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Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC 50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781-97. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 ( CDKN2A ) locus and mutations in NF-κB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-naïve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFβ pathway activation signature. Implications: This study represents the first integrated analysis of mutation, copy number, and gene expression data in NPC and suggests how tumor genetics and EBV infection influence the tumor microenvironment in this disease. These insights should be considered for guiding immunotherapy treatment strategies in this disease. Mol Cancer Res; 15(12); 1722-32. ©2017 AACR ., (©2017 American Association for Cancer Research.)

SNPs in IKZF1 are associated with inherited susceptibility to B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Besides, somatic copy number abnormalities (CNA) in genes related to lymphopoiesis (e.g., IKZF1, CDKN2A/B, BTG1 ) impact patient's outcome. Therefore, this study aimed to investigate an association between germline susceptibility and CNAs in BCP-ALL. The IKZF1 SNPs (rs11978267 and rs4132601) were genotyped in 276 cases and 467 controls. Bone marrow samples were used to determine the presence of somatic abnormalities. The IKZF1 transcript levels were quantified and associated with the SNPs and CNAs. Categorical variables were compared by χ 2 test. ORs were estimated with unconditional logistic regression with 95% confidence interval (CI). The variant allele of IKZF1 rs4132601 conferred increased risk of BCP-ALL (OR, 2.09; 95% CI, 1.16-3.74). Individuals with either rs11978267 or rs4132601 had an increased risk for harboring IKZF1 deletion (OR, 2.80; 95% CI, 1.25-6.23 and OR, 2.88; 95% CI, 1.24-6.69, respectively). Increased risks were observed for individuals harboring both IKZF1 and BTG1 deletions (OR, 4.90; 95% CI, 1.65-14.55, rs11978267 and OR, 5.80; 95% CI, 1.94-17.41, rs4132601). Germline genetic variation increases the risk for childhood ALL in general, but also acts as a susceptibility factor bound for risk of specific somatic alterations. These findings provide new insight into the development of childhood ALL regarding causal variants and the biological basis of the risk association, offering the opportunity for future tailored research. Cancer Prev Res; 10(12); 738-44. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Unlabelled: ANRIL, a long noncoding RNA (lncRNA), has recently been reported to have a direct role in recruiting polycomb repressive complexes PRC2 and PRC1 to regulate the expression of the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. Expression analysis of ANRIL, EZH2, SUZ12, EED, JARID2, CBX7, BMI1, p16, p15, and p14/ARF genes was evaluated in a large cohort of invasive breast carcinomas (IBC, n = 456) by qRT-PCR and immunohistochemistry (IHC) was performed on CBX7, EZH2, p14, p15, p16, H3K27me3, and H3K27ac. We observed significant overexpression in IBCs of ANRIL (19.7%) and EZH2 (77.0%) and an underexpression of CBX7 (39.7%). Correlations were identified between these genes, their expression patterns, and several classical clinical and pathologic parameters, molecular subtypes, and patient outcomes, as well as with proliferation, epithelial-mesenchymal transition, and breast cancer stem cell markers. Multivariate analysis revealed that combined EZH2/CBX7 status is an independent prognostic factor (P = 0.001). In addition, several miRNAs negatively associated with CBX7 underexpression and EZH2 overexpression. These data demonstrate a complex pattern of interactions between lncRNA ANRIL, several miRNAs, PRC2/PRC1 subunits, and p15/CDKN2B-p16/CDKN2A-p14/ARF locus and suggest that their expression should be considered together to evaluate antitumoral drugs, in particular the BET bromodomain inhibitors., Implications: This study suggests that the global pattern of expression rather than expression of individual family members should be taken into account when defining functionality of repressive Polycomb complexes and therapeutic targeting potential. Mol Cancer Res; 14(7); 623-33. ©2016 AACR., (©2016 American Association for Cancer Research.)

