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January and February 2013

Posted By Connections Editor, Wednesday, March 6, 2013
Updated: Wednesday, February 27, 2013

Take a quick tour of the January and February issues and get a feel for the publication's cutting-edge scientific content. Read the full issues online.

In this Issue – January 2013

Role of Wnt antagonists in the regulation of hematopoietic stem cells fate decisions
Cain and Manilay

The Wingless (Wnt) are secreted lipid-modified signaling proteins of 350–400 amino acids in length. The Frizzled (FZD) proteins are seven-pass transmembrane receptors to which Wnts bind. The Wnt signaling is involved in cell fate decisions during hematopoietic differentiation in the bone marrow, in the maintenance of bone homeostasis, and in hematopoietic differentiation in a non-cell autonomous manner. There are 19 different Wnt ligands and 10 FZD receptors identified in humans and mice. In addition, Wnts utilize distinct co-receptor proteins that initiate "canonical” and "noncanonical” Wnt signaling pathways. Additional levels of complexity are added by secreted Wnt antagonists expressed in bone and bone marrow stromal cells that either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. In this timely review, Cain et al. compared and contrasted the roles of these Wnt antagonists and their effects on hematopoietic development in mice and also discussed the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease. This manuscript provides an interesting and comprehensive reading for anybody looking to update their knowledge of the field.

An improved method to track kinetics of hematopoietic stem cell reconstitution
Cornils et al.

Integrating viral vectors provide a unique and heritable genomic mark for the identification of single stem cell–derived clones. Linear amplification–mediated (LAM) PCR is an integration-site directed PCR method to sequence the unknown DNA flanking sequences down to the single cell. It has enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. In this report, Cornils et al. presented a time-course clonal analysis of hematopoietic reconstitution following transplantation of retrovirus and lentivirus transduced cells using a multiarm, 2 restriction enzymes LAM-PCR analysis coupled to next generation sequencing (NGS). They found that the number of clones contributing to hematopoiesis over time were relatively stable for both vectors; however, the change of size of individual clones marked with the lentiviral vector followed wave-like kinetics, whereas retrovirally tag clones showed a tendency for continuous expansion. They also detected that long-term reconstituting clones marked with the retroviral vector had a tendency toward overrepresentation of insertions in the vicinity of proto-oncogenes, which supports the notion that clonal dominance due to insertional mutagenesis is more likely to develop after transduction with retroviral than with lentiviral vector. Finally they performed ligation-mediated (LM)-PCR, a method of sequencing directly from genomic DNA using a specific primer for the region of interest, and found that quantification of LAM-products did not always correlate with results from qPCR. As shown in this and other studies, even the use of two different restriction enzymes for LAM-PCR may not strictly ensure a complete figure of gene-marked clones. Thus, improved technologies are needed to benefit many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment.

Gpr171: a possibly new regulator of HSC differentiation
Rossi et al.

The G protein-coupled receptor (GPR) 171 is structurally and evolutionally related to a subfamily of the GPR family of purinergic receptors (P2YRs) for extracellular nucleotides, a group of mediators that regulate hematopoietic progenitor cells. GPR171 is an orphan receptor, and its signaling pathway and biological role remain unknown. In this study, Rossi et al. investigated the role of Gpr171 in the murine hematopoietic system. Their phylogenetic analysis confirmed that Gpr171 is evolutionally related to members of a P2Y gene-cluster that is localized on mouse chromosome 3. Interestingly, examination of its expression profile showed that, as opposed to other P2YRs, Gpr171 is downregulated in monocytes and granulocytes. Moreover, its enforced over-expression resulted in decreased expression of myeloid markers and increased the generation of colonies in vitro. Conversely, Gpr171 silencing diminished the expression of myeloid markers and the clonogenic potential of cells. In addition, mice transplanted with HPCs over-expressing Gpr171 displayed a significant reduction in the percentage of myeloid cells. Together, these elegant experiments demonstrated that Gpr171 plays a role in myeloid-monocytic lineage development and suggested that Gpr171 may have followed a separate evolutionary pathway as compared to other P2YRs belonging to the same gene-cluster. This work provides the framework for the identification of Gpr171 agonists and antagonists, which will not only help achieve a deeper understanding of the role of this orphan receptor, but also of the molecular mechanisms underlying the multi-step process that leads from committed progenitors to terminally differentiated cells.

Sca-1 as a possible early-response marker to identify hematopoietic stem/progenitor cells with increased self-renewal capacity in response to HDIs in vitro
Walasek et al.

