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Supplementary Methods, Supplementary Tables S1-S5, and Supplementary Figures S1-S15

MALAT1is a long non-coding RNA with oncogenic roles in cancer but poorly studied in indolent B-cell neoplasms. Here,MALAT1expression was analyzed using RNA-seq, microarrays or qRT-PCR in primary samples from various clinico-biological subtypes of chronic lymphocytic leukemia (CLL, n=266) and follicular lymphoma (FL, n=61). In peripheral blood (PB) CLL samples, highMALAT1expression was associated with a significantly shorter time to treatment, independently from other known prognostic factors, such as IGHV mutational status. Coding genes whose expression levels were associated withMALAT1in CLL were predominantly related to oncogenic pathways stimulated in the lymph node (LN) microenvironment. Further analysis ofMALAT1expression by microarrays in paired CLL samples from PB/LN showed that its levels were maintained between both anatomical compartments, supporting that the clinical value ofMALAT1expression found in PB is mirroring expression differences already present in LN. Similarly, highMALAT1expression in FL predicted for a shorter progression-free survival, and its correlated expressed genes were associated with pathways promoting FL pathogenesis. In summary,MALAT1expression is related to pathophysiology and clinical behavior of indolent B-cell neoplasms. Particularly in CLL its levels could be a surrogate marker of the microenvironment stimulation and may contribute to refine the clinical management of these patients.

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11– MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

Purpose: Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression. Cyclin D1 is a major cell-cycle regulator and also regulates transcription, but the impact of cyclin D1–mediated transcriptional dysregulation on MCL pathogenesis remains poorly understood. The aim of this study was to define a cyclin D1–dependent gene expression program and analyze its prognostic value. Experimental Design: We integrated genome-wide expression analysis of cyclin D1–silenced and overexpressing cells with cyclin D1 chromatin-binding profiles to identify a cyclin D1–dependent transcriptional program in MCL cells. We analyzed this gene program in two MCL series of peripheral blood samples (n = 53) and lymphoid tissues (n = 106) to determine its biological and clinical relevance. We then obtained a simplified signature of this program and evaluated a third series of peripheral blood MCL samples (n = 81) by NanoString gene expression profiling to validate our findings. Results: We identified a cyclin D1–dependent transcriptional program composed of 295 genes that were mainly involved in cell-cycle control. The cyclin D1–dependent gene program was overexpressed in MCL tumors directly proportional to cyclin D1 levels. High expression of this program conferred an adverse prognosis with significant shorter overall survival of the patients. These observations were validated in an independent cohort of patients using a simplified 37-gene cyclin D1 signature. The cyclin D1–dependent transcriptional program was also present in multiple myeloma and breast tumors with cyclin D1 overexpression. Conclusions: We identified a cyclin D1–dependent transcriptional program that is overexpressed in MCL and predicts clinical outcome.

Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cancer stem cells (CSC) able to self-renew and responsible for drug resistance. SOX11 embryonic transcription factor is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-CSC have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL clinical features and aggressive behavior remains unknown. We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. Here, we integrated the differential gene expression profile (GEP) between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, and specific SOX11 ChIP-chip and epigenetic data in MCL cell lines and primary cases, to identify potential mediators of CSC directly regulated by SOX11 in MCL. We observed an enrichment of hematopoietic (HSC) and leukemic stem cells (LSC) gene signatures in SOX11+ compared to SOX11- MCL primary cases. MSI2, an RNA binding protein that maintains self-renewal and prevents differentiation in HSC, emerged as one of the most significant upregulated CSC-related gene in SOX11+ compared to SOX11- MCLs, positively correlating with SOX11 expression in MCL primary cases. Our in vitro experiments showed that SOX11 binds to MSI2 regulatory regions increasing its expression in MCL cells. MSI2 enhancers located in intronic regions were activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/TP53, 11q/ATM and 9p/CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models. Overall, our results suggest that SOX11+ MCL cases acquire stemness features through the upregulation of MSI2 expression, which promotes chemoresistance, self-renewal and tumor survival. MSI2 represents a novel biomarker for MCL-CSC and therapeutic target for relapsed MCL. Citation Format: Marta Sureda-Gómez, Patricia Balsas, Marta Leonor Rodríguez, Ferran Nadeu, Anna De Bolòs, Álvaro Eguileor, Marta Kulis, Giancarlo Castellano, José Ignacio Martin-Subero, Santiago Demajo, Pedro Jares, Eva Giné, Elias Campo, Virginia Amador. Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 777.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy, but some patients have a very indolent evolution. This heterogeneous course is related, in part, to the different biological characteristics of conventional MCL (cMCL) and the distinct subgroup of leukemic nonnodal MCL (nnMCL). Robust criteria to distinguish these MCL subtypes and additional biological parameters that influence their evolution are not well defined. We describe a novel molecular assay that reliably distinguishes cMCL and nnMCL using blood samples. We trained a 16-gene assay (L-MCL16 assay) on the NanoString platform using 19 purified leukemic samples. The locked assay was applied to an independent cohort of 70 MCL patients with leukemic presentation. The assay assigned 37% of cases to nnMCL and 56% to cMCL. nnMCL and cMCL differed in nodal presentation, lactate dehydrogenase, immunoglobulin heavy chain gene mutational status, management options, genomic complexity, and CDKN2A/ATM deletions, but the proportion with 17p/TP53 aberrations was similar in both subgroups. Sequential samples showed that assay prediction was stable over time. nnMCL had a better overall survival (OS) than cMCL (3-year OS 92% vs 69%; P = .006) from the time of diagnosis and longer time to first treatment. Genomic complexity and TP53/CDKN2A aberrations predicted for shorter OS in the entire series and cMCL, whereas only genomic complexity was associated with shorter time to first treatment and OS in nnMCL. In conclusion, the newly developed assay robustly recognizes the 2 molecular subtypes of MCL in leukemic samples. Its combination with genetic alterations improves the prognostic evaluation and may provide useful biological information for management decisions.