The morphogen and mitogen Sonic Hedgehog (Shh) activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNP) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Shh signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice. However, enforced expression of Atoh1 in primary GNPs enhances the oncogenicity of cells overexpressing Gli1 by almost three orders of magnitude. Unlike Gli1, Atoh1 cannot support GNP proliferation in the absence of Shh signaling and does not govern expression of canonical cell cycle genes. Instead, Atoh1 maintains GNPs in a Shh-responsive state by regulating genes that trigger neuronal differentiation, including many expressed in response to bone morphogenic protein-4. Therefore, by targeting multiple genes regulating the differentiation state of GNPs, Atoh1 collaborates with the pro-proliferative Gli1-dependent transcriptional program to influence medulloblastoma development., (Copyright 2010 AACR.)

RhoA, a member of the Rho GTPase family, has been extensively studied in the regulation of cytoskeletal dynamics, gene transcription, cell cycle progression, and cell transformation. Overexpression of RhoA is found in many malignancies and elevated RhoA activity is associated with proliferation phenotypes of cancer cells. We reported previously that RhoA was hyperactivated in gastric cancer tissues and suppression of RhoA activity could partially reverse the proliferation phenotype of gastric cancer cells, but the underlying mechanism has yet to be elucidated. It has been reported that RhoA activation is crucial for the cell cycle G(1)-S procession through the regulation of Cip/Kip family tumor suppressors in benign cell lines. In this study, we found that selective suppression of RhoA or its effectors mammalian Diaphanous 1 and Rho kinase (ROCK) by small interfering RNA and a pharmacologic inhibitor effectively inhibited proliferation and cell cycle G(1)-S transition in gastric cancer lines. Down-regulation of RhoA-mammalian Diaphanous 1 pathway, but not RhoA-ROCK pathway, caused an increase in the expression of p21(Waf1/Cip1) and p27(Kip1), which are coupled with reduced expression and activity of CDK2 and a cytoplasmic mislocalization of p27(Kip1). Suppression of RhoA-ROCK pathway, on the other hand, resulted in an accumulation of p15(INK4b), p16(INK4a), p18(INK4c), and p19(INK4d), leading to reduced expression and activities of CDK4 and CDK6. Thus, RhoA may use two distinct effector pathways in regulating the G(1)-S progression of gastric cancer cells.

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Glioblastoma multiforme (GBM) is one of the most dreaded cancer diagnoses due to its poor prognosis and the limited treatment options. Homozygous deletion of the p16(INK4a)/p14(ARF)/p15(INK4b) locus is among the most common genetic alterations in GBM. Two recent studies have shown that deletion and mutation of another INK4 family member, p18(INK4c), also drives the pathogenesis of GBM. This minireview will discuss the known roles for p18(INK4c) in the initiation and progression of cancer and suggest opportunities for future studies.

Genomic alterations leading to aberrant activation of cyclin/cyclin-dependent kinase (cdk) complexes drive the pathogenesis of many common human tumor types. In the case of glioblastoma multiforme (GBM), these alterations are most commonly due to homozygous deletion of p16(INK4a) and less commonly due to genomic amplifications of individual genes encoding cyclins or cdks. Here, we describe deletion of the p18(INK4c) cdk inhibitor as a novel genetic alteration driving the pathogenesis of GBM. Deletions of p18(INK4c) often occurred in tumors also harboring homozygous deletions of p16(INK4a). Expression of p18(INK4c) was completely absent in 43% of GBM primary tumors studied by immunohistochemistry. Lentiviral reconstitution of p18(INK4c) expression at physiologic levels in p18(INK4c)-deficient but not p18(INK4c)-proficient GBM cells led to senescence-like G(1) cell cycle arrest. These studies identify p18(INK4c) as a GBM tumor suppressor gene, revealing an additional mechanism leading to aberrant activation of cyclin/cdk complexes in this terrible malignancy.

Cell cycle progression from G(1) to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16(Ink4a) and p18(Ink4c) showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16(Ink4a) and p18(Ink4c) resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16(Ink4a-/-);p18(Ink4c-/-) mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18(Ink4c-/-) mice and in MEFs from p16(Ink4a-/-), p18(Ink4c-/-), or p16(Ink4a-/-);p18(Ink4c-/-) mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16(Ink4a) and p18(Ink4c) coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice.

