p16INK4a Deletion Research Articles

Transcriptome analysis reveals genes associated with stem cell activation by physical exercise in the dentate gyrus of aged p16Ink4a knockout mice.

Throughout adulthood neural stem cells divide in neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone-producing progenitor cells and new neurons. Stem cells self-renew, thus preserving their pool. Furthermore, the number of stem/progenitor cells in the neurogenic niches decreases with age. We have previously demonstrated that the cyclin-dependent kinase inhibitor p16Ink4a maintains, in aged mice, the pool of dentate gyrus stem cells by preventing their activation after a neurogenic stimulus such as exercise (running). We showed that, although p16Ink4a ablation by itself does not activate stem/progenitor cells, exercise strongly induced stem cell proliferation in p16Ink4a knockout dentate gyrus, but not in wild-type. As p16Ink4a regulates stem cell self-renewal during aging, we sought to profile the dentate gyrus transcriptome from p16Ink4a wild-type and knockout aged mice, either sedentary or running for 12days. By pairwise comparisons of differentially expressed genes and by correlative analyses through the DESeq2 software, we identified genes regulated by p16Ink4a deletion, either without stimulus (running) added, or following running. The p16Ink4a knockout basic gene signature, i.e., in sedentary mice, involves upregulation of apoptotic, neuroinflammation- and synaptic activity-associated genes, suggesting a reactive cellular state. Conversely, another set of 106 genes we identified, whose differential expression specifically reflects the pattern of proliferative response of p16 knockout stem cells to running, are involved in processes that regulate stem cell activation, such as synaptic function, neurotransmitter metabolism, stem cell proliferation control, and reactive oxygen species level regulation. Moreover, we analyzed the regulation of these stem cell-specific genes after a second running stimulus. Surprisingly, the second running neither activated stem cell proliferation in the p16Ink4a knockout dentate gyrus nor changed the expression of these genes, confirming that they are correlated to the stem cell reactivity to stimulus, a process where they may play a role regulating stem cell activation.

Oxylipin-PPARγ-initiated adipocyte senescence propagates secondary senescence in the bone marrow

The chronic use of glucocorticoids decreases bone mass and quality and increases bone-marrow adiposity, but the underlying mechanisms remain unclear. Here, we show that bone-marrow adipocyte (BMAd) lineage cells in adult mice undergo rapid cellular senescence upon glucocorticoid treatment. The senescent BMAds acquire a senescence-associated secretory phenotype, which spreads senescence in bone and bone marrow. Mechanistically, glucocorticoids increase the synthesis of oxylipins, such as 15d-PGJ2, for peroxisome proliferator-activated receptor gamma (PPARγ) activation. PPARγ stimulates the expression of key senescence genes and also promotes oxylipin synthesis in BMAds, forming a positive feedback loop. Transplanting senescent BMAds into the bone marrow of healthy mice is sufficient to induce the secondary spread of senescent cells and bone-loss phenotypes, whereas transplanting BMAds harboring a p16INK4a deletion did not show such effects. Thus, glucocorticoid treatment induces a lipid metabolic circuit that robustly triggers the senescence of BMAd lineage cells that, in turn, act as the mediators of glucocorticoid-induced bone deterioration.

P16 I NK 4a Deletion Ameliorates Damage of Intestinal Epithelial Barrier and Microbial Dysbiosis in a Stress-Induced Premature Senescence Model of Bmi-1 Deficiency.

This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1–deficient (Bmi-1–/–), Bmi-1 and p16INK4a double-knockout (Bmi-1–/–p16INK4a–/–), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)–dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1–/– and Bmi-1–/–p16INK4a–/– mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1–/– mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α–dependent barrier permeability and aging. Accumulated p16INK4a combined with occludin at the 1st–160th residue in cytoplasm of intestinal epithelium cells from Bmi-1–/– mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16INK4a deletion could maintain barrier function and microbiota balance in Bmi-1–/– mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16INK4a accumulation. The clearance of p16INK4a-positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1–160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16INK4a with occludin for protecting TJ.

