The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition

Abstract

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.

Figure 1

CSF3R^T618I causes a fatal disorder reminiscent of neutrophilic leukemia. Mice were transplanted with bone marrow expressing CSF3R^WT (n = 5) or CSF3R^T618I (n = 5). (A) CSF3R^T618I causes leukocytosis. Total WBCs were measured over time in both the WT and T618I groups using an animal blood counter (scil Vet ABC). (B) Leukocytosis of CSF3R^T618I is caused by granulocytic expansion. The percentage of granulocytes was measured using an animal blood counter and plotted over time. (C) CSF3R^T618I results in an expansion of the granulocyte lineage at the expense of lymphocytes. Flow cytometry analysis was performed on peripheral blood from CSF3R^WT and CSF3R^T618I mice over time. The percentage of granulocytes relative to B-cell and T-cell lineages was increased in CSF3R^T618I mice relative to CSF3R^WT mice. Gating schemes used for analysis are shown in supplemental Figure 1. (D) CSF3R^T618I mutation is uniformly lethal. For CSF3R^T618I mice, events represent mouse death (n = 3) or euthanization as a result of moribund appearance (n = 2). The CSF3R^WT mice were sacrificed at day 90 for histologic analysis; all mice appeared healthy. (E) CSF3R^T618I mice have increased levels of phospho-STAT3. Blood from CSF3R^WT or CSF3R^T618I mice was pooled and pSTAT3 levels were measured by phospho-flow cytometry. Mean fluorescence intensity (MFI) of pSTAT3 was 604.5 for CSF3R^WT and 1354.3 for CSF3R^T618I.

Figure 2

The JAK1/2 inhibitor ruxolitinib decreases WBC count and reduces splenomegaly in CSF3R^T618I mice. A second cohort of mice was transplanted with bone marrow expressing CSF3R^T618I (n = 18); treatment with 90 mg/kg ruxolitinib twice per day (n = 9) or vehicle (n = 9) was initiated at day 12 post transplant. (A) Treatment with ruxolitinib results in prompt reduction in WBC count. Peripheral blood was collected on all mice 1 to 2 times per week. (B) Ruxolitinib decreases spleen weight. Mice were sacrificed on day 21 or 24 to evaluate spleen size. (C) Representative images of spleens from ruxolitinib- or vehicle-treated mice at time of necropsy. (D) Ruxolitinib-treated mice have significantly increased body weight compared with vehicle-control mice. Body weight was recorded on day 24 post transplant.