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Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14−CD15+CD33+HLADR− cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC–related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.

Summary Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses., Highlights • Patient-specific PKDiPSCs are generated from PB-MNCs by a non-integrative system • PKDiPSCs are gene edited to insert a partial co-RPK in the PKLR locus mediated by TALEN • An SNP in the homology arm leads to allele-specific homologous recombination • Gene-edited PKDiPSCs generate a high number of metabolically corrected erythroid cells, Patient-specific induced pluripotent stem cells (iPSCs) are the perfect platform to study erythroid metabolic diseases and test innovative treatments. Segovia, Quintana-Bustamante, and colleagues showed the correction of pyruvate kinase deficiency (PKD) by combining iPSC and gene-editing technologies and provide an approach to generate the large number of erythroid cells required for comprehensive biochemical and metabolic analyses of this disease and its treatment.

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Fanconi anemia (FA) is an inherited genetic disorder associated with BM failure and cancer predisposition. In the present study, we sought to elucidate the role of microRNAs (miRNAs) in the hematopoietic defects observed in FA patients. Initial studies showed that 3 miRNAs, hsa-miR-133a, hsa-miR-135b, and hsa-miR181c, were significantly down-regulated in lymphoblastoid cell lines and fresh peripheral blood cells from FA patients. In vitro studies with cells expressing the luciferase reporter fused to the TNF alpha 3'-untranslated region confirmed in silico predictions suggesting an interaction between hsa-miR-181c and TNF alpha mRNA. These observations were consistent with the down-regulated expression of TNF alpha mediated by hsa-miR-181c in cells from healthy donors and cells from FA patients. Because of the relevance of TNF alpha in the hematopoietic defects of FA patients, in the present study, we transfected BM cells from FA patients with hsa-miR-181c to evaluate the impact of this miRNA on their clonogenic potential. hsa-miR-181c markedly increased the number and size of the myeloid and erythroid colonies generated by BM cells from FA patients. Our results offer new clues toward understanding the biologic basis of BM failure in FA patients and open new possibilities for the treatment of the hematologic dysfunction in FA patients based on miRNA regulation. (Blood. 2012;119(13):3042-3049)

The nucleoporin 98 gene (NUP98) has been reported to be fused to 13 partner genes in hematological malignancies with 11p15 translocations. Twelve of them have been identified in patients with myeloid neoplasias and only 1, RAP1GDS1 (4q21), is fused with NUP98 in five patients with T-cell acute lymphoblastic leukemia (T-ALL). Three of these patients coexpressed T and myeloid markers, suggesting the specific association of t(4;11)(q21;p15) with a subset of T-ALL originating from an early progenitor, which has the potential to express mature T-cell antigens as well as myeloid markers. We describe here a new NUP98 partner involved in a t(10;11)(q25;p15) in a patient with acute biphenotypic leukemia, showing coexpression of mature T and myeloid markers. The gene involved, located in 10q25, was identified as ADD3 using 3'-RACE. ADD3 codes for the ubiquitous expressed subunit gamma of the adducin protein, and it seems to play an important role in the skeletal organization of the cell membrane. Both NUP98-ADD3 and ADD3-NUP98 fusion transcripts are expressed in the patient. This is the second partner of NUP98 described in T-ALL. Adducin shares with the product of RAP1GDS1, and with all of the nonhomeobox NUP98 partners, the presence of a region with significant probability of adopting a coiled-coil conformation. This region is always retained in the fusion transcript with the NH(2) terminus FG repeats of NUP98, suggesting an important role in the mechanism of leukemogenesis.

36 p.-6 fig., The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival and resistance to different anti‐MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4β1 integrin is a main adhesion receptor mediating MM cell‐stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion‐mediated drug resistance (CAM‐DR) and MM cell apoptosis. Whether decreased α4β1 expression and activity is maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4β1 function could boost MM cell survival and disease progression. Using BTZ‐resistant MM cells, we found that they not only rescue their α4β1 expression, but its levels were higher than in parental cells. Increased α4β1 expression in resistant cells correlated with enhanced α4β1‐mediated cell lodging in the BM, and with disease progression. BTZ‐resistant MM cells displayed enhanced NF‐κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4β1‐dependent MM cell adhesion. These data emphasize the upregulation of α4β1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM‐DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin β1) expression in MM and poor progression‐free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high‐risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma., This work was supported by grants SAF2014-53059-R and SAF2017-85146-R from the Ministerio de Ciencia, Innovación y Universidades (MCIU) to JT; SAF2017-89672-R from MCIU to FM; by the Research Institute Hospital 12 de Octubre (i+12) and grants from Instituto de Salud Carlos III (ISCIII) and CIBERONC to JML; by grants from ISCIII PI16/02024,PI17/00701 and PI19/01352, TRASCAN (EPICA and Immunocell), Fundació La Marató de TV3, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, CIBERONC(CB16/12/00489) and co-financed with FEDER funds MINECO Explora (RTHALMY),Gobierno de Navarra, Departamento de Salud 40/2016 and Departamento de Industria (Proyecto Estrategico, Reto Genomica, DIANA) and Fundación Ramón Areces (PREMAMM) to FP and XA The study was also supported by the Multiple Myeloma Research Foundation Networks of excellence, the International Myeloma Foundation (Brian van Novis), and the Qatar National Research Fund award 7-916-3-237.

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10−16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial–mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing., This study was supported by the Centro de Investigación Biomédica en Red —Área de Oncología— del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369, CB16/12/00489, and CB16/12/00400), Cancer Research UK, FCAECC and AIRC under the Accelerator Award Programme, Instituto de Salud Carlos III and Asociación Española Contra el Cáncer by ERA-NET TRANSCAN-2 Programme (AC17/00101), the Black Swan Research Initiative of the International Myeloma Foundation, the European Research Council (ERC) 2015 Starting Grant (MYELOMANEXT, 680200), the Czech Science Foundation through Project No. 19-25354Y, the European Regional Development Fund—Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868), and the Ministry of Health of the Czech Republic (15-29667A).