PDGFR\u03b2 is an essential therapeutic target for BRCA1-deficient mammary tumors

BackgroundBasal-like breast cancers (BLBCs) are a leading cause of cancer death due to their capacity to metastasize and lack of effective therapies. More than half of BLBCs have a dysfunctional BRCA1. Although most BRCA1-deficient cancers respond to DNA-damaging agents, resistance and tumor recurrence remain a challenge to survival outcomes for BLBC patients. Additional therapies targeting the pathways aberrantly activated by BRCA1 deficiency are urgently needed.MethodsMost BRCA1-deficient BLBCs carry a dysfunctional INK4-RB pathway. Thus, we created genetically engineered mice with Brca1 loss and deletion of p16INK4A, or separately p18INK4C, to model the deficient INK4-RB signaling in human BLBC. By using these mutant mice and human BRCA1-deficient and proficient breast cancer tissues and cells, we tested if there exists a druggable target in BRCA1-deficient breast cancers.ResultsHeterozygous germline or epithelium-specific deletion of Brca1 in p18INK4C- or p16INK4A-deficient mice activated Pdgfrβ signaling, induced epithelial-to-mesenchymal transition, and led to BLBCs. Confirming this role, targeted deletion of Pdgfrβ in Brca1-deficient tumor cells promoted cell death, induced mesenchymal-to-epithelial transition, and suppressed tumorigenesis. Importantly, we also found that pharmaceutical inhibition of Pdgfrβ and its downstream target Pkcα suppressed Brca1-deficient tumor initiation and progression and effectively killed BRCA1-deficient cancer cells.ConclusionsOur work offers the first genetic and biochemical evidence that PDGFRβ-PKCα signaling is repressed by BRCA1, which establishes PDGFRβ-PKCα signaling as a therapeutic target for BRCA1-deficient breast cancers.

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

TGF-β1/IL-11/MEK/ERK signaling mediates senescence-associated pulmonary fibrosis in a stress-induced premature senescence model of Bmi-1 deficiency

To study whether TGF-β1/IL-11/MEK/ERK (TIME) signaling mediates senescence-associated pulmonary fibrosis (SAPF) in Bmi-1-deficient (Bmi-1−/−) mice and determines the major downstream mediator of Bmi-1 and crosstalk between p16INK4a and reactive oxygen species that regulates SAPF, phenotypes were compared among 7-week-old p16INK4a and Bmi-1 double-knockout, N-acetylcysteine (NAC)-treated Bmi-1−/−, Bmi-1−/−, and wild-type mice. Pulmonary fibroblasts and alveolar type II epithelial (AT2) cells were used for experiments. Human pulmonary tissues were tested for type Ι collagen, α-smooth muscle actin (α-SMA), p16INK4a, p53, p21, and TIME signaling by using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that Bmi-1 deficiency resulted in a shortened lifespan, ventilatory resistance, poor ventilatory compliance, and SAPF, including cell senescence, DNA damage, a senescence-associated secretory phenotype and collagen overdeposition that was mediated by the upregulation of TIME signaling. The signaling stimulated cell senescence, senescence-related secretion of TGF-β1 and IL-11 and production of collagen 1 by pulmonary fibroblasts and the epithelial-to-mesenchymal transition of AT2 cells. These processes were inhibited by anti-IL-11 or the MEK inhibitor PD98059. NAC treatment prolonged the lifespan and ameliorated pulmonary dysfunction and SAPF by downregulating TIME signaling more than p16INK4a deletion by inhibiting oxidative stress and DNA damage and promoting ubiquitin-proteasome degradation of p16INK4a and p53. Cytoplasmic p16INK4a accumulation upregulated MEK/ERK signaling by inhibiting the translocation of pERK1/2 (Thr202/Tyr204) from the cytoplasm to the nucleus in senescent fibroblasts. The accumulation of collagen 1 and α-SMA in human lungs accompanied by cell senescence may be mediated by TIME signaling. Thus, this signaling in aging fibroblasts or AT2 cells could be a therapeutic target for preventing SAPF.

Hoxa9 silencing of p16 ink4a is essential for murine lymphopoiesis

Abstract The homeo-domain transcription factor Hoxa9 is required for normal B and T lymphocyte development in bone marrow (BM) and thymus, respectively. Here we show that Hoxa9-deficiency is accompanied by increased expression of the cdkn2a tumor suppressor locus that encodes the cell cycle inhibitor p16Ink4a. Furthermore, we found that somatic deletion of p16Ink4a is sufficient to rescue the B and T cell progenitor deficiencies, as well as the BM lymphoid progenitor pools, in hoxa9−/− mice. In contrast, the hematopoietic stem cell (HSC) and multipotential progenitor (MPP) abnormalities characteristic of Hoxa9-deficient mice were not corrected by loss of p16Ink4a. These new data reveal that Hoxa9 repression of p16ink4a is essential for lymphoid lineage development in BM and thymus. Importantly, they highlight distinct functions for Hoxa9 in regulation of primitive hematopoietic progenitors versus those committed to the B or T cell fates.

