Impeding DNA Repair Offers a Novel Possibility for Personalized Therapy in AML Patients, Who Are Carrier of the Growth Factor Independence 1 Variant “GFI1-36N”

Background: Growth factor independence 1 (GFI1) is a hematopoietic transcriptional repressor, which regulates proliferation and differentiation of hematopoietic stem cells (HSCs). GFI1 plays an important role in the self-renewal of healthy HSCs and the development of acute myeloid leukemia (AML). We have previously shown that the GFI1-36N variant (Serine 36 is replaced with Asparagine 36) is present in 3-5% of the healthy population and 9-12% in myelodysplastic syndrome (MDS) and AML patients. GFI1-36N carriers have a 1.6 fold increased risk of MDS and AML development, therefore GFI1-36N predisposes to the development of MDS and AML. Genomic instability is a key driver of leukemia and recent reports from our group have underscored an important role for GFI1 in DNA repair.

Aim: We investigated if the GFI1-36N variant affects the genomic integrity and DNA damage-repair mechanisms leading to leukemogenesis. We further aimed to exploit therapeutic vulnerabilities associated with the GFI1-36N variant by employing DNA repair inhibitors in GFI1-36N expressing leukemic and non-leukemic cells.

Methods: Using previously described murine models of human AML (GFI1-36S (WT/control) and GFI1-36N knock-in mice), we investigated, the influence of the GFI1-36N variant on genomic stability and molecular aberrations associated with DNA repair pathways. We, therefore, used Array-CGH, RNA-Seq, transposon mouse model, and performed functional DNA repair-assays to examine the influence of GFI1-36N variant on genomic alterations in murine models as well as primary MDS and AML patients samples. We have additionally performed proteome analyses in GFI1-36S and GFI1-36N leukemic cells. To validate our observations and hypotheses we performed a variety of in vitro and in vivo assays and further exploited the suitability of DNA-repair inhibitors to therapeutically target GFI1-36N expressing AML cells.

Results: In a serial transplantation murine model of human leukemia, the presence of the GFI1-36N variant was associated with a significant increase in chromosomal aberrations, including insertions, deletions, and single nucleotide polymorphisms as compared to GFI1-36S cells (50-fold, p< 0.01). Gene set enrichment analysis (GSEA) further identified that the GFI1-36N cells were negatively enriched for hallmark DNA repair gene set. In line with this observation, human GFI1-36N AML cells were also associated with a 3-4 times higher rate of cytogenetic aberrations.

On mRNA level, GFI1-36N was associated with altered expression and splicing of genes involved in DNA repair, cell cycle progression, and significantly correlated with changes in the expression of 44 DNA repair proteins, including methylguanine-methyltransferase (MGMT). MGMT was significantly downregulated both on mRNA and protein levels. The results were more pronounced upon irradiation of the cells. Supporting this observation, the ability to repair O6-MeG lesions via the MGMT pathway was significantly lower in GFI1-36N cells than control GFI1-36S cells (p<0.05). Furthermore, the comparison between differentially expressed genes and proteomics data identified an overlap of five DNA repair proteins involved in BER, two interesting candidates being Apex1 and Parp1.

Based on these findings and recent publications, we evaluated the suitability of Temozolomide (TMZ, an alkylating agent that induces O6-MeG lesions, repaired by MGMT and BER pathways) alone or in combination with Olaparib (a DNA repair inhibitor) in vitro and murine models of AML and primary human AML cells. When treated alone, a low dose of TMZ negatively affected proliferation and differentiation of murine GFI1-36N leukemic cells but not in non-leukemic cells and GFI1-36S leukemic cells (IC₅₀=38.01 vs 287.20 µg/ml). Furthermore, TMZ in combination with Olaparib (0.2 µM) selectively and synergistically targeted leukemic cells from murine models of human leukemia and primary human AML cells but not non-malignant GFI1-36N cells.

Conclusion: In summary, we here provide evidence for the GFI1-36N variant induced alterations of genomic integrity and DNA repair capacity of murine and human AML cells. The results could in-part explain the AML-predisposing function of the GFI1-36N variant. Furthermore, our data underscored the possibility of selectively targeting GFI1-36N leukemic cells using two well-established drugs, TMZ and Olaparib.