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238 Part IV: Molecular and Cellular Hematology Chapter 16: Cell-Cycle Regulation and Hematologic Disorders 239

epigenetic modifications. In addition, at least two epigenetic mech- kinases, for example, ATM, ATR, Chk1 and Chk2, which phosphorylate
anisms—the hypermethylation of CpG islands in the promoter region p53. Abnormalities of p53 are found in slightly more than 50 percent
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and the aberrant acetylation of histones (especially histone H4)—can of all human tumors and, surprisingly, even in some normal cells. It is
silence tumor-suppressor genes in a variety of human cancer cell lines unclear if these “normal” cells represent a pool of premalignant cells
and primary tumors. in an otherwise healthy individual or, more likely, p53 changes are just
The products of three important tumor-suppressor genes (RB, P53, one step in multistage tumorigenesis. Two different p53 homologues,
and p16 INK4A ) are interconnected biochemically. The RB gene maps to p63 and p73, have been described, which show DNA binding, transac-
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chromosome 13q14 and has several downstream effectors, among which tivation, and oligomerization domains similar to p53. This similarity
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the transcription factor E2F is the best characterized. The RB gene in the DNA binding domain allows p63 and p73 to regulate p53 target
family consists of three closely related proteins, RB, p107, and p130. All genes, induce cell-cycle arrest and apoptosis, and therefore act as tumor
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three proteins are able to interact with several E2F family members. suppressors. The p73 gene has been localized to chromosome 1p36, a
Transcriptional activation and repression are mediated via com- common region of cytogenetic changes in cancer. p73 protein can also
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plexes consisting of RB family members, E2F family members, and bind p53, inhibiting its transcriptional regulatory activity. Although
so-called DP proteins. Besides its role in cell-cycle control, RB can p53 mutations are found frequently in almost all cancers, p63 and p73
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modulate RNA polymerase activity, thus linking cell-cycle progression mutations are much more rare. 264,266 However, the p73 gene is inacti-
to transcriptional regulation. Many cellular proteins have been identi- vated by hypermethylation of CpG islands in its promoter region in
fied that bind to RB. These proteins can be divided into different groups, both leukemias and lymphomas. 267
including transcription factors, growth factors, protein kinases, protein Homozygous deletions of the p16 INK4A /p14 ARF gene locus on human
phosphatases, and nuclear matrix proteins. Mutations of RB are fre- chromosome 9p21 have been detected in gliomas, 107,268 primary can-
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quent in cancer e.g., leukemias; soft-tissue sarcomas; and breast, esoph- cers of lung, 107,269 bladder, head and neck, as well as in acute T-cell
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agus, prostate, and renal carcinomas. Several viral or oncoproteins leukemias 272,273 and mesotheliomas. Because inherited mutations of
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can bind to and inactivate RB. 112,237 p16 INK4A exon 2 may interfere with its expression and/or function, with-
The p53 gene has been called a “guardian” of the genome because it out causing an amino acid change in p14 ARF , it is clear that p16 INK4A inac-
transmits signals arising from various forms of DNA damage, leading to tivation alone is an important step in the evolution of malignant disease.
cell-cycle arrest or apoptosis. p53 protein is also the target of leukemo- However, in established tumor cell lines, nearly all chromosome 9p21
genic mutations. Damaging factors, such as hypoxic stress, chemicals, deletions disable the entire p16 INK4A /p14 ARF locus. Both proteins act as
or irradiation either alter the p53 protein itself or can stabilize its cellu- suppressors of the G -S transition, even though they function in two dif-
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lar inhibitor, MDM2 (in humans the homologue is termed HDM2). ferent pathways: p16 INK4A acts as an inhibitor of cyclin D /cdk4/6 com-
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The MDM2 protein inhibits p53 transcription and stimulates p53 deg- plexes whereas p14 ARF stabilizes p53 by inhibition of MDM2. Several
radation. 239,240 For the former, MDM2 is able to bind to the transactiva- models provide insight into the different modes of action of p16 INK4A
tion domain of p53 through a p53-interacting domain on the MDM2 and p14 ARF on cell-cycle regulation. Interestingly, if the entire p19 ARF /
amino terminus, which inhibits p53 from binding to its transcriptional p16 INK4A locus is disrupted in mice (the mouse homologue of p14 ARF is
coactivators, thereby preventing activation of p53 transcriptional targets. p19 ARF ), the mice develop lymphomas, lymphoid leukemias, and sarco-
For the latter, MDM2 binds to p53 through a RING-domain and ubiq- mas, suggesting that these tumor-suppressor genes do not act in a lin-
uitinates p53 through the E3 ubiquitin ligase activity of MDM2, caus- eage-specific manner on cell-cycle regulation but in a more general way.
