PRECEDENTES EVOLUCIÓN DE MI PINTURA

Initially developed to modulate chromatin condensation to affect transcriptional activation and gene expression, HDACi compounds like soduim butyrate (NaB) were

found to have anti-tumor capabilities when shown to induce terminal differentiation in erythroleukemia (Leder et al., 1975; Richon et al., 1998; Tanaka et al., 1975). It was later discovered that NaB-induced histone hyperacetylation (Riggs et al., 1977), which caused inhibition of tumor cell growth and survival (Richon et al., 1998; Yoshida et al., 1990). Today, there are a number of structurally diverse synthetic and natural product HDACi compounds, 11 of which are in clinical trials (Table 1.3). HDACi’s restore the balance between differentiation and proliferation, forcing tumor cells to differentiate into more mature cell types and relieving their constant proliferative potential (Hagelkruys et al., 2011). This is achieved by regulating the acetylation status of histones and non-histone proteins that ultimately affect the expression of genes or the activity of proteins

associated with inhibition of angiogenesis, the endoplasmic stress response, cellular differentiation, cell cycle arrest at both G1/S and G2/M phase transitions, BCL-2 family- mediated apoptosis, and tumor suppressor genes or oncongenes in a number of cancer types, including a variety of lymphomas and leukemias (Bolden et al., 2006; Cotto et al., 2010; Piekarz and Bates, 2009; Wiegmans et al., 2011; Zain and O’Connor, 2010).

Gene expression profiling using microarrays has shown that up to 22% of genes are altered by HDACi as early as 4 h post treatment several cell types (Halsall et al., 2012; Peart et al., 2003; Van Lint et al., 1996). A major conclusion from these profiling studies is that HDACi treatment results in expression of pro-apoptotic genes and

suppression of anti-apoptotic genes (Inoue et al., 2007a; Mitsiades et al., 2004; Zhang et al., 2006; Zhang et al., 2012b). Apoptosis can be achieved through two different

BCL-2-mediated mitochondrial permeablization and cytochrome C release (Shamas-Din et al., 2011; Strasser et al., 2011; Youle and Strasser, 2008). Specific to this thesis, inhibition of HDAC activity results in a change in BCL-2 family expression to favor a pro-apoptotic state. The extrinsic pathway involves binding of ligands to their appropriate death receptors (Fas, TNF, TRAIL), leading to the release and activation of caspase-8 and -10, and subsequent activation of effector caspases (reviewed by Hagelkruys et al., 2011; Zain and O’Connor, 2010).

GEP studies of various lymphoma and leukemia cell lines have shown similar results across studies. CTCL cell lines treated with vorinostat showed that HDACi

treatment led to hyperacetylation of all core histones, which is associated with changes in the expression of genes involved in cell cycle regulation of the G1/S and G2/M

transitions, apoptosis, antiproliferation, and MAPK signaling (Richon et al., 2000). Treatment of the promyelocytic leukemia cell line HL60 with TSA, vorinostat, or VPA showed that all three HDACi’s induced G2/M cell cycle arrest, while only vorinostat and TSA induced apoptosis (Halsall et al., 2012). Microarry results showed that an 8-h treatment with either 2.5 µM vorinostat, 5 mM VPA, or 165 nM TSA changes only 258 genes (5%), 369 genes (7%), and 675 genes (13%), respectively. Surprisingly, only seven of the 1167 genes were dysregulated by all three HDACi treatments, although histone acetylation status was increased to a similar degree, suggesting a pathway specific effect for each HDACi. Another study treated the K562 human chronic myelogenic leukemia cell line with 2 mM VPA for 12 h (Zhang et al., 2012b). The microarray identified 706 altered transcripts, 34 of which are involved in apoptosis, including up-regulation of pro-

apoptotic protein BAX and down-regulation of the BH3-only apoptosis modulator protein BID. GO analysis showed that genes involved in cytochrome C release were enriched. In another study (Mitsiades et al., 2004), vorinostat was shown to induce irreversible apoptosis in multiple myeloma cell lines, and gene expression profiling revealed an up- regulation of p53 and the pro-apoptotic proteins Apaf-1 and caspase-9 while there was a down-regulation of the anti-apoptotic genes FLIP, survivin, Apo2L/TRAIL, and XBP-1, priming the cells for apoptosis. There was also an up-regulation in growth arrest and anti- proliferative genes like p19, p21, p57, and BTG-1 while proliferation genes like CDK4, cyclins, and others were down-regulated. A number of other HDACi-regulated genes fall into groups including cytokine-induced proliferative/survival signaling cascades,

oncogenes/tumor suppressor genes, regulators of apoptosis, DNA synthesis/repair and cell cycle, and proteasome/ubiquitin function.

