Cancer Res Treat.  2022 Jan;54(1):301-313. 10.4143/crt.2020.1371.

Role of Roflumilast Combined with ESHAP Chemotherapy in Relapsed/Refractory Patients with Diffuse Large B-Cell Lymphoma

• Kim DY
• Nam J
• Chung Js
• Kim SW
• Shin HJ

• KMID: 2524611
• DOI: http://doi.org/10.4143/crt.2020.1371

Abstract

Purpose
There are unmet needs associated with the current treatment strategies for relapsed/refractory diffuse large B-cell lymphoma (DLBCL) due to the poor treatment outcomes of these strategies. Roflumilast, a selective phosphodiesterase-4 inhibitor used for treating chronic obstructive pulmonary disease, is effective against B-cell malignancy via phosphoinositide 3-kinase (PI3K)–activity suppression. We analyzed the effects of roflumilast combined with ESHAP (etoposide, cisplatin, methylprednisolone, and cytarabine) chemotherapy in experimental and clinical settings.
Materials and Methods
An in vitro study using lymphoma cell lines and a pilot study on relapsed/refractory DLBCL patients were conducted to investigate the effects and mechanism of the combination of roflumilast and chemotherapy. The complete response (CR), overall response rate (ORR), and 1-year progression-free survival (PFS) were analyzed.
Results
We found that roflumilast is efficient when combined with other chemotherapy drugs, especially cytarabine. Synergistic effects between these two drugs influence the translation of mammalian target of rapamycin and myeloid cell leukemia 1, resulting in apoptosis and inhibition of B-cell lymphoma proliferation. In clinical setting, the roflumilast group showed better rates of CR (46.2% vs. 34.6%), ORR (76.9% vs. 53.8%), and 1-year PFS (50.0% vs. 25.9%) compared with the control group, though not statistically significant. The roflumilast group showed a higher incidence of asthenia and gastrointestinal adverse events. However, grade 3 or 4 adverse events were similar in both groups.
Conclusion
We found that roflumilast, when combined with ESHAP chemotherapy, for relapsed/refractory DLBCL was clinically active and well tolerated. This combined treatment was able to suppress PI3K activity, which is correlated with the degree of clinical response.

Keyword

Relapsed/refractory DLBCL; Roflumilast; ESHAP; Chemotherapy; Cytarabine

Figure

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  Fig. 1 Synergistic effect of roflumilast with chemotherapy drugs. A PDE4B-high cell line, OCI-Ly1, was treated with was treated with roflumilast (0 or 40 μM) in combination with forskolin (0, 10, 20, or 40 μM) and one of the six chemotherapy agents (cytarabine, cisplatin, mitoxantrone, etoposide, carboplatin, ifosfamide) that are commonly used for salvage chemotherapy for diffuse large B-cell lymphoma. The treatment was conducted for 48 hours, as indicated and the MTS assay were performed to measure cell viability. The results are presented as mean±standard deviation and are representative of three independent experiments (*p < 0.05, One-way ANOVA). Frs, forskolin; Rf, roflumilast.

• 

  Fig. 2 Combination of roflumilast and cytarabine have synergistic effects on cell death. (A) Three PDE4B-high cell lines (Ly10, Ly1, and Ramos) were treated with forskolin (0, 10, 20, or 40 μM), cytarabine (0, 0.5, 1, or 2 μM) and roflumilast (0 or 40 μM) for 48 hours, as indicated and the MTS assay were performed to measure cell viability. The results are presented as mean±standard deviation and are representative of three independent experiments (*p < 0.05, One-way ANOVA). (B) To evaluate the levels of drug synergy, combination index (CI) values were estimated using the CompuSyn software. The percent inhibition (fraction affected, Fa) from drug combinations was compared with that from individual drugs. Synergy levels are as follows: < 0.1 very strong synergy; 0.1–0.3, strong synergy; 0.3–0.7, synergy; 0.7–0.85, moderate synergy; 0.85–0.90, slight synergy; 0.90–1.10, nearly additive; 1.10–1.20, slight antagonism; 1.20–1.45, moderate antagonism; and 1.45–3.30, antagonism. Cyt, cytarabine; Frs, forskolin; Rf, roflumilas.

• 

  Fig. 3 Roflumilast and cytarabine regulate the translation of mammalian target of rapamycin and myeloid cell leukemia 1 (MCL1). p4EBP1 and MCL1 protein levels were analyzed by western blotting. β-Actin was used as an internal control. Cyt, cytarabine; Frs, forskolin; Rf, roflumilast.

• 

  Fig. 4 Synergistic effect of roflumilast and cytarabine on mitochondrial membrane potential. (A) To identify the potential role of disruption of the mitochondrial membrane potential (MMP) in apoptosis, an MMP assay was performed. First, all three cell lines (Ly1, Ly10, and Ramos) were treated with forskolin/roflumilast (40 μM), cytarabine (0.5 μM), or their combination for 48 hours and stained with the membrane-permeant dye JC-1. Subsequently, mitochondrial states were observed using fluorescence microscopy. Red-stained cells indicate live cells and green-stained cells indicate dead cells. (B) To quantify the number of cells with MMP disruption, all stained cells were counted and the ratio of red-stained cells to all stained cells was determined. The ratio was normalized to the control (DMSO) group. The results are presented as mean±standard deviation and are representative of three independent experiments (*p < 0.05, One-way ANOVA). Frs, forskolin; Rf, roflumilast.

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  Fig. 5 Kaplan-Meier curves showing the progression-free survival (PFS) (A) and overall (OS) (B) of the patients.

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  Fig. 6 Swimmer plot of 13 patients who received ESHAP (etoposide, cisplatin, methylprednisolone, and cytarabine) chemotherapy combined with roflumilast.

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