Role of Cdkn2a in the Emery–Dreifuss Muscular Dystrophy Cardiac Phenotype

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Genetic Alterations in the INK4a/ARF Locus: Effects on Melanoma Development and Progression

Genetic alterations in the INK4a/ARF (or CDKN2A) locus have been reported in many cancer types, including melanoma; head and neck squamous cell carcinomas; lung, breast, and pancreatic cancers. In melanoma, loss of function CDKN2A alterations have been identified in approximately 50% of primary melanomas, in over 75% of metastatic melanomas, and in the germline of 40% of families with a predisposition to cutaneous melanoma. The CDKN2A locus encodes two critical tumor suppressor proteins, the cyclin-dependent kinase inhibitor p16INK4a and the p53 regulator p14ARF. The majority of CDKN2A alterations in melanoma selectively target p16INK4a or affect the coding sequence of both p16INK4a and p14ARF. There is also a subset of less common somatic and germline INK4a/ARF alterations that affect p14ARF, while not altering the syntenic p16INK4a coding regions. In this review, we describe the frequency and types of somatic alterations affecting the CDKN2A locus in melanoma and germline CDKN2A alterations in familial melanoma, and their functional consequences in melanoma development. We discuss the clinical implications of CDKN2A inactivating alterations and their influence on treatment response and resistance.

Emerging Roles for the INK4a/ARF (CDKN2A) Locus in Adipose Tissue: Implications for Obesity and Type 2 Diabetes

Besides its role as a cell cycle and proliferation regulator, the INK4a/ARF (CDKN2A) locus and its associated pathways are thought to play additional functions in the control of energy homeostasis. Genome-wide association studies in humans and rodents have revealed that single nucleotide polymorphisms in this locus are risk factors for obesity and related metabolic diseases including cardiovascular complications and type-2 diabetes (T2D). Recent studies showed that both p16INK4a-CDK4-E2F1/pRB and p19ARF-P53 (p14ARF in humans) related pathways regulate adipose tissue (AT) physiology and adipocyte functions such as lipid storage, inflammation, oxidative activity, and cellular plasticity (browning). Targeting these metabolic pathways in AT emerged as a new putative therapy to alleviate the effects of obesity and prevent T2D. This review aims to provide an overview of the literature linking the INK4a/ARF locus with AT functions, focusing on its mechanisms of action in the regulation of energy homeostasis.

P14ARF: The Absence that Makes the Difference

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

Targeting MAP2K1 mutation with trametinib in a triple wild-type melanoma patient.

e21027 Background: MAP2K1 inhibitors combined with BRAF inhibitors provide a prolonged progression-free survival for BRAF mutant patients. However, the impact of MEK inhibition of MAP2K1 mutant and BRAF/NRAS/NF1 wild type melanoma patients remains elusive. Methods: We performed next-generation sequencing using an in-house developed 400 full exon panel for one of our triple wild type (BRAF/NRAS/NF1) melanoma patient. The patient received off-label, trametinib at a dose of 2 mg o.d. We performed additional sequencing at progression. After progression, the patient was treated off-label with combined trametinib and palbociclib therapy. Furthermore, we screened the TCGA melanoma and MSK-IMPAKT databases for patients with similar mutation profiles. Results: Our patient presented with dual somatic MAP2K1 mutations at Cys121Ser and Pro124Arg with a 68% allele frequency. The tumor also harbored a minor EGFR mutant clone (11% allele frequency) and a complete loss of the CDKN2A locus. The patient received trametinib and after two months presented a partial response as per RECIST1.1. However, after two additional months, the patient progressed. A new biopsy was performed, and the treatment was modified to combine trametinib and palbociclib to co-target MAP2K1 and the loss of the CKDN2A locus. The patient progressed under dual trametinib/palbociclib therapy without any objective benefit. The on-progression biopsy showed the absence of a gatekeeper mutation of MAP2K1 and the persistence of the loss of the CDKN2A locus. We detected a large number of novel copy number variations, most notable amplification of MITF (9x), FGFR1 (10X) and of MDM2 (16x). We also detected a loss of the minor, EGFR mutant clone. The analysis of large-scale state transitions (LSTs) for deficiency in homolog recombination (HRDness) was negative in pre and post-treatment biopsies. Conclusions: Our patient provides unique evidence to target solitary MAP2K1 mutations in triple wild melanoma by MEK1 inhibitors. Resistance to MEK1 inhibition developed rapidly and likely involved FGFR1 and MDM2, which have not been previously associated with resistance to MEK inhibitor monotherapy. The addition of palbociclib could not rescue these potential resistance mechanisms.

