**5. Oncogenic proteoforms leading to leukemia**

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.

#### **6. High-throughput proteomics methodologies**

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

*Proteoforms - Concept and Applications in Medical Sciences*

patients.

**4.3 Non-coding microRNAs**

leukemic subtypes.

**4.4 Post-translational modifications**

both ALL and AML cases (manuscript in preparation).

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

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

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

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

**66**

provide useful information about both protein abundance and activity, as regulated by the PTM, the protein-protein and protein-DNA interactions. Nowadays, two of the most commonly used high-throughput techniques to study the proteome in leukemia are mass-spectrometry (MS)-based techniques and antibody-based techniques.

#### **6.1 MS-based**

MS is a high-throughput technique uses the formation of ions (charged fragments) from the protein analyte to distinguish between proteoforms. Those ions can be sorted and measured using electrical and/or magnetic fields based on their mass-to-charge ratio (m/z), and identification of the protein follows based on the abundance of those m/z-fragments [40]. Globally, proteins can be ionized with two distinct methods: matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In MALDI the protein sample is mixed with an energy absorbing matrix. Irradiation of this matrix causes vaporization of the matrix together with the sample, resulting in the formation of ions [41]. ESI creates ions using electrospray to dissolve the protein lysate, by applying high-voltage to the dissolvent to create an aerosol of small charged fragments. When a protein sample is highly complex, samples may require separation prior to MS analysis using 1D or 2D gel electrophoresis, high-pressure liquid chromatography (LC-MS), or gas chromatography (GC-MS) to maximize the sensitivity. Because proteoforms are derived from a single gene, they often contain homologous sequence regions, and because of the digestion step, information about the relationship between amino acid sequence and the PTM often lacks, this significantly complicates the process of identifying proteoforms. Several overviews have been published that discuss recent technological developments of MS to enable analysis distinct proteoforms [42–44].

#### **6.2 Antibody-based**

Another high-throughput approach is the protein microarray (PMA), of which two different types exist: forward phase protein arrays (FPPA) and reverse phase protein arrays (RPPA). Given that antibodies can be raised to specifically recognize sequence variations or PTM, they enable measurement of selected proteoforms. In FPPA, protein antibodies are immobilized on an array in known positions, and samples are then printed on the array. If a particular proteoform is present in the sample, the proteoform binds to the antibody and after exposure to a secondary antibody, the abundance can be measured. Each slide is incubated with a single protein sample, but multiple proteins can be measured simultaneously depending on the number of antibodies printed on the slide.

The "reverse" version of the FPPA is the RPPA methodology. In RPPA, samples are first printed on the array, and subsequently each slide is stained with a single protein antibody, followed by a secondary antibody to amplify the signal. The downsides of RPPA are that all samples must be printed at the same time to avoid methodological barriers due to printing irregularities between batches, and that RPPA can only be used to detect proteins for which a strictly validated antibody is available. As there is no separation of the proteins according to molecular weight, it is crucial that antibodies are proven to be highly specific, selective and reproducible. Plus, RPPA is biased to proteins and isoforms for which a strictly validated antibody is available. On the other hand, RPPA requires only a small number of cells (approximately 3 × 105 cells to test 400 different antibodies), making it highly suitable for retrospective clinical applications. As it in addition analyzes all samples at once, it allows a direct comparison of protein abundance across samples.

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

**7.1 Disease-specific proteoform landscape of acute myeloid leukemia and acute** 

Acute leukemia is a heterogeneous group of diseases both in terms of biology and prognosis. Classification into those arising from the myeloid or the lymphoid lineage is based on cytomorphology and cytochemistry, with further differentiation into specific subgroups based on morphology, immunophenotyping, cytogenetics, and molecular genetics of the acute leukemia cells. However, present classification systems are not adequate to differentiate between all subtypes and do not always accurately predict the clinical outcome. Whether changes in the leukemic cells that cause those differences are due to developmental, genetic, or environmental effects, they all are ultimately mediated by changes in protein abundance or modification. Therefore, we hypothesize that systematic comparative or differential proteomics can discover changes in the presence and quantity of individual proteoforms that underlie these cellular changes, and can add to current diagnostics, prognostics and

Assessment of the "diseased"-proteome compared to the proteome of the "normal/healthy" cells (e.g. CD34+, CD38+CD34+, CD38−CD34+; a discussion about the optimal normal comparator is discussed elsewhere [45]) can identify proteins that are aberrantly expressed or activated compared to normal, as well as can identify different forms of the same protein that differ between the diseased cell and the healthy comparator. This enables recognition of pathways utilization of cells present within a certain set of patients or related to a specific clinical feature. In addition, proteins or sets of proteins that are differentially expressed, may aid for

Furthermore, detailed proteomic profiling can help identifying differences between subgroups of diseases, including ALL and AML, and also between subgroups within one of both. It may be important (informative) to know how these two diseases are similar as well as how they differ. As ALL and AML are both dominated by immature malignant hematopoietic cells, they can serve as lineageindependent control for each other. Defining which proteins display similar expression in ALL and AML, but which are different compared to the "normal" healthy control, or to more mature cells, are likely to be related to a block in differentiation, whereas other proteins patterns that are similar in both, could be related to the hallmark of uncontrolled proliferation, resistance to cell death, or other shared

As example, Cui et al. performed proteomic analysis using 2D-MS for 61 bone marrow biopsies from patients diagnosed with French-American-British (FAB) M1-M5 AML or ALL [46, 47]. Comparative analysis, identified 27 proteins with lineage-specific expression. Among them, myeloperoxidase was already known to be highly expressed in AML compared to ALL, but they also recognized heat shock factor binding protein 1 (HSBP1) as being high in ALL. In addition, they found proteins that were higher expressed in M2 and M3 AML compared to M1, and 23 proteins that were differentially expressed between granulocytic lineage (M1, M2, M3) AML, and AML derived from the monocytic lineage (M5). To prove clinical usefulness, Cui et al. also applied proteomic analysis to an AML-M3 bone marrow (which was classified based on morphology by the presence of atypical granules) from a patient who did not respond to the standard differentiation-inducing therapy with all-*trans* retinoic acid or As2O3. Their analysis showed that this sample exhibited a "protein expression profile" specific to M1, and not to M3, and after

confirmatory diagnostic purposes and early disease detection.

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

**7. Proteomics in acute leukemia**

**lymphoid leukemia**

therapeutics.

deregulations.
