The Role of hnRNPs in Acute Myeloid Leukemia Thesis

The blood system unites all the organs involved in hematopoiesis and implementing blood roles and functions. This system includes blood, red bone marrow, thymus, spleen, lymph nodes, lymphoid tissue of non-hematopoietic organs, and blood cells in connective and epithelial tissues (Maloy & Hughes, 2013). The elements of the system closely interact and obey the general laws of neurohumoral regulation, are genetically and functionally related. Understanding the blood system involves the functions and roles, the development and structure of system elements.

The lymphatic and immune systems are closely related to the blood system. Since immunocytes are formed in the hematopoietic organs, they circulate and recirculate in the peripheral blood and lymph. Blood and lymph are tissues of mesenchymal origin, which consist of plasma and corpuscles suspended in it and form the internal environment of the body (Maloy & Hughes, 2013). In the blood and lymph, the elements exchange constantly, including substances in the plasma, since these two systems are closely connected. Lymphocytes recirculate from blood to lymph and from lymph to blood, and all blood cells are created from pluripotent stem cells.

Hematopoiesis

The development of blood cells from a pluripotent stem cell occurs during embryogenesis and after birth. These stages are called embryonic hematopoiesis and postembryonic hematopoiesis. Embryonic hematopoiesis occurs during the embryonic period when the blood develops as a tissue. At this stage, different organs of the body are involved in the creation of blood cells. Then, postembryonic hematopoiesis occurs after birth and is a blood regeneration process (Maloy & Hughes, 2013). Postembryonic hematopoiesis includes the development of erythrocytes, granulocytes, platelets, monocytes, lymphocytes, and immunocytes – erythropoiesis, granutocytopoiesis, thrombocytopoiesis, monocytopoiesis, lymphocytopoiesis, and immunocytopoiesis.

Overview of Hematopoiesis

Hematopoiesis is a process that continues throughout a person’s life. Its main function is the formation and renewal of blood cells that would meet daily needs or help mitigate risks such as injury or infection (Maloy & Hughes, 2013). Interestingly, hematopoiesis is a very active and comprehensive process, and every day one trillion blood cells are produced in the body of an adult, including 200 billion red blood cells and 70 billion neutrophils (Maloy & Hughes, 2013). The process of hematopoiesis is regulated by various mechanisms studied separately, including stochastic and instructive mechanisms.

Hematopoietic Stem and Progenitor Cells

Particular attention should be paid to the haematopoietic stem and progenitor cells, which play a central role in the hematopoietic process. Hematopoietic stem cells (HSC) can be symbolically placed at the top of the hematopoietic pyramid. They have fundamental qualities: the ability to self-renewal and the ability to a multipotent differentiation into all mature bloodlines (Maloy & Hughes, 2013). Self-renewal occurs through division leading to the creation of two HSCs, and differentiation is the creation of cells from all mature bloodlines, i.e. erythrocytes, platelets, lymphocytes, monocytes, and granulocytes.

Scientists have been actively investigating hematopoiesis by HSC since the 1950s. Thanks to experiments on mice, it was proved that hematopoiesis of irradiated animals is restored with the participation of spleen and bone marrow cells (Maloy & Hughes, 2013). Later, the concept of chimerism was introduced, that is, the restoration of host cells from donor cells (Maloy & Hughes, 2013). Further, in the 1960s, a revolutionary discovery was made about the fact of self-renewal of individual clonogenic cells inside the bone marrow, which, after self-renewal, restored hematopoiesis. This discovery postulated the existence of HSCs in vivo (Till et al., 1964; Till & McCulloch, 1980). Equally important, this research became fundamental for the subsequent clinical transplantation of hematopoiesis.

Hematopoietic Regulation by Transcription Factors

Once again, it should be emphasized that HSCs are located in the bone marrow and are responsible for maintaining the entire ventilation system, being at the top of the blood pyramid. During embryonic hematopoiesis, the final HSCs are produced from the mesoderm and inoculate the fetal liver before migrating to the brain. Scientists came to this conclusion following the data obtained as a result of observations of hematopoiesis in mice (Wilkinson & Göttgens, 2013). In the postembryonic period of hematopoiesis, HSCs are at rest and retain their qualities of potential for self-renewal, division, proliferation, and differentiation into any mature type of hematopoietic cells (Wilkinson & Göttgens, 2013). The specification of HSCs during development and their cellular selection is controlled at the transcriptional level. Transcriptional regulators are present at the level of HSC specification, expansion, homeostasis, and differentiation. These include transcription factors, signaling pathway effectors, and epigenetic modifiers.