P16Ink4a deletion in cells of the intervertebral disc affects their matrix homeostasis and senescence associated secretory phenotype without altering onset of senescence

Intervertebral disc degeneration is an important contributor to chronic low back and neck pain. Although many environmental and genetic factors are known to contribute to disc degeneration, age is still the most significant risk factor. Recent studies have shown that senescence may play a role in age-related disc degeneration and matrix catabolism in humans and mouse models. Clearance of p16Ink4a-positive senescent cells reduces the degenerative phenotype in many age-associated diseases. Whether p16Ink4a plays a functional role in intervertebral disc degeneration and senescence is unknown. We first characterized the senescence status of discs in young and old mice. Quantitative histology, gene expression and a novel p16tdTom reporter mice showed an increase in p16Ink4a, p21 and IL-6, with a decrease in Ki67 with aging. Accordingly, we studied the spinal-phenotype of 18-month-old mice with conditional deletion of p16Ink4a in the disc driven by Acan-CreERT2 (cKO). The analyses of discs of cKO and age-matched control mice showed little change in cell morphology and tissue architecture. The cKO mice exhibited changes in functional attributes of aggrecan as well as in collagen composition of the intervertebral disc. While cKO discs exhibited a small decrease in TUNEL positive cells, lineage tracing experiments using ZsGreen reporter indicated that the overall changes in cell fate or numbers were minimal. The cKO mice maintained expression of NP-cell phenotypic markers CA3, Krt19 and GLUT-1. Moreover, in cKO discs, levels of p19Arf and RB were higher without alterations in Ki67, γH2AX, CDK4 and Lipofuscin deposition. Interestingly, the cKO discs showed lower levels of SASP markers, IL-1β, IL-6, MCP1 and TGF-β1. These results show that while, p16Ink4a is dispensable for induction and maintenance of senescence, conditional loss of p16Ink4a reduces apoptosis, limits the SASP phenotype and alters matrix homeostasis of disc cells.

Deletion of the p16INK4a tumor suppressor and expression of the androgen receptor induce sarcomatoid carcinomas with signet ring cells in the mouse prostate.

The tumor suppressor p16Ink4a, encoded by the INK4a gene, is an inhibitor of cyclin D-dependent kinases 4 and 6, CDK4 and CDK6. This inhibition prevents the phosphorylation of the retinoblastoma protein (pRb), resulting in cellular senescence through inhibition of E2F-mediated transcription of S phase genes required for cell proliferation. The p16Ink4a plays an important role in tumor suppression, whereby its deletion, mutation, or epigenetic silencing is a frequently observed genetic alteration in prostate cancer. To assess its roles and related molecular mechanisms in prostate cancer initiation and progression, we generated a mouse model with conditional deletion of p16Ink4a in prostatic luminal epithelium. The mice underwent oncogenic transformation and developed prostatic intraepithelial neoplasia (PIN) from eight months of age, but failed to develop prostatic tumors. Given the prevalence of aberrant androgen signaling pathways in prostate cancer initiation and progression, we then generated R26hARL/wt:p16L/L: PB-Cre4 compound mice, in which conditional expression of the human AR transgene and deletion of p16Ink4a co-occur in prostatic luminal epithelial cells. While R26hARL/wt:PB-Cre4 mice showed no visible pathological changes, R26hARL/wt:p16L/L: PB-Cre4 compound mice displayed an early onset of high-grade PIN (HGPIN), prostatic carcinoma, and metastatic lesions. Strikingly, we observed tumors resembling human sarcomatoid carcinoma with intermixed focal regions of signet ring cell carcinoma (SRCC) in the prostates of the compound mice. Further characterization of these tumors showed they were of luminal epithelial cell origin, and featured characteristics of epithelial to mesenchymal transition (EMT) with enhanced proliferative and invasive capabilities. Our results not only implicate a biological role for AR expression and p16Ink4a deletion in the pathogenesis of prostatic SRCC, but also provide a new and unique genetically engineered mouse (GEM) model for investigating the molecular mechanisms for SRCC development.

