Abstract A comprehensive understanding of the molecular mechanisms underlying multiple myeloma (MM) pathogenesis is essential for developing therapeutic strategies that overcome disease heterogeneity and treatment relapse. In this review, we focus on transcription factors (TFs), key regulators of gene expression that play critical roles in normal hematopoiesis and MM biology. We first discuss the physiological functions of TFs in lymphoid lineage commitment and terminal B‐cell differentiation into plasma cells. This provides insights into the contribution of key TFs, such as IRF4 and PRDM1, to development of MM. We then examine the regulatory mechanisms of TFs in MM, exploring how their deregulation participates in the pathogenesis of the disease. We summarize the roles of major TF families, highlighting both well‐established TFs essential for MM pathogenesis and emerging TFs with potential clinical relevance. In addition to their functional roles, several TFs show promise as biomarkers for patient stratification and risk assessment. Finally, we discuss recent advances that challenge the notion of TFs as “undruggable”, including siRNA‐loaded lipid nanoparticles or Proteolysis‐Targeting Chimeras (PROTACs), which offer novel opportunities to therapeutically modulate TF activity. These strategies could enable the development of novel interventions aimed at improving clinical outcomes and quality of life for MM patients. Collectively, this review integrates physiological and pathological insights into TF function in MM and underscores their potential as biomarkers and actionable targets in precision medicine.

Epigenetic modulators in combination with proapoptotic drugs have become the standard of care treatment in hematological malignancies. Conversely, these combinations have failed to demonstrate clinical efficacy in solid tumors. To address this discrepancy, we conducted a comprehensive analysis of the anti-tumor activity of epigenetic inhibitors in combination with BH3 mimetics that block anti-apoptotic proteins BCL-XL, BCL2 or MCL1 in a large set of solid tumor cell lines derived from patients and mouse models. Treatment with epigenetic drugs targeting DNA methyltransferase, histone methyltransferase, and histone deacetylase enzymes in combination with a BCL-XL inhibitor resulted in marked synergistic in vitro responses both in human and mouse solid tumor cell lines. This unique BCL-XL dependency was in clear contrast to hematological malignancies, which are largely dependent on BCL2 or MCL1 inhibition under epigenetic drug treatment. Mechanistically, co-targeting of epigenetic regulators and BCL-XL induced expression of endogenous retroelements that led to immunogenic cell death. We thus hypothesized that this response may sensitize tumor cells to immune checkpoint blockade (ICB). Accordingly, treatment with a triple combination of epigenetic and BCL-XL inhibitors with an anti-PD-1 monoclonal antibody in vivo reduced tumor growth and prolonged overall survival in a panel of murine syngeneic and orthotopic models of lung, colorectal and breast carcinomas, melanoma, and glioblastoma, as well as in an immunocompetent human colon cancer model. Using flow cytometry and single-cell RNA sequencing of the tumor microenvironment, we found that the broad activity of the triple therapy relied on the expansion of T and NK cells with cytotoxic potential, an increase in the M1/M2 macrophage ratio, and a reduction of immunosuppressive Treg cells, dendritic cells, and B lymphocytes. In conclusion, we report a novel regimen combining epigenetic and BCL-XL inhibitors with ICB that produces potent anti-tumor responses in multiple preclinical models of solid tumors.

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

The cover image is based on the Research Article Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors by Marta H. G. Costa et al., https://doi.org/10.1002/bit.28378.

