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Background: Outcomes for children with relapsed B-ALL remain poor, in part due to genetic and epigenetic lesions that confer drug resistance to one or more classes of agents used in therapy. SETD2, an epigenetic modifier, commonly harbors loss of function mutations in relapsed pediatric B-ALL. Prior studies have demonstrated the tumor suppressor function of SETD2 in an AML model as well as its role in resistance to DNA damaging agents, but the role of SETD2 mutations in relapsed B-ALL is not yet understood.
Objective(s): Determine the role of relapse-specific SETD2 loss-offunction mutations in disease progression by measuring proliferation and drug sensitivity in isogenic B-ALL cell lines that recapitulate these mutations. Design/Method: A panel of isogenic B-ALL cell lines (697 and KOPN-8) were generated with knockout of SETD2 using the CRISPR/Cas9 system. Conversely, B-ALL cell lines already harboring SETD2 heterozygous mutations (REH and RCH) had SETD2 expression restored using an inducible vector system. Western blot analysis was used to confirm the relative expression of SETD2 and downstream markers of DNA damage response in engineered cell lines. Isogenic cell lines were plated with or without HEK 293 stromal cells and then exposed to vincristine, etoposide, cytarabine, prednisone, or mercaptopurine for 72 hours. Cell viability of cells plated without stroma was measured using CellTiter-Glo. Apoptosis of cells plated with stroma was measured using flow cytometric analysis of apoptosis markers Annexin V and 7AAD. Relative proliferation of all untreated cell lines were measured over 168 hours using an automated cell counter.
Result(s): 697 and KOPN-8 clones with either a SETD2 heterozygous mutation or compound heterozygous mutations exhibited similar rates of proliferation compared to their respective isogenic controls. The half-maximal inhibitory concentrations (IC50) of all chemotherapy agents were similar in mutant clones and their isogenic controls, regardless of the presence of stromal cells. REH and RCH with reexpression of SETD2 also had similar rates of proliferation and IC50 compared to their isogenic controls. No increase in DNA damage was observed upon knockout of SETD2.
Conclusion(s): Loss of SETD2 expression in B-ALL cell lines does not confer increased resistance to conventional chemotherapy agents, either in isolation orwhen grown in a stromal microenvironment. Conversely, re-expression of SETD2 in lines that already harbored SETD2 mutations does not restore chemosensitivity. We postulate that SETD2 deletions alone may not confer a clonal advantage, but may operate in collaboration with other genetic alterations in a cell context-specific manner