Formin-like 1 Research Articles

Unveiling promising drug targets for autism spectrum disorder: insights from genetics, transcriptomics, and proteomics.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which current treatments are limited and drug development costs are prohibitive. Identifying drug targets for ASD is crucial for the development of targeted therapies. Summary-level data of expression quantitative trait loci obtained from GTEx, protein quantitative trait loci data from the ROSMAP project, and two ASD genome-wide association studies datasets were utilized for discovery and replication. We conducted a combined analysis using Mendelian randomization (MR), transcriptome-wide association studies, Bayesian colocalization, and summary-data-based MR to identify potential therapeutic targets associated with ASD and examine whether there are shared causal variants among them. Furthermore, pathway and drug enrichment analyses were performed to further explore the underlying mechanisms and summarize the current status of pharmacological targets for developing drugs to treat ASD. The protein-protein interaction (PPI) network and mouse knockout models were performed to estimate the effect of therapeutic targets. A total of 17 genes revealed causal associations with ASD and were identified as potential targets for ASD patients. Cathepsin B (CTSB) [odd ratio (OR) = 2.66 95, confidence interval (CI): 1.28-5.52, P = 8.84 × 10-3], gamma-aminobutyric acid type B receptor subunit 1 (GABBR1) (OR = 1.99, 95CI: 1.06-3.75, P = 3.24 × 10-2), and formin like 1 (FMNL1) (OR = 0.15, 95CI: 0.04-0.58, P = 5.59 × 10-3) were replicated in the proteome-wide MR analyses. In Drugbank, two potential therapeutic drugs, Acamprosate (GABBR1 inhibitor) and Bryostatin 1 (CASP8 inhibitor), were inferred as potential influencers of autism. Knockout mouse models suggested the involvement of the CASP8, GABBR1, and PLEKHM1 genes in neurological processes. Our findings suggest 17 candidate therapeutic targets for ASD and provide novel drug targets for therapy development and critical drug repurposing opportunities.

FMNL1 and mDia1 promote efficient T cell migration through complex environments via distinct mechanisms.

Lymphocyte trafficking and migration through tissues is critical for adaptive immune function and, to perform their roles, T cells must be able to navigate through diverse tissue environments that present a range of mechanical challenges. T cells predominantly express two members of the formin family of actin effectors, Formin-like 1 (FMNL1) and mammalian diaphanous-related formin 1 (mDia1). While both FMNL1 and mDia1 have been studied individually, they have not been directly compared to determine functional differences in promoting T cell migration. Through in vivo analysis and the use of in vitro 2D and 3D model environments, we demonstrate that FMNL1 and mDia1 are both required for effective T cell migration, but they have different localization and roles in T cells, with specific environment-dependent functions. We found that mDia1 promotes general motility in 3D environments in conjunction with Myosin-II activity. We also show that, while mDia1 is almost entirely in the cytoplasmic compartment, a portion of FMNL1 physically associates with the nucleus. Furthermore, FMNL1 localizes to the rear of migrating T cells and contributes to efficient migration by promoting deformation of the rigid T cell nucleus in confined environments. Overall, our data indicates that while FMNL1 and mDia1 have similar mechanisms of actin polymerization, they have distinct roles in promoting T cell migration. This suggests that differential modulation of FMNL1 and mDia1 can be an attractive therapeutic route to fine-tune T cell migration behavior.

Pan-cancer analysis revealing DAAM1 as a novel predictive biomarker for PD-1/PD-L1 blockade in clear cell renal cell carcinoma.

Pan-cancer analysis revealing DAAM1 as a novel predictive biomarker for PD-1/PD-L1 blockade in clear cell renal cell carcinoma.

Discovery of anti-Formin-like 1 protein (FMNL1) antibodies in membranous nephropathy and other glomerular diseases

Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.

Formin-like 1 mediates effector T cell trafficking to inflammatory sites to enable T cell-mediated autoimmunity.