Association of the independent polymorphisms in CDKN2A with susceptibility of acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and alterations in CDKN2A were considered to play an important role on leukemogenesis. Two single nucleotide polymorphisms (SNPs) at CDKN2A locus were identified to impact on ALL susceptibility via genome wide association studies, and followed by multiple subsequent replication studies at the specific hits. Here, we conducted a systematic review and meta-analysis to re-evaluate the association of both SNPs (rs3731217 and rs3731249) with ALL susceptibility by gathering the data from 24 independent studies, totally containing 7922 cases/21503 controls for rs3731217 and 6295 cases/24191 controls for rs3731249. Both SNPs were significantly associated with ALL risk (odds ratio [OR] = 0.72 and 2.26 respectively), however, exhibit race-specific pattern. In summary, our meta-analysis indicated that two SNPs at CDKN2A locus are associated with ALL susceptibility independently mainly in Caucasians. Future large-scale studies are required to validate the associations in other ethnicities.

Upregulation of Vav3 Is Required for Leukemogenesis By BCR-ABL through Polycomb Repression Complex Dependent De-Repression of the Cdkn2a Locus

Activating mutations of Rho-family of small GTPases have been linked to lymphoproliferative disorders, although the pathogenesis mechanism involved is unknown. BCR-ABL (p190) B-cell acute lymphoblastic leukemia (B-ALL) arises from the expression of the oncofusion protein BCR-ABL in a B-cell progenitor. The transforming effect of BCR-ABL is dependent on the tyrosine kinase (TK) activity of the fusion protein that leads to autophosphorylation, recruitment of adaptor proteins, and subsequent activation of downstream signaling. TK inhibitors (TKIs) have been used as frontline treatment for Ph+ B-ALL patients. However, relapse is common in Ph+ B-ALL despite high rates of complete response with initial therapy, probably because of survival of leukemic progenitors. These BCR-ABL+ progenitors appear to develop additional epigenetic and genetic alterations that result in proliferative advantage frequently associated with silencing of the cyclin dependent kinase inhibitor Cdkn2a, even before mutant Cdkn2a gene deleted cells are selected during clonal evolution. Recent work by our group (Chang KH et al., Blood 2012) identified the Rho GTPase guanine nucleotide exchange factor Vav3 in BCR-ABL mediated lymphoid leukemogenesis. We showed that the deficiency of the guanosine nucleotide exchange factor Vav3 delays leukemogenesis and phenocopies the effect of Rac2 (and combined Rac2/Rac1) deficiency (Thomas EK et al., Cancer Cell 2007; Sengupta A et al., Blood 2010), a downstream effector of Vav3. Upregulated Vav3 expression and activation only partly depend on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to impair leukemogenesis in vitro and in vivo through impaired proliferation and survival. On the other hand, our group has demonstrated that Bmi1 overexpression frequently found in BCR-ABL+ B-ALL results in B-cell progenitor reprogramming through acquisition of a stem cell-like phenotype (Sengupta A et al., Blood 2012). Bmi1 forms part of the classical polycomb repression complex 1 (PRC1) where its component Ring1A/B catalyzes histone H2A mono-ubiquitination at lysine 119, which in conjunction with the PRC2 complex activity leads to chromatin compaction and repression of target genes. Through epistasis experiments, we found that Vav3 or Rac2 deficiency abrogates the oncogenic effect of Bmi1 overexpression. Co-immunoprecipitation experiments in nuclear and cytoplasmic cell extracts demonstrated that Vav3 and Rac1/Rac2 co-immunoprecipitate with Bmi1 in the nucleus but not in the cytosol of BCR-ABL+ leukemic cells. Interestingly, control non-BCR-ABL expressing nuclear extracts show minimal, if any, level of co-immunoprecipitation. This co-immunoprecipitation is not directly induced by BCR-ABL since BCR-ABL does not co-immunoprecipitate with Vav3/Rac1/Rac2 but does with Bmi1, suggesting that nuclear Vav3 activity may be dissected from the TK activity of BCR-ABL. Biochemically, the overexpression of Bmi1 results in increased activation of nuclear Rac which is practically abrogated by the deficiency of Vav3 as assessed in cellular pulldown assays of primary leukemic B-cell progenitors. As expected, downstream expression of Cdkn2a is repressed by overexpression by Bmi1. Deficiency of Vav3 restores the expression of Cdkn2a to control levels. This data suggests a transcriptional regulatory role of the signaling proteins Vav3/Rac2 in the nucleus. Chromatin immunoprecipitation (ChIP)-qPCR for Bmi1, Ring1B and polycomb repressive histone marks (H2AK119 and H3K27me3) and the assay for Tn5-transposase accessible chromatin (ATAQ)-qPCR for the Cdkn2a locus in Vav3- or Rac2-deficient, BCR-ABL+ primary B-cell progenitors were compared with their BCR-ABL, Vav3/Rac2 expressing counterparts. These assays confirmed that Vav3 and Rac2 are essential for PRC dependent transcriptional repression of Cdkn2a through occupancy of the Cdkn2a promoter and decreased accessibility to Cdkn2a chromatin. In conclusion, our studies establish for the first time an association between nuclear Vav3/Rac and polycomb repressive activities in p190-BCR-ABL+ leukemogenesis through their activity on the Cdkn2a locus. Vav3 may represent a novel target for adjuvant therapy with TKI in BCR-ABL+ lymphoblastic leukemia. Disclosures No relevant conflicts of interest to declare.