Histone deacetylase inhibitors (HDIs) are epigenetic modifiers that control gene expression by altering the acetylation status of chromatin and other non-histone proteins. These small molecules can enhance the ex vivo self-renewal and propagation of hematopoietic stem/progenitor cells (HSPCs) as well as modify the process of differentiation. In a paper published in Blood in March 2012, this group reported the molecular and functional changes induced on HSPCs by a 7-day treatment with valproic acid (VPA), a widely used HDI. In the current study, Walasek et al. performed microarrays after short-term, 24h VPA stimulation, to identify early response VPA-targets in HSPCs. Their genome-wide gene expression studies identified the well-known and widely used HSPC marker Sca-1 as an early VPA-responsive gene. Early up-regulation of Sca-1 was also seen with two additional HDIs, Apicidin and MS-275, which differ from VPA in their chemical structure and their specificity. Strikingly, VPA not only strongly preserved Sca-1 expression and the LSK (Lin-Sca1+cKit+) phenotype of isolated HSPCs after 24h culture, but it also re-induced Sca-1 on committed common myeloid and granulocyte macrophage progenitor cells that had lost its expression in the process of differentiation, reverting those cells to a more immature LSK phenotype. Moreover, Sca-1 re-induction coincided with induced self-renewal capacity as measured by in vitro re-plating assays, while Sca-1 itself was dispensable for the functional effects of VPA. Together, the authors provided the first analysis of direct VPA targets in primitive hematopoietic cells and showed that Sca-1 is a faithful marker to identify cells with increased self-renewal capacity in response to HDIs in vitro. Studies like this one, aimed at understanding the spectrum of both biological and molecular effects of VPA, are crucial to determine the potential of this HDI to improve hematopoietic reconstitution and engraftment of ex vivo expanded HSPCs, a vital goal for transplantation, gene, and cellular therapies

In this Issue – February 2013

Improved therapeutic outcome of patients with Multiple Myeloma requires novel treatments and a better understanding of the mechanistic underlying drug resistance
See Salem et al. and Wolschke et al.

Multiple myeloma (MM) is a rare but devastating plasma cell malignancy that affects mainly older adults. In the last ten years, induction regimens that combine the glucocorticoid-analog dexametasone (Dex) with novel chemotherapeutic agents, such as the proteasome-inhibitor bortezomib (BTZ) or the antiangiogenetic and immunomodulator drugs thalidomide and its derivative lenalidomide, have significantly increased the complete response (CR) rate before and after Allogenic Stem Cell Transplantation (ASCT) with a positive impact on progression-free survival. Maintenance therapy with thalidomide and lenalidomide may further prolong remission duration. However, in spite of these advances, MM remains incurable, as the disease ultimately acquires resistance to therapies. Thus, novel treatment approaches and a better understanding of the mechanism(s) underlying resistance are essential to improve the therapeutic outcome of patients with relapsed MM.

The manuscript by Salem et al. provides a mechanistic rationale for combining Dex and BTZ, which is one of the most effective and widely used treatments for MM. Both drugs are inhibitors of the nuclear factor (NF)-κB pathway, which is chronically active in myeloma cells and can induce stromal cells to produce myeloma growth factors such as interleukin (IL)-6. Concomitantly, aberrant NF-kB activation has been associated with the emergence of resistance to anti-cancer drugs and radiation in MM, and elevated IL-6 serum levels correlate with poor prognosis. Dex and BTZ inhibit NF-κB by different pathways, and the authors found that the combination potentiated direct cell death of both the drug-sensitive, and, importantly, drug-resistant MM cells. Furthermore, combination therapy down-regulated the NF-κB targeted gene expression of IL-6 and manganese superoxide dismutase, which can induce chemo- and radio-resistance in MM, supporting potential combinations of these drugs with radiotherapy and additional chemotherapeutic drugs for the clinical benefit of MM patients.

The manuscript by Wolschke et al. reports a prospective phase I/II study to define the dose-limiting toxicity and the immunological effects of lenalidomide given to MM patients early post-ASCT. Lenalidomide is a more potent analog of thalidomide, developed to enhance immunomodulatory properties with improved safety. Its antitumor activity seems to be mediated through the modulation of cytokines and activation of T- and natural killer (NK) cell-mediated antitumor response. Increasing evidence of NK cell allo-reactivity in MM patients after ASCT provides a rationale for an early application of lenalidomide after transplantation. In this context, two recent clinical trials published by McCarthy et al. and Attal et al. in May 2012 in the New England Journal of Medicine showed that lenalidomide maintenance therapy significantly lengthens disease-free progression and improves overall survival among patients with MM. However, as described in the first study, this was associated with more toxicity and second cancers. Undesired side effects were also found in the HOVON 76 Trial published in Blood in September 2011, which excluded the feasibility of using lenalidomide as maintenance after ASCT due to Graft versus Host Disease (GvHD). Although encouraging, these clinical trials underscore the need to determine lenalidomides maximum tolerated dose as well as the mechanism underlying its unwanted side effects. In this study, the authors found that 5 mg of lenalidomide given daily for 21 days was the maximal tolerable dosage early after ASCT, with the major limiting factor being the occurrence of acute liver and gut toxicity due to GvHD, supporting the results of the HOVON trial. In agreement with the two New England Journal of Medicine papers, they also reported that lenalidomide treatment strongly increased complete remission rate, which they linked to its immunomodulatory effect.