Multiple myeloma (MM) is a hematologic neoplasm characterized by a clonal expansion of malignant plasma cells (PCs) in the bone marrow, showing clinical, genetic, and epigenetic heterogeneity. Chromosomal translocations are one of the hallmarks of MM, and mainly involve the immunoglobulin heavy chain locus (IGH). These translocations usually result in the placement of various oncogenes under the control of IGH, leading to the up-regulation of genes that provide a selective growth advantage to MM cells.1 Five recurrent IGH translocations have been described in MM; however, in many cases, the second gene involved is not defined in routine clinical analyses. Besides, recent studies have reported novel recurrent fusion partners and novel non-IGH fusions beyond well-known translocations.2, 3 Nevertheless, these approaches did not consider the normal counterpart of B-cells, which may provide new insights regarding the role of fusion transcripts (FT) in MM. Furthermore, MM is also associated with deregulation of long noncoding RNAs (lncRNA), a group of genes with increasing relevance in cancer.4 Various studies suggest the involvement of lncRNAs in chromosomal translocations; however, this has not been assessed in MM. Here, to define the landscape of expressed FTs in MM, we analyzed the strand-specific RNA-seq (ssRNA-seq) data of 35 samples obtained from 6 different B-cell subpopulations (5 naïve, 7 centroblast, 7 centrocyte, 8 memory, 5 tonsillar PC, and 3 bone marrow [BM] PC samples) obtained from 11 healthy donors (8 tonsil and 3 BMPCs) and PCs from 37 MM patients, paying particular attention to FTs involving lncRNAs (lncFT). Using the STAR-Fusion algorithm, we initially identified 2169 FTs. After applying several computational filtering steps, we defined 1454 FTs expressed in B-cells and MM samples (Figure S1). The highest numbers of FTs were detected in healthy donor PCs (tonsillar plasma cells [TPC] and BMPC) (Figure S2A-B), and based on the biological relevance of IG genes in B-cells and malignant PC, detected FTs were classified into IG and REST (none of the associated genes corresponded to an IG gene) categories (Figure S2C–E). The 82.5% of FTs detected in healthy PCs occurred with IG genes, harbored very few reads per transcript, and were only supported by junction reads without any spanning reads covering the non-IG partner gene. Therefore, FTs that were not supported by at least one spanning read were filtered out, resulting in the final detection of 208 expressed FTs in normal B-cells and MM cells (Figure S1A). To validate our results and identify FTs consistently detected, we also applied ARRIBA and STAR-SEQR algorithms to our cohort with the same filters described above. One hundred and fifty-eight FTs were detected by at least two algorithms and were selected for further analyses after a quality check step (Appendix S1; Figures S1A, S2F–I; Table S1). These expressed FTs were detected in every cell population, with a significantly higher number of FTs in MM cells (median of 3 ± 2.97) (Wilcoxon p-value <.001) (Figure S2G). A similar number of reads per FT was detected in all cell subpopulations (analysis of variance [ANOVA] p-value .382), suggesting that FTs are expressed consistently at low levels (Figure S2G), and most of the expressed FTs occurred between two non-IG partners (Figure S2H). Characteristic features of B-cells include IG gene rearrangement and active transcription of IG genes, leading to the transcription of thousands of similar transcripts from these loci,5 which could be misidentified as FTs in these cells. Thus, cell-specific features should be considered when implementing an adequate FT detection pipeline for each cell type to exclude false positive events. Furthermore, the presence of FTs in normal B-cells indicates that FTs are not exclusive to tumor cells, suggesting that FTs may contribute to transcriptional diversity in healthy tissues. From the 158 FTs, we filtered out those detected in at least one normal B-cell sample to focus on MM-specific FTs, leading to the identification of 79 expressed FTs (61 unique) (Figure S1A, Table S2), 29.5% of which had not been previously described (Figure 1A). At least one expressed FT was identified in 75.7% of the MM samples (Figure 1B), indicating that some FTs emerge specifically after malignant transformation. A Human Phenotype ontology analysis of coding genes involved in these 61 unique FTs showed a significant enrichment of genes associated with B lymphocyte dysfunction phenotypes (p-adj <.05), suggesting that important genes for B-cell abnormalities and MM pathogenesis may be more prone to FT formation. Most of the MM-expressed FTs showed an overall low read count, with some exceptions (Figure 1C). As previously described,2 85.3% of MM FTs were patient-specific, but 9 were recurrently expressed FTs (Figure 1D). A total of 88.5% of MM-specific FTs were derived from the fusion between 2 non-IG partners and IG FT percentages were lower than those previously reported2, 3 