Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia.

Approximately 50% of acute myeloid leukemia (AML) patients 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 paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [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 CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956.

Circulating Tumor DNA as a Predictive Biomarker for Clinical Outcomes With Margetuximab and Pembrolizumab in Pretreated HER2-Positive Gastric/ Gastroesophageal Adenocarcinoma.

PURPOSE: To assess the ability of circulating tumor DNA (ctDNA)-based testing to identify patients with HER2 (encoded by ERBB2)-positive gastric/gastroesophageal adenocarcinoma (GEA) who progressed on or after trastuzumab-containing treatments were treated with combination therapy of anti-HER2 and anti-PD-1 agents. METHODS: ctDNA analysis was performed retrospectively using plasma samples collected at study entry from 86 patients participating in the phase 1/2 CP-MGAH22-05 study (NCT02689284). RESULTS: Objective response rate (ORR) was significantly higher in evaluable ERBB2 amplification-positive vs - negative patients based on ctDNA analysis at study entry (37% vs 6%, respectively; P = .00094). ORR was 23% across all patients who were evaluable for response. ERBB2 amplification was detected at study entry in 57% of patients (all HER2 positive at diagnosis), and detection was higher (88%) when HER2 status was determined by immunohistochemistry fewer than 6 months before study entry. ctDNA was detected in 98% (84/86) of patients tested at study entry. Codetected ERBB2-activating mutations were not associated with response. CONCLUSIONS: Current ERBB2 status may be more effective than archival status at predicting clinical benefit from margetuximab plus pembrolizumab therapy. ctDNA testing for ERBB2 status prior to treatment will spare patients from repeat tissue biopsies, which may be reserved for reflex testing when ctDNA is not detected.

The PD-1- and LAG-3-targeting bispecific molecule tebotelimab in solid tumors and hematologic cancers: a phase 1 trial.

Tebotelimab, a bispecific PD-1×LAG-3 DART molecule that blocks both PD-1 and LAG-3, was investigated for clinical safety and activity in a phase 1 dose-escalation and cohort-expansion clinical trial in patients with solid tumors or hematologic malignancies and disease progression on previous treatment. Primary endpoints were safety and maximum tolerated dose of tebotelimab when administered as a single agent (n = 269) or in combination with the anti-HER2 antibody margetuximab (n = 84). Secondary endpoints included anti-tumor activity. In patients with advanced cancer treated with tebotelimab monotherapy, 68% (184/269) experienced treatment-related adverse events (TRAEs; 22% were grade ≥3). No maximum tolerated dose was defined; the recommended phase 2 dose (RP2D) was 600 mg once every 2 weeks. There were tumor decreases in 34% (59/172) of response-evaluable patients in the dose-escalation cohorts, with objective responses in multiple solid tumor types, including PD-1-refractory disease, and in LAG-3+ non-Hodgkin lymphomas, including CAR-T refractory disease. To enhance potential anti-tumor responses, we tested margetuximab plus tebotelimab. In patients with HER2+ tumors treated with tebotelimab plus margetuximab, 74% (62/84) had TRAEs (17% were grade ≥3). The RP2D was 600 mg once every 3 weeks. The confirmed objective response rate in these patients was 19% (14/72), including responses in patients typically not responsive to anti-HER2/anti-PD-1 combination therapy. ClinicalTrials.gov identifier: NCT03219268 .

Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice.

Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.

Telomeres, aging and cancer: in search of a happy ending.

Telomeres are distinctive structures, composed of a repetitive DNA sequence and associated proteins, that cap the ends of linear chromosomes. Telomeres are essential for maintaining the integrity and stability of eukaryotic genomes. In addition, under some circumstances, telomeres can influence cellular gene expression. In mammals, the length, structure, and function of telomeres have been proposed to contribute to cellular and organismal phenotypes associated with cancer and aging. Here, we discuss what is known about the basis for the links between telomeres, aging and cancer, and some of the known and proposed consequences of telomere dysfunction and maintenance for mammalian cells and organisms.

Is Akt the mastermind behind age-related heart disease?

