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HEMATOPOIESIS: Edited by Hal E. Broxmeyer

Small molecule regulation of normal and leukemic stem cells

Fares, Imana; Rivest-Khan, Lauraa; Cohen, Sandrab; Sauvageau, Guya,b,c

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Current Opinion in Hematology: July 2015 - Volume 22 - Issue 4 - p 309-316
doi: 10.1097/MOH.0000000000000151
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Abstract

INTRODUCTION

Hematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate into all blood cell lineages, thereby maintaining blood cell homeostasis throughout the lifetime of an organism. Accordingly, HSCs are widely used in transplantation and gene therapies. Cord blood has become an attractive source of hematopoietic stem and progenitor cells (HSPCs) for transplantation because of its tolerance for human leucocyte antigen mismatches, improved access for ethnic minorities, low incidence of chronic graft-versus-host disease and rapid availability when compared with other sources [1]. Cord blood may also be associated with lower disease relapse rate [2]. The main problem in using cord blood, especially for adult recipients, is the limited number of HSPCs resulting in delayed engraftment, thereby increasing early infections and transplant-related mortality [3,4].

Experiments in which cord blood-derived HSPCs are barcoded with retroviral libraries and transplanted in immunodeficient mice revealed the presence of different engrafted clones with variable repopulating potential both short-term and long-term [5]. The short-term repopulating clones depict a rapid reconstitution potential, detectable at 4 weeks posttransplantation and in some cases lasting up to 16 weeks. Long-term repopulating clones become evident after 16 weeks and persist beyond 26 weeks. In contrast to the mouse model, it remains difficult to prospectively distinguish human long-term HSCs (LT-HSCs) vs. short-term HSCs (ST-HSCs) [6] although markers of fresh LT-HSCs were recently identified [7]. Unfortunately, with the exception of CD34, CD45RA and CD90, most markers of LT-HSCs are not reliable after culture. Cell surface markers that better identify and distinguish the different cellular populations are needed to ascertain the quality of expanded cells and identify additional molecules/factors that govern their self-renewal program.

Leukemic stem cells (LSCs) are responsible for acute myeloid leukemia (AML) maintenance in vivo and relapse after therapy [8]. They are notoriously difficult to keep in culture, hence the difficulties in finding LSC-specific therapies whose development would be greatly enhanced if this hurdle was overcome.

Various approaches and reagents have been tested to improve culture conditions for HSPCs and LSCs. These include the use of cytokine combinations [9], improved bioreactor-based methodologies [10], mesenchymal stromal cell cocultures [11–13] and immobilized biologicals such as delta 1 notch ligand [14]. New methodologies to identify potent small molecules active on human HSPCs were pioneered by Boitano et al.[15]. Small molecules discussed in this article are very attractive as they can be optimized; they are usually more stable than biologicals and can be produced in large amounts for therapeutic and laboratory utilization. Moreover, they can be exploited as tool compounds/probes for target identification and cell labeling.

Box 1
Box 1:
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SMALL MOLECULES THAT ENHANCE HEMATOPOIETIC STEM AND PROGENITOR CELL HOMING

A stable prostaglandin E2 (PGE2) derivative, 16,16-dimethyl PGE2 (dmPGE2) has been shown to regulate HSPC homeostasis in zebrafish and in mice [16,17]. Recent findings show that dmPGE2 treatment also enhances migration and homing of human and nonhuman primate HSPCs (Table 1) by upregulating expression of the chemokine receptor type 4 and survivin [17,18,24]. dmPGE2 also increases cyclic adenosine monophosphate activity, which influences HSPCs through enhanced Wnt signaling [18,25]. Short exposure of cord blood cells to dmPG2 was recently tested in a phase I double-cord based clinical trials [26], revealing a modest 3.5 day reduction to neutrophil engraftment compared with historical controls with no effect on platelets (Table 2). Interestingly, 10 of the 12 patients sustained long-term engraftment with the dmPGE2-treated unit [26]. Future adjustments in clinical trials may potentially reveal the benefits of this approach.

Table 1
Table 1:
Small molecules that enhance hematopoietic stem and progenitor cell homing and expansion
Table 1
Table 1:
(Continued) Small molecules that enhance hematopoietic stem and progenitor cell homing and expansion
Table 2
Table 2:
Published clinical trials in which small molecules were used

Diprotin A is a small molecule that also enhances HSPC homing by inhibiting the enzyme dipeptidyl peptidase (DPP4). By cleaving the stromal derived factor-1 which can no longer activate chemokine receptor type 4, DPP4 interferes with HSPC chemotaxis. Inhibiting DPP4 in cord blood-derived Lin or CD34+ cells, by brief exposure to diprotin A, enhanced human engraftment in immunocompromised mice (see Table 1 and [19]). The Food and Drug Administration-approved DPP4 inhibitor (sitagliptin) was recently evaluated in a pilot clinical trial [27]. Unfortunately, no improvement in neutrophil engraftment was observed in this study (Table 2).

