Matko Kalac

HDAC inhibitors and decitabine are highly synergistic and associated with unique gene-expression and epigenetic profiles in models of DLBCL.

Interactions between histone deacetylase inhibitors (HDACIs) and decitabine were investigated in models of diffuse large B-cell lymphoma (DLBCL). A number of cell lines representing both germinal center B-like and activated B-cell like DLBCL, patient-derived tumor cells and a murine xenograft model were used to study the effects of HDACIs and decitabine in this system. All explored HDACIs in combination with decitabine produced a synergistic effect in growth inhibition and induction of apoptosis in DLBCL cells. This effect was time dependent, mediated via caspase-3 activation, and resulted in increased levels of acetylated histones. Synergy in inducing apoptosis was confirmed in patient-derived primary tumor cells treated with panobinostat and decitabine. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. We analyzed the molecular basis for this synergistic effect by evaluating gene-expression and methylation patterns using microarrays, with validation by bisulfite sequencing. These analyses revealed differentially expressed genes and networks identified by each of the single treatment conditions and by the combination therapy to be unique with few overlapping genes. Among the genes uniquely altered by the combination of panobinostat and decitabine were VHL, TCEB1, WT1, and DIRAS3.

Sirtuin and pan-class I/II deacetylase (DAC) inhibition is synergistic in preclinical models and clinical studies of lymphoma

Understanding the molecular pathogenesis of lymphoma has led to paradigm-changing treatment opportunities. One example involves tailoring specific agents based on the cell of origin in aggressive lymphomas. Germinal center (GC)-derived diffuse large B-cell lymphoma (DLBCL) is known to be driven by an addiction to Bcl6, whereas the activated B-cell (ABC) subtype is driven by nuclear factor κB. In the GC subtype, there is a critical inverse relationship between Bcl6 and p53, the functional status of which is linked to each transcription factors degree of acetylation. Deacetylation of Bcl6 is required for its transcriptional repressor effects allowing for the oncogene to drive lymphomagenesis. Conversely, acetylation of p53 is activating when class III deacetylases (DACs), or sirtuins, are inhibited by niacinamide. Treatment of DLBCL cell lines with pan-DAC inhibitors in combination with niacinamide produces synergistic cytotoxicity in GC over ABC subtypes. This correlated with acetylation of both Bcl6 and p53. This combination also produced remissions in a spontaneous aggressive B-cell lymphoma mouse model expressing Bcl6. In a phase 1 proof-of-principle clinical trial, 24% of patients with relapsed or refractory lymphoma attained a response to vorinostat and niacinamide, and 57% experienced disease stabilization. We report herein on the preclinical and clinical activity of this targeted strategy in aggressive lymphomas. This trial was registered at www.clinicaltrials.gov as #NCT00691210.

New Drugs for the Treatment of Lymphoma

Historically, most drugs developed for treatment of leukemias, lymphomas, and myeloma had already been studied in the solid tumor setting. Nearly 10 years ago, chronic myelogenous leukemia (CML) forever changed this paradigm. Imatinib showed that it was possible to nullify the pathognomic genetic lesion in a hematologic malignancy. Since the approval of imatinib for CML, a host of new drugs active in blood cancers have emerged. This article highlights some areas of innovative drug development in lymphoma where possible; it emphasizes the biologic basis for the approach, linking this essential biology to the biochemical pharmacology. The article focuses on the many new targets including Syk, Bcl-2, CD-40, and the phosphoinositide-3 kinase/AKT/mammalian target of rapamycin pathway.

Development and Characterization of a Novel CD19CherryLuciferase (CD19CL) Transgenic Mouse for the Preclinical Study of B-Cell Lymphomas

