Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo

Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells

Inhibition of BCR signaling using the Syk inhibitor TAK-659 prevents stroma-mediated signaling in chronic lymphocytic leukemia cells

Altres ajuts: This work was cofinanced by the European Regional Development Fund (ERDF) and Asociación Española Contra el Cáncer (AECC, M.C). N.P. is a recipient of a PhD fellowship granted by Institut de Recerca Vall d'Hebron. C.C. is supported by a grant from Sociedad Española de Hematología y Hemoterapia (SEHH).Proliferation and survival of chronic lymphocytic leukemia (CLL) cells depend on microenvironmental signals coming from lymphoid organs. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the B-cell receptor (BCR). Syk protein, a tyrosine kinase essential for BCR signaling, is therefore a rational candidate for targeted therapy in CLL. Against this background, we tested the efficacy of the highly specific Syk inhibitor TAK-659 in suppressing the favorable signaling derived from the microenvironment. To ex vivo mimic the microenvironment found in the proliferation centers, we co-cultured primary CLL cells with BM stromal cells (BMSC), CD40L and CpG ODN along with BCR stimulation. In this setting, TAK-659 inhibited the microenvironment-induced activation of Syk and downstream signaling molecules, without inhibiting the protein homologue ZAP-70 in T cells. Importantly, the pro-survival, proliferative, chemoresistant and activation effects promoted by the microenvironment were abrogated by TAK-659, which furthermore blocked CLL cell migration toward BMSC, CXCL12, and CXCL13. Combination of TAK-659 with other BCR inhibitors showed synergistic effect in inducing apoptosis, and the sequential addition of TAK-659 in ibrutinib-treated CLL cells induced significantly higher cytotoxicity. These findings provide a strong rationale for the clinical development of TAK-659 in CLL

Microenvironment regulates the expression of miR-21 and tumor suppressor genes PTEN, PIAS3 and PDCD4 through ZAP-70 in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.This work was supported by research funding from the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI14/00055, F.B. and PI13/00279, M.C.), cofinanced by the European Regional Development Fund (ERDF) and Asociación Española Contra el Cáncer (AECC Barcelona, M.C. and P.A.). M.C. holds a contract from Ministerio de Economía y Competitividad (MINECO) (RYC-2012-12018). Authors thank the Cellex Foundation for providing research facilities and equipmen

Repolarization of tumor infiltrating macrophages and increased survival in mouse primary CNS lymphomas after XPO1 and BTK inhibition

Altres ajuts: This work was supported by research funding from the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias cofinanced by the European Regional Development Fund (ERDF); Fundación Asociación Española Contra el Cáncer (M.C. and P.A.) and Gilead Fellowships (GLD16/00144, GLD18/00047, F.B). M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades. S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero.Patients diagnosed with primary central nervous system lymphoma (PCNSL) often face dismal outcomes due to the limited availability of therapeutic options. PCNSL cells frequently have deregulated B-cell receptor (BCR) signaling, but clinical responses to its inhibition using ibrutinib have been brief. In this regard, blocking nuclear export by using selinexor, which covalently binds to XPO1, can also inhibit BCR signaling. Selinexor crosses the blood-brain barrier and was recently shown to have clinical activity in a patient with refractory diffuse large B-cell lymphoma in the CNS. We studied selinexor alone or in combination with ibrutinib in pre-clinical mouse models of PCNSL. Orthotopic xenograft models were established by injecting lymphoma cells into the brain parenchyma of athymic mice. Tumor growth was monitored by bioluminescence. Malignant cells and macrophages were studied by immunohistochemistry and flow cytometry. Selinexor blocked tumor growth and prolonged survival in a bioluminescent mouse model, while its combination with ibrutinib further increased survival. CNS lymphoma in mice was infiltrated by tumor-promoting M2-like macrophages expressing PD-1 and SIRPα. Interestingly, treatment with selinexor and ibrutinib favored an anti-tumoral immune response by shifting polarization toward inflammatory M1-like and diminishing PD-1 and SIRPα expression in the remaining tumor-promoting M2-like macrophages. These data highlight the pathogenic role of the innate immune microenvironment in PCNSL and provide pre-clinical evidence for the development of selinexor and ibrutinib as a new promising therapeutic option with cytotoxic and immunomodulatory potential. The online version of this article (10.1007/s11060-020-03580-y) contains supplementary material, which is available to authorized users

