietic progenitors resulted in hematopoietic expansion, lineage skewing, and for some fusion partners, self-renewal in vitro; co-expression with common concomitant lesions, such as NRAS G12D, was necessary in order to develop a fully penetrant mouse leukemia in vivo. In contrast, ZNF384 fusions alone drive B/myeloid leukemia when expressed in human hematopoietic stem and progenitor cells and transplanted into NSG-SGM3 mice, highlighting the benefits of using human models to investigate human oncogenes. Importantly, these experiments confirm that hematopoietic stem cells are the most sensitive to cellular transformation by ZNF384 fusions. Mechanistic studies integrating gene expression and chromatin occupancy data revealed that fusions bind canonical ZNF384 sites with greater intensity than wild type protein. A subset of regions with increased fusion binding also had increased H3K27Ac and were intronic or intergenic suggesting they are putative enhancer regions. These findings support that ZNF384 fusions occur in an early hematopoietic stem or progenitor cell which leads to skewed hematopoiesis and leukemic transformation in the presence of additional lesions, proliferative stress, or cytokine stimulation. This is likely caused by inappropriate extended activation of stem and progenitor enhancer regions along with deregulation of lineage-specific genes by altered binding of ZNF384 fusions. Together, these results demonstrate an intersection of cell of origin and expression of fusion oncoproteins as necessary prerequisites for generating lineage ambiguous leukemia."

Distinct gene expression profiles in leukocortical demyelinated white and grey matter areas of Multiple Sclerosis patients

ABSTRACTDemyelination of the CNS is a prominent pathological hallmark of Multiple Sclerosis (MS) and affects both white (WM) and grey matter (GM). However, demyelinated WM and GM areas exhibit clear pathological differences, most notably the presence or absence of inflammation and activated glial cells in WM and GM, respectively. In order to gain more insight into the differential pathology of demyelinated WM and GM areas, we micro-dissected neighbouring WM and GM demyelinated areas as well as normal appearing matter from leukocortical lesions of human post-mortem material and used these samples for RNA-sequencing. Our data show that even neighbouring WM and GM demyelinated areas share only 10% overlap in gene expression, implying a distinct gene expression profile, which is extending to a specific glial cell related signature. We propose that, based on their distinct expression profile, pathological processes in neighbouring WM and GM are likely different which could have implications for the efficacy of current MS treatments.

Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the causative transcriptional mechanisms remain unclear. Here we used chromatin run-on sequencing to discover approximately 7,000 enhancers and 141 enhancer hotspots activated in FLC relative to non-malignant liver. Detailed bioinformatic analyses revealed aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also defined the genes most strongly associated with aberrant FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduced cell viability and/or significantly enhanced the effect of MEK inhibitor cobimetinib. These findings highlight new molecular targets for drug development as well as novel drug combination approaches.

Placental gene expression and antibody levels of mother-neonate pairs reveal an enhanced risk for inflammation in a helminth endemic country

In utero exposure to environmental factors can modify the development of allergies later in life whereby the mechanisms of the feto-maternal crosstalk still remain largely unknown. Murine studies revealed that inflammatory maternal signals elicited by chronic helminth infection within the placenta imprint a distinct gene expression profile related to the Vitamin-D-receptor (VDR)-inflammation-axis. We thus investigated whether pro- or anti- inflammatory immune responses as well as VDR and related gene expression within the placenta differ between women from helminth-endemic and non-endemic areas. A prospective pilot study was conducted in Munich, Germany (helminth non-endemic) and Lambaréné, Gabon (helminth-endemic). At delivery, clinical information alongside placenta tissue samples and maternal and cord blood were obtained for further laboratory analysis. Schistosoma haematobium infection was detected in 13/54 (23%) Gabonese women. RT PCR revealed significantly lower gene expression of VDR, Cyp27b1, Foxp3 and IL10 in Gabonese compared to German placentae as well as significantly lower levels of plasma IgG4 in newborns resulting in a significantly higher IgE/IgG4 ratio. These findings demonstrate that exposure in utero to different environments alters placental gene expression and thus possibly plays a role in the development and modulation of the immune system of the offspring.

Pre-existent adult sox10+ cardiomyocytes contribute to myocardial regeneration in the zebrafish

During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from pre-existing ones. It is unclear whether the heart is comprised of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identified a subset of pre-existent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded massively after cryoinjury. Genetic ablation of sox10+ cardiomyocytes severely impaired cardiac regeneration revealing that they play a crucial role for heart regeneration.

