Elena Cubedo

HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway

HGAL is a germinal center (GC)-specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism - direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.

Identification of LMO2 transcriptome and interactome in diffuse large B-cell lymphoma

LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis.

PRDM1/Blimp1 downregulates expression of germinal center genes LMO2 and HGAL

Human germinal center‐associated lymphoma (HGAL) and LIM domain only‐2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC‐derived lymphomas and distinguish biologically distinct subgroups of diffuse large B‐cell lymphomas (DLBCL) associated with improved survival. However, little is known about their regulation. PRDM1/Blimp1 is a master regulator of terminal B cell differentiation and may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. We now demonstrate that both HGAL and LMO2 are directly regulated by the transcription repressor PRDM1. In vivo studies demonstrate that PRDM1 directly binds to the recognition sites within the upstream promoters of both HGAL and LMO2. PRDM1 binding suppresses endogenous protein and mRNA levels of HGAL and LMO2. In addition, promoter analysis reveals that site‐specific binding of PRDM1 to the promoters is capable of repressing transcriptional activity. This inhibitory effect of PRDM1 suggests that it has a key role in the loss of HGAL and LMO2 expression upon differentiation of GC B cells to plasma cells and may also contribute to absence of HGAL and LMO2 expression in post‐GC lymphoid tumors.

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer

Triple negative breast cancer (TNBC) frequently relapses locally, regionally or as systemic metastases. Development of targeted therapy that offers significant survival benefit in TNBC is an unmet clinical need. We have previously reported that blocking interactions between PAH2 domain of chromatin regulator Sin3A and the Sin3 interaction domain (SID) containing proteins by SID decoys result in EMT reversal, and re-expression of genes associated with differentiation. Here we report a novel and therapeutically relevant combinatorial use of SID decoys. SID decoys activate RARα/β pathways that are enhanced in combination with RARα-selective agonist AM80 to induce morphogenesis and inhibit tumorsphere formation. These findings correlate with inhibition of mammary hyperplasia and a significant increase in tumor-free survival in MMTV-Myc oncomice treated with a small molecule mimetic of SID (C16). Further, in two well-established mouse TNBC models we show that treatment with C16-AM80 combination has marked anti-tumor effects, prevents lung metastases and seeding of tumor cells to bone marrow. This correlated to a remarkable 100% increase in disease-free survival with a possibility of “cure” in mice bearing a TNBC-like tumor. Targeting Sin3A by C16 alone or in combination with AM80 may thus be a promising adjuvant therapy for treating or preventing metastatic TNBC.

Abstract 1906: The role of immunogenic SPANX antigens in distinct cancer stem cell subsets within triple negative breast cancers

Breast cancer is the leading cause of death in women, primarily due to metastatic disease rather than the primary tumor. Median survival with metastatic breast cancer is 3 years, with no statistically significant change in survival in over 20 years. Triple-negative breast cancer (TNBC) lacks estrogen and progesterone receptor expression and ERBB2 amplification and is the most lethal form of breast cancer. It is resistant to endocrine therapy and chemo-resistance and metastasis invariably emerge. Increasing evidence indicates that cancer stem cells (CSCs) are chemo-resistant and initiate metastasis. We have previously identified and characterized distinct subsets of CSCs within TNBC cell lines and patient-derived tumors. Within the surface CD44+ populations, CD24 expression defines two subsets of CSCs: mesenchymal-like CD24neg and epithelial-like CD24low+. Epithelial-like CD24low+ cells are more aggressive than CD24neg cells, with increased self-renewal, migration, invasion, tumor-initiation and metastatic potential. In addition, CD24low+ cells are enriched following chemo- and radiation therapies; greater than 80% of CD24neg cells die and the majority of cells that survive are CD24low+. Hence, therapies that selectively eliminate the CD24low+ population in TNBC have the potential to be of enormous benefit to cancer patients. Therapeutic targeting of surface CD24 using antibodies have not been successful due to the expression of CD24 on many different cell types including B cells. Thus, there is a need for more selective strategies to target CSC populations without affecting normal cells. Here, we show for the first time, increased expression of a family of cancer/testis antigens (CTAs) namely SPANX in the most aggressive CD24low+ CSC population. CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. Thus, SPANX may serve as a selective marker for targeting CSCs. In addition, we demonstrate a functional role for SPANX in mediating CSC phenotype. Knockdown of SPANX decreased the percent of CD24low+ CSCs in TNBCs. It also attenuated the proliferation, migration and invasion of CD24low+ cells. Radiation treatment increased SPANX expression levels and enriched for CD24low+ cells. Loss of SPANX resulted in increased cell death of CD24low+ cells after radiation treatment. Hence, we show that SPANX may be promoters of the most aggressive CSC subset of TNBC. Since SPANX is highly immunogenic, our data provide rationale for further testing of combined radiation and immunotherapy approaches in the treatment of this deadly cancer. It also supports the use of protective and therapeutic SPANX vaccines against the most aggressive CSC subset in TNBC. Citation Format: Lauren Shahin, Elena Cubedo, Ebony Coats, Jeff Boyd, Diana Azzam. The role of immunogenic SPANX antigens in distinct cancer stem cell subsets within triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1906. doi:10.1158/1538-7445.AM2017-1906

