In Vivo Analyses of Leukemia Stem Cells in Genetically Defined Murine Models of Chronic and Blast Crisis CML.
Authors: Jordan Craig T. , Neering Sarah J. , Wang Pin-Yi , Rossi Randall M. , Bushnell Timothy 
Published: October 2019

Abstract
Studies to date have shown that primary human leukemia stem cells (LSC) are resistant to standard chemotherapy agents and are likely to be a major cause of drug refractory disease and relapse. Therefore, elucidating the in vivo biology of LSC is critical in order to develop more effective therapeutic regimens. To this end, we report the first genetically defined model of LSC, using syngeneic murine systems in which the biological features of human LSC are recapitulated. The approach employs retroviral vectors to transduce normal murine hematopoietic stem cells with either BCR/ABL-GFP alone, or in combination with Nup98/HoxA9-YFP. Expression of BCR/ABL creates a well-described model of chronic phase CML, whereas expression of BCR/ABL in combination with Nup98/HoxA9 induces acute disease that mimics blast crisis CML. Analysis of the normal cell competent to generate LSC indicates that the BCR/ABL mutation must occur in primitive HSC in order to manifest disease, however, subsequent progression to blast crisis can occur through mutation in cells at the myeloid progenitor stage. Characterization of stem cells in these models revealed several striking features. First, chronic phase stem cells are1 phenotypically identical to normal hematopoietic stem cells (lin−, Sca-1+, c-kit+) and display cell cycle rates (percentage of cells in S or G2 phase) that are nearly double normal controls. However, the overall frequency of such cells is not elevated. In contrast, blast crisis stem cells show a distinct immunophenotype (lin−, Sca-1+, c-kit-lo, Flt3+, CD150−) and cycle rates nearly identical to normal controls, but are approximately 10-fold increased numbers. These data indicate that BCR/ABL alone functions as a stem cell mitogen, but does not enhance self-renewal, whereas added expression of Nup98/HoxA9 is sufficient to increase self-renewal, but return cell cycle regulation to normal levels. Furthermore, analysis of co-resident non-leukemic cells in each model shows that while the cycle activity of normal stem cells (HSC) was not affected, the cycle rates of normal progenitors (lin−, c-kit+) were substantially reduced. Thus, in either disease, active suppression of normal progenitors is evident and thereby increases the growth advantage of malignant populations. To test methods for modulation of normal vs. leukemic cells in vivo, we challenged blast crisis animals with ara-C (single dose, 100mg/kg) or imatinib mesylate (200mg/kg/day for 3 consecutive days) and assessed the consequences in primitive populations. The data indicate that ara-C reduced frequency and cycle rate of progenitor cells in vivo, but that the effects were identical between normal and malignant populations. Thus, at least for short-term studies there was no therapeutic index for ara-C at the level of primitive cells. In contrast, treatment with imatinib induced a 50% increase in the cycle rate and a 2–4 fold increase in numbers of progenitor cells. These findings imply a homeostatic mechanism in blast crisis leukemia, where pressure towards the malignant population may induce increased activity of stem and progenitor cells. In summary, this model provides a novel means by which the biology of LSCs may be directly characterized and the consequences of candidate treatment regimens can be assessed with regard to normal vs. leukemia stem cells in vivo.