Search

RNAseq Gene Lists

S1. Schematic diagram of NSD2 isoform indicating domains, the location of amino acid E1099 within the SET domain, and the targets of the shRNAs. S2. NSD2 Overexpression in B-ALL Cell Lines Does Not Impart Clonal Advantage. S3. Overexpression of REIIBP in B-ALL Cell Lines Does Not Impart Clonal Advantage. S4. Knockdown of WT NSD2 in B-ALL Cell Lines Does Not Lead to Phenotypic Differences. S5. Knockdown of NSD2 in RS4;11 and RCH-ACV lines leads to increased chemosensitivity. S6. Sensitivity to 6-MP Upon Knockdown of NSD2 in RS4;11 is Not Depedent of Level of Knockdown. S7. Knockdown of NSD2 in WT B-ALL Cell Lines Does Not Lead to Increased Sensitivity. S8. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S9. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Epigenetic Signature Resulting in Gene Expression Changes. S10. NSD2 Knockdown in Mutant B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S11. ATAC peaks significantly correlate with gene expression for RS4;11 and RCH-ACV cell lines.

Cell line IC50 values

Supplementary materials and methods and supplementary figure legends.

Although B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children and while highly curable, it remains a leading cause of cancer-related mortality. The outgrowth of tumor subclones carrying mutations in genes responsible for resistance to therapy has led to a Darwinian model of clonal selection. Previous work has indicated that alterations in the epigenome might contribute to clonal selection, yet the extent to which the chromatin state is altered under the selective pressures of therapy is unknown. To address this, we performed chromatin immunoprecipitation, gene expression analysis, and enhanced reduced representation bisulfite sequencing on a cohort of paired diagnosis and relapse samples from individual patients who all but one relapsed within 36 months of initial diagnosis. The chromatin state at diagnosis varied widely among patients, while the majority of peaks remained stable between diagnosis and relapse. Yet a significant fraction was either lost or newly gained, with some patients showing few differences and others showing massive changes of the epigenetic state. Evolution of the epigenome was associated with pathways previously linked to therapy resistance as well as novel candidate pathways through alterations in pyrimidine biosynthesis and downregulation of polycomb repressive complex 2 targets. Three novel, relapse-specific superenhancers were shared by a majority of patients including one associated with S100A8, the top upregulated gene seen at relapse in childhood B-ALL. Overall, our results support a role of the epigenome in clonal evolution and uncover new candidate pathways associated with relapse. Significance: This study suggests a major role for epigenetic mechanisms in driving clonal evolution in B-ALL and identifies novel pathways associated with drug resistance.

The NSD2 p.E1099K (EK) mutation is observed in 10% of acute lymphoblastic leukemia (ALL) samples with enrichment at relapse indicating a role in clonal evolution and drug resistance. To discover mechanisms that mediate clonal expansion, we engineered B-precursor ALL (B-ALL) cell lines (Reh, 697) to overexpress wildtype (WT) and EK NSD2, but observed no differences in proliferation, clonal growth, or chemosensitivity. To address whether NSD2 EK acts collaboratively with other pathways, we used short hairpin RNAs to knockdown expression of NSD2 in B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM). Knockdown resulted in decreased proliferation in all lines, decreased clonal growth in RCH-ACV, and increased sensitivity to cytotoxic chemotherapeutic agents, although the pattern of drug sensitivity varied among cell lines implying that the oncogenic properties of NSD2 mutations are likely cell context specific and rely on cooperative pathways. Knockdown of both Type II and REIIBP EK isoforms had a greater impact than knockdown of Type II alone, suggesting that both SET containing EK isoforms contribute to phenotypic changes driving relapse. Furthermore, in vivo models using both cell lines and patient samples revealed dramatically enhanced proliferation of NSD2 EK compared with WT and reduced sensitivity to 6-mercaptopurine in the relapse sample relative to diagnosis. Finally, EK-mediated changes in chromatin state and transcriptional output differed dramatically among cell lines further supporting a cell context–specific role of NSD2 EK. These results demonstrate a unique role of NSD2 EK in mediating clonal fitness through pleiotropic mechanisms dependent on the genetic and epigenetic landscape. Implications: NSD2 EK mutation leads to drug resistance and a clonal advantage in childhood B-ALL.