Scientists are analyzing transcription factors during the embryonic period of hematopoiesis by analyzing the pathways of HSC formation during stages of fetal development and stages of hematopoiesis. There is a transcriptional network that regulates the appearance of blood cells, taking into account the transcription factors that are necessary for the process of hematopoiesis. Many factors are involved in the transcriptional network, the purpose of which is to regulate hematopoiesis. The central components of these factors are ETV2, SCL and RUNX1 (Wilkinson & Göttgens, 2013). They are essential for the formation of blood cells and therefore cannot be ruled out. Each factor controls a certain precise stage in the specification of the hematopoietic process, for example, the germ layer of the mesoderm or fully functional blood precursors.

An understanding of the process of hematopoiesis and, in particular, transcriptional control was necessary for scientists to develop protocols for the generation of blood cells de novo. The process has also been studied by “reprogramming somatic cells, or by direct programming of pluripotent stem cells” (Menegatti et al., 2019, p. 3304). Remarkably, the process of creating cells in the laboratory is completely different from a similar physiological process.

Hematopoietic Regulation by Growth Factors

Hematopoietic cells are multipotent because they give rise to cell lines. The final elements of these cell lines form the blood and immune system cells. In other words, hematopoietic cells are proteins that regulate the growth and differentiation of red and white blood cells (Lee et al., 2020). The growth regulatory factors of these cells include the stem cell factor (cytokine SCF) involved in the proliferation and self-assessment of hematopoietic cells, erythropoietin, and colony-stimulating factors (CSFS) that stimulate the production of blood cells (Lee et al., 2020). HSCs continually produce new blood cells, and this process continues throughout the life of the body due to their qualities of self-renewal and differentiation. The process of hematopoiesis is controlled by the complex microenvironment of the bone marrow niche. This microenvironment consists of different types of bone marrow cells and is regulated by growth factors and cytokines. The stage of hematopoiesis within this microenvironment is called bone marrow niche homeostasis.

Dysregulation in Haematopoietic

Hematopoietic stem cells (HSC) provide the blood system with mature hematopoietic cells throughout life. They also form a reservoir for replenishing the hematopoietic system if the body urgently needs blood in the event of acute blood loss. To ensure the continuity of the production of hematopoietic cells through evolution, complex regulatory programs have arisen. These programs control differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) (Basilico & Göttgens, 2017). Dysregulation can occur if leukemogenic mutations disrupt regulatory programs. Such mutations can lead to the blocking of differentiation with a simultaneous increase in spread (Basilico & Göttgens, 2017). In particular, the regulatory programs of HSPCs can be disrupted by leukemogenic fusion genes that contain a mixed leukemia gene (MLL). In normal hematopoiesis, regulatory programs and HSPC progenitor cells are in the bone marrow, at rest, and contribute to the stability of hematopoiesis.

Acute Myeloid Leukemia

Acute myeloid leukemia or AML is an aggressive myeloid cell cancer. Leukemia is a cancer of white blood cells, and the term acute leukemia is used to describe the rapid and aggressive progression of this cancer. Therefore, leukemia requires immediate treatment, since the risks of death due to the rapid development of the disease are very high (“Acute myeloid leukemia,” 2021). There is a classification of acute leukemia depending on the type of leukocytes affected. The two main types of white blood cells are lymphocytes and myeloid cells. Lymphocytes fight viral infections, and myeloid cells have more functions, including fighting bacterial infections, preventing tissue damage, and protecting the body from parasites.

Overview of AML

First, the symptoms of AML should be described, which arise from an increase in the number of immature white blood cells, develop over several weeks, and quickly become more severe. Symptoms include pale skin, shortness of breath, fatigue, heavy sweating, weight loss, high fever, fever, frequent infections, frequent bleeding such as from the gums or nose, and flat red or purple patches on the skin (“Acute myeloid leukemia,” 2021). Other symptoms also include bruising easily, bone and joint pain, abdominal discomfort, swollen glands in the neck, armpits, or groin, and patients may experience pain when the glands are touched.

Etiology of the Acute Myeloid Leukemia

Etiology is the science of the origin of diseases, the conditions, and the causes of their occurrence. Acute myeloid leukemia has a complex etiology, which will be outlined below. AML is caused by a DNA mutation in bone marrow stem cells (HSC). As a result of the mutation, HSCs begin to produce more white blood cells than required, which are immature and cannot fight infections (“Acute myeloid leukemia,” 2021). Due to the increased flow of immature cells, the level of healthy red blood cells and platelets in the blood decreases, and this causes the symptoms listed above. At the same time, the reasons for the genetic mutation in AML are still unknown. Scientists have identified the existence of several risk factors for the development of AML.

