Michaël H. Meel

Normal hematopoietic stem cells within the AML bone marrow have a distinct and higher ALDH activity level than co-existing leukemic stem cells.

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDHbright). The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.

Preclinical evaluation of convection-enhanced delivery of liposomal doxorubicin to treat pediatric diffuse intrinsic pontine glioma and thalamic high-grade glioma

OBJECTIVE Pediatric high-grade gliomas (pHGGs) including diffuse intrinsic pontine gliomas (DIPGs) are primary brain tumors with high mortality and morbidity. Because of their poor brain penetrance, systemic chemotherapy regimens have failed to deliver satisfactory results; however, convection-enhanced delivery (CED) may be an alternative mode of drug delivery. Anthracyclines are potent chemotherapeutics that have been successfully delivered via CED in preclinical supratentorial glioma models. This study aims to assess the potency of anthracyclines against DIPG and pHGG cell lines in vitro and to evaluate the efficacy of CED with anthracyclines in orthotopic pontine and thalamic tumor models. METHODS The sensitivity of primary pHGG cell lines to a range of anthracyclines was tested in vitro. Preclinical CED of free doxorubicin and pegylated liposomal doxorubicin (PLD) to the brainstem and thalamus of naïve nude mice was performed. The maximum tolerated dose (MTD) was determined based on the observation of clinical symptoms, and brains were analyzed after H & E staining. Efficacy of the MTD was tested in adult glioma E98-FM-DIPG and E98-FM-thalamus models and in the HSJD-DIPG-007-Fluc primary DIPG model. RESULTS Both pHGG and DIPG cells were sensitive to anthracyclines in vitro. Doxorubicin was selected for further preclinical evaluation. Convection-enhanced delivery of the MTD of free doxorubicin and PLD in the pons was 0.02 mg/ml, and the dose tolerated in the thalamus was 10 times higher (0.2 mg/ml). Free doxorubicin or PLD via CED was ineffective against E98-FM-DIPG or HSJD-DIPG-007-Fluc in the brainstem; however, when applied in the thalamus, 0.2 mg/ml of PLD slowed down tumor growth and increased survival in a subset of animals with small tumors. CONCLUSIONS Local delivery of doxorubicin to the brainstem causes severe toxicity, even at doxorubicin concentrations that are safe in the thalamus. As a consequence, the authors could not establish a therapeutic window for treating orthotopic brainstem tumors in mice. For tumors in the thalamus, therapeutic concentrations to slow down tumor growth could be reached. These data suggest that anatomical location determines the severity of toxicity after local delivery of therapeutic agents and that caution should be used when translating data from supratentorial CED studies to treat infratentorial tumors.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum‐free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method. HighlightsDIPG cells can be cultured as either adherent monolayers or neurospheres.Culture conditions induce major changes in pathway activation of DIPG cells.Culture conditions induce major changes in gene expression of DIPG cells.These induced changes influence the response of DIPG cells to targeted therapies.

An efficient method for the transduction of primary pediatric glioma neurospheres

Graphical abstract

Signaling pathways and mesenchymal transition in pediatric high-grade glioma

Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal types of cancer in children. In recent years, it has become evident that these tumors are driven by epigenetic events, mainly mutations involving genes encoding Histone 3, setting them apart from their adult counterparts. These tumors are exceptionally resistant to chemotherapy and respond only temporarily to radiotherapy. Moreover, their delicate location and diffuse growth pattern make complete surgical resection impossible. In many other forms of cancer, chemo- and radioresistance, in combination with a diffuse, invasive phenotype, are associated with a transcriptional program termed the epithelial-to-mesenchymal transition (EMT). Activation of this program allows cancer cells to survive individually, invade surrounding tissues and metastasize. It also enables them to survive exposure to cytotoxic therapy, including chemotherapeutic drugs and radiation. We here suggest that EMT plays an important, yet poorly understood role in the biology and therapy resistance of pHGG and DIPG. This review summarizes the current knowledge on the major signal transduction pathways and transcription factors involved in the epithelial-to-mesenchymal transition in cancer in general and in pediatric HGG and DIPG in particular. Despite the fact that the mesenchymal transition has not yet been specifically studied in pHGG and DIPG, activation of pathways and high levels of transcription factors involved in EMT have been described. We conclude that the mesenchymal transition is likely to be an important element of the biology of pHGG and DIPG and warrants further investigation for the development of novel therapeutics.