The current five-year survival rate for patients with MM is 40% and thus, studies such as these that search for mechanistic insights of current therapies, drug resistance and side effects, offer great hope for MM patients who are in desperate need of treatments with a better response and survival rate. Such studies might also provide better target therapies and new immunomodulator regimes to supply physicians with a wider variety of treatment options to tailor the most appropriate and efficacious treatment to their patients' disease.

Azacitidine and decitabine are "coming of age” in treatment, but great challenges in understanding how they work remain
See Tsujioka et al.and Bernal et al.

Low dose regimes of the DNA demethylating agents Azacytidine (AZA) and its derivative decitabine (DAC) were approved in 2004 and 2006, respectively, by the US Food and Drug Administration for the treatment of myelodysplastic syndrome (MDS), a group of hematopoietic disorders characterized by ineffective hematopoiesis and increased risk of leukemic progression. In Europe, AZA is the only drug approved for the treatment of high-risk MDS not eligible for stem cell transplantation. These drugs are proposed to work by inducing functional re-expression of aberrantly hypermethylation-silenced tumor suppressor genes. However, whether epigenetic effects of the drugs account for all, or even some, of their clinical efficacy is still under debate. The mechanisms of resistance to AZA and DAC, which develops during treatment, are also still unclear, with some studies suggesting methylation-independent pathways. The importance of unravelling the mechanisms underlying AZA and DAC clinical efficacy and resistance is underscored by a recent clinical trial sponsored by Lung Cancer SPORE and Stand Up to Cancer. The results of the trial, published by Jurgens et al. in Cancer Discovery in December 2011, demonstrate the benefit of low doses of the drugs in patients with refractory lung cancer, and thus support the potential use of DAC and AZA for a wider management of cancer. Moreover, clinical trials in breast cancer have already begun in patients with advanced disease, and trials in colon cancer are planned. Searching for mechanistic insights, the paper by Tsujioka et al. show a positive relationship between growth suppression and demethylation by DAC or AZA in the MDS-derived cell line developed in their lab. Interestingly, DAC-induced cell death was preceded by DNA damage-induced G2 arrest via a p53-independent pathway, whereas AZA did not influence the pattern of the cell cycle. Their gene expression profiling suggest that at very low concentration, DAC significantly affect various gene biogroups, which include categories such as cellular movement, inflammatory response, hematological system development and function, hematopoiesis, and cell death. This work supports the finding of a recent important study by Tsai et al., published in Cancer Cell in March 2012, showing that transient low doses of AZA and DAC had antitumor effects on leukemic and epithelial tumor cells, which were accompanied by genome-wide changes in promoter DNA methylation and gene expression, which affected multiple key regulatory pathways.

Looking for mechanisms of drug resistance, the paper by Bernal et al. analyzes the effect of AZA on Matrix-Metalloproteinase (MMP)-9 expression. MMP-9 is a member of the MMP family of zinc-dependent proteolytic enzymes, which play a key role in angiogenesis and metastasis. MMP-9 regulates extramedullary disease in acute leukemia, and it is overexpressed in MDS cells, a phenomenon that in other cancers has been linked to its aberrant methylation. This article describes a dose-response increase of MMP-9 mRNA expression in two human cell lines established from patients with acute leukemia evolved from MDS and in six patients with MDS that relapsed after AZA treatment. This increment was associated with decreased DNA methylation levels in the MMP-9 promoter region and correlated with a higher invasiveness of the cells in vitro. Although the number of patient's samples analysed was small, the findings are interesting for their practical and speculative aspects, providing further evidence of the epigenetic regulation of MMP9, and suggesting that its aberrant methylation may play a role in the resistance to azacitidine in MDS patients. If these findings are confirmed in a larger cohort of patients, the role of MMP-9 in the therapeutic management of MDS or as a prognosis tool will warrant further investigation.

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