Cooperative Repression of IRF4 and Induction of IRF7 By CDK4/6 Inhibition Sensitizes Mantle Cell Lymphoma Cells to IMiD-Mediated Apoptosis Independent of p16 Deletion or c-Myc Overexpression

Lenalidomide (Len) and pomalidomide (Pom) are standard of care therapies for multiple myeloma (MM) and also exhibit clinical efficacy in some lymphomas such as diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). As single agents, each rarely achieves complete remission despite a high overall response rate and durability; however, they potentiate the clinical response to diverse partners including Rituximab in MCL. As immunomodulatory drugs (IMiDs), Len and Pom target multiple cell types in vivo and have been shown to promote the expansion of NK cells in MCL. Len in combination with BTK inhibitors apparently leads to synthetic lethality in the activated B cell-like DLBCL cell lines, but the mechanism that mediates IMiD sensitivity in MCL cells remains largely unknown.We found that Len markedly enhanced ACP196 (second generation BTK inhibitor) killing of the CCMCL1 MCL cell line, despite c-Myc overexpression and deletion of p16INK4a, but not the JEKO-1 cells, which harbor TP53 deletion. Therefore Len appears to enhance killing by BTK inhibition but does not confer sensitivity to resistant MCL cells. Next, we investigated how inhibition of CDK4 might sensitize MCL cells to Len or Pom killing, given that dysregulation of cyclin D1 and CDK4 (CDK6 is silenced in MCL) underlies uncontrolled proliferation and disease progression, and inhibition of CDK4 is clinically efficacious in MCL. Indeed, priming of CCMCL1 cells by prior induction of G1 arrest with the selective CDK4/6 inhibitor palbociclib markedly sensitized them to Len-induced apoptosis, but only marginally so in JEKO-1 cells. Kinetic studies revealed that Len reduced the IRF4 protein markedly in CCMCL1 cells arrested in G1 by CDK4 inhibition, but marginally so in cycling CCMCL1 cells and not at all in Len-resistant JEKO-1 or Maver-1 cells. Reduction of IRF4 protein by Len in prolonged G1 arrest was concomitant with induction of IRF7 protein and tightly associated with apoptosis, as evidenced by PARP and caspase-3 cleavage. Moreover, inhibition of CDK4 conferred sensitivity to Pom in the Len-resistant JEKO-1 cells, and it was concurrent with reduction of IRF4 and induction of IRF7.The triple combination of Len, Ibrutinib, and Rituximab in MCL has been shown to be clinically active in relapsed MCL. To explore the underpinning, we found by RNA-seq that CD20 mRNA expression was downregulated in normal primary human peripheral B cells when they were activated into the cell cycle and in primary MCL cells, suggesting an inverse correlation between cell cycling and CD20 mRNA expression. However, surface CD20 expression was not collinear with CD20 mRNA expression and there was no evidence for upregulation of CD20 by BTK inhibition. On this basis, upregulation of the rituximab target CD20 is unlikely to be the primary mechanism for improved clinical response to this triple combination in MCL.In summary, our data suggest cooperative repression of IRF4 and induction of IRF7 as a central mechanism that underlies enhancement of Len and Pom killing by inhibition of BTK or CDK4, independent of deletion of the physiologic CDK4/6 inhibitor p16INK4a or overexpression of c-Myc. Furthermore, it appears that induction of prolonged early G1 arrest by Palbociclib confers Pom sensitivity independent of p53, implicating a potential to combine Palbociclilb with Pom for treatment of MCL with p53 loss or mutation. DisclosuresNo relevant conflicts of interest to declare.

Correlations between p16 Protein Expression and Genomic Profile in Mantle Cell Lymphoma and Impact on Survival, a Lyma-Genomic Project Conducted on Behalf of the Lysa Group

