SETD2 mutations do not contribute to clonal fitness in response to chemotherapy in childhood B cell acute lymphoblastic leukemia.

Mutations in genes encoding epigenetic regulators are commonly observed at relapse in B cell acute lymphoblastic leukemia (B-ALL). Loss-of-function mutations in SETD2, an H3K36 methyltransferase, have been observed in B-ALL and other cancers. Previous studies on mutated SETD2 in solid tumors and acute myelogenous leukemia support a role in promoting resistance to DNA damaging agents. We did not observe chemoresistance, an impaired DNA damage response, nor increased mutation frequency in response to thiopurines using CRISPR-mediated knockout in wild-type B-ALL cell lines. Likewise, restoration of SETD2 in cell lines with hemizygous mutations did not increase sensitivity. SETD2 mutations affected the chromatin landscape and transcriptional output that was unique to each cell line. Collectively our data does not support a role for SETD2 mutations in driving clonal evolution and relapse in B-ALL, which is consistent with the lack of enrichment of SETD2 mutations at relapse in most studies.

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

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.