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

Myeloproliferative neoplasms (MPNs) are clonal blood cancers marked by excessive production of mature myeloid cells. The JAK2-V617F (JAK2^VF) 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 JAK2^VF. Yet, the precise interaction between FAK and JAK2^VF remains unclear. Targeting this axis may improve JAKi efficacy and offer novel strategies to overcome resistance.

Murine 32D myeloid cells expressing erythropoietin receptor (EpoR) and wild-type JAK2 (32D-WT) or mutant JAK2^VF (32D-VF) were cultured in RPMI-1640 with 10% FBS; 32D-WT cells additionally received erythropoietin. The JAK2^VF-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).

The JAK2^VF 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 JAK2^VF remain undefined. We hypothesized a functional crosstalk between FAK and JAK2^VF. 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 JAK2^VFdependent activation. Co-immunoprecipitation showed that JAK2^VF 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. JAK2^VF also elevates PD-L1, suppressing T-cell activation. We therefore investigated whether the JAK2^VF/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 JAK2^VF/FAK axis potentially promotes immune evasion and may serve as a therapeutic target in MPNs.

Our findings establish FAK as a functional collaborator of JAK2^VFkinase, promoting both cell survival and immune evasion in MPNs. We demonstrate that JAK2^VF 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 JAK2^VF-positive MPNs.