P45 high-dose carfilzomib achieves superior anti-tumor activity over low-dose and recaptures response in relapsed/refractory multiple myeloma resistant to low-dose carfilzomib by co-inhibiting the b2 and b1 proteasome subunits

The proteasome is a multi-subunit complex responsible for intracellular protein degradation, while only 3 subunits harbor proteolytic activity, the. β1, β2, and β5 subunits. Carfilzomib (CFZ), a second-generation proteasome inhibitor (PI), induces cell death in multiple myeloma (MM) by selective and irreversible β5 inhibition. Currently, the optimal CFZ dosing is still controversial, with the approved dosage ranging from 20 to 70mg/m2 in different regimens. Moreover, it remains to be explored whether high-dose CFZ can achieve superior anti-MM efficacy over low-dose and recapture response in relapsed/refractory (RR) MM patients progressing under low-dose CFZ. To address these issues, we analyzed the clinical data and the inhibition profiles of proteolytic proteasome subunits β1, β2, and β5 of RRMM patients treated with different CFZ doses. We prospectively collected clinical data and peripheral blood mononuclear cells (PBMC) of 103 patients with RRMM before and 3 hours after CFZ. PBMC were lysed and labelled for the activity of individual proteasome subunits using activity based proteasome probes and the proteasome subunits were separated using SDS-PAGE. The activity of constitutive and immunoproteasome β1, β2 and β5 subunits was evaluated by densitometry analysis. We then investigated the clinical data of 114 patients treated with CFZ combinations. Overall, 23, 27, 38, and 15 patients received 20, 27, 36, and 56 mg/m2 of CFZ, respectively. β5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas β2 and β1 were co-inhibited only by 36 and 56 mg/m2, respectively. Co-inhibition of β2 (P=0.0001) and β1 activity (P=0.0005) differed significantly between high-dose (36 or 56 mg/m2) and low-dose (20 or 27 mg/m2) CFZ. Subsequently, high-dose CFZ showed significantly more effective proteasome inhibition than low-dose drug in vivo (P=0.0003). We then investigated the clinical data of 114 MM patients treated with CFZ combinations Kd, KRD, and D-Kd. In the entire group, high-dose CFZ demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose. In the subgroup analysis of Kd, high-dose CFZ likewise showed improved PFS over low-dose (P=0.0006). In patients treated with KRD, PFS was significantly longer in patients who had received high-dose CFZ than low-dose (P=0.02), while lenalidomide dose did not affect PFS in our cohort. In light of this finding, we escalated the dose of CFZ to ≥ 36 mg/m2 in 16 patients who progressed during low-dose CFZ-containing therapies, and the doses of agents other than CFZ in the combination regimens remained the same. High-dose CFZ recaptured response (≥ partial remission) in 9 (56%) patients with a median PFS of 4.4 months. Here, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose CFZ (≥ 36 mg/m2) differs from that of low-dose CFZ by co-inhibition of β2 and β1 proteasome subunits and, consequently, high-dose CFZ achieves a superior anti-MM effect than low-dose and recaptures response in RRMM being resistant to low-dose CFZ.