IntroductionMantle cell lymphoma (MCL) is a heterogeneous disease with a complex genetic landscape. Among genetic anomalies, alterations of several tumor suppressor genes are prognostic markers. The p16INK4A protein, encoded by CDKN2A, is known to bind and inactivate the cyclin-dependent kinase CDK4/6, blocking the phosphorylation of the retinoblastoma protein Rb and inducing cell cycle arrest. The p16INK4A and p53 overexpression are associated with poor prognosis (D. Canioni et al. ASH 2017). Here we compared the expression levels of p16INK4A and p53 in immunohistochemistry (IHC) with the profile (copy number alterations, CNAs) of the genes encoding these proteins, on diagnosis MCL lymph nodes. Results were correlated with patients' outcome in order to identify prognostic biomarkers in MCL.MethodsAll samples (n=86) used in the present work were collected from untreated MCL patients enrolled in the LyMa trial (S. Le Gouill et al. NEJM 2017). IHC was performed for p16INK4A and p53 protein expression assessment on formalin fixed paraffin embedded diagnostic Tissue Micro Arrays. Cut-offs for over expression of p16INK4A and p53 proteins were 10% and 30% respectively (D. Canioni et al. ASH 2016). A pan-genomic copy number analysis was performed with the Oncoscan® SNP-array on DNA extracted from the same samples. Data were compared using chi² tests. Progression free survival (PFS) and overall survival (OS) were studied by log rank test and Kaplan Meier representation.ResultsPatients characteristics (n=86) were similar to the whole LyMa trial population (n= 299) regarding age, gender, Ann Arbor stage and blastoid morphology. Overexpression of p16INK4A was observed in 11% of the patients and was not associated with any deletion of CDKN2A.There was a significant association between p16INK4A protein overexpression and TP53 mono-allelic deletion (38% vs 7%; p<0.05). CNAs of the CDK4 and RB genes were not associated with p16INK4A protein expression level. Mono and bi-allelic losses of CDKN2A were observed in 19% and 8% of the cases respectively. As expected, bi-allelic loss of CDKN2A (n=7) was associated with a weak p16INK4A expression <5% (p<10-6). However, a similar p16INK4A expression was observed between patients with mono-allelic losses (n=16) and those retaining both copies of CDKN2A (n=65) (p=NS). The 3q26 (BCL6) gains (n=32) were also associated with a higher p16 expression (70% vs 33%; p=0.04). Overexpression of the p53 protein (55% of the patients) was negatively associated with the 15q11 deletion (4% versus 29%; p=0.005) and positively associated with the 1q23 deletion (22% vs 4%; p=0.04) but not with the 17p13 (TP53) deletion.Regarding patients' outcome, early relapse or progression (<1y) were associated with TP53 deletion (HR=5.8; 95%CI 1.0-33.4), CDKN2A deletion (HR=4.0; 95%CI 0.8-22.5), p53 overexpression (HR=7.6; 95%CI 0.9-354) and p16INK4A overexpression (HR=9.0; 95%CI 1.4-56.9) (p<0.05). Overexpression of p16INK4A (p= 0.009) and TP53 deletion (p=0.05) were both found to be associated with a shorter OS in univariate analysis. Only p16INK4A overexpression had an independent impact on OS after multivariate analysis including TP53 and CDKN2A deletion, p16 and p53 expression, (p=0.03; HR=4.3; CI95%: 1.2-15.0). Patients with p16INK4A overexpression or double CDKN2A deletion displayed a worse PFS (p=0.05; HR= 2.7; 95%CI 0.8-8.6) and OS (p=0.02; HR=2.7; 95%CI 0.8-8.6) (Figure).ConclusionThis work shows that p16INK4A protein expression is correlated with TP53 deletion and BCL6 gain. The p16INK4A overexpression or CDKN2A double deletion could be used as prognostic biomarkers at diagnosis to predict poor response in first line treatment. [Display omitted] DisclosuresHermine:Hybrigenics: Research Funding; Erythec: Research Funding; Novartis: Research Funding; Celgene Corporation: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding.

Identification of Genetic Pathways Controlling Resistance to Standard Combination Chemotherapy in Acute Myeloid Leukemia

