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218 Part IV: Molecular and Cellular Hematology Chapter 16: Cell-Cycle Regulation and Hematologic Disorders 219

which block S-phase entry (the G /S-phase checkpoint), delay S-phase increased cytarabine-induced apoptosis, with negligible impact on nor-
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progression (the intra–S- or S-phase checkpoint), or prevent mitotic mal myeloid progenitors, leading to a phase I trial of the combination
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entry (the G /M-phase checkpoint). These events direct phase-specific in patients with relapsed and refractory acute leukemias. Chk1 inhibi-
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DNA repair mechanisms through repair-specific gene transcription. tors increase HDACI lethality in human leukemia cells. This may reflect
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If repair fails, checkpoints trigger apoptosis. Checkpoints are thus the ability of HDACIs to induce DNA damage in AML cells, inhibit
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important quality control measures that ensure the proper sequence of both homologous recombination and nonhomologous end-joining
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cell-cycle events and allow cells to respond to DNA damage. 160 mechanisms of DNA repair, and downregulate/inactivate Chk1.
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The G /S checkpoint is the first defense against genomic stress in However, clinical development of MK-8776 has been halted. Based upon
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cycling cells. In response to DNA damage, the G /S checkpoint prevents the ability of Hsp90 inhibitors to downregulate Chk1, a phase I study of
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cells from entering the S-phase by inhibiting the initiation of DNA rep- the combination of cytarabine and the Hsp90 inhibitor tanespimycin
lication. At this checkpoint, the checkpoint kinase Chk2 is activated by was conducted in adults with recurrent or refractory acute leukemia;
the proximal transducer ATM to phosphorylate (and thereby inhibit) however, the combination exhibited limited clinical activity. 169
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the cdc25A phosphatase, thus preventing activation of cyclin E(A)/cdk2 Wee1 is a validated therapeutic target in AML. The combination
and temporarily halting the cell cycle. G arrest is sustained by ATM/ of cytarabine and the Wee1 inhibitor AZD1775 synergistically induces
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Chk2-mediated phosphorylation of murine double minute protein 2 apoptosis in myeloid cell lines. Similarly, genome-wide short hairpin
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(MDM2) and p53, resulting in p53 stabilization and accumulation. p53 RNA screens strongly implicate cell-cycle checkpoint proteins, partic-
transcriptionally activates the endogenous cdk inhibitor p21, which, in ularly Wee1, as critical mediators of AML cell survival after cytarabine
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turn, inhibits cyclin E(A)/cdk2 and preserves the association of Rb with exposure. In AML cell lines, synergistic inhibition of proliferation by
E2F. 160 the combination of AZD1775 and Ara-C occurred regardless of p53
The intra-S or S-phase checkpoint is activated in response to struc- functionality. Another combinatorial strategy in AML involves coad-
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tural DNA damage as well as stalled replication forks. Upon activation ministration of AZD1775 with HDACIs. Besides inducing DNA dam-
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of this checkpoint, ATR/Chk1 and ATM/Chk2 phosphorylate cdc25A, age and inhibiting DNA repair, HDACIs downregulate several proteins
resulting in enhanced proteolysis of the phosphatase and inhibition critical to checkpoint function, such as ATR, Chk1, and Wee1, 168,175–177
of its function through 14-3-3 σ binding. Cyclin E(A)/cdk2 is thereby and Wee1 inhibition promotes premature mitotic entry of cells bearing
inhibited, and progression through the S-phase halted. 158 unrepaired DNA damage. Efforts to extend these pre-clinical findings
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The G /M checkpoint prevents mitotic entry of cells that have to the clinic are underway.
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either incurred DNA damage during G , or that have escaped the G /S
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and intra-S checkpoints despite earlier genomic insults. The key down-
stream target of the G /M checkpoint is the promitotic cyclin B/cdk1 ONCOGENES
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(cdc2) complex. During interphase, this complex is inactivated through
phosphorylation by Myt1 and Wee1. Chk1 may phosphorylate Wee1, Table 16–2 lists the common genomic aberrations seen in hemato-
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mediating binding of Wee1 to 14-3-3 proteins, which, in turn, may stim- logic malignancies. Tables 16–3 and 16–4 list common somatic muta-
ulate the kinase activity of Wee1 against cdk1 (cdc2). Thus, both Chk1 tions encountered in the major myeloid and lymphoid malignancies,
and 14-3-3 proteins may act together as positive regulators of Wee1. respectively.