probably due to our smaller cohort size (Figure 1E). Nevertheless, we identified the IGH-NSD2 expressed FT derived from t(4;14) in two patients and two MM cell lines (Figure S3A–C), and described a novel FT between the genes GBE1 and KIF20B in one MM cell line (Figure S3D–F). Recent studies have shown the implication of lncFTs in MM, such as lncFTs with PVT1, but a complete characterization of the lncFT transcriptome is still pending.3 We observed that the 27.9% of MM-specific expressed FTs were lncFTs (Figure 1F), some of them leading to the overexpression of the associated lncRNA (Figure 1G,H), as in the case of FTs involving oncogenes.2, 3 Interestingly, a relevant fraction of MM-specific FTs occurred between two adjacent genes in the same DNA strand, being defined as transcription read-throughs (RT) (Figure 1I), a novel class of MM-specific FT. A total of 64.3% of RTs involved a lncRNA as a fusion partner gene (Figure 1J), some of them were detected in both MM patient samples and cell lines with a low expression and were validated in cell lines through real-time quantitative reverse transcription PCR (qRT-PCR) and Sanger sequencing (Figure S3G–I). Furthermore, we found recurrences for three RTs with lncRNAs, such as the FT between AC092691.1 and LSAMP (Figure 1K), showing an increased expression of the AC092691.1 in comparison to normal PCs and MM samples without the FT (Figure 1L). The presence of functional oncogenic lncFTs and RTs has been reported for other tumor types,6 suggesting that lncFTs could be important in MM, but additional studies will be needed to determine their role in MM. Finally, we analyzed whether lncFTs might have an impact on the outcome of MM patients by analyzing expressed FTs in 599 MM patients included in the MMRF CoMMpass data set release IA15. We used the intersection of STAR-Fusion and ARRIBA, identifying 556 expressed lncFTs. Interestingly, we found that 35% of MM-specific unique lncFTs defined in our cohort were present in the CoMMpass data set. We observed various FTs between lncRNAs and IG genes (IGK-FAM230C, IGH-LINC-PINT), suggesting that MM patients with IG translocations could involve both coding and noncoding partner genes, and that lncRNAs could explain some of the MM cases in which the associated IG gene in translocations remained unknown. We validated the robustness of our algorithm by comparing the number of patients in which we detected the IGH-NSD2 FT with those patients in which t(4;14) was detected by whole-genome sequencing (WGS), identifying IGH-NSD2 FT in 75 of the 79 samples positive by WGS, and additionally, detecting the expression of this FT in 2 other MM samples where WGS for t(4;14) was negative (Fisher's exact test p-value = 6.7e-88). To assess whether the lncFTs could be associated with prognosis in MM, we selected those lncFTs that were detected in more than 2% of MM patients (Figure S4A), and we evaluated the combination of lncFTs and the defined high-risk genetic markers1 (International Staging System [ISS] stage, t(4;14), t(14;16), t(14;20), del(17p), deletion of CDKN2C, del(1p), amp(1q), and mutations of TP53) using a multivariate coxph model and BIC to select the optimal number of variables. We discovered that the expression of 3 lncFTs (TEX35-AL37796.1, AL050309.1-KLF8, and PVT1-IGL), together with the ISS stage and TP53 mutations resulted in a significantly lower progression-free survival (PFS) (global p < .0001) and the model stratifies the MM patients according to the number of events they have (an event consists of having any of the five risk factors) into four risk groups (Figure 1M,N). Similarly, expression of 1 lncFT (TEX35-AL37796.1) together with the ISS stage, del(17p) or amp(1q) also resulted in statistically significant worse overall survival (OS) (global p < .0001), identifying five groups with significant differences in their OS (Figure 1O,P). An ANOVA test comparing the models derived from high-risk genetic factors only or combining them with lncFTs resulted in a significant improvement for the combination of both risk factors for PFS (p-value = 6.3e−5, Figure S4B) and OS (p-value = .019, Figure S4C). These findings should be validated in other MM cohorts, but our results suggest that lncFTs in MM could contribute to a better patient stratification, impacting in patient management in terms of treatment choice or contributing to the identification of specific subgroups of patients suitable for personalized therapies. In summary, this study provides the first comprehensive landscape of expressed lncFTs and RTs in MM, demonstrating that FTs may also be expressed in normal B-cells and that expression of recurrent lncFTs may have a significant impact in PFS and OS in MM patients. This research was funded by grants from Instituto de Salud Carlos III and co-financed by European Regional Development Fund-FEDER "A way to make Europe" (PI16/02024, PI17/00701 and PI19/01352, CIBERONC CB16/12/00489, Spanish Ministry of Economy, Industry and Competitivity (RTHALMY