ALBERT

Description: Abstract 1770 Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature CD5+/CD23+ B lymphocytes, with a highly variable clinical course. Gene expression profiling studies identified SLAMF-1 (signaling lymphocytic activation molecule aka CD150) as part of the genetic signature characterizing CLL patients with favorable prognosis. Human SLAMF-1 is the prototype member of a family of receptors that act as co-activators through self-interactions on hematopoietic cell surface. SLAMF-1 activation is essential for full T cell functions, including cytokine secretion and development. The role of SLAMF-1 in antigen presenting cells, including B cells, is less well characterized, though it is known that the molecule initiates a signaling pathway that leads to B cell proliferation or CD95-mediated apoptosis. More recently, SLAMF-1 has been attributed a novel function as a microbial sensor that regulates bacterial phagosome functions by recruiting a supra-molecular complex, part of the ubiquitous cellular autophagic machinery. The analysis on the CD19+ fraction of 292 clinically and molecularly characterized CLL patients revealed highly variable levels of SLAMF-1 expression (1–95%). Statistical analyses of the data indicated that patients characterized by a good prognosis (in terms of disease stage at diagnosis or treatment requirements) express higher levels of SLAMF-1 compared to the other subgroups. Moreover, patients with 〉 6% SLAMF-1+/CD19+ CLL cells had a significantly longer treatment free survival (median 6.4 in SLAMF-1+vs 1.2 years in SLAMF-1− patients, P=.002). Consistently, SLAMF-1 expression inversely correlates with CD38 and CD49d, two molecular markers of unfavorable prognosis, and positively associates with the presence of somatic mutations in the IgHV genes. Functional experiments showed that the engagement of SLAMF-1 by an agonistic mAb started a well-characterized signaling pathway. Co-immunoprecipitation experiments demonstrated a direct interaction between the receptor and the adaptor molecule EAT-2, with its consequent phosphorylation and the following downstream activation of Vav-1, p38 and JNK. Moreover, co-crosslinking of SLAMF-1 and sIgM prolonged the phosphorylation of p38 and JNK and resulted in an increased percentage of CLL cells undergoing apoptosis, as compared to either signals alone or the basal condition. Furthermore, the engagement of SLAMF-1 for a period of 6 hours led to an increased appearance of autophagic vesicles, as confirmed by confocal and transmission electron microscopy. The modulation of apoptosis and autophagy was mediated by the sequential phosphorylation of JNK and Bcl-2: the final result is the activation of Bcl-2 (phospho-Ser70) and the release of Beclin-1, an essential member of the autophagic complex. In conclusion, i) SLAMF-1 is expressed at higher levels by patients with a good prognosis, ii) and performs as an immunoreceptor on CLL cells, starting a signaling pathway that involves the adaptor molecule EAT-2, Vav-1 and MAP kinases, like p38 and JNK. iii) SLAMF-1+ CLL patients respond to receptor engagement modulating an autophagic pathway mediated by the phosphorylation of JNK and Bcl-2. Taken together, these results suggest that SLAMF-1 could represent a novel marker for the subset of CLL patients characterized by an indolent clinical course and highlight a hypothetical link between the activation of the autophagic process and a better clinical outcome in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Human SLAMF1 (signaling-lymphocytic-activation-molecule-family1, CD150) is a self-ligand adhesion/co-stimulatory molecule wich belongs to a family of 9 receptors. SLAMF1 is also a microbial sensor, as it regulates Gram- bacterial phagosome functions through an ubiquitous cellular autophagic machinery and serves as a receptor for Measles virus. In this work, we investigated expression and function of SLAMF1 in chronic lymphocytic leukemia (CLL) cells. Results indicate that expression of SLAMF1 is lost in a subset of patients with chronic lymphocytic leukemia characterized by an aggressive form of the disease, with shorter time to first treatment (median 2.2 years in SLAMF1- vs 7.6 in SLAMF1+ patients, P=.001) and overall survival (77.5% survival rate at 10 years in SLAMF1- vs 94.7% years in SLAMF1+ patients, P=.036). Consistently, SLAMF1low CLL patients are characterized by clinical or molecular markers of a more aggressive disease. Stable silencing of SLAMF1 in the CLL-like Mec-1 cell line (constitutively SLAMF1+) modulated pathways related to cell migration, cytoskeletal organization and intracellular vesicle formation/recirculation. Decreased expression