Synergistic correction of polycythemia vera phenotype via dual targeting of JAK2 and Gas6 in Jak2 exon 12 mutant mice Free

Introduction: Polycythemia vera (PV) is a chronic BCR-ABL1-negative myeloproliferative neoplasm driven by JAK2 mutations, causing excessive red cell production. Despite treatments like phlebotomy, cytoreduction, and JAK2 inhibitors (e.g., ruxolitinib, RUX), disease control is often insufficient. Patients remain at risk for progression to myelofibrosis or acute leukemia, and for vascular complications such as thrombosis and bleeding.These complications significantly affect outcomes, yet their mechanisms remain poorly understood. PV mouse models closely replicate human disease and are useful for studying pathogenesis and evaluating new therapies.

Growth arrest-specific gene 6 (Gas6), a ligand of the TAM receptor family (Tyro3, Axl, Mertk), regulates processes relevant to PV pathogenesis, including erythropoiesis and hemostasis. Although dispensable under normal conditions, Gas6 becomes crucial during pathological stress, making the Gas6/TAM axis a potential therapeutic target in PV.

Aim: To assess the role of Gas6 in PV progression and vascular complications, and to evaluate whether Gas6 inhibition by batiraxcept can enhance the efficacy of RUX.

Methods:A mouse model carrying the most common JAK2 exon 12 mutation with inducible SclCre, with (Jak2Ex12) and without Gas6 (Gas6^-/-Jak2Ex12), was generated. PV was induced at 5–7 weeks of age with tamoxifen. Disease progression was assessed at 2, 4, and 6 weeks post-induction via full blood counts, spleen weight, and histological analysis. Erythroid differentiation in spleen and bone marrow was analyzed by flow cytometry (CD71, Ter119).

Bleeding was evaluated by saphenous vein puncture (SVP), and thrombus formation was assessed using FeCl₃-induced injury in the vena cava, monitored by high-frequency ultrasound. For therapeutic studies, Jak2Ex12 mice received either RUX alone or in combination with the Gas6 inhibitor batiraxcept for 4 weeks.

Results: Six weeks after PV induction, Jak2Ex12 mice showed pronounced erythrocytosis and splenomegaly compared to Gas6^-/-Jak2Ex12 mice, which had ~26% smaller spleens (wildtype, WT: 114±38 mg; Jak2Ex12: 919±86 mg, p= <0.001; Gas6^-/-Jak2Ex12: 678±32 mg, p=<0.001; n=4-12), indicating a milder phenotype. Histology confirmed reduced extramedullary hematopoiesis and better-preserved splenic architecture in Gas6^-/-Jak2Ex12 mice. RUX reduced spleen weight by 46%, while combination with batiraxcept led to a 72% reduction (680±85 mg p= 0.07 vs. 188±64 mg p=0.003; n=4-12), indicating superior efficacy. Combination therapy also corrected abnormal erythroid differentiation, reducing expanded progenitor populations (CD71^highTer119⁺ from 71.7±6% to 44±12% p=0.02; n=4-5) and normalizing the distribution of erythroid precursors (proerythroblasts: 6.6±0.6% to 4±0.7% p=0.02; basophilic erythroblasts: 29.2±1.2% to 13±4.9% p=0.02; polychromatic erythroblasts: 12.5±1% to 9.5±4.1%, p=0.2 and orthochromatic erythroblasts: 22.2±1% to 9±1.9%, p=0.02, erythrocytes: 25.8±3% to 62.4±11.3%, p=0.02; n=4-5)

In Jak2Ex12 mice, plasma Gas6 levels were lower than in WT but increased with RUX, suggesting that RUX may upregulate Gas6 expression or stability, potentially sustaining erythropoiesis (WT: 17±5 ng/mg; Jak2Ex12: 12±7 ng/mg, p= 0.03; Jak2Ex12+RUX: 27±5 ng/mg, p= 0.03; n=5-8).

As assessed by SVP, the average bleeding episode (ABE) was prolonged in both Jak2Ex12 and Gas6^-/-Jak2Ex12 mice compared to WT (65±57 sec, 56±47 sec, vs. 31±9 sec), accompanied by fewer hemostatic events (WT: 17±2; Jak2Ex12: 15±6; Gas6^-/-Jak2Ex12: 13±6). RUX normalized both parameters (ABE: 31±16 sec; HE: 16±1).

Preliminary thrombus analysis revealed reduced vessel occlusion in Jak2Ex12 mice treated with RUX (18 ± 8%) compared to those without RUX treatment (27 ± 20%). Ongoing studies are evaluating whether the addition of batiraxcept provides a greater reduction in thrombus formation than RUX alone.

Conclusion: Gas6 deletion mitigates PV, supporting its role in controlling disease proliferation. Batiraxcept plus RUX reversed splenomegaly and normalized erythroid differentiation. Persistent high hematocrit despite normalized reticulocytes suggests longer treatment is needed. Preliminary data suggest Gas6 also contributes to clotting abnormalities. Ongoing studies in the Jak2V617F model are evaluating the combined therapy's effects on erythropoiesis, thrombosis, and hemostasis.