The NSD2 p.E1099K Mutation is Enriched at Relapse and Confers Drug Resistance in a Cell Context Dependent Manner in Pediatric Acute Lymphoblastic Leukemia (ChIP-Seq)

The NSD2 p.E1099K (EK) mutation increases methyltransferase activity and 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 the clonal expansion, we engineered 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 shRNAs 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 three lines, decreased clonal growth in RCH-ACV, and increased sensitivity to chemotherapeutic agents routinely used in ALL therapy, 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 to exert its effect. 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 to 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. Reh B-ALL cell lines engineered to overexpress WT or EK NSD2 were sequenced by ChIPseq; only one replicate was submitted for sequencing. RS4;11 B-ALL cell lines engineered to knockdown NSD2 (NT control and sh2 knockdown) were also sequenced; two replicated were submitted per cell line. The following histone marks were analyzed: H3K9ac, H3K27ac, H3K27me3 and H3K36me2. Input files were used as a reference.