In vitro and in vivo interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib in non-Hodgkin lymphoma cells

Abstract

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2-M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34(+) cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib-ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL.

Figure 1. ACY1215 interacts synergistically with CFZ in multiple NHL models and primary DLBCL cells, but not in normal cells

(A–B) Cells were treated with minimally toxic concentrations of CFZ (SUDHL16-2.5 nM, SUDHL4, U2932 and Granta 519-3.5 nM, Rec-1–15 nM, OCI-LY18-3.0 nM) in the presence or absence of ACY1215 (1.5–2.0 µM) for 48h, after which cell death was monitored by 7-AAD staining and flow cytometry (C) Fractional Effect (FA) values were determined by comparing results obtained for untreated controls and treated SUDHL16 cells following exposure to agents (36h) administered at a fixed ratio (CFZ:ACY1215::2.5:1500), as per Median Dose Effect analysis. Combination Index (C.I.) values less than 1.0 denote a synergistic interaction. (D) SUDHL16 cells were treated with varying CFZ (1.5–3.5 nM) concentrations in the presence or absence of fixed concentrations of ACY1215 (1.5–2.0 µM); alternatively, (E) cells were treated with varying ACY1215 (0.5–2.0 µM) concentrations ± fixed concentrations of CFZ (2.0–2.5 nM) for 48h, after which cell death was monitored by flow cytometry and AnnexinV/PI staining. (F) Primary human DLBCL (ABC subtype) mononuclear cells (90% purity) were isolated as described in Methods and resuspended in medium containing 10% FCS at a density of 0.6 × 10⁶ cells/mL. Bone marrow CD34⁺ cells were isolated as described in Methods. Both samples were exposed to CFZ (10 nM) ± ACY1215 (1.25 µM) for 14h. Cell death was monitored by Annexin V/PI staining. For all studies, values represent the means for 3 independent experiments performed in triplicate ± S.D. For A–B * = significantly more than values obtained for CFZ or ACY1215 treatment alone; P < 0.02. D–F, * = significantly greater than values obtained for CFZ or ACY1215 treatment alone P < 0.04.

Figure 2. Combined ACY1215/CFZ exposure activates stress pathways and increases DNA damage in multiple NHL cell types

(A–C) Granta519, SUDHL4 and U2932 cells were treated (24h) with CFZ (3.5 nM) ± ACY1215 (1.5µM) (D) OCI-LY18 cells were treated (24h) with CFZ (3.0 nM) ± ACY1215 (2.0 µM). (A–D) Protein expression was determined by Western blotting using indicated antibodies. Results are representative of three independent experiments. Expression of cytochrome C was studied in cytosolic cell fraction. Expression of total proteins like JNK. P44/42 remain unchanged in U2932 and OCI-LY18 (data not shown) CF- cleaved fragment

Figure 3. JNK and p38 activation, but not ERK1/2 inactivation, play functional roles in ACY1215/CFZ synergism

(A) SUDHL16 cells stably transfected with JNK1 shRNA or scrambled sequence were exposed to CFZ (2.5 nM) + ACY1215 (1.5 µM). After 36h, cell death was monitored by 7AAD staining and flow cytometry. Inset: relative expression of JNK1 protein in SUDHL16-scrambled sequence and shJNK clones. (B) SUDHL16 cells stably transfected with JNK-DN cDNA or empty vector (pcDNA3.1) were exposed to CFZ (2.5 nM) + ACY1215 (1.5 µM). After 48h, cell death was monitored by 7AAD staining and flow cytometry. Inset: JNK protein expression in SUDHL16 empty vector and JNK-DN clones. (C) Following 20h of drug exposure as in (A) above, Western blot analysis was employed to monitor expression of the indicated proteins. (D) SUDHL4 cells pre-treated with the selective p38 inhibitor SB203580 (10 µM) for 2h were exposed to CFZ (3.0 nM) + ACY1215 (2.0 µM) for 48h. After drug exposure, cell death was monitored by 7AAD staining and flow cytometry. (E) Following 24h of drug exposure as described above (D), Western blot analysis was employed to monitor expression of the indicated proteins. All values represent the means of triplicate experiments performed on three separate occasions ± S.D. For A and B * = significantly less than values for scrambled or empty vector control lines; P < 0.05. For D, * = significantly less than values for cells treated with CFZ + ACY1215; P < 0.05

