Restoring the Functional Immunogenicity of Chronic Lymphocytic Leukemia Using Epigenetic Modifiers.

Chronic lymphocytic leukemia (CLL) is a malignancy arising from immune cells (B-lymphocytes) endowed with intrinsic antigen-presenting capabilities. Such a function however is lost during malignant transformation and CLL cells are well known for their inability to process and present antigens to the T-cell arm of the immune system. Instead, malignant CLL cells elicit a vast array of immune regulatory mechanisms conducive to T-cell dysfunction and immunosupression. Recent evidence suggests that DNA methylation inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression.

To investigate the potential efficacy of a combined therapy of DNMTi (5-aza-2'-deoxycytidine) and HDACi (LAQ) in human CLL we performed a gene expression microarray profile and further confirmed the expression of CTAs. Furthermore we investigated phenotypic alterations due to changes in costimulatory molecules (CD40, 80, and 86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. To confirm that alterations in molecule surface expression correlated to more potent immune stimulation we characterized the formation of the immune-synapse between the CLL antigen presenting cell and healthy T-lymphocytes in treated and untreated samples using confocal microscopy and flow conjugation assay. To demonstrate functional significance we subjected treated and untreated CLL cell lines to mixed lymphocyte proliferation assays with CD4, CD8, and NK cell subsets.

As expected we demonstrate by gene expression profile and RT-PCR that many highly immunogenic CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2 and NXF2) are significantly upregulated after treatment with an HDACi and DNMTi in combination. Interestingly, preliminary data showed interferon gamma mRNA upregulation. Consistent with this information, our data also indicates that a more proinflammatory signaling pathway is developed between the CLL B-cell and the T-lymphocyte after combination treatment demonstrated by upregulated surface expression of CD86, 80, 40, HLA-A,B,C, and HLA-DR as well as the formation of significantly more, robust, numerous, and organized actin-mediated immune-synapses, changes which were not duplicated using either drug alone. Functionally, only the combined HDACi/DNMTi treatment of CLL cells led to increased allogenic T and NK cell proliferation and cytotoxicity.

Taken together these data indicate that combined HDACi/DNMTi may benefit current antigen specific vaccine strategies for CLL by inducing the expression of a highly antigenic CTAs, increasing CLL costimulatory capacity, repairing immunological synapse, and functionally increasing the proinflammatory status of the CLL APC.

Pinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.