The NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance.
To uncover 3D ...chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction.
In spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.
Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous ...deletions in
, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether
deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down
in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold)
, as well as increased resistance to 6-mercaptopurine treatment
No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070
1722 fmol/μg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure.
knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant
adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.
The hypothalamus plays a central role in regulating appetite and metabolism. However, the gene networks within the hypothalamus that regulate feed intake and metabolism, and the effects of fasting on ...those pathways are not completely understood in any species. The present experiment evaluated global hypothalamic gene expression in newly hatched chicks using microarray analysis to elucidate genes and pathways regulated by feeding, fasting, and delayed feeding. Ten groups of chicks were sampled over four days post-hatch, including fed, fasted, and 48 h fasted followed by access to feed for 4 h, 24 h, and 48 h. Hypothalamic samples were collected for microarray analysis (n = 4). Expression patterns of selected genes were confirmed by quantitative real-time PCR. Pathway analysis of the microarray results predicted a network of genes involved in neuropeptide or neurotransmitter signaling. To confirm the functionality of this predicted gene network, hypothalamic neurons from fed and fasted chicks were isolated and cultured in the presence of neuropeptide Y, somatostatin, alpha-melanocyte stimulating hormone, norepinephrine, and L-phospho-serine. Results confirmed functional relationships among members of the predicted gene network. Moreover, the effects observed were dependent upon the nutritional state of the animals (fed vs. fasted).
Differences in gene expression (> or = 1.6 fold) were detected in 1,272 genes between treatments, and of those, 119 genes were significantly (P < 0.05) different. Pathway Miner analysis revealed that six genes (SSTR5, NPY5R, POMC, ADRB2, GRM8, and RLN3) were associated within a gene network. In vitro experiments with primary hypothalamic neurons confirmed that receptor agonists involved in this network regulated expression of other genes in the predicted network, and this regulation within the network was influenced by the nutritional status and age of the chick.
Microarray analysis of the hypothalamus during different nutritional states revealed that many genes are differentially regulated. We found that functional interactions exist among six differentially regulated genes associated within a putative gene network from this experiment. Considering that POMC, an important gene in controlling metabolism, was central to this network, this gene network may play an important role in regulation of feeding and metabolism in birds.
Introduction: While the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically, the prognosis for those who relapse remains poor. One of the most common alterations ...found at relapse is the p.E1099K missense change within the SET domain of NSD2, a histone methyltransferase that di-methylates histone 3 lysine 36 (H3K36). NSD2 has 3 isoforms, two of which, Type II (canonical) and REIIBP (C-terminal), contain the SET domain, and another, Type I (N-terminal), that does not. The p.E1099K mutation leads to increased enzymatic activity, but pathways leading to a clonal advantage are unknown in ALL.
Methods: We used short hairpin RNAs (shRNAs) to target knockdown of two combinations of NSD2 isoforms: shI/II targets Types I and II, shII/RE targets Type II and REIIBP. Three different B-cell lines (Reh, 697, and KOPN-8) with 2 wildtype (WT) copies of NSD2 were stably transduced with shII/RE. Two B-Cell lines, RS4;11 and RCH-ACV, heterozygous for the NSD2 p.E1099K mutation, were transduced with shI/II and shII/RE. As a control, each B-cell line was stably transduced with a scrambled non-targeting (NT) shRNA. NSD2 knockdown was confirmed by Western Blots.
Cell lines were treated for 5 days with chemotherapy agents commonly used in pediatric ALL treatments (mercaptopurine (MP), cytarabine, methotrexate, prednisone, and doxorubicin). Cytotoxicity was assessed by CellTiter- Glo® and significance between IC50s was determined by ANOVA and post hoc Tukey test. Cell proliferation was measured by cell counting with trypan blue. Cell cycle progression in RS4;11 lines was monitored with Edu staining and flow cytometry with and without exposure to MP.
Results: Similar to previously reported results, knockdown of NSD2 in the 3 WT B-cell lines had no effect on cell proliferation. However, shI/II reduced growth by 40% in RS4;11 and 20% in RCH-ACV, while shII/RE decreased proliferation by 45% in RS4;11 and 55% in RCH-ACV when compared to their NT control. In RS4;11, both shI/II and shII/RE led to a similar 10% decrease in cells progressing through S phase compared to NT, which could be due to either a slower progression through cell cycle or less cells entering the cell cycle.
Knockdown of NSD2 resulted in sensitivity to 6MP compared to NT in both RS4;11 and RCH-ACV lines. RS4;11 shII/RE had an IC50 3.2-fold more sensitive ( p<.01) and the RS4;11 shI/II IC50 was 1.25-fold more sensitive (NS) versus the NT control. Similarly, RCH-ACV shII/RE had an IC50 3.4-fold more sensitive (p<.01) and the RCH-ACV shI/II IC50 was 2.6-fold more sensitive (p<.01) compared to the NT control. No significant changes in drug sensitivity were noted for the 3 WT NSD2 knockdown B-cell lines compared to their NT controls.
