Thrombopoietin inhibition abrogates JAK2V617F clonal expansion in a murine model of myeloproliferative neoplasm Free

Background: Myeloproliferative neoplasms (MPN) are a heterogenous group of hematopoietic stem cell (HSC)-derived disorders resulting from somatic mutations that activate the thrombopoietin (TPO) receptor (MPL) signaling. MPL is the only hematopoietic growth factor receptor expressed on HSCs and is important for HSC homeostasis as well as megakaryocyte progenitor proliferation. JAK2 is essential for MPL signaling and activating JAK2 mutations such as JAK2V617F are common drivers of MPNs, resulting in constitutive MPL signaling. Sustained MPL/JAK2 signaling leads to megakaryocyte hyperplasia and clustering in bone marrow (BM) as well as excess circulating TPO which reinforces MPN HSC proliferation. Targeting the TPO/MPL/JAK2 axis may therefore be a therapeutic strategy for MPN. Here we determined the effect of reducing TPO gene (Thpo) expression using a Thpo antisense oligonucleotide (ASO) in murine MPN models compared to and in combination with the JAK1/2 inhibitor ruxolitinib (rux).

Methods: We used a transgenic JAK2V617F mouse model with MPL manipulation (PLoS One. 2020 Jun 1;15(6):e0232801), and unmanipulated transgenic JAK2V617F BM cells to examine the effect of THPO-ASO +/- rux on both the MPN phenotype in the steady state transgenic model and a competitive transplantion model. For BM transplantation, a 2:1 ratio of Ly5.1 and JAK2V617F BM cells were injected into Ly5.1 mice. Eight weeks after transplant, injections of ASO (20 mg/kg every 2 weeks) or saline were administered in combination with rux chow (2g/kg) or control chow for 24 weeks. Peripheral blood flow cytometry was performed to determine JAK2V617F clonal expansion. Peripheral blood cell counts, liver Thpo, and plasma TPO levels were measured. Quantification of megakaryocyte density and clustering was performed on femurs photographed at 20x. Eight mice (n=4/sex) per treatment group were studied.

Results: In JAK2V617F/Mpl mice, ASO reduced Thpo expression by 83% and plasma TPO by 100%. In the competitive transplant model at 24 weeks, mean liver THPO expression dropped by 68% with ASO and 72% with ASO + rux, but was unchanged with rux alone compared to saline. Plasma TPO decreased by 44% with ASO and 22% with ASO + rux while rux alone increased plasma TPO by 71%. At 24 weeks, mean platelet counts (+/-SD) were 1,836 + 1283x10³/μL in saline-treated mice and reduced by ASO (640 +/- 394x10³/μL, p=0.0038), rux (642 +/- 440x10³/μL, p=0.0009), and combination treatment (363 +/- 296x10³/μL, p<0.0001). Mean white blood cells (+/-SD) in saline-treated mice were 11 +/- 3x10³/μL and were reduced by ASO (6 +/- 2x10³/μL, p=0.0171), rux (3 +/- 1x10³/μL, p=<0.001), and combination treatment (2 +/- 0.3x10³/μL, p<0.0001). Mean percentage of circulating JAK2V617F leukocytes was 69% +/- 16 in saline-treated mice which was suppressed by ASO (39% +/- 15, p=0.0006) or ASO + rux (50% +/- 18, p=0.0206), but not rux alone (54% +/- 14, p=0.0937). Splenomegaly was unchanged by ASO treatment but was dramatically reduced by rux alone or ASO + rux. BM megakaryocyte density was 32 +/- 12 (mean +/-SD) in saline-treated mice and reduced by ASO (13 +/- 4, p<0.001), rux (12 +/- 4, p<0.001), and combination (7 +/- 2, p<0.001). BM megakaryocyte clusters were 4.9 +/- 1.6 in vehicle-treated mice and reduced by ASO (1.5 +/- 1.3, p<0.001), rux (1.2 +/- 0.9, p<0.001), and ASO + rux (0.4 +/- 0.3, p<0.001). MPL cell surface expression can be reduced in MPN, so we also evaluated the effect of THPO suppression in the transgenic JAK2V617F mouse heterozygous for Mpl knockout and found consistent results for improvement in BM megakaryocyte hyperplasia and clustering and spleen size. Finally, we evaluated the reversibility of platelet count reduction by ASO treatment in wild type mice with the TPO mimetic romiplostim. Treatment with romiplostim (300µg/kg, s.c.) rapidly reversed platelet count reduction induced by ASO administration.

Conclusions: In both steady state and transplant JAK2V617F-driven mouse MPN models, ASO, rux and ASO+rux treatments had consistent effects on blood counts, spleen size and BM histology. However, in the transplant model,Thpo-ASO treatment blunted JAK2V617F clonal expansion more effectively than rux alone or rux + ASO. This is possibly explained by elevated plasma TPO levels with rux treatment. These results suggest that targeting TPO may be an effective strategy to reduce JAK2V617F HSC clonal expansion, and may complement JAK2 inhibition in the treatment of MPN.