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Epidermal growth factor receptor (EGFR) represents the most attractive target for personalized medicine of glioblastoma (GBM) due to its frequency in and specificity for the disease, and the availability of multiple inhibitors that target its tyrosine kinase domain. Despite its therapeutic attractiveness, several EGFR-targeted tyrosine kinase inhibitors (TKI) have failed clinically, in part due to multiple molecular mechanisms of resistance. To dissect the genetic contributions to EGFR TKI resistance, we examined the sensitivity of Ink4a/Arf (CDKN2A)-null murine astrocytes engineered to harbor EGFRvIII, the most common activating mutation in GBM where exons 2–7 of the extracellular domain are deleted, to a panel of first (gefitinib, erlotinib), second (lapatinib, afatinib), and third (AZD3759) generation EGFR TKI in vitro. Moreover, we examined the role of PTEN deletion mutations on drug sensitivity. We found that EGFRvIII conferred sensitivity to 4/5 drugs (ΔIC50 1.5-6.5-fold) in the presence of wild-type Pten and 5/5 (ΔIC50 1.5-78.0-fold) when Pten was deleted. In contrast, Pten deletion conferred resistance to 4/5 drugs (ΔIC50 5.6-17.7-fold) in Ink4a/Arf-null astrocytes with wild-type Egfr and 2/5 (ΔIC50 1.4-5.4-fold) when these cells harbored EGFRvIII. Moreover, Ink4a/Arf-null, EGFRvIII-mutated astrocytes developed cross resistance to the other 4 TKI (ΔIC50 2.6-11.0-fold) when grown continuously in the presence of increasing gefitinib (≤ 2 µM). We have previously shown that dynamic kinome responses may be responsible for TKI resistance. Therefore, we are currently using a chemical proteomics method consisting of multiplex inhibitor beads and mass spectrometry (MIB-MS) to assess the functional state of the kinomes of these non-germline genetically engineered mouse (nGEM) models en masse, both in the presence (dynamic profiling) and absence (baseline profiling) of EGFR TKI. Thus, functional kinome analysis using targeted EGFR TKI and MIB-MS will help define the kinase networks required for EGFRvIII-driven GBM pathogenesis and may aid in the identification of novel treatment combinations.

The protein products of the INK4a/ARF gene locus, p16INK4a and ARF, activate distinct protective checkpoints that inhibit cell proliferation and maintain genomic stability in response to cellular stresses. As such, both factors play a central role in tumor suppression and their inactivation is one of the most frequent events in human cancer. Unique roles for p16INK4a and ARF in other key biological processes, such as aging and development, are emerging.

SummaryHistiocytic sarcoma (HS) is a rare malignant proliferation of histiocytes of uncertain molecular pathogenesis. Here, genetic analysis of coincident loss of Pten and Ink4a/Arf tumor suppressors in the mouse revealed a neoplastic phenotype dominated by a premalignant expansion of biphenotypic myelolymphoid cells followed by the development of HS. Pten protein loss occurred only in the histiocytic portion of tumors, suggesting a stepwise genetic inactivation in the generation of HS. Similarly, human HS showed genetic or epigenetic inactivation of PTEN, p16INK4A, and p14ARF, supporting the relevance of this genetically engineered mouse model of HS. These genetic and translational observations establish a cooperative role of Pten and Ink4a/Arf in the development of HS and provide mechanistic insights into the pathogenesis of human HS.

The control exerted by the INK4a/ARF locus on cellular proliferation is crucial to restrict tumor development. In agreement with this, mice with defects in this locus are highly tumor prone. However, the potential contribution of other pathways in modulating tumorigenesis in the absence of INK4a/ARF is largely unexplored. In the present study, we investigated the consequences of the combined loss of either of two cyclin-dependent kinase inhibitors, p21 and p27, in cooperation with deletion of the INK4a/ARF locus. Our results show a clear differential effect in tumorigenesis depending on the CKI that is absent. The absence of p21 produced no overt alteration of the lifespan of the INK4a/ARF-null mice, although it modified their tumor spectrum, causing a significant increase in the incidence of fibrosarcomas and the appearance of a small number of rhabdomyosarcomas. In contrast, deficiency of p27 resulted in a significant increase in lethality due to accelerated tumor development, especially in the case of T-cell lymphomas. Finally, combined deficiency of INK4a/ARF and p27 resulted in a significant increase in the number of metastatic tumors. These results demonstrate genetically the oncogenic cooperation between defects on INK4a/ARF and p27, which are common alterations in human cancer.

