Kelly Zullo

Dual Targeting of Protein Degradation Pathways with the Selective HDAC6 Inhibitor ACY-1215 and Bortezomib Is Synergistic in Lymphoma

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 used to confirm in vitro findings. A collection of primary lymphoma samples were surveyed for markers of the unfolded protein response (UPR). Results: Concentration–effect relationships defined maximal cytotoxicity at 48 hours with IC50 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 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 with reactive lymphoid tissue. Conclusions: 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. Clin Cancer Res; 21(20); 4663–75. ©2015 AACR.

Preclinical Pharmacologic Evaluation of Pralatrexate and Romidepsin Confirms Potent Synergy of the Combination in a Murine Model of Human T-cell Lymphoma

Purpose: T-cell lymphomas (TCL) are aggressive diseases, which carry a poor prognosis. The emergence of new drugs for TCL has created a need to survey these agents in a rapid and reproducible fashion, to prioritize combinations which should be prioritized for clinical study. Mouse models of TCL that can be used for screening novel agents and their combinations are lacking. Developments in noninvasive imaging modalities, such as surface bioluminescence (SBL) and three-dimensional ultrasound (3D-US), are challenging conventional approaches in xenograft modeling relying on caliper measurements. The recent approval of pralatrexate and romidepsin creates an obvious combination that could produce meaningful activity in TCL, which is yet to be studied in combination. Experimental Design: High-throughput screening and multimodality imaging approach of SBL and 3D-US in a xenograft NOG mouse model of TCL were used to explore the in vitro and in vivo activity of pralatrexate and romidepsin in combination. Corresponding mass spectrometry–based pharmacokinetic and immunohistochemistry-based pharmacodynamic analyses of xenograft tumors were performed to better understand a mechanistic basis for the drug:drug interaction. Results: In vitro, pralatrexate and romidepsin exhibited concentration-dependent synergism in combination against a panel of TCL cell lines. In a NOG murine model of TCL, the combination of pralatrexate and romidepsin exhibited enhanced efficacy compared with either drug alone across a spectrum of tumors using complementary imaging modalities, such as SBL and 3D-US. Conclusions: Collectively, these data strongly suggest that the combination of pralatrexate and romidepsin merits clinical study in patients with TCLs. Clin Cancer Res; 21(9); 2096–106. ©2015 AACR.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma

Purpose: Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. Experimental Design: A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. Results: In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell–specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. Conclusions: These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development. Clin Cancer Res; 21(18); 4097–109. ©2015 AACR.

The combination of hypomethylating agents and histone deacetylase inhibitors produce marked synergy in preclinical models of T-cell lymphoma

T‐cell lymphomas (TCL) are aggressive lymphomas usually treated with CHOP (cyclophsophamide, doxorubicin, vincristine, prednisolone)‐like regimens upfront. Recent data suggest that TCL are driven by epigenetic defects, potentially rendering them sensitive to epigenetic therapies. We explored the therapeutic merits of a combined epigenetic platform using histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMT) in in vitro and in vivo models of TCL. The 50% inhibitory concentration (IC50) values revealed romidepsin was the most potent HDACI, with an IC50 in the low nanomolar range. The combination with a hypomethylating agent produced synergy across all cell lines, which was confirmed in cytotoxicity and apoptosis assays. An in vivo xenograft study demonstrated inhibition of tumour growth in the combination cohort compared to the single agent. Gene expression array and global methylation profiling revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. Most of the effects induced by the single agent treatment were maintained in the combination group. In total, 944 unique genes were modulated by the combination treatment, supporting the hypothesis of molecular synergism. These data suggest combinations of hypomethylating agents and HDACIs are synergistic in models of TCL, which is supported at the molecular level.

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib

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- 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. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: 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. Clin Cancer Res; 23(12); 3084–96. ©2016 AACR.

Murine models in mantle cell lymphoma

Mantle cell lymphoma (MCL), an aggressive, heterogeneous B-cell lymphoma associated with a relatively short survival has been challenging to study in the laboratory due to the lack of in vitro and in vivo models that accurately recapitulate the disease. Advancement has been made in the characterization of MCL cell lines through the generation of the ATCC MCL bank, enabling their use in xenograft murine models. These models provide valuable but limited information for the preclinical evaluation and development of targeted therapies for MCL despite their deficiencies of a functioning immune system and correct micro-environment. Currently, there is only one double transgenic murine model known to develop spontaneous MCL. There is an urgency to develop innovative transgenic murine models that could be used to better predict therapeutic responses and precisely decipher mechanisms of action, to foster refinement of novel therapeutics for mantle cell lymphoma.

The novel kinesin spindle protein (KSP) inhibitor SB-743921 exhibits marked activity in in vivo and in vitro models of aggressive large B-cell lymphoma

The kinesin spindle protein (KSP) is a mitotic protein essential for cell cycle control and motility. SB-743921 (hereafter SB-921) is an inhibitor that selectively targets the ATP-binding domain of the KSP. The preclinical activity of SB-921 was evaluated in models of diffuse large B-cell lymphoma (DLBCL). The cytotoxicity of SB-921 was evaluated in a series of germinal center (GC-DLBCL) and post-germinal center (ABC-DLBCL) DLBCL cell lines and a murine lymphoma xenograft model. GC-DLBCL lines generally demonstrated greater sensitivity to SB-921. IC50 values ranged between 1 nM and 900 nM for GC-DLBCL compared to 1 nM to 10 μM for ABC lines. SB-921 demonstrated marked activity in a xenograft model of Ly-1 (GC-DLBCL). While SB-921 was relatively more active in GC derived cell lines, ABC-derived lines still underwent apoptosis at higher concentrations. These results demonstrate that SB-921 inhibits proliferation and induces apoptosis in both GC-DLBCL and ABC-DLBCL.