Parallel Transcriptional Analysis of Multiple Stem and Progenitor Populations Identifies Novel Commonly Dysregulated and Functionally Relevant Targets in AML
Bibliographic record
Abstract
Abstract Abstract 1875 Recent experimental evidence suggests that acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells (HSPC) following the acquisition of multiple genetic or epigenetic changes that initially give rise to pre-leukemic HSPC (pre-LSC) and then to fully transformed leukemia stem cells (LSC). Relapse continues to be the major cause of death in most subtypes of AML, suggesting that current therapies are largely ineffective in eliminating LSC and pre-LSC. Cellular heterogeneity and the recent observation that LSC are contained within different phenotypic cellular compartments are challenges for the identification of pathways contributing to the initiation and maintenance of AML. To address these challenges we employed a novel strategy of parallel transcriptional analysis of multiple phenotypic HSPC populations from individuals with AML with normal karyotype (N=5), -7/7q- (N=6) and complex karyotype (N=5), including long-term HSC, short-term HSC, and granulocyte-monocyte progenitors (GMP), and comparison to corresponding cell populations from age-matched healthy controls (HC) (N=6). Specifically, we sorted Lin−/CD34+/CD38−/CD90+ (LT-HSC), Lin−/CD34+/CD38−/CD90− (ST-HSC), and Lin−/CD34+/CD38+/CD123+/CD45RA+ (GMP), and hybridized RNA to Affymetrix GeneST 1.0 expression arrays. Differential gene expression was determined within each compartment by direct comparison of AML LT-HSC vs. HC LT-HSC, AML ST-HSC vs. HC ST-HSC, and AML GMP vs. HC GMP. Subsequent intersection of all differentially expressed genes revealed that only a relatively small number of 6 to 11 genes were consistently dysregulated in all examined leukemic stem and progenitor cell compartments. Interleukin 1 receptor accessory protein (IL1RAP) was one of the most significantly upregulated genes in LT-HSC, ST-HSC, and GMP in all examined subtypes of AML. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family, including IL-1R1 and ST2. We detected significant overexpression of IL1RAP protein on HSC and progenitor cells of AML patients. Interestingly, CD34+/Lin+ precursor cells showed only a marginal increase of IL1RAP at the protein level in AML, underscoring the importance of purifying HSPC with stringent lineage depletion. We performed fluorescence in situ hybridization in sorted IL1RAP+ and IL1RAP− cells from -7 AML. We observed that the -7 clone was restricted to IL1RAP+ cells, while IL1RAP- cells did not display monosomy 7, demonstrating that IL1RAP overexpression is a distinguishing feature of the cells of the -7 clone. Patients with normal karyotype AML showed a wider range of IL1RAP expression levels; some were as high as in -7 AML and others were as low as in HC. We asked whether IL1RAP expression levels were associated with known clinical or molecular parameters. We analyzed two published datasets of patients with normal karyotype AML (Metzeler, Blood. 2008;112:4193–4201; Tomasson, Blood. 2008;111:4797–4808). Patients with high IL1RAP levels showed inferior overall survival than patients with lower IL1RAP (p=2.2×10−7; median survival: 7.82 mo. for IL1RAP high, 20 mo. for IL1RAP low). Multivariate analysis using a Cox regression model showed that high IL1RAP status was an independent prognostic factor (p=0.002), and even stronger than FLT3 mutation status (p=0.006). In addition, we analyzed data from 183 patients with MDS and found IL1RAP expression to be specifically elevated in cases with RAEB-2, suggesting a role of IL1RAP in MDS and in the progression to AML. Downregulation of IL1RAP protein expression in 4 AML cell lines (THP1, OCI-AML3, HL60, HEL) led to a significant 45–98% decrease in clonogenic growth and increased apoptosis in vitro. To assess the effects of IL1RAP downregulation in vivo, we performed xenotransplants into immunodeficient NOD/SCID/IL2Rg-null mice. THP-1 AML cells showed a 92% reduced proliferation and infiltration of hematopoietic organs upon IL1RAP knockdown in comparison to a non-silencing control in vivo. Genetic studies to assess the role of IL1RAP in the initiation and maintenance of AML in an IL1RAP−/− mouse model are currently ongoing. In summary, our study provides a map of consistently dysregulated transcripts across multiple fractionated stem and progenitor cell types from patients with AML, and identifies IL1RAP as a putative new therapeutic and prognostic target in stem cells in AML and MDS. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".