BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

A BAG's life: Every connection matters in cancer

The members of the BCL-2 associated athanogene (BAG) family participate in the regulation of a variety of interrelated physiological processes, such as autophagy, apoptosis, and protein homeostasis. Under normal circumstances, the six BAG members described in mammals (BAG1-6) principally assist the 70 kDa heat-shock protein (HSP70) in protein folding; however, their role as oncogenes is becoming increasingly evident. Deregulation of the BAG multigene family has been associated with cell transformation, tumor recurrence, and drug resistance. In addition to BAG overexpression, BAG members are also involved in many oncogenic protein\u2013protein interactions (PPIs). As such, either the inhibition of overloading BAGs or of specific BAG\u2013client protein interactions could have paramount therapeutic value. In this review, we will examine the role of each BAG family member in different malignancies, focusing on their modular structure, which enables interaction with a variety of proteins to exert their pro-tumorigenic role. Lastly, critical remarks on the unmet needs for proposing effective BAG inhibitors will be pointed out

BAG-1 IN ACUTE LEUKEMIA: HUNDRED FACES OF A SINGLE PROTEIN

Introduction. Bcl-2 associated athanogene-1 (Bag-1), a founding member of BAG protein family, is a multifunctional protein which has a role in a wide range of cellular processes including apoptosis, cell survival, transcription, cell motility and proliferation. The involvement of Bag-1 in different cellular pathways can be in part examined by the sub-cellular compartmentalisation of the three major Bag-1 isoforms (Bag-1L, Bag-1M and Bag-1S), generated by alternative translation from a single mRNA, and in part by its interaction with a large number of disparate proteins, including Bcl-2, Raf-1, nuclear hormone receptors, subunits of the ubiquitinylation proteasome complex, Hsc70 and Hsp70. Aims. The elevated level of Bag-1 protein has been confirmed as a considerable index in several malign diseases. To determine significance Bag-1 might have in the processes of leukemogenesis a sequence of human leukemic cell lines and pediatric bone marrow samples with confirmed acute myeloid leukemia were included in our research. Our goal was to clarify the molecular mechanisms potentially in charge for Bag-1 action, in either leukemic cell lines or primary cell cultures. Methods. In vitro studies were based on a small-interfering RNA (siRNA) approach and the results were validated using standard techniques for mRNA and protein expression study. Assays for cell cycle and apoptosis detection were performed. Results. The protein study revealed elevated Bag-1 levels in human leukemic cell lines of both myeloid (ML2, THP1, NOMO1, NB4, MV4;11 and HL60) and lymphoid (REH, RS4;11, 697 and JURKAT) origin. A different expression pattern of Bag-1 protein isoforms was noted for two considered groups of patients, AML or ALL, with changes in protein expression profile at different point of the disease. After Bag-1 was knock-down, a modest effect on cell death or cell cycle profile was observed for the human cell lines while primary cultures showed to be more sensitive to Bag-1 silencing. However, significant decrease was confirmed at the expression level of a wide range of proteins, specially the ones involved in the regulation of apoptosis (Bcl-2, PARP, Caspase-3), cell cycle (p27, CDK2, Cyclin D1) and autophagy (LC3, p62), without affecting the mRNA levels. When double silencing experiments of Bag-1 and Bag-3 (a family co-member) were performed, the effect on cell death and cell cycle arrest were found enforced, suggesting a connection between two proteins to be significant for cells faith in leukemia. Conclusions. Results indicate that the role of Bag-1 in cell death prevention might be more related to lymphoid leukemia, while it might be considered more significant for cell differentiation in myeloid cells. At the same time, elevated Bag-1 protein expression levels in acute leukemia indicates its possible significance for AML and ALL growth. A different expression profile of Bag-1L, Bag-1M and Bag-1S isoforms in myeloid with respect to lymphoid leukemia could lead to the hypothesis that Bag-1 might play a role in leukemia switch, triggering to either ALL or AML phenotype

NOVEL Recurrent Genetic Aberrations in Pediatric AML: An AIEOP AML-2002 Study Group

