Laura Barreyro

Stem and progenitor cells in myelodysplastic syndromes show aberrant stage-specific expansion and harbor genetic and epigenetic alterations

Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage(-)/CD34(+)/CD38(-)/CD90(+)) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.

Mutational Cooperativity Linked to Combinatorial Epigenetic Gain of Function in Acute Myeloid Leukemia

Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.

Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS

Cellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (-7/7q-) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the -7/7q- anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10(-7)). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in -7/7q- AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.

Eltrombopag inhibits the proliferation of leukemia cells via reduction of intracellular iron and induction of differentiation

Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EPs antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Satb1 regulates the self-renewal of hematopoietic stem cells by promoting quiescence and repressing differentiation commitment

How hematopoietic stem cells (HSCs) coordinate the regulation of opposing cellular mechanisms such as self-renewal and differentiation commitment remains unclear. Here we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of HSC fate. HSCs lacking Satb1 had defective self-renewal, were less quiescent and showed accelerated lineage commitment, which resulted in progressive depletion of functional HSCs. The enhanced commitment was caused by less symmetric self-renewal and more symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed sets of genes encoding molecules involved in HSC activation and cellular polarity, including Numb and Myc, which encode two key factors for the specification of stem-cell fate. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.

IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34(+) cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML.

Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia

Modest transcriptional changes caused by genetic or epigenetic mechanisms are frequent in human cancer. Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces acute myeloid leukemia (AML) in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet, moderate PU.1 inhibition is common in AML patients. We assessed functional consequences of modest reductions in PU.1 expression on leukemia development in mice harboring DNA lesions resembling those acquired during human stem cell aging. Heterozygous deletion of an enhancer of PU.1, which resulted in a 35% reduction of PU.1 expression, was sufficient to induce myeloid-biased preleukemic stem cells and their subsequent transformation to AML in a DNA mismatch repair–deficient background. AML progression was mediated by inhibition of expression of a PU.1-cooperating transcription factor, Irf8. Notably, we found marked molecular similarities between the disease in these mice and human myelodysplastic syndrome and AML. This study demonstrates that minimal reduction of a key lineage-specific transcription factor, which commonly occurs in human disease, is sufficient to initiate cancer development, and it provides mechanistic insight into the formation and progression of preleukemic stem cells in AML.

Concise Review: Preleukemic Stem Cells: Molecular Biology and Clinical Implications of the Precursors to Leukemia Stem Cells

Recent experimental evidence has shown that acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells and committed progenitors. The series of transforming events initially gives rise to preleukemic stem cells (pre‐LSC), preceding the formation of fully transformed leukemia stem cells (LSC). Despite the established use of poly‐chemotherapy, relapse continues to be the most common cause of death in AML and MDS. The therapeutic elimination of all LSC, as well as pre‐LSC, which provide a silent reservoir for the re‐formation of LSC, will be essential for achieving lasting cures. Conventional sequencing and next‐generation genome sequencing have allowed us to describe many of the recurrent mutations in the bulk cell populations in AML and MDS, and recent work has also focused on identifying the initial molecular changes contributing to leukemogenesis. Here we review recent and ongoing advances in understanding the roles of pre‐LSC, and the aberrations that lead to pre‐LSC formation and subsequent LSC transformation.

HSC commitment–associated epigenetic signature is prognostic in acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitment–associated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitment–associated methylome that is independently prognostic of poorer overall survival in AML.