Drug-Insensitive CML Stem/Progenitor Cells Highly Express Key Regulators of the Hedgehog Pathway and Inhibition of the Hedgehog Pathway Enhances Their Response to TKI Treatment
Bibliographic record
Abstract
Abstract Chronic myeloid leukemia (CML) stem/progenitor cells are relatively insensitive to tyrosine kinase inhibitor (TKI) monotherapies, and may not exclusively rely on BCR-ABL activity for survival. This population rapidly generates therapy-resistant clones and is often responsible for relapse after TKI therapy discontinuation. It has been reported that Hedgehog (HH) signalling in hematopoietic stem/progenitor cells is essential for CML induction by BCR-ABL in a mouse model. Inhibition of Smoothened (SMO), a key regulator of the HH pathway, reduces the number of BCR-ABL+ stem cells in vitro and slows disease progression in vivo. However, it is not known if SMO and other key regulators are differentially expressed in stem/progenitor cells derived from imatinib (IM)-nonresponders and IM-responders. If so, they may serve as potential biomarkers and/or therapeutic targets. We have assessed gene expression changes in the HH pathway using RNA sequencing (RNA-seq) in 6 highly purified CD34+ stem/progenitor samples from newly-diagnosed chronic phase (CP)-CML patients. After IM therapy was initiated, 3 patients were classified retrospectively as IM-responders, and 3 as IM-nonresponders. This study identified 27 differentially expressed HH genes between healthy and CML patients (>1.5-fold). In particular, SMO and GLI2, key components of the HH pathway,were upregulated in CP-CML patients relative to healthy bone marrow (HBM) controls (n=3).In particular, GLI2 was highly overexpressed in CP-CML compared to HBM (48-fold upregulated in IM-responders and 166-fold upregulated in IM-nonresponders). To validate the RNA-seq results, expression of the principal HH genes (PTCH1, GLI1, GLI2, SMO) was assessed in 18 CD34+ CP-CML patient samples and 8 HBM samples using qRT-PCR. Interestingly, the expression levels correlated with patients' TKI resistance status, with IM-nonresponders expressing significantly higher levels of SMO (p<0.01)and GLI2 (p<0.05) compared with IM-responders. GLI2 and SMO expression was then assessed in CD34-subpopulations in 5 IM-responders and 4 IM-nonresponder samples. GLI2 was exclusively and highly expressed in the most primitive population (lin-CD34+CD38-), while its expression was very low in more mature populations (lin-CD34+38+ and CD34-). This effect was amplified in IM-nonresponder samples, where GLI2 is greater than 40-fold upregulated in the lin-CD34+CD38- subpopulation compared with IM-responders. In contrast, SMO was more uniformly expressed in both stem and progenitor subpopulations, but was more highly expressed in IM-nonresponders than IM-responders.These results suggest that key regulators of the HH pathway are highly expressed in IM-nonresponder stem cells and that these cells may be more responsive to SMO inhibition than IM-responder cells. We then performed a viability and apoptosis analysis of 5 CD34+ CP-CML patient samples. IM-nonresponder samples were more sensitive to SMO inhibition using a highly selective SMO inhibitor (PF-04449913) compared with IM-responders, and this effect was enhanced in combination with the new TKI bosutinib (BOS). To investigate the effects of BOS and PF-04449913 on CML progenitor cells, colony forming cell assays were performed on CD34+ CP-CML cells from 4 IM-responders and 3 IM-nonresponders. Overall, combination treatment with BOS and PF-04449913 only modestly reduced colony forming ability compared to either agent alone. The inhibitory effects of combination therapy were, however, strikingly enhanced in a replating experiment, resulting in a greatly reduced replating efficiency compared to BOS treatment alone (15% vs. 30%), suggesting that the combination specifically targets primitive CP-CML cells. Taken together, we have demonstrated that both SMO and GLI2 are highly expressed in primitive CP-CML cells, and that the transcript levels of GLI2 are significantly increased in CP-CML stem cells from IM-nonresponders, which may serve as a new biomarker to predict patient TKI responsivity. We also found that IM-nonresponder stem/progenitor cells are more sensitive to SMO suppression in short- and long-term assays compared with IM-responders, suggesting that HH pathway activation may comprise a potential mechanism of resistance during TKI therapy, and that dual inhibition of the BCR-ABL and HH pathways may constitute a rational approach to abrogate drug resistance and disease progression in CML. Disclosures Woolfson: Pfizer Inc: Employment.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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) | 0.002 | 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".