CYTOKINES ARE ESSENTIAL MITOGENS FOR HSPC EXPANSION

Various cytokine combinations have been tested to maximize HSPC expansion ex vivo[30]. The safety of transplanting growth factor-expanded CB CD34+ cells to humans was previously documented [31]. Growth factors such as stem cell factor, thrombopoietin, Flt-3 ligand, and granulocyte colony-stimulating factor improve total cell numbers and output of myeloid progenitors, often at the expense of LT-HSCs [32]. One hypothesis in the field is that small molecules which inhibit HSPC differentiation could be introduced in such cultures and provide benefits to cord blood transplant patients.

SMALL MOLECULES THAT ENHANCE HEMATOPOIETIC STEM AND PROGENITOR CELL EXPANSION

Several small molecules, summarized in Table 1, have the potential to enhance HSPC activity in vitro. The copper chelator [tetraethylenepentamine (TEPA)] [20] and Sirtuin 1 deacetylase inhibitor [nicotinamide (NAM)] [21] appear to preserve immature phenotype of cells kept in growth factor-supplemented cultures. Importantly, CD34+ cell expansion in such cultures is similar to that found in control conditions (Table 1). However TEPA or NAM treatments appear to expand cells with short-term repopulating potential (Table 1). The recently published NAM and TEPA trials (Table 2, see also StemEx trial, Stiff et al., Abstract 295, ASH meeting 2013) showed that recipients of expanded cord blood units had neutrophil and platelet engraftment earlier than that seen with historical controls [28,29]. The contribution of NAM or TEPA over that of growth factor-induced HSPC expansion to this clinical benefit remains unclear.

Histone deacetylase inhibitors such as valproic acid (VPA) may also play an important role in HSPC expansion [22]. HSPC exposed to VPA upregulate several stem cell specific transcription factors such as NANOG and OCT4. Convincing recent preclinical data suggest that VPA treatment leads to massive expansion of CD34+ cells with a better retention of the primitive stem cell phenotype and improvement in the number and activity of SRCs (Table 1).

Two small molecules were recently characterized from phenotypical screens exploiting primary human CD34+ cells. The first one, StemRegenin1 (SR1), was identified by Novartis, and the second one, UM171, by our group. Ongoing clinical trials with SR1 are very encouraging as a subset of patients showed neutrophil engraftment within 10 days (Wagner et al., Abstract 728, ASH meeting 2014).

Both SR1 and UM171 suppress cellular differentiation and enhance CD34+ cell production over control cultures. Interestingly, the two molecules appear to potently affect different cell populations, hinting at different mechanisms. UM171 has a robust impact on cells with more primitive phenotypes, including the CD34+CD45RA population, but SR1 better expands the more mature CD34+CD45RA+ cells [23▪▪,33,34]. Importantly, combination of SR1 and UM171 enhances the production of multilineage colony forming cells to levels not encountered when each molecule is used separately.

Comparison of UM171 and SR1 effects on SRC is more difficult since different culture conditions were used in the two publications. For example, Boitano et al. found a 17-fold expansion of SRCs in their original article, although these cells were kept in culture for longer periods than cultures performed by Fares et al. and analyzed at much earlier time points after transplantation (3 months for SR1 vs. 6 months for UM171). In similar conditions as used for UM171 (12 day fed-batch cultures supplemented with three growth factors (removing interleukin-6, used by Boitano et al.), we found that SR1 expands LT-HSCs by only two-fold, but confirmed its profound impact on progenitor cell output somehow compromising LT-HSC activity, a finding not observed with UM171. Interestingly, UM171 was able at least partially to revert the negative impact of SR1 on LT-HSCs. Although extensive studies will likely reveal that these two molecules differentially affect ST-HSCs and LT-HSCs, it is tempting to speculate that they would strongly complement each other in clinical trials, probably leading to shorter culture time and sparing effect on proliferating LT-HSCs. This would be particularly important when small single cords are used in clinical trials.

