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Arf Tumor Suppression: Stem Cells and Cancer

Research Summary

Charles Sherr studies tumor-suppressor genes that counter uncontrolled cell proliferation in response to mutagenic oncogenic insults. A tumor-suppressor network dominated by the retinoblastoma protein (Rb), p53, and the Ink4-Arf locus may have evolved to limit stem cell self-renewal and maintain tissue homeostasis. This concept explains why three chromosomally linked and coregulated tumor-suppressor genes (Ink4a, Ink4b, and Arf) are conserved in mammals, despite their frequent codeletion in cancers.

Mutations that activate oncogenes in cancer cells stimulate uncontrolled cell proliferation. Tumor-suppressor genes oppose the actions of oncogenes, and their inactivation predisposes to cancer. The realization that oncogenes and tumor-suppressor genes govern diverse biological processes, such as gene expression, cell differentiation, tissue development, and responses to environmental stress, provided key mechanistic insights into tumor formation.

A Hallmark of Cancer: Inactivation of the Rb and p53 Network
Transformation of normal cells to cancer cells commonly involves the disruption of signaling networks regulated by two canonical tumor-suppressor genes encoding the retinoblastoma protein (Rb) and the p53 transcription factor. Although the functions of Rb and p53 are not normally required for cell division, both proteins govern protective "checkpoint" responses that are triggered by DNA damage or by oncogene stimulation. Activation of Rb and p53 induces global changes in gene expression, which either arrest cell division or induce the death of damaged cells. Disruption of Rb and p53 compromises these safeguards, allowing incipient cancer cells to continue to divide, to consolidate mutational damage, and, ultimately, to form tumors.

Lesions affecting other genes governing the Rb and p53 signaling network can mimic Rb and p53 loss of function. Prominent among these are mutations that disrupt the Ink4-Arf gene cluster, a compact genetic locus that specifies three additional tumor-suppressor proteins: p16Ink4a, p15Ink4b, and p19Arf (p14ARF in humans; Figure 1). Expression of the p16Ink4a and p15Ink4b proteins in response to oncogenic stress activates Rb, whereas synthesis of p19Arf induces p53 (Figure 2). Therefore, chromosomal deletions that encompass the entire Ink4-Arf locus simultaneously compromise the activities of both Rb and p53. The Ink4-Arf locus is not highly expressed under most normal physiologic conditions in young mammals. Notably, Ink4-Arf is actively silenced en bloc in embryonic, fetal, and adult stem cells but becomes poised to respond to oncogenic stress signals as stem cells lose their self-renewal capacity and differentiate, thereby providing a potent barrier to tumor formation. The Ink4-Arf locus may have evolved to physiologically restrict the self-renewal capacities and numbers of stem and progenitor cells, with the attendant consequence of limiting tissue regenerative capacity in aging animals. Deletion of Ink4-Arf contributes to the aberrant self-renewal capacity of tumor cells and occurs frequently in many forms of human cancer.

Arf Tumor Suppression in Acute Lymphoblastic Leukemia
The Philadelphia chromosome (Ph), the first tumor-specific cytogenetic anomaly identified, results from a translocation that adjoins the human ABL oncogene on chromosome 9 to a breakpoint cluster region (BCR) on chromosome 22 to produce the BCR-ABL fusion protein, a constitutively active protein tyrosine kinase. This translocation is the founding lesion of two distinct hematological malignancies—chronic myelogenous leukemia (CML), which initially presents as a relatively benign myeloproliferative disorder, and Ph+ acute lymphoblastic leukemia (ALL), a more aggressive B cell tumor. CML affects immature hematopoietic progenitor cells in which the human INK4-ARF locus is normally silenced and INK4-ARF is seldom deleted. In contrast, in more mature lymphoblastic cells that arise in Ph+ ALL, INK4-ARF is induced and plays a protective role in the early stages of disease. However, rare cells that sustain INK4-ARF deletions emerge as highly aggressive malignant clones that have acquired a capacity for continuous self-renewal. A survey of patients with newly diagnosed Ph+ ALL revealed that almost two-thirds of them had already sustained biallelic INK4-ARF deletions in their leukemic blasts before therapy could be initiated. This suggests that inactivation of the locus contributes to the more aggressive features found in Ph+ ALL than in CML and to reduced therapeutic responsiveness.

Despite the dramatic clinical efficacy of BCR-ABL kinase inhibitors (imatinib, dasatinib, and nilotinib) in treating CML patients, these drugs fail to provide durable therapeutic benefit in those with Ph+ ALL. Our investigations of BCR-ABL–driven B cell ALL in a mouse model system that recapitulates the underlying genetics and therapeutic responsiveness of Ph+ ALL indicated that Arf inactivation enhances the biological "fitness" of Ph+ ALL cells in the hematopoietic microenvironment through tumor cell–autonomous and –nonautonomous mechanisms. Arf deficiency diminishes the effectiveness of targeted therapy in eradicating minimal residual disease and facilitates the subsequent emergence of drug-resistant clones that have acquired BCR-ABL kinase mutations. Hence, deletion of INK4-ARF and mutations in the BCR-ABL kinase collaborate through separate mechanisms to hamper the potentially beneficial effects of targeted therapy in Ph+ ALL.

BCR-ABL circumvents the normal requirement of B lymphoid cells for supportive hematopoietic cytokines, but targeted inhibition of the kinase with dasatinib restores their dependence on cytokines for survival. Salutary cytokine signaling in the hematopoietic microenvironment maintains minimal residual disease, but treatment of leukemic mice with dasatinib together with a second drug that interferes with cytokine receptor signaling, provokes a significantly improved therapeutic response. These preclinical data have now formed the basis for a phase I/II clinical trial combining dasatinib, a JaK kinase inhibitor, and selected chemotherapeutic agents for treatment of human patients with Ph+ ALL.

Disparate Physiological Functions of Arf Suggest Another Role for Limiting Tumor Progression
Arf is not generally expressed in normal tissues but is induced in response to activated oncogenes, thereby triggering p53-dependent elimination of incipient cancer cells (Figure 2). Paradoxically, we detected Arf expression in three tissues during early mouse development: (1) in the hyaloid vasculature of the eye, (2) in male germ cells (spermatogonia) in seminiferous tubules, and (3) in the fetal yolk sac, a tissue which arises from extraembryonic endoderm. Inactivation of Arf in these three tissues results in focal developmental defects that respectively lead to blindness, impaired spermatogenesis, and delayed formation of extraembryonic endoderm in the earliest stages of embryogenesis. Studies in which Arf deletions were specifically targeted to affected tissues, combined with the use of engineered "indicator strains," in which engagement of the Arf promoter was monitored throughout mouse development, revealed that Arf inactivation provokes alterations in cell-to-cell adhesion and migration that underlie each of these disparate defects. Our working hypothesis is that Arf inactivation in tumor cells similarly promotes aberrant cell migration and tumor cell metastasis, providing a conceptual link between Arf's developmental and tumor-suppressive functions.

As of March 11, 2013

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St. Jude Children's Research Hospital
Cancer Biology, Medicine and Translational Research