of CXCR3 and an increased expression of CXCR4, CD38 and CD44 were maintained at the molecular level, likely explaining why SLAMF1- cells show enhanced chemotactic responses to CXCL12. This phenotype was confirmed in primary cells, by comparing a cohorts of SLAMF1high to one of SLAMF1low patients. Gene expression profiling also indicates profound modulation of pathways connected with vesicle formation and recirculation. Consistently, cross-linking of SLAMF1 with an agonisic mAb in primary cells and in the Mec-1 cell line enhanced the generation of autophagic vesicles and their fusion with the lysosomes. Ligation of SLAMF1 with this agonistic monoclonal antibody promoted the autophagic flux, by increasing accumulation of reactive oxygen species (ROS) and inducing phosphorylation of p38, JNK1/2 and bcl-2. The direct consequence was the formation of the autophagy macro-complex containing SLAMF1, the scaffold protein beclin1 and the enzyme Vps34. In agreement with the observation that many drugs used in CLL have autophagy-mediated effects, including fludarabine and the BH3 mimetic ABT-737, SLAMF1-silenced Mec-1 cells or SLAMF1low primary CLL cells were resistant to treatment with both agents. These results indicate that SLAMF1 plays as a critical role in CLL homeostasis. Loss of SLAMF1 expression modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, thus providing a likely explanation for the unfavorable clinical outcome experienced by this patient subset. Restoring SLAMF1 expression in CLL cells would therefore be of therapeutic value for patients with aggressive CLL. Disclosures Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

Description: Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors. Chronic lymphocytic leukemia (CLL) cells expressing the ectoenzymes CD39 and CD73 can actively produce adenosine, activating an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells. Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment. Results indicate that culture of CLL cells under hypoxic conditions, such as those observed in lymph nodes from CLL patients, boosts adenosine production, mainly because of the significant increase in the mRNA and protein levels of CD73, the rate-limiting enzyme in adenosine synthesis. CLL also underwent a robust up-regulation of CD26, which functions as an adenosine-deaminase scaffold protein, in keeping with the hypothesis that extracellular nucleotides enter a scavenging pathway, with conversion to inosine and re-uptake by the leukemic cells. Confirmation was obtained using HPLC assays, which showed increased inosine generation under hypoxia. Consistently, expression of membrane nucleoside transporters was also significantly up-regulated. However, hypoxic CLL cells also expressed high levels of the A2A adenosine receptor, which delivered cytoprotective signals and which supported CLL proliferation in response to TLR signaling. Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by a pharmacological agonist favored NLC generation, with overexpression of indoleamine 2,3-dioxygenase (IDO) and of the M2 macrophage markers CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC differentiation, expansion, expression of immunosuppressive molecules and secretion of cytokines and chemokines. In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of A2A, without significantly affecting the enzymes that generate adenosine, which were anyway restricted to the regulatory T cell (Treg) compartment. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by A2A receptor antagonists. Furthermore, hypoxic T cells underwent a metabolic switch, with increased expression of nucleoside transporters and enzymes involved in glucose metabolism, suggesting a Warburg effect. This was accompanied by the differentiation of a population of Tr1 cells, characterized by the expression of LAG3 and CD49b and by the secretion of high levels of IL-10 and VEGF. Expression of the PD-1 immuno-inhibitory receptor was enhanced in hypoxic T cells, suggesting that multiple inhibitory mechanisms are activated. We also observed expansion of classical Tregs, defined on the basis of a CD4+/CD25high/CD127low/foxp3+ phenotype. Blockade of the A2A receptor prevented this phenotype, partially restoring T cell proliferation and immune competence. Together, these findings indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that pharmacological inhibition of the adenosinergic signals may counteract some of the effects mediated by an hypoxic environment, contributing to disrupt the leukemic niche and to restore the immune system. Disclosures Gaidano: Celgene: Research Funding; MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards.

