The hallmarks of cancer. Cell 100:57-70, 2000 Acquired Capabilities of Cancer: We suggest that most if not all cancers have acquired the same set of functional capabilities during their development, albeit through various mechanistic strategies. Hanahan & Weinberg: The hallmarks of cancer. Cell 100:57-70, 2000 Hanahan & Weinberg: The hallmarks of cancer. Cell 100:57-70, 2000

ONCOGENE ACTIVATION Genetic mechanisms in human tumours that lead to uncontrolled activation of oncogenic tyrosine kinases.   a | Reciprocal chromosomal translocations generate fusion proteins, the amino-terminal portion of which is responsible for oligomerization, allowing constitutive activation of the catalytic activity of a kinase domain that is located on the carboxy-terminal portion. b | Overexpression of a cell-membrane receptor tyrosine kinase leads to spontaneous (or autocrine ligand-dependent) dimerization and constitutive kinase activation. c | Point mutation in the juxtamembrane region of a receptor tyrosine kinase causes constitutive ligand-independent dimerization of the receptor and activation of its kinase activity. d | Truncation of the carboxy-terminal portion prevents phosphorylation of a tyrosine residue that is involved in protein folding and stabilizes tyrosine kinase in the active conformation. ABL, Abelson leukaemia; ALK, anaplastic lymphoma kinase; FGFR, fibroblast growth-factor receptor; KD, kinase domain; PDGFR , platelet-derived growth-factor receptor- ; TRKC, neurotrophin 3 receptor. Nature Reviews Cancer 2, 351-360 (2002); doi:10.1038/nrc799 ONCOGENIC TYROSINE KINASES AND THE DNA-DAMAGE RESPONSE

ONCOGENE ACTIVATION Genetic mechanisms in human tumours that lead to uncontrolled activation of oncogenic tyrosine kinases.   a | Reciprocal chromosomal translocations generate fusion proteins, the amino-terminal portion of which is responsible for oligomerization, allowing constitutive activation of the catalytic activity of a kinase domain that is located on the carboxy-terminal portion. b | Overexpression of a cell-membrane receptor tyrosine kinase leads to spontaneous (or autocrine ligand-dependent) dimerization and constitutive kinase activation. c | Point mutation in the juxtamembrane region of a receptor tyrosine kinase causes constitutive ligand-independent dimerization of the receptor and activation of its kinase activity. d | Truncation of the carboxy-terminal portion prevents phosphorylation of a tyrosine residue that is involved in protein folding and stabilizes tyrosine kinase in the active conformation. ABL, Abelson leukaemia; ALK, anaplastic lymphoma kinase; FGFR, fibroblast growth-factor receptor; KD, kinase domain; PDGFR , platelet-derived growth-factor receptor- ; TRKC, neurotrophin 3 receptor. Nature Reviews Cancer 2, 351-360 (2002); doi:10.1038/nrc799 ONCOGENIC TYROSINE KINASES AND THE DNA-DAMAGE RESPONSE

TUMOR SUPPRESSOR INACTIVATION 1) Point mutation p53

TUMOR SUPPRESSOR INACTIVATION 1) Point mutation: p53, BRCA1 etc. 2) Deletion 3) Epigenetic silencing

Locus 9p21 a | The 9p21 locus has a highly unusual genomic organization. The CDKN2A gene contains two upstream exons, 1 and 1. These two exons are driven by separate promoters, which result in alternative transcripts that share common downstream exons 2 and 3. Although a common acceptor site in the second exon is used by both first exons, the open-reading frames remain distinct in the shared exon 2, and result in two distinct protein products. The transcript initiated from the proximal promoter (1) encodes INK4A12, the founding member of the INK4-family proteins. The second transcript, initiated from the upstream exon 1, encodes ARF11. b | INK4A controls the RB-regulated G1–S transition. By inhibiting CDK4/6–cyclin-D-mediated hyperphosphorylation of RB, INK4A ensures that RB remains in complex with the E2F transcription factor142. These RB–E2F complexes recruit histone deacetylase to promote and repress transcription of target genes, leading to G1 arrest143. In the absence of inhibition by INK4A, CDK4/6–cyclin-D phosphorylates RB, which results in the release of E2F. E2F then activates genes that are necessary for progression into S phase142. c | ARF functions as a potent growth suppressor11, 144, blocks oncogenic transformation and sustains p53-dependent apoptosis in RB-null cells that have re-entered the cell cycle in vivo or in the setting of hyperproliferative oncogenic signals. Biochemically, ARF stabilizes and enhances p53 levels by blocking MDM2-mediated p53 ubiquitylation and degradation

