Preclinical modeling of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological clonal disorders. Here, we have tested the bone marrow (BM) cells from 38 MDS patients covering all risk groups in two immunodeficient mouse models: NSG and NSG-S. Our data show comparable level of engraftment in both models. The level of engraftment was patient specific with no correlation to any specific MDS risk group. Furthermore, the co-injection of mesenchymal stromal cells (MSCs) did not improve the level of engraftment. Finally, we have developed an in vitro two-dimensional co-culture system as an alternative tool to in vivo. Using our in vitro system, we have been able to co-culture CD34+ cells from MDS patient BM on auto- and/or allogeneic MSCs over 4 weeks with a fold expansion of up to 600 times. More importantly, these expanded cells conserved their MDS clonal architecture as well as genomic integrity.

Rationale and efficacy of proteasome inhibitor combined with arsenic trioxide in the treatment of acute promyelocytic leukemia.

Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia-retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL.

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages.

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.

Lithium chloride antileukemic activity in acute promyelocytic leukemia is GSK-3 and MEK/ERK dependent.

We recently identified that the MEK/ERK1/2 pathway synergized with retinoic acid (RA) to restore both transcriptional activity and RA-induced differentiation in RA-resistant acute promyelocytic leukemia (APL) cells. To target the MEK/ERK pathway, we identified glycogen synthase kinase-3ß (GSK-3ß) inhibitors including lithium chloride (LiCl) as activators of this pathway in APL cells. Using NB4 (RA-sensitive) and UF-1 (RA-resistant) APL cell lines, we observed that LiCl as well as synthetic GSK-3ß inhibitors decreased proliferation, induced apoptosis and restored, in RA-resistant cells, the expression of RA target genes and the RA-induced differentiation. Inhibition of the MEK/ERK1/2 pathway abolished these effects. These results were corroborated in primary APL patient cells and translated in vivo using an APL preclinical mouse model in which LiCl given alone was as efficient as RA in increasing survival of leukemic mice compared with untreated mice. When LiCl was combined with RA, we observed a significant survival advantage compared with mice treated by RA alone. In this work, we demonstrate that LiCl, a well-tolerated agent in humans, has antileukemic activity in APL and that it has the potential to restore RA-induced transcriptional activation and differentiation in RA-resistant APL cells in an MEK/ERK-dependent manner.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Self-renewal gene tracking to identify tumour-initiating cells associated with metastatic potential.

Tumour-initiating cells (TICs) are rare cancer cells isolated from tumours of different origins including high-grade tumours that sustain neoplasic progression and development of metastatic disease. They harbour deregulated stem cells pathways and exhibit an unchecked ability to self-renew, a property essential for tumour progression. Among the essential factors maintaining embryonic stem (ES) cells properties, OCT-4 (also known as POU5F1) has been detected in tumours of different origins. Although ectopic expression results in dysplasic growth restricted to epithelial tissues, overexpression expands the proportion of immature cells in teratomas. However, OCT-4-expressing cells have not been purified from spontaneously occurring tumours, thus information concerning their properties is rather scant. Here, using p53-/- mice expressing green fluorescent protein and the puromycin resistance gene under the control of the Oct-4 promoter, we show that OCT-4 is expressed in 5% onwards of the undifferentiated tumour cell populations derived from different organs. OCT-4 expression was low as compared with ES cells, but was associated with a 'stemness' signature and expression of the chemokine receptor CXCR4. These cells displayed cancer stem cell features, including increased self-renewal and differentiation ability in vitro and in vivo. They not only formed allografts containing immature bone regions but also disseminated into different organs, including lung, liver and bone. Experiments based on RNA interference revealed that Oct-4 expression drives both their engraftment and metastasis formation. This work points out the crucial contribution of Oct-4-expressing TICs in the hierarchical organization of the malignant potential, leading to metastasis formation. Consequently, it provides an appropriate model to develop novel therapies aiming to strike down TICs by targeting self-renewal genes, therefore efficient to reduce tumour growth and metastatic disease.

New role for granulocyte colony-stimulating factor-induced extracellular signal-regulated kinase 1/2 in histone modification and retinoic acid receptor α recruitment to gene promoters: relevance to acute promyelocytic leukemia cell differentiation.

