Sunday, April 13, 2014

Introduction to the Stem Cell Origin of Acute Myeloid Leukemia (AML)

I. Purpose: In our cancer project, we will be focusing on the role of DNMT3A gene mutation in the progression of Acute Myeloid Leukemia. As these studies have been published recently in the past few months, we will be analyzing how this new data fits in with previous studies on the prevalence of cancer stem cells leading to development of AML cancer. Further research into the differentiation of pre-leukemic stem cells would improve treatment methods by targeting the root causes of AML.

II. Introduction: 

Adult Acute Myeloid Leukemia (AML) is a cancer of the blood and bone marrow and it is the most common type of acute leukemia in adults. Normally, the bone marrow makes immature, blood stem cells that become mature blood stem cells later over time. A blood stem cell, that is essentially a multipotent stem cell derived from a hemotopoietic stem cell (see discussion below), can become a myeloid stem cell or a lymphoid stem cell. The myeloid stem cell can give rise to one of three types of mature blood cells: red blood cells, white blood cells that fight infection and disease, and platelets that form blood clots to stop bleeding. The lymphoid stem cell becomes a white blood cell. In AML, myeloid stem cells usually become immature blood cells called myeloblasts (myeloid blasts). These myeloid blasts are abnormal and do not become healthy, normal white blood cells. If too many of these stem cells become abnormal red and white blood cells or platelets, then they are called leukemia cells or blasts. These leukemia cells can then spread to other parts of the body, including the central nervous system, skin and gums. 

What are Hematopoietic Stem Cells?
Hematopoietic Stem Cells (HSCs) are stem cells that reside primarily in the bone marrow of adult mammals but that can also be found in the spleen, in peripheral blood circulation, and other tissues. They are important in the constant renewal of the blood, capable of giving rise to billions of new blood cells each day that include macrophages, erythrocytes (red blood cells), platelets, leukocytes (white blood cells), B and T cells, and Natural Killer (NK) cells. Scientists have defined the four actions of an HSC:

(1) it can renew itself;

(2) proliferate and differentiate to replenish all functional types of blood cells
(3) it can mobilize out of the bone marrow into circulation (or the reverse)
(4) it can undergo programmed cell death, or apoptosis

Studies have also revealed there to be two kinds of HSCs: (1) long-term stem cells that are capable of self-renewal, and (2) short-term progenitor or precursor cells that can proliferate but that cannot differentiate into more than one cell type. For example, a blood progenitor cell may only be able to make another red blood cell.

Source: Ref 2

Over the past few decades, scientists could not identify the single, unifying origin of stem cells that gave rise to the human AML disease due to the immense heterogeneity of these cells - in other words - their capability to proliferate and differentiate into numerous specialized cells in tissues of the body. They were aware that the two potential sources of cells from which AML could arise were the primitive, multipotent HSCs or the mature committed myeloid precursor cells downstream of the HSCs, and from studies done on mice and humans, they even knew that it were these primitive, small populations of self-renewing leukemic stem cells that gave rise to the large population of mature leukemic blasts which lacked self-renewal capacity. Until recently this year, however, researchers did not know that these ancestral leukemic stem cells can competitively out compete non-leukemic HSCs leading to clonal expansion, and that these leukemic stem cells were found in high proportion of AML patients that carry mutations in DNMT3A among other genes that, unlike AML blasts, survive chemotherapy and persist in the bone marrow at remission, providing a reservoir of cells that can cause AML progression.