TUMOR SUPPRESSOR INACTIVATION 1) Point mutation: p53, BRCA1 etc. 2) Deletion 3) Epigenetic silencing 4) Protein-protein interactions

TUMOR SUPPRESSOR INACTIVATION Protein-protein interactions

Framework for developing anticancer drugs with a high therapeutic index.   An anticancer drug might have a high therapeutic index because its target is uniquely present in cancer cells (a), or because the requirement for its target is quantitatively or qualitatively different in cancer cells than in normal cells (b and c). This differential requirement might be because of intrinsic differences in the cells (b), such as genetic (red) and epigenetic (blue) differences, or extrinsic differences in the cells (c), such as loss of survival signals provided by normal cell–cell and cell–matrix interactions. Modified with permission from Ref. 2 © (2002) Elsevier Science.

Framework for developing anticancer drugs with a high therapeutic index.   An anticancer drug might have a high therapeutic index because its target is uniquely present in cancer cells (a), or because the requirement for its target is quantitatively or qualitatively different in cancer cells than in normal cells (b and c). This differential requirement might be because of intrinsic differences in the cells (b), such as genetic (red) and epigenetic (blue) differences, or extrinsic differences in the cells (c), such as loss of survival signals provided by normal cell–cell and cell–matrix interactions. Modified with permission from Ref. 2 © (2002) Elsevier Science.

Survival signals in tumour cells: an Achille's heel?  The issue of the degree of the therapeutic window that will be provided by drugs that target the RAS pathways is a crucial one. All cells use RAS signalling pathways to some extent, so there is a danger that inhibitors will have severe effects on normal cells as well as tumour cells. Potent inhibition of RAS function through the expression of dominant-negative mutants or microinjection of neutralizing antibodies has long been known to block normal cell proliferation. Although, ultimately, each drug target has to be validated experimentally for its differential effect on tumour versus normal cells, there are conceptual reasons for believing that certain types of signalling inhibitors, in particular those that inhibit survival pathways, might selectively disadvantage tumour cells. As represented in the figure by the 'scales' of life and death signals that the cell is experiencing, a normal cell requires a continuous low level of survival signal to remain alive85. These survival signals emanate from various different sources, including adhesion to extracellular matrix, soluble factors in the extracellular environment and interactions between cells. Each of these acts to instruct the cell that it is in an appropriate environment. The cells are also exposed to a low level of cell-death signals, perhaps due to occasional DNA damage or oxidative stress, but the balance of signals favours survival. In the tumour cells, microevolutionary processes have led to the selection of cells with greatly increased survival signalling — for example, by the loss of PTEN. Once a mutation has given a cell a survival advantage, it is then able to tolerate more death signals. For example, this allows it to survive notable DNA damage as a result of loss of cell-cycle control and rapid proliferation, and to be less easily killed by hypoxia and by immune system attack; all of these events result in the tumour cell's increasingly antisocial behaviour. It can also afford to dispense with previously required survival signals that were provided by matrix and other cells and to grow in a completely independent manner that is characteristic of tumour cells. This disorganized growth is therefore occurring at high levels of both apoptotic and survival signals, with the cells being dependent on one or a few strongly activated survival pathways, compared with a more complex pattern of survival signals for the normal cell. If both cell types are now treated with a survival-pathway inhibitor that targets the pathway on which the tumour cell is reliant, rapid death of the tumour cell will result, driven by the damage it has accumulated and formerly been able to ignore. The normal cell, by contrast, might survive, having much lower intrinsic levels of death signals and receiving a wider range of survival signals.

attività PI3K/Akt/mTOR effetti lievi Cellula normale PTEN attività PI3K/Akt/mTOR INIBITORI DI mTOR Cellula tumorale PTEN attività PI3K/Akt/mTOR

Table 1 Examples of oncogene addiction: studies in mice Weinstein IB and Joe AK (2006) Mechanisms of Disease: oncogene addiction—a rationale for molecular targeting in cancer therapy Nat Clin Pract Oncol 3: 448–457 10.1038/ncponc0558