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice and showed that p18, but not p27, functionally collaborates with Men1 in suppressing lung tumorigenesis. Lung tumors developed in both Men1(+/-) and p18(-/-);Men1(+/-) mice at a high penetrance and contain both neuroendocrine and nonneuroendocrine cells. The remaining wild-type Men1 allele was lost in most lung tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-);Men1(+/-) mice, showing a functional collaboration between p18 and Men1 in lung tumor suppression. Phosphorylation of Rb protein at both CDK2 and CDK4/CDK6 sites were significantly increased in normal bronchial epithelia and tumor cells derived from p18(-/-);Men1(+/-) mice compared to those from single p18(-/-) or Men1(+/-) mice. Lung tumors developed in p18(-/-);Men1(+/-) mice were multifocal, more heterogeneous, and highly invasive compared to those developed in either p18(-/-) or Men1(+/-) mice. Bronchioalveolar stem cells are expanded in normal and tumorigenic lungs of p18(-/-) mice and are further expanded in p18(-/-);Men1(+/-) lung tumors. These results reveal a previously unrecognized function of p18 in lung tumor suppression through collaboration with Men1 to control lung stem cell proliferation.

Mice lacking p53 and one or two alleles of the cyclin D-dependent kinase inhibitor p18(Ink4c) are prone to medulloblastoma development. The tumor frequency is increased by exposing postnatal animals to ionizing radiation at a time when their cerebella are developing. In irradiated mice engineered to express a floxed p53 allele and a Nestin-Cre transgene, tumor development can be restricted to the brain. Analysis of these animals indicated that inactivation of one or both Ink4c alleles did not affect the time of medulloblastoma onset but increased tumor invasiveness. All such tumors exhibited complete loss of function of the Patched 1 (Ptc1) gene encoding the receptor for sonic hedgehog, and many exhibited other recurrent genetic alterations, including trisomy of chromosome 6, amplification of N-Myc, modest increases in copy number of the Ccnd1 gene encoding cyclin D1, and other complex chromosomal rearrangements. In contrast, medulloblastomas arising in Ptc1(+/-) mice lacking one or both Ink4c alleles retained p53 function and exhibited only limited genomic instability. Nonetheless, complete inactivation of the wild-type Ptc1 allele was a universal event, and trisomy of chromosome 6 was again frequent. The enforced expression of N-Myc or cyclin D1 in primary cerebellar granule neuron precursors isolated from Ink4c(-/-), p53(-/-) mice enabled the cells to initiate medulloblastomas when injected back into the brains of immunocompromised recipient animals. These "engineered" tumors exhibited gene expression profiles indistinguishable from those of medulloblastomas that arose spontaneously. These results underscore the functional interplay between a network of specific genes that recurrently contribute to medulloblastoma formation.

AIMP3 (previously known as p18) was shown to up-regulate p53 in response to DNA damage. Here, we show that AIMP3 couples oncogenic stresses to p53 activation to prevent cell transformation. Growth factor- or Ras-dependent induction of p53 was blocked by single allelic loss of AIMP3 as well as by suppression of AIMP3. AIMP3 heterozygous cells became susceptible to cell transformation induced by oncogenes such as Ras or Myc alone. The transformed AIMP3+/- cells showed severe abnormality in cell division and chromosomal structure. Thus, AIMP3 plays crucial roles in p53-mediated tumor-suppressive response against oncogenic stresses via differential activation of ATM and ATR, and in the maintenance of genomic stability.

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G(0)/G(1) to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18(Ink4c) and p27(Kip1). Moreover, Men1 excision results in decreased expression of p18(Ink4c) and p27(Kip1) in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G(0)/G(1) to S transition.

Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18(INK4C) (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo, whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18-/- origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia-initiating cells (mainly in a CD3(lo) cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3+ CD8+ cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18.