A gene expression assay based on chronic lymphocytic leukemia activation in the microenvironment to predict progression

Gene expression; Chronic lymphocytic leukemiaExpresión génica; Leucemia linfocítica crónicaExpressió gènica; Leucèmia limfocítica crònicaSeveral gene expression profiles with a strong correlation with patient outcomes have been previously described in chronic lymphocytic leukemia (CLL), although their applicability as biomarkers in clinical practice has been particularly limited. Here we describe the training and validation of a gene expression signature for predicting early progression in patients with CLL based on the analysis of 200 genes related to microenvironment signaling on the NanoString platform. In the training cohort (n = 154), the CLL15 assay containing a 15-gene signature was associated with the time to first treatment (TtFT) (hazard ratio [HR], 2.83; 95% CI, 2.17-3.68; P < .001). The prognostic value of the CLL15 score (HR, 1.71; 95% CI, 1.15-2.52; P = .007) was further confirmed in an external independent validation cohort (n = 112). Notably, the CLL15 score improved the prognostic capacity over IGHV mutational status and the International Prognostic Score for asymptomatic early-stage (IPS-E) CLL. In multivariate analysis, the CLL15 score (HR, 1.83; 95% CI, 1.32-2.56; P < .001) and the IPS-E CLL (HR, 2.23; 95% CI, 1.59-3.12; P < .001) were independently associated with TtFT. The newly developed and validated CLL15 assay successfully translated previous gene signatures such as the microenvironment signaling into a new gene expression–based assay with prognostic implications in CLL.This work was supported by research funding from the Asociación Española Contra el Cáncer grant [5U01CA157581-05, ECRIN-M3 - A29370] and in part by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [PI17/00950, M.C., PI17/00943, F.B, PI18/01392, P.A.], and the Spanish Ministry of Economy and Competitiveness [CIBERONC-CB16/12/00233], the Education Council or Health Council of the Junta de Castilla y León [GRS 2036/A/19], and Gilead Sciences [GLD15/00348]. This work was supported by research funding from the Asociación Española Contra el Cáncer grant [5U01CA157581-05, ECRIN-M3 - A29370] and in part by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [PI17/00950, M.C., PI17/00943, F.B, PI18/01392, P.A.], and the Spanish Ministry of Economy and Competitiveness [CIBERONC-CB16/12/00233], the Education Council or Health Council of the Junta de Castilla y León [GRS 2036/A/19], Gilead Sciences [GLD15/00348] and Gilead Fellowships [GLD16/00144, GLD18/00047, F.B.], and Fundació la Marató de TV3 [201905-30-31 F.B]. All Spanish funding was cosponsored by the European Union FEDER program “Una manera de hacer Europa”. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades [RYC-2012-2018]

Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Progressió clínica; Evasió immuneProgresión clínica; Evasión inmuneClinical progression; Immune evasionBackground Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.This work was supported by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI17/00950, M.C., PI18/01392, P.A. and PI17/00943, F.B.) and co-financed by the European Regional Development Fund (ERDF) and Fundación Asociación Española Contra el Cáncer (M.C. and P.A.), Gilead Fellowships (GLD16/00144, GLD18/00047, F.B.) and Fundació la Marató de TV3 (201905–30-31 F.B). S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero. R.V-M. is supported by a Torres Quevedo fellowship from the Spanish Ministry of Science and Innovation (PTQ-16-08623). A.E-C. is funded by ISCIII/MINECO (PT17/0009/0019) which is co-funded by FEDER. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-2018)

Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas

Limfoma no Hodgkin agressiu; Limfoma del SNCLinfoma no Hodgkin agresivo; Linfoma del SNCAggressive Non-Hodgkin's Lymphoma; CNS lymphomaThe levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas.This work was supported by research funding from Fundación Asociación Española contra el Cáncer (AECC) (to JS, MC and PA); FERO (to JS), laCaixa (to JS), BBVA (CAIMI) (to JS), the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI16/01278 to JS; PI17/00950 to MC; PI17/00943 to FB) cofinanced by the European Regional Development Fund (ERDF) and Gilead Fellowships (GLD16/00144, GLD18/00047, to FB). MC holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-12018). SB received funding from Fundación Alfonso Martin Escudero. LE received funding from the Juan de la Cierva fellowship. We thank CERCA Programme / Generalitat de Catalunya for institutional support

