Siglec-6 as a therapeutic target for cell migration and adhesion in chronic lymphocytic leukemia

Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.For quantitation, a two tailed student t test was performed for these two group.Proteins with P value less than 0.05 (or 5%) were considered to be significantly changed.Proteins with a fold change >2 or <0.5 were considered as up-and down-regulation.Statistical analysis: All analyses were performed by the OSU Center for Biostatistics using previously described models using SAS/STAT software, v9.4 of the SAS System for Windows (SAS Institute Inc., Cary, NC).This proteomics dataset was generated for this study.

Data Policy information about availability of data
All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability Research involving human participants, their data, or biological material Policy information about studies with human participants or human data.See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.

Reporting on sex and gender
Reporting on race, ethnicity, or other socially relevant groupings

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Data exclusions
Protocol was approved by the Ohio State University All patients examined had CLL which was defined by the 2008 IWCLL criteria.Covariate-relevant characteristics collected include: IGHV mutation status (un-mutated/mutated), 17p deletion (yes/no), 13q deletion (yes/no), previous treatment (yes/no).
Peripheral blood or lymph node biopsy from CLL patients was obtained after written informed consent in accordance with the Declaration of Helsinki, under a protocol approved by the institutional review board (IRB) at The Ohio State University.Participants were not prospectively identified for the current study.The population chosen is representative for a tertiary referral center without any identifiable bias.
All the CLL and healthy donor samples were de-identified and as a result, information regarding race and ethnicity was not available.
All the CLL and healthy donor samples were de-identified and as a result, information regarding sex and age was not available.

X
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files.All the raw data generated in this study are provided in the Source Data file.The mass spectrometry publicly available data used in this study have been deposited in the ProteomeXchange database (Project accession: PXD039892).The complete list of proteins identified in the mass spectrometry experiment is provided in the Supplementary Information.Other relevant data that further support the findings of this study are available from the corresponding author upon request.Source data are provided with this paper.
No datapoints were excluded from any of the experiments.
No statistical tests were done to pre-determine samples sizes.Based on previous publications from our group and upon recommendation from Dr. Xiaokui Mo at the OSU Center for Biostatistics, a sample size of n ≥ 3 for experiments with cell lines and n ≥ 5 for experiments with primary cells was chosen and was sufficient to detect a response between groups.For all mouse experiments, 4-9 mice were used/group to allow sufficient power analysis.
All replication attempts to verify experimental findings, including engraftment and therapeutic studies in the in vivo experiments, were successful.
In engraftment studies with immunocompetent huCD3 mice, mice were randomly assigned to treatment groups according to the date on which their circulating leukemia exceeded the 5 % threshold and evenly distributed according to disease severity at that date.For the in vivo migration experiment, NSG mice were randomly assigned to each group and there were no covariates since they were genetically similar.For the CLL PDX model, 2 mice received one CLL patient donor engraftment (with a total of 6 mice and 3 patients) and were then randomly assigned into control or treated group.
For the proteomics analysis, the investigator performing the analysis was blinded to the isotype versus Siglec-6 antibody pull down group.For statistical analysis, the biostatistician was blinded to group allocation.For all therapeutic and other mouse experiments, veterinary mouse technicians determining euthanasia criteria were blinded to the treatment enrollment group.

Validation
Flow cytometry gating strategies were followed according to published data and technical resource publications.They were adapted to allow exclusion and interrogation of CD19+CD5+ CLL-like populations.
Fluorescence-minus-one or isotype controls were used for gate positions.

X
Mass spectrome try database searching -All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.6.2).Mascot was set up to search the UNIPROT_HUMAN_Reviewed_20181126 database (49259 entries) assuming the digestion enzyme stricttrypsin.Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 5.0 PPM.Carbamidomethyl of cysteine was specified in Mascot as a fixed modification.Deamidated of asparagine and glutamine and oxidation of methionine were specified in Mascot as variable modifications.Criteria For Protein Identification-Scaffold (version Scaffold_4.11.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications.Peptide identifications were accepted if they could be established at greater than 91.0%probability to achieve an FDR less than 1.0% by the Peptide Prophet algorithm (Keller, A et al Anal.Chem.2002;74(20):5383-92) with Scaffold delta-mass correction.Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides.Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal.Chem.2003;75(17):4646-58).
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.