Lymphocyte migration is essential for the function of the adaptive immune system, and regulation of T cell entry into tissues is an effective therapy in autoimmune diseases. Little is known about the specific role of cytoskeletal effectors that mediate mechanical forces and morphological changes essential for migration in complex environments. We developed a new Formin-like-1 (FMNL1) knock-out mouse model and determined that the cytoskeletal effector FMNL1 is selectively required for effector T cell trafficking to inflamed tissues, without affecting naïve T cell entry into secondary lymphoid organs. Here, we identify a FMNL1-dependent mechanism of actin polymerization at the back of the cell that enables migration of the rigid lymphocyte nucleus through restrictive barriers. Furthermore, FMNL1-deficiency impairs the ability of self-reactive effector T cells to induce autoimmune disease. Overall, our data suggest that FMNL1 may be a potential therapeutic target to specifically modulate T cell trafficking to inflammatory sites.

Formin-like-1 mediates effector T cell extravasation through restrictive endothelial barriers enabling T cell entry into peripheral tissues and autoimmunity induction

Abstract To perform their functions, T cells migrate from the blood vasculature into tissues by traversing the vascular wall through a process known as transendothelial migration (TEM). While many of the extracellular cues that guide this process have been identified, the role of downstream cytoskeletal effectors that mediate force generation and morphological changes required for TEM remains an area of active research. Formin-like-1 (FMNL1) is a cytoskeletal effector that mediates cytoskeletal remodeling. However, the role of FMNL1 in T cell functions, and migration in particular, is mostly unknown. The goal of our study was to examine the role of FMNL1 in T cell extravasation and trafficking. We generated a new FMNL1 knock-out (KO) mouse and identified a novel role for FMNL1 in promoting T cell migration through restrictive endothelial barriers during TEM. In vitro, FMNL1-deficient T cells had reduced actin polymerization in response to chemokine signaling. Furthermore, FMNL1 KO T cells had a selective impairment in chemotaxis through restrictive 3μm pores vs permissive 5μm pores. Using time-lapse microscopy, we determined that FMNL1-deficient T cells are severely impaired in their capacity to complete TEM through endothelial barriers. In vivo, we found that FMNL1 was dispensable for T cell extravasation through permissive high endothelial venules and homeostatic trafficking to lymphoid organs. Instead, FMNL1 regulated the ability of activated T cells to extravasate into inflamed peripheral tissues and enabled self-reactive T cells to induce autoimmune disease. Overall, our data identify FMNL1 as a key regulator of lymphocyte migration through restrictive barriers and as a potential target for modulating effector T cell trafficking.

Formin-like 1 (FMNL1) Is Associated with Glioblastoma Multiforme Mesenchymal Subtype and Independently Predicts Poor Prognosis.

Glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults, is characterized by rapid proliferation, aggressive migration, and invasion into normal brain tissue. Formin proteins have been implicated in these processes. However, the role of formin-like 1 (FMNL1) in cancer remains unclear. We studied FMNL1 expression in glioblastoma samples using immunohistochemistry. We sought to analyze the correlation between FMNL1 expression, clinicopathologic variables, and patient survival. Migration and invasion assays were used to verify the effect of FMNL1 on glioblastoma cell lines. Microarray data were downloaded from The Cancer Genome Atlas and analyzed using gene set enrichment analysis (GSEA). FMNL1 was an independent predictor of poor prognosis in a cohort of 217 glioblastoma multiforme cases (p < 0.001). FMNL1 expression was significantly higher in the mesenchymal subtype. FMNL1 upregulation and downregulation were associated with mesenchymal and proneural markers in the GSEA, respectively. These data highlight the important role of FMNL1 in the neural-to-mesenchymal transition. Conversely, FMNL1 downregulation suppressed glioblastoma multiforme cell migration and invasion via DIAPH1 and GOLGA2, respectively. FMNL1 downregulation also suppressed actin fiber assembly, induced morphological changes, and diminished filamentous actin. FMNL1 is a promising therapeutic target and a useful biomarker for GBM progression.