Paper de la via de senyalització del receptor de cèl·lules B (BCR) en la interacció entre les cèl·lules de leucèmia limfàtica crònica i el microambient tumoral

La leucèmia limfàtica crònica (LLC) és la leucèmia més freqüent en adults als països occidentals. El curs clínic de la malaltia és extremadament variable i la supervivència dels pacients oscil·la entre pocs mesos i vàries dècades. Aquesta neoplasia es caracteritza per la proliferació i l’acumulació progressiva de limfòcits B madurs i clonals amb un immunofenotip característic (CD5+, CD19+, CD20 dèbil, CD23+, Ig superfície dèbil) en sang perifèrica, medul·la òssia, ganglis limfàtics i melsa. Concretament és als ganglis limfàtics i a la medul·la òssia on les cèl·lules de LLC es troben amb un microambient favorable que afavoreix la seva supervivència, proliferació i quimioresistència. Una de les principals molècules involucrades en la interacció entre les cèl·lules de LLC i les cèl·lules accessòries del microambient és el BCR. La proteïna Syk, una tirosina quinasa fosforilada immediatament després de l’activació del BCR, és essencial per la senyalització d’aquest receptor i per tant és una excel·lent diana terapèutica. En base a aquest context, el primer estudi es centra en l’estudi de l’eficàcia de la TAK-659, un nou inhibidor altament específic de Syk. Els resultats mostren que la TAK-659 és capaç de suprimir eficaçment la inducció de la supervivència, proliferació i migració de les cèl·lules de LLC per part del microambient contribuint així en el desenvolupament clínic d’aquesta teràpia en pacients amb LLC. ZAP-70 és una tirosina quinasa homologa a Syk. La seva expressió en LLC està associada amb un increment de la capacitat de senyalització del BCR així com amb un increment de resposta als estímuls del microambient. A més, és un factor de mal pronòstic molt ben definit en la LLC. El microRNA-21 (miR-21), es troba sobreexpressat en mostres procedents de ganglis limfàtics de pacients amb LLC i es relaciona amb la resistència a la fludarabina, una menor supervivència global i una major probabilitat de progressió. Per això el segon estudi es basa en l’estudi del possible paper de ZAP-70 en la regulació del miR-21 i la possible influència del microambient. Els resultats mostren que el microambient tumoral és capaç de regular l’expressió del miR-21 i dels seus gens diana (PTEN, PIAS3 i PDCD4) a través de la via de senyalització que involucra les proteïnes ZAP-70 i MAPK i el factor de transcripció STAT3.Chronic lymphocytic leukemia (CLL) is the most common leukemia found in adults in Western countries. The clinical course is heterogeneous and survival can vary from months to decades. This neoplasm is characterized by the proliferation and progressive accumulation of monoclonal mature B lymphocytes with a typical immunophenotype (CD5+, CD19+, CD20 low, CD23+, surface Ig low) in peripheral blood, bone marrow, lymph nodes and spleen. Microenvironment found in bone marrow and lymph nodes supports survival, proliferation and drug resistance. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the BCR. Syk protein, a tyrosine kinase immediately phosphorylated after BCR activation which is essential for this signaling pathway, is therefore a rational candidate for targeted therapy in CLL. Against this background, in the first study we tested the efficacy of the highly specific Syk inhibitor TAK-659. The results showed that TAK-659 is able to efficiently suppress the pro-survival, proliferative and chemoresistant effects promoted by the microenvironment providing a strong rationale for the clinical development of TAK-659 in CLL. Expression of ZAP-70 in CLL is related to enhanced response to BCR stimulation, as well as to increased response to microenvironment stimuli. Moreover, high expression of ZAP-70 is associated with an adverse prognosis in CLL. MicroRNA-21 (miR-21) is overexpressed in lymph nodes from patients with CLL compared to peripheral blood and it is associated to refractoriness to fludarabine, shorter overall survival and higher probability of progression. The aim of the second study was to study the ZAP-70 involvement in miR-21 regulation and how it would be influenced by the microenvironment. The results showed that stimuli from the microenvironment regulate the expression of miR- 21 and the tumor suppressor genes (PTEN, PDCD4 and PIAS3) via a signaling pathway involving ZAP-70, MAPK and STAT3 transcription factor

