Search

Despite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B–T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL–FDC and FL–macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) cases have a poor outcome. Here we analysed clinico-biological features in 373 DLBCL patients homogeneously treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), in order to identify variables associated with early failure to treatment (EF), defined as primary refractoriness or relapse within 12 months from diagnosis. In addition to clinical features, mutational status of 106 genes was studied by targeted next-generation sequencing in 111 cases, copy number alterations in 87, and gene expression profile (GEP) in 39. Ninety-seven cases (26%) were identified as EF and showed significantly shorter overall survival (OS). Patients with B symptoms, advanced stage, high levels of serum lactate dehydrogenase (LDH) or beta 2-microglobulin, low lymphocyte/monocyte ratio and higher Revised International Prognostic Index (R-IPI) scores, as well as those with BCL2 rearrangements more frequently showed EF, with R-IPI being the most important in logistic regression. Mutations in NOTCH2, gains in 5p15 center dot 33 (TERT), 12q13 (CDK2), 12q14 center dot 1 (CDK4) and 12q15 (MDM2) showed predictive importance for EF independently from R-IPI. GEP studies showed that EF cases were significantly enriched in sets related to cell cycle regulation and inflammatory response, while cases in response showed over-representation of gene sets related to extra-cellular matrix and tumour microenvironment.

Simple Summary Follicular lymphoma is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Although huge efforts have been made in the last 10 years, this pathology is still considered as incurable, leaving open the discovery and testing of new therapeutic targets requiring relevant preclinical models. Here, we report a realistic 3D model of t (14;18)-positive B-NHL cell culture (ultra-low attachment (ULA)-multicellular aggregates of lymphoma cells (MALC)), which monitored by state-of-the-art 2D and 3D imaging, allows more robust drug testing. Abstract Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

Simple Summary Follicular lymphoma is a paradigm of tumors that require the interaction between tumor and microenvironment cells to foster their development from initial steps to progression. Recent large-scale genome studies have uncovered multiple genetic alterations that cooperate with the lymphoma microenvironment to promote cell survival, proliferation and to facilitate tumor evasion from host immune system. Understanding the crosstalk between tumor B-cells and the microenvironment is fundamental to identify vulnerabilities that may offer novel therapeutic targets. This review highlights recent findings showing the effect of common genetic mutations modulating the cell composition and phenotype of the tumor microenvironment and the novel therapeutic perspectives to target these interactions. Abstract Follicular Lymphoma (FL), the most common indolent non-Hodgkin’s B cell lymphoma, is a paradigm of the immune microenvironment’s contribution to disease onset, progression, and heterogeneity. Over the last few years, state-of-the-art technologies, including whole-exome sequencing, single-cell RNA sequencing, and mass cytometry, have precisely dissected the specific cellular phenotypes present in the FL microenvironment network and their role in the disease. In this already complex picture, the presence of recurring mutations, including KMT2D, CREBBP, EZH2, and TNFRSF14, have a prominent contributory role, with some of them finely tuning this exquisite dependence of FL on its microenvironment. This precise characterization of the enemy (FL) and its allies (microenvironment) has paved the way for the development of novel therapies aimed at dismantling this contact network, weakening tumor cell support, and reactivating the host’s immune response against the tumor. In this review, we will describe the main microenvironment actors, together with the current and future therapeutic approaches targeting them.

Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures (n = 19) and in vivo mouse co-xenografts demonstrated that FL-FDC crosstalk favors tumor growth and, via the secretion of CCL2 and CSF-1, promotes monocyte recruitment, differentiation, and polarization towards an M2-like protumoral phenotype. Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy. Grants that contributed to this work included: Gilead Sciences Research Funded Agreement, Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa” for SAF2014/57708R and SAF2017/ 88275R to PP-G and MCC, RTI2018-094584-B-I00 to DC, CIBERONC (CB16/12/00334 and CB16/12/00225), and finally Generalitat de Catalunya support for AGAUR 2017SGR1009 to DC

