Enrica Marchi

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.

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.

Brentuximab vedotin plus bendamustine in relapsed or refractory Hodgkin's lymphoma: an international, multicentre, single-arm, phase 1–2 trial

BACKGROUNDnBrentuximab vedotin is currently approved for patients with relapsed or refractory Hodgkins lymphoma who previously received an autologous stem cell transplant or two previous multiagent chemotherapy regimens, and for patients with relapsed or refractory systemic anaplastic large-T-cell lymphoma who previously received at least one chemotherapy regimen. A high proportion of patients with CD30-expressing relapsed or refractory lymphomas have durable responses to single-agent brentuximab vedotin and show longer progression-free survival than do patients treated with chemotherapy. In patients with Hodgkins lymphoma and peripheral T-cell lymphoma, treatment with bendamustine alone only achieves modest improvements in progression-free survival compared with that for chemotherapy. The objective of this study was to explore the safety and clinical activity of the combination of brentuximab vedotin plus bendamustine in heavily pretreated patients with relapsed or refractory Hodgkins lymphoma and anaplastic large-T-cell lymphoma.nnnMETHODSnIn this international, multicentre, single-arm, phase 1-2 trial, eligible patients were aged 18 years or older, had histologically confirmed relapsed or refractory Hodgkins lymphoma or anaplastic large-T-cell lymphoma, had biopsy-proven CD30-positive tumours, had an Eastern Cooperative Oncology Group performance status of 2 or less, and received at least one previous multiagent chemotherapy regimen. In phase 1, patients were assigned following a 3+3 dose-escalation design to one of four cohorts to receive one dose of either 1·2 mg/kg or 1·8 mg/kg of brentuximab vedotin intravenously on day 1 of a 21 day cycle, plus one dose of bendamustine (70 mg/m2, 80 mg/m2, or 90 mg/m2) on days 1 and 2 of the treatment cycle. In phase 2, all patients were assigned to receive brentuximab vedotin plus bendamustine at the recommended phase 2 dose from phase 1. The primary endpoints were maximum tolerated dose and dose-limiting toxicity for phase 1, and the proportion of patients achieving an overall response in phase 2. For both phases 1 and 2, all patients receiving at least one dose of study drug were evaluable for toxicity and all patients completing at least one cycle of therapy were evaluable for response. The study is ongoing but no longer recruiting patients. This trial is registered with ClinicalTrials.gov, number NCT01657331.nnnFINDINGSnBetween July 26, 2012, and May 31, 2017, we enrolled and assigned 65 patients to treatment (64 [98%] with Hodgkins lymphoma and one [2%] with anaplastic large-T-cell lymphoma; 28 [43%] during phase 1 and 37 [57%] during phase 2). In the phase 1 part, the maximum tolerated dose of the combination was not reached. Dose-limiting toxicities were observed in three (11%) of 28 patients, including grade 4 neutropenia at 1·8 mg/kg brentuximab vedotin plus 80 mg/m2 of bendamustine in two (7%) patients and diffuse rash at 1·2 mg/kg brentuximab vedotin plus 70 mg/m2 of bendamustine in one (4%) patient. The recommended phase 2 dose was deemed to be 1·8 mg/kg of brentuximab vedotin and 90 mg/m2 of bendamustine, which are the standard doses of the drugs when given as single agents. In the phase 2 part, an overall response was achieved in 29 (78% [95% CI 62-91]) of 37 patients. Serious adverse events included grade 3 lung infection in five (14%) of 37 patients in the phase 2, and grade 3-4 neutropenia in 16 (25%) of 65 patients across phases 1 and 2. There were no treatment-related deaths.nnnINTERPRETATIONnThis study shows that brentuximab vedotin plus bendamustine, with a favourable safety profile, is an active salvage regimen for heavily pretreated patients with relapsed or refractory Hodgkins lymphoma. This salvage regimen can potentially serve as an efficacious and safe alternative to platinum-based chemotherapy before autologous stem cell transplant.nnnFUNDINGnSeattle Genetics, Lymphoma Research Fund of Columbia University and National Center for Advancing Translational Sciences, and National Institutes of Health.

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.

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.

Novel Agents in the Treatment of Relapsed or Refractory Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of mature T-cell malignancies associated with exceptionally poor prognoses. Currently, chemotherapy remains the standard of care, but outcomes are suboptimal, with 5-year survival rates ranging from 15% to 25%. In recent years, several novel agents, including pralatrexate, romidepsin, belinostat, and brentuximab vedotin, have been approved for the treatment of relapsed/refractory PTCL. In addition, numerous other therapies with different mechanisms of action and targets are currently under investigation. This article discusses in detail agents currently available, those currently under investigation, and active combination trials.

Abstract 4530: Pralatrexate-induced apoptosis in multiple myeloma cells correlates with RFC-1 and DHFR expression

Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a novel anti-folate agent that is approved for use in relapsed peripheral T cell lymphoma (PTCL). It is distinguished from the parent drug methotrexate (MTX) by having significantly greater affinity for the reduced folate carrier (RFC)-1, the principle transporter that internalize anti-folates, as well as broader efficacy, which strongly suggests that PDX has mechanisms of action beyond inhibition of nucleotide synthesis. Given the activity of PDX in aggressive lymphoid malignancies such as PTCL, we examined the activity of this agent in preclinical models of multiple myeloma (MM), a plasma cell malignancy that is currently incurable. We examined the cytotoxic activity of anti-folates in vitro against a panel of human multiple myeloma cell lines (HMCL) and showed that PDX induced dose-dependent apoptotic cell death in a subset of these. In sensitive cell lines (MM.1s and ARH-77) PDX demonstrated significantly greater potency at 48 hours compared to MTX (LD50 PDX = 2-3 nM vs. LD50 MTX = 25-30 nM). PDX also induced apoptosis in a polyclonal population of primary myeloma cells isolated from a patient specimen, whereas MTX had no effect on viability in these cells. PDX-induced apoptosis in sensitive HMCL was characterized by cleavage and activation of caspase-3 and -9, accompanied by the loss of the anti-apoptotic Bcl-2 family protein Mcl-1. Quantitative RT-PCR analysis of a panel of folate metabolism genes, including RFC-1, folyl polyglutamate synthetase (FPGS), gamma-glutamyl hydrolase (GGH), thymidylate synthase (TS), and dihydrofolate reductase (DHFR) revealed that RFC-1 expression was significantly higher in sensitive HMCL compared to resistant. Dose-dependent up-regulation of DHFR protein expression was observed by western blot in PDX-resistant cell lines, but not in PDX-sensitive ones. Incubation of HMCL with the proteasome inhibitor bortezomib or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA), both of which demonstrate activity in MM, did not affect expression of RFC-1 in PDX-resistant HMCL and did not synergize with PDX to induce apoptosis in vitro. These results demonstrate that PDX induces apoptosis in sensitive HMCL, and that RFC-1 expression and DHFR up-regulation are functional biomarkers for susceptibility to this anti-folate. These data support further exploration of PDX therapy in MM, including combination therapy with agents that can increase expression of RFC-1 and/or block up-regulation of DHFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4530. doi:10.1158/1538-7445.AM2011-4530