Players in Drug-Resistant Leukemia Stem/Initiating Cells and Immunity in Patients with CML in Context of Oxygen Levels: Would Collecting/Processing Cells in Hypoxia Offer Additional Information? A Next Frontier of Investigation

As noted by Silvestri and colleagues (1), chronic myelogenous leukemia (CML) treatment does not result in cured disease for many patients (2, 3). Thus, therapeutic challenges remain. Challenges include resistance to ABL tyrosine kinase inhibitors (TKI), which apparently spares survival of quiescent CML leukemia stem/initiating cells (LS/IC), and that of impaired immunity.

In this in-depth mechanistic study on TKI resistance involving CML LS/ICs and impaired natural killer (NK)-cell activity using semi- and much more highly purified human cells, the authors (1) report a number of interesting and relevant findings. In summary (See Fig. 1), this includes: (i) Involvement of MIR300 in the progression of CML bone marrow (BM) CD34⁺ cells during the chronic and blastic phases of CML. Expression of MIR300 was found to be up to 800-fold lower in dividing normal cord blood CD34⁺ progenitors than in CD34⁺CD38⁻ LS/ICs. By restoring appropriate levels of expression of MIR300 in CML LS/ICs by CpG-based oligonucleotides, in a remarkable study, they greatly reduced proliferation and clonogenic potential of CML LS/ICs while enhancing programmed cell death (apoptosis). These effects were not seen with normal human cord blood cells which were used as controls for normal hematopoietic stem (HSC) and progenitor (HPC) cells. Effects were dose-dependent within relatively narrow (lower or higher) levels of MIR300 expression. These narrow ranges of activity, while experimentally of interest, may make it somewhat difficult to translate clinically for treatment when modulating MIR300 for therapeutic value. However, it is a starting point for better understanding the intimate dose–response relationships of pro- and con-activities. Of importance, MIR300 was shown to act as a dual antiproliferative and proapoptotic miRNA; (ii) interestingly, targets for MIR300 are also validated targets for PP2A. Targeting of SET induced protein phosphatase 2A (PP2A)-dependent inactivation genes/proteins involved in G₁–S cell-cycle transition and survival of quiescent CML LS/ICs and progenitors were evaluated. These data suggest that MIR300-induced inactivation or down regulation is mediated by PP2A, occurs upon inhibition of SET, and reflects levels of MIR300 higher than that which inhibit cell-cycle genes/proteins and trigger PP2A-independent cell-cycle arrest; (iii) LS/ICs reside in an in vivo BM microenvironmental niche (4). Relating induction of MIR300 to the BM microenvironment niche was, in the opinion of this author, a highlight of the paper, as it may allow for a more tissue-specific targeted approach for future treatment. Exposure to hypoxia of primary CD34⁺ cells from patients with blastic phase CML or a relevant cell line, as well as that of primary enriched normal human mesenchymal stem/stromal cells (MSC) and an established MSC cell line, increased expression of MIR300 to levels of quiescent LS/ICs or even higher. Hypoxia and MSC-induced expression of MIR300 was associated with reduced cell division of quiescent LS/ICs, but resulted in a dichotomy of effects on cell-cycle exit and lack of PP2A-mediated triggering of apoptosis; (iv) enhancement of MIR300 expression in blastic phase CML LS/ICs induced by hypoxia required activity of C/EBP/β. C/EBP/β binding to a region 109 base pairs upstream of the human MIR300 gene was an essential regulatory element for transactivation in hypoxia, but not in ambient air (normoxia) cultured CD34⁺ blastic phase CML cells. It was suggested that the in vivo hypoxic BM niche may increase levels of MIR300 in CML LS/ICs by simultaneously inducing C/EBPβ/LAP1-dependent transcription of MIR300, and by exosomal MIR300 derived from MSCs; (v) MIR300 was also linked with impaired cell immune responses of NK cells, suggesting that, in the context of an hypoxic environment, modulation of MIR300 expression may have dual effects on proliferation/apoptosis of CML LS/ICs and immune system cells. The authors suggested that impaired NK-cell activity, which affect the growth and cytotoxicity of quiescent CML LS/ICs and proliferating blast cells, is a MIR300-dependent effect. The MIR300 effects involve hypoxia- and MSC products (in MSC-conditioned medium and associated MSC exosomes). These generate signals increasing C/EBP/β and MIR300 expression. Levels of TUG1, which in CML is regulated in a manner similar to MIR300, was also implicated in proliferation/apoptosis of quiescent CML LS/ICs; and (vi) using human CML cells to engraft a PDX1 mouse model, with or without saturation of TUG1 MIR300 activity was shown to kill most or all in vivo engrafting LS/ICs. These events were associated with barely detectable BCR-ABL1 transcripts and increased the number of human Philadelphia chromosome–negative BM cells.

The article by Silvestri and colleagues (1) is a “tour-de-force” revealing insights into potentially new and useful targetable intracellular players to ameliorate at least some of the clinical problems involved in drug resistance of CML. There is much “food-for-thought” in this article, which I recommend be read in detail. The conclusions will need to be further evaluated by this and other groups interested in this most clinically relevant pursuit. There is, however, one insight that particularly caught my attention, the apparent link of oxygen tension to many of the above noted effects.