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Activation of cyclin B/cdk1 (cdc2) requires dephosphorylation by the The complicated cell-cycle network has its parallel in the several
cdc25 phosphatases (A, B, and C). Notably, phosphorylation/inactiva- different oncogenes and tumor-suppressor genes that influence car-
tion of cdk1 (cdc2) involves two inhibitory sites, for example, Tyr15 cinogenesis and tumor progression. The products of oncogenes, the
and Thr14, and dephosphorylation of both sites is necessary for full oncoproteins, lead to or facilitate the transformation of a normal into a
cdk1 (cdc2) activation. Initiation of the G /M checkpoint is mediated malignant cell. Oncogenes can be carried into the cell by viruses or they
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by ATR/Chk1, which phosphorylates (and thereby inhibits) cdc25A, can arise from mutations in normal cellular genes. In addition, they can
B, and C, whereas maintenance of this checkpoint requires p53 and its also arise from leukemia- or lymphoma-associated translocations where
downstream effectors p21, 14-3-3 σ, and growth arrest and DNA dam- two usually separated genes are fused together and form a novel fusion
age (GADD) 45. 158 protein. The familiar concept of this kind of protooncogene activation
Checkpoint dysfunction is common in human cancers and is can be blurred by the fusion proteins because they possess unique capa-
considered a pathologic hallmark of neoplastic transformation. bilities not shared by either of the individual fusion partners. Oncopro-
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Conversely, agents used for cancer treatment, such as cytotoxic che- teins can interact directly with cell-cycle regulatory proteins or control
motherapy and ionizing radiation, activate cell-cycle checkpoints. their activity through phosphorylation and dephosphorylation. Not
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Cancer cells are particularly dependent upon the S- and G /M-phase all mutations in oncogenes lead to an altered function of the resulting
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checkpoints for repair of DNA damage because of preexisting defects in product. The nomenclature in the oncogene and tumor-suppressor gene
G /S checkpoint mechanisms, such as p53 and RB mutations. Because field is not always clear. As a general guideline, if a mutation causes a
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the S-phase checkpoint facilitates slowing, rather than arrest, of the cell functional loss of the gene product (loss of function), and the recessive
cycle, a cancer cell harboring DNA damage may progress through the loss of function leads directly to uncontrolled cell division, the under-
S-phase checkpoint, only to halt at the G /M checkpoint. The latter is, lying gene can be named a tumor-suppressor gene. On the other hand,
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therefore, a key guardian of the cancer cell genome, and its abrogation if the mutation leads to an altered gene product (gain of function) that
can lead to enhanced tumor cell death while sparing normal cells, which interacts abnormally with other proteins to influence the cell cycle, this
maintain an intact G /S-phase checkpoint. G /M checkpoint abrogation gene is an oncogene, acting in a dominant fashion. Translocations are
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prevents cancer cells from repairing DNA damage, forcing them into a typical of oncogenes, whereas homozygous deletions and hypermeth-
premature and lethal mitosis (“mitotic catastrophe”). 160 ylation of CpG-nucleotide repeats in the promoter regions (“epigenetic
Based upon these concepts, synergism between DNA-damaging silencing”) are characteristic features of tumor-suppressor genes.
agents (cytotoxic chemotherapy) and G /M checkpoint abrogators have Numerous oncogenes/oncogene candidates are described in
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been examined both preclinically and in clinical studies. In human the literature, and most of them are involved in the pathogene-
AML cell lines, the Chk1 inhibitor MK-8776 (SCH900776) markedly sis and development of many different tumors types, especially the
Kaushansky_chapter 16_p0213-0246.indd 219 9/18/15 11:57 PM