Bayard Clarkson: Overview

Comparative Studies of Normal and Cancer Stem Cells

There is increasing evidence that most lethal cancers are initiated by oncogenic mutations in adult stem cells or in progenitors with innate potential stem cell properties, which, when mutated, are able to function as de facto cancer stem cells. Requisite stem cell properties include extensive self-renewing ability, relative resistance to apoptosis and senescence, multilineage differentiation potential of varying degree, and the necessity of spending a substantial part of their life span in a dormancy state to lessen the risk of errors from repeated divisions, and to allow time for repair and elimination of noxious chemicals and toxins, including cytotoxic drugs. Quiescent cancer stem cells are not killed by drugs designed to kill proliferating cells, and their survival is a major reason for failure to cure even most highly chemosensitive tumors.

The quiescent cancer stem cells in some malignant tumors, such as acute lymphoblastic leukemia in children, acute promyelocytic leukemia, rapidly growing lymphomas, and testicular cancers, are very sensitive to certain non-cell cycle specific drugs (NCCSDs), such as alkylating agents, anthracyclines, arsenic trioxide or platinum derivatives. These cancers can often be cured, but in most cancers there is insufficient differential sensitivity between the normal and cancer stem cells to eliminate the latter without prohibitive toxicity.Another hallmark of cancer stem cells is their failure to respond to normal regulatory mechanisms that curtail cell production when a normal homeostatic cell density equilibrium is reached, balancing cell production and cell death. While it is known that many cell cycle and apoptotic genes and signaling pathways controlling proliferation are dysregulated in cancer cells, little is known about the specific molecular abnormalities responsible for their inability to maintain normal homeostatic cell densities that might be targetable, nor how bacterial quorum sensing (QS) systems regulating cell densities may resemble mammalian QS systems.

The central focus of our research is to search for selective targetable differences between normal and CML quiescent stem cells. We chose CML as a prototype because without effective treatment it regularly undergoes malignant progression from a relatively benign chronic phase to a rapidly fatal blastic phase and because highly effective drugs are now available that inhibit the causative oncogenic stem cell mutation (bcr-abl fusion gene). These drugs (Tyrosine Kinase Inhibitors or TKIs) can eliminate the great majority of proliferating cells in early (chronic) stage disease with relatively little toxicity, but they are not curative and the disease almost always recurs if the treatment is stopped, presumably because the quiescent CML stem cells are not killed by TKIs or most other drugs and resume proliferation. Once blastic transformation occurs, the response to TKIs or any other treatment at best is partial and usually short-lived.

Using a variety of methods, we have isolated highly purified subpopulations of proliferating and quiescent normal and CML stem and progenitor cells. The quiescent CML stem and progenitor cells (CD34+/GO) are at a slightly later stage of development and are more easily triggered into cycle by cytokines than the comparable normal cells. In quiescent normal but not CML cells compared to cycling cells (CD34+/G1SG2M), there is down-regulation of CD38 and up-regulation of stem cell-associated genes including ALDH, HLF, and GATA3. In comparing CML and normal CD34+/GO cells, a number of stem cell associated genes are down-regulated in CML GO cells, including CD133 («20 fold), MSI2, HLF, GBP2, and DLG7, and there is up-regulation of genes characteristic of erythro-megakaryocyte development (CD36, KLF-Hemoglobin β, δ, γ, TFR2 and CD41, providing additional evidence that the majority of GO CML cells belong to a more differentiated compartment than the normal GO cells. Of all the genes up-regulated in CML GO cells we first focused on the Leptin receptor (LepR) because the Ra isoform is one of the most over-expressed genes (20 fold), and, being a receptor, it might be used to distinguish quiescent CML cells from normal ones and possibly serve as a target for a drug or armed antibody. We are currently further enriching and characterizing normal and CML stem cells (i.e. Lin- CD34+CD38-Thy1+CD45RA-) and also examining other differences in signaling molecules and pathways (e.g. CD133, Akt-FOXO, PI3K, lipid raft formation) that we suspect may explain why CML stem cells continue cycling and producing cells long beyond normal homeostatic limits. We are also examining differences between chronic and blastic phase stem and progenitor cells to seek explanations why the usually slower proliferating blastic phase cells inevitably replace the faster growing chronic phase cells. If we can identify targetable differences and developing effective drugs to selectively kill the quiescent and blastic phase CML stem/progenitor cells, it is likely this research will be applicable to many other types of cancer.