SAF2017-92632-EXP), with support from 616/C/2019 Fundació La Marató de TV3, Departamento de Salud-Gobierno de Navarra 40/2016 and Fundación Ramón Areces (PREMAMM). The study was also supported by Cancer Research UK [C355/A26819] and FC AECC and AIRC under the Accelerator Award Programme, the Multiple Myeloma Research Foundation Networks of excellence 2017 Immunotherapy Program Grant Award, the International Myeloma Foundation (Brian van Novis), the Qatar National Research Fund award 7-916-3-237, and Paula and Rodger Riney Foundation.Ane Amundarain was supported by FPU (FPU17/02733) grant from Ministerio de Ciencia, Innovación y Universidades, Gobierno de España. Luis V. Valcárcel and Arantxa Carrasco-Leon were supported by PFIS (FI17/00297 and FI16/00275, respectively) award from Instituto de Salud Carlos III (ISCIII). Xabier Cendoya was supported by Basque Government with the grant promoting doctoral theses to young predoctoral researchers (PRE_2018.2.0297). These data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (https://research.themmrf.org and www.themmrf.org). The authors declare no competing financial interests. Conception and design: Ane Amundarain, Luis V. Valcárcel, Felipe Prósper, Xabier Agirre. Development of methodology: Ane Amundarain, Luis V. Valcárcel, Raquel Ordoñez, Felipe Prósper, Xabier Agirre. Acquisition of data and assistance with experiments: Ane Amundarain, Luis V. Valcárcel, Raquel Ordoñez, Leire Garate, Estíbaliz Miranda, Xabier Cendoya, Arantxa Carrasco-Leon, María José Calasanz, Cem Meydan, Christopher E. Mason, Ari Melnick, Francisco J. Planes, Felipe Prósper, Xabier Agirre. Analysis and interpretation of data: Ane Amundarain, Luis V. Valcárcel, Leire Garate, Estíbaliz Miranda, Xabier Cendoya, Bruno Paiva, Cem Meydan, Ari Melnick, Paula Rodriguez-Otero, José I. Martín-Subero, Jesús San Miguel, Francisco J. Planes, Felipe Prósper, Xabier Agirre. Writing, review, and/or revision of the manuscript: Ane Amundarain, Luis V. Valcárcel, Felipe Prósper, Xabier Agirre and all authors reviewed and approved final version of the manuscript. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Luis V. Valcárcel, Cem Meydan, Christopher E. Mason, Francisco J. Planes. Study supervision: Felipe Prósper and Xabier Agirre. ssRNA-seq data of MM patients is available at GEO under accession number GSE151063, and ssRNA-seq data of healthy samples are available under accession codes GSE114816 and GSE114803. Figure S1. FT detection pipeline and initial homology filtering. Figure S2. FT in healthy B differentiation and MM samples. Figure S3. Validation of expressed FT detected in MM cohort. Figure S4. FT detection in the CoMMpass dataset. Table S1. Spanning and junction reads, similarity score, and GC percentage of FTs detected in B cell differentiation and MM patient samples. Table S2. Expressed FTs detected in MM samples. Appendix S1. Supplementary Data. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Clonal hematopoiesis of indeterminate potential (CHIP) is defined as a clonal accumulation of somatic mutations in hematopoietic stem cells in the absence of hematologic malignancy or other clonal disorders. CHIP has been associated with a higher risk of developing hematologic malignancies. Recently, CHIP has been linked to a family history of lung cancer (FHLC) in lung cancer patients. In the present study, we evaluated the association between clinical factors and CHIP in individuals enrolled in a lung cancer screening program. Thirty-two asymptomatic individuals with lung cancer enrolled in the International-Early Lung Cancer Action Program (I-ELCAP) at the Clínica Universidad de Navarra, were matched by sex, age, COPD, smoking history and FHLC with 32 screened controls without lung cancer. CHIP was evaluated with the PMPv2 NGS panel, an in-house panel designed for the evaluation of SNV/INDELs in 56 genes associated with myeloid malignancies. CHIP was defined by the presence of cancer-associated somatic mutations with a minimum of 1000 reads and a variant allele frequency (VAF) ≥ 2% and < 40%. Statistical differences were analyzed with the Fisher's exact test. CHIP was found in 13 individuals (20%). Two individuals had two mutations. Mutated genes included DNMT3A (6), TET2 (3), ASXL1 (2), PPM1D (2), BCOR (1) and SH2B3 (1). The presence of CHIP was significantly associated with age (<60 vs. ≥60 years; p=0.022) and smoking history (<50 vs. ≥50 pack-years; p=0.009), but not with sex, COPD or FHLC. No statistically significant differences in CHIP frequencies were found between lung cancer patients (22%) and controls (19%). Mutated genes in cancer patients included DNMT3A (2), PPM1D (2), ASXL1, TET2, BCOR and SH2B3 (1 each), while control subjects had mutations in DNMT3A (4), TET2 (2) and ASXL1 (1). CHIP was found in 20% of asymptomatic individuals screened for lung cancer. It was more common in older individuals and in those with greater tobacco exposure. The prevalence of CHIP was similar in screened individuals with or without lung cancer.

