Unbiased Pharmacological Screening Identified New Therapeutic Strategies For Peripheral T-Cell Lymphomas (PTCLs)

T-cell non-Hodgkin lymphoma (T-NHL) responds poorly to standard chemotherapeutic regimens like CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone). In order to identify better therapeutic approaches we conducted a viability screening using a library of known anti-neoplastic agents.

We performed a first screening in the PTCL-NOS cell line OCI-Ly12 using 101 approved oncology drugs obtained from the National Cancer Institute in 3 concentrations (10, 1, 0.1 μM). We measured cell viability after 48 h of exposure and calculated 50% growth inhibitory concentrations (GI50s). We found 30 drugs with GI50s< 10 μM that were further tested at 0.1 μM in 3 additional cell lines: anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL): Karpas299, ALK-negative ALCL: OCI-Ly13.2, and cutaneous T-cell lymphoma (CTCL): HuT78. In this secondary screening, we found 3 drugs that killed > 70% of cells in all 4 cell lines: bortezomib (proteasome inhibitor), romidepsin (histone deacetylase -HDAC- inhibitor) and dactinomycin (RNA polymerase -RNA-Pol- inhibitor). We then tested these 3 compounds with additional HDAC inhibitors (panobinostat, vorinostat, trichostatin A), at 6 concentrations, in an extended panel of 7 T-NHL cell lines (tertiary screening). We found that only romidepsin and dactinomycin had GI50s lower than their respective maximum plasma concentration (Cmax) in humans; we therefore focused further mechanistic studies in these two compounds.

Romidepsin GI50 ranged from 10 nM (in Karpas299) to 200 nM (in Mac2A and SU-DHL1), which were lower than its Cmax (1850 nM). Administering the drug one or two times over the 48 h exposure yielded equivalent anti-lymphoma effect. To further characterize this effect and determine whether the biological change was transient or permanent, we treated cells with romidepsin once with their GI50 dose and measured cell proliferation, apoptosis and extent of histone 3 lysine 27 acetylation (H3K27Ac) by immunoblotting at 4, 8, 12 h, then daily for 7 days. We found that the main cellular effect of romidepsin was inhibition of cell proliferation rather than inducing cell death. This effect was associated with increasing H3K27Ac as early as 8 h, peaking at 24 h (∼30 fold to control), maintaining for 3 days with subsequently slow declining until day 7 (∼15 fold to control). Since romidepsin response rate in relapsed/refractory PTCL patients is ∼30%, we wished to determine whether it could be more active when combined with chemotherapy. We found that in all T-NHL cell lines, concomitant administration with doxorubicin or vincristine yielded synergistic killing effect by isobologram analysis. This data suggested that romidepsin could be a potent chemosensitizer in PTCLs.

Dactinomycin is a weak reversible RNA-Pol elongation inhibitor that affects all 3 RNA-Pols, which confers broad anti-neoplastic activity, but also significant toxicity. To determine whether more selective RNA-Pol2 inhibitors are active in PTCL, we first analyzed the expression of cyclin-dependent kinase (CDK) complexes, CDK7/CCNH and CDK9/CCNT, responsible for RNA-Pol2 activation through phosphorylation, in T-NHL cell lines. We found overexpression of CDK7 by 3.06 fold (± 0.3) and CCNH by 2.1 fold (± 0.2) in all 7 PTCLs cell lines compared to tonsilar normal T cells. This prompted us to investigate the anti-proliferative effect of a recently described CDK7 inhibitor (CDK7i). CDK7i showed anti-proliferative effect in all 7 T-NHL cell lines tested with GI50 ranging from 0.2 μM (OCI-Ly13.2) to 2.3 μM (Mac2A), which were 5-10 times lower than the CDK7i chemical control. We found that this anti-proliferative effect correlated with a primary decrease in phospho-Ser5 RNA-Pol2 followed by a decrease in phospho-Ser2 RNA-Pol2 ultimately causing transcriptional inhibition of active loci. This effect led to a decrease in BCL2 and MYC mRNA levels in treated cells, which suggested that CDK7i could represents the first of a novel class of selective transcriptional inhibitors for PTCL treatment.

In sum, we identified romidepsin as the most active drug for T-NHL and described a potential chemosensitizing effect. We also uncovered a potential novel target, CDK7, for the treatment of this disease.