The INK4a/ARF locus on chromosome 9p21 is one of the important defenses against tumor development and engages both the Rb and the p53 tumor suppressor pathways through its capacity to encode two distinct proteins, p16(INK4a) and p14(ARF). Despite controversial reports, the body of present data suggests that tumor suppressors p16(INK4a) and p14(ARF) are targets of in-activation in GEP-NETs. Moreover, tumor type-specific aberrant p16(INK4a) silencing appears to be associated with advanced tumor stage and may function as a predictor of patients' outcome after surgical resection. Since conventional histological and biochemical assessment are limited with respect to predicting GEP-NET behavior or outcome, methylation profiles including INK4a/ARF might offer a tool to refine future diagnosis and therapeutic management of GEP-NET patients.

To characterize further the role of the INK4a-ARF locus in the multistep process of skin carcinogenesis, we performed a mutational analysis of this locus in skin lesions from hairless mice either irradiated with UVB alone or with a solar simulator delivering UVA + B. INK4a-ARF mutations were present in five of 57 squamous cell carcinomas (9%), but no mutation was detected in precancerous lesions. All mutations were C:G > T:A transitions located at dipyrimidic sites, the hallmark of UVB mutagenesis. Three mutations affected only the p19(ARF) reading frame, whereas two mutations affected only the p16(INK4a) transcript. This study demonstrates for the first time UV-induced mutations of INK4a-ARF that occur in a small percentage in late stages skin tumors.

Summary The INK4a/ARF locus encodes two proteins whose expression limits cellular proliferation. Whilst the biochemical activities of the two proteins appear very different, they both converge on regulating the retinoblastoma and p53 tumour suppressor pathways. Neither protein is required for normal development, but lack of either predisposes to the development of malignancy. Both proteins have also been implicated in the establishment of senescence states in response to a variety of stresses, signalling imbalances and telomere shortening. The INK4a/Arf regulatory circuits appear to be partially redundant and show evidence of rapid evolution. Especially intriguing are the large number of biological differences documented between mice and man. We review here the brief history of INK4a/Arf and explore possible links with organismal aging and the evolution of longevity.

We apologize to colleagues whose work was not discussed or cited owing to space constraints. We thank Norman Sharpless, Steven Artandi and Ron DePinho for critical reading of the manuscript. L.C. is a V foundation scholar and is supported by grants from the NIH and NCI.

Inactivation of the INK4a/ARF locus is a frequent event in non-Hodgkin's lymphomas (NHLs), which may be attributed to deletion, point mutation, and 5' CpG methylation at its promoter region. In the present study we evaluated the occurrence of deletions and genetic instability of INK4a/ARF locus in 30 paired normal and tumor samples of B cell NHLs by conducting an allelotypic analysis with two new polymorphic markers, one located at the intron 1 of p16INK4a gene and the other one placed downstream exon 1beta of p19ARF. Comparison of these results with those obtained in a previous paper using flanking markers (D9S171, D9S942, D9S958 and IFNA) allowed us to detect two new cases of microsatellite instability (L-446 and L-442), and to confirm the occurrence of LOH at the INK4a/ARF locus in one tumor (M-3770). On the contrary, this locus is not affected in three different tumors (L-421, L-272 and L-159) which exhibited LOH at some of the flanking markers.

Although several biomarkers of aging have been described in the literature, it is only recently that gerontologists have started to search for molecular biomarkers of aging. A gene or a set of genes that are expressed in a wide range of tissues and exhibit an age-dependent, easily quantifiable increase in their expression represent a possible molecular biomarker of aging. Because the physiology of an organism is profoundly affected by the pattern of gene expression, it is hoped that molecular biomarkers of aging will more accurately predict the physiological age of an organism than the chronological age. A recent report from Sharpless's laboratory examines the possibility that the tumor suppressors p16 and ARF (encoded by the INK4a/ARF locus) represent molecular biomarkers of aging in rodent models.