FMNL1 down-regulation suppresses bone metastasis through reducing TGF-β1 expression in non-small cell lung cancer (NSCLC)

Approximately 40% of patients with non-small cell lung cancer (NSCLC) develop bone metastasis. The formin protein formin-like 1 (FMNL1) plays a key role in the pathogenic processes of hematopoietic malignancies, and has been reported to be associated with the progression of multiple types of cancer. In the study, we found that FMNL1 expression was markedly up-regulated in primary NSCLC samples, and stronger expression of FNML1 was detected in bone metastasis. Reducing FMNL1 expression significantly suppressed cell proliferation in NSCLC cells. We also investigated the functional effects of FMNL1 knockdown on the inhibition of migration and invasion by meditating the expression of epithelial to mesenchymal transition (EMT)-associated signals in NSCLC cells. The transforming growth factor-β1 (TGF-β1)/SMADs signaling pathway was repressed in FMNL1-knockdown NSCLC cells. Further studies indicated that additional treatment with TGF-β1 could markedly abrogate FMNL1 knockdown-induced suppression of migration and invasion in NSCLC cells. In addition, NSCLC cell-induced osteoclastogenesis was also inhibited by FMNL1 deletion, as evidenced by the down-regulated expression of tartrate-resistant acid phosphatase (TRAP) and NFATc1. In vivo studies confirmed the results that FMNL1 knockdown markedly limited tumor growth. Importantly, decreasing FMNL1 reduced bone metastasis ability in vivo. Therefore, our results demonstrated that suppressing FMNL1 expression could inhibit bone metastasis in NSCLC through blocking TGF-β1 signaling, and FMNL1 might be a novel target for developing effective therapeutic strategy to limit the bone metastasis of NSCLC.

FMNL1 (formin like 1)

FMNL1 (formin like 1)

FMNL1 promotes T cell extravasation and trafficking to sites of inflammation

Abstract To execute their effector function, activated T cells traffic through the blood stream to sites of inflammation. To enter tissues, T cells extravasate through the vascular endothelial cell wall by a process known as transendothelial migration (TEM). While many of the adhesion molecules and chemokine signaling pathways required for TEM have been previously characterized, little is known about how downstream cytoskeletal effectors mediate the mechanical forces and shape changes needed for T cell TEM. Formin-like 1 (FMNL1) is a terminal cytoskeletal effector directly involved in actin network remodeling. It has been associated with the formation of membrane protrusions that likely function in migration and cell-cell interactions, and is highly expressed by activated T cells. The goal of our study was to examine the role of FMNL1 in activated T cell extravasation and trafficking to sites of inflammation. While activated FMNL1 deficient T cells had normal migration in response to chemokine across 5μm pore transwell membranes, they displayed an impaired ability to migrate through more restrictive 3μm pores. In vitro time-lapse microscopy data suggest that FMNL1 deficient T cells have a reduced ability to complete TEM across brain endothelium. FMNL1 deficient T cells were also impaired in their ability to traffic to the inflamed central nervous system in vivo. Finally, adoptively transferred autoreactive FMNL1 deficient T cells were impaired in their ability to induce Experimental Autoimmune Encephalitis. Together these data suggest that FMNL1 is required for the efficient migration of activated T cells though restrictive endothelial barriers and may be a promising therapeutic target for regulating T cell trafficking in autoimmunity.

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.

Identification of biomarkers for metastatic osteosarcoma based on DNA microarray data.