ATRT-05. PRECLINICAL EFFICACY OF RADIATION-FREE TREATMENT OF ATYPICAL TERATOID/RHABDOID TUMORS BY COMBINED MEK AND MELK INHIBITION

AbstractAtypical teratoid/rhabdoid tumors (AT/RT) are highly malignant tumors that occur both in- and outside the central nervous system, predominantly in young children. Although therapeutic interventions, combining surgery with intensive chemotherapy and conformal radiotherapy, have shown curative potential, the long-term consequences of these treatment modalities in young children are often severe. Therefore, there is a dire need for novel, targeted therapeutic strategies that potentially reduce or eliminate chemo- and radiotherapy, especially for children under three years of age. Genetically, AT/RT is characterized by a biallelic inactivation of SMARCB1 or SMARCA4, causing aberrant chromatin remodeling and thereby expression of a variety of oncogenes. Recently, the importance of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of AT/RT has been demonstrated, with preclinical efficacy of MEK inhibition in AT/RT cells. Preclinical patient-derived AT/RT models are largely lacking. We developed a patient-derived primary cell line and xenograft model, VUMC-AT/RT-01, from a surgical specimen of an AT/RT patient that had not received prior therapy. Immunohistochemistry was performed on xenograft tumors, revealing typical loss of SMARCB1 expression as well as heterogeneous expression of glial, epithelial, mesenchymal and neuronal differentiation markers. In silico analysis of gene expression revealed strong upregulation of MELK in AT/RT tumors compared to normal brain tissues. These high levels of MELK were subsequently confirmed in our xenograft model. Inhibition of MELK with the small molecule OTSSP167 effectively reduced proliferation and induced cell death in primary AT/RT neurospheres. Combined treatment of primary AT/RT cells with OTSSP167 and the MEK inhibitor Trametinib showed strongly synergistic cytotoxicity at low nanomolar concentrations. Treatment of mice carrying VUMC-AT/RT-01-Fluc xenografts with OTSSP167 and Trametinib confirmed this synergy in vivo. We conclude that combined MEK and MELK inhibition represents a promising targeted therapy strategy for AT/RT that might ultimately reduce or eliminate the need for radiation and intensive chemotherapy.

Abstract LB-45: High aldehyde dehydrogenase activity is a marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia: novel therapeutic implications.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Only a minority of cells, the leukemic stem cells (LSC), within AML are responsible for tumor growth and maintenance. Many patients experience relapse after therapy which originates from outgrowth of therapy resistant LSC. Therefore, eradication of LSC is necessary to cure AML. Both the normal hematopoietic stem cells (HSC) and LSC co-exist in the bone marrow (BM) of AML patients and success of anti-LSC strategies relies on specific elimination of LSC while sparing HSC. LSC are contained within the CD34+CD38-, the side population (SP) and the high aldehyde dehydrogenase (ALDH) activity compartments. ALDH is a detoxifying enzyme responsible for oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as cyclophosphamide. It has been shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blasts. In view of applicability of LSC specific therapies the detoxification by ALDH is clinically very important. A difference in ALDH activity between HSC and LSC might be used to preferentially kill LSC while sparing HSC. To establish ALDH activity differences between HSC and LSC it should be possible to discriminate between them. We have shown that LSC can be identified and discriminated from HSC using stem cell-associated cell surface markers, such as CLL-1, lineage markers (CD7, CD19, CD56) and recently CD34/CD45 expression and cell size characteristics (Terwijn, Blood 111: 487, 2008). This offers the opportunity to identify co-existing LSC and HSC in the AML BM. We now show that, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that HSC that co-exist with LSC in BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. We have analyzed ALDH activity in HSC and LSC, both present in the BM from 18 AML patients. In nine BM AML samples, defined as CD34negative (<1%CD34+ blasts), the CD34+ compartment contained only normal CD34+CD38− HSC. The ALDH activity in these CD34+ HSC, is a factor 4,4 (range 1,7–18,9) higher than in LSC. In nine BM AML samples, defined as CD34positive AML, the CD34+CD38- HSC have a 7,7 fold (range 1,73–29,2 fold) higher ALDH activity as compared to putative LSC. In both CD34-positive and CD34-negative AML, we confirmed the identity of HSC and LSC by screening for molecular aberrancies present in AML blasts. The level of the ALDH activity of HSC within the AML BM is similar to that of HSC in NBM of healthy donors. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype) at diagnosis. Consequently, AML patients with high ALDH activity in HSC might benefit from treatment with agents that will be converted by ALDH enzymes, such as cyclophosphamide, whereby the difference between the activity in LSC and HSC will define the therapeutic window. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2011-LB-45