Purpose: To generate a transgenic mouse that when crossed with spontaneous mouse models of lymphoma will allow for quantitative in vivo measurement of tumor burden over the entire spectrum of the disease and or response to therapy in a “disease” or lymphoma subtype-specific manner. Experimental Design: We developed a novel genetically engineered transgenic mouse using a CherryLuciferase fusion gene targeted to the CD19 locus to achieve B-cell–restricted fluorescent bioluminescent emission in transgenic mouse models of living mice. The use of a dual function protein enables one to link the in vivo analysis via bioluminescence imaging to cell discriminating ex vivo analyses via fluorescence emission. Results: The spatiotemporal tracking of B-cell lymphoma growth and the response of an established B-cell lymphoma to a drug known to induce remission was evaluated in a double transgenic animal obtained by crossing the CD19CherryLuciferase transgenic mouse to a mouse model of an aggressive B-cell lymphoma. The observations validated the use of the CD19CherryLuciferase transgenic mouse in the assessment of an active drug routinely used in the treatment of lymphoproliferative malignancies. Conclusions: The transgenic mouse described here is the first of its kind, intended to be used to hasten translational studies of novel agents in lymphoma, with the intent that understanding the relevant pharmacology before clinical study will accelerate successful development in clinical studies. Clin Cancer Res; 18(14); 3803–11. ©2012 AACR.

A phase I study of romidepsin and pralatrexate reveals marked activity in relapsed and refractory T-cell lymphoma

Peripheral T-cell lymphomas (PTCL) are a group of rare malignancies characterized by chemotherapy resistance and poor prognosis. Romidepsin and pralatrexate were approved by the US Food and Drug Administration for patients with relapsed/refractory PTCL, exhibiting response rates of 25% and 29% respectively. Based on synergy in preclinical models of PTCL, we initiated a phase 1 study of pralatrexate plus romidepsin in patients with relapsed/refractory lymphoma. This was a single institution dose-escalation study of pralatrexate plus romidepsin designed to determine the dose-limiting toxicities (DLTs), maximum tolerated dose, pharmacokinetic profile, and response rates. Patients were treated with pralatrexate (10 to 25 mg/m2) and romidepsin (12 to 14 mg/m2) on 1 of 3 schedules: every week × 3 every 28 days, every week × 2 every 21 days, and every other week every 28 days. Treatment continued until progression, withdrawal of consent, or medical necessity. Twenty-nine patients were enrolled and evaluable for toxicity. Coadministration of pralatrexate and romidepsin was safe, well tolerated, with 3 DLTs across all schedules (grade 3 oral mucositis × 2; grade 4 sepsis × 1). The recommended phase 2 dose was defined as pralatrexate 25 mg/m2 and romidepsin 12 mg/m2 every other week. Twenty-three patients were evaluable for response. The overall response rate was 57% (13/23) across all patients and 71% (10/14) in PTCL. The phase 1 study of pralatrexate plus romidepsin resulted in a high response rate in patients with previously treated PTCL. A phase 2 study in PTCL will determine the efficacy of the combination. This trial was registered at www.clinicaltrials.gov as #NCT01947140.

Abstract 4705: Dual targeting of epigenetic pathways and NF-kappaB in diffuse large B-cell lymphoma

INTRODUCTION: Current immunochemotherapy can cure approximately two-thirds of patients with diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin9s lymphoma. Drugs that selectively target specific mechanisms of pathogenesis to which the disease is known to be addicted, offer a unique opportunity to intervene in the natural history of the disease, and may hold the key to future breakthroughs in treating DLBCL. While several unique pathogenetic events have been recently identified in driving lymphomagenesis, two potentially interrelated events contributing to the underlying molecular pathogenesis of DLBCL have come to light. The first involves constitutive activation of NF-kappaB, as noted by Rosenwald and colleagues in their identification of the activated B-cell (ABC) subtype of DLBCL, while the second involves dysregulation of the protein acetylation-deacetylation balance due to either inactivating mutations of histone acetyltransferases (HATs) like p300 and CBP, or overexpression of histone deacetylases (HDACs). HATs and HDACs operate epigenetic pathways to modulate gene expression and to control the activity of important oncoproteins such as Bcl6 through posttranslational modification. While Bcl6 and NF-kappaB are known to be closely associated with the pathogenesis of GCB (germinal center B-cell) and ABC subtypes of DLBCL respectively, it has become clear that many ABC lymphomas can have dysregulation of Bcl6, and many cases of GCB DLBCL can have dysregulation of NF-kappaB. Therefore, strategies oriented toward modulation of these biological traits could offer a unique opportunity to treat DLBCL at its molecular roots, potentially sensitizing them to traditional chemotherapy approaches. RESULTS: We have conducted pharmacological and molecular studies of a novel IKKbeta inhibitor, LY2409881, in DLBCL cell lines. We found that LY2409881 specifically inhibited the activation of NF-kappaB and increased apoptosis, and inhibited both ABC and GCB cells that are addicted to NF-kappaB, with moderate potentcy. Furthermore, LY2409881 demonstrated marked anti-lymphoma synergism with HDAC inhibitors; the synergism varied significantly in a cell line and HDAC inhibitor-dependent manner. The discrete patterns of synergism among different HDAC inhibitors with LY2409881 were not solely attributable to acetylation of p65/RelA mediated by HDAC inhibitors, as these inhibitors all stimulated binding of p65/RelA to its target DNA. We will determine the mechanism of the drug: drug synergism by investigating whether knocking down HDAC molecules in lymphoma cells make them hypersensitive to IKK inhibitors. We are using xenograft lymphoma models in mice to confirm the in vivo activity of LY2409881, as a single agent and in combination with HDAC inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4705. doi:1538-7445.AM2012-4705