Cyclin D1 is an oncogene frequently overexpressed in human cancers that has a dual function as cell cycle and transcriptional regulator, although the latter is widely unexplored. Here, we investigated the transcriptional role of cyclin D1 in lymphoid tumor cells with cyclin D1 oncogenic overexpression. Cyclin D1 showed widespread binding to the promoters of most actively transcribed genes, and the promoter occupancy positively correlated with the transcriptional output of targeted genes. Despite this association, the overexpression of cyclin D1 in lymphoid cells led to a global transcriptional downmodulation that was proportional to cyclin D1 levels. This cyclin D1-dependent global transcriptional downregulation was associated with a reduced nascent transcription and an accumulation of promoter-proximal paused RNA polymerase II (Pol II) that colocalized with cyclin D1. Concordantly, cyclin D1 overexpression promoted an increase in the Poll II pausing index. This transcriptional impairment seems to be mediated by the interaction of cyclin D1 with the transcription machinery. In addition, cyclin D1 overexpression sensitized cells to transcription inhibitors, revealing a synthetic lethality interaction that was also observed in primary mantle cell lymphoma cases. This finding of global transcriptional dysregulation expands the known functions of oncogenic cyclin D1 and suggests the therapeutic potential of targeting the transcriptional machinery in cyclin D1-overexpressing tumors.

Acute myeloid leukemia (AML) is frequently linked to epigenetic abnormalities and deregulation of gene transcription, which lead to aberrant cell proliferation and accumulation of undifferentiated precursors. ZRF1, a recently characterized epigenetic factor involved in transcriptional regulation, is highly overexpressed in human AML, but it is not known whether it plays a role in leukemia progression. Here, we demonstrate that ZRF1 depletion decreases cell proliferation, induces apoptosis and enhances cell differentiation in human AML cells. Treatment with retinoic acid (RA), a differentiating agent currently used to treat certain AMLs, leads to a functional switch of ZRF1 from a negative regulator to an activator of differentiation. At the molecular level, ZRF1 controls the RA-regulated gene network through its interaction with the RA receptor α (RARα) and its binding to RA target genes. Our genome-wide expression study reveals that ZRF1 regulates the transcription of nearly half of RA target genes. Consistent with our in vitro observations that ZRF1 regulates proliferation, apoptosis, and differentiation, ZRF1 depletion strongly inhibits leukemia progression in a xenograft mouse model. Finally, ZRF1 knockdown cooperates with RA treatment in leukemia suppression in vivo. Taken together, our data reveal that ZRF1 is a key transcriptional regulator in leukemia progression and suggest that ZRF1 inhibition could be a novel strategy to be explored for AML treatment.

Endometrial carcinoma (EC) is the most common gynecological malignancy in the western world. A widely accepted dualistic model, which has been established on a morphological basis, differentiates EC into two broad categories: Type I oestrogen-dependent adenocarcinoma with an endometrioid morphology and Type II non-oestrogen-dependent EC with a serous papillary or clear cell morphology. Molecular genetic evidence indicates that endometrial carcinoma, as described in other malignancies, likely develops as the result of a stepwise accumulation of alterations in cellular regulatory pathways, such as oncogene activation and tumor suppressor gene inactivation, which lead to dysfunctional cell growth. These molecular alterations appear to be specific in Type I and Type II cancers. In type I endometrioid endometrial cancer, PTEN gene silencing in conjunction with defects in DNA mismatch repair genes, as evidenced by the microsatellite instability phenotype, or mutations in the K-ras and/or beta-catenin genes, are recognized major alterations, which define the progression of the normal endometrium to hyperplasia, to endometrial intraepithelial neoplasia, and then on to carcinoma. In contrast, Type II cancers show mutations of TP53 and Her-2/neu and seem to arise from a background of atrophic endometrium. Nevertheless, despite the great effort made to establish a molecularly-based histological classification, the following issues must still be clarified: what triggers the tumor cells to invade the myometrium and what causes vascular or lymphatic dissemination, finally culminating in metastasis? RUNX1, a transcription factor, was recently identified as one of the most highly over-expressed genes in a microarray study of invasive endometrial carcinoma. Another candidate gene, which may be associated with an initial switch to myometrial infiltration, is the transcription factor ETV5/ERM. These studies, as well as those conducted for other genes possibly involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, could help in understanding the differences in the biology and the clinical outcome among histological types.

