The Proteasome Inhibitor Carfilzomib Functions Independently of p53 to Induce Cytotoxicity and an Atypical NF-κB Response in Chronic Lymphocytic Leukemia Cells

Abstract

Purpose The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2 and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. Experimental Design Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction and electrophoretic mobility shift assays. Additionally, a p53 dominant-negative construct was generated in a human B-cell line. Results Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells vs. CLL. and the cytotoxicity of CFZ correlates with baseline CT-L activity. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B-cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IkBα, phosphorylation of IκBα and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. Conclusions Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support Phase I investigation of CFZ in this disease.

PI3KCA plays a major role in multiple myeloma and its inhibition with BYL719 decreases proliferation, synergizes with other therapies and overcomes stroma-induced resistance

“…Previous studies have shown that BM stroma induced resistance to carfilzomib in Waldenstrom Macroglobulinaemia and in CLL (Gupta et al, 2013). Therefore, we tested the effect of BM stroma on the sensitivity of MM cells to carfilzomib, and here we first report that, similar to other haematological malignancies, BM stroma induced drug resistance to carfilzomib.…”

Proteasome inhibitor carfilzomib complements ibrutinib’s action in chronic lymphocytic leukemia

“…These observations are consistent with the report that carfilzomib is less cytotoxic to normal lymphocytes than CLL cells. 9 We next used western blot analysis to assess the effects of carfilzomib treatment on CLL cells isolated before and after ibrutinib treatment from 2 patients, 1 with therapy-related lymphocytosis (CLL-967) and 1 without (CLL-630). Cells from both patients responded in a similar manner to carfilzomib treatment, irrespective of ibrutinib therapy, as indicated by the accumulation of polyubiquitinated proteins, p-IkBa, CHOP, and Noxa ( Figure 1G).…”

Phase I dose escalation trial of the novel proteasome inhibitor carfilzomib in patients with relapsed chronic lymphocytic leukemia and small lymphocytic lymphoma

“…(25) CLL cells exposed to increasing doses of carfilzomib showed an accumulation of pIκB and decreased IκB in the cytoplasm, along with nuclear accumulation of NF-κB. Similarly, p53 and select downstream targets of p53 such as p21, NOXA and PUMA were also found to be consistently up-regulated in patient CLL cells(24). …”

“…(19, 23) Carfilzomib was also shown to more potently induce apoptosis of CLL cells compared to bortezomib, and unlike bortezomib, was equally effective in media with human versus fetal bovine serum. (24) Furthermore, carfilzomib can induce cell death in bortezomib-resistant cells. (22) One potential mechanism of cell death is through the NF-kB pathway, which is well-established to promote survival signaling in CLL.…”