Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression.

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38- cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9-218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36-99%) and 71% (95% CI: 58-81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker.

Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters.

Although chemotherapy induces complete remission in the majority of acute myeloid leukemia (AML) patients, many face a relapse. This relapse is caused by survival of chemotherapy-resistant leukemia (stem) cells (measurable residual disease; MRD). Here, we demonstrate that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing histone 3 lysine 27 (H3K27) and H3K4 tri-methylation. Within a doxorubicin-sensitive leukemia cell population, we identified a subpopulation of reversible anthracycline-tolerant cells (ATCs) with leukemic stem cell (LSC) features lacking doxorubicin-induced H3K27me3 or H3K4me3 upregulation. These ATCs have a distinct transcriptional landscape than the leukemia bulk and could be eradicated by KDM6 inhibition. In primary AML, reprogramming the transcriptional state by targeting KDM6 reduced MRD load and survival of LSCs residing within MRD, and enhanced chemotherapy response in vivo. Our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of transcriptional reprogramming by epigenetic-based therapeutics to target chemotherapy-resistant AML cells.

The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells.

Acute myeloid leukemia (AML) is a disease characterized by transcriptional dysregulation that results in a block in differentiation and aberrant self-renewal. Inhibitors directed to epigenetic modifiers, aiming at transcriptional reprogramming of AML cells, are currently in clinical trials for AML patients. Several of these inhibitors target bromodomain and extraterminal domain (BET) proteins, cyclic AMP response binding protein-binding protein (CBP), and the E1A-interacting protein of 300 kDa (p300), affecting histone acetylation. Unfortunately, single epigenetic inhibitors showed limited efficacy due to appearance of resistance and lack of effective eradication of leukemic stem cells. Here, we describe the efficacy of 2 novel, orally available inhibitors targeting both the BET and CBP/p300 proteins, NEO1132 and NEO2734, in primary AML. NEO2734 and NEO1132 efficiently reduced the viability of AML cell lines and primary AML cells by inducing apoptosis. Importantly, both NEO drugs eliminated leukemic stem/progenitor cells from AML patient samples, and NEO2734 increased the effectiveness of combination chemotherapy treatment in an in vivo AML patient-derived mouse model. Thus, dual inhibition of BET and CBP/p300 using NEO2734 is a promising therapeutic strategy for AML patients, making it a focus for clinical translation.

IGFBP7 activates retinoid acid-induced responses in acute myeloid leukemia stem and progenitor cells.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) in combination with low doses of arsenic trioxide or chemotherapy leads to exceptionally high cure rates (>90%). ATRA forces APL cells into differentiation and cell death. Unfortunately, ATRA-based therapy has not been effective among any other acute myeloid leukemia (AML) subtype, and long-term survival rates remain unacceptably low; only 30% of AML patients survive 5 years after diagnosis. Here, we identified insulin-like growth factor binding protein 7 (IGFBP7) as part of ATRA-induced responses in APL cells. Most importantly, we observed that addition of recombinant human IGFBP7 (rhIGFBP7) increased ATRA-driven responses in a subset of non-APL AML samples: those with high RARA expression. In nonpromyelocytic AML, rhIGFBP7 treatment induced a transcriptional program that sensitized AML cells for ATRA-induced differentiation, cell death, and inhibition of leukemic stem/progenitor cell survival. Furthermore, the engraftment of primary AML in mice was significantly reduced following treatment with the combination of rhIGFBP7 and ATRA. Mechanistically, we showed that the synergism of ATRA and rhIGFBP7 is due, at least in part, to reduction of the transcription factor GFI1. Together, these results suggest a potential clinical utility of IGFBP7 and ATRA combination treatment to eliminate primary AML (leukemic stem/progenitor) cells and reduce relapse in AML patients.

Uncoupling DNA damage from chromatin damage to detoxify doxorubicin.

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

IGFBP7 Induces Differentiation and Loss of Survival of Human Acute Myeloid Leukemia Stem Cells without Affecting Normal Hematopoiesis.

Leukemic stem cells (LSCs) are thought to be the major cause of the recurrence of acute myeloid leukemia (AML) due to their potential for self-renewal. To identify therapeutic strategies targeting LSCs, while sparing healthy hematopoietic stem cells (HSCs), we performed gene expression profiling of LSCs, HSCs, and leukemic progenitors all residing within the same AML bone marrow and identified insulin-like growth factor-binding protein 7 (IGFBP7) as differentially expressed. Low IGFBP7 is a feature of LSCs and is associated with reduced chemotherapy sensitivity. Enhancing IGFBP7 by overexpression or addition of recombinant human IGFBP7 (rhIGFBP7) resulted in differentiation, inhibition of cell survival, and increased chemotherapy sensitivity of primary AML cells. Adding rhIGFBP7 reduced leukemic stem and/or progenitor survival and reversed a stem-like gene signature, but it had no influence on normal hematopoietic stem cell survival. Our data suggest a potential clinical utility of the addition of rhIGFBP7 to current chemotherapy regimens to decrease AML relapse rates.

Primary acute myeloid leukemia cells with overexpression of EVI-1 are sensitive to all-trans retinoic acid.

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION: Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS: For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE: Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Attenuation of microRNA-126 expression that drives CD34+38- stem/progenitor cells in acute myeloid leukemia leads to tumor eradication.

Despite high remission rates after therapy, 60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC), which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs, we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34(+)CD38(-) hematopoietic stem cells (HSC), CD34(+)CD38(-) LSCs, and CD34(+)CD38(+) LPs, all derived from the same patients' bone marrow (BM) specimens. In this manner, we identified multiple differentially expressed miRNAs, in particular miR-126, which was highly expressed in HSCs and increased in LSCs compared with LPs, consistent with a stem-like cell function. High miR-126 expression in AML was associated with poor survival, higher chance of relapse, and expression of genes present in LSC/HSC signatures. Notably, attenuating miR-126 expression in AML cells reduced in vitro cell growth by inducing apoptosis, but did not affect the survival of normal BM in which it instead enhanced expansion of HSCs. Furthermore, targeting miR-126 in LSCs and LPs reduced their clonogenic capacity and eliminated leukemic cells, again in the absence of similar inhibitory effects on normal BM cells. Our results define miR-126 as a therapeutic focus to specifically eradicate LSCs and improve AML outcome.