The Zuotin-related factor 1, ZRF1, has recently been identified as an epigenetic regulator of gene transcription in stem cells and cancer. During differentiation of human teratocarcinoma cells, ZRF1 promotes transcriptional induction of developmental genes that are repressed by Polycomb complexes. Importantly, ZRF1 has recently been shown to be required for both neural differentiation of embryonic stem cells (ESCs) and for maintenance of neural progenitor cell (NPC) identity. Moreover, a dual role has now emerged for ZRF1 in cancer: on the one hand, ZRF1 plays a crucial role in oncogene-induced senescence (OIS) by activating the INK4/ARF locus, thus working as a tumor suppressor; on the other hand, ZRF1 promotes leukemogenesis in acute myeloid leukemia (AML) in a Polycomb-independent fashion. Therefore, increasing evidence points to ZRF1 as a novel target for therapy of neurodegenerative diseases and cancer. This work was supported by grants from the Spanish “Ministerio de Educación y Ciencia” (SAF2013-48926-P),/nAGAUR, AICR (10-0177), from the Fundación Vencer El Cáncer (VEC), and the European Commission's 7th Framework Program 4DCellFate grant number 277899 to L.D.C.

The introduction of new methods for genome-wide analyses of the chromatin state, together with the power of refined techniques for mass spectrometry and biochemistry, has provided an unprecedented view on the complexity of eukaryotic gene regulation. Chromatin structure, the state of histone modifications and DNA methylation are highly dynamic and subject to various levels of regulation. In addition, the subunit compositions of the protein complexes that bring about these changes appear to be assembled in a combinatorial manner that is specific for the cell type and developmental stage, providing increased specificity to these complexes. Here we discuss recent evidence regarding the combinatorial control of chromatin regulatory complexes.

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

Covalent modification of histones is fundamental in orchestrating chromatin dynamics and transcription. One example of such an epigenetic mark is the mono-ubiquitination of histones, which mainly occurs at histone H2A and H2B. Ubiquitination of histone H2A has been implicated in polycomb-mediated transcriptional silencing. However, the precise role of the ubiquitin mark during silencing is still elusive. Here we show in human cell lines that ZRF1 (zuotin-related factor 1) is specifically recruited to histone H2A when it is ubiquitinated at Lys 119 by means of a novel ubiquitin-interacting domain that is located in the evolutionarily conserved zuotin domain. At the onset of differentiation, ZRF1 specifically displaces polycomb-repressive complex 1 (PRC1) from chromatin and facilitates transcriptional activation. A genome-wide mapping of ZRF1, RING1B and H2A-ubiquitin targets revealed its involvement in the regulation of a large set of polycomb target genes, emphasizing the key role ZRF1 has in cell fate decisions. We provide here a model of the molecular mechanism of switching polycomb-repressed genes to an active state.

Endometrial carcinoma is the most common gynaecological malignancy in the western world and the most frequent among infiltrating tumours of the female genital tract. Despite the characterisation of molecular events associated with the development of endometrial carcinoma, those associated with the early steps of infiltration and invasion in endometrial cancer are less known. Deep myometrial invasion correlates with more undifferentiated tumours, lymph-vascular invasion, node affectation and decreased global survival. In this review we present an overview of the molecular pathology of myometrial infiltration that defines the initial steps of invasion in endometrial cancer. Down-regulation of E-cadherin as a main player of epithelial to mesenchymal transition, as well as modifications on other molecules involved in cell-cell contacts, render cells with a migratory phenotype. In addition, altered signalling pathways and transcription factors associate with myometrial invasion, histologic grade and metastasis. Fil: Abal, M.. Hospital Universitari Vall d; España Fil: Llaurado, M.. Hospital Universitari Vall d; España Fil: Dolla, A.. Hospital Universitari Vall d; España Fil: Monge, M.. Hospital Universitari Vall d; España Fil: Colas, E.. Hospital Universitari Vall d; España Fil: Gonzalez, M.. Hospital Universitari Vall d; España Fil: Rigau, M.. Hospital Universitari Vall d; España Fil: Allazzouzi, H.. Hospital Universitari Vall d; España Fil: Demajo, S.. Hospital Universitari Vall d; España Fil: Castellví, J.. Hospital Universitari Vall d; España. Universitat Autònoma de Barcelona; España Fil: Garcia, A.. Hospital Universitari Vall d; España. Universitat Autònoma de Barcelona; España Fil: Ramon y Cajal, S.. Hospital Universitari Vall d; España. Universitat Autònoma de Barcelona; España Fil: Xercavins, J.. Hospital Universitari Vall d; España. Universitat Autònoma de Barcelona; España Fil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Alameda, F.. Universitat Autònoma de Barcelona; España. Hospital del Mar; España Fil: Gil Moreno, A.. Hospital Universitari Vall d; España. Universitat Autònoma de Barcelona; España Fil: Reventos, J.. Universitat Autònoma de Barcelona; España. Hospital Universitari Vall d; España