Abstract
CSF3R is the receptor for granulocyte colony stimulating factor and plays a critical role in the proliferation and maturation of granulocytes. Although rare in most hematologic malignancies, we have discovered CSF3R mutations in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) (Maxson et al, NEJM 2013). These mutations render the receptor constitutively active, leading to unrestrained neutrophil production. Although initially felt to be specific to CNL, through a collaboration with the National Cancer Institute/Children's Oncology Group TARGET Initiative, we identified a small fraction of patients with pediatric Acute Myeloid Leukemia (AML) that also have CSF3R mutations (Maxson et al, Blood, 2016). To interrogate this observation, we looked at co-occurring genetic lesions for the two malignancies. Interestingly, we found that the cooperating lesions are very different between the CNL and pediatric AML patient cohorts. For instance, ~25% of patients with CNL also have a mutation in SETBP1, and nearly half of patients with CSF3R-mutated pediatric AML have mutations in CEBPA. Furthermore, Lavallee et al identified CSF3R mutations as being specifically enriched within the cohort of AML patients with bi-allelic CEBPA mutations (Blood, 2016).
We therefore explored whether these co-occurring mutations might synergize with CSF3R mutations in primary mouse bone marrow cells. We found that when WT CEBPA is co-expressed with the CSF3R T618I mutation, it strongly suppressed mouse bone marrow colony formation, while a CEBPA mutant enhanced colony formation. In addition, mutated SETBP1, which leads to protein overexpression, was co-expressed with CSF3R T618I. Interesting, WT SETBP1 enhanced colony formation, with a further increase in colony number with the SETBP1 G870S mutant. These studies suggest that there is a synergistic relationship between the CSF3R T618I mutation and two commonly co-mutated genes in CNL and pediatric AML. Since the CSF3R T618I mutation is very strongly activating, we wanted to use a model in which combinations of mutations could be expressed at endogenous levels to further study their interactions at the transcriptional level. To this end we are using CRISPR-Cas9 to generate myeloid cellular models. Two CRISPR-Cas9 gene-editing techniques are being employed to generate our mutations of interest following a Cas9-generated double-stranded break: (1) homology directed repair to insert point mutations into the CSF3R and SETBP1 loci, and (2) non-homologous end joining which results in frameshift mutations in CEBPA. We have completed validation of guide RNAs and HDR templates in 293T17 cells, with successful generation of both frameshift and specific point mutations. We have applied these tools to HL-60 cells, a human myeloid leukemia cell line, with successful isolation of clones with frameshift mutations. Investigation of these combinations of mutations in cell lines will allow us to determine how they cooperate to regulate transcriptional programs and produce distinct cellular identities. Our hope is that the principles learned from these well-defined diseases will provide a conceptual framework that will help us understand other myeloid malignancies with more complex clonal architecture and mutation combinations.
No relevant conflicts of interest to declare.
