Activation of the PP2A-B56α heterocomplex synergizes with venetoclax therapies in AML through BCL2 and MCL1 modulation.

Venetoclax combination therapies are becoming the standard of care in acute myeloid leukemia (AML). However, the therapeutic benefit of these drugs in older/unfit patients is limited to only a few months, highlighting the need for more effective therapies. Protein phosphatase 2A (PP2A) is a tumor suppressor phosphatase with pleiotropic functions that becomes inactivated in ∼70% of AML cases. PP2A promotes cancer cell death by modulating the phosphorylation state in a variety of proteins along the mitochondrial apoptotic pathway. We therefore hypothesized that pharmacological PP2A reactivation could increase BCL2 dependency in AML cells and, thus, potentiate venetoclax-induced cell death. Here, by using 3 structurally distinct PP2A-activating drugs, we show that PP2A reactivation synergistically enhances venetoclax activity in AML cell lines, primary cells, and xenograft models. Through the use of gene editing tools and pharmacological approaches, we demonstrate that the observed therapeutic synergy relies on PP2A complexes containing the B56α regulatory subunit, of which expression dictates response to the combination therapy. Mechanistically, PP2A reactivation enhances venetoclax-driven apoptosis through simultaneous inhibition of antiapoptotic BCL2 and extracellular signal-regulated kinase signaling, with the latter decreasing MCL1 protein stability. Finally, PP2A targeting increases the efficacy of the clinically approved venetoclax and azacitidine combination in vitro, in primary cells, and in an AML patient-derived xenograft model. These preclinical results provide a scientific rationale for testing PP2A-activating drugs with venetoclax combinations in AML.

Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DNA methylation profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Acquired mutations in BCL2 family proteins conferring resistance to the BH3 mimetic ABT-199 in lymphoma.

Acquired resistance to targeted drugs is emerging as an obstacle to successful cancer treatment. Recently, a BCL2-selective BH3 mimetic termed ABT-199 showed promising therapeutic results in BCL2-dependent tumors. Based on its high affinity for BCL2, we studied potential mechanisms conferring resistance upon ABT-199 therapy, aiming to anticipate its occurrence in the clinic. Two models of resistant lymphomas were established by continuous ABT-199 exposure. In resistant Bcl2-expressing mouse lymphoma cells, 2 missense mutations within the Bcl2 BH3 domain were identified. Both F101C and F101L mutations impeded ABT-199 binding to the BH3 domain, therefore suppressing mitochondrial apoptosis. In resistant human lymphoma cells, a missense mutation in the C-terminal transmembrane domain of proapoptotic BAX (G179E) was found, which abrogated BAX anchoring to mitochondria and blocked ABT-199-induced apoptosis both in vitro and in vivo. Importantly, G179E BAX mutation also induced partial cross-resistance to other antineoplastic drugs. Our study reveals the acquisition of mutations in BCL2 family proteins as a novel mechanism of apoptosis resistance in cancer. These results anticipate the potential development of such mutations in patients treated with ABT-199, providing a basis to preventing their occurrence and to designing drugs able to circumvent the acquired resistance.

Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations.

BACKGROUND: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. RESULTS: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). CONCLUSION: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.

Downregulation of FOXP1 is required during germinal center B-cell function.

B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginal-zone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding γ1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by B-cell activation, potentially contributing to B-cell lymphomagenesis.

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice.

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

The origin and targeting of mucosa-associated lymphoid tissue lymphomas.

PURPOSE OF REVIEW: Extranodal mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct clinical-pathological entity that can be distinguished from other lymphomas by a number of unique features, including their location in various extranodal sites, being preceded by chronic inflammatory or infection processes; a characteristic histopathological picture; and the presence of exclusive chromosomal translocations which increase MALT1 proteolytic activity to promote constitutive NF-κB signaling and eventually drive lymphomagenesis. RECENT FINDINGS: This review explores the major molecular and cellular events that participate in MALT lymphoma pathogenesis, focusing on gastric MALT lymphoma as a model of chronic inflammation-induced tumor development. In addition, the pivotal roles of activated MALT1 protease, its substrate TNFAIP3/A20, and the MyD88 adaptor protein in abnormally triggering downstream NF-κB pathway are overviewed. These new insights provide a mechanistic basis for using novel therapies targeting MALT1 protease or IRAK4 kinase activities. Finally, the putative cellular origin of MALT lymphomas is also discussed. SUMMARY: Over the last decade, unraveling the biological complexity of MALT lymphomas has shed light on the fundamental cellular and molecular aspects of the disease that are to be translated into clinical diagnostics and therapy.

