Is Genetic Instability the Key Driver or Progression?

The main functional changes that occur with progression of CML are marked changes in proliferation, differentiation, apoptosis, and adhesion. These functional changes accompanying progression are accompanied by profound changes in treatment response. But what drives progression?

There is growing evidence that BCR-ABL itself is a direct cause of the genetic instability seen in progression. While the contributions of these pathways in the initiation CML are logical, it is not clear that these pathways alone drive progression from chronic phase to advanced phase. However, insights that BCR-ABL causes genetic instability may prove to be a unifying theme in starting the basis for progression at the very beginning of CML. When considering the effect of BCR-ABL on genetic instability, one must confront a seeming paradox: cell lines with activated tyrosine kinases, such as BCR-ABL, accumulate more DNA damage than those cell lines without activated kinases, yet these cell lines also repair the DNA damage faster. The combination of more DNA damage and repair activity may lead to less accurate repair. As BCR-ABL upregulates antiapoptosis genes BCL2 and BCL-Xl, thus causing G2/M delay, cells accumulate DNA damage but are not targeted for cell death. It also appears that BCR-ABL directly causes DNA damage by increasing reactive oxygen species (ROS) ; ROS leads to DNA base-pair transversions (GC→TA) and transitions (GC→AT). Thus, unbridled BCR-ABL activity may contribute to the genetic instability leading to chromosomal aberrations, mutations, and changes in gene expression that cause progression.

Genetic Structural Changes

Chromosomal changes in addition to the Ph are the norm in blast crisis. Isochromosome i(17q) is a relatively common occurrence (∼20% of cases of blast-crisis cases), and this is likely associated with progression, as it means the loss of a copy of the p53 tumor suppressor gene. However, the remaining p53 allele does not seem to be mutated in these cases, so a direct link of p53 inactivation and progress to blast crisis is not so clear, though the reduction of the total cellular p53 level may upset the integration of genetic repair and apoptosis and thus contribute to progression. Alternatively, there may be critical but yet unknown genes on 17q contributing to progression. Trisomy 8 is also common in blast crisis (∼40%), which is interesting because Myc is located at 8q24. There are several lines of evidence linking MYC to progression. In vitro inhibition of c-Myc with antisense oligonucleotides, or dominant-negative constructs, can inhibit BCR-ABL transformation or leukemogenesis. Myc is often overexpressed in blast crisis compared with chronic phase ; whereas in acute myelogenous leukemia (AML) cases with trisomy 8, c-Myc is downregulated, but other genes on chromosome 8 are upregulated. However, trisomy 8 is a common feature of cases of CML that exhibit clonal evolution Ph-negative cells while in a CCyR (see later discussion). These cases of Ph-negative, trisomy 8 appear to have a benign course, suggesting that trisomy 8 may not be leukemogenic.

Translocations of known oncogenes in blast crisis occur relatively rarely (<5%). The most notable of these recurrent translocations are t(3;21) and t(7;11), involving the AML-1/EVI-1 and NUP98/HOXA9 genes, respectively. Evi-1 and HOXA9 are both transcription factors, and their aberrant expression in the context of these fusion proteins causes differentiation arrest in the case of AML-1/EVI-1, and increased proliferation in the case of NUP98/HOXAP. In mouse models, BCR-ABL and AML-1/EVI-1 coexpression creates a disease produces a picture of myeloid leukemia, whereas mice transplanted with cells transfected with NUP98/HOXAP develop a myeloproliferative disease that evolves into an acute leukemia. Recently it was shown that NUP98-HOXA9 increased the expression of the RNA binding protein Musashi 2 (Msi2), which in turn repressed the Numb expression, involved in the control of Notch signaling. Msi2 was found to be overexpressed in blast-crisis cases compared with chronic phase; moreover, increased Msi2 expression was found to be associated with poorer survival in all phases of CML following allogeneic transplantation.

Disruption of Critical Pathways in Progression

Activation of the MAPK proliferation pathway is central in myeloid leukemia (BCR-ABL in CML, RAS, FLT3, kit mutations in AML). Thus, it is not surprising that RAS and FLT3 mutations are rare in CML progression, because their activation would not add to the selective advantage already conferred by BCR-ABL. The tumor suppressor p53, inactivated in many solid tumors, is occasionally involved in blast crisis (∼20%–30%); it is not known if there are aberrations in genes involved in p53-mediated function. In lymphoid blast crisis (but not myeloid), a homozygous deletion of exon 2 of INK4A/ARF occurs commonly (∼50%). This deletion eliminates both p16 and p19, two proteins that normally check G1/S cell cycle progression and upregulate p53.

The activity of the tumor suppressor PP2A may be involved in the pathogenesis of CML progression and may provide a new drug target. PP2A activity is involved in regulating proliferation, survival, and differentiation, and is involved in the proteosomal degradation of BCR-ABL. Increasing BCR-ABL levels (induced in vitro or in human leukemia) increases the expression of the phosphoprotein SET, a negative regulator of PP2A. Thus, progression may set up a feedback loop whereby increasing BCR-ABL increases SET, decreasing PP2A, which further ensures the persistence of BCR-ABL. In in vitro and mouse models, restoration of PP2A activity by the activator forskolin appeared to decrease BCR-ABL leukemic potential, thus suggesting a potential therapeutic target to arrest or regress CML progression.

A block in myeloid differentiation occurs in progression, contributing to the accumulation of immature blasts. A search for mutations differentiation genes has been relatively unrewarding. For example, the CCAAT/enhancer binding protein α (CEBPA) is a member of the basic leucine zipper family of transcription factors essential to the control of granulocytic differentiation, regulating several genes necessary for orderly differentiation, including CSFR3. Inhibition of CEBPA in t(8;21) AML occurs through repression on CEBPA by the fusion AML1-ETO protein. In blast-crisis CML, high levels of BCR-ABL induce the MAPK phosphorylation of the poly(rC) binding protein hnENPE2, which then causes the translation block of CEBPA mRNA. However, complete loss of CEBPA, experimentally performed by transducing BCR-ABL into primitive murine cells engineered to have CEPBA deleted, confers an erythroid leukemia rather than a myeloproliferative disease. This implies that some residual CEPBA activity must be present, even in blast crisis, and that disturbances in proliferation in CML progression may result from subtle changes in the regulation of factors critical in controlling the flow of hematopoietic differentiation.

Genes associated with the maintenance of stem cell renewal have been logically associated with progression. Data from several sources seem to be converging on the Wnt/β-catenin pathway as critical for the evolution of CML. The Wnt/β-catenin signaling pathway has come front and center in normal and abnormal hematopoiesis. Wnt/β-catenin signaling is thought to be important in cell self-renewal, and mutations in β-catenin have been found in various epithelial solid tumors. Aberrant Wnt/β-catenin signaling has thus been demonstrated in CML and T-lineage acute lymphoblastic leukemia (ALL). In CML, activation of the Wnt/β-catenin pathway was observed in primary cell samples from patients with CML, with levels increasing with progression. β-Catenin activation in CML seemed to reside predominately in granulocyte-macrophage progenitor cells, and the self-renewal of these progenitors could be reduced by overexpression of Axin, which modulates free β-catenin.

Several transcription factors appear to be involved in CML progression. Jun B knock-out mice develop a myeloproliferative disorder similar to CML. Jun B has been shown to be downregulated in CML progression, and the decrease in junB does not appear to be mediated by methylation changes of junB promoters. Genes controlled by the transcription factors MZF1 and delta EF1 appear to be deregulated in CML progression. MZF1 is a member of the Kruppel family of zinc finger proteins originally cloned from a cDNA library from a blast-crisis CML patient, and plays a critical role in hematopoietic stem cell differentiation, including modulation of CD34 and c-myb expression, and MZF1 ^−/− knock-out mice display an increase in hematopoietic progenitor proliferation, which continues in long-term culture conditions. Both MZF1 and delta EF1 have been shown to influence cadherin expression. Moreover, MDFI, an inhibitor of myogenic basic helix-loop-helix transcription factors found overexpressed in CML progression, both interacts with axin and influences Jun signaling, thus perhaps linking these two pathways implicated in CML progression.