Cardiovascular diseases are the leading cause of death globally and more than four out of five cases are due to ischemic events. Cardiac fibroblasts (CF) contribute to normal heart development and function, and produce the post-ischemic scar. Here, we characterize the biochemical and functional aspects related to CF endurance to ischemia-like conditions. Expression data mining showed that cultured human CF (HCF) express more BCL2 than pulmonary and dermal fibroblasts. In addition, gene set enrichment analysis showed overrepresentation of genes involved in the response to hypoxia and oxidative stress, respiration and Janus kinase (JAK)/Signal transducer and Activator of Transcription (STAT) signaling pathways in HCF. BCL2 sustained survival and proliferation of cultured rat CF, which also had higher respiration capacity and reactive oxygen species (ROS) production than pulmonary and dermal fibroblasts. This was associated with higher expression of the electron transport chain (ETC) and antioxidant enzymes. CF had high phosphorylation of JAK2 and its effectors STAT3 and STAT5, and their inhibition reduced viability and respiration, impaired ROS control and reduced the expression of BCL2, ETC complexes and antioxidant enzymes. Together, our results identify molecular and biochemical mechanisms conferring survival advantage to experimental ischemia in CF and show their control by the JAK2/STAT signaling pathway. The presented data point to potential targets for the regulation of cardiac fibrosis and also open the possibility of a general mechanism by which somatic cells required to acutely respond to ischemia are constitutively adapted to survive it., This research was funded by Ministerio de Ciencia e Innovación (MICINN), Gobierno de España, grant numbers SAF2013-44942-R and PID2019-104509RB-I00 to DS; Fundació La Marató TV3, grant number 20153810 to D.S; A.B. holds a contract from Fundació La Marató TV3 and IRBLleida/Diputació de Lleida; Generalitat de Catalunya, (AGAUR) grant number 2017SGR996 to DS; PP-G Laboratory support was obtained through research grants from MICINN (SAF2017/88275R) and CIBERONC (CB16/12/00334); JI and MR-M Laboratory support was obtained from Instituto de Salud Carlos III (ISCIII-FIS) grant PI19-01196; AZ Laboratory support was obtained through research grants from MICINN (PID2019-106209RB-I00), and the Generalitat de Catalunya, (AGAUR) grant number 2017SGR1015. AZ is a recipient of an ICREA ‘Academia’ Award (Generalitat de Catalunya). We gratefully acknowledge institutional funding from the MINECO through the Centres of Excellence Severo Ochoa Award, and from the CERCA Programme of the Generalitat de Catalunya.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, mostly known as double-hit lymphoma (DHL), is a rare entity characterized by morphologic and molecular features between Burkitt lymphoma and the clinically manageable diffuse large B-cell lymphoma (DLBCL). DHL patients usually undergo a rapidly progressing clinical course associated with resistance to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival inferior to 1 year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of a specific subtype of lymphoid neoplasm. In this study, we demonstrate that single-agent ABT-199 efficiently displaces BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+DHL cell lines and primary cultures, as well as in a xenograft mouse model of the disease. We further identify the accumulation of the BCL2-like protein BFL-1 to be a major mechanism involved in acquired resistance to ABT-199. Noteworthy, this phenomenon can be counteracted by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1, the BFL-1 coding gene, as one of the top apoptosis-related gene modulated by this compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 further overcomes resistance to ABT-199 both in vitro and in vivo, engaging synergistic caspase-mediated apoptosis in DHL cultures and tumor xenografts. Together, these findings highlight the relevance of BFL-1 in DH lymphoma-associated drug resistance and support the combined use of a BCL-2 antagonist and a BET inhibitor as a promising therapeutic strategy for patients with aggressive DHL.