CTCF and cohesin play a key role in organizing chromatin into topologically associating domain (TAD) structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma, a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2, resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, here we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A logistic regression model reveals that differentially expressed genes are significantly enriched within the same insulated domain as altered H3K27ac and CTCF peaks. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation., In multiple myeloma, a 4;14 translocation induces overexpression of histone methyltransferase NSD2, resulting in expansion of H3K36me2 and shrinkage of H3K27me3 domains. Here the authors find that CTCF, H3K27ac and gene expression changes cluster within a subset of insulated domains implicating 3D chromosome organization as a key factor in the NSD2-mediated phenotype.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low input, low sequencing depth method, EpiMethylTag that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

Introduction: Outcomes for children with relapsed acute lymphoblastic leukemia (ALL) remain poor. Mutations in epigenetic modifiers are commonly found at relapse, including the activating p.E1099K (EK) mutation within the conserved SET domain of NSD2, which has been reported in 10% of patients at relapse. NSD2 has 3 isoforms, two of which (Types II and REIIBP) contain the SET domain. Our aim was to determine the role of NSD2 in driving relapse and further elucidate the prevalence of NSD2 EK in patient samples. Methods: Whole genome/exome sequencing was performed on diagnosis/relapse ALL pairs and data were aggregated for somatic SNVs/indels. NSD2 EK positive cases at relapse were subject to ultra-deep targeted re-sequencing to determine the presence of small subclones at diagnosis. To investigate the role of NSD2 and its isoforms in mediating relapse, short hairpin RNAs (shRNAs) were used to knockdown (KD) NSD2 in three NSD2 EK mutant cell lines with non-targeting (NT) shRNA used as controls. Cells were treated with chemotherapy commonly used in pediatric ALL and assessed for cytotoxicity by CellTiter- Glo®. To determine NSD2's impact on gene expression and the epigenetic landscape, RNAseq, ChIPseq, ATACseq, and Hi-C were performed using standard techniques. Results: Examination of paired ALL patient samples revealed enrichment of NSD2 EK clones at relapse. B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM) revealed that KD led to decreased proliferation in all lines and decreased clonal growth in RCH-ACV relative to NT. Furthermore, increased sensitivity to various chemotherapeutic agents was observed in the KD lines compared to NT controls, but the pattern of drug sensitivity varied among lines implying cell context specific drug responses. Simultaneous knockdown of Type II and REIIBP had a greater impact than Type II alone, indicating both SET containing EK isoforms contribute to phenotypic changes. When comparing gene expression changes in NT vs KD, minimal overlap in differential genes was noted among the three cell lines (4.4% upregulated, 1.7% downregulated) indicating significant diversity in transcriptional reprogramming. ATACseq of the NT and KD for all three lines revealed intergenic regions as the most affected by NSD2 with RCH-ACV and RS4;11 NT lines having more open chromatin compared to their KD whereas SEM NT displayed marked peak loss compared to its KD. Less than 2% of differential intergenic regions were shared among the lines. Hi-C analysis demonstrated disparity in chromosome architecture among NSD2 EK lines as well. Shifts in intergenic regions was further supported by ChIPseq, which revealed increased H3K36me2 across intergenic regions in RS4;11 NT compared to KD. Lastly, we noted a strong correlation of increased gene expression with increased H3K36me2, H3K9ac, and H3K27ac at promoters and gene bodies. Conclusions: NSD2 EK plays a unique role in mediating clonal fitness through pleiotropic mechanisms that are dependent on the underlying cellular context. Citation Format: Joanna Pierro, Jason Saliba, Sonali Narang, Gunjan Sethia, Shella Saint Fleur-Lominy, Ashfiyah Chowdhury, Takaya Moriyama, Kjeld Schmiegelow, Jun J. Yang, Mignon L. Loh, Patrick A. Brown, Jinghui Zhang, Xiaotu Ma, Aristotelis Tsirigos, Nikki A. Evensen, William L. Carroll. The NSD2 p.E1099K mutation is enriched at relapse and confers drug resistance in a cell context dependent manner in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5399.

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution., A fluorescent reporter system reveals that copy number variants (CNVs) are repeatedly generated and selected during the early stages of adaptive evolution, resulting in initially predictable dynamics with thousands of independent CNV-containing lineages competing within populations., Author summary Duplications and deletions of genomic sequence, known as copy number variants, are a common source of genetic diversity across all domains of life. Copy number variants play a crucial role in driving evolutionary processes but can also cause genetic disease and cancer. Although copy number variants are important drivers of diversity, adaptation, and disease, the underlying dynamics of their formation and selection are poorly understood. Copy number variants are difficult to detect, especially when present at low frequencies in heterogenous evolving populations. To overcome this challenge, we developed a novel fluorescent reporter that allows us to visualize copy number variants as they emerge and to track them throughout hundreds of generations of laboratory evolution. We show that copy number variants arise early and repeatedly, that they are diverse in size and copy number, and that they are generated at a high rate, leading to competition among cells containing different copy number variants. Molecular characterization of copy number variants indicates that many of them are likely generated by errors during DNA replication. This method is broadly applicable to studying the molecular mechanisms underlying formation of copy number variants, as well as their role in driving evolutionary processes and cancer.

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.