Abstract A12: Validation of a novel bioluminescent mouse model of Sezary syndrome for preclinical drug screening.

Background: Sezary syndrome (SS) is an aggressive leukemic form of cutaneous T cell lymphoma (CTCL). Until now no true animal model for SS exists which could be used for the screening of novel compounds against the disease. We successfully developed a bioluminescent xenograft mouse model for SS to noninvasively monitor tumor cell engraftment and progression and to measure the effects of treatments on tumor burden. Methods: A fusion protein was constructed consisting of the monomeric mutant red fluorescent mCherry, and the synthetic-firefly Luciferase by cloning the mCherry gene into the plasmid vector pGL4.13 [luc2/SV40] (Promega) carrying the luciferase gene, thus obtaining the pGLCherryLuciferase plasmid, where the Cherry and the luciferase genes formed one open reading frame. H9 cell line derived from a SS patient was transfected with this plasmid using the transfection reagent, Effectene. Transfected H9 cells were injected subcutaneously into the 5–7 week old female SCID-Beige and NOG (NOD/Shi-scid/IL-2R null) mice. After confirmation of a bioluminescent signal, NOG mice were assigned to the vehicle control group or treatment groups, which received romidepsin (1.2 or 2.3mg/kg on day1, 4, 8 and 11, ip) or pralatrexate (30mg/kg on day1, 4, 8 and 11, ip). In vivo bioluminescence imaging over 3 weeks was performed using an IVIS imaging system (Caliper Life Sciences). Results: SCID-Beige mice did not permit any engraftment of the H9 injected cells whereas all the NOG mice exhibited tumor growth and progression. This suggests that the NOG mice are superior animal recipients for xenotransplantation of Sezary cells, potentially making them a preclinical tool to understand tumorigenesis and drugs effects in this malignancy. Sequential quantitative signals from bioluminescent imaging over the 3-week period were significantly lower in the mice treated with pralatexate compared with the control group. The mice treated with 1.2mg/kg of romidepsin had no effect on tumor suppression whereas the mice that received the higher dose of romidepsin demonstrated decrease of tumor growth suggesting dose-dependent tumor inhibition. Mice treated with pralatrexate demonstrated diminished bioluminescent signal as early as after the first dose compared to the mice treated with higher dose of romidepsin which produced a slower time to onset of activity. No mice treated with pralatrexate or with the higher dose of romidepsin exhibited toxicity. Conclusion: This novel bioluminescent xenograft mouse model of SS enables non-invasive, sensitive, quantitative evaluation of disease progression in living animals and evaluation of pharmacologic factors in real time. We are able to detect and monitor lymphoma cell growth before the presentation of clinical manifestations. Further this model recapitulates our understanding of behavior of drugs used in the treatment of lymphomas such as pralatrexate which has a rapid onset of action compared to romidepsin that has a delayed time to onset of activity. This represents the first bioluminescent animal model of human CTCL that is intended to be used to investigate novel treatment platforms in preclinical studies. This preclinical model also compliments the ongoing phase 2 trial of pralatrexate in relapsed or refractory CTCL. Further in vivo studies to evaluate synergy of promising new agents in this novel mouse model of SS have begun and will be reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A12.