These are risk factors for radiation exposure at high levels, such as when receiving radiation therapy as part of cancer treatment. Benzene, which is found in gasoline and is usually released in the rubber industry, is another serious risk factor. Equally important, benzene is found in cigarette smoke, which is why smokers have an increased risk of AML (“Acute myeloid leukemia,” 2021). Treating other cancers with chemotherapy drugs is also a risk factor. Other risk factors are blood disorders such as myelodysplasia, myelofibrosis or polycythemia vera, and genetic disorders, including Down syndrome and Fanconi’s anemia.

Diagnosis of Acute Myeloid Leukemia

Diagnosis of AML can involve multiple tests, as it is a complex disease. In the early stages of diagnosis, the therapist checks for physical signs, including those listed above, and requires a blood test. In the case of abnormal white blood cells or a very low blood count, the test indicates the possibility of leukemia. In such a situation, the therapist refers the patient to a hematologist who performs additional tests to clarify the diagnosis (“Acute myeloid leukemia,” 2021). A bone marrow biopsy is a next step in the tests that are done to confirm the diagnosis of AML. For this test, the doctor uses a thin needle to draw out a sample of fluid bone marrow, which is removed from the back of the thigh bone. The bone marrow sample is then checked for cancer cells. If these cells are present, the doctor uses the same sample to determine the type of leukemia.

As the disease progresses, doctors use tests such as genetic testing, scans, and lumbar puncture to get more information about the course of the disease and the extent of AML. Genetic testing helps determine the type of AML, and blood and bone marrow samples are tested (“Acute myeloid leukemia,” 2021). An X-ray or ultrasound scan of the heart is needed to check the health of the lungs and heart, and to assess overall health to determine the type of further treatment. A lumbar puncture tests the risk of AML spreading to the nervous system, and the doctor uses a needle to draw out cerebrospinal fluid and check for cancer cells. If cancer cells are found, this affects the type of further treatment.

Classification and Clinical Features of Acute Myeloid Leukemia

The AML classification differs from the traditional classification of other cancers according to stages. Traditionally, the stage is considered as it determines how far cancer has spread. The stage can affect the general wellbeing of a person, including emotional and mental state, and this information is needed to choose further treatment (Hwang, 2020). Acute myeloid leukemia usually does not lead to the formation of tumors, and is usually widespread in the bone marrow, and, in some cases, in the liver and spleen (Hwang, 2020). Therefore, AML is classified not according to stages, but within the subtypes of AML that are determined by laboratory tests. The age and general health of the patient and additional tests also affect the prognosis, as does the AML subtype.

The medications used to treat the different subtypes of AML usually differ. Two main systems are used to classify AML: the French-American-British (FAB) classification and the World Health Organization (WHO) classification. FAB was developed in the 1970s, and divides the disease into subtypes from M0 to M7, distinguishing between the types of cells from which leukemia originated, and indicators of cell maturity (Hwang, 2020). This system was formed by examining cells under a microscope. According to this classification, M0 is undifferentiated acute myeloid leukemia, M1 is acute myeloblastic leukemia with minimal maturation, M2 is acute myeloblastic leukemia with maturation, M3 is acute promyelocytic leukemia, M4 is acute myelocytic leukemia, and M4 eos is acute myelomonocytic leukemia with eosinophilia. Then, M5 is acute monocytic leukemia, M6 is acute erythroid leukemia, M7 is acute megakaryoblastic leukemia.

The WHO classification, in contrast to the FAB, takes into account the factors affecting the prognosis and was determined in 2016. Therefore, the classification includes determining factors, which makes it more convenient to understand the types of disease (Hwang, 2020). According to the WHO classification, there are AML with a translocation between chromosomes 8 and 21, AML with a translocation or inversion on chromosome 16 (Hwang, 2020). These are also APL with a hybrid PML-RARA gene, AML with a translocation between chromosomes 9 and 11, AML with a translocation between chromosomes 6 and 9, AML with a translocation or inversion of chromosome 3, AML (megakaryoblastic) with a translocation between chromosomes 1 and 22, AML with a mutated NPM1 gene.

