Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

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.

Software and code
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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
All data are available from the authors upon request. Source data are provided with this paper for Figures 1-7 and Supplementary Figures 1-9. RNA microarray dataset on shControl and shCALCRL MOLM14 cells in independent triplicates were deposited at GEO with accession code GSExxxxx. All publicly accessible transcriptomic databases of AML patients used in this study: GSE30377: Eppert K, Takenaka  No data obtained in this study were excluded.
To verify the reproducibility experimental findings were replicated several times with at least three independent experiments for both in vitro and in vivo conditions.
For in vivo studies, mice grafted with primary samples (PDXs) and cell lines (CLDXs) were randomly distributed into cages. While disease established, mice were randomly distributed between arms (AraC vs Placebo) taking into account the peripheral blood engraftment, the weight of mice and their gender.
The investigator responsible for in vivo experiments was blinded for the group allocation and at point of analysis, but blinding was not possible during AraC since this compound is opaque and white while the vehicle is transparent. For expression analysis, blinding was not possible to enable orderly loading of the gels. Other experiments presented in this study not required blinding.  DSMZ and ATCC provide authenticated cell lines by cytochrome C oxidase I gene analysis and short tandem repeat profiling. The names of the used cell lines are authentic and previously published. Nevertheless, all cell lines were sequenced regularly in order to avoid cross-contamination or other mechanisms by STR technique.
All cell lines have been routinely tested for Mycoplasma contamination in the laboratory and were negative for contamination.
No commonly misidentified lines were used in this study. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation NOD/LtSz-SCID/IL-2R#chain null (NSG) mice. Animals were used for transplantation of AML cell lines and primary AML patients cells in accordance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Région Midi-Pyrénées (France). NSG mice were produced at the Genotoul Anexplo platform at Toulouse (France) using breeders obtained from Charles River Laboratories. Mice were housed in sterile conditions using HEPA-filtered microisolators and fed with irradiated food and sterile water. 8 weeks old male and female mice (1:1) were sublethally treated with busulfan (20 mg/kg) 24 hours before injection of AML cells.
The study did not involve wild animals.
The study did not involve samples collected from field.
This study was approved by the Institutional Animal Care and Use Committee of Re" gion Midi-Pyre" ne" es (France). AML cells in culture or from murine bone marrow and spleen were spun down and processed for staining as described in the Material and Methods section.