A significant proportion of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) patients harbor a gain-of-function, heterozygous somatic mutations of the methyltransferase gene EZH2. Despite acceptable safety profile and early signs of activity in clinical trials, single agent treatment with EZH2 inhibitors is unlikely to be curative in aggressive lymphomas. In an effort to established novel rational combinations, we have evaluated the activity and mechanism of action of the EZH2 small molecule inhibitor CPI169 as single agent and in combination with BET bromodomain inhibition, using preclinical models of DLBCL and FL with distinct EZH2 mutational status. CPI169 anti-tumor activity and specificity was assessed in vitro in 10 DLBCL and FL cell lines, including cells expressing basal or ectopic EZH2mut. Molecular bases of its activity were determined by gene expression profiling (GEP), qPCR and western blot, followed by automated exploratory data analysis. Biomarkers validation was made in vitro and in vivo, using a mouse xenotransplant model of EZH2mut DLBCL, considering both exposure to CPI169 single agent and/or combination treatment with a BRD4 inhibitor, CPI203. CPI169 induced dose-dependent proliferation blockade in EZH2mut, but not EZH2wt DLBCL and FL cell lines, independently of EZH2 expression level or basal methyltransferase activity. Loss of H3K27me3 mark upon CPI169 treatment was associated with upregulation of gene sets related to G1 cell cycle blockade, mTOR and P53 pathways, and MYC signaling. Accordingly, combination with the MYC-interfering drug, BET inhibitor CPI203, achieved a synergistic anti-proliferating activity in EZH2 mutated cases and in mice bearing EZH2mut DLBCL tumors. Activity of EZHi/BRD4i combo was characterized by lower mitotic index, increased loss of H3K27me3 mark, in association with MYC downregulation. GEP analysis, followed by automated exploratory data analysis and validation by a siRNA screening, further identified the PI3K/AKT-regulated gene and mitosis regulator, YPEL2, and the regulator of innate-like B lymphocyte maturation, KLHL14, as crucial factors involved in the efficacy of MYC/EZH2 dual targeting. In conclusion, CPI169 shows significant activity and safety as single agent in EZH2 mutated-DLBCL and FL cases and displays synergistic interaction in vitro and in vivo with BRD4 inhibition, mediated by the modulation of a limited set of EZH2-regulated genes. Citation Format: Aranzazu Chamorro-Jorganes, Marcelo L. Ribeiro, Nuria Profitos-Peleja, Diana Reyes-Garau, Clara Recasens-Zorzo, Juan G. Valero, Marc Armengol, Patricia Perez-Galan, Ray Butler, Antonio Postigo, Francesc Bosch, Gael Roue. Safety and efficacy of EZH2 and BRD4 dual targeting in EZH2Y641mut germinal centre-derived lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2925.

Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids.

11 páginas, 4 figuras., BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases., This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN - BIO2007-60066, SAF2008-00048, SAF30542-C01-01 and SAF2010-15512), Laboratorios SALVAT, S.A., Fundacio´n Renal Toma´s de Osma, Generalitat Valenciana Prometeo 2010/005 (partially funded with ERDF), Consolider-Ingenio 2010 (MICINN - CSD2008-00005C) and by the Generalitat Valenciana through Prometeo 2014/061. Moure was funded by a predoctoral fellowship from JAE-pre CSIC.

Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors. Contrary to currently popular models, MCL1ΔNΔC did not require becoming embedded into the membrane to inhibit membrane permeabilization, and interaction with cBID was more productive for MCL1ΔNΔC inhibitory activity than interaction with BAX. We also report that membranes rich in cardiolipin (CL), but not phosphatidylinositol (PI), trigger a profound conformational change in MCL1ΔNΔC leading to membrane integration and unleashment of an intrinsic lipidic pore-forming activity of the molecule. Cholesterol (CHOL) reduces both the conformational change and the lipidic pore-forming activity of MCL1ΔNΔC in CL-rich membranes, but it does not affect the interaction of MCL1ΔNΔC with proapoptotic partners in MOM-like liposomes. In addition, we identified MCL1α5 as the minimal domain of the protein responsible for its membrane-permeabilizing function both in model membranes and at the mitochondrial level. Our results provide novel mechanistic insight into MCL1 function in the context of a membrane milieu and add significantly to a growing body of evidence supporting an active role of mitochondrial membrane lipids in BCL2 protein function.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

SUMMARYAlthough many cancer cells are primed for apoptosis, they usually develop resistance to cell death at multiple levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members like Bax, is considered as a point-of-no-return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on using minimal active version of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, able to target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived poropeptide produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins evolutionary functionalized to form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

International audience; BACKGROUND:The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction.METHODOLOGY/PRINCIPAL FINDINGS:Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death.CONCLUSION/SIGNIFICANCE:BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.