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

To the editor:

Ibrutinib, an orally bioavailable Bruton tyrosine kinase (BTK) inhibitor,¹  has shown significant clinical benefit for chronic lymphocytic leukemia (CLL) patients.^2,3  However, ibrutinib monotherapy often leads to partial responses and extended lymphocytosis,⁴  which led us to question whether combination therapy could provide complete clinical responses. To identify pharmacologic agents that complement ibrutinib therapy, we performed an ex vivo drug screen using targeted agents on CLL cells isolated from ibrutinib-treated patients (Table 1). We identified carfilzomib (PR-171), a second-generation proteasome inhibitor⁵  and ABT-199⁶  (a Bcl-2 antagonist) as the most cytotoxic agents, as indicated by increased annexin V/propidium iodide (PI) double positivity after 24-hour incubation (Figure 1A,⁷ ). Results were consistent in samples from 7 additional patients (data not shown). Carfilzomib cytotoxic effect after 24-hour treatment was confirmed in samples from 23 patients treated with ibrutinib for 2 to 23 weeks (Figure 1B).

Next, we evaluated carfilzomib and ibrutinib synergetic cytotoxic effect on ibrutinib-naive CLL cells during in vitro incubation. Cells isolated from 7 patients (Table 1) were treated for 16 hours with concentrations of ibrutinib and carfilzomib that resulted in modest toxicity individually but, when combined, showed at least an additive cytotoxic effect (Figure 1C). The response to single agents and the combination varied between patients. Western blot analysis of CLL cells isolated from 2 patients confirmed the inhibitory effect of carfilzomib on the proteasome machinery, as illustrated by the accumulation of polyubiquitinated proteins and the stabilization of a short-lived protein (ie, p-IκBα) in a dose-dependent manner (Figure 1D,⁸ ). Furthermore, carfilzomib induced a dose-dependent activation of caspase 3 and accumulation of cleaved PARP and Bcl-2 (Figure 1D), both of which were consistent with a previous study where CLL samples with 17p del also responded well to the cytotoxic effect of carfilzomib.⁹  Ibrutinib treatment resulted in decreased p-BTK at tyr-223, with a minor effect on caspase activation; however, when combined with carfilzomib, an enhancement of cleaved caspase 3 was notably observed with low doses of carfilzomib (Figure 1D). Notably, increasing concentrations of carfilzomib caused accumulation of the transcription factor CHOP and the proapoptotic BH3-only protein Noxa (Figure 1D), which is consistent with evidence that proteasome inhibition leads to activation of the endoplasmic reticulum stress response and accumulation of apoptotic regulators.^10,11  Interestingly, Noxa was previously shown to play a critical role in bortezomib-induced apoptosis in CLL; however, treatment with this reversible inhibitor did not result in increased of CHOP protein,¹²  suggesting different modes of action between bortezomib and carfilzomib. Here, ibrutinib cotreatment did not influence carfilzomib’s ability to induce accumulation of polyubiquitinated proteins, p-IκBα, Noxa, and CHOP (Figure 1D).

To compare carfilzomib-induced cytotoxic responses between untreated and ibrutinib-treated samples, we isolated CLL cells from 8 patients prior to and 4 weeks after the initiation of ibrutinib therapy, which was previously reported as the time line for maximum transient lymphocytosis.²  With ibrutinib therapy, 4 patients exhibited signs of lymphocytosis as previously reported² : 2 remained unchanged and 2 patients showed a >80% decrease in absolute lymphocyte count (ALC) (Figure 1E). No notable differences were observed in carfilzomib responses between CLL cells isolated before and after ibrutinib treatment, except 3 patients (CLL-967, CLL-864, and CLL-488) whose cells showed increased sensitivity to carfilzomib in ibrutinib-treated samples (Figure 1F). In contrast, the ibrutinib-treated sample from patient CLL-683 had a marked decrease in apoptosis (Figure 1F), possibly due to a >80% reduction in his ALC (Figure 1E), and similar results were observed in patient CLL-826, 12 weeks following ibrutinib treatment (Figure 1E). 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-IκBα, CHOP, and Noxa (Figure 1G). Additionally, carfilzomib treatment in both untreated and ibrutinib-treated cells showed caspase 3 activation and accumulation of cleaved PARP and Bcl-2 (Figure 1G). We extended these findings with cells isolated from 3 patients before and after 2, 4, or 12 weeks of ibrutinib treatment, respectively; in all cases, the cells exhibited decreased phosphorylation of BTK (Figure 1H). Furthermore, both the extrinsic and the intrinsic apoptotic pathways were activated by carfilzomib treatment, as indicated by increases in cleaved caspase 8 and caspase 9, respectively (Figure 1H), and to some extent the cells appeared more resistant to spontaneous endogenous apoptosis ex vivo following ibrutinib therapy.

In summary, this pilot study provides some foundation to further investigate carfilzomib-ibrutinib combination therapy for CLL.