Search

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

Acute lymphoblastic leukemia (ALL) is the most common diagnosed pediatric cancer. Despite improvements in chemotherapy that have increased the 5-year survival rate to close to 90%, 15-20% of these patients may relapse with the majority of such children succumbing to this disease. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biological processes mediated by mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in two B-ALL cell lines (RCH-ACV and SEM) and one T-ALL cell line (RPMI-8402) and inserted the E1099K mutation into three ALL cell lines (697, CEM, MOLT4). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells compared to cells in which the mutation is removed demonstrate enhanced cell growth, colony formation and migration. NSD2 mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system (CNS) in xenografts. The NSD2 mutation is found prominently in children who relapse early from therapy for ALL, and NSD2E1099K cells are particularly resistant to glucocorticoids (GC). Reversion of NSD2E1099K mutation to wild type NSD2 conferred glucocorticoid sensitivity to both B and T cell lines. GC response upon disruption of mutant NSD2 was accompanied by cell cycle arrest and apoptosis. Mice xenografted with NSD2E1099K cells were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2 wild-type cells led to significant tumor reduction and survival extension. RNA-Seq analysis showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2 wild-type cells. Furthermore, in NSD2 mutant cells, there was lower basal expression level of glucocorticoid receptor (GR) and GR levels were not significantly induced by GC. Accordingly, after treatment with GC, there was significantly less DNA-binding activity of the GR in NSD2E1099K cells than that of NSD2 wild-type cells. The key pro-apoptotic regulators Bim and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. In conclusion, these studies demonstrate that the NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by causing GC resistance. Future studies will determine how NSD2 which generally activates genes paradoxically blocks the ability of GC and the GR to induce critical pro-death genes. Disclosures Licht: Celgene: Research Funding.