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GE-based biomarkers in CML
ciated silencing in BC progression were enriched for down- regulated genes identified in the datasets of both McWeeney et al. and Yong et al.10 The cross-validation of these three independent datasets suggests important les- sons for the development of GE-based risk assessment: (i) the discovery of reproducible GE-based biomarkers is pos- sible when homogeneous CD34+ populations are used; (ii) the processes of TKI resistance and BC transformation are biologically convergent despite genetic heterogeneity:10 and (iii). PRC-regulated processes contribute to silencing of prognostically informative genes.
Contribution of somatic mutations to gene expression signatures
Recent reviews have described the range and frequency of specific genetic mutations in patients who developed TKI resistance and/or BC.37 For many of these genes, there is strong preclinical information indicating that their asso- ciated mutations contribute to or are even sufficient to pro- duce TKI resistance or transformation phenotypes (sum- marized in Table 3).38-48 These studies imply that genetic mutations alter GE profiles, and here we review their con- tributions to GE changes in CML since these changes may represent useful GE-based biomarkers.
For RUNX1 mutations, the Mustjoki group identified an accompanying GE signature in BC samples.49 They found that RUNX1 mutations were associated with the upregula- tion of stemness, B-cell markers, interferon and immune signaling and transcription factors regulating plasmacytoid dendritic cell development.
In analogous work, the overexpression of an IKZF1 dominant-negative mutant in CD34+ cells from CP patients increased STAT5 expression, a pathway associated with imatinib resistance,50 and enhanced transformation.51 RAG expression status was recently assessed in diagnostic sam- ples, given the role of RAG recombination as a mediator of IKZF1 deletions.52 Notably, RAG1/2 and DNTT upregula- tion at diagnosis suggested imminent lymphoid BC trans- formation within 12 months (8/8 patients), demonstrating that GE signatures can reliably predict transformation.
Despite limited functional interrogation of ASXL1 using CML patient material, insertion sites within ASXL1 pro- moted BC progression in a CP mouse model subjected to transposition-based mutagenesis.53 Transgenic expression of truncated protein ASXL1aa1-587 in mice increased HSC self-renewal, and Brd4 occupancy and chromatin accessi- bility around genes required for stemness, and predisposed mice to myeloid malignancies.54 However, the clinical rele- vance of diagnostic ASXL1 mutations is still unclear because some patients with ASXL1 variants at diagnosis can achieve a MMR after TKI therapy.14 Furthermore, ASXL1 mutations frequently disappeared when monitored in the long-term during TKI therapy (personal observation by Dr. Dennis Kim). Meanwhile, direct evidence for contribu- tions of other mutations to CML GE signatures is currently lacking, and we have to infer them from studies in other malignancies (Table 3).
Lessons from clonal hematopoiesis
Clonal hematopoiesis is the clinical phenomenon by which populations of hematopoietic cells expand and
carry a somatic mutation that is at least 2% of the variant allele fraction.55 The common genes comprise DNMT3A, TET2, and ASXL1, and others also found in CML individ- uals, including RUNX1, BCORL1, and TP53.56 Individuals with clonal hematopoiesis are at increased risk of devel- oping hematologic malignancies, and it is therefore per- tinent to ask whether clonal hematopoiesis-related mutations also confer increased risk of TKI resistance or progression. A study by Kim et al. has highlighted impor- tant features of clonal hematopoiesis-related mutations in CML.57 Firstly, they may occur in a non-Philadelphia chromosome-positive clone and predate the develop- ment of CML, and are unrelated to the CML clone. Secondly, even when a specific mutation occurs in the Philadelphia chromosome-positive clone, it only confers a relative risk of TKI resistance or progression. Indeed, patients with RUNX1 mutations have been documented to achieve MMR (personal observation, Dr. Dennis Kim). Nevertheless, Kim et al. concluded that mutations in genes regulating epigenetic function (TET2, ASXL1 among them) were associated with a higher risk of infe- rior TKI responses.
There are also strong preclinical data indicating that clonal hematopoiesis-related mutations result in subtle but important changes in GE in HSC. For example, Dnmt3a-deficient HSC show a loss of DNA methylation in regions enriched for self-renewal genes such as Meis1, Evi1 and HoxA9.58 In Tet2-deficient mice, the loss of DNA demethylation is accompanied by an expansion of the stem and progenitor cell compartments, and eventual myeloproliferation.45 In ASXL1-deficient mice, an increase in self-renewal capacity of stem cells is observed, through the loss of PRC1-mediated gene repression.59 Another interesting aspect of hematopoietic stem and progenitor cells harboring inactivating muta- tions of DNMT3A and TET2 is that they both led to increased cytokine production in peripheral myeloid cells, including interleukin-6 and interleukin-1b.60,61 Furthermore, mutations associated with clonal hematopoiesis are frequently found in monocytes, gran- ulocytes, and natural killer cells compared to B or T cells, suggesting that their effects may also be manifest in mul- tiple differentiated cell types within the hematopoietic compartment.62 Together, these observations are relevant to the search for prognostic GE signatures in CML for the following reasons: (i) increased inflammation and cytokine production is associated with LSC persistence,32 and disease progression;10,63 (ii) prognostic GE changes may be found in both CD34+ and CD34– fractions of peripheral blood or bone marrow mononuclear cells; and (iii) changes in natural killer cell function and number may predict treatment-free remissions, and presumably contain informative natural killer cell GE signatures.64-66
Epigenetic contributions to gene expression signatures
Polycomb repressive complex-associated gene expression changes
Among the most well studied epigenetic complexes in CML are the polycomb group (PcG) proteins.67 The poly- comb group proteins assemble into two complexes, PRC2 and PRC1, which modify histones through repressive H3K27 trimethylation (H3K27me3) and H2AK119
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