Another approach to eliminating LSCs is to determine the pathways that are aberrantly expressed and identify drugs, which selectively target LSCs while leaving normal HSCs alone, hence reducing toxicity. Although much remains to be elucidated about the regulation of HSCs and LSCs, several genes are recognized as being important in the regulation of HSCs. These include HoxB4
, which have both been implicated in the expansion of hematopoietic cells both in vivo
and in vitro
The Bmi-1 protein, which is necessary for the self-renewal of HSCs, also has potential as a target of therapeutic intervention. Bmi-1
is a member of the Polycomb Group (PcG) gene family and its expression is limited to immature HSC and the CD34+
fraction of AML patients.99,100
Studies with Bmi-1
-knockout mice have shown its requirement in blood cell development by demonstrating the progressive loss of all hematopoietic lineages. When Bmi-1-/-
cells were transplanted into lethally irradiated normal mice, the cells were able to partially reconstitute myeloid cells and lymphocytes. They did not persist for a prolonged time, as they were unable to self-renew.99,100
To understand the reason behind this effect, Park et al.100
compared the gene expression profiles of Bmi-1-/-
HSCs with normal HSCs. Bmi-1-/-
HSCs have increased levels of cell cycle inhibitors, such as p16INK4α
and p53. p53 induced Wig1 and downregulated apoptosis inhibitors such as AI-6. The research further supports the theory that cancer is essentially a disease of stem cells. In addition, the PcG genes have long been suspected of being involved in oncogenesis and the study performed with Bmi-1 knockout mice further adds support for this hypothesis. Finally, these studies demonstrate the critical importance of Bmi-1 in stem cell self-renewal and point to a potential target of intervention that can be used to combat a variety of cancers.99
As stated earlier, the Wnt signaling pathway may be elevated in LSCs.50
This may arise from mutations in FLT-3 and chimeric transcription factors, such as promyelocytic leukemia-retinoic acid receptor-α t(15;17) (PML-RARα) and acute myeloid leukemia-1 gene (8;21) (AML-ETO). FLT-3 is mutated in over 30% of cases of AML and associated with poor prognosis and increased relapse rates.101
Internal tandem duplications of the FLT-3 gene have been detected in AML LSC.83,101
An end result of these mutations is increased expression of the Wnt signaling pathway, which may result in increased growth of the LSC. In addition, cells stimulated with the Wnt ligand are known to stabilize β-catenin. β-Catenin then translocates to the nucleus and activates specific gene transcription. Overexpression of mutant β-catenin induces the expansion of HSC.5
For these reasons, targeting of various components in these pathways (for example, FLT-3) may inhibit LSC growth. Indeed the FLT-3 inhibitor CEP-701 suppressed the engraftment of FLT-3/ITD LSC.83,101
In addition, the Notch pathway may be deregulated in LSC. Genomic analysis from AML stem cells determined that the Jagged-2 gene, a Notch ligand is overexpressed in LSC. Inhibition of γ-secretase by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) inhibits LSC growth. γ-secretase is necessary for Jagged and Notch signaling.102
Likewise, this group also determined that certain genes are detected at lower levels in LSCs, including genes involved in DNA repair, signal transduction and cell cycle progression, which is consistent with the quiescent nature of LSC.
The Wilms' tumor (WT1) gene has also been observed to be expressed at elevated levels in LSCs.103
WT1 was not expressed at high levels in normal long-term HSCs or in multipotent progenitor cells. In contrast, in AML1-ETO+TEL-PDGFRbR or BCR-ABL murine leukemias, WT1 was expressed in approximately 50% of the transplantable LSC.99
Hence, the WT1 protein may be a target for the successful treatment of LSCs.
Transcription factors have been implicated in regulating the LSC. Meis1 has recently been shown to be an essential and ratelimiting regulator of MLL LSC potential.104
Furthermore, Wong et al.
demonstrated that Meis plays a major function in establishing LSC potential by regulating self-renewal, differentiation arrest and cycling. In contrast, other transcription factors (for example, PU.1 and JunB) may display reduced expression in AML stem cells. Although targeting of transcription factors has proven problematic, it is an area with great potential.
The PI3K/PTEN (phosphatase and TENsin homolog deleted on chromosome 10)/Akt/mammalian target of rapamycin (mTOR) is another cell signaling pathway of immense importance with regard to a variety of cancers. This pathway has been determined to be consistently activated in AML cell lines and strongly contributes to the proliferation, survival and drug resistance.107
In addition, its upregulation has been specifically observed in LSCs transplanted in NOD-SCID mice and shown to provide an anti-apoptotic response.108
Although this upregulation is associated with increased drug resistance and a poor prognosis, a surprising new study by Tamburini et al.109
demonstrated the upregulation of this pathway is actually a favorable factor in de novo
cases of AML. This is perhaps because the upregulation of this pathway moves immature leukemic cells, including LSCs, into S phase and therefore makes them more susceptible to certain chemotherapeutic agents, especially those targeting actively replicating cells.110
An overview of the potential for targeting signal pathways in LSCs is present in .
Sites of interaction of signal transduction pathway inhibitors. Various types of inhibitors have been developed to target different molecules involved in signal transduction.
The PI3K/PTEN/Akt/mTOR pathway is of critical importance because of its effects on downstream proteins, including Bad, nuclear factor-kappa B (NF-κB) and MDM2. Inhibitors for several of these proteins, such as NF-κB and MDM2, have been developed and their efficacies in treating various leukemias and myelomas are being evaluated.111
Although its specific role remains unclear, the NF-κB transcription factor is of particular interest because it is found to be particularly active in LSCs but not in HSCs, making it a point of selective intervention. Proteasome inhibitors, such as salinosporamide A and bortezomib, are currently being examined in phase I/II trials and show potential.5
Furthermore, inhibitors of mTOR show promise in LSC therapy. These inhibitors used in conjunction with conventional therapies induce apoptosis and specifically have been shown to reduce the abundance of LSCs.5
Upstream of Akt, there also exists the tumor suppressor PTEN, which has been a further point of intervention, as it is frequently mutated or silenced in human cancer, including leukemia.112
In addition, recent evidence indicates a relationship between the PI3K/PTEN/Akt/mTOR pathway and expression of the MRP1, another ABC transporter protein that is also linked with multidrug resistance. Upregulation of this pathway was found to increase levels of the MRP1 protein.113
These data suggest a p53-dependant mechanism of MRP1, as the inhibition of MRP1 was linked with a concurrent increase in p53 levels. Inhibition of the ubiquitous ligase, MDM-2, with Nutlin-3a increases p53 stability and promotes apoptosis of leukemia, which are WT at p53.114-126
p53 has been shown to be an important target in leukemia. In addition, the connection between MRP1 and this pathway shows another possible reason for multidrug resistance in leukemias.
This connection between multidrug resistance and the PI3K/PTEN/mTOR/Akt pathway also shows the potential of targeting this cascade. Studies using inhibitors to PI3K, such as Wortmannin and LY294002 in combination with conventional chemotherapeutic drugs, demonstrated an increased sensitivity of the cells to more readily undergo apoptosis.125,126
Furthermore, as normal hematopoietic progenitors are less affected by these inhibitors, the potential to selectively target LSCs and AML cells and reduce toxicity is a possibility.110,111