Quinacrine-CASIN combination overcomes chemoresistance in human acute lymphoid leukemia

Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidence that prolonged and intensive chemotherapy often fails to eradicate leukemic stem cells, which are protected by the bone marrow niche and can induce relapse. Thus, new therapeutic approaches to overcome chemoresistance are urgently needed. By conducting an ex vivo small molecule screen, here we have identified Quinacrine (QC) as a sensitizer for Cytarabine (AraC) in treating acute lymphoblastic leukemia (ALL). We show that QC enhances AraC-mediated killing of ALL cells, and subsequently abrogates AraC resistance both in vitro and in an ALL-xenograft model. However, while combo AraC+QC treatment prolongs the survival of primary transplanted recipients, the combination exhibits limited efficacy in secondary transplanted recipients, consistent with the survival of niche-protected leukemia stem cells. Introduction of Cdc42 Activity Specific Inhibitor, CASIN, enhances the eradication of ALL leukemia stem cells by AraC+QC and prolongs the survival of both primary and secondary transplanted recipients without affecting normal long-term human hematopoiesis. Together, our findings identify a small-molecule regimen that sensitizes AraC-mediated leukemia eradication and provide a potential therapeutic approach for better ALL treatment.


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No Sample size calculation was performed. Sample size was determined according to previous published literature. References: Himburg HA, 2016;Di Tullio A, 2017;Guo et al., 2014 When applicable, positive and negative controls were used in each experiment. If these controls failed, the whole set of data was excluded.
Replicate numbers for individual experiments were listed in figure legend and supplementary. Briefly, all NSGS experiments have at least 3 biological replicates, growth curve OD measurement have 4 technical replicates for each genotype. all attempts on replication were successful. In general, all experiments were generated from 2-3 biological replicate samples. Biological replicate samples were generated independent of each other. All attempts on replication were successful.
No randomization was performed. All strains used in this study are isogenic and are therefore considered identical. Any genetic manipulation and chemical treatment is therefore intrinsically randomized.
Investigators were not blinded as this was not relevant to the analysis of the data generated here, and the same pipelines and scripts were used to analyze all samples.
For analysis or sorting of ALL and HSPCs derived from human cord blood or adult BM cells, we used: hCD45 microbeads (Miltenyi Biotec, Auburn CA). Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

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antibodies used for western blotting include: LC3A/B (#4108S, Cell Signaling technology) b-actin (Clone AC-74, Cat # A2228, sigma-Aldrich) p65 (#8242S; Cell Signaling technology) p62 (#5114, Cell Signaling technology) phosph-p65 (#3033, Clone 93H1, Cell Signaling technology) All antibodies used have been validated by the manufacturer and used according to the manufacturer's instructions. the manufacturer's websites staining the validation of each antibody are listed above. antibodies have been re-validated by titrating their concentration. Optimal antibody concentration was used, depicted in the method section of the manuscript. Isotypes and FMO were included in the experiments whenever appropriate. Policy information about dual use research of concern Hazards Could the accidental, deliberate or reckless misuse of agents or technologies generated in the work, or the application of information presented in the manuscript, pose a threat to:

No Yes
Public health National security Crops and/or livestock Ecosystems Any other significant area Experiments of concern Does the work involve any of these experiments of concern: No Yes Non-viable cells were excluded by DAPI staining. Appropriate isotype-matched antibodies were used as controls. Flow cytometry analysis was performed using an LSRII "ow cytometer (BD Biosciences, San Jose, CA). Cell sorting was performed using a FACS Aria or INFLUX (BD Biosciences, San Jose, CA). FACSDiva software v 6.1.3 was used for data acquisition (BD Biosciences, San Jose, CA). Cells were subjected to the indicated mice followed by flow cytometry analysis to determine cell apoptotic status following Annexin V and 7-AAD staining, or cell cycling analysis using Ki67 and DAPI staining. For BrdU incorporation assay, Bromodeoxyuridine (BrdU, 150 ul of 10 mg/ml) were intraperitoneally (i.p.) injected to subjected mice followed by BM cells isolation 14 hours later. BrdU incorporated cells (S phase) were analyzed with the APC BrdU Flow Kit (BD Biosciences, San Jose, CA), following the manufacturer's instructions. Briefly, cells were surface stained then fixed and permeabilized using BD Cytofix/Cytoperm Buffer. After 1 hour incubation with DNase at 37°C, cells were stained with APC-conjugated anti-BrdU monoclonal antibody. 7-aminoactinomycin (7-AAD) was added to each sample right before Flow Cytometry analysis (BD Biosciences, San Jose, CA). For mitoSOX staining, treated cells were stained with MitoSOX (5 uM, Molecular Pribes, Waltham. MA) at 37°C for 10 mi in the dark then washed with pre-warmed PBS. cell pellets were suspended in pre-warmed PBS followed by Flow cytometry analysis. For intracellular phos-p65 staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm Buffer and stained with pho-p65 antibody (#3033S, Clone 93H1, Cell Signaling Tech, Beverly, MA) for 30 min followed by secondary Antibody incubation. PBS washed cells were then subjected to flow cytometry analysis.