Marc H.G.P. Raaijmakers

Breast Cancer Resistance Protein in Drug Resistance of Primitive CD34+38− Cells in Acute Myeloid Leukemia

Purpose: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38− stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. Experimental Design: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38− cells. Results: BCRP was preferentially expressed in leukemic CD34+38− cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38− cells. Conclusions: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.

Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction.

OBJECTIVEnQuantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations.nnnMATERIALS AND METHODSnReal-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques.nnnRESULTSnValidation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells.nnnCONCLUSIONSnReal-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.

Niche contributions to oncogenesis: emerging concepts and implications for the hematopoietic system

The field of hematopoietic oncology has traditionally focused on the study of hematopoietic cell autonomous genetic events in an effort to understand malignant transformation and develop therapeutics. Although highly rewarding in both aspects, this cell autonomous approach has failed to fully satisfy our need to understand tumor cell behavior and related clinical observations. In recent years, it has been increasingly recognized that the tumor microenvironment plays a pivotal role in cancer initiation and progression. This review will discuss recent experimental evidence in support of this view derived from investigations in both epithelial and hematopoietic systems. Based on this, conceptual views and therapeutic implications will be provided on the emerging role of the bone marrow microenvironment in leukemogenesis.

ABCB1 Modulation Does Not Circumvent Drug Extrusion from Primitive Leukemic Progenitor Cells and May Preferentially Target Residual Normal Cells in Acute Myelogenous Leukemia

Purpose: Acute myelogenous leukemia (AML) is a disease originating from normal hematopoietic CD34+CD38− progenitor cells. Modulation of the multidrug ATP-binding cassette transporter ABCB1 has not resulted in improved outcome in AML, raising the question whether leukemic CD34+CD38− cells are targeted by this strategy. Experimental Design: ABCB1-mediated transport in leukemic CD34+CD38− cells compared with their normal counterparts was assessed by quantitating the effect of specific ABCB1 modulators (verapamil and PSC-833) on mitoxantrone retention [defined as efflux index (EI), intracellular mitoxantrone fluorescence intensity in the presence/absence of inhibitor]. Results: ABCB1 was the major drug transporter in CD34+CD38− cells in normal bone marrow (n = 16), as shown by the abrogation of mitoxantrone extrusion by ABCB1 modulators (EI, 1.99 ± 0.08). Surprisingly, ABCB1-mediated drug extrusion was invariably reduced in CD34+CD38− cells in AML (n = 15; EI, 1.21 ± 0.05; P < 0.001), which resulted in increased intracellular mitoxantrone retention in these cells (mitoxantrone fluorescence intensity, 4.54 ± 0.46 versus 3.08 ± 0.23; P = 0.004). Active drug extrusion from these cells occurred in the presence of ABCB1 modulators in the majority of samples, pointing in the direction of redundant drug extrusion mechanisms. Residual normal CD34+CD38− cells could be identified by their conserved ABCB1-mediated extrusion capacity. Conclusion: ABCB1-mediated drug extrusion is reduced in leukemic CD34+CD38− progenitor cells compared with their residual normal counterparts. Redundant drug transport mechanisms confer mitoxantrone transport from leukemic progenitors. These data argue that ABCB1 modulation is not an effective strategy to circumvent drug extrusion from primitive leukemic progenitor cells and may preferentially target residual normal progenitors in AML.

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Disease progression in myelodysplastic syndromes: do mesenchymal cells pave the way?

Early events driving the initiation and evolution of neoplasms remain poorly defined but involvement of an instructive or permissive niche has been implicated. In this issue of Cell Stem Cell, Medyouf et al. (2014) provide insights into the role of the niche in myelodysplastic syndromes, the principle preleukemic disorder of the hematopoietic system.

Epigenetically Aberrant Stroma in MDS Propagates Disease via Wnt/β-Catenin Activation

The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow-derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine-treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98-HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846-57. ©2017 AACR.

Regulating traffic in the hematopoietic stem cell niche

Cells in multicellular organisms do not live in a vacuum. Coordinated responses of individual cells to challenges at the level of the organism are instructed by integrated prompts from their environment. This environmental signaling determines the cells’ properties and behavior to a considerable

Persistent Hematologic Dysfunction after Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE: Incidence, Course, and Predicting Factors in Patients with Gastroenteropancreatic Neuroendocrine Tumors

Peptide receptor radionuclide therapy (PRRT) may induce long-term toxicity to the bone marrow (BM). The aim of this study was to analyze persistent hematologic dysfunction (PHD) after PRRT with 177Lu-DOTATATE in patients with gastroenteropancreatic neuroendocrine tumors (GEP NETs). Methods: The incidence and course of PHD were analyzed in 274 GEP NET patients from a group of 367 patients with somatostatin receptor–positive tumors. PHD was defined as diagnosis of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), myeloproliferative neoplasm (MPN), MDS/MPN, or otherwise unexplained cytopenia (for >6 mo). Using data from The Netherlands Cancer Registry, the expected number of hematopoietic neoplasms (MDS, AML, MPN, and MDS/MPN) was calculated and adjusted for sex, age, and follow-up period. The following risk factors were assessed: sex, age over 70 y, bone metastasis, prior chemotherapy, prior external-beam radiotherapy, uptake on the [111In-DTPA0]octreotide scan, tumor load, grade 3–4 hematologic toxicity during treatment, estimated absorbed BM dose, elevated plasma chromogranin A level, baseline blood counts, and renal function. Results: Eleven (4%) of the 274 patients had PHD after treatment with 177Lu-DOTATATE: 8 patients (2.9%) developed a hematopoietic neoplasm (4 MDS, 1 AML, 1 MPN, and 2 MDS/MPN) and 3 patients (1.1%) developed BM failure characterized by cytopenia and BM aplasia. The median latency period at diagnosis (or first suspicion of a PHD) was 41 mo (range, 15–84 mo). The expected number of hematopoietic neoplasms based on The Netherlands Cancer Registry data was 3.0, resulting in a relative risk of 2.7 (95% confidence interval, 0.7–10.0). No risk factors for PHD could be identified for the GEP NET patients, not even bone metastasis or estimated BM dose. Seven patients with PHD developed anemia in combination with a rise in mean corpuscular volume. Conclusion: The prevalence of PHD after PRRT with 177Lu-DOTATATE was 4% in our patient population. The median time at which PHD developed was 41 mo after the first PRRT cycle. The relative risk for developing a hematopoietic neoplasm was 2.7. No risk factors were found for the development of PHD in GEP NET patients.