BackgroundEpigenetic code modifications by histone deacetylase inhibitors (HDACi) have been proposed as potential new therapies for lymphoid malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma for which standard first line treatment is the chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP). The HDACi valproate, which has for long been utilized in anti-convulsive therapy, has been shown to sensitize to chemotherapy in vitro. Valproate upregulates expression of CD20 in lymphoma cell lines; therefore, 48 hour pre-treatment with valproate before first line R-CHOP in DLBCL stages II-IV is evaluated in the phase I clinical trial VALFRID; Valproate as First line therapy in combination with Rituximab and CHOP in Diffuse large B-cell lymphoma.FindingsPretreatment with valproate at oral doses comparable to anti-convulsive therapy, resulted in upregulation of CD20 mRNA and CD20 protein on the cell surface as measured by qPCR and FACS analysis in lymphoma biopsies from three evaluated patients from the VALFRID study. Valproate-treatment corresponded to increased acetylation of Histone3Lysine9 (H3K9ac) in peripheral blood mononuclear cells (PBMCs), which were employed as surrogate tissue for valproate-related epigenetic modifications.ConclusionsValproate treatment at pharmacologically relevant doses resulted in upregulation of CD20 in vivo, and also in expected epigenetic modifications. This suggests that pre-treatment with valproate or other HDACis before anti-CD20 therapy could be advantageous in CD20-low B-cell lymphomas. Further studies are warranted to evaluate this conclusion.

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies.

Chronic lymphocytic leukaemia is the most common leukaemia in the Western countries. Treatment of CLL and other lymphomas is based on cytostatic drugs in combination with rituximab or other monoclonal antibodies targeting the B-cell surface marker CD20. Although the CD20-antibodies have revolutionized the therapeutic perspectives of indolent B-cell lymphomas, the treatment of CLL with CD20-antibodies is often inefficient, a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown previously that CD20 is epigenetically regulated in some lymphoma cell lines and that some histone deacetylase inhibitors (HDACi) can increase CD20 expression in B-cell lymphomas both in vitro and in vivo. Therefore, we utilized the HDACi valproate to upregulate CD20 both in CLL cell lines and in patients.Two CLL patients with del13q were treated with valproate at serum levels around 800 μM for three days in three 21-day cycles. CD20 expression was analyzed by Real time-PCR and flowcytometry on the B-CLL cells isolated from peripheral blood.In addition, the valproate-induced histone modifications and changes in repressor/activator complexes of the CD20 promoter were investigated in the CLL cell lines and in patients.In the patient 1, CD20 expression was increased 2 times after 2 weeks of treatment, suggesting long-term effects by valproate. Neither the mRNA stability nor the protein stability seems to be affected by Valproate, suggesting transcriptional effects. However, no effects were observed in the patient 2. In contrast to patient results, Valproate increases CD20 transcript and protein levels by 2,5 times in the CLL-cell line I83-E95 after 48 hours. Here, the activating histone mark, acetylated H3K9, is enriched on the CD20 promoter suggesting valproate-related effects on the epigenome. Our study will continue the characterization of valproate-related effects on the epigenetic signatures of the CD20 promoter in CLL cells in vitro and in vivo, dissecting the mechanisms underling low CD20 expression in CLL. Citation Format: Annarita Scialdone, Jesper Kofoed Damm, Urban Gullberg, Kristina Drott. Overcoming the resistance to the anti CD20 treatments in chronic lymphocytic leukaemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4471.