In line with the complementary effects of these two molecules, transcription profiling of CD34+ cells exposed to SR1 or UM171 reveals only a few commonly regulated genes. As expected from the original study and unlike UM171, SR1 treatment remarkably suppresses the downstream targets of the aryl hydrocarbon receptor (AhR). UM171 uniquely reduces the expression of transcripts associated with erythroid and megakaryocytic differentiation and enhances that of several cell surface molecules [23▪▪].

The ability of UM171 to amplify human LT-HSCs without suppressing AhR activity suggests that LT-HSC self-renewal in vitro does not require AhR inhibition. Accordingly, the preferential amplification of CD34+CD45RA+ cells by SR1 suggests that a clinically important cell with short-term repopulation potential is responsive to AhR suppression. Hence, our current interpretation is that a form of LT-HSC is responsive to UM171 and a ST-HSC to SR1.

SMALL MOLECULE REGULATION OF LEUKEMIC STEM CELLS

This section focuses on small molecules, which affect self-renewal regulation of LSCs and permit their maintenance ex vivo. Recent findings on the use of small molecules targeting LSCs for their eradication in AML are reviewed by Al-Hussaini and DiPersio [35]. Although recent success in expanding HSCs ex vivo has been achieved, maintaining LSCs ex vivo remains a challenge. When LSCs were first described, they were thought to result from genetically modified LT-HSCs (the so-called cell of origin). However, groups have shown that LSCs could also arise from committed progenitors that reacquire a self-renewal program from various genetic alterations [36]. To date, xenografts in immunodeficient mice remain the optimal test to assess LSC activity and numbers. However, evidence suggests that the measured engraftment may still depict an in-vivo selection of certain clones, which do not necessarily represent the clonal complexity of the original disease [37,38].

In order to improve culture conditions for LSCs, we recently performed a high-throughput phenotypical screen of 6000 small molecules to identify compounds that would suppress differentiation of a primary human AML specimen, selected for its high expression level of CD34 and low expression of CD15 [39▪▪]. Surprisingly, the majority of identified hits were AhR suppressors. Indeed, addition of SR1 or other newly identified AhR antagonists was beneficial in preventing in-vitro differentiation of genetically diverse AML specimens. Limit dilution assay in immunodeficient mice with fresh AML cells, or cultured cells with or without SR1, showed that LSC frequency was much higher when cells were cultured in the presence of SR1, underlying a requirement of AhR suppression for LSC maintenance ex vivo. Importantly, most SR1 supplemented cultures contained lower LSC numbers than the uncultured cells, possibly suggesting clonal selection in our experimental conditions.

Another finding was that addition of UM729 (the only non-AhR suppressor hit and an analog of UM171) alone in AML cultures prevented differentiation of a small subset of specimens and, with the exception of one sample, to a much lesser extent than SR1. However, in combination with SR1, UM729 exhibited an additive/synergetic effect on most AML specimens pointing to the great heterogeneity of this disease. These results suggest that unlike normal LT-HSCs which are best supported in vitro by UM171, LSCs in culture mostly depend on AhR suppression, providing ground for the hypothesis that LSCs are potentially derived from a cell type most sensitive to AhR activation, referred to above as a possible ST-HSC. However, the heterogeneity in LSC response to UM729 may suggest that a proportion of human AML may originate from LT-HSCs as postulated by Stubbs and Armstrong [40]. Regardless, these results provide a proof of principle that LSC cultures can be pharmacologically optimized by combinations of small molecules. Although these conditions represent a great improvement, in contrast to what is found with normal LT-HSCs, they fail to support a net ex-vivo expansion of LSCs. Improvements of such cultures will represent the focus of future work by our group until all LSCs can be expanded in which case clonal diversity of the disease should be maintained. This combined with better markers for cultured LSCs will open unprecedented possibilities for screening compounds that selectively eliminate these cells.

CONCLUSION

Small molecules are highly efficacious modulators of HSC and LSC activity and self-renewal. Although several of these molecules have already reached clinical trials, more (e.g., UM171) will be tested in 2015. Ultimately and considering the important costs associated with clinical trials, it will be important to rapidly define the best combination of such compounds to provide the ideal graft in a timely fashion.

These molecules also provide important tools for basic discoveries of the molecular basis of self-renewal and pave the way for a new chemical identification/classification of HSC/LSC subsets, a new branch of ‘Stemistry’ [41].

Acknowledgements

The authors thank J. Krosl and J. Chagraoui for critical reading of the article.

Financial support and sponsorship

This work was supported by grants from the Stem Cell Network of Canada and from Genome Canada/Génome Québec.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest

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Keywords:

hematopoietic stem cell; leukemic stem cell; self-renewal; small molecules

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