Table 2 Examples of oncogene addiction: studies in human cancer cell lines Weinstein IB and Joe AK (2006) Mechanisms of Disease: oncogene addiction—a rationale for molecular targeting in cancer therapy Nat Clin Pract Oncol 3: 448–457 10.1038/ncponc0558

Table 3 Clinical evidence of oncogene addiction Weinstein IB and Joe AK (2006) Mechanisms of Disease: oncogene addiction—a rationale for molecular targeting in cancer therapy Nat Clin Pract Oncol 3: 448–457 10.1038/ncponc0558

MODELS OF ONCOGENE ADDICTION Figure 4 | Models of oncogene addiction. a | Many oncogenes paradoxically induce pro-mitogenic signals as well as antimitogenic (or pro-apoptotic) signals. Growth stimulation results from oncogene activation presumably because the former is dominant to the latter. However, acute inactivation of the oncogene might cause growth cessation or death if the anti-mitogenic/pro-apoptotic signals decay more slowly than the mitogenic signals (for example, because of differences in mRNA and protein halflife). Adapted from REF. 53.

MODELS OF ONCOGENE ADDICTION Figure 4 | Models of oncogene addiction. c | Activation (indicated by bold arrow) of an oncogenic pathway diminishes selection pressure to maintain collateral signalling pathways. Silencing of these collateral pathways over time, because of genetic or epigenetic changes, leads to oncogene dependency. Adapted from REF. 57.

MODELS OF ONCOGENE ADDICTION Figure 4 | Models of oncogene addiction. b | Oncogene dependency due to gene–gene interactions. Cancer cells accumulate mutations (arrows) over time that cumulatively lead to a transformed phenotype. Selection favours acquisition of mutations that are neutral or beneficial (adaptive) in the context of the mutations that preceded them. However, some of these changes might be deleterious (red arrow) were it not for the changes that preceded them. If true, correcting early genetic changes (yellow arrow) will unmask these deleterious effects. In this model, cancer cells behave like a molecular ‘house of cards’.

Two genes (‘A’ and ‘B’) are said to be synthetic lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes causes death.

 espressione gene topoisomerasi II SOPRAVVIVENZA Cellula normale blocco attività E2F Rb VELENI DELLA TOPO II Cellula tumorale Rb  espressione gene A  espressione gene topoisomerasi II  attività E2F  espressione gene B  espressione gene C

B is an extragenic suppressor of A if mutation of B suppresses the phenotype observed when A is mutated.

% B inhibition % B inhibition Theoretical fitness curves for wild-type and A-/- cells in response to a drug that inhibits the B gene product.   A reading of 0% fitness denotes death, whereas 100% fitness denotes the wild-type state (for simplicity, fitness >100% is not considered in these examples). In the middle panel, a therapeutic window is created by a shift in the fitness curve when gene A is absent. In the left and right panels the therapeutic window is created by changes in the shapes of the fitness curves when gene A is absent.

Synthetic lethal screening with chemical or interfering RNA libraries Synthetic lethal screening with chemical or interfering RNA libraries.   Isogenic cell-line pairs that do or do not harbour a cancer-relevant mutation (in the case illustrated, the cell-line pair differs only with respect to a particular tumour-suppressor gene (TSG)) are grown in multiwell plates to which different chemical or genetic (short interfering RNAs, short hairpin RNAs or other interfering RNAs) perturbants are added. In time, such assays might be carried out using microarrays spotted with chemicals or siRNA species104, 105. A 'hit' is a perturbant that is cytostatic or cytotoxic to the cell with the cancer-relevant mutation (arrow). It should be noted that the interpretation of such assays needs to consider potentially confounding effects, such as differences in proliferation rate and cell-cycle distribution.

Fluorescence-based mammalian synthetic lethal assay Fluorescence-based mammalian synthetic lethal assay.   a | The Kinzler method. Isogenic cell-line pairs that do/do not harbour a cancer-relevant mutation are engineered to produce blue fluorescent protein (BFP) and yellow fluorescent protein (YFP), respectively, and are co-cultured in multiwell plates to which different chemicals are added. Selective killing of blue cells is indicative of a synthetic lethal interaction (yellow well).

HOW CAN WE SELECTIVELY TARGET TUMOR RELEVANT DEFECTS? INIBIT GENE EXPRESSION INIBIT PROTEIN FUNCTION Small molecule inhibitors AS-ODN siRNA mAbs