In Vitro Growth Inhibition, Target Modulation and Drug Synergy In Pediatric Leukemia By The Novel Proteasome Inhibitor Carfilzomib

Abstract Introduction Despite the considerable progress that has been made in past two decades, relapsed or refractory leukemia still remains a leading cause of death in children. Acute and long term toxicities prevent significant further intensification of current chemotherapeutic regimens. Hence, effective pre-clinical data on new agents and novel therapeutic approaches are urgently needed. Recent studies have established proteasome inhibition as a distinctive and effective way to induce cytotoxicity in tumor cells that have acquired resistance to conventional chemotherapy. Although they represent a major advancement in the treatment of various hematological malignancies, the first generation proteasome inhibitors exhibit measurable off-target toxicities and the eventual development of resistance. Carfilzomib (CFZ) is a selective proteasome inhibitor that is structurally distinct from bortezomib and has shown efficacy and favorable toxicity profile in multiple myeloma patients. Mechanistically, carfilzomib has been shown to irreversibly bind and inhibit the chymotrypsin-like activity of the 20S proteasome and to cause the accumulation of polyubiquinated proteins resulting in cell cycle arrest, apoptosis, and suppression of tumor growth. Methods A diverse panel of pediatric leukemia derived cell lines and primary leukemia specimens (n = 12) were used to evaluate CFZ induced in vitro cytotoxicity using the Alamar blue assay. These cell lines include leukemia with abnormal FLT3 (FLT3-ITD and over-expression), Bcr-Abl fusion, extremely high white blood cell count variants, mixed lineage leukemia and Juvenile Myelomonocytic Leukemia (JMML) cells that are hyper-stimulated with exogenous GM-CSF. A luminescent based technique that individually measures protease activities associated with the proteasome complex in cultured cells (Promega, Cell-Based Proteasome-Glo Assay) was used to evaluate the mechanism of CFZ activity in these cells. Drug combination studies were carried out with etoposide, cytarabine, sorafenib and mefloquine using Chou and Talalay methodology. Target modulation, induction of apoptosis and the modulation of autophagy were evaluated by Western Blot analysis of cells treated with CFZ at defined time periods. Results Carfilzomib induced effective cytotoxicity in all leukemia cells tested (IC50 mean = 7 nM, range = 0.2 – 10 nM). Cell based proteasome assays confirmed the targeted and specific activity of CFZ in these cells. Infant leukemia cells with FLT3 over-expression were highly sensitive to CFZ followed by cells with ITD. Primary JMML cells that showed high growth stimulation with GM-CSF were also significantly affected by CFZ (IC50 = 0.2 nM). Although the extent of drug synergy varied between AML and ALL cells, CFZ synergized with all four agents (Combination Index (CI) mean = 0.47, range = 0.2 – 0.9). Induction of apoptosis by CFZ was evidenced by the increase in the active fragments of caspase 7 and 8 and PARP cleavage. CFZ also modulated autophagy by showing concentration regulated changes in p62 and LC3B. However, this effect appears to be restricted to AML cells. In vitro clonogenic assays using normal human CD34+ cells showed that even at 10 nM concentration, CFZ has no detectable inhibition on erythroid or myeloid colony formation. Discussion Carfilzomib is a potent, selective and irreversible inhibitor of the ubiquitin-proteasome pathway in cancer cells and has shown an acceptable toxicity profile in adult clinical trials. Our current data substantiates its potential as an active anti-leukemic agent in currently difficult to cure pediatric leukemia subtypes. Furthermore, we provide evidence on useful drug combinations and target modulation data to characterize the molecular mechanisms and biological correlates of distinct proteasome inhibitors in pediatric leukemia. This information provides key primary data for further in vivo studies and to design effective early phase clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.

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