Introduction. Acute myeloid leukemia (AML) is an heterogeneous disease with known specific recurrent genetic aberrations. The continuous and increasing identification of new genetic mutations has permitted to identify new subgroups with different prognosis. In the present work we evaluated the incidence of rare genetic abnormalities in pediatric AML such as del(4)(q12)FIP1L1-PDGFRA, t(16;21)(p11;q22)FUS-ERG, t(8;16)(p11;p13)MOZ-CBP, t(11;17)(q23;q12-21)MLL-AF17, t(4;11)(q35;q23)MLL-ArgB2, t(5;11)(q35;p15.5)NUP98-NSD1, t(3;5)(q25;q34)NPM1-MLF1, and MLLPTD. Methods. We selected 306 patients with AML other than acute promyelocytic leukemia, negative for known recurrent genetic abnormalities involving MLL, CBF-beta and FLT3genes. RNA was extracted from fresh bone marrow at diagnosis, and multiplex RT-PCR was employed. Sequencing by Sanger method was applied to all positive cases to characterize breakpoints of fusion. The Kaplan-Meier method was used for estimating the probability of event-free survival (EFS). Results. We identified one patient each positive for t(16;21)(p11;q22)FUS-ERG, t(11;17)(q23;q12-21)MLL-AF17, and t(4;11)(q35;q23)MLL-ArgB2, respectively, this suggesting that these rearrangements are rare in pediatric AML. 2/306 patients had del(4)(q12)FIP1L1/PDGFRA, and 4/306 the t(8;16)(p11;p13)MOZ-CBP; both these anomalies should be investigated in larger cohorts for definining their prognostic value. Interestingly 6/306 (2%) patients had the t(3;5)(q25;q34)NPM1-MLF1, 6/306 (2%) the MLLPTD, and 8/306 (2.6%) were found to carry the t(5;11)(q35;p15.5)NUP98-NSD1. Since the t(5;11) fusion was recently associated to FLT3ITD, we enlarged the screening to 42 de novo AML harbouring FLT3ITD mutation enrolled in the AIEOP-LAM 2002 protocol finding that 6 of them (14%) had the NUP98-NSD1fusion gene. We documented a poor EFS for patients with t(5;11)NUP98-NDS1 (n=12) as compared to patients negative for molecular lesions and enrolled in the LAM 2002-AIEOP protocol (25% vs 53.1% at 3 years, p<0.01, n=154). We did not find differences in clinical or biological features of the isolated t(5;11) and t(5;11)+FLT3ITD positive patients (Table 1). We then evaluated the prognostic impact of the t(5,11) in the FLT3ITD+ cohort of AML, finding that the NUP98/NSD1 identifies a previously unrecognized subgroup of FLt3ITD patients with worse prognosis (EFS 33.3% vs 42.7 at 8y, p= 0.2). Furthermore, to analyze whether MLLPTD might also have a role in the progression to relapse, we screened 40 AML at relapse; however, we did not find the abnormality in this cohort. By contrast, 4 patients harbored at relapse the same MLLPTD found at diagnosis, suggesting the stability of this mutation. Conclusions. We provide evidence that NUP98-NSD1 may be considered a recurrent translocation in pediatric AML with poor prognosis. Being cryptic to conventional karyotyping, we confirmed the need of using molecular approaches for a proper identification of this anomaly. We also suggest that the NUP98-NSD1 fusion gene be considered for a better evaluation of the FLT3ITD+ patients. Disclosures: No relevant conflicts of interest to declare

MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia.

Arare location, t(6;11)(q27;q23) (MLL-AF6), is associated with poor outcome in childhood acute myeloid leukemia (AML). The described mechanism by which MLL-AF6, through constitutive self-association and in cooperation with DOT-1L, activates aberrant gene expression does not explain the biological differences existing between t(6;11)-rearranged and other MLL-positive patients nor their different clinical outcome. Here, we show that AF6 is expressed in the cytoplasm of healthy bone marrow cell sand controls rat sarcoma viral oncogene (RAS)-guanosine triphosphate (GTP) levels. By contrast, in MLL-AF6-rearranged cells, AF6 is found localized in the nucleus, leading to aberrant activation of RAS and of its downstream targets. Silencing MLL-AF6, we restored AF6 localization in the cytoplasm, thus mediating significant reduction of RAS-GTP levels and of cell clonogenic potential. The rescue of RAS-GTP levels after MLL-AF6 and AF6 co-silencing confirmed that MLL-AF6 oncoprotein potentiates the activity of the RAS pathway through retention of AF6 within the nucleus. Exposure of MLL-AF6-rearranged AML blasts to tipifarnib, a RAS inhibitor, leads to cell autophagy and apoptosis, thus supporting RAS targeting as a novel potential therapeutic strategy in patients carrying t(6;11). Altogether, these data point to a novel role of the MLL-AF6 chimera and show that its gene partner, AF6, is crucial in AML development