Therapeutic targeting of focal adhesion kinase (FAK) modulates oncogenic and immune pathways in myeloid progenitor cells expressing oncogenic Janus kinase 2-V617F.

Abstract

Abstract Introduction: Myeloproliferative neoplasms (MPNs) are clonal blood cancers marked by excessive production of mature myeloid cells. The JAK2-V617F (JAK2VF) mutation, present in >95% of polycythemia vera (PV) and ~50–60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF), drives constitutive JAK/STAT signaling and contributes to inflammation, thrombosis, and disease progression. Although JAK inhibitors (JAKi) such as ruxolitinib (Rux) provide symptom relief and reduce splenomegaly, they have limited disease-modifying activity, and resistance or relapse is frequent. Emerging evidence suggests focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase involved in integrin and growth factor signaling, may be modulated by JAK2VF. Yet, the precise interaction between FAK and JAK2VF remains unclear. Targeting this axis may improve JAKi efficacy and offer novel strategies to overcome resistance. Methods Murine 32D myeloid cells expressing erythropoietin receptor (EpoR) and wild-type JAK2 (32D-WT) or mutant JAK2VF (32D-VF) were cultured in RPMI-1640 with 10% FBS; 32D-WT cells additionally received erythropoietin. The JAK2VF-positive human cell line UKE-1 was used for validation. Protein expression and phosphorylation were analyzed by Western blot. Co-immunoprecipitation of 32D-WT and 32D-VF lysates assessed protein interactions. Apoptosis was measured by flow cytometry using Annexin V/ Pacific Blue staining. Drug synergy was evaluated using the Coefficient of Drug Interaction (CDI, CDI < 1 indicates synergy). Statistical significance was determined by an unpaired Student's t-test (p < 0.05). Results The JAK2VF mutation activates integrin β1 signaling, implicating FAK in MPN pathogenesis. While FAK's role in tumor survival is established in other cancers, its involvement in MPNs and interaction with JAK2VF remain undefined. We hypothesized a functional crosstalk between FAK and JAK2VF. Transcriptomic analysis (GSE54644; 93 MPNs, 11 healthy donors [HD]) revealed significant upregulation of FAK (PTK2) across PV, ET, and PMF subtypes compared to HD. Immunoblotting confirmed elevated phosphorylated FAK (Tyr397) in 32D-VF versus 32D-WT cells. Treatment with Rux (2 µM) reduced FAK phosphorylation, indicating JAK2VFdependent activation. Co-immunoprecipitation showed that JAK2VF and FAK coexist in a complex, supporting direct signaling interaction. Functionally, 32D-VF cells showed strong dependency on FAK activation for survival. Treatment with the FAK inhibitor defactinib (Def) significantly reduced viability in a dose-dependent manner (1–3 µM, 10–55%, p < 0.01). Rux (0.5 µM) also impaired viability. Combined Def (1–3 µM) and Rux (0.5 µM) treatment led to 20–85% loss of viability (p < 0.01), with synergy indicated by CDI < 1. These findings were independently confirmed in UKE-1 cells using colony formation assays, which demonstrated reduced clonogenic potential. Mechanistically, Def disrupted the p53-MDM2 regulatory axis, evidenced by increased p53 phosphorylation. This was accompanied by downregulation of c-Myc and MCL1, especially with combination treatment. FAK regulates immune checkpoints like PD-L2, and its inhibition synergizes with PD-1 blockade. JAK2VF also elevates PD-L1, suppressing T-cell activation. We therefore investigated whether the JAK2VF/FAK axis modulates PD-L1 expression using Rux (0.25 µM) and Def (1-3 µM). Consistent with previous reports, Rux treatment reduced PD-L1 expression. Interestingly, Def also reduced PD-L1 expression (20–30%, p < 0.05). Another immune checkpoint, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), which promotes T-cell exhaustion, was also reduced with Def (25–50% reduction, p < 0.01). Co-treatment with Rux and Def only modestly reduced TIM-3 expression. These findings suggest the JAK2VF/FAK axis potentially promotes immune evasion and may serve as a therapeutic target in MPNs. Conclusion Our findings establish FAK as a functional collaborator of JAK2VFkinase, promoting both cell survival and immune evasion in MPNs. We demonstrate that JAK2VF physically interacts with and activates FAK, revealing a previously unrecognised signalling axis. Targeting FAK with Def synergises with Rux, enhancing cytotoxicity and disrupting key survival pathways, including the p53-MDM2 axis. Additionally, FAK inhibition reduces immune checkpoint molecules PD-L1 and TIM-3. These findings support dual JAK2 and FAK inhibition as a novel therapeutic strategy in JAK2VF-positive MPNs.