The induction of the granulocytic differentiation of leukemic cells by all-trans retinoic acid (RA) has been a major breakthrough in terms of survival for acute promyelocytic leukemia (APL) patients. Here we highlight the synergism and the underlying novel mechanism between RA and the granulocyte colony-stimulating factor (G-CSF) to restore differentiation of RA-refractory APL blasts. First, we show that in RA-refractory APL cells (UF-1 cell line), PML-RA receptor alpha (RARα) is not released from target promoters in response to RA, resulting in the maintenance of chromatin repression. Consequently, RARα cannot be recruited, and the RA target genes are not activated. We then deciphered how the combination of G-CSF and RA successfully restored the activation of RA target genes to levels achieved in RA-sensitive APL cells. We demonstrate that G-CSF restores RARα recruitment to target gene promoters through the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the subsequent derepression of chromatin. Thus, combinatorial activation of cytokines and RARs potentiates transcriptional activity through epigenetic modifications induced by specific signaling pathways.

Germline mutations of the CBL gene define a new genetic syndrome with predisposition to juvenile myelomonocytic leukaemia.

BACKGROUND: CBL missense mutations have recently been associated with juvenile myelomonocytic leukaemia (JMML), an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterised by excessive macrophage/monocyte proliferation. CBL, an E3 ubiquitin ligase and a multi-adaptor protein, controls proliferative signalling networks by downregulating the growth factor receptor signalling cascades in various cell types. METHODS AND RESULTS: CBL mutations were screened in 65 patients with JMML. A homozygous mutation of CBL was found in leukaemic cells of 4/65 (6%) patients. In all cases, copy neutral loss of heterozygosity of the 11q23 chromosomal region, encompassing the CBL locus, was demonstrated. Three of these four patients displayed additional features suggestive of an underlying developmental condition. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient. The germline mutation represents the first hit, with somatic loss of heterozygosity being the second hit positively selected in JMML cells. The three patients display a variable combination of dysmorphic features, hyperpigmented skin lesions and microcephaly that enable a 'CBL syndrome' to be tentatively delineated. Learning difficulties and postnatal growth retardation may be part of the phenotype. CONCLUSION: A report of germline mutations of CBL in three patients with JMML is presented here, confirming the existence of an unreported inheritable condition associated with a predisposition to JMML.

CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts.

CD44 is a marker of tumour-initiating cells and is upregulated in invasive breast carcinoma; however, its role in the cancer progression is unknown. Here, we show that antibody-mediated CD44-targeting in human breast cancer xenografts (HBCx) significantly reduces tumour growth and that this effect is associated to induction of growth-inhibiting factors. Moreover, treatment with this antibody prevents tumour relapse after chemotherapy-induced remission in a basal-like HBCx.

Interferon-alpha therapy in bcr-abl-negative myeloproliferative neoplasms.

Interferon (IFN) was the first cytokine discovered 50 years ago, with a wide range of biological properties, including immunomodulatory, proapoptotic and antiangiogenic activities, that rapidly raised interest in its therapeutic use in malignancies. IFN-receptor characterization was also pivotal in the discovery of the JAK/STAT signaling pathway. Among the large IFN family, mainly one of the type I IFN, IFN-alpha2, is used in therapy. Many clinical trials have shown remarkable efficacy of IFN-alpha in bcr-abl-negative myeloproliferative neoplasms (MPNs), especially polycythemia vera (PV), and essential thrombocythemia (ET). IFN-alpha induces about 80% of hematological responses in those diseases and is able to reduce splenomegaly, as well as relieve pruritus and other constitutional symptoms. Yet its use was limited by toxicity, leading to early treatment discontinuation in about 20% of the patients. However, its lack of leukemogenic potential and its possible use during pregnancy have already made IFN-alpha the drug of choice for younger MPN patients. In addition, several studies have shown a probably selective effect of IFN-alpha on PV and ET clones, as shown by cytogenetic remissions, reversions to polyclonal hematopoiesis, and more recently by induction of JAK2V617F complete molecular remissions in PV which may widen the indications of IFN-alpha in JAK2-mutated MPN.