Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n=11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs

Daratumumab in combination with urelumab to potentiate anti-myeloma activity in lymphocytedeficient mice reconstituted with human NK cells

Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNɣ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival

A new regulatory mechanism of protein phosphatase 2A activity via SET in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy. Although novel emerging drugs are available, the overall prognosis remains poor and new therapeutic approaches are required. PP2A phosphatase is a key regulator of cell homeostasis and is recurrently inactivated in AML. The anticancer activity of several PP2A-activating drugs (e.g., FTY720) depends on their interaction with the SET oncoprotein, an endogenous PP2A inhibitor that is overexpressed in 30% of AML cases. Elucidation of SET regulatory mechanisms may therefore provide novel targeted therapies for SET-overexpressing AMLs. Here, we show that upregulation of protein kinase p38 beta is a common event in AML. We provide evidence that p38 beta potentiates SET-mediated PP2A inactivation by two mechanisms: facilitating SET cytoplasmic translocation through CK2 phosphorylation, and directly binding to and stabilizing the SET protein. We demonstrate the importance of this new regulatory mechanism in primary AML cells from patients and in zebrafish xenograft models. Accordingly, combination of the CK2 inhibitor CX-4945, which retains SET in the nucleus, and FTY720, which disrupts the SET-PP2A binding in the cytoplasm, significantly reduces the viability and migration of AML cells. In conclusion, we show that the p38 beta/CK2/SET axis represents a new potential therapeutic pathway in AML patients with SET-dependent PP2A inactivation

IL-10 expression defines an immunosuppressive dendritic cell population induced by antitumor therapeutic vaccination

Vaccination induces immunostimulatory signals that are often accompanied by regulatory mechanisms such as IL-10, which control T-cell activation and inhibit vaccine-dependent antitumor therapeutic effect. Here we characterized IL- 10-producing cells in different tumor models treated with therapeutic vaccines. Although several cell subsets produced IL-10 irrespective of treatment, an early vaccine-dependent induction of IL-10 was detected in dendritic cells (DC). IL-10 production defined a DC population characterized by a poorly mature phenotype, lower expression of T-cell stimulating molecules and upregulation of PD-L1. These IL-10+ DC showed impaired in vitro T-cell stimulatory capacity, which was rescued by incubation with IL-10R and PD-L1-inhibiting antibodies. In vivo IL-10 blockade during vaccination decreased the proportion of IL-10+ DC and improved their maturation, without modifying PD-L1 expression. Similarly, PD-L1 blockade did not affect IL-10 expression. Interestingly, vaccination combined with simultaneous blockade of IL-10 and PD-L1 induced stronger immune responses, resulting in a higher therapeutic efficacy in tumor-bearing mice. These results show that vaccine-induced immunoregulatory IL-10+ DC impair priming of antitumor immunity, suggesting that therapeutic vaccination protocols may benefit from combined targeting of inhibitory molecules expressed by this DC subset

Immunological Biomarkers of Fatal COVID-19: A Study of 868 Patients

Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients

Differentiation stage of myeloma plasma cells: biological and clinical significance

[EN] The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n = 20) that the CD19 − CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19 − CD81+ and CD19 − CD81 − BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19 − CD81 −) clones, 38% intermediate-differentiated (CD19 − CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P = 0.005) and overall survival (HR: 2.1; P = 0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n = 40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles

Estudio del perfil genético en pacientes con Amiloidosis de cadenas ligeras

Oral Presentation [CO-130] Introducción: Los estudios de secuenciación masiva (NGS) han permitido profundizar en el conocimiento de las gammapatías monoclonales tales como el mieloma múltiple (MM) y la macroglobulinemia de Waldesntröm’s (WM). Desafortunadamente, la baja incidencia de la amiloidosis de cadenas ligeras (AL) y la baja carga tumoral que presenta, a menudo enmascarada por un fondo policlonal de células plasmáticas (PC), explica la poca información que hay sobre la biología de la célula tumoral. Por ello, se desconoce si la AL presenta alguna mutación común como ocurre en la WM, si existen mutaciones recurrentes, y si estas podrían coincidir con las observadas en MM. Por lo tanto, el objetivo de este trabajo es realizar una secuenciación de exoma (WES) en una serie de pacientes con AL y comparar su perfil mutacional con el de MM. Métodos: En este estudio se incluyeron 28 pacientes con AL. Se realizó un WES, incluyendo las regiones reguladoras UTR (SureSelect Human All Exon V6 + UTRs (Agilent)) en 56 muestras pareadas sorteadas de células plasmáticas patológicas y sangre periférica como muestra control. Cada muestra tumoral fue capturada por triplicado y secuenciada en la plataforma NextSeq 500 (Illumina). Para el análisis de variantes somáticas se utilizaron los programas Strelka y ANNOVAR. . Las firmas mutacionales se analizaron con el software DeconstructSigs. Para comparar el perfil mutacional de AL con MM se utilizó la base de datos MMRF CoMMpass con 895 pacientes. Además, se han determinado los reordenamientos de los genes de las inmunoglobulinas (Igs) mediante NGS. Resultados: La cobertura media de secuenciación para las muestras de control y tumor fue de 64x y 186x, respectivamente. Se detectaron un total de 1983 SNV y 133 INDEL con una media de 71 (20-281) SNV y 5 (0-25) INDEL por paciente. Al comparar con MM (media 66 SNV y 2.5 INDEL) se observó una carga mutacional similar. Los únicos genes mutados tanto en AL como en MM fueron MUC16 (recurrencia 17% y 8%, respectivamente) e IGLL5 (recurrencia 17%, en ambas), siendo además los genes más frecuentemente mutados en AL Las firmas mutacionales más frecuentes que se identificaron fueron la 1 (desaminación espontánea de citosinas metiladas en sitios CpG), la 3 (fallo en la reparación de la ruptura de la doble cadena de ADN mediante recombinación homóloga), y la 9 (transveriones T> G en trinucleótidos ApTpN y TpTpN), identificadas en el 96%, 54% y 46% de los pacientes, respectivamente. Respecto al repertorio de los genes de las Igs, se observó que el 26% de los pacientes con AL presentan más de un clon, siendo esta heterogeneidad clonal similar a la encontrada en MM (23%). El gen IGHV3-30 fue identificado con mayor frecuencia tanto en AL como en MM, 10% y 12% de recurrencia, respectivamente. Conclusiones: Este es el primer estudio de WES en una serie de pacientes con AL. Los resultados muestran que no hay una mutación común driver en esta enfermedad, que podrían estar implicados múltiples procesos mutacionales, y que los genes descritos más frecuentemente mutados en AL y MM no coinciden. En conjunto, estos resultados suponen un avance en el entendimiento de la patogénesis de la AL

In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis

Bulk ex vivo and single-cell in vivo CRISPR knockout screens are used to characterize 680 chromatin factors during mouse hematopoiesis, highlighting lineage-specific and normal and leukemia-specific functions. Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis

The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma

The development of sensitive and non-invasive ‘‘liquid biopsies’’ presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs) is prognostic in multiple myeloma (MM), but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM) tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs