Marius Bill

Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Significance In this work we report on the previously uncharacterized clinical and biological role for EGFL7 in acute myeloid leukemia (AML). Patients with increased EGFL7 mRNA expression had lower complete remission rates and shorter overall and event-free survival, demonstrating the clinical relevance of EGFL7 expression in cytogenetically normal AML. Our results show that AML blasts are able to synthesize and secrete EGFL7 protein, promoting autocrine blast cell growth. Inhibition of EGFL7 results in decreased proliferation and induces apoptosis of AML cells. Taken together, our data provide the rationale for targeting EGFL7 using blocking antibodies as a therapy for patients with AML. Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.

Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable (“minimal”) residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers—also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients’ outcomes.

Genome-wide association study identifies an acute myeloid leukemia susceptibility locus near BICRA

Christopher J. Walker1, Christopher C. Oakes1, Luke K. Genutis1, Brian Giacopelli1, Sandya Liyanarachchi1, Deedra Nicolet1,2, Ann-Kathrin Eisfeld1, Markus Scholz3,4, Pamela Brock1, Jessica Kohlschmidt1,2, Krzysztof Mrózek1, Marius Bill1, Andrew J. Carroll5, Jonathan E. Kolitz6, Bayard L. Powell7, Eunice S. Wang8, Dietger W. Niederwieser9, Richard M. Stone10, John C. Byrd1, Sebastian Schwind9, Albert de la Chapelle#1, and Clara D. Bloomfield#*,1 1The Ohio State University Comprehensive Cancer Center, Columbus OH, USA.

MicroRNA-143 targets ERK5 in granulopoiesis and predicts outcome of patients with acute myeloid leukemia

Hematopoiesis, the formation of blood cells from hematopoietic stem cells (HSC), is a highly regulated process. Since the discovery of microRNAs (miRNAs), several studies have shown their significant role in the regulation of the hematopoietic system. Impaired expression of miRNAs leads to disrupted cellular pathways and in particular causes loss of hematopoietic ability. Here, we report a previously unrecognized function of miR-143 in granulopoiesis. Hematopoietic cells undergoing granulocytic differentiation exhibited increased miR-143 expression. Overexpression or ablation of miR-143 expression resulted in accelerated granulocytic differentiation or block of differentiation, respectively. The absence of miR-143 in mice resulted in a reduced number of mature granulocytes in blood and bone marrow. Additionally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia (AML). Overexpression of miR-143 in AML cells impaired cell growth, partially induced differentiation, and caused apoptosis. Argonaute2-RNA-Immunoprecipitation assay revealed ERK5, a member of the MAPK-family, as a target of miR-143 in myeloid cells. Further, we observed an inverse correlation of miR-143 and ERK5 in primary AML patient samples, and in CD34+ HSPCs undergoing granulocytic differentiation and we confirmed functional relevance of ERK5 in myeloid cells. In conclusion, our data describe miR-143 as a relevant factor in granulocyte differentiation, whose expression may be useful as a prognostic and therapeutic factor in AML therapy.

Prognostic relevance of DNMT3A R882 mutations in AML patients undergoing non-myeloablative conditioning hematopoietic stem cell transplantation

Mutations in the epigenetic modifying gene DNA methyltransferase 3A (DNMT3A) are found in 14–35% of acute myeloid leukemia (AML) patients [1–6]. The majority of DNMT3A mutations cluster at codon R882 in exon 23 [1–6]. Most of the studies that have investigated the prognostic impact of these alterations reported inferior outcomes associated with the presence of DNMT3A mutations—particularly in codon R882 [1–4, 6–9]. However, the negative impact on outcomes may be dependent on the clinical and biological context, such as the presence of concurrent gene mutations (i.e. NPM1 mutations and/or presence of FLT3ITD) and the DNMT3A mutation type (i.e. R882 vs. nonR882) [1–6, 9]. In older AML patients, DNMT3A mutations are more prevalent [7] and have been associated with poor outcomes [1, 6, 8]. DNMT3A mutations are often found to persist during remission (possibly due to a preexisting clonal hematopoiesis) and at AML relapse, and might contribute to chemotherapy resistance [10]. This raises the issue of allogeneic hematopoietic stem cell transplantation (HSCT) as a therapeutic approach specifically for DNMT3A mutated AML. Studies that included HSCT treated patients—most of which focused on younger AML patients—observed no influence of an HSCT on the negative prognostic impact of DNMT3A mutations [3–5, 9, 11]. For example, Gaidzik et al. [5]. described a shorter relapse-free survival in younger DNMT3A R882 mutated AML patients independent of the use of HSCT. Similarly, Ahn et al. [9]. and Xu et al. [11]. reported a negative impact of DNMT3A R882 mutations after HSCT in mainly younger patients with normal karyotype (NK) AML. Since DNMT3A R882 mutations occur more frequently with age [7] and data on the influence of DNMT3A R882 mutations on outcome in older AML patients receiving HSCT are limited, we analyzed the prognostic impact of DNMT3A R882 mutations at diagnosis among mainly older (>60 years) AML patients who underwent nonmyeloablative (NMA)-HSCT. NMA-HSCT relies on an immunological donor leukocyte-mediated GvL effect and, because of the lower treatment-related toxicity, represents a feasible consolidating therapy for older AML patients and/or those with comorbidities [12, 13]. The patients studied here were treated with cytarabinebased chemotherapies followed by conditioning with fludarabine and 2 Gy TBI or 2 Gy TBI only before NMAHSCT (for further details, see Supplementary Information) at the University Hospital Leipzig between February 2003 and December 2012. We retrospectively analyzed the presence of DNMT3A R882 mutations in pre-treatment bone marrow (n= 98) or peripheral blood (n= 10) samples of 108 AML patients (for further details, see Supplementary Information). All patients gave written informed consent in accordance with the declaration of Helsinki. Pre-treatment cytogenetics and the mutation status of NPM1, CEBPA, and the presence of FLT3-ITD were determined as previously Laura K. Schmalbrock and Lynn Bonifacio contributed equally to this work.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC/ABL1 copy numbers was determined and applied to define patients with high or low BAALC/ABL1 copy numbers. High pre-HSCT BAALC/ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC/ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC/ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC/ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT.

Prognostic Impact of the CD34+/CD38‐ Cell Burden in Patients with Acute Myeloid Leukemia receiving Allogeneic Stem Cell Transplantation

In acute myeloid leukemia (AML), leukemia‐initiating cells exist within the CD34+/CD38− cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38− cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft‐versus‐leukemia effects. Percentage of bone marrow CD34+/CD38− cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%–89% of all mononuclear cells). A high CD34+/CD38− cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38− cell burden had shorter relapse‐free and overall survival which may be mediated by residual leukemia‐initiating cells in the CD34+/CD38− cell population, escaping the graft‐versus‐leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38− cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia‐initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.