The human ink4a/arf locus encodes two cell cycle regulatory proteins - the cyclin-dependent kinase inhibitor (p16(ink4a)), and the p53 activator (ARF) - through the use of alternative first exons. This genomic organization is unique in eukaryotes, with two different proteins obtained using different reading frames. The divergence between mouse or opossum and human ARF is very high, whereas proteins have the same nucleolar localization and function. To gain further insights into the relative importance of ARF in different settings, we characterized here the exon 1beta of ARF in 12 different species of primates. We did not find any polymorphism in studied species (monkeys, apes, and humans). These sequences are very similar, with few amino acids substitutions compared to the human sequence. It is strange to find such a high degree of conservation among primates when there is such a low degree of conservation between the human pig, rat, or mouse, chicken exon 1beta sequences. More surprisingly, we observe a threonine at position 31 in all human sequences, whereas an alanine is always present in other sequences. We suggest that when the radiation human/simian appeared or after, a selection of threonine occurred. Moreover, the modifications detected could play a role in different interactions between ARF and other proteins to stabilize or not these complexes.

The INK4a/ARF locus encodes two physically linked tumor suppressor proteins, p16(INK4a) and ARF, which regulate the RB and p53 pathways, respectively. The unusual genomic relationship of the open reading frames of these proteins initially fueled speculation that only one of the two was the true tumor suppressor, and loss of the other merely coincidental in cancer. Recent human and mouse genetic data, however, have firmly established that both proteins possess significant in vivo tumor suppressor activity, although there appear to be species- and cell-type specific differences between the two. For example, ARF plays a clear role in preventing Myc-induced lymphomagenesis in mice, whereas the role for p16(INK4a) is human carcinomas is more firmly established. In this review, I discuss the evolutionary history of the locus, the relative importance of these tumor suppressor genes in human cancer, and recent information suggesting novel biochemical and physiologic functions of these proteins in vivo.

Senescence is a potent anticancer mechanism, representing a barrier that most, if not all, would-be tumor cells must traverse on their path to malignant transformation. In this Perspective, I discuss two recent publications ( 1 , 2 ) that deal with the durability of senescence. These findings are of interest not only to those who study aging, but to those who study cancer as well.

R.K. is an INSERM research fellow and thanks J. Weitzman, T. Chang and M. Buckingham for helpful comments.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16 INK4a and p14 ARF . Inactivation of the p16 INK4a ( MTS1 ) tumor suppressor gene by mutations, promoter methylation or gene deletions is a common event in the development of many different human tumors. The present report describes a novel polyA mononucleotide repeat situated 7·2 kb on the telomeric side of the INK4a/ARF locus. This highly polymorphic microsatellite marker (heterozygote frequency: 0.78) proved to be efficient for p16 allele loss and microsatellite instability analyses in human colon cancer.

Stem cell self-renewal capacity declines with age. In a recent issue of Cell, Nishino and colleagues (2008) show that Hmga2 maintains neural stem cell (NSC) function in young mice through repression of the Ink4a/Arf locus; in contrast, during aging, elevated let-7b blocks Hmga2 and contributes to declining NSC function.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Kras mutations are observed in 80%-90% of pancreatic cancer. Oncogenic Kras is involved with the initiation or early phase of PDAC. To understand the molecular mechanisms, several mouse models of PDAC have been generated by targeting a conditionally mutated KrasG12D to recapitulate the progression of PDAC. It was previously shown that one mouse model which activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic PDAC. However, the molecular mechanism(s) by which Kras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated. Here, we assessed the molecular alterations in mice which are activated Kras and Ink4a/Arf deficiency. For the first time, we demonstrated that deletion of Ink4a/Arf in Kras expressing mice leads to PDAC partly through Notch and NF-κB signaling pathways. Moreover, we show that several miRNAs, such as miR-200 family and miR-34 could partially play roles in tumorigenesis in activated Kras and Ink4a/Arf deficiency mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2011-2385