Abstract LB-160: HDAC9 expression is deregulated in malignant B-cell lymphomas in particular in diffuse large B-cell lymphoma and mantle cell lymphoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme that, within the hematopoietic system, is preferentially expressed in the B-cell lineage. In our previous works, in order to identify HDAC9 function in the B cell lineage we developed mice that constitutively expressed human HDAC9 from early stages of B-cell development, under the control of the immunoglobulin heavy chain (IgH) enhancer. These mice developed lymphoproliferative disorders, including indolent Marginal Zone Lymphoma (MZL) and more aggressive post-Germinal Center (GC) lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells. In order to examine the relationship between diseases observed in the mouse model and human primary lymphoma, we have examined, using immunohistochemistry (IHC) the expression of full length HDAC9 isoform in a panel of various B-cell malignancies from human tumor samples. The study group included 59 non-Hodgkin lymphomas (NHL), and 3 classical HL. Non-HL consisted of 34 diffuse large B cell lymphoma (DLBCL), 9 follicular lymphoma (FL), 5 marginal zone lymphoma (MZL), 6 mantle cell lymphoma (MCL), and 2 small lymphocytic lymphomas (SLL). HDAC9 expression was assessed by IHC using tissue microarray and/or routine tissue sections. Protein expression was scored as negative (0), low (1), or high (2) depending on the staining signal intensity. Expression of HDAC9 in the nuclei of the tumor cells was compared with that seen in adenocarcinoma cells; if equal or higher, then expression of HDAC9 was considered high and if lower, then expression of HDAC9 was considered low. Five reactive lymph nodes were studied to assess the baseline expression of HDAC9. Rectal adenocarcinomas were used as positive controls. In reactive lymph nodes, HDAC9 was weakly expressed in a subset of germinal center cells, a subset of lymphoid cells in the paracortex as well as in endothelial cells. HDAC9 expression was detected in all subsets of B-cell lymphomas analyzed and in most cases with a level of expression higher than those seen in reactive lymph nodes. DLBCL and MCL tumors had the highest frequency of high HDAC9 expression among the B-cell lymphomas analyzed, 77 and 83% (Fishers exact test P = 1.0), respectively. No differences in HDAC9 expression were detected in DLBCL of GC and non-GC type. In contrast, most (69%) of the low-grade B cell lymphomas showed no or lower expression of HDAC9 (Fishers exact test P = 0.004; as compared to DLBCL). Classical HL showed frequently low-expression of HDAC9 in the tumor cells. In summary, HDAC9 is frequently expressed in B-cell lymphomas with the highest level of expression found in the most aggressive lymphomas such as DLBCL and MCL. These findings support the biological role of HDAC9 in the pathobiology of aggressive B cell neoplasms and highlight the need to further study HDAC9 function in these malignancies as well as its importance as a therapeutic target. Citation Format: Elena Cubedo, Veronica Gil, Chae Hwa Kim, German Campuzano-Zuluaga, Nitin Kumar Agarwal, Louise Howell, Kevin R Petrie, Francisco Vega, Arthur Zelent. HDAC9 expression is deregulated in malignant B-cell lymphomas in particular in diffuse large B-cell lymphoma and mantle cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-160. doi:10.1158/1538-7445.AM2015-LB-160