Maritza Lienlaf

Contribution of E3-ubiquitin ligase activity to HIV-1 restriction by TRIM5alpha(rh): structure of the RING domain of TRIM5alpha.

ABSTRACT TRIM5αrh is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5αrh is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5α exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5αrh restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5αrh RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5α and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5α RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5αrh, such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5α molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5α self-ubiquitylation in the ability of TRIM5α to restrict HIV-1.

RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 From Inhibition of Reverse Transcription and Acceleration of Uncoating

ABSTRACT Rhesus TRIM5α (TRIM5αrh) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5αrh to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5αrh RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5αrh RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5αrh RING domain variants also prevented the degradation of TRIM5αrh that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5αrh blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.

TNPO3 is Required for HIV-1 Replication After Nuclear Import but Prior to Integration and Binds the HIV-1 Core

ABSTRACT TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.

Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL‐10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage‐restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

Functional Analysis of Histone Deacetylase 11 (HDAC11).

The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.

Expression and Function of Histone Deacetylase 10 (HDAC10) in B Cell Malignancies.

Histone deacetylase 10 (HDAC10) belongs to the class IIb HDAC family and its biological role remains mostly unidentified. A decreased HDAC10 expression has been reported in patients with aggressive solid tumors (Osada et al. Int J Cancer 112: 26-32, 2004; Jin et al. Int J Clin Exp Pathol 7: 5872-5879, 2014), suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Consequently, results from our lab suggests that overexpression of HDAC10 in aggressive mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) Z138c and MEC1 cells, respectively, resulted in a rapid induction of cell death in vitro with only 5 % of cells being alive at 48 h, cell cycle arrest, and up-regulation of co-stimulatory molecules. Here we present several standard methods to study the function of HDAC10 in B cell malignancies.

Abstract 2331: HDAC6, new role as master regulator of PD-L1 and immune-related pathways

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immunobiology is still not completely understood, and the reported immunological outcomes when using HDACi are heterogeneous. Our group recently reported that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modulates the expression of immuno-regulatory proteins, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. We primarily focused in PD-L1, which is an important negative regulator of T-cell function and often over-expressed in cancer cells. In a mechanistic point of view, we have found that the pharmacological inhibition and genetic abrogation of HDAC6 inactivates the STAT3 pathway, impairs the nuclear translocation and the recruitment of STAT3 to the PD-L1 promoter and subsequently down-regulates the expression of PD-L1. Moreover, the in vivo abrogation of HDAC6 reduces tumor growth in melanoma models, effect that is enhanced in the presence of the immune check-point blocking antibodies anti-PD-1 and anti-CTLA4. These results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immunomodulatory agents in cancer. Citation Format: Tessa Knox, Maritza Lienlaf, Patricio Perez, Mibel Pabon, Calvin Lee, Fengdong Cheng, Eva Sahakian, John Powers, Susan Deng, Smalley Keiran, Alan Kozikowski, Javier Pinilla, Amod Sarnaik, Ed Seto, Jeffrey Weber, Eduardo Sotomayor, Alejandro Villagra. HDAC6, new role as master regulator of PD-L1 and immune-related pathways. [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 2331.

Abstract 4086: Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment. This includes the deacetylation of multiple non-histone targets, such as proteins involved in cell cycle/apoptosis and immune regulation. Specifically, HDACs have garnered significant interest due to the availability of drugs that selectively inhibits HDACs. We recently identified that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modifies the immunogenicity and proliferation of melanoma in both in vitro and in vivo models. Using specific HDAC6 inhibitors (HDAC6i) we observed decreased proliferation and G1 cell cycle arrest in all melanoma cell lines measured by MTS assay and flow cytometry. These results were also observed in stable HDAC6 knockdown melanoma cell lines (HDAC6KD) generated by specific lentiviral shRNA for HDAC6. In addition to the effects observed in proliferation and apoptosis after inhibiting HDAC6, we found important changes in the expression of immune-related pathways, including increased expression of MHC, co-stimulatory molecules, and specific melanoma tumor associated antigens such as gp100, MART-1, Tyrp1 and Tyrp2. Our in vitro results were further supported by in vivo tumor growth studies. We observed a delayed tumor growth of inoculated B16 melanoma cells in C57BL/6 mice treated with selective HDAC6i. A similar outcome was identified after inoculation of HDAC6KD B16 melanoma cells in C57BL/6 mice. Such an effect was reverted partially in CD4+ and CD8+ depleted C57BL/6 mice challenged with HDAC6KD cells, suggesting that the disruption of HDAC6 enhances immune system recognition of melanoma cells. This delay in tumor growth could be a reflection of their diminished proliferation and an increase in their immunogenicity leading to improved immune recognition and clearance. These studies provide critical insights into the molecular pathways that are involved in the regulatory role of HDAC6 in cell proliferation, survival, and cytokine signaling of human melanoma cells. Collectively, our data has identified HDAC6 as an attractive therapeutic target in melanoma. Citation Format: Patricio Perez-Villarroel, Maritza Lienlaf, Calvin Lee, Fengdong Cheng, David Woods, Kelly Barrios, Karrune Woan, Jorge Canales, Tessa Knox, Danay Marante, Hongwei Wang, Pedro Horna, Keiran Smalley, Esteban Celis, Ed Seto, Jeffrey S. Weber, Eduardo M. Sotomayor, Alejandro Villagra. Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2014-4086

