Abstract
Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be inferred when leukemia cells persist after 28 days of initial treatment. Survival of these long-term persister (LTP) / minimal residual disease (MRD) cells is partly due to bone marrow stromal cells that protect them under conditions of chemotherapy stress. We used RNA-seq to analyse BCP-ALL cells that survived a long-term, 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. RNAs of as many as 10% of the protein-encoding genes were differentially expressed. There was substantial overlap with genes associated with MRD cell persistence reported in other studies. The top pathway regulated in the LTP cells was that involving p53, a master regulator of a spectrum of responses relevant to drug resistance and cytotoxic drug exposure including control of autophagy. We tested a select number of genes for contribution to BCP-ALL cell survival using Cas9/CRISPR in a 2-step selection, initially for overall effect on cell fitness, followed by 21 days of exposure to vincristine. Many genes involved in autophagy and lysosomal function were found to contribute to survival both at steady-state and during drug treatment. We also identified MYH9, NCSTN and KIAA2013 as specific genes contributing to fitness of BCP-ALL cells. CD44 was not essential for growth under steady state conditions but was needed for survival of vincristine treatment. Finally, although the drug transporter ABCC1/MRP1 is not overexpressed in BCP-ALL, a functional gene was needed for DTP cells to survive treatment with vincristine. This suggests that addition of possible ABCC1 inhibitors during induction therapy could provide benefit in eradication of minimal residual disease in patients treated with a chemotherapy regimen that includes vincristine.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Email addresses: Mingfeng Zhang mingfengzhang{at}coh.org, Lu Yang luyang{at}coh.org, David Chen cweichen{at}coh.org
Declaration of interests: None
Submission declaration: The work described has not been published previously, is not under consideration for publication elsewhere, its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out. If accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.
Role of funding source: This study was partly supported in 2016/2017 by a New Idea Award to NH from the Leukemia Lymphoma Society, by NIH RO1 CA090321 and CA172040 to NH. The funding organizations had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Abbreviations
- BCP-ALL
- precursor B-cell acute lymphoblastic leukemia
- DE
- differential expression
- DTP
- drug-tolerant persister
- EMDR
- environment-mediated drug resistance
- GSEA
- Gene Set Enrichment analysis
- logFc
- log2-fold change
- MAGeCK
- Model-based Analysis of Genomewide CRISPR/Cas9 Knockout
- MFI
- mean fluorescent intensity
- MRD
- minimal residual disease
- rpkm
- reads per kilobase million