There are also AML with a mutated RUNX1 gene, AML with changes associated with myelodysplasia, AML associated with previous chemotherapy or radiation therapy. Other types are AML unless otherwise noted, AML with minimal differentiation (FAB M0), AML without maturation (FAB M1), AML with maturation (FAB M2), acute myelomonocytic leukemia (FAB M4), acute monoblastic/monocytic leukemia (FAB M5), pure erythroid leukemia (FAB M6), acute megakaryoblastic leukemia (FAB M7) (Hwang, 2020). The classification also includes acute basophilic leukemia, acute panmyellosis with fibrosis, myeloid sarcoma, and myeloid proliferation associated with Down’s syndrome. Not included in the classification or undifferentiated types are leukemias, which are called mixed phenotype leukemias, they have lymphocytic and myeloid features.

Treatment of the Acute Myeloid Leukemia

Due to the nature of the disease, which is rapidly progressing, treatment is started as soon as the diagnosis is confirmed. AML is a complex disease and is treated by a multidisciplinary team of specialists (“Acute myeloid leukemia,” 2021). The treatment plan consists of two stages, including induction and consolidation. During induction, doctors aim to destroy as many leukemia cells in the blood and bone marrow as possible to stop symptoms and premature death. During the consolidation stage, doctors set the task of preventing recurrence and killing the remaining leukemia cells. In some cases, the induction phase must be completed several times before consolidation can begin.

If there is a high risk of complications, less intensive chemotherapy or alternative treatments are given. The induction stage depends on the absence of the risk of complications and the possibility of intensive chemotherapy, that is, when the patient receives a high dose of the drug, in the form of a combination of 2 or more drugs (“Acute myeloid leukemia,” 2021). As a rule, patients undergo 2 courses of induction, that is, intensive chemotherapy, at a hospital or other medical facility. During the courses, health workers carry out medical supervision, regularly transfuse the patient’s blood so that it contains enough healthy blood cells. The patient at this time is vulnerable to infections and is kept in a clean, stable environment with medical control.

Conventional Therapeutic in AML

Traditional therapy is based on methods developed in the 1970s and includes an intensive induction regimen of cytarabine and anthracycline. In the case of good indicators, after the induction stage, patients proceed to the consolidation stage, which includes chemotherapy based on high doses of cytarabine (“Acute myeloid leukemia,” 2021). In the case of poor indicators, that is, high or intermediate risks, patients are referred for allogeneic HSCT, given the high risk of relapse when treated with chemotherapy alone. This approach helps to cure 35-45% of patients under the age of 60 and less than 15% of patients aged 60 and over. At the same time, the average age at the time of AML diagnosis is 68 years (“Acute myeloid leukemia,” 2021). Therefore, a very large group of patients cannot undergo intensive therapy or allogenic HSCT due to the excessively increased risk of mortality.

Therefore, generally effective treatment options for this group include low-dose cytarabine (LDAC) or DNA methyltransferase inhibitors (“Acute myeloid leukemia,” 2021). It is noteworthy that with such therapy, the average survival in the group is 6-10 months. Therefore, scientists recommend exploring the possibilities of targeted therapy, since some regimens without chemotherapy “can provide a complete remission rate of almost 100% and a long-term survival rate of more than 98%” (“Acute myeloid leukemia,” 2021). As mentioned above, conventional treatment includes the stages of induction and consolidation.

Targeted Therapy in AML

Targeted therapy has this name because of its specificity and applicability only to certain types of mutations. The most common types of targeted therapy are FLT3 inhibitors, IDH1, and IDH2 inhibitors, RAS inhibitors, KIT inhibitors, targeting the apoptotic pathway, BCL2, and MCL1 inhibitors, MDM2 inhibitors, immune therapy, and mAbs targeting leukemia surface antigens. It is noteworthy that mutations in the FLT3 gene are characteristic for almost 33% of all patients with AML diagnosed for the first time (“Acute myeloid leukemia,” 2021). Mutations IDH1 and IDH2 are present in 5-15% and 10-15% of newly diagnosed AML. Equally important, mutations in KRAS or NRAS are found in 10-25% of patients at diagnosis, and KIT mutations in up to 25% of patients (“Acute myeloid leukemia,” 2021).

Heterogeneous Nuclear Ribonucleoprotein (hnRNP)

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group or family of RNA-binding proteins. These proteins are multifunctional due to the complexity and diversity of hnRNPs. They are involved in the processing of heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs (Short et al., 2020). hnRNPs are also involved in the regulation of gene expression as trans factors. These predominantly nuclear RNA-binding proteins typically form complexes with RNA polymerase II transcripts. They perform a myriad of cellular activities, including transcription and processing of pre-mRNA in the nucleus. They are also involved in the translation and exchange of cytoplasmic mRNA.