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ing p53 nuclear export and ultimate degradation. Moreover, MDM2 Retroviral expression of p16 INK4A restores the normal phenotype in some
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is able to affect chromosomal stability independently of p53. The cell types underlining the strong tumor-suppressor potency of p16 INK4A .
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MDM2 binding region contains several phosphorylation sites. These The p15 INK4B gene, also located on chromosome 9p21, approximately 20
residues (e.g., serine 395) are phosphorylated by DNA damage-ac- kb centromeric of p16 INK4A , is deleted somewhat less frequently. Analyses
tivated kinases (e.g., ATM and Chk2), which is required for p53 acti- of primary tumors, however, show that not all 9p21 deletions encompass
vation. The p14 ARF tumor-suppressor gene, which is encoded within these three tumor suppressor genes. One mechanism for disruption of
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the p16 INK4A locus by alternate splicing, controls MDM2 activity. In the p15 INK4B /p14 ARF /p16 INKA region in T-cell leukemias may be the action
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de novo AML, although p53 mutations are not common, overexpres- of an illegitimate variable diversity joining (V[D]J) recombinase. Sev-
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sion of MDM2 is frequently encountered, and there is current interest eral other binding partners of p16 INK4A have been identified. The RB
in exploring MDM2 antagonists, for example, RO5503781, in combi- gene interacts with one of these factors, BRG1, to remodel chromatin
nation with cytarabine in patients with relapsed or refractory AML. structures. BRG1 also acts upstream of RB with p16 INK4A and functions
Additionally, the MDM2 antagonist nutlin-3A causes p53-dependent as a tumor suppressor. 276
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apoptosis in AML cells by disrupting the p53-MDM2 interaction and The p15 INK4B /p16 INK4A /p14 ARF locus on chromosome 9p21 is a
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synergizes with BH3-mimetics, MEK/ERK inhibitors, 250,251 aurora hotspot in the development of human cancer and approximately 50
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kinase inhibitors, cdk1 inhibitors, XIAP antagonists, FLT3 inhib- percent of all human malignancies show abnormalities in at least one
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itors, 254,255 and inhibitors of nuclear export, such as CRM1. The p14 ARF of these tumor-suppressor genes. Another gene lies about 100 kb telo-
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gene shares exons 2 and 3 with p16 INK4A but has a distinct exon 1. The meric of p16 INK4A , and this gene, methylthioadenosine phosphorylase
discovery that two important tumor-suppressor genes are encoded by (MTAP), encodes an important enzyme in purine metabolism. Some
the same chromosomal locus and share several exons was unexpected early gliomas show MTAP deletions without deletions of other genes
and is unique in human biology. The p16 INK4A gene function depends on 9p21, suggesting that MTAP by itself has tumor-suppressor proper-
on p53, because overexpression of p16 INK4A causes cell-cycle arrest in ties. The reexpression of MTAP in breast cancer cells severely inhibits
p53 wild-type cells but not in p53-deficient cells. The transcription their ability to form colonies in soft agar or collagen, supporting this
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of p16 INK4A is regulated by E2F, which is under the control of RB. This hypothesis. In addition, MTAP-expressing cells are suppressed for
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indicates the existence of yet another feedback loop, which links the RB tumor formation when implanted into severe combined immune defi-
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pathway to p53. The Ras protein is another identified p16 INK4A fac- ciency (SCID) mice. Recent findings suggest that the enzyme ornithine
tor involved in MDM2-p53-p21-RB regulation. 260,261 Different signaling decarboxylase (ODC) is overexpressed in MTAP-deleted tumors, pro-
routes that connect DNA damage with p53 include a cascade of Ser/Thr viding evidence for a new pathway in tumorigenesis. Overexpression of
Kaushansky_chapter 16_p0213-0246.indd 238 9/18/15 11:58 PM