DP133 | CIP2A IS IMPLICATED IN RUXOLITINIB RESISTANCE IN MYELOFIBROSIS

Although ruxolitinib (Ruxo) is the standard of care for patients (pts) with myelofibrosis (MF), therapeutic resistance develops in approximately 50% within 3-5 years, posing a clinical challenge. We investigated molecular mechanisms of Ruxo resistance, focusing on protein phosphatase 2A (PP2A) due to its broad role in cancer cells and evidence of downregulation by JAK2V617F, a key MF driver (PMID: 23926298). We compared mRNA level and protein activity (by enzymatic assay) of PP2A in 14 granulocyte samples of MF pts versus 5 healthy donors (HD), finding significantly lower baseline PP2A activity (3.55-fold, p<0.001) in MF pts despite the increase in the mRNA levels of its catalytic subunits (6.4- and 4.7-fold for PP2ACA and PP2ACB subunits, respectively). Given that PP2A activity is directly regulated by two inhibitors, SET and CIP2A, their expression was evaluated by real-time PCR and immunohistochemistry (IHC). CIP2A mRNA was significantly higher (5.7-fold, p=0.001) in MF pts vs. HD, while SET mRNA showed no change, confirmed by IHC analysis (5 BM biopsies). We then assayed PP2A and inhibitor levels at baseline (T0), response (T1), and loss of response (T2) in GN cells from 14 pts who received Ruxo. The levels of PP2A activity significantly increased at treatment response (2-fold, p=0.047, T1vs.T0) and returned to baseline at relapse (2-fold lower, p=0.005, T2vs.T1). Also, IHC revealed decreased Serine/Threonine phosphorylation in responding patients compared to baseline, unlike in pts who did not respond to Ruxo. Levels of CIP2A mRNA inversely correlated with PP2A activity, decreasing at T1 (1.6-fold, p=0.001 vs.T0) and increasing at T2 (2.2-fold, p=0.001 vs.T1), confirmed by protein levels with IHC. SET mRNA levels showed no significant changes. The role of CIP2A in response/resistance to Ruxo was also investigated in Ruxo-resistant cell lines derived from JAK2V617F-mutated SET2 and HEL. CIP2A knockdown induced by CIP2A siRNA effectively silenced protein expression and reversed resistance to Ruxo in these cell lines. While Ruxo was completely ineffective in parental resistant cells, cell growth was reduced by 50% upon CIP2A protein silencing using 0.25 and 1µM doses of Ruxo, respectively in SET2 and HEL cell lines (p<0.001 for SET2 and p=0.002 for HEL). These data suggest that PP2A is involved in resistance to Ruxo and its inhibition through CIP2A downregulation might reverse such resistance in MF patients who have lost treatment response.