Abstract 4089: Histone deacetylase 6 (HDAC6) as a regulator of PD-L1 expression through STAT3 modulation in melanoma

In spite of the progress made in the understanding of the cell biology, genetics and immunology of melanoma, the outcome for patients with advanced-stage disease has remained poor with a median survival ranging from 2-16 months. Some optimism was recently provided in metastatic melanoma by the improved clinical outcomes observed in patients receiving PD-L1 blocking antibodies. A better understanding of the environmental, genetic and epigenetic factors limiting the efficacy of melanoma immunotherapy will provide appropriate partner(s) for combination with Ipilimumab or PD1/PDL1 antibodies. Among the epigenetic factors, we have found that one member of the histone deacetylase family, HDAC6, plays a critical role not only in the regulation of survival/apoptosis of melanoma cells but also in limiting their immunogenicity and recognition by immune effector cells. Particularly, we found a major role of HDAC6 as a modulator of the immunosuppresive STAT3/IL-10 pathway and down-regulation of tolerogenic PD-L1 molecules in melanoma cells. By analyzing HDAC6 knock-down melanoma cell lines (HDAC6KD) we demonstrated the inactivation of the STAT3 pathway and the subsequent down-regulation of its target genes, including the expression of PD-L1. We also observed that the PD-L1 expression and phosphorylation of STAT3 was decreased in melanoma isolated from xenograph tumor growth models after in vivo treatment with specific HDAC6 inhibitors Fortunately, there are multiple HDAC6-selective inhibitors available to mechanistically study the role of HDAC6 on these processes and provide a viable therapeutic avenue, which may minimize undesirable side effects that are characteristic of pan-HDACi such as SAHA. By building on our understanding of HDAC6 and applying these findings to novel experimental design, we hope to identify innovative therapeutic options to benefit cancer patients. Citation Format: Maritza Lienlaf, Patricio Perez-Villarroel, Calvin Lee, Fengdong Cheng, Jorge Canales, Tessa Knox, Danay Marante, Amod Sarnaik, Pedro Horna, Ed. Seto, Keiran Smalley, Jeffrey S. Weber, Eduardo M. Sotomayor, Alejandro Villagra. Histone deacetylase 6 (HDAC6) as a regulator of PD-L1 expression through STAT3 modulation in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4089. doi:10.1158/1538-7445.AM2014-4089

Abstract 3554: The histone deacetylase inhibitor LBH589 augments anti-tumor immunity through direct effects on tumor and immune cells leading to impaired tumor progression in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Overcoming immune tolerance is critical for the development of effective immunotherapy against cancer. Tumor cells induce tolerance through a variety of modalities including the secretion of IL-10 and TGF-β, the expression of inhibitory ligands, and the generation of cells with regulatory properties. Epigenetic modifiers have gained special attention due to their ability to modify immune regulatory pathways. In this context, a group of chemical compound collectively named histone deacetylase inhibitors (HDACi) have recently been shown to modulate tumor cell immunogenicity, adding a new property to their already well-documented cytotoxic effect against transformed cells. LBH589 is a potent, pan-HDACi that has been used in clinical trials for the treatment of several hematological malignancies. Here, we provide evidence of the anti-melanoma effect of LBH589 through its direct effects upon tumor cells as well as its modulatory effects upon immune cells. Indeed, treatment of human and murine melanoma cells in vitro with LBH589 resulted in inhibition of melanoma proliferation, characterized by cell cycle arrest in G1. Moreover, we found that LBH589 enhances the expression of MHC class I and II molecules on melanoma cells, which is critical for immune recognition. In addition, LBH589 treatment inhibits the production of IL-10, while enhancing the secretion of pro-inflammatory cytokines such as IL-12 and the expression of the costimulatory molecule B7.2 on antigen-presenting cells (APCs). LBH-treated APCs also displayed an enhanced capability to activate naive antigen-specific T-cells as evidenced by their increased production of IL-2 and IFN-γ. More importantly, LBH589-treated APCs also restored the function of anergic T-cells isolated from tumor bearing mice. Finally, in vivo treatment of B16 melanoma bearing mice with LBH589 resulted in a substantial decrease in tumor growth when compared to vehicle-control treated mice (mean 602mm3 vs. 1316mm3, respectively). In conclusion, the dual ability of the pan-HDACi LBH589 to target both melanoma cells and to enhance anti-tumor immune responses provides the rationale for evaluating this compound either alone or in combination with immune enhancing therapeutic approaches (eg, CTLA4 blockade) for melanoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3554. doi:1538-7445.AM2012-3554