CD30 Downregulation, MMAE Resistance, and MDR1 Upregulation Are All Associated with Resistance to Brentuximab Vedotin

Abstract

Brentuximab vedotin (BV) is an antibody-drug conjugate that specifically delivers the potent cytotoxic drug monomethyl auristatin E (MMAE) to CD30-positive cells. BV is FDA approved for treatment of relapsed/refractory Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV-resistant disease. We selected for BV-resistant Hodgkin lymphoma (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (Hodgkin lymphoma) exposure to BV. We confirmed drug resistance by MTS assay and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. In addition, tissue biopsy samples from 10 Hodgkin lymphoma and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the Hodgkin lymphoma cell line, demonstrated downregulated CD30 expression compared with the parental cell line. In contrast, the Hodgkin lymphoma cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared with the parental line. For both Hodgkin lymphoma and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One Hodgkin lymphoma patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possible modes of BV resistance for Hodgkin lymphoma both in vitro and in patients.

Figure 1. BV-resistant ALCL and HL in vitro cell models

Proliferation experiments were performed in duplicate wells and averaged over 2 separate experiments, and a repeated measures ANOVA model is applied to show that time, group, and their interactions are all very significant (Panel A and B). Viable cells counts were performed by hemocytometer with methylene blue. Panel A: L428 and L428-R were seeded at 100,000 cells per well and incubated with BV at 80 μg/ml. L428-R cells are able to proliferate at this concentration whereas L428 can not. (p < 0.0001). Panel B: Karpas-299 and Karpas-R were seeded at 40,000 cells per well and incubated with BV at 30 ng/ml. Karpas-R cells are able to prliferate at this concentration whereas Karpas-299 can not (p < 0.0003). MTS assays were performed in triplicate wells and averaged over 3 separate experiments, and cells were seeded in 96-well plates at 5,000 cells (L428) and 10,000 cells (Karpas-299) per well. Panel C: A four-parameter log-logistic model was fitted to assess inhibitory effect of L428-P and L428-R, respectively. The estimated IC50 (standard error)s are 27.46 (4.76) and 236.08 (22.15) respectively. Panel D: A four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P and Karpas-R, respectively. The estimated IC50 (standard error)s are 0.0029 (0.021) and 19.45 (1.87) respectively.

Figure 2. BV-resistance ALCL and HL in vitro cell models

Panel A–D. Flow cytometry showing surface CD30 expression in Karpas P, Karpas-R, and Karpas-R CD30+ and Karpas-R CD30− cells after cell sorting. Panel E shows CD30 mRNA expression by qRT-PCR.(qRT-PCR performed with triplicate wells and repeated twice). A two-sample t-test showed that there are significant expression differences for comparisons Karpas-P vd Karpas-R (P-value = 0.003) and Karpas-R CD30+ vs Karpas-R CD30− (P-value = 0.002). Panel F: MTS assay were performed with triplicate wells and averaged over 3 experiments. A four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P, Karpas-R, Karpas-R CD30+, and Karpas-R CD30-, respectively. The estimated IC50 (standard error)s are 0.0058 (0.0071), 14.18 (1.46), 10.82 (0.89), and 16.99 (2.06) respectively.

Figure 3. CD30 staining in tissue from ALCL patients resistant to BV

Please see methods section on CD30 immunohistochemical staining. Biopsy post BV was done at the time of disease relapse while off BV or disease progression while on BV. CD 30 expression is retained in both scenarios.

Figure 4. MMAE resistance and intracellular accumulation

Panel A shows MTS assay performed on L428P and L428 R cells using MMAE. L428 R is resistant to MMAE as compared to L428 P (MTS assay performed in triplicate wells and repeated three times). A four-parameter log-logistic model was fitted to assess inhibitory effect of L428-P and L428-R, respectively. The estimated IC50 (standard error)s are 0.63 (0.07) and 24.66 (4.87) respectively. Panel B shows MTS assay performed on Karpas-P and Karpas-R cells using MMAE. Karpas R is equally sensitive to MMAE as compared to Karpas-P (MTS assay performed in triplicate wells and repeated three times). A four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P and Karpas-R, respectively. The estimated IC50 (standard error)s are 0.28 (0.05) and 0.52 (0.05) respectively. Panels C–E shows intracellular MMAE concentration from 0–48 hours of exposure with BV (experiments done in duplicate wells and repeated twice). Data are expressed as mean curves with 95% confidence interval and a repeated measures ANOVA model applied to show that th time, group, and their interactions are significant. In Panel C, L428P had much more intracellular MMAE as compared to L428 R (p < 0.0001). Panel D shows Karpas R and P had same intracellular concentration of MMAE when incubated with low concentration of BV. Panel E shows Karpas-P had more intracellular concentration of MMAE as compared to Karpas-R when incubated with higher concentration of BV (p ≤ 0.0001).

Figure 5. MDR1/P-glycoprotein Drug Exporter Expressed in L428 BV-resistant model

Panel A shows MDR1 mRNA expression in L428-P, L428-R, and L428-R off drug for 5 months. qRT-PCR for MDR1 mRNA expression performed in triplicate wells and repeated three times. A two-sample t-test showed that there is significant expression level difference for comparisons L 428-P vs L 428-R (p-value<0.0001), L 428-P vs L 428-R Off (p-value<0.001), and L 428-R Off vs L 428-R (p-value = 0.003). Panel B shows Western blot for P-glycoprotein (PgP, protein product of MDR1 mRNA) in L428-P, L428-R, and L428-R off drug for 5 months. L428-R had more MDR1 mRNA and PgP as compared to L428-R off drug or L428-P. Panel C shows MTS assays performed in triplicate wells and averaged over 3 separate experiments. Cells were seeded in 96-well plates at 5,000 cells per well. A four-parameter log-logistic model was fitted to assess inhibitory effect of L428-R with and without verapamil, respectively. The estimated IC50s (±standard error) are 76 (± 23) and 297 (±12), respectively.