A Refined Single Cell Landscape of Haematopoiesis in the Mouse Foetal Liver.

During prenatal life, the foetal liver is colonised by several waves of haematopoietic progenitors to act as the main haematopoietic organ. Single cell (sc) RNA-seq has been used to identify foetal liver cell types via their transcriptomic signature and to compare gene expression patterns as haematopoietic development proceeds. To obtain a refined single cell landscape of haematopoiesis in the foetal liver, we have generated a scRNA-seq dataset from a whole mouse E12.5 liver that includes a larger number of cells than prior datasets at this stage and was obtained without cell type preselection to include all liver cell populations. We combined mining of this dataset with that of previously published datasets at other developmental stages to follow transcriptional dynamics as well as the cell cycle state of developing haematopoietic lineages. Our findings corroborate several prior reports on the timing of liver colonisation by haematopoietic progenitors and the emergence of differentiated lineages and provide further molecular characterisation of each cell population. Extending these findings, we demonstrate the existence of a foetal intermediate haemoglobin profile in the mouse, similar to that previously identified in humans, and a previously unidentified population of primitive erythroid cells in the foetal liver.

Co-targeting leukemia-initiating cells and leukemia bulk leads to disease eradication.

According to a hierarchical model, targeting leukemia-initiating cells (LICs) was speculated to achieve complete remission (CR) or cure. Nonetheless, increasing evidence emphasized the plasticity of differentiated blasts undergoing interconversion into LICs. We exploited murine models of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia driven by the promyelocytic leukemia/retinoic acid receptor (PML-RARα) oncofusion protein, which recruits histone deacetylase (HDAC)-containing complexes. We studied APLs with different LIC frequencies and investigated the effect of two HDAC inhibitors: valproic acid (VPA), with relative selectivity towards class I HDAC enzymes and vorinostat/suberoylanilide hydroxamic acid (SAHA) (pan-HDAC inhibitor) in combination with all-trans retinoic acid (ATRA), on the bulk APL cells and APL LICs. Indeed, we found that while VPA differentiates the bulk APL cells, SAHA selectively targets LICs. ATRA + VPA + SAHA combination efficiently induced CR in an APL model with lower LIC frequency. Substituting ATRA with synthetic retinoids as etretinate which promotes APL differentiation without downregulating PML/RARα compromised the therapeutic benefit of ATRA + VPA + SAHA regimen. Altogether, our study emphasizes the therapeutic power of co-targeting the plasticity and heterogeneity of cancer -herein demonstrated by tackling LICs and bulk leukemic blasts - to achieve and maintain CR.

Pharmacological inhibition of LSD1 triggers myeloid differentiation by targeting GSE1 oncogenic functions in AML.

The histone demethylase LSD1 is over-expressed in hematological tumors and has emerged as a promising target for anticancer treatment, so that several LSD1 inhibitors are under development and testing, in preclinical and clinical settings. However, the complete understanding of their complex mechanism of action is still unreached. Here, we unraveled a novel mode of action of the LSD1 inhibitors MC2580 and DDP-38003, showing that they can induce differentiation of AML cells through the downregulation of the chromatin protein GSE1. Analysis of the phenotypic effects of GSE1 depletion in NB4 cells showed a strong decrease of cell viability in vitro and of tumor growth in vivo. Mechanistically, we found that a set of genes associated with immune response and cytokine-signaling pathways are upregulated by LSD1 inhibitors through GSE1-protein reduction and that LSD1 and GSE1 colocalize at promoters of a subset of these genes at the basal state, enforcing their transcriptional silencing. Moreover, we show that LSD1 inhibitors lead to the reduced binding of GSE1 to these promoters, activating transcriptional programs that trigger myeloid differentiation. Our study offers new insights into GSE1 as a novel therapeutic target for AML.

LSD1-directed therapy affects glioblastoma tumorigenicity by deregulating the protective ATF4-dependent integrated stress response.

Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, poor blood-brain barrier penetration, and resistance to therapy highlight the need for new targets and clinical treatments. A step toward clinical translation includes the eradication of GBM tumor-initiating cells (TICs), responsible for GBM heterogeneity and relapse. By using patient-derived TICs and xenograft orthotopic models, we demonstrated that the selective lysine-specific histone demethylase 1 inhibitor DDP_38003 (LSD1i) is able to penetrate the brain parenchyma in vivo in preclinical models, is well tolerated, and exerts antitumor activity in molecularly different GBMs. LSD1 genetic targeting further strengthens the role of LSD1 in GBM TIC maintenance. GBM TIC plasticity supports their adaptation and survival under a plethora of environmental stresses, including nutrient deficiency and proteostasis perturbation. By mimicking these stresses in vitro, we found that LSD1 inhibition hampers the induction of the activating transcription factor 4 (ATF4), the master regulator of the integrated stress response (ISR). The resulting aberrant ISR sensitizes GBM TICs to stress-induced cell death, hampering tumor aggressiveness. Functionally, LSD1i interferes with LSD1 scaffolding function and prevents its interaction with CREBBP, a critical ATF4 activator. By disrupting the interaction between CREBBP and LSD1-ATF4 axis, LSD1 inhibition prevents GBM TICs from overcoming stress and sustaining GBM progression. The effectiveness of the LSD1 inhibition in preclinical models shown here places a strong rationale toward its clinical translation for GBM treatment.

KIT Is Required for Fetal Liver Hematopoiesis.

In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial-to-hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed in hematopoietic progenitors that arise via endoHT from yolk sac hemogenic endothelium to generate early, transient hematopoietic waves. However, it remains unclear whether KIT has essential roles in early hematopoiesis. Here, we have combined single-cell expression studies with the analysis of knockout mice to show that KIT is dispensable for yolk sac endoHT but required for transient definitive hematopoiesis in the fetal liver.

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.

Tuning mTORC1 activity dictates the response of acute myeloid leukemia to LSD1 inhibition.

Lysine specific demethylase-1 (LSD1) has been shown to be critical in acute myeloid leukemia (AML) pathogenesis and this has led to the development of LSD1 inhibitors (LSD1i) which are currently tested in clinical trials. Nonetheless, preclinical studies reported that AML cells frequently exhibit intrinsic resistance to LSD1 inhibition, and the molecular basis for this phenomenon is largely unknown. We explored the potential involvement of mammalian target of rapamycin (mTOR) in mediating the resistance of leukemic cells to LSD1i. Strikingly, unlike sensitive leukemias, mTOR complex 1 (mTORC1) signaling was robustly triggered in resistant leukemias following LSD1 inhibition. Transcriptomic, chromatin immunoprecipitation and functional studies revealed that insulin receptor substrate 1(IRS1)/extracellular-signal regulated kinases (ERK1/2) signaling critically controls LSD1i induced mTORC1 activation. Notably, inhibiting mTOR unlocked the resistance of AML cell lines and primary patient-derived blasts to LSD1i both in vitro and in vivo In conclusion, mTOR activation might act as a novel pro-survival mechanism of intrinsic as well as acquired resistance to LSD1i, and combination regimens co-targeting LSD1/mTOR could represent a rational approach in AML therapy.

Self-renewal of tumor cells: epigenetic determinants of the cancer stem cell phenotype.

Among the functional subpopulations that coexist within the tumor, 'cancer stem cells' are characterized by increased self-renewal and the ability to derive all of the other subpopulations of tumor cells ('bulk'). The functional heterogeneity among cancer stem cells and bulk cells must reflect distinct cellular epigenetic landscapes, but - due to the difficulty to isolate bona fide cancer stem cells with a high degree of purity - those different epigenetic landscapes, and the molecular mechanisms underlying them, remain largely unknown. Cues of intratumor phenotypic plasticity complicate the interpretation of the cancer stem cell phenotype: we contend that, however, the concept of cancer stem cell has crucial therapeutic implication, and remains a key target for the exploration of the cancer epigenome.

Inhibition of histone deacetylases in cancer therapy: lessons from leukaemia.

Histone deacetylases (HDACs) are a key component of the epigenetic machinery regulating gene expression, and behave as oncogenes in several cancer types, spurring the development of HDAC inhibitors (HDACi) as anticancer drugs. This review discusses new results regarding the role of HDACs in cancer and the effect of HDACi on tumour cells, focusing on haematological malignancies, particularly acute myeloid leukaemia. Histone deacetylases may have opposite roles at different stages of tumour progression and in different tumour cell sub-populations (cancer stem cells), highlighting the importance of investigating these aspects for further improving the clinical use of HDACi in treating cancer.