Over the past several years, the insulin/insulin-like growth factor (IGF) signaling pathway has become a central figure in the study of organismal aging. Mutations in components of this pathway have led to enhanced longevity in several organisms, but it is still not clear whether and how this pathway contributes to human aging and aging-related diseases. In a new study, Miyauchi and colleagues propose that Akt, a member of the phosphatidylinositol 3-kinase family and a downstream component of the insulin/IGF pathway, plays a central role in the life span of endothelial cells. These findings implicate the insulin/IGF pathway in the development and progression of cardiovascular disease.

A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.

Telomeres are specialized heterochromatin at the ends of linear chromosomes. Telomeres are crucial for maintaining genome stability and play important roles in cellular senescence and tumor biology. Six core proteins-TRF1, TRF2, TIN2, POT1, TPP1 and Rap1 (termed the telosome or shelterin complex)-regulate telomere structure and function. One of these proteins, TIN2, regulates telomere length and structure indirectly by interacting with TRF1, TRF2 and TPP1, but no direct function has been attributed to TIN2. Here we present evidence for a TIN2 isoform (TIN2L) that differs from the originally described TIN2 isoform (TIN2S) in two ways: TIN2L contains an additional 97 amino acids, and TIN2L associates strongly with the nuclear matrix. Stringent salt and detergent conditions failed to extract TIN2L from the nuclear matrix, despite removing other telomere components, including TIN2S. In human mammary epithelial cells, each isoform showed a distinct nuclear distribution both as a function of cell cycle position and telomere length. Our results suggest a dual role for TIN2 in mediating the function of the shelterin complex and tethering telomeres to the nuclear matrix.

Telomere dysfunction and cell survival: roles for distinct TIN2-containing complexes.

Telomeres are maintained by three DNA-binding proteins (telomeric repeat binding factor 1 [TRF1], TRF2, and protector of telomeres 1 [POT1]) and several associated factors. One factor, TRF1-interacting protein 2 (TIN2), binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether subcomplexes also exist in vivo. We provide evidence for two TIN2 subcomplexes with distinct functions in human cells. We isolated these two TIN2 subcomplexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13 and TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

Vitamin K3 (menadione)-induced oncosis associated with keratin 8 phosphorylation and histone H3 arylation.

The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 microM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.

Higher-order nuclear organization in growth arrest of human mammary epithelial cells: a novel role for telomere-associated protein TIN2.

Nuclear organization, such as the formation of specific nuclear subdomains, is generally thought to be involved in the control of cellular phenotype; however, there are relatively few specific examples of how mammalian nuclei organize during radical changes in phenotype, such as those occurring during differentiation and growth arrest. Using human mammary epithelial cells in which growth arrest is essential for morphological differentiation, we show that the arrest of cell proliferation is accompanied by a reorganization of the telomere-associated protein, TIN2, into one to three large nuclear subdomains. The large TIN2 domains do not contain telomeres and occur concomitant with the continued presence of TIN2 at telomeres. The TIN2 domains were sensitive to DNase, but not RNase, occurred frequently, but not exclusively near nucleoli, and overlapped often with dense domains containing heterochromatin protein 1gamma. Expression of truncated forms of TIN2 simultaneously prevented the formation of TIN2 domains and relaxed the stringent morphogenesis-induced growth arrest in human mammary epithelial cells. Here we show that a novel extra-telomeric organization of TIN2 is associated with the control of cell proliferation and identify TIN2 as an important regulator of mammary epithelial differentiation.

ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation.

The BLM helicase, a deficiency that markedly increases cancer incidence in humans, is required for optimal repair during DNA replication. We show that BLM rapidly moves from PML nuclear bodies to damaged replication forks, returning to PML bodies several hours later, owing to activities of the DNA damage response kinases ATR and ATM, respectively. Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different sub-cellular compartments after replication stress. Unexpectedly, fibroblasts lacking BLM were deficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failed to form foci following replication stress. Expression of a dominant p53 mutant or helicase-deficient BLM restored replication stress-induced 53BP1 foci, but only mutant p53 restored optimal ATM activation. Thus, optimal repair of damaged replication fork lesions likely requires both ATR and ATM. BLM recruits 53BP1 to these lesions independent of its helicase activity, and optimal activation of ATM requires both p53 and BLM helicase activities.

TIN2 mediates functions of TRF2 at human telomeres.

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.