Single-cell multi-omics analyses reveal EZH2 as a main driver of retinoic acid resistance in PLZF-RARA leukemia.

Cancer relapse is caused by a subset of malignant cells that are resistant to treatment. To characterize resistant cells and their vulnerabilities, we studied the retinoic acid (RA)-resistant PLZF-RARA acute promyelocytic leukemia (APL) using single-cell multi-omics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells and identified a subset of cells resistant to RA that depend on a fine-tuned transcriptional network targeting the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2). Epigenomic and functional analyses validated EZH2 selective dependency of PLZF-RARA leukemia and its driver role in RA resistance. Targeting pan-EZH2 activities (canonical/non-canonical) was necessary to eliminate leukemia relapse initiating cells, which underlies a dependency of resistant cells on an EZH2 non-canonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach.

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Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Blood ◽

10.1182/blood-2021-146777 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2205-2205

Author(s):

Mathilde Poplineau ◽

Nadine Platet ◽

Adrien Mazuel ◽

Léonard Hérault ◽

Shuhei Koide ◽

...

Keyword(s):

Retinoic Acid ◽

Single Cell ◽

Acid Resistance ◽

Promising Therapeutic Approach ◽

Main Driver ◽

M3 Subtype ◽

Chemical Corporation ◽

Retinoic Acid Resistance

Abstract Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA. We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature ("Rep" cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance. These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. Disclosures Iwama: Nissan Chemical Corporation: Research Funding.

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A novel mechanism of retinoic acid resistance in acute promyelocytic leukemia cells through a defective pathway in telomerase regulation

Leukemia ◽

10.1038/sj.leu.2402470 ◽

2002 ◽

Vol 16 (5) ◽

pp. 826-832 ◽

Cited By ~ 17

Author(s):

F Pendino ◽

T Sahraoui ◽

M Lanotte ◽

E Ségal-Bendirdjian

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Acid Resistance ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Retinoic Acid Resistance ◽

Telomerase Regulation

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Constitutive Degradation of PML/RAR Through the Proteasome Pathway Mediates Retinoic Acid Resistance

Blood ◽

10.1182/blood.v93.5.1477 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1477-1481 ◽

Cited By ~ 46

Author(s):

Mirco Fanelli ◽

Saverio Minucci ◽

Vania Gelmetti ◽

Clara Nervi ◽

Carlo Gambacorti-Passerini ◽

...

Keyword(s):

Retinoic Acid ◽

Proteasome Inhibitors ◽

Acid Resistance ◽

Parental Cell ◽

Parental Cell Line ◽

In Vitro Degradation ◽

Nb4 Cells ◽

Proteasome Pathway ◽

Retinoic Acid Resistance

Abstract PML/RAR is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RAR, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RAR protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RAR is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RAR protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RAR, but not RAR, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RAR protein may lead to RA resistance and that PML/RAR expression is crucial to convey RA sensitivity to APL cells.

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