Purpose Pan-class histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform selective HDAC inhibitors are emerging as potentially more targeted agents. HDAC6 is a class IIb deacetylase that facilitates misfolded protein transport to the aggresome for degradation. We investigated the mechanism and therapeutic impact of the selective HDAC6 inhibitor ACY-1215 alone and in combination with bortezomib in preclinical models of lymphoma. Experimental Design Concentration : effect relationships were defined for ACY-1215 across 16 lymphoma cell lines and for synergy with bortezomib. Mechanism was interrogated by immunoblot and flow cytometry. An in vivo xenograft model of DLBCL was utilized to confirm in vitro findings. A collection of primary lymphoma samples were surveyed for markers of the UPR. Results Concentration : effect relationships defined maximal cytotoxicity at 48 hours with IC50 values ranging from 0.9—4.7 μM. Strong synergy was observed in combination with bortezomib. Treatment with ACY-1215 led to inhibition of the aggresome evidenced by acetylated α-tubulin and accumulated poly-ubiquitinated proteins, and up-regulation of the UPR. All pharmacodynamic effects were enhanced with the addition of bortezomib. Findings were validated in vivo where mice treated with the combination demonstrated significant tumor growth delay and prolonged overall survival. Evaluation of a collection of primary lymphoma samples for markers of the UPR revealed increased HDAC6, GRP78 and XBP-1 expression as compared to reactive lymphoid tissue. Conclusion These data are the first results to demonstrate that dual targeting of protein degradation pathways represents an innovative and rational approach for the treatment of lymphoma.
Purpose Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first in class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue and a xenograft mouse model. Results Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC50 values 10-20 fold greater than that for parental lines. GEP revealed up-regulation of MAPK10, HELIOS, HDAC9 and FYN, as well as down-regulation of SH3BP5 and LCK. Gene set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of anti-tumor synergy was demonstrated with a xenograft of DLBCL. Conclusion The development of this ACY-1215 resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway.
<p>Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll-Paque layering. (A) Concentration : effect relationship was determined for ACY-1215 at 24 and 48 hours in PBMCs. No cytotoxicity was observed after 24 and 48 hours of exposure. The IC50 was not reached at 24 and 48 hours. (B) Treatment of PBMCs with ACY-1215 1500 nM led to no induction of the UPR as demonstrated by Western blot analysis. (C) Dual targeting of protein degradation pathways with ACY-1215 and bortezomib led to no cytotoxicity in PBMCs and the combination is not synergistic. Cells were treated with increasing concentrations of ACY-1215 (0, 500, 1000, and 1500nM) plus bortezomib 3 nM for 48 hours. Synergy was calculated using Excess over Bliss where values greater than 10 represent synergy.</p>
<div>Abstract<p><b>Purpose:</b> Pan-class histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. HDAC6 is a class IIb deacetylase that facilitates misfolded protein transport to the aggresome for degradation. We investigated the mechanism and therapeutic impact of the selective HDAC6 inhibitor ACY-1215 alone and in combination with bortezomib in preclinical models of lymphoma.</p><p><b>Experimental Design:</b> Concentration–effect relationships were defined for ACY-1215 across 16 lymphoma cell lines and for synergy with bortezomib. Mechanism was interrogated by immunoblot and flow cytometry. An <i>in vivo</i> xenograft model of DLBCL was used to confirm <i>in vitro</i> findings. A collection of primary lymphoma samples were surveyed for markers of the unfolded protein response (UPR).</p><p><b>Results:</b> Concentration–effect relationships defined maximal cytotoxicity at 48 hours with IC<sub>50</sub> values ranging from 0.9 to 4.7 μmol/L. Strong synergy was observed in combination with bortezomib. Treatment with ACY-1215 led to inhibition of the aggresome evidenced by acetylated α-tubulin and accumulated polyubiquitinated proteins and upregulation of the UPR. All pharmacodynamic effects were enhanced with the addition of bortezomib. Findings were validated <i>in vivo</i> where mice treated with the combination demonstrated significant tumor growth delay and prolonged overall survival. Evaluation of a collection of primary lymphoma samples for markers of the UPR revealed increased HDAC6, GRP78, and XBP-1 expression as compared with reactive lymphoid tissue.</p><p><b>Conclusions:</b> These data are the first results to demonstrate that dual targeting of protein degradation pathways represents an innovative and rational approach for the treatment of lymphoma. <i>Clin Cancer Res; 21(20); 4663–75. ©2015 AACR</i>.</p></div>
<div>Abstract<p><b>Purpose:</b> Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215.</p><p><b>Experimental Design:</b> The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model.</p><p><b>Results:</b> Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC<sub>50</sub> values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. <i>In vivo</i> confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL.</p><p><b>Conclusions:</b> The development of this ACY-1215–resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. <i>Clin Cancer Res; 23(12); 3084–96. ©2016 AACR</i>.</p></div>
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