Introduction: Resistance to chemotherapy, manifesting as refractory or relapsed disease, remains the largest cause of mortality in acute myeloid leukemia (AML). Clonal evolution through the acquisition of new mutations, or selection of resistant clones, appears responsible in many cases of relapsed disease. We used a genome-wide CRISPR-Cas9 loss of function screen to identify deleted genes, which mediate resistance to Cytarabine (AraC) and anthracycline, Doxorubicin (Dox) in AML.Methods: Dose response was defined using MTS viability assays, with synergism determined using the Chou-Talalay method. For the screen, Cas9-expressing OCI-AML3 cells were transduced with the Brunello gRNA library, which contains 76,441 gRNAs targeting 19,114 genes (Doench et al. 2016, Nat Biotech). Cells were treated with continuous, intermediate-dose AraC/Dox (A/D) or with intermittent, high-dose A/D. Cell viability was monitored by trypan blue exclusion. gRNA representation was measured prior to, and at the end of treatment, using next-generation sequencing. Individual genomic targets were examined sequentially, using multiple gRNAs and correlated with effects on proliferation and cell cycle. Validation was performed in vivo, in independent cell lines (MV4;11) and in silico using bioinformatics on human clinical datasets. Differences in mean values were compared with t-tests.Results: AraC and Dox were synergistic at ratios 10:1, 20:1 and 40:1; the most synergistic ratio (40:1) was used for the screen. By 12 days, both continuous and intermittent dosing eliminated non-transduced controls and the bulk of library-transduced cells. Rapid resistance was seen in AraC monotherapy (10 days), however using combined AraC and Dox, a resistant population emerged by 20 days, mimicking dynamics of relapsed or refractory disease. Resistant populations also showed increased IC50 in vitro and resistance to in vivo A/D when transplanted into immunodeficient NRGS mice, compared to native cell lines (higher white cell counts (3.9 vs 1.9 x 109/L, p = 0.013), lower platelets (597 vs 978 x 109/L, p = 0.024) and higher bone marrow human CD45 chimerism (12.6 vs 1.7%, p = 0.007)).Deoxycytidine kinase (DCK) and cyclin-dependent kinase inhibitor 2a (CDKN2A) were the top hits, out of ~10 genes identified. DCK catalyses the rate-limiting step in metabolising AraC to its active form, and accordingly, AML cells with CRISPR-mediated DCK deletion were resistant to AraC, but also to combination A/D, demonstrating that resistance to a single agent may result in therapeutic failure of combination regimens.CDKN2A transcriptionally encodes p14ARF and p16INK4A, using common exons 2 and 3, but a distinct exon 1 and alternate reading frame. Enriched CDKN2A gRNAs identified in the screen were directed at common exon 2 and p16INK4A unique exon 1, with a p14ARF exon 1 gRNA not being represented in the Brunello library. p16INK4A regulates cell cycle and proliferation by inhibiting G1-S transition and p14ARF prevents p53 degradation, promoting apoptosis. CDKN2A is frequently deleted in multiple types of cancer. Functionally, CDKN2A exon 2 knockout, expected to disrupt both p14ARF and p16INK4A functions, led to chemotherapy resistance through enhanced proliferation and prevention of cell cycle arrest after low-dose and high-dose chemotherapy treatment. To delineate differential contributions of p14ARF and p16INK4A deletion to this resistance phenotype, we deleted p16INK4A unique exon 1. p16INK4A exon 1 deletion also conferred a proliferative advantage in low-dose chemotherapy, but not high-dose, suggesting that concurrent impaired function of p14ARF is required to confer resistance to high-dose chemotherapy. Finally, gene expression data from 3 independent AML patient cohorts (Verhaak, et al. 2009, Haematologica; Metzeler, et al. 2008, Blood; Bullinger, unpublished) demonstrated that low CDKN2A expression conferred inferior survival, confirming the clinical relevance of CDKN2A loss-of-function in AML.Conclusion: This study demonstrates the utility of genome-wide CRISPR screens to functionally capture genetic heterogeneity and evolution through chemotherapy treatment in AML. This approach can identify clinically relevant gene mutations and therapeutic vulnerabilities. DisclosuresBullinger:Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lane:Novartis: Consultancy; Celgene: Consultancy; Janssen: Consultancy, Research Funding.

Abstract 2302: Preclinical evaluation of CDK4/6 inhibitor and biomarker exploration in gastric cancer

