In addition to genes that encode “core” components of the apoptosis machinery, tumor-associated alterations have been identified in genes that provide important regulatory inputs into the expression of apoptosis genes or the regulation of their encoded proteins. This includes many transcription factors and growth factor receptors. However, ultimately, many of these signaling molecules funnel into common pathways linked to the apoptosis machinery. Two prominent examples are described here, namely, NFκB and Akt. For more comprehensive summaries of signal transduction inputs into the core apoptosis machinery, the reader is referred to recent reviews.^{182, 203–206}

NFκB

NFκB represents a family of transcription factors, comprising the REL gene family. NFκB directly binds the promoters and induces expression of several antiapoptotic genes, including the BCL-2 family members BCL-X and BFL-1, the IAP-family member C-IAP2, and the DED-family gene C-FLIP.²⁰⁷ Thus, elevations in NFκB activity can increase cellular resistance to apoptosis, affecting (a) the Intrinsic (mitochondrial) pathway through elevations in antiapoptotic Bcl-2 family proteins, (b) the Extrinsic (TNF-family death receptor) pathway through upregulation of FLIP, and (c) downstream common pathways involving effector Caspases, as a result of over-expression of cIAP2. Recent data demonstrate hyper-activity of NFκB in many solid and hematopoietic malignancies.²⁰⁸

The first example of NFκB involvement in malignancy was provided by studies of the avian Rev-T retrovirus, a transforming retrovirus which causes rapidly fatal lymphomas in young chickens and which carries the v-Rel oncogene. The cellular homolog of this viral oncogene is C-REL, which encodes the p65 subunit of NFκB. Amplification of the C-REL gene has been reported in NHLs, occurring in ˜23 % of diffuse large B-cell lymphoma and commonly associated with extranodal presentation.²⁰⁹ Other genetic alterations associated with dysregulation of NFκB include chromosomal translocations involving the IκB family member BCL-3 in B-CLL.²¹⁰ IκB family proteins bind and sequester NFκB complexes, preventing the transcription factor from entering the nucleus. Typically, IκB is regulated by ubiquitin-mediated turnover by the 26s proteasome. Mutations in IκB thus may enhance NFκB activity, either by producing unstable proteins or reducing the affinity of IκB for NFκB. The anticancer activity of drugs that inhibit the proteasome, currently in human clinical trials, may be attributable in part to suppression of IκB degradation, thereby inhibiting NFκB induction.²¹¹ Kinases that induce phosphorylation of IκB, a prerequisite for Ubiquitination and subsequent degradation, also have emerged as attractive drug-discovery targets (Figure 4-8).

Figure 4-8

Apoptosis-suppressing mechanisms of NFκB. NFκB-family transcription factors induce the expression of multiple antiapoptotic genes, including BCL-2 family members BCL-X and BFL1 (A1), IAP family member C-IAP2, and Caspase-8 antagonist, (more...)

AKT

The protein kinase Akt (PKB) plays important roles in linking growth factor receptors and oncoproteins to apoptosis pathways (Figure 4-9). The murine gene encoding Akt was first discovered by virtue of its similarity to the v-akt oncogene found in some murine leukemia viruses and through its activation in thymomas caused by retrovirus insertions near the c-akt gene.²¹² Humans contain three AKT genes. Akt can phosphorylate multiple proteins within the core apoptosis machinery, some of which have been mentioned above. For example, the pro-apoptotic Bcl-2 family member BAD is a target of Akt, where phosphorylation of BAD inhibits its ability to heterodimerize with Bcl-X_L.²⁰⁰ Akt also can phosphorylate human Caspase-9, blocking apoptosis down-stream of mitochondria.¹¹⁷ Another substrate of Akt that is relevant to apoptosis is Forkhead Transcription Factors (FKHD). Some FKHD family members appear to control apoptosis, perhaps by affecting transcription of the gene encoding FasL.²¹³ Phosphorylation of FKHD by Akt prevents its entry into the nucleus. Akt may also play a role in NFκB activation.²¹⁴ Moreover, Akt has been implicated in suppression of Nur77 (TR3), an orphan member of the retinoid/steroid family of transcription factors implicated in apoptosis induction.²¹⁵ Akt also opposes apoptosis induced by the kinase Ask1 ²¹⁶ and promotes p53 degradation through effects on Mdm2.²¹⁷

Figure 4-9

Apoptosis substrates of Akt. The protein kinase Akt (PKB) is activated in response to second-messengers produced by PI3K, a lipid kinase that is activated by many growth factor receptors and oncoproteins. PTEN is a lipid phosphatase that prevents accumulation (more...)

In recent years, evidence of hyper-activity of kinase Akt has been found for many types of human cancers.^{200, 218} The Akt protein contains a PH domain which binds phospholipid second-messengers produced by Phosphatidyl-Inositol 3′ Kinase (PI3K). Elevations in the levels of these lipid second-messengers result in recruitment of Akt to the plasma membrane, where it becomes activated by phosphorylation.²¹⁸ These phospholipid second-messengers are destroyed by PTEN, a lipid phosphatase and important tumor suppressor. Deletions and somatic point mutations that inactivate PTEN occur commonly in cancers.²¹⁸ Other mechanisms for deregulating Akt activity have also been described in cancers, including amplification of the AKT2 gene in breast and ovarian cancers, and over-production of an Akt-binding protein called Tcl1, as a result of chromosomal translocations in T-cell pro-lymphocytic leukemia (T-PLL). Consequently, Akt has emerged as an attractive candidate for drug discovery, using small-molecule compounds to target the ATP-binding site of the kinase, thereby inhibiting its catalytic activity.