Identification of a Unique Autophagy Gene Expression Signature in CD34+ CML Stem/Progenitor Cells That Correlates with Clinical Response to Imatinib Mesylate
Bibliographic record
Abstract
Abstract Abstract 1662 Chronic myeloid leukemia (CML) stem cells are biologically insensitive to ABL tyrosine kinase inhibitor (TKI) monotherapy and are genetically unstable and rapidly generate imatinib (IM)-resistant mutants in vivo and in vitro. Therefore, there is clearly a need to develop new diagnostic and therapeutic strategies to identify biomarkers to predict patients' response to TKI therapy, and to develop potential therapeutics to target CML stem cells to prevent acquisition of resistance. It has been reported that the induction of macroautophagy (autophagy) may play a critical role in the persistence of primitive CML cells, but how this process is regulated is largely unknown. To investigate whether CML stem/progenitor cells harbor a unique autophagy gene expression profile that could be predictive of patient response to TKI therapy, we have now examined transcript levels of several key autophagy and autophagy-related genes (ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG7, ATG12, BECLIN-1, and LC3B) in CD34+ subpopulations obtained at diagnosis from chronic phase (CP) CML patients who were retrospectively classified, after initiation of IM therapy, as IM-responders (n=14) and IM-nonresponders (n=14), as well as normal healthy donors (n=8). Q-RT-PCR analysis revealed that CD34+ CML cells display significantly higher expression levels of ATG4A, ATG4B, ATG4C, and BECLIN-1(p<0.05) compared to normal bone marrow (NBM) cells. Increased transcript levels of ATG4 family members were further observed in CD34+ CML cells upon TKI treatment (IM, dasatinib and nilotinib) in serum-free culture conditions in vitro. Most interestingly, transcript levels of ATG4B were significantly higher in CD34+ CML cells isolated from IM-nonresponders as compared to the same cells from IM-responders (p=0.011), whereas ATG5 was significantly lower in CD34+ CML cells from IM-nonresponders than IM-responders (p=0.003). Moreover, we observed that the stem cell-enriched CD34+CD38− subpopulation from IM-nonresponders expressed higher levels of several ATG genes, including ATG4 family members, than the same cells from IM-responders. In addition, increased transcript levels of ATG4 family members were also observed in peripheral blood (PB) samples of accelerated phase (AP) CML patients (n=7) compared to PB samples from normal individuals (n=4), but there were no significant differences between samples from CP (n=7) vs. AP (n=7). Importantly, our studies show that expression changes in ATG4B and ATG4D in CD34+ CML cells vs. NBM cells correlated with the transcript levels of miR-34a and miR-152, which are predicted to target the ATG4 family. MicroRNA sequence profiling also confirmed that these miRNAs are differentially expressed in CD34+ cells from IM-nonresponders. To further investigate the biological importance of ATG4B, a key cysteine protease involved in the regulation of the autophagy process, IM-sensitive and IM-resistant K562 cells were transduced with an inducible ATG4B shRNA lentiviral vector and knockdown of ATG4B protein expression (∼80%) was confirmed in these cells. Interestingly, the knockdown of ATG4B decreased the viability of these cells (as compared to the scramble control), reduced their proliferative capacity and inhibited the formation of colonies in a colony-forming cell (CFC) assay in the presence or absence of IM. In particular, a reduction in both CFC numbers and colony size were observed in IM-resistant cells with suppression of ATG4B in response to IM treatment, suggesting that ATG4B plays a critical role in IM-induced autophagy. This is the first report investigating potential differences in autophagy gene transcript levels in CD34+ subpopulations from IM-responders vs. IM-nonresponders. The unique gene expression signature identified, particularly differentially expressed ATG4B and ATG5 in IM-nonresponders vs. IM-responders, may serve as a novel, clinically useful biomarker for predicting future TKI therapy response and ATG4B may be a new drug target for directing treatment at CML stem cells. 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 | 0.001 | 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".