Figure 4. Simultaneous CFZ and ACY1215 exposure induces oxidative injury-mediated cell death in both parental and bortezomib-resistant cells

(A) SUDHL4 and (B) SUDHL16-10BR cells were treated with CFZ (3.5 nM) ± ACY1215 (2.0 µM) and CFZ (5 nM) ± ACY1215 (1.5 µM) respectively (± pre-treatment with 400 µM TBAP for 3h) for 12h, after which ROS generation was monitored. (C) SUDHL4 and (D) SUDHL16-10BR cells were treated with CFZ (3.0 nM) ± ACY1215 (2.0 µM) and CFZ (5 nM) ± ACY1215 (1.5 µM) respectively (± pre-treatment with 400 µM TBAP for 3h) for 48h, after which cell death was monitored by 7AAD. Values represent means ± S.D. for triplicate determinations from 3 independent experiments. (E) Following 24h of drug exposure as in (A), expression of the indicated proteins was monitored by Western blotting. For C–D ** = significantly more versus CFZ or ACY1215 single-agent treatment; P < 0.01, * = significantly less in the presence of TBAP versus CFZ+ACY1215 treatment alone; P < 0.05

Figure 5. Combined ACY1215/CFZ exposure down-regulates the cargo protein HR23B, contributing to cell death

(A) Cells were treated with CFZ (SUDHL16 - 2.5 nM, SUDHL4 and OCI-LY7 - 3.5 nM) in the presence or absence of ACY1215 (1.5–2.0 µM) for 14h (SUDHL16 cells) to 24h (SUDHL4 and OCI-LY7 cells). Protein expression was determined by Western blotting. Each lane was loaded with 20 µg of protein; blots were stripped and re-probed with antibodies to tubulin to ensure equivalent loading and transfer. Results are representative of three independent experiments. (B) SUDHL4 cells were transiently transfected with shHR23B or scrambled control sequence for 24h, and then exposed to drug concentrations as in (A) for an additional 48h. Cell death was determined by flow cytometry with 7AAD staining. (C) SUDHL4 cells were transfected and treated as described in (B) above for 24h and expression of the indicated proteins was monitored by Western blotting. For B, * = significantly more for shHR23B sublines compared to scrambled controls; P < 0.05.

Figure 6. CFZ and ACY1215 co-treatment reduces tumor growth and significantly improves survival of mice vs single agent treatment in a mantle cell lymphoma xenograft model

(A) Granta 519 cells (5 ×10⁶) were injected in the flanks of Fox Chase SCID^®, C.B-17/Icr-Prkdc^scid mice in the presence of matrigel as described in Methods. Once tumors formed, mice were grouped with average tumor sizes of approximately 120 mm³ and treated with CFZ (1.0 mg/kg) ± ACY1215 (50 mg/kg) as per the schedule described in Methods. Tumor volumes were measured twice weekly and mean tumor volumes (± SEM) were plotted against days of treatment. Combination treatment with CFZ ± ACY1215 significantly reduced tumor burden relative to treatment with either single agent (P <0.001). (B) Survival curves of individual groups of mice were evaluated using Kaplan–Meier analysis from the first day of treatment until sacrifice (P < 0.05). (C) Tumor samples were excised from mice after 21 days of drug treatment, and lysed with lysis buffer followed by sonication. Western blotting was performed using the extracted proteins, which were probed with the indicated antibodies. (D) Mouse weights following various treatment regimens were monitored twice weekly and the mean weight of each group (± SEM) was plotted against days of treatment