DUSP1 Specific Protac Selectively Targets the JAK2 V617F Expressing Cells

Constitutive JAK-STAT signaling resulting from mutations in JAK2 V617F and certain growth-factor receptors has provided a strong rationale for targeting JAK2 kinase activity in myeloproliferative neoplasms (MPNs). Although several JAK2 inhibitors have been approved for MPN treatment, their clinical efficacy is limited to alleviating the constitutional symptoms associated with inflammatory cytokine signaling. These inhibitors are not cytotoxic and only exert a cytostatic response, failing to eradicate the malignant clone, induce molecular remission, or prevent progression to acute myeloid leukemia (AML). The inability of JAK2 TKIs to fully suppress the inflammatory cytokine overproduction, a key feature of MPN development and transition to AML, contributes to clonal persistence and TKI refractoriness. Recently, we reported that the cytokine signaling, in the context of JAK2 V617F signaling induces the expression of DUSP1, which enhances P53 degradation that leads to TKI refractoriness. Enhanced DUSP1 expression blocks P53 phosphorylation at Serine 15 (S15 phosphorylation prevents its interaction with MDM2) by inhibiting the P38 kinase activity. Consequently, reduced phosphorylation under JAK2 V617F signaling enhances P53 turnover. Deletion of DUSP1 enhances P38 kinase activity, leading to greater P53 stabilization and expression, which selectively induce cell death in JAK2 V617F expressing MPN cells while sparing normal cells. However, chemical inhibition of DUSP1 using a small molecule inhibitor (BCI) was ineffective in achieving synthetic lethality to JAK2 V617F cells, mainly due to pERK1/2 rebound caused by the off-target inhibition of DUSP6. Reintroduction of DUSP6 and BCI treatment restored JAK2 V617F clonal selectivity. This study provided the first mechanistic basis of TKI refractoriness in JAK2 targeted MPNs and suggested that selectively targeting DUSP1 could offer a stable or curative response in JAK2 V617F -driven MPNs. To address this, we developed a DUSP1-protac, a novel and highly potent DUSP1 selective PROTAC. Treatment of BaF3 cells expressing JAK2 V617F with DUSP1-protac resulted in dose- and time-dependent degradation of DUSP1, accompanied by increased P38 activity and P53 stabilization. The maximal level of DUSP1 degradation (Dmax) achieved was 95% with a DC50 of 70 nM/L within 24 hours. Pre-treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, reversed DUSP1 degradation, confirming that degradation occurs via the 26S-proteasome. As expected, DUSP1-protac induced cytotoxicity specifically in JAK2 V617F expressing cells, with a cellular IC50 of 50 nM. No toxicity was observed in BaF3 cells or those expressing MPL or CALR variants at concentrations up to 1000 nM, confirming its selectivity. In conclusion, our data suggest that DUSP1-protac is an exceptionally potent first-in-class DUSP1 selective PROTAC. It exerts a cytotoxic response specifically in JAK2 V617F expressing cells by enhancing P53 expression. Cellular uptake experiments and efficacy studies in primary cells are currently underway and will be presented in subsequent reports. The development of DUSP1-protac represents a promising approach for targeting JAK2 V617F -driven MPNs, offering potential benefits in achieving a stable or curative therapeutic response.