Obesity-induced galectin-9 is a therapeutic target in B-cell acute lymphoblastic leukemia

The incidence of obesity is rising with greater than 40% of the world’s population expected to be overweight or suffering from obesity by 2030. This is alarming because obesity increases mortality rates in patients with various cancer subtypes including leukemia. The survival differences between lean patients and patients with obesity are largely attributed to altered drug pharmacokinetics in patients receiving chemotherapy; whereas, the direct impact of an adipocyte-enriched microenvironment on cancer cells is rarely considered. Here we show that the adipocyte secretome upregulates the surface expression of Galectin-9 (GAL-9) on human B-acute lymphoblastic leukemia cells (B-ALL) which promotes chemoresistance. Antibody-mediated targeting of GAL-9 on B-ALL cells induces DNA damage, alters cell cycle progression, and promotes apoptosis in vitro and significantly extends the survival of obese but not lean mice with aggressive B-ALL. Our studies reveal that adipocyte-mediated upregulation of GAL-9 on B-ALL cells can be targeted with antibody-based therapies to overcome obesity-induced chemoresistance.

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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.

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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.

Software and code
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Data analysis
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Curtis J. Henry Jan 8, 2022 The software programs used for data analysis are listed below. No software was used for data collection.
2. All flow samples were analyzed by using Flowjo Portal Software Version 9 (Ashland, Oregon).
3. GraphPad Prism (San Diego, California) Software Version 9.1.1 was used to determine statistical significance for the data presented in this manuscript. 4. Image J (Version 1.53n) was used for all microscopy experiments presented in this manuscript. 5. BioRender.com (Toronto, Ontario) was used to create all model images used in this manuscript.

October 2018
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 list of figures that have associated raw data -A description of any restrictions on data availability 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. 2. Patient derived LGALS9 (GAL-9) and HAVCR2 (TIM-3) gene expression levels were determined using the St. Jude Cloud Pecan Bioportal (https:// pecan.stjude.cloud/).
4. The datasets generated during and/or analyzed in the current study are available from the corresponding author on reasonable request (for Table 1 and Figure  4). The raw data and processed RNA-seq data used in this study are deposited in the GEO repository under accession number GSE183062 and can be accessed without restrictions. Source data are provided with this paper.
For RNA-seq experiments, sample size was not determined. For mouse experiments, power analysis was performed based on preliminary estimates of leukemia burden and variance. The number of animals per group was chosen so as to provide more than 90% statistical power at an alpha levelof 0.05 to detect the difference between treatment groups. All tissue culture experiments were performed in triplicates to allow for calculation of standard deviation, standard error of the mean and t-statistics for use in two-tailed Student's t-test or ANOVA.
No data was excluded from the analysis.
All experiments were performed in triplicates with some experiments performed with greater than 10 independent experiments. We did not exclude any replicates. We performed one experiment in human B-ALL cells treated with DMSO or MTX for the RNA-sequencing studies presented in this manuscript. Information pertaining to the number of independent replications for each experiment are included in the figure legends.
Both male and female mice were used in the studies presented in this manuscript. Mice were randomized based on diet and treatment groups (vehicle control, MTX, and anti-Galectin-9). Using both male and female mice in these studies ensured that sex as a biological variable was addressed for leukemia progression, as well as, treatment responses. All other experiments were randomized using randomly assigned plates of cells per condition.
For survival experiments, mice were removed for these studies at the first sign of morbidity. The treatment groups were unknown to the investigator removing mice for euthanasia due to signs of illness or distress; thereby, avoiding bias in these experiments. For all microscope images, slides were double blinded prior to analysis. For all other experiments, sample were harvested and processed by scientists without knowledge of hypothesized treatment effects. After processing, results were properly labeled once data processing had been completed.  Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics Recruitment Anti-Cyclin A (cat# sc-271682, Santa Cruz Biotechnology). 77 citations for validation on the manufacture's website. Validated for western blot in SK-BR-3, A-431, HeLa, K562, and HuT 78 cells. Molecular weight is 54 kDa and reactive with human. Similar results were obtained in our studies.
Anti-E2F1 (cat# sc-56661, Santa Cruz Biotechnology). 8 citations for validation on the manufacture's website. Validated for western blot in Jurkat, WEHI-23, SUP-T1, CCRF-CEM, and TK-1 cells. Molecular weight is 47 kDa and reactive with human. Similar results were obtained in our studies.