Overview of hnRNPs

Some of the common functions of hnRNPs include preventing the folding of pre-mRNA into secondary structures, transporting mRNA from the nucleus, regulating cell surface glycoprotein CD44, and interacting with telomeres. Several human genes encode hnRNPs, and the types of hnRNPs’ families determine their functional diversity (Short et al., 2020). Studies are being carried out for each hnRNP family, based on the determination of the processes of every hnRNP’s functioning, and in the search for the causes of their role in DNA damage.

hnRNPs Structure and Function

hnRNPs have a role in the cell cycle and DNA damage. Functions include recruiting, splicing, and co-regulation of the array of proteins that control the cell cycle. Because hnRNPs are so important for cell cycle control, they play the role of an oncogene (Short et al., 2020). This means that the loss of the function of hnRNPs leads to several common cancers. Typically, due to the destruction of the ability to execute hnRNPs functions, errors occur during splicing. However, some hnRNPs are responsible for the recruitment and management of proteins.

The Role of hnRNPs in Normal Hematopoiesis

According to recent studies, hnRNPs have several fundamental characteristics that explain the cause and nature of their participation in a variety of regulatory pathways (Short et al., 2020). hnRNPs are RNA-protein complexes and are present in the cell nucleus during gene transcription. After gene transcription, post-transcriptional modification of the newly synthesized RNA, also called pre-mRNA, occurs (Short et al., 2020). The presence in the process of these proteins bound to the pre-mRNA molecule indicates the immaturity of the pre-mRNA – that it is not yet ready for export to the cytoplasm as it is not completely processed. Proteins that participate in hnRNPs complexes are called heterogeneous ribonucleoproteins.

The Role of hnRNPs in Acute Myeloid Leukemia

Scientists are focusing on the study of hnRNPs and a vision of their role in DNA damage. This approach can be a breakthrough in determining the exact causes of AML in different types and will allow the development of more advanced and justified therapies (Short et al., 2020). The study of hnRNPs has a particularly high potential, as they have been proven to play a key role in DNA destruction and the interruption of key functions of HSCs leading to the increased production of immature leukocytes, which is the main characteristic of AML disease.

Aims and Objectives

This thesis aims to study the role of hnRNPs in acute myeloid leukemia. Research in this area is actively continuing, and it is of extreme importance, given the relatively low percentage of patients in whom complete remission occurs. The percentage of remission is particularly low among the elderly, who are the main group in which AML is diagnosed for the first time. Therefore, the purpose of this thesis is to provide an overview of the articles that study the role of hnRNPs in acute myeloid leukemia, summarize their main concepts, and organize the information for future research, for example, in the form of tables or diagrams.

References

Acute myeloid leukemia. (2021). NHS. Web.

Basilico, S., & Göttgens, B. (2017). Dysregulation of hematopoietic stem cell regulatory programs in acute myeloid leukemia. Journal of Molecular Medicine, 95(7), 719-727.

Hwang, S. M. (2020). Classification of acute myeloid leukemia. Blood Research, 55(1), 1-4.

Lee, D., Kim, D. W., & Cho, J. Y. (2020). Role of growth factors in hematopoietic stem cell niche. Cell Biology and Toxicology, 36(2), 131-144.

Maloy, S., & Hughes, K. (Eds.). (2013). Brenner’s encyclopedia of genetics. Academic Press.

Menegatti, S., de Kruijf, M., Garcia‐Alegria, E., Lacaud, G., & Kouskoff, V. (2019). Transcriptional control of blood cell emergence. FEBS Letters, 593(23), 3304-3315.

Short, N. J., Konopleva, M., Kadia, T. M., Borthakur, G., Ravandi, F., DiNardo, C. D., & Daver, N. (2020). Advances in the treatment of acute myeloid leukemia: New drugs and new challenges. Cancer Discovery, 10(4), 506-525.

Till, J. E., McCulloch, E. A., & Siminovitch, L. (1964). A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proceedings of the National Academy of Sciences of the United States of America, 51(1), 29.

Till, J. E., & McCulloch, E. A. (1980). Hemopoietic stem cell differentiation. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, 605(4), 431-459.

Wilkinson, A. C., & Göttgens, B. (2013). Transcriptional regulation of haematopoietic stem cells. Transcriptional and Translational Regulation of Stem Cells, 187-212.