Paper de la via de senyalització del receptor de cèl·lules B (BCR) en la interacció entre les cèl·lules de leucèmia limfàtica crònica i el microambient tumoral

La leucèmia limfàtica crònica (LLC) és la leucèmia més freqüent en adults als països occidentals. El curs clínic de la malaltia és extremadament variable i la supervivència dels pacients oscil·la entre pocs mesos i vàries dècades. Aquesta neoplasia es caracteritza per la proliferació i l’acumulació progressiva de limfòcits B madurs i clonals amb un immunofenotip característic (CD5+, CD19+, CD20 dèbil, CD23+, Ig superfície dèbil) en sang perifèrica, medul·la òssia, ganglis limfàtics i melsa. Concretament és als ganglis limfàtics i a la medul·la òssia on les cèl·lules de LLC es troben amb un microambient favorable que afavoreix la seva supervivència, proliferació i quimioresistència. Una de les principals molècules involucrades en la interacció entre les cèl·lules de LLC i les cèl·lules accessòries del microambient és el BCR. La proteïna Syk, una tirosina quinasa fosforilada immediatament després de l’activació del BCR, és essencial per la senyalització d’aquest receptor i per tant és una excel·lent diana terapèutica. En base a aquest context, el primer estudi es centra en l’estudi de l’eficàcia de la TAK-659, un nou inhibidor altament específic de Syk. Els resultats mostren que la TAK-659 és capaç de suprimir eficaçment la inducció de la supervivència, proliferació i migració de les cèl·lules de LLC per part del microambient contribuint així en el desenvolupament clínic d’aquesta teràpia en pacients amb LLC. ZAP-70 és una tirosina quinasa homologa a Syk. La seva expressió en LLC està associada amb un increment de la capacitat de senyalització del BCR així com amb un increment de resposta als estímuls del microambient. A més, és un factor de mal pronòstic molt ben definit en la LLC. El microRNA-21 (miR-21), es troba sobreexpressat en mostres procedents de ganglis limfàtics de pacients amb LLC i es relaciona amb la resistència a la fludarabina, una menor supervivència global i una major probabilitat de progressió. Per això el segon estudi es basa en l’estudi del possible paper de ZAP-70 en la regulació del miR-21 i la possible influència del microambient. Els resultats mostren que el microambient tumoral és capaç de regular l’expressió del miR-21 i dels seus gens diana (PTEN, PIAS3 i PDCD4) a través de la via de senyalització que involucra les proteïnes ZAP-70 i MAPK i el factor de transcripció STAT3.Chronic lymphocytic leukemia (CLL) is the most common leukemia found in adults in Western countries. The clinical course is heterogeneous and survival can vary from months to decades. This neoplasm is characterized by the proliferation and progressive accumulation of monoclonal mature B lymphocytes with a typical immunophenotype (CD5+, CD19+, CD20 low, CD23+, surface Ig low) in peripheral blood, bone marrow, lymph nodes and spleen. Microenvironment found in bone marrow and lymph nodes supports survival, proliferation and drug resistance. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the BCR. Syk protein, a tyrosine kinase immediately phosphorylated after BCR activation which is essential for this signaling pathway, is therefore a rational candidate for targeted therapy in CLL. Against this background, in the first study we tested the efficacy of the highly specific Syk inhibitor TAK-659. The results showed that TAK-659 is able to efficiently suppress the pro-survival, proliferative and chemoresistant effects promoted by the microenvironment providing a strong rationale for the clinical development of TAK-659 in CLL. Expression of ZAP-70 in CLL is related to enhanced response to BCR stimulation, as well as to increased response to microenvironment stimuli. Moreover, high expression of ZAP-70 is associated with an adverse prognosis in CLL. MicroRNA-21 (miR-21) is overexpressed in lymph nodes from patients with CLL compared to peripheral blood and it is associated to refractoriness to fludarabine, shorter overall survival and higher probability of progression. The aim of the second study was to study the ZAP-70 involvement in miR-21 regulation and how it would be influenced by the microenvironment. The results showed that stimuli from the microenvironment regulate the expression of miR- 21 and the tumor suppressor genes (PTEN, PDCD4 and PIAS3) via a signaling pathway involving ZAP-70, MAPK and STAT3 transcription factor