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Unraveling the function of m6A modification in acute myeloid leukemia
No full textN6-methyladenosine (m6A) is a well-known RNA modification that can affect mRNA stability and translation (Yue et al., Genes Dev. 2015). In mammals, the m6A writer is a multicomponent complex composed of the two methyltransferases METTL3 and METTL14 and the regulatory protein WTAP. WTAP has been recently described as an oncogenic factor in AML suggesting that m6A modification might play crucial role in leukemogenesis (Bansal et al., Leukemia 2014). Notably, we also found that METTL3 and METTL14 are upregulated in primary AML samples. Here, we analyzed the functional role of m6A during myeloid differentiation of AML cell lines. Impairing the expression of the methylation complex components by RNAi affected consistently myeloid differentiation and induced massive apoptosis. Moreover, in AML cell lines METTL3 mislocalized in the cytoplasm and associated with polysomes. These data indicate that the misregulation of m6A methylation may contribute to leukemogenesis, but also highlight a putative m6A-independent role for METTL3. Our data also pave the way to the development of new therapies for AML through the inhibition of the methylome complex
Retinoic Acid strongly sensitizes Acute Myeloid Leukemia cells to ER stress
No full textAcute myeloid leukemia (AML) is caused by the blockade of hematopoietic myeloid precursors at different stages of differentiation. A subtype of AML, acute promyelocytic leukemia (APL), is a paradigm of differentiation therapy since all-trans-retinoic acid (ATRA)-based treatments are able to induce leukemic blast terminal differentiation, leading to clinical remission in the majority of APL patients. However, ATRA can lead to systemic toxicity and relapses after initial remission followed by resistance. Furthermore APL accounts for about 10-15% of AML cases and non-APL AML respond only very slightly to ATRA. Thus the search for a strategy to further sensitize AML cells to ATRA is highly needed. Retinoic acid induces differentiation of APL blasts to granulocytes, cells characterized by accumulation of secretory granules containing peptides assembled in the ER. Generally, increased protein folding demand in the ER activates a series of intracellular signal transduction pathways, the unfolded protein response (UPR). The UPR intervenes in reliving ER stress, but if such stress is too strong or prolonged it triggers pro-apoptotic pathways. We set out to investigate if the UPR plays a role in RA-dependent APL differentiation and possibly to exploit RA-induced differentiation to sensitize APL and non-APL AML cells to ER stress.
We found that RA-triggered differentiation deeply altered APL cells sensitivity to ER stress. Indeed doses of tunicamycin or thapsigarging that did not adversely affect proliferating cells resulted in proliferation arrest, cell death and increased differentiation of RA stimulated APL and non-APL cell lines and, most importantly, of human primary leukemic blasts. We show that CHOP is strongly up-regulated upon induction of ER stress in RA-treated APL cells and its down-regulation by shRNA partially restored resistance. Our work suggests that modulation of the UPR in combination with RA based differentiation therapy could be an interesting strategy to target AML cells
Development of a combination strategy based on ER and oxidativa stress to target acute myeloid leukemia
No full textIntroduction
Acute Myeloid Leukemia (AML) is a heterogeneous disease caused by different molecular genetic aberrations. These result in the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. We previously showed that APL cell lines and primary blasts induced to differentiate by Retinoic Acid (RA) become highly sensitive to amounts of ER stress not detrimental for the same cells in the absence of RA. Furthermore the same cells resulted sensitive to a combination of ER stress inducers with Arsenic Trioxide (ATO) that generates oxidative stress. Importantly we observed that ER stress caused increased amounts of disulphide-bound high molecular weight aggregates of PML-RARα and PML, exacerbating the alteration of cellular proteostasis already generated by induction of ER stress. This observation provides the rationale to translate the findings we observed in APL to other types of AML characterized by fusion or mutant proteins. The presence of mutant proteins that are easily prone to aggregation or mis-folding, because of their mutant structure or because of mis-localization, could render the cells sensitive to levels of ER and oxidative stress that could be recovered in their absence.
Methods
We treated AML cell lines and AML primary leukemic blasts with RA and ER and oxidative stress inducers, evaluating cell proliferation and death, activation of the ER/oxidative stress responses, localization and possible aggregation of the mutant proteins by confocal microscopy, colony forming capacity.
Results
We first tested a panel of AML cell lines characterized by different oncogenic fusion or mutant proteins and we found that ML-2 cells, bearing the MLL-AF6 fusion protein, and MV-4-11 cells, expressing the fusion protein MLL-AF4 and FLT3-ITD are highly sensitive to the combination of sub-lethal amounts of RA, Tm and ATO. In the cells undergoing ER and oxidative stress in combination, we found prolonged activation of the antioxidant response and of the unfolded protein response (UPR), activated by ER stress, as indicated by the expression of HMOX, CHOP, BiP and sXBP1. The antioxidant agent N-acetyl-cystenine and the inhibitor of the UPR player GADD34 determine resistance of the cells to the treatments. Furthermore, an inhibitor of the PERK branch of the UPR dramatically exacerbates the sensitivity to the combination of ER and oxidative stress pointing to this pathway as a possible new therapeutic molecular target. Importantly, the combination of ER and oxidative stress significantly reduces the colony forming capacity of primary leukemic blasts isolated from the bone marrow of FLT3-ITD positive patients.