With the frenetic growth of high-dimensional datasets in different biomedical domains, there is an urgent need to develop predictive methods able to deal with this complexity. Feature selection is a relevant strategy in machine learning to address this challenge. We introduce a novel feature selection algorithm for linear regression called BOSO (Bilevel Optimization Selector Operator). We conducted a benchmark of BOSO with key algorithms in the literature, finding a superior accuracy for feature selection in high-dimensional datasets. Proof-of-concept of BOSO for predicting drug sensitivity in cancer is presented. A detailed analysis is carried out for methotrexate, a well-studied drug targeting cancer metabolism.

LXR stimulates a metabolic switch and reveals cholesterol homeostasis as a statin

Clinical and genetic risk factors are currently used in multiple myeloma (MM) to stratify patients and to design specific therapies. However, these systems do not capture the heterogeneity of the disease supporting the development of new prognostic factors. In this study, we identified active promoters and alternative active promoters in 6 different B cell subpopulations, including bone-marrow plasma cells, and 32 MM patient samples, using RNA-seq data. We find that expression initiated at both regular and alternative promoters was specific of each B cell subpopulation or MM plasma cells, showing a remarkable level of consistency with chromatin-based promoter definition. Interestingly, using 595 MM patient samples from the CoMMpass dataset, we observed that the expression derived from some alternative promoters was associated with lower progression-free and overall survival in MM patients independently of genetic alterations. Altogether, our results define cancer-specific alternative active promoters as new transcriptomic features that can provide a new avenue for prognostic stratification possibilities in patients with MM.

The use of allogeneic adipose-derived mesenchymal stromal cells (alloADSCs) represents an attractive approach for treating myocardial infarction (MI). Furthermore, adding a natural support improves alloADSCs engraftment and survival in heart tissues, leading to a greater therapeutic effect. We aimed to examine the safety and immunological reaction induced by epicardial implantation of a clinical-grade collagen scaffold (CS) seeded with alloADSCs for its future application in humans. Thus, cellularized scaffolds were myocardially or subcutaneously implanted in immunosuppressed rodent models. The toxicological parameters were not significantly altered, and tumor formation was not found over the short or long term. Furthermore, biodistribution analyses in the infarcted immunocompetent rats displayed cell engraftment in the myocardium but no migration to other organs. The immunogenicity of alloADSC-CS was also evaluated in a preclinical porcine model of chronic MI; no significant humoral or cellular alloreactive responses were found. Moreover, CS cellularized with human ADSCs cocultured with human allogeneic immune cells produced no alloreactive response. Interestingly, alloADSC-CS significantly inhibited lymphocyte responses, confirming its immunomodulatory action. Thus, alloADSC-CS is likely safe and does not elicit any alloreactive immunological response in the host. Moreover, it exerts an immunomodulatory action, which supports its translation to a clinical setting.

