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.
ARTICLE ABSTRACTThe 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.
NSD2 EK mutation leads to drug resistance and a clonal advantage in childhood B-ALL.