2018 David F. Silbert Summer Fellowship Research

Summer research project abstract

Mutant U2AF1(Q157P) expression alters hematopoiesis in vivo
Zhu M; Alberti MO; Walter MJ

Introduction: Myelodysplastic Syndromes (MDS) are the most common myeloid malignancies among the elderly. Mutations of U2AF1, a spliceosome gene, are identified in 11% of MDS patients. The two most common mutants, S34F and Q157P, have been shown to alter the splicing of two distinct sets of pre-mRNA targets in vitro and are hypothesized to effect MDS pathogenesis differently. While U2AF1(S34F) causes MDS-like phenotypes such as reduced B-cell numbers, no such study has been done on U2AF1(Q157P). We aim to study the U2AF1(Q157P) hematopoietic phenotypes in vivo using transgenic mice that inducibly express mutant U2AF1(Q157P) or U2AF1 wild-type (WT).

Methods: We set up non-competitive BM transplants to understand how U2AF1(Q157P) alters peripheral blood (PB) lineages, and competitive transplants to examine the stem cell function of U2AF1(Q157P) BM. In the non-competitive model, BM from U2AF1(Q157P)/rtTA or U2AF1(WT)/rtTA doubly transgenic mice was transplanted into lethally irradiated congenic WT mice. In the competitive model, “test” BM from U2AF1(Q157P)/rtTA or U2AF1(WT)/rtTA mice was pooled 1:1 with “competitor” wild-type BM and transplanted into lethally irradiated WT mice. Six weeks were allowed for engraftment before transgene induction. PB was drawn from recipients at 6 and 12 weeks post-transplant for flow cytometry.

Results: In non-competitive transplants, PB lineage distribution was unaffected at 12 weeks post-transplant. Notably,
U2AF1(Q157P) recipients did not have reduced B-cells compared with U2AF1(WT) recipients (46.9% vs. 50.7%, p=0.235). In competitive transplants, U2AF1(Q157P) BM recipients showed lower levels of “test” BM-derived cells than U2AF1(WT) recipients in total PB, B-cells, T-cells, granulocytes, and monocytes, indicating reduced stem cell function in U2AF1(Q157P) BM.

Conclusions: Hematopoietic expression of U2AF1(Q157P) causes a multi-lineage competitive disadvantage of BM stem cells, similar to previous reports on U2AF1(S34F). However, at this early time-point, PB counts are not affected, indicating that the two common U2AF1 mutations Q157P and S34F produce different hematopoietic phenotypes and may have different roles in MDS pathogenesis.

Summer research project abstract

Characterizing the role of CHD4 in glioblastoma treatment response
LeClair JW; McKenzie LD; Chheda MG

Introduction: Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. Most patients die within 2 years of initial diagnosis and nearly all patients have tumor recurrence, which is primarily caused by resistance to DNA damaging treatments. Prior work from our lab demonstrated that CHD4, an ATPase and member of the nucleosome remodeling and deactetylase (NuRD) complex, drives a component of this resistance by repairing double stranded DNA breaks, and may do so by regulating RAD51, a protein involved in homologous recombination. The purpose of this research is to examine how CHD4 protein levels change in response to external DNA damage and whether or not CHD4 suppression reduces cell viability in different genetic and treatment contexts.

Methods: GBM cells were exposed to radiation and temozolomide and CHD4 protein levels were measured at different time points after treatment. Treatment sensitivity assays in GBM cells were performed by counting viable cells after CHD4 suppression with a lentiviral shRNA and treatment with temozolomide or radiation. Cell viability assays were performed in immortalized astrocytes by counting viable cells after CHD4 suppression in cells with either an IDH1WT or IDH1R132H plasmid added.

Results: CHD4 protein levels increase in GBM cells in response to external DNA damage from both temozolomide and radiation. In treatment sensitivity assays, CHD4 suppression sensitizes GBM cells to radiation treatment but not to temozolomide treatment. Additionally, there is no added reduction in the viability of IDH1R132H mutant immortalized human astrocytes compared to wild-type immortalized astrocytes after suppression with CHD4.

Conclusions: These findings reinforce prior work that demonstrated that CHD4 is critical in the DNA damage response and suggest that suppression of CHD4 could be used alongside radiation to improve sensitivity to treatment and reduce recurrence. Our results also indicate that there are cell and treatment specific contexts of CHD4 suppression among GBM and immortalized astrocyte cell lines and these need to be studied further to maximize therapeutic benefit.