Abstract Dysregulation of cell cycle is a hallmark of cancer and the p16INK4a-CDK4/6-Rb pathway is often disrupted in many types of cancer including gastric cancer. Inhibition of CDK4/6 in cancer cells has become a promising therapeutic strategy, but not tested in gastric cancer. In this study, we investigated the effect of abemaciclib (LY2835219), a dual inhibitor of CDK4 and CDK6, and identified potential biomarkers in 49 gastric cancer cell lines. The cytotoxicity of abemaciclib was assessed by CCK-8 assay. For exploration of predictive biomarkers, status of single nucleotide variants (SNVs) and copy number variations (CNVs) were analyzed by whole exome sequencing (WES). Also, RNA and protein expression levels of cell cycle related molecules were determined by RNA sequencing and Western Blot, respectively. In TCGA study (The Cancer Genome Atlas, Nature 2017) approximately 37% of stomach adenocarcinoma patients have defects in the p16INK4a-CDK4/6-Rb pathway (143/388patients); notable signatures include alterations of p16INK4a (14%), Rb1 (7%), CDK4 (4%), CDK6 (8%), cyclinD1 (6%), and cyclinE1 (12%). In our 49 gastric cancer cell lines, we confirmed 25 cell lines have defects in this pathway (51%) including alterations of p16INK4a (38%), Rb1 (10%), CDK6 (8%), cyclinD1 (4%), and cyclinE1 (2%). Based on the sensitivity of abemaciclib, we could divide 49 gastric cancer cell lines into: sensitive (IC50&amp;lt;0.5uM, 28/49 cell lines) and resistant (IC50≥0.5uM, 21/49cell lines) groups with the cut off 0.5uM which was determined by clinically achievable concentration from phase I trial. As a result of relation analysis between protein expression and sensitivity of abemaciclib, p16INK4a deletion did not correlate with sensitivity. Then, correlation analysis between sensitivity and RNA expressions of p16INK4a-CDK4/6-Rb pathway related genes were determined. Significant correlations were found in p16INK4a (r=0.4783, p=0.0016), Rb1 (r=-0.3206, p=0.0410), and cyclinE1 (r=0.5378, p=0.0003) whereas not in cyclinD1, CDK4, and CDK6. Protein expression levels of p16INK4a and cyclinD1 were not related to sensitivity of abemaciclib. In addition, with reproduction of the GDSC (Genomics of Drug Sensitivity in Cancer) information, focal recurrent copy number alterations of regions of chromosome 9p24.3-p21.1 (cnaPANCAN144) were correlated with CDK4/6 inhibitor sensitivity. Among ninety-one genes within these regions, 16 genes from cnaPANCAN144 were correlated with sensitivity of abemaciclib (p≤0.05). Our results indicate that abemaciclib is a potential therapeutic agent for gastric cancer and demonstrate that RNA expressions of p16INK4a, Rb1, and cyclinE1 would be predictive biomarkers based on molecular genetic profiling. Citation Format: Hyun Joo Bae, Woo Sun Kwon, Hyun Jeong Kim, Sun Kyoung Kang, Inhye Jeong, Tae Soo Kim, Hyun Cheol Chung, Sun Young Rha. Preclinical evaluation of CDK4/6 inhibitor and biomarker exploration in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2302.

Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice

AbstractThe hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf. However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)–dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal–repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor–independent using a constitutively active IL-3 receptor common β subunit (βcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

P16INK4a Deletion Ameliorated Renal Tubulointerstitial Injury in a Stress-induced Premature Senescence Model of Bmi-1 Deficiency

To determine whether p16INK4a deletion ameliorated renal tubulointerstitial injury by inhibiting a senescence-associated secretory phenotype (SASP) in Bmi-1-deficient (Bmi-1−/−) mice, renal phenotypes were compared among 5-week-old Bmi-1 and p16INK4a double-knockout, and Bmi-1−/− and wild-type mice. Fifth-passage renal interstitial fibroblasts (RIFs) from the three groups were analyzed for senescence and proliferation. The effect of Bmi-1 deficiency on epithelial-to-mesenchymal transition (EMT) was examined in Bmi-1-knockdown human renal proximal tubular epithelial (HK2) cells, which were treated with concentrated conditioned medium (CM) from the fifth-passage renal interstitial fibroblasts (RIFs) of above three group mice or with exogenous TGF-β1. Our results demonstrated that p16INK4a deletion largely rescued renal aging phenotypes caused by Bmi-1 deficiency, including impaired renal structure and function, decreased proliferation, increased apoptosis, senescence and SASP, DNA damage, NF-κB and TGF-β1/Smad signal activation, inflammatory cell infiltration, and tubulointerstitial fibrosis and tubular atrophy. P16INK4a deletion also promoted proliferation, reduced senescence and SASP of RIFs and subsequently inhibited EMT of Bmi-1-knockdown HK2 cells. TGF-β1 further induced the EMT of Bmi-1-knockdown HK2 cells. Thus, p16INK4a positive senescent cells would be a therapeutic target for preventing renal tubulointerstitial injury.

MOZ-Mediated Repression of p16INK4a Is Critical for the Self-Renewal of Neural and Hematopoietic Stem Cells

Although inhibition of p16INK4a expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16INK4a expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16INK4a. In the absence of the HAT activity of MOZ, expression of p16INK4a is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16INK4a reverses the proliferative defect in both MozHAT−/− hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16INK4a expression and to protect stem cells from early entrance into replicative senescence.

