Telomeres are DNA-protein structures that cap linear chromosomes and are essential for maintaining genomic stability and cell phenotype. We identified a novel human telomereassociated protein, TIN2, by interaction cloning using the telomeric DNA-binding-protein TRF1 as a bait. TIN2 interacted with TRF1 in vitro and in cells, and co-localized with TRF1 in nuclei and metaphase chromosomes. A mutant TIN2 that lacks amino-terminal sequences effects elongated human telomeres in a telomerase-dependent manner. Our findings suggest that TRF1 is insufficient for control of telomere length in human cells, and that TIN2 is an essential mediator of TRF1 function.

Despite recent advancements, approximately 50% of patients with acute myeloid leukemia (AML) do not respond to induction therapy (primary induction failure, PIF) or relapse after <6 months (early relapse, ER). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART® antibody-based molecule to CD3ε and CD123. This study reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in adults with relapsed/refractory AML. Eighty-eight AML patients were enrolled, 42 in dose-finding and 46 at the recommended phase 2 dose (RP2D) of 500ng/kg/day. Consistent with flotetuzumab's mode of action, the most frequent adverse events were infusion-related reactions (IRR)/cytokine release syndrome (CRS), the majority as grade 1-2. Stepwise dosing during week 1, pre-treatment dexamethasone, prompt use of tocilizumab and temporary dose reductions/interruptions successfully prevented severe IRR/CRS, resulting in acceptable tolerability. Clinical benefit accrued to PIF/ER AML patients, who showed an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the CR/CRh rate was 26.7%, with an overall response rate (CR/CRh/CRi) of 30.0%. In PIF/ER patients who achieved CR/CRh, median OS was 10.2 months (range 1.87-27.27), with 6- and 12-month survival rates of 75% (95%CI, 0.450-1.05) and 50% (95%CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted complete responses to flotetuzumab (AUROC=0.904 versus 0.672 for the ELN risk classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER AML patients. Trial registration number: NCT02152956.

Telomeres are protective structures at chromosome ends and are crucial for genomic stability. Mammalian TRF1 and TRF2 bind the double-stranded telomeric repeat sequence and in turn are bound by TIN2, TANK1, TANK2, and hRAP1. TRF1 is a negative regulator of telomere length in telomerase-positive cells, whereas TRF2 is important for telomere capping. TIN2 was identified as a TRF1-interacting protein that mediates TRF1 function. We show here that TIN2 also interacts with TRF2 in vitro and in yeast and mammalian cells. TIN2 mutants defective in binding of TRF1 or TRF2 induce a DNA damage response and destabilize TRF1 and TRF2 at telomeres in human cells. Our findings suggest that the functions of TRF1 and TRF2 are linked by TIN2.Telomeres are the DNA protein structures that cap the ends of linear chromosomes thereby protecting them from degradation and fusion by cellular DNA repair systems (1, 2). In mammals, telomeres consist of several kilobase pairs of doublestranded DNA that have the repeat sequence TTAGGG followed by 100 -150 nucleotides of a single-stranded TTAGGG 3Ј-overhang. The telomeric DNA tract is thought to loop back on itself, thereby protecting the overhang, in a structure termed the t-loop (3). This protected or capped telomeric structure can be lost or disrupted by DNA damage, malfunctions in telomere-associated proteins, or as a consequence of the gradual attrition of telomeric DNA that occurs when cells proliferate in the absence of telomerase. Dysfunctional telomeres activate a DNA damage response, resulting in the binding of DNA damage response proteins to the chromosome end and induction of apoptosis or cellular senescence (4 -7).Telomere-associated proteins are crucial for forming and maintaining the protective telomeric structure in vivo. Some telomere-associated proteins bind directly to telomeric DNA, whereas others associate with telomeres via protein-protein interactions. Among the direct telomere-binding factors is a family of Myb domain-containing proteins that specifically bind double-stranded telomeric DNA. These proteins include Rap1p in Saccharomyces cerevisiae (8), Taz1 in Schizosaccharomyces pombe (9), and TRF1 and TRF2 in mammals (10 -12). TRF1 reduces telomere length, whereas a dominant-negative TRF1 mutant increases telomere length when overexpressed in telomerase-positive human cells (13). These results suggest that TRF1 controls the extent to which telomerase can access and hence elongate the telomeres, presumably by modulating the telomeric structure. TRF2 is essential for t-loop formation in vitro (14) and telomere integrity in vivo (3). It has been proposed that t-loop formation by TRF2 provides a mechanism to sequester the telomeric 3Ј-overhang, thereby preventing its degradation and/or fusion to another telomere by DNA repair processes. Disruption of TRF2 function induces (ataxia-telangiectasia-mutated) (ATM)/p53-dependent apoptosis in human tumor cells (4) and premature senescence in normal human and mouse cells (5).Several TRF1-and TRF2-interacting factors have been id...

Tankyrase (TANK1) is a human telomere-associated poly(ADP-ribose) polymerase (PARP) that binds the telomere-binding protein TRF1 and increases telomere length when overexpressed. Here we report characterization of a second human tankyrase, tankyrase 2 (TANK2), which can also interact with TRF1 but has properties distinct from those of TANK1. TANK2 is encoded by a 66-kilobase pair gene (TNKS2) containing 28 exons, which express a 6.7-kilobase pair mRNA and a 1166-amino acid protein. The protein shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile ␣-motif, and PARP catalytic domains but has a unique N-terminal domain, which is conserved in the murine TNKS2 gene. TANK2 interacted with TRF1 in yeast and in vitro and localized predominantly to a perinuclear region, similar to the properties of TANK1. In contrast to TANK1, however, TANK2 caused rapid cell death when highly overexpressed. TANK2-induced death featured loss of mitochondrial membrane potential, but not PARP1 cleavage, suggesting that TANK2 kills cells by necrosis. The cell death was prevented by the PARP inhibitor 3-aminobenzamide. In vivo, TANK2 may differ from TANK1 in its intrinsic or regulated PARP activity or its substrate specificity.Telomeres are the repetitive DNA sequences and specialized proteins that cap the ends of linear chromosomes and protect them from end-to-end fusion. In mammalian cells, loss or disruption of a telomere can cause cellular senescence, cell death, or genomic instability, depending on the genotype and cell context. A variety of events can lead to dysfunctional telomeres. Telomeres can be damaged directly by genotoxic agents and/or faulty DNA repair processes. In addition, the telomeric structure can be disrupted by changes in the expression or function of certain telomere-associated proteins. Finally, telomeres can erode owing to the biochemistry of DNA replication, which leaves 50 -200 bp 1 of 3Ј-telomeric DNA unreplicated upon completion of each S phase. Thus, in the absence of the enzyme telomerase, or another mechanism to replenish telomeric DNA, proliferating cells progressively lose telomeric DNA and eventually acquire one or more critically short or dysfunctional telomeres (1-3).Most normal mammalian cells respond to a critically short or dysfunctional telomere by undergoing cellular senescence (4 -7). This process results in an irreversible arrest of cell proliferation and striking changes in cell function (8). Dysfunctional telomeres can also induce apoptotic cell death, particularly in cells that harbor mutations in one or more cell cycle or DNA damage checkpoints (9 -11). Very little is known about how telomeres signal cells to undergo senescence or apoptosis. However, the recent discovery of a telomere-associated poly(ADPribose) polymerase (PARP) (12) provides a potential mechanism by which telomeres transmit signals to cellular proteins that regulate the senescence and apoptotic responses.PARPs catalyze the formation of branched chains of ADPribose polymers on selected proteins, using NAD ϩ ...

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