NSD2 Mutations in Pediatric ALL Leads to a Distinct Gene Expression Profile and Epigenetic Landscape That Is Cell Context Specific

Background: Outcomes for children with acute lymphoblastic leukemia (ALL) have dramatically improved, but survival for patients who relapse remains poor. Mutations in genes encoding epigenetic modifiers are present in the majority of patients at relapse. In particular, activating mutations in NSD2 (MMSET, WHSC1), namely the glutamate to lysine substitution at amino acid 1099 (p.E1099K), are among the most common such mutations in epigenetic regulators. NSD2 converts histone 3 lysine 36 (H3K36) into its dimethylated form (H3K36me2) which in turn leads to stereotactic inhibition of EZH2 mediated H3K27me3. We and others have established that this leads to changes in chromatin state and gene expression. However, the pathways by which this leads to a clonal advantage remains elusive. Design/Method: We previously reported that overexpression of wild-type (WT) and p.E1099K mutant (EK) NSD2 in B-ALL cell lines led to unique cell context specific chromatin alterations and altered gene expression but did not lead to changes in proliferation or intrinsic drug resistance in vitro (Pierro et. al. Blood 2017 130:2474). We reasoned that these observations could be explained by the need for cooperating pathways that together with NSD2 EK lead to a clonal advantage. Thus we modulated expression of NSD2 using short hairpin RNAs (shRNAs) in the B-ALL cell line RS4;11 which harbors a heterozygous NSD2 EK mutation (NSD2 low). As a control, RS4;11 was also stably transduced with a non-targeting shRNA sequence (NSD2 high). Knockdown of NSD2 as well as decrease in H3K36me2 in NSD2 low lines was confirmed by Western Blot. Differences in gene regulation in NSD2 low cells were assessed by ChIPseq for CTCF, H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3, and the results were correlated with RNAseq data. This data was then compared to RNAseq and ChIPseq data from REH and 697 NSD2 WT and EK overexpression cell lines in an effort to identify candidate genes or pathways preferentially regulated by the NSD2 EK mutation. Results: NSD2 low cells displayed a distinct gene expression profile compared to NSD2 high with 301 upregulated and 573 downregulated genes (LFC 0.58, P = 0.05). When compared to gene expression data from our previously reported NSD2 overexpression cell lines, there was minimal overlap across cell lines with only 15 differentially expressed genes shared between RS4;11 NSD2 knockdown and REH EK overexpression cell lines and only 24 genes shared between RS4;11 NSD2 knockdown and 697 EK overexpression cell lines. Across all cell lines (RS4;11, REH and 697), only three genes (NSD2, SCN8 and PCNXL2) overlapped, all of which were upregulated in NSD2 high cell lines. Using less stringent criteria (LFC 0.26, P = 0.1), we observed greater overlap with 34 shared up and downregulated genes among lines. Of the shared genes, only ZNF521 which is overexpressed in NSD2 high cell lines, is known play a role in leukemogenesis. Moreover, RS4;11 pathway analysis revealed several biologically relevant pathways modulated by the NSD2 EK mutation such as Ras, integrin signaling, cholesterol/steroid biosynthesis, apoptosis and cell proliferation. Significant differences were also observed across epigenetic marks between RS 4;11 NSD2 high and low cells. In accordance with previously published data, we observed a global decrease in the H3K36me2 mark in RS4;11 NSD2 low lines. When aligned with changes in histone marks, among genes downregulated in NSD2 low cells there was a clear correlation with acquisition of the repressive H3K27me3 mark (and a decrease in the H3K9Ac mark). However among genes upregulated in NSD2 low cells we saw paradoxical increases in the H3K36me2 mark and decreases in the H3K27me3. Furthermore, gene expression was also influenced by marks not directly regulated by NSD2, namely H3K27ac and H3K9ac, indicating that local NSD2 mediated epigenetic changes are not the sole regulator of gene expression. Conclusion: The activating p.E1099K substitution in NSD2 leads to a distinct gene expression profile in B-ALL cell lines that is cell context dependent. Moreover, while there is significant overlap in the transcriptional profile between WT and EK overexpression, there are distinct differences possibly indicating novel properties of the pE1099K substitution beyond enzyme hyperactivation. Our findings also imply that NSD2 EK collaborates with other leukemia associated alterations that result in clonal selection. Disclosures No relevant conflicts of interest to declare.