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.

Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVß3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, ß3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVß3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.

A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma.

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G(1)-S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors.

LITAF, a BCL6 target gene, regulates autophagy in mature B-cell lymphomas.

We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.

PIM2 inhibition as a rational therapeutic approach in B-cell lymphoma.

PIM serine/threonine kinases are overexpressed, translocated, or amplified in multiple B-cell lymphoma types. We have explored the frequency and relevance of PIM expression in different B-cell lymphoma types and investigated whether PIM inhibition could be a rational therapeutic approach. Increased expression of PIM2 was detected in subsets of mantle cell lymphoma, diffuse large B-cell lymphoma (DLBLC), follicular lymphoma, marginal zone lymphoma-mucosa-associated lymphoid tissue type, chronic lymphocytic leukemia, and nodal marginal zone lymphoma cases. Increased PIM2 protein expression was associated with an aggressive clinical course in activated B-like-DLBCL patients. Pharmacologic and genetic inhibition of PIM2 revealed p4E-BP1(Thr37/46) and p4E-BP1(Ser65) as molecular biomarkers characteristic of PIM2 activity and indicated the involvement of PIM2 kinase in regulating mammalian target of rapamycin complex 1. The simultaneous genetic inhibition of all 3 PIM kinases induced changes in apoptosis and cell cycle. In conclusion, we show that PIM2 kinase inhibition is a rational approach in DLBCL treatment, identify appropriate biomarkers for pharmacodynamic studies, and provide a new marker for patient stratification.

Chronic docosahexaenoic acid supplementation improves metabolic plasticity in subcutaneous adipose tissue of aged obese female mice.

This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice.

Venetoclax improves CD20 immunotherapy in a mouse model of MYC/BCL2 double-expressor diffuse large B-cell lymphoma.

BACKGROUND: Approximately one-third of diffuse large B cell lymphoma (DLBCL) patients exhibit co-expression of MYC and BCL2 (double-expressor lymphoma, DEL) and have a dismal prognosis. Targeted inhibition of the anti-apoptotic protein BCL2 with venetoclax (ABT-199) has been approved in multiple B-cell malignancies and is currently being investigated in clinical trials for DLBCL. Whether BCL2 anti-apoptotic function represents a multifaceted vulnerability for DEL-DLBCL, affecting both lymphoma B cells and T cells within the tumor microenvironment, remains to be elucidated. METHODS: Here, we present novel genetically engineered mice that preclinically recapitulate DEL-DLBCL lymphomagenesis, and evaluate their sensitivity ex vivo and in vivo to the promising combination of venetoclax with anti-CD20-based standard immunotherapy. RESULTS: Venetoclax treatment demonstrated specific killing of MYC+/BCL2+ lymphoma cells by licensing their intrinsically primed apoptosis, and showed previously unrecognized immunomodulatory activity by specifically enriching antigen-activated effector CD8 T cells infiltrating the tumors. Whereas DEL-DLBCL mice were refractory to venetoclax alone, inhibition of BCL2 significantly extended overall survival of mice that were simultaneously treated with a murine surrogate for anti-CD20 rituximab. CONCLUSIONS: These results suggest that the combination of anti-CD20-based immunotherapy and BCL2 inhibition leads to cooperative immunomodulatory effects and improved preclinical responses, which may offer promising therapeutic opportunities for DEL-DLBCL patients.

Differential remodeling of subcutaneous white and interscapular brown adipose tissue by long-term exercise training in aged obese female mice.

Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Reversion of epigenetically mediated BIM silencing overcomes chemoresistance in Burkitt lymphoma.

In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2(-/-)γc(-/-) mice and of murine B220(+)IgM(+) B-cell lymphomas generated in Eµ-MYC and Eµ-MYC-BIM(+/-) transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL.

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies.

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.