Osteosarcoma (OS) is amalignant bone tumor very often with pulmonary metastasis and is the main cause of OS mortality. The objective of this study was to screen for possible biomarkers of metastatic OS to explore the mechanisms of pulmonary metastasis of OS through network construction. GSE14359 was downloaded from the Gene Expression Omnibus database, which included 5 samples from conventional OS group with 2 replicates and 4 samples from OS pulmonary metastasis group in duplicate. Differentially expressed genes (DEGs) between two groups were identified by limma packages in Rand classical t-test with the threshold of the false discovery rate (FDR) <0.05. The Database for Annotation, Visualization and Integrated Discovery (DAVID) were then used to perform functional annotation (FDR < 0.01). Differential coexpression network was constructed with subspace differential coexpression analysis (SDC), and genes with high degrees in the differential coexpression network were identified. A total of 1344 genes were screened as DEGs, including 677 up- and 667 down-regulated DEGs in the pulmonary metastasis of OS. Thirty-one significantly enriched functions were obtained, such as blood vessel morphogenesis, defense response, cell death and so on. DEGs with high degrees (brain-specific angiogenesis inhibitor 2 (BAI2), formin-like 1 (FMNL1), dual-specificity phosphatase 7 (DUSP7), transient receptor potential melastatin 2 (TRPM2), CBP80/20-dependent translation initiation factor (KIAA0427) and C120rf35) in the differential coexpression network were found. BAI2, FMNL1, DUSP7 and TRPM2 may be useful markers for predicting tumor metastasis and therapeutic targets for the treatment of OS patients with metastasis.

Proteomic investigation of the interactome of FMNL1 in hematopoietic cells unveils a role in calcium-dependent membrane plasticity

Formin-like 1 (FMNL1) is a formin-related protein highly expressed in hematopoietic cells and overexpressed in leukemias as well as diverse transformed cell lines. It has been described to play a role in diverse functions of hematopoietic cells such as phagocytosis of macrophages as well as polarization and cytotoxicity of T cells. However, the specific role of FMNL1 in these processes has not been clarified yet and regulation by interaction partners in primary hematopoietic cells has never been investigated. We performed a proteomic screen for investigation of the interactome of FMNL1 in primary hematopoietic cells resulting in the identification of a number of interaction partners. Bioinformatic analysis considering semantic similarity suggested the giant protein AHNAK1 to be an essential interaction partner of FMNL1. We confirmed AHNAK1 as a general binding partner for FMNL1 in diverse hematopoietic cells and demonstrate that the N-terminal part of FMNL1 binds to the C-terminus of AHNAK1. Moreover, we show that the constitutively activated form of FMNL1 (FMNL1γ) induces localization of AHNAK1 to the cell membrane. Finally, we provide evidence that overexpression or knock down of FMNL1 has an impact on the capacitative calcium influx after ionomycin-mediated activation of diverse cell lines and primary cells.

Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.

Formin-Like 1 (FMNL1) Associates with Rac1 and Negatively Regulates Neoplastic Growth and Migration in Leukemia Cell Lines.

AbstractAbstract 1030FMNL1 belongs to a conserved family of formin-related proteins, has high restricted expression in malignant lymphoid derived cells and has been described as a tumor associated antigen in chronic lymphoid leukemia (CLL). Overexpression of FMNL1 in CLL has already been show to correlate with adverse clinical and biological parameters, particularly in young patients (E Calpe et al, ASH 2006, abstract submitted). Formins are known for their ability to act as effectors of Rho family GTPases, proteins that regulate multiple pathways involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as leukemia. FMNL1 function and regulation have not yet been well characterized; its restricted expression suggests that FMNL1 represents an attractive target for novel immunotherapies in hematopoietic malignant disorders. Herein, we evaluated the association of FMNL1 with the three most studied Rho GTPase proteins (Rac1, RhoA and Cdc42) and the role of FMNL1, by gene silencing, in proliferation, migration and cell cycle in acute lymphoblastic leukemia cell lines. We also tested the cumulative effect of FMNL1 silencing and rapamycin treatment in proliferation assays. FMNL1 association with Rho GTPases proteins was analyzed by immunoprecipitation, followed by western blot. To inhibit FMNL1, specific shRNA-expressing lentiviral vector targeting the FMNL1 gene or no specific sequence (control) were used. Cell growth was measured using the MTT colorimetric reduction method in cells treated or not with different concentrations of rapamycin (10 or 100nM) in five replicates. Migration assays were performed in triplicate using 5-mm Transwells and the lower compartment was filled with 600 mL 0.5% BSA/RPMI containing 100ng/mL SDF-1. The experiments were performed in two cell lines: Jurkat and Namalwa. P value <0.05 was considered statistically significant. The levels of FMNL1 mRNA and protein in FMNL1 knockdown cells were reduced by approximately 70% for both cell lines tested. Inhibition of FMNL1 in Jurkat and Namalwa cells resulted in a significant decrease of proliferation by 40% for both cell lines when compared with control cells (P<0.01). Interesting, in both cell lines, the combination of FMNL1 inhibition/rapamycin treatment showed higher reduction in cell proliferation when compared with FMNL1 inhibited cells alone (P=0.008) or rapamycin treated cells (P=0.007). Inhibition of FMNL1 resulted in an accumulation of Namalwa cells in the S phase and a consequent decrease of cells in G2/M. FMNL1 silencing also resulted in a significant decrease by 62% of cell migration for both cell lines when compared to control cells (P=<0.01). Among the Rho GTPase proteins tested, FMNL1 was found to be associated with Rac1, but not with RhoA and Cdc42. The immunoprecipitation results suggest FMNL1 as an effector of Rac1. Indeed, similar effect obtained with FMNL1 silencing has already been shown with Rac1 knockdown, which also suppresses leukemia cell migration and growth. Our findings indicate that FMNL1 participates in the regulation of proliferation and migration of Jurkat and Namalwa cell lines, which suggests that FMNL1 represents an attractive therapeutic target. Based on the result of FMNL1 inhibition/rapamycin combination, we hypothesized that combinatorial inhibition of these pathways would be effective for the treatment of lymphoid malignancies. Supported by Fapesp and CNPq. Disclosures:No relevant conflicts of interest to declare.

Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1gamma is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1DeltaDAD), indicating that deregulation of autoinhibition is effective in FMNL1gamma. Expression of both FMNL1gamma and FMNL1DeltaDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.

Formins Regulate the Actin-Related Protein 2/3 Complex-Independent Polarization of the Centrosome to the Immunological Synapse

T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. We therefore examined the requirement for Arp2/3- and formin-dependent F-actin nucleation during T cell activation. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing. Altogether, our results have identified Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells.

FMNL-1 (Formin-Like 1) Gene in Chronic Lymphocytic Leukemia (CLL) Is Overexpressed in Young Patients with Adverse Prognostic Factors and Poor Outcome.

Prognosis of patients with CLL has been traditionally assessed by using clinical parameters. Although useful, such parameters are a mere reflection of the biological diversity of CLL. In this regard, the mutational status of VH genes or ZAP-70 expression separates CLL into two clinical forms with different presenting features and outcome. Formins are multidomain proteins characterized by the presence of two conserved prolin-rich regions, namely formin homology 1 and 2. These proteins are implicated in a wide range of processes, including regulation of the cytoskeleton and in the regulation of the signal for cell survival. Formin is normally expressed in spleen, lymph node, and bone marrow cells, and it has been recently found to be overexpressed in T-cell lymphomas. The aim of this study was to analyze the expression of FMNL-1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL-1 expression was analyzed by Western Blot in separate subpopulations and by quantitative RT-PCR using expression in Jurkat as baseline. Among normal lymphocytes, FMNL-1 was only expressed in memory (CD19+CD27+) B-cells and in T-cells. In CLL cases with a low percentage of T-cells, mean of FMNL-1 expression was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL-1 were associated with a younger age at diagnosis (< 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL-1 expression. No relationship was observed with genetic abnormalities (table). In summary, among B-cell lymphoproliferative disorders, FMNL-1, a gene that regulates cell survival, is found only in CLL and its overexpression correlates with adverse clinical and biological parameters, particularly in young patients.VariableFMNL-1 normalFMNL-1 increasedp valueAge < 50 yrs15%86%0.0001Lymphocyte count > 50.000/ μL14%57%0.018Increasedβ2-microglobulin17%66%0.038CD38 > 30%28%100%0.001ZAP-70≥20%50%100%0.014Time to progression (median)5.1 yrs0.5 yrs0.003Survival (median)16.4 yrs8.5 yrs0.009