Deregulation of long non-coding RNAs (lncRNAs) is emerging as a common feature of human tumors, suggesting that their investigation may uncover novel oncogenic mechanisms. Previous studies suggested that the alteration of some lncRNAs might play an important role in multiple myeloma (MM); however, the complete expression landscape of lncRNAs has not been elucidated. In the present work we characterized the lncRNAs transcriptome of MM determining their potential involvement in this disease. Firstly, we performed paired-end strand-specific RNA-seq (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, 3 bone marrow PCs (BMPCs) of healthy donors, and in distinct normal B-cell populations (Naïve, Centroblasts, Centrocytes, Memory and Tonsilar PCs). We identified 40,511 novel lncRNAs, representing more than half of MM transcriptome (56%) and which, together with the previously annotated lncRNAs, comprised most of the MM transcriptome (82%). We studied the transcriptional heterogeneity in MM, observing that lncRNAs showed a more heterogeneous expression than coding genes, suggesting that these elements could contribute to the heterogeneity of MM. To determine differentially expressed genes, each MM patient was compared to normal BMPCs, detecting 10,351 lncRNAs overexpressed and 9,535 downregulated in more than 50% of patients, focusing on a group of 989 lncRNAs specifically upregulated in MM (MM-specific lncRNAs), considering the B-cell populations. Next, we aimed to determine whether upregulation of those lncRNAs was under epigenetic control, analyzing the distribution of six histone marks (H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3, and H3K9me3) by ChIP-seq. We compared MM cases to normal B-cell subtypes and detected 89 lncRNAs with de novo epigenomic activation and expression in MM, suggesting an epigenetic rewiring in MM. We focused on LINC-SMILO, de novo epigenetically active and expressed lncRNA in MM. Knockdown of LINC-SMILO in 3 different MM cell lines (MM.1S, MM.1R and KMS-11) by 2 different shRNAs resulted in reduced proliferation and induction of apoptosis, associated with activation of ERVs (Endogenous retroviruses) and increase in interferon induced genes (measured by MARS-seq), which results in the activation of Interferon pathways, essential for MM cells survival. Finally, we aimed to determine whether lncRNAs could improve the current prognostic of MM patients. Using the IA11 release of CoMMpass data, we analyzed lncRNAs by COX regression and Backward elimination of Stepwise regression analysis, obtaining that the overexpression of the lncRNA PDLIM1P4 together with 1q amplification and 17p deletion stratified MM patients in three different risk groups. In summary, our study shows the complexity of lncRNA transcriptome in MM, and suggests that some of these lncRNAs have prognostic influence or can be used as potential therapeutic targets for MM.

Tissue engineering applies the principles of engineering, medicine and life sciences to the generation of biological substitutes (artificial tissues, bioengineered tissues or tissue constructs) with the aim of restoring, maintaining, or improving tissue function. A complex regulatory frame controls the clinical application and commercialization of bioengineered tissues. In this review, we highlight the state of the art and recent advances on the three main components that are used to build artificial tissues, including cells-especially adult stem cells-, extracellular matrices and morphogens or molecular signals. In particular, we discuss the limitations and challenges of the field and the different causes that may justify this limited situation.

AUTHORS: Paula Río, Xabier Agirre, Leire Garate, Rocío Baños, Lara Álvarez, Edurne San José-Enériz, Isabel Badell, José A. Casado, Marina Garín, Felipe Prósper and Juan A. Bueren INSTITUTIONS: Hematopoiesis and Gene Therapy Division. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, Spain.

