IPO11 Regulates the Nuclear Import of BZW1/2 and Is Necessary for AML Cells and Stem Cells
Bibliographic record
Abstract
While most patients with acute myeloid leukemia (AML) achieve remission with initial therapy, the majority relapse leading to poor overall survival. Relapse is frequently driven by a rare subset of leukemic stem cells (LSC). Understanding biological mechanisms that maintain LSCs will help identify new therapeutic strategies for this disease. To identify such vulnerabilities, we overlaid the results of a genome-wide CRISPR screen with the expression of genes enriched in functionally defined LSCs. Through our CRISPR screen, we identified 570 genes whose knockout reduced the growth and viability of OCI-AML2 cells. Essential genes for growth and viability by our CRIPSR screen were enriched in the LSC+ population. By overlaying the hits from our CRISPR screen with genes upregulated in LSCs, we identified IPO11, as a top hit, with 7.5-fold increase in the LSC+ fraction compared to the LSC- fraction. IPO11 is a member of the importin-β family of proteins and facilitates the import of protein cargo into the nucleus. Further analysis showed that IPO11 was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) primary AML samples, CD34+ vs CD34- AML samples, undifferentiated progenitor vs. myeloid cluster AML samples, and relapse vs de novo AML. IPO11 was increased in AML cells compared to normal hematopoietic cells and increased IPO11 expression was associated with decreased overall survival in AML. By immunoblotting, IPO11 protein was increased in primary AML (n=4) compared to normal hematopoietic cells (n=4). To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen, did not reduce AML growth and viability. Knockdown of IPO11 increased differentiation of AML cells as evidenced by the changes in gene expression, decreased chromatin accessibility, increased CD11b expression and increased non-specific esterase staining. Finally, knockdown of IPO11 reduced the engraftment of TEX cells and the low passage primary AML 8227 cells into immune deficient mice by over 90%. Importantly, IPO11 knockdown reduced engraftment of primary AML cells into mouse marrow. To identify novel cargos of IPO11, we performed proximity-dependent biotin labeling (BioID) coupled with mass spectrometry and identified proteins that interacted with IPO11. Among the top hits were BZW1 and BZW 2 (Basic leucine zipper and W2 domains 1 and 2). BZW1 and BZW2 are members of the bZIP super family of transcription factors. Knockdown of IPO11 reduced levels of BZW1 in the nucleus detected by immunoblotting and confocal microscopy. Commercial antibodies could not detect BZW2. To determine if the nuclear import of BZW1 and 2 were functionally important for the effects of IPO11 on AML stem cell function and differentiation, we knocked down BZW1 and BZW2. Dual knockdown of BZW1 and BZW2 (but not individual) mimicked the effects of IPO11 inhibition and decreased the growth and viability of AML cells. Changes in gene expression after BZW1/2 knockdown were similar to IPO11 knockdown with enrichment in myeloid-differentiated genes. By pathway analysis, we identified that IPO11 knockdown, as well as BZW1/2 knockdown decreased expression of MYC target genes, suggesting a mechanism by which these proteins regulate AML stem cell function. Thus, in summary, we identified IPO11 as an essential gene for the viability of AML cells and stem cells. This work highlights a previously unappreciated role of the protein import pathway in regulating AML stem cell function and highlights a potential new therapeutic target for AML. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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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) | 0.002 | 0.001 |
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".