Joby L. McKenzie

Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.

Although genetic abnormalities associated with hematological malignancies are readily identified, the natural history of human leukemia cannot be observed because initiating and subsequent transforming events occur before clinical presentation. Furthermore, it has not been possible to study leukemogenesis in vitro as normal human cells do not spontaneously transform. Thus, the nature and sequence of genetic changes required to convert human hematopoietic cells into leukemia cells have never been directly examined. We have developed a system where the first step in the leukemogenic process is an engineered disruption of differentiation and self-renewal due to expression of the TLS-ERG oncogene, followed in some cases by overexpression of hTERT. In two of 13 experiments, transduced cells underwent step-wise transformation and immortalization through spontaneous acquisition of additional changes. The acquired karyotypic abnormalities and alterations including upregulation of Bmi-1 and telomerase all occur in acute myeloid leukemia (AML), establishing the relevance of this system. One resultant cell line studied in depth exhibits cellular properties characteristic of AML, notably a hierarchical organization initiated by leukemic stem cells that differentiate abnormally. These findings provide direct evidence for multiple cooperating events in human leukemogenesis, and provide a foundation for studying the genetic changes that occur during leukemic initiation and progression.

58 – High Resolution Clonal Marking-Analysis

An in vivo repopulation assay for human HSCs (hematopoietic stem cells) was only recently developed. HSCs are challenging to study because they are rare, rendering HSC analysis equivalent to looking for a needle in a haystack. Most stem cell assays are geared toward cell population studies. However, to gain a detailed analysis of the functional capacity of an entire HSC pool, analysis must be done at the level of the individual cell. An individual HSC can be studied by transplantation with a single purified HSC or with a population of uniquely marked HSCs. The field of stem cell biology has evolved greatly since the early 1960s, when Till and McCulloch developed the first quantitative assay for individual stem cells. HSC evaluation can only take place in the context of an in vivo assay system, since current in vitro assays are unable to maintain intrinsic stem cell characteristics. The murine model was a convenient system to begin analysis of the HSC; thus, there is a marked difference in ones knowledge of the murine HSC in comparison to the human HSC. Human HSCs must be uniquely tagged to track stem cell behavior at the single-cell level. By contrast, murine HSCs can be purified and transplanted at the single-cell level.