Detection of FHIT and p16 mRNA Deletion in Biopsy Specimens Obtained by Bronchoscopy for the Diagnosis of Lung Cancer

The purpose of this study was to investigate the diagnostic value of the deletion of fragile histidine triad (FHIT) and p16INK4a (p16) mRNA in biopsies obtained by bronchoscopy. Biopsies were analyzed using RT-PCR in 52 patients with lung cancer and 19 patients with benign lung disease. The results showed that the detection rates of FHIT and p16 gene transcript deletion were significantly higher in lung cancer patients than in patients with benign lung disease (65.4% versus 10.5%, p=0.001 and 59.6% versus 5.3%, p<0.001, respectively). The sensitivities for detecting FHIT and p16 transcript deletion in biopsies were 65.4% and 59.6% (combined 80.8%), respectively, which were markedly better than those of histology and cytology (42.3% and 34.6%, respectively; combined 57.7%). In 22 lung cancer patients with negative histology and cytology at initial bronchoscopy, FHIT and p16 mRNA loss was detected in 40.9% (9/22) and 36.4% (8/22) cases, respectively. FHIT mRNA loss was associated with smoking status in lung cancer patients. In conclusion, deletion of FHIT and p16 mRNA can be identified in biopsies obtained during bronchoscopic procedures. FHIT and p16 mRNA deletion can be used as biomarkers in the clinical diagnosis of lung cancer and may serve as adjuncts to histology and cytology in lung cancer diagnosis.

Abstract 1087: Differential oncogenicity of N-RAS mutations in melanoma.

Abstract In human cancer, N-RAS mutations resulting in constitutive, oncogenic signaling are predominately localized to codons 12, 13 or 61. Traditionally, activating RAS mutations have been considered oncogenically equivalent, yet recent studies suggest important clinical distinctions between colon cancers containing K-RAS codon 12 vs. codon 13 mutations. More than 20% of human melanomas harbor N-RAS mutations, the vast majority of which target codon 61, as opposed to N-RAS mutations of codon 12 or 13, which are common in other tumor types. To address this issue, we characterized syngeneic knock-in mouse models conditionally expressing either N-RasG12D or N-RasQ61R under the control of the endogenous locus. In primary melanocyte cultures, activation of either N-Ras allele reduced cellular proliferation even in the presence of concomitant p16INK4a deletion. Correspondingly, melanocyte-specific N-RasG12D expression combined with p16INK4a loss failed to efficiently promote melanoma formation in mice. In contrast, melanocyte-specific N-RasQ61R induction readily combined with p16INK4a loss to promote melanoma formation in vivo with short latency and high penetrance. These results provide a first murine model of melanoma featuring knock-in N-Ras mutationQ61R, and suggest that the preference for codon 61 mutations in human tumors is conserved in mice. Citation Format: Christin Burd, William R. Jeck, Kailing Fu, Kelly S. Clark, Jessie C. Xiong, George P. Souroullas, Norman E. Sharpless. Differential oncogenicity of N-RAS mutations in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2013-1087

Amplification of the STOML3, FREM2, and LHFP Genes Is Associated with Mesenchymal Differentiation in Gliosarcoma

Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas that display either glial (glial fibrillary acidic protein-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses have shown identical genetic alterations in glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array comparative genomic hybridization in glial and mesenchymal tumor areas of 13 gliosarcomas. The patterns of gain and loss were similar, except that the gain at 13q13.3-q14.1 (log(2) ratio >3.0), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2, and LHFP genes in 14 (22%), 10 (16%), and 7 (11%) mesenchymal tumor areas, respectively, but not in glial tumor areas. Results of IHC analysis confirmed that overexpression of STOML3 and FREM2 was more extensive in mesenchymal than in glial tumor areas. These results suggest that the mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations.

Chromosome 7p11.2 (EGFR) variation influences glioma risk

While gliomas are the most common primary brain tumors, their etiology is largely unknown. To identify novel risk loci for glioma, we conducted genome-wide association (GWA) analysis of two case-control series from France and Germany (2269 cases and 2500 controls). Pooling these data with previously reported UK and US GWA studies provided data on 4147 glioma cases and 7435 controls genotyped for 424 460 common tagging single-nucleotide polymorphisms. Using these data, we demonstrate two statistically independent associations between glioma and rs11979158 and rs2252586, at 7p11.2 which encompasses the EGFR gene (population-corrected statistics, P(c) = 7.72 × 10(-8) and 2.09 × 10(-8), respectively). Both associations were independent of tumor subtype, and were independent of EGFR amplification, p16INK4a deletion and IDH1 mutation status in tumors; compatible with driver effects of the variants on glioma development. These findings show that variation in 7p11.2 is a determinant of inherited glioma risk.