The cloning of portions of the human BCL-2 gene was first reported in 1985 by Tsujimoto et al., who cloned the breakpoints from t(14;18) chromosomal translocations observed in follicular lymphoma. In these translocations, the BCL-2 gene becomes fused with the immunoglobulin heavy-chain locus (IgH), bringing the juxtaposed BCL-2 gene under the control of the IgH enhancer and thereby dysregulating BCL-2 gene expression at a transcriptional level. These data established the biological significance of programmed cell death in lymphoma, and subsequently in virtually every cancer. Over the succeeding decades, our understanding of this complex biology has created the prospect of therapeutically manipulating various components of the pathway, which has begun to change our perspectives on how to best tailor new therapeutic strategies for the molecule phenotype of different diseases. Dysregulation of the many proteins that govern control of programmed cell death poise the cell to resist traditional chemotherapy regimens, and contribute to their mortality. While many conventional drugs clearly have the potential to induce ’cell death’, relatively few directly modulate the balance of proand antiapoptotic forces, which collectively set the threshold for programmed cell death. One of the fist drugs to selectively target these pathways was oblimerson, an antisense molecule that directly targeted and degraded Bcl-2, an anti-apoptotic protein. Since then, a host of small molecules functioning as BH3 only mimetics have entered the clinic, including AT-101, a stereoisomer isolated from gossypol; obatoclax and ABT-263/737. These molecules, which have completed phase 1 testing as single agents, have produced responses in various disease settings, including CLL and follicular lymphoma, and are now being studied in combination with other antineoplastic agents. Beyond targeting the regulation of proteins that govern mitochondrial membrane depolarization leading to cytochorme C release, new molecules targeting the extrinsic pathway, including TRAIL and other Fas signaling pathways have also completed early phase clinical testing. While these agents have not produced substantial single agent signals to date, the idea of combining them with other agents to aid in lowering the threshold required to induce apoptosis is the most viable strategy for their development moving forward. Similarly, new molecules targeting survivin, and other IAP (inhibitors of apoptosis) family members, including YM 155, have been studied in relatively chemotherapy resistant lymphomas, including refractory diffuse large B-cell lymphoma. Again, while limited single agent signals have been reported, increasing emphasis is being placed on the study of these agents in combination. While many of these targeted Bcl-2 directed agents have been designed to directly modulate a very discrete aspect of the apoptotic pathway, it is also clear that several other new classes of drugs, including the proteasome inhibitor bortezomib and the histone deacetylase inhibitors, also have the potential to modulate these pathways. There is considerable enthusiasm that the integration of these agents into our present treatment regimens will afford new opportunities to overcome the survival instincts of many cancers, that will hopefully lead to improved outcomes.

While leukemia-originating stem cells are critical in the initiation and maintenance of leukemias, the existence of similar cell populations that may generate B-cell lymphoma upon mutation remains uncertain. Here we propose that committed lymphoid progenitor/precursor cells with an active V-D-J recombination program are the initiating cells of follicular lymphoma and mantle cell lymphoma when targeted by immunoglobulin (IG)- gene translocations in the bone marrow. However, these pre-malignant lymphoma-initiating cells cannot drive complete malignant transformation, requiring additional cooperating mutations in specific stem-cell programs to be converted into the lymphoma-originating cells able to generate and sustain lymphoma development. Conversely, diffuse large B-cell lymphoma and sporadic Burkitt's lymphoma derive from B lymphocytes that acquire translocations through IG-hyper-mutation or class-switching errors within the germinal center. Although secondary reprogramming mutations are generally required, some cells such as centroblasts or memory B cells that have certain stem cell-like features, or lymphocytes with MYC rearrangements that deregulate self-renewal pathways, may bypass this need and directly function as the lymphoma-originating cells. An alternative model supports an aberrant epigenetic modification of gene sets as the first occurring hit, which either leads to retaining stem-cell features in hematopoietic stem or progenitor cells, or reprograms stemness into more committed lymphocytes, followed by secondary chromosomal translocations that eventually drive lymphoma development. Isolation and characterization of the cells that are at the origin of the different B-cell non-Hodgkin's lymphomas will provide critical insights into the disease pathogenesis and will represent a step towards the development of more effective therapies.

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