III. Research:

According to a report published in Nature in February, John E. Dick, PhD, a researcher at the Princess Margaret Cancer Center in Toronto and the University of Toronto in Canada, and his colleagues sequenced 103 commonly mutated leukemia genes in peripheral blood samples from 83 patients at diagnosis. They identified mutations in the DMNT3A gene in AML cells in roughly 25% of the samples (mutant allele frequency ~50%). The researchers also found another mutation in the NPM1c gene, which occurred in 88% of the samples that contained mutations in DMNT3A. These findings were consistent with previous published data, but what the researchers didn't expect to find was that in the T cells of 15 patients, mutations in DMNT3A were found (low mutant allele frequency 1-20%) but with no evidence of mutations in NPM1c. It should be noted that two types of cells were involved in this study: normal T cells  obtained from non-leukemic tissue that are of the lymphoid lineage, and AML cells of the myeloid lineage. The fact that mutated DNMT3A was found in both the T cells and the AML cells, whereas mutated NPM1c was found only in the latter (refer to Table b), led the researchers to conclude the order in which mutations arise in the disease, with mutated DNMT3A arising the earliest followed by mutations in NPM1c as well as in FLT3-ITD, another type of mutated gene found only in AML blasts but not in T cells.The important finding to take away from this study is that mutated DMNT3A is present in the pre-leukemic ancestral cells which give rise to both the T cells and the AML cells that are observed during diagnosis. 

Table 1b.  Frequency (%) of both mutated DNMT3A and NPM1c in isolated CD33+ AML blasts and T cells. A procedure called ddPCR (droplet digital PCR) was used to determine the mutant allele frequencies corresponding to the length of the bars in 17 patients with normal karotype AML. (Source: Ref 4)

The findings presented in Table1b seem valid. However, while the table depicts that mutations in DNMT3A occur at an allele frequency of approximately 50% or greater in patients with AML, it fails to acknowledge what the exact nature or type of mutation is present in that particular gene that could adversely affect, say, the translation process that would result in abnormal levels of protein products. Are the mutations in DNMT3A largely due to missense, frameshift, nonsense, or splice-site mutations and at what rate do they occur in a particular subset of patients? Another question the article should have tried to answer is whether the overall survival rate in patients with such DNMT3A is significantly lower than in patients without these mutations. Also, how do the particular mutations in DNMT3A come about, or in other words, what are their cause(s)? Are there any other genes that have been or could potentially be discovered that are similar to DNMT3A in the pre leukemic stem cell pool in AML patients? It seems to be that this would be highly likely due to the complexity and heterogeneity of the AML disease that manifests itself both biologically and clinically. Multiple genes would influence the number of distinct genetic abnormalities that are typically observed. The discovery and knowledge of these genes would be of immense importance as they would serve as developing therapeutic targets for AML.

The topic of this article that focuses on the origin of stem cells that causes AML is related to our class discussion about the tumor microenvironment. Initially, the cancer cells that constituted this environment in early tumor were portrayed as homogenous cell populations until relatively late in the course of the tumor progression, as increased genetic instability and effects of hyper proliferation were then able to give rise to distinct clonal subpopulations. However, the research presented in this article forces us to reconsider our initial thoughts and evidence gathered thus far because even in the early stages of tumor pathogenesis, and especially in the case of hematopoietic malignancies such as AML, there exists a multitude of diverse cells with varied cell-surface markers, ability to differentiate, proliferate and invade surrounding tissue. Our lack of deeper knowledge and understanding about the way cancer stem cells work and behave is responsible, to a great extent, for our inability to properly diagnose, treat, and prevent the recurrence of many cancers including acute myeloid leukemia. 


1. Hanahan, Douglas, and Robert Weinberg. "Hallmarks of Cancer: The Next Generation." Cell. 144.5 (2011): 646-74. Web. 13 Apr. 2014.

2. Hematopoietic Stem Cells . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2011 [cited Sunday, April 13, 2014] Available at

3. National Cancer Institute: PDQ® Adult Acute Myeloid Leukemia Treatment. Bethesda, MD: National Cancer Institute. Date last modified <12/26/2013>. Available at: Accessed <04/13/2014>.

4. Shlush, Liran, Sasan Zandi, et al. "Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia." Nature. 506.February (2014): 328-33. Web. 13 Apr. 2014.