Description: BACKGROUND . NOTCH1 PEST domain mutations were among the first to be identified using whole exome sequencing and their prevalence and prognostic power for CLL patients has since been extensively studied. Nevertheless, the functional contribution of NOTCH1 and the impact of its mutations in CLL remain poorly understood. This is mainly due to the intrinsic limits in using primary CLL cells and to the rapid inactivation of the NOTCH1 pathway in vitro. AIM. The aim of the study was to highlight the NOTCH1-dependent mechanisms contributing to CLL pathogenesis and progression and the effects of PEST mutations in this disease context. RESULTS. By using the CRISPR/Cas9 technology, we first generated a cellular model either lacking NOTCH1 (Mec-1/KO) or expressing it in its wild-type or mutated forms. RNA-seq analysis of the transcriptome of Mec-1/WT and /KO cells, highlighted signaling and migration as the most prominently down-regulated pathways in KO cells. These findings were confirmed by showing that Mec-1/KO cells migrated significantly less towards CCL19 than their WT counterparts, as the result of the down-modulation of CCR7, the CCL19 receptor. Mechanistically, Mec-1/KO were characterized by increased expression of the tumor suppressor gene DUSP22, a phosphatase that negatively regulates MAPK and STAT3 signaling, in turn responsible for CCR7 gene transcription. Up-regulation of DUSP22 in Mec-1/KO was caused by decreased methylation of the gene promoter, as shown using a methylation specific PCR. Re-expression of the NOTCH1 Intracellular Domain (NICD) rescued the phenotype, increasing methylation of the DUSP22 promoter, which in turn led to decreased gene and protein expression. Mec-1 cells with reconstituted NICD expression showed increased STAT3 phosphorylation, CCR7 expression and, ultimately, migrated more actively in response to CCL19. Importantly, reconstitution with an NICD carrying the PEST domain mutation most commonly found in patients (ΔCT_7541-7542), significantly enhanced all these events, including DUSP22methylation, STAT3 phosphorylation, CCR7 expression, generating cells with the highest chemotactic responses to CCL19. NOTCH1 directly participates to the epigenetic regulation of DUSP22, by conditioning the activity of the DNA methyltransferase 3A (DNMT3A) on the gene promoter. By binding RBPJk, the NICD displaces HDAC1 from the repressor complex and initiates NOTCH1-dependent signaling. Consequently, free HDAC1 binds to and stabilizes DNMT3A, promoting its activity on DUSP22promoter. These results were first corroborated in a large cohort of CLL (n=113) carrying mutations in NOTCH1 PEST domain (ΔCT_7541-7542)at the clonal (〉12%) or subclonal (≤12%) level. The clonally mutated subset showed significant downregulation of DUSP22expression and gene promoter hypermethylation compared to subclonal samples. Consistently, the clonal subset displayed higher constitutive STAT3 phosphorylation, expressed higher levels of CCR7 and migrated more efficiently to CCL19 than subclonally mutated CLL. The second confirmation was obtained by studying a Mec-1 variant with PEST domain mutation (Mec-1/PEST), generated using the CRISPR/Cas9 system. Like primary cells, Mec-1/PEST needed the ligand to initiate signaling. As predicted, the mutant NICD was more stable and more transcriptionally active. Consistently, these cells had the lowest DUSP22 levels and the highest CCR7 expression. When Mec-1/PEST were xenografted in immunocompromised mice, activation of the NOTCH1 pathway was more pronounced than that of Mec-1/WT cells. In keeping with their minimal expression of DUSP22, Mec-1/PEST cells were characterized by markedly increased metastatic properties, with extensive colonization of the liver, the spleen, and the brain, at variance with the other cell variants. CONCLUSIONS . Considered together, these results show that PEST mutations increase NICD stability, in turn affecting a complex nuclear balance. The final outcome for the CLL cell is decreased expression of the tumor suppressor gene DUSP22 and increased chemotaxis towards CCL19. As this chemokine regulates homing to secondary organs, conceivably NOTCH1 mutations might favor CLL recirculation to lymph node and spleen, where the local environment triggers proliferation and protects from apoptosis, two conditions that are associated with a more aggressive disease and an unfavorable prognosis. Disclosures Coscia: Karyopharm: Research Funding; Janssen: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; ROCHE: Honoraria, Other: Advisory board. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria.