**4.2 Chromosomal translocations**

Chromosomal abnormalities, gene fusions and copy number aberrations are more common in the younger patient population [28]. The ratio of structural variation to mutational burden decreases continuously with age, with the most chromosomal translocations in infants (<1 year) compared to all other ages. Within this young age-group, the most common fusion involves *KMT2A* (also known as *MLL1*), present in 38% of the infants [28]. A second age-peak is recognized in young to middle aged AML adults. Overall, more than 80 fusion partners of KMT2A are described and it is the protein partner of KMT2A that determine characteristics specific to age and disease. Interestingly, 50% of the infants younger than 1 year with ALL contain the specific MLL-AF4 fusion protein caused by the t(4;11)(q21,q23) translocation [29], whereas in AML, the most common MLL rearrangement is the MLL-AF9 that arises from a t(9;11)(p22,q23) translocation. In both populations, MLL leukemia confers poor prognosis and identification of unique proteoforms in this subtype leukemia may guide treatment stratification by providing targetable leads. For instance, downstream proteomic targets mediated by MLL-AF4 include *HOX*, *EPHA7*, *MEIS*, *PBX* and *GSK-3* and these are already considered or investigated as therapeutic targets in the context of MLL-rearranged

leukemia. In addition, *RAS*, *DOT1L*, and *HSP-90* also have been described as potential targets in MLL leukemia [30]. As those genes and their protein products are in particular involved in transcription regulation, we hypothesize that patients that expression differences in those conserved genes, likely also harbor differences in abundance, or proteoforms of its downstream proteins, compared to wild-type patients.

### **4.3 Non-coding microRNAs**

MicroRNAs (miRNA) are small non-coding RNAs that affect the proteome through their binding to mRNA influencing/inhibiting the translation to proteins. Aberrant miRNA expression is associated with leukemogenesis [31], and multiple miRNAs are found to be expressed differently upon age. A study by Noren Hooten et al. showed downregulation of miRNA expression in peripheral blood of healthy individuals with advancing age. Cancer is often age-related and five out of nine downregulated miRNAs in this study were related to cancer pathogenesis [32]. Another study compared miRNA profiles between pediatric and adult patients with AML and again, identified significant lower miRNA expression in adults compared to children. In addition, they found distinct miRNA expression patterns in both t(8;21) and t(15;17) translocated pediatric AML, but not in adults. Also, nine-fold upregulation of miR-21 was identified in the MLL-rearranged pediatric patients compared to others and this finding was also not reflected by the MLL-rearranged adult population [33]. The identification of age-specific miRNA specific expressing in leukemia together with the fact that miRNA will affect the final proteomic state, indicates that further proteomic approaches could likely unravel differences in proteoforms between younger and older patients within leukemic subtypes.

#### **4.4 Post-translational modifications**

DNA is wrapped around histone to form a compact chromatin structure and PTMs on histone tails, such as the addition or removal of methyl or acetyl groups on lysine residues, or direct DNA methylation regulate chromatin accessibility and initiate and maintain gene expression patterns that account for specific cell lineage differentiation and development [34]. Packaging of the chromatin structure changes with age and include global loss of heterochromatin resulting in a more open chromatin state in the elderly. Reduction of heterochromatin due to increased histone acetylation during aging is also well-established [35, 36], but less well-characterized is the role of histone methylation. Since the prevalence of AML increases with age, we asked ourselves if histone methylation profiles are different between pediatric and adult AML. We recently applied RPPA-based profiling using antibodies against multiple histone methylation sites which enabled us to define disease and age characteristic patterns of histone modification. In agreement with our hypothesis, a significant decline in histone methylation was seen upon age in both ALL and AML cases (manuscript in preparation).

As mentioned, MLL-rearrangements are specific to age and disease, and are frequently altered in leukemia. As MLL fusion proteins modulate the chromatin structure by histone tail modifications, MLL-rearranged leukemia is considered as epigenetic malignancy. In addition, mutations in proteins that modify the histone PTM process (e.g. writers, erasers and readers) are more frequently found in T-ALL compared to other childhood malignancies, and distinct DNA methylation patterns were recognized among different subtypes of ALL. Those patterns correlated with changed transcriptomes. Aberrant DNA methylation is associated with silencing of

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*Proteoforms in Acute Leukemia: Evaluation of Age- and Disease-Specific Proteoform Patterns*

genes that involved in lymphoid development, and contribute to leukemogenesis. By combining DNA methylation and transcriptome analysis, transcriptional silencing via promotor hypermethylation was recently identified in pediatric AML [28],

Since hypomethylating agents have been widely used to treat, in particular the older leukemia patients, we hypothesize that proteomics can help to identify more refined subgroups (maybe even from the younger population) that can be treated with certain treatment regimens that alter the epigenome. For instance, the discovery of a specific protein or proteomic signature (either related to the epigenome or not) that is correlated with sensitivity to hypomethylating agents, can potentially act as biomarker in MLL-rearranged leukemia, to select patients that can benefit from those agents. In the experimental setting, therapies with hypomethylating agents have already showed re-expression of the hypermethylated genes along with restored chemosensitivity, and in relapsed ALL, increased promotor methylation was found to be related to increased chemoresistance [37]. If it is possible to identify a set of proteins that is specific for relapsed MLL-rearrangement AML and/or ALL, rapid tests (e.g. ELISA, IHC or FPPA) could be developed to quickly provide information about the protein abundance in relapsed patients, to identify those who will benefit from additional treatment, as well as, *a priori*, predict which newly diagnosed patients are most likely to relapse, and treat those with additional

Mutations in the DNA of the hematopoietic stem cells play a pivotal role in leukemogenesis and within single genes, multiple mutations have been identified that results in different forms of the protein. One example involves transcription factor CCAAT/enhancer binding protein A (CEBPA) mutated AML patients, which is known to regulate growth arrest and differentiation in hematopoiesis by promoting granulocyte lineage differentiation in common myeloid progenitor cells, and disruption of normal CEBPA expression in myeloid progenitors may lead to a block in granulopoiesis resulting in erythropoiesis in its place [38]. As critical regulator of myeloid lineage development it is not surprising that CEBPA is mutated in ~10% of AML patients and most frequently classified as myeloblastic AML subtype M1 of M2 according the French-American-British (FAB) classification. CEBPA transcript translates for a full-length (CEBPA-p42) or shorter isoform (CEBPA-p30). CEBPA-p30 isoforms contain the DNA binding domain but lack the N-terminal transactivation domain. However, CEBPA-p30 is dominant negative by reducing transcriptional activity after heterodimerization with full-length CEBPA-p42. About half of CEBPA mutated AML patients have one allele with a N-terminal mutation and one allele with a C-terminal mutation. The N-terminal mutant results in translational termination of the fulllength isoform and increase truncated CEBPA-p30 expression. In contrast, C-terminal mutations in CEBPA-p42 are mostly characterized by in-frame basic region leucine zipper (bZIP) variants inhibiting normal CEBPA function by disrupting DNA binding and dimerization [39]. CEBPA mutated patients might be candidates for inhibition of

the oncogenic CEBPA-p30 isoform to recover the disrupted p42/p30-ratio.

Proteomics may be the least developed and investigated "-omics" approach, it is likely one of the most informative for understanding of cellular behavior as it can

**6. High-throughput proteomics methodologies**

*DOI: http://dx.doi.org/10.5772/intechopen.90329*

treatment to prevent relapse.

and correlated with age, karyotype and outcome.

**5. Oncogenic proteoforms leading to leukemia**

*Proteoforms in Acute Leukemia: Evaluation of Age- and Disease-Specific Proteoform Patterns DOI: http://dx.doi.org/10.5772/intechopen.90329*

genes that involved in lymphoid development, and contribute to leukemogenesis. By combining DNA methylation and transcriptome analysis, transcriptional silencing via promotor hypermethylation was recently identified in pediatric AML [28], and correlated with age, karyotype and outcome.

Since hypomethylating agents have been widely used to treat, in particular the older leukemia patients, we hypothesize that proteomics can help to identify more refined subgroups (maybe even from the younger population) that can be treated with certain treatment regimens that alter the epigenome. For instance, the discovery of a specific protein or proteomic signature (either related to the epigenome or not) that is correlated with sensitivity to hypomethylating agents, can potentially act as biomarker in MLL-rearranged leukemia, to select patients that can benefit from those agents. In the experimental setting, therapies with hypomethylating agents have already showed re-expression of the hypermethylated genes along with restored chemosensitivity, and in relapsed ALL, increased promotor methylation was found to be related to increased chemoresistance [37]. If it is possible to identify a set of proteins that is specific for relapsed MLL-rearrangement AML and/or ALL, rapid tests (e.g. ELISA, IHC or FPPA) could be developed to quickly provide information about the protein abundance in relapsed patients, to identify those who will benefit from additional treatment, as well as, *a priori*, predict which newly diagnosed patients are most likely to relapse, and treat those with additional treatment to prevent relapse.
