Noncanonical EZH2 drives retinoic acid resistance of variant acute promyelocytic leukemias.

Cancer cell heterogeneity is a major driver of therapy resistance. To characterize resistant cells and their vulnerabilities, we studied the PLZF-RARA variant of acute promyelocytic leukemia, resistant to retinoic acid (RA), using single-cell multiomics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells. We identified a subset of cells resistant to RA with proliferation, DNA replication, and repair signatures that depend on a fine-tuned E2F transcriptional network targeting the epigenetic regulator enhancer of zeste homolog 2 (EZH2). Epigenomic and functional analyses validated the driver role of EZH2 in RA resistance. Targeting pan-EZH2 activities (canonical/noncanonical) was necessary to eliminate leukemia relapse-initiating cells, which underlies a dependency of resistant cells on an EZH2 noncanonical 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. Beyond RA resistance and acute promyelocytic leukemia context, our study also demonstrates the power of single-cell multiomics to identify, characterize, and clear therapy-resistant cells.

Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells.

Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.

Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche.

Hematopoietic stem cells (HSCs) exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation, with age. The HSC niche, which is essential for the maintenance of HSCs, also undergoes marked changes with aging. However, it has been technically challenging to directly evaluate the contribution of niche aging to age-associated HSC alterations without niche-damaging myeloablation in HSC transplantation assays. We herein transplanted an excess of aged HSCs into young mice without preconditioning. Although aged HSCs successfully engrafted in the intact young bone marrow niche, they poorly regenerated downstream progenitors and exhibited persistent myeloid-biased differentiation, resulting in no significant functional rejuvenation. Transcriptome and methylome analyses revealed that the young niche largely restored the transcriptional profile of aged HSCs, but not their DNA methylation profiles. Therefore, the restoration of the young niche is insufficient for rejuvenating HSC functions, highlighting a key role for age-associated cell-intrinsic defects in HSC aging.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Bmi1 restricts the adipogenic differentiation of bone marrow stromal cells to maintain the integrity of the hematopoietic stem cell niche.

The polycomb group protein Bmi1 maintains hematopoietic stem cell (HSC) functions. We previously reported that Bmi1-deficient mice exhibited progressive fatty changes in bone marrow (BM). A large portion of HSCs reside in the perivascular niche created partly by endothelial cells and leptin receptor+ (LepR+) BM stromal cells. To clarify how Bmi1 regulates the HSC niche, we specifically deleted Bmi1 in LepR+ cells in mice. The Bmi1 deletion promoted the adipogenic differentiation of LepR+ stromal cells and caused progressive fatty changes in the BM of limb bones with age, resulting in reductions in the numbers of HSCs and progenitors in BM and enhanced extramedullary hematopoiesis. This adipogenic change was also evident during BM regeneration after irradiation. Several adipogenic regulator genes appeared to be regulated by Bmi1. Our results indicate that Bmi1 keeps the adipogenic differentiation program repressed in BM stromal cells to maintain the integrity of the HSC niche.

Impact of the SCF signaling pathway on leukemia stem cell-mediated ATL initiation and progression in an HBZ transgenic mouse model.

Adult T-cell leukemia (ATL) is a malignant disease caused by human T-lymphotropic virus type 1. In aggressive ATL, the response to chemotherapy is extremely poor. We hypothesized that this poor response is due to the existence of chemotherapy-resistant cells, such as leukemic stem cells. Previously, we successfully identified an ATL stem cell (ATLSC) candidate as the c-kit+/CD38-/CD71- cells in an ATL mouse model using Tax transgenic mice. Here, with a new ATL mouse model using HBZ-transgenic mice, we further discovered that the functional ATLSC candidate, which commonly expresses c-kit, is drug-resistant and has the ability to initiate tumors and reconstitute lymphomatous cells. We characterized the ATLSCs as c-kit+/CD4-/CD8- cells and found that they have a similar gene expression profile as T cell progenitors. Additionally, we found that AP-1 gene family members, including Junb, Jund, and Fosb, were up-regulated in the ATLSC fraction. The results of an in vitro assay showed that ATLSCs cultured with cytokines known to promote stem cell expansion, such as stem cell factor (SCF), showed highly proliferative activity and maintained their stem cell fraction. Inhibition of c-kit-SCF signaling with the neutralizing antibody ACK2 affected ATLSC self-renewal and proliferation. Experiments in Sl/Sld mice, which have a mutation in the membrane-bound c-kit ligand, found that ATL development was completely blocked in these mice. These results clearly suggest that the c-kit-SCF signal plays a key role in ATLSC self-renewal and in ATL initiation and disease progression.