Molecular studies of HDACi treatment in a variety of different cancer types have confirmed the GEP results, showing that there is a set of common HDACi-regulated genes including the cyclins A, E, B1, D1, and D3, p21, p53, Bax, BCL-2, c-myc, PKCδ, ICAM-1, IL-6 receptor, IL-2, IL-8, IL-10, VEGF, Notch, GADD45α and GADD45β, TGFβ receptor, CTP synthase, and thymidylate synthase (reviewed by Bolden et al., 2006; de Ruijter et al., 2003; Zain and O’Connor, 2010). These types of molecular studies have shown that HDACi’s can induce cell cycle arrest by a p53-dependent induction of p21 in order to repress cyclin D and A, which contributes to the loss of CDK2 and 4 activity and up-regulation of cell cycle regulators GADD45α and GADD45β (reviewed by Bolden et al., 2006; Zain and O’Connor, 2010). HDACi

treatment also leads to hyperaceylation of p53, which stabilizes the protein and increases expression of pro-apoptotic target genes like BCL-2 family members BAX, PUMA, and NOXA (Xu, 2003). Additionally, p21 is up-regulated by the HDACi compounds

vorinostat and VPA in many cancer types, including Hodgkin lymphoma (Buglio et al., 2008), multiple myeloma (Mitsiades et al., 2004), and other human lymphoid cancers (Sakajiri et al., 2005), and is thought to be the important molecular event that makes HDACi treatment toxic to lymphomas and leukemias. Furthermore, vorinostat and VPA can decrease STAT6 and BCL-XL levels in Hodgkin lymphoma (Buglio et al., 2008) and cyclin D1/D2 in mantle cell lymphoma (Sakajiri et al., 2005) promoting cell cycle arrest and apoptosis. Vorinostat has also been shown to repress expression of TCR signaling genes including ZAP70, CD3DIL4, IL5, IL10, FOXP3 and to increase expression of FYN, IFNG, IL12A (reviewed by Zain and O’Connor, 2010).

Important to this thesis, HDACi treatment of tumor cells has been strongly linked to the modulation of BCL-2 family expression to favor a pro-apoptotic expression pattern (reviewed by Bolden et al., 2006; Hagelkruys et al., 2011; Zain and O’Connor, 2010). There are three classes of BCL-2 family proteins: anti-apoptotic, BH3-only modulators of apoptosis, and pro-apoptotic activators (reviewed by Shamas-Din et al., 2011; Strasser et al., 2011). In many cases, HDACi-induced apoptosis has been shown to act through the up-regulation of the pro-apoptotic BH3-only protein BIM (reviewed by Bolden et al., 2006; Chen et al., 2009). Tumor cells attempt to protect themselves from BCL-2- mediated apoptosis with increased expression of anti-apoptotic proteins or decreased

expression of pro-apoptotic proteins, thus shifting the balance of BCL-2 proteins to an anti-apoptotic state.

Ultimately, HDACi treatment results in p21-mediated cell cycle arrest, increased apoptosis, mitochondrial permeablization, release of cytochrome C, and BIM-mediated caspase-9 and -3 activation (reviewed by Bolden et al., 2006; Zain and O’Connor, 2010). The HDACi compounds MS-275 and vorinostat are also associated with hyperacetylation and increased nuclear localization of p65 in B- and T-cell leukemia cell lines (Dai et al., 2005). The use of an NF-κB inhibitor with HDACi treatment also potentiates apoptosis and increases ROS production (Dai et al., 2005; Dasmahapatra et al., 2010; Moreira et al., 2003).

A common translocation found in B-cell lymphomas is the t(14,18) translocation that places the BCL-2 gene downstream of the IgH promoter (Graninger et al., 1987). This translocation is found in 85% of follicular lymphomas and 20% of DLBCLs. When treated with NaB or trichostatin A (TSA), cell lines harboring this translocation show an increase in c-Myc and NF-κB activity, undergo G0/G1 cell cycle arrest, transcriptional down-regulation of BCL-2, and apoptosis (Duan et al., 2005). There is also increased acetylation of Sp1 and C/EBPα, resulting in decreased DNA binding at the BCL-2 promoter with decreased interactions with CBP and HDAC2.