CSF3R T618I Mouse Bone Marrow Transplant Model Of Neutrophilic Leukemia

We have recently identified mutations in CSF3R in the majority of patients with chronic neutrophilic leukemia (CNL). The most common mutation identified thus far is the T618I membrane proximal mutation, which confers rapid transformation capacity and ligand-independence to the receptor. We also observed less common mutations that result in truncation of the cytoplasmic domain of CSF3R. These truncation mutations, which have been studied in the context of severe congenital neutropenia progression to acute myeloid leukemia, lack the capacity for inducing disease in transgenic mouse models but enhance the progression to leukemia in the presence of a second genetic driver. The CSF3R T618I mutation transforms cells at a substantially faster rate than the truncation mutation, but the capacity of this mutation to induce a hematologic disorder in mice is not yet known. We created a CSF3R T618I mouse bone marrow transplant model, which resulted in a lethal myeloproliferative disorder characterized by high levels of granulocytes.

Mice were transplanted with donor bone marrow infected with a murine retrovirus expressing either wild type CSF3R or the CSF3R T618I mutation. White blood cell counts were measured 1-2 times per week for 90 days. The percentages of monocytes, granulocytes, T-cells and B-cells were analyzed by flow cytometery. The levels of pSTAT3 were measured by phospho-flow. Hematoxylin and eosin staining was performed on fixed and sectioned spleens, livers and bone marrow.

Mice transplanted with CSF3R T618I showed an initial transient granulocytic leukocytosis that normalized by 33 days post transplant. Subsequently, at day 47 the CSF3R T618I mice exhibited a dramatic and persistent rise in white blood cell counts. The elevated white blood cells were primarily mature granulocytes. By day 90, all of the mice transplanted with CSF3R T618I had died, while the mice transplanted with cells expressing wild type CSF3R did not exhibit morbidity or mortality. The granulocytes from CSF3R T618I mice also exhibited elevated levels of pSTAT3—a marker of JAK signaling downstream of CSF3R—relative to CSF3R wild type granulocytes. Histology revealed that both CSF3R wild type and T618I mice had hypercellular bone marrow, however the hypercellularity was more extreme in the CSF3R T618I mice. Mice harboring the CSF3R T618I and to a lesser extent wild type CSF3R exhibited infiltration of the spleen and liver by mature granulocytic cells and disrupted tissue architecture.

The CSF3R T618I mouse bone marrow transplant provides a tractable model of Neutrophilic Leukemia. In contrast to CSF3R truncation mutations, which do not cause leukemia in isolation, the T618I mutation causes a fatal expansion of granulocytes. This model provides a useful tool for studying the biology of granulocytes and myeloproliferative neoplasms.

Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Fleischman:Incyte: Speakers Bureau. Druker:Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other. Tyner:Incyte Corporation: Research Funding.