Oxygen sensing received its due with recent Nobel Prizes to three outstanding investigators for their critical work in this area. The effects of oxygen tension have been studied in the context of normal hematopoietic stem cells (HSCs)/hematopoietic progenitor cells (HPCs) and LS/ICs and of leukemia progenitor cells dating to at least the late 1970s (5). Normal HSCs/HPCs and LS/ICs grow better in vitro under hypoxic conditions of 1% to 5% oxygen tension which are found in vivo within the BM. Other in vivo sources of these immature cell populations, including circulating blood and extramedullary tissues that support these cells, also manifest oxygen tensions much lower than that of ambient air (∼21% oxygen). Yet essentially all studies that evaluate in vitro growth patterns, cell responsiveness, and gene expression patterns and in vivo engraftment of cells into mice (normal or immune-deficient, including PDX models) collect, isolate, and process cells entirely under the normoxic ambient air. This likely allows ambient air-induced artifacts as a potential block to better understanding cell growth and gene expression patterns as well as responsiveness in vitro to reagents used to alter these parameters. This is regardless of whether or not the cells removed in ambient air are then subsequently placed for further processing and study in a hypoxic environment. Air-induced changes in collected stem-cell populations occur rapidly by a process referred to as extra physiological shock/stress (EPHOSS; refs. 6, 7). Within minutes of collection of cells from mice or humans in ambient air, the cells are subjected to EPHOSS effects induced by a high oxygen containing ambient air environment. This phenomenon has now been reported by us for normal and gene modified mouse BM (e.g., cyclophilin D^−/−, p53^−/−, hypoxia inducing factor (hif)-1a^−/−, and MiR210^−/− mice) and human cord blood HSCs (6). Our study (6) demonstrated that cells collected under hypoxia (3% oxygen), so that the cells never experience ambient air oxygen, preserve normal mouse, and human HSC number and function, previously underestimated, that are decreased by ambient air EPHOSS-induced differentiation (not cell death) of HSCs to HPCs. EPHOSS has so far been linked to a p53/cyclophilin D/mitochondria permeability transition pore (MPTP) axis in which ambient air induces opening of the MPTP and increased release of mitochondrial reactive oxygen specifies. This is also mediated through an under-explored link with HIF-1α and the hypoxamir miR-210. The ability to detect and block the effects of EPHOSS on HSC differentiation requires very stringent methodology in which everything to be used (e.g., glassware, plastic ware, media, reagents, antibodies, etc.) must be preequilibrated within the hypoxia chamber at 3% oxygen for at least 16 hours, with care to make sure that while the actual studies are being done there is no or very minimal air oxygen leakage into the chamber. Moreover, cells collected in hypoxia are also injected into mice that are placed into a special holder within the hypoxia chamber that allows them to breath air but with their tails still within an atmosphere of 3% oxygen. Any break in this procedure, intentional or not, negates the ability to observe and block air-induced EPHOSS effects on tested cells (human or mouse) and the capability of these cells to engraft in mice. Other means to mimic EPHOSS-blocking effects of air collected cells have been published for mice using combinations of antioxidants and/or epigenetic enzyme inhibitors (8), but this has not yet been evaluated in human cells. Work in progress in our lab and in collaboration with other investigators at the Indiana University School of Medicine suggests a similar phenomenon in granulocyte colony stimulating factor- and Plerixafor/AMD3100-mobilized mouse peripheral blood, and in other mouse models of leukemia and preleukemia LS/ICs (Aljoufi and colleagues, manuscripts in preparation), for breast and ovarian cancer cells (Nakshatri and colleagues, manuscript in preparation) and for many mouse tissue immune cells (Kai and colleagues, manuscript in preparation), amongst other ongoing studies including a reevaluation of reported defects in hematopoiesis of aged (24–28 month old) mice (Capitano and colleagues, manuscript submitted).

Whether or not using stringent low oxygen collection/processing procedures will make a difference in gaining an enhanced understanding of LS/ICs and other cancer stem/initiating cells (CS/IC) remains to be determined. But it makes sense that if one truly wants to better understand these cells in the context of their in vivo growth environment, treatment responsiveness, and immune cell patterns, this should be seriously considered. Other factors that one should also consider for future efforts are the in vivo cellular microenvironmental niche components ex vivo in the context of their isolation/processing in low oxygen tension. In this way, no components in these assessments are subjected, even for minutes, to ambient air.

It will be of interest to see how more stringent low oxygen assessments might affect the results reported by Silvestri and colleagues (1) and others for LS/ICs and other CS/ICs in terms of ex vivo growth characteristics, gene and protein expression patterns, and responsiveness to agents used to modulate these activities for eventual health benefit. More importantly, will this and the studies of Silvestri and colleagues (1) eventually be able to be used in a clinical setting? We all certainly hope so.

No potential conflicts of interest were disclosed.

Some of the studies referenced by the author were supported by US Public Health Service Grants to the author: R35 (Outstanding Investigator Award) 139599 and U54 DK106846.