During a 120 hour exposure to MP, 34% more RS4;11 shII/RE cells were arrested in the G phase than NT controls, while 26% more RS4;11 shI/II cells were arrested in G phase relative to NT controls. This result indicates MP exposure leads to a reduced percentage of knockdown cells able to progress through the cell cycle. Overall, simultaneously reduced expression of Type II and REIIBP had a greater effect of on cell proliferation and MP response compared to the co-reduction of Types I and II NSD2 in the p.E1099K heterozygous cell lines.
Conclusion: The p.E1099K mutation confers a growth advantage and resistance to MP, a cornerstone of ALL therapy. Concurrent reduction of Type II and REIIBP expression by shII/RE resulted in the largest impact on proliferation and MP sensitivity. Both of these isoforms include the SET domain containing the p.E1099K mutation, which indicates one or both isoforms could be responsible for changes in the chromatin state and other possible alterations that lead to a clonal advantage. Based on our findings, determining the mechanism of resistance to MP imparted by NSD2 p.E1099K is now a top priority.
No relevant conflicts of interest to declare.
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.
No relevant conflicts of interest to declare.
Background: While outcomes for pediatric patients with newly-diagnosed acute B-lymphoblastic leukemia (ALL) have dramatically improved over the past 20 years, relapse remains a major cause of ...treatment failure and death. Discovering the underlying biological pathways that lead to drug resistance and relapse is therefore a top priority. MMSET (NSD2 / WHSC1) encodes a histone methyltransferase that dimethylates histone 3 lysine 36 (H3K36me2) with subsequent decrease in histone 3 lysine 27 trimethylation (H3K27me3). Overexpression of MMSET, as in the t(4;14) translocation in multiple myeloma (MM), has been shown to affect proliferation, gene expression, chromatin accessibility and DNA damage repair. Mutations in the SET domain of MMSET resulting in a glutamate to lysine substitution at amino acid 1099 (p.E1099K) have been recognized as one of the most common mutations in relapsed ALL in children. While it has been shown that this leads to increased enzyme activity, the downstream effects of this mutation and the underlying mechanisms of tumor escape are poorly understood. Understanding the biological pathways affected by this mutation is key to discovering new therapies to improve outcomes.
Objective: To determine the role of the MMSET p.E1099K mutation in cell proliferation, drug resistance and gene regulation.
Design/Method: B-lineage REH and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) or mutant (p.E1099K) MMSET or an empty vector (EV) control. Western Blot analysis confirmed overexpression of MMSET as well as increase in H3K36me2 and decrease in H3K27me3 in overexpressed WT and mutant cell lines. The generated cell lines were then plated with and without stroma and evaluated for differences in apoptosis and proliferation in response to chemotherapeutic agents commonly used in the treatment of ALL (e.g. mercaptopurine, thioguanine, methotrexate, prednisone, cytarabine, etoposide, doxorubicin and L-asparaginase). Clonogenic growth in vitro was also assessed using MethoCultTM media. Differences in gene regulation in the REH cell lines were assessed by performing ChIPseq for CCCTC-binding factor (CTCF) and histone marks (H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3) and the results were correlated with RNAseq data.
Results: The transduced WT, mutant and EV cell lines did not display differences in proliferation, cell cycle regulation or adhesion. Moreover we were unable to detect any intrinsic drug resistance between the cell lines with respect to apoptosis or proliferation both with and without stroma in vitro . There was, however, a marked increase in clonogenicity in the cell lines overexpressing WT or mutant MMSET compared to the EV control (348% and 285% increased colony number, respectively) when plated in MethoCultTM media.
Significant differences in the epigenetic landscape were observed between WT and mutant vs. EV control. As previously described in t(4;14) MM cell lines, over 99% of peaks for the H3K27me3 and H3K36me2 were lost in both the overexpressed WT and mutant cell lines compared to the EV control. However, whereas the decrease in peaks in H3K27me3 represents an overall loss of the mark, the lower peak number for H3K36me2 is actually due to widespread enrichment of the mark throughout the genome.
Moreover, RNAseq data revealed significant differences in gene expression between the three cell lines. Notably, the overexpressed WT and mutant cell lines revealed 83 and 90 upregulated genes and 44 and 99 downregulated genes, respectively, compared to EV control. Notably, 70% of upregulated genes and 80% of downregulated genes were shared between cell lines. When compared with previously published data on t(4;14) MM cell lines, the genes upregulated by overexpressed WT or mutant MMSET appear to be unique to leukemia. Interestingly, ETV5, which is involved in transcriptional regulation , is upregulated in both our cell lines and MM cell lines.
Conclusion: Catalytically active MMSET rendered by the p.E1099K substitution in ALL leads to an altered chromatin landscape and distinct gene expression signature (distinct from the t(4;14)) but does not result in intrinsic drug resistance in vitro . The increased clonogenic potency may suggest an increased leukemic stem cell population but understanding the precise mechanism for clonal expansion and relapse is the subject of additional in vitro and in vivo experiments.
No relevant conflicts of interest to declare.
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