Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792 ± 86 cells/μL versus 485 ± 46 cells/μL, P=0.003). Higher numbers of non-classical CD14(+)CD16(++) and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated "education" of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.

No prospective data were available prior to 2021 to inform selection between combination BRAF and MEK inhibition versus dual blockade of programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) as first-line treatment options for BRAFV600-mutant melanoma. SECOMBIT (NCT02631447) was a randomized, three-arm, noncomparative phase II trial in which patients were randomized to one of two sequences with immunotherapy or targeted therapy first, with a third arm in which an 8-week induction course of targeted therapy followed by a planned switch to immunotherapy was the first treatment. BRAF/MEK inhibitors were encorafenib plus binimetinib and checkpoint inhibitors ipilimumab plus nivolumab. Primary outcome of overall survival was previously reported, demonstrating improved survival with immunotherapy administered until progression and followed by BRAF/MEK inhibition. Here we report 4-year survival outcomes, confirming long-term benefit with first-line immunotherapy. We also describe preliminary results of predefined biomarkers analyses that identify a trend toward improved 4-year overall survival and total progression-free survival in patients with loss-of-function mutations affecting JAK or low baseline levels of serum interferon gamma (IFNy). These long-term survival outcomes confirm immunotherapy as the preferred first-line treatment approach for most patients with BRAFV600-mutant metastatic melanoma, and the biomarker analyses are hypothesis-generating for future investigations of predictors of durable benefit with dual checkpoint blockade and targeted therapy.

The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3′UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.

Lenalidomide is an immunomodulatory agent clinically active in chronic lymphocytic leukemia patients. The specific mechanism of action is still undefined, but includes the modulation of microenvironment. In chronic lymphocytic leukemia patients, nurse-like cells differentiate from CD14+ mononuclear cells and protect chronic lymphocytic leukemia cells from apoptosis. Nurse-like cells resemble M2 macrophages with potent immunosuppressive functions. Here, we examined the effect of lenalidomide on the monocyte/macrophage population from chronic lymphocytic leukemia patients. We found that lenalidomide induces high actin polymerization on CD14+ monocytes through activation of small GTPases, RhoA, Rac1 and Rap1 that correlated with increased adhesion and impaired monocytes migration in response to CCL2, CCL3 and CXCL12. In presence of lenalidomide, we observed an increased number of nurse-like cells that lost the ability to nurture chronic lymphocytic leukemia cells and acquired properties of phagocytosis and induction of T cell proliferation. Gene expression signature, induced by lenalidomide in nurse-like cells, indicated a reduction of pivotal pro-survival signals for chronic lymphocytic leukemia, such as CCL2, IGF1, CXCL12, HGF1, and supported a modulation towards M1 phenotype with high IL2 and low IL10, IL8 and CD163. Our data provide new insights into the mechanism of action of lenalidomide that mediates a pro-inflammatory switch of nurse-like cells affecting the protective microenvironment generated by chronic lymphocytic leukemia into tissues.

Background Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia. Design and Methods We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture. Results We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both β₁- and β₂- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFβ and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin. Conclusions Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.

The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ET_AR and ET_BR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ET_AR axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ET_A receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ET_AR triggering. Moreover, the blockade of ET_AR by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ET_AR via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ET_AR blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ET_AR signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ET_AR inhibition.