Conclusions
Altogether our data suggest that the combination of low levels of ER and oxidative stress leads to apoptosis rather than recovery, achieved instead when the same stresses are induced alone
Retinoic acid sensitizes acute myeloid leukemia cells to ER stress
No full textAcute myeloid leukemia (AML) is caused by the blockade of hematopoietic myeloid precursors at different stages of differentiation. A subtype of AML, acute promyelocytic leukemia (APL), is a paradigm of differentiation therapy since retinoic acid (RA) is able to induce leukemic blast terminal differentiation leading to cure rates exceeding 80% when administered in combination with chemotherapy. Although APL patients refractory to RA or who relapsed are very effectively treated with arsenic trioxide (ATO) in combination with RA, the elevated costs limit its use in developing countries and in first line therapy so that RA plus chemotherapy currently remain the standard of care (1, 2). Most importantly non-APL acute myeloid leukemia do not respond to RA indicating the need for novel strategies to sensitize AML cells to RA. Here we show that RA-triggered differentiation of APL cells induces endoplasmic reticulum (ER) stress slightly activating the unfolded protein response (UPR). This is sufficient to render leukemic cell lines and human primary blasts very sensitive to doses of ER stress inducing drugs, like tunicamycin (Tm), that are not toxic for the same cells in the absence of RA or for most cell types. Furthermore we observed that low doses of Tm, even in the absence of RA, are sufficient to strongly increase ATO toxicity. Indeed both RA-sensitive and RA-resistant APL cell lines resulted sensitive to Tm-ATO combined treatment at low doses of ATO that are ineffective in the absence of ER stress. The use of inhibitors targeting specific UPR branches indicate that the Protein Kinase RNA-like Endoplasmic Reticulum kinase (PERK) pathway protects differentiating APL cells from ER stress rendering it an interesting therapeutic molecular target. Finally, we extended our observations in a non-APL model, assessing that RA sensitize the non-APL cell line HL60 to ER stress. Altogether our data indicate ER stress as a possible target for designing novel combination therapeutic strategies in AML
Retinoic Acid and Arsenic Trioxide sensitize Acute Promyelocytic Leukemia cells to ER stress
No full textPromyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15:17) that results in the expression of the chimeric protein PML-RARα. The fusion of PML, a tumor suppressor that is the major component of the PML-nuclear bodies, with the Retinoic Acid Receptor-α arrests the differentiation program driven by RARα, blocking the leukemic blasts at the promyelocytic stage. Pharmacological doses of Retinoic Acid (RA) are able to remove the block, resume granulocytic differentiation and partially degrade PML-RARα leading to reformation of nuclear bodies. The association of RA with chemotherapy or with arsenic trioxide (ATO), the latter efficiently targeting PML-RARα for degradation, results in high cure rates of acute promyelocytic leukemia (APL). Despite showing a considerably improved safety profile, either RA or ATO are not devoid of toxicity, with the most important and potentially life-threatening one being the so-called retinoic acid differentiation syndrome. We show here that RA-induced differentiation of human APL cell lines and primary blasts dramatically increases their sensitivity to ER stress inducing drugs, like Tunicamycin (Tm), at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response, a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. Although mild, the ER stress induced by RA is sufficient to render differentiating APL cells very sensitive to low doses of Tm. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. Moreover we found that low amounts of pharmacologically induced ER stress are also able to strongly increase ATO toxicity even in the absence of RA. Indeed the combination of ATO with Tm efficiently induced apoptosis in RA-sensitive and RA resistant APL cell lines, at doses ineffective in the absence of ER stress. Eventually, we demonstrate that insurgency of oxidative stress, tightly linked with the UPR, is at the basis of the toxicity induced by Tm in combination with RA and/or ATO. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML
Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress.
No full textPromyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15:17). This translocation results
in the expression of the chimeric protein PML-RARα that arrests the differentiation program driven by RARα,
blocking the leukemic blasts at the promyelocytic stage. Pharmacological doses of Retinoic Acid (RA) are able to
resume granulocytic differentiation and partially degrade PML-RARα. The association of RA with chemotherapy or
with arsenic trioxide (ATO), which efficiently targets PML-RARα for degradation, results in high cure rates. Despite
showing a considerably improved safety profile RA and ATO are not completely devoid of toxicity. We show here
that granulocytic differentiation of human APL cells, driven by RA, generates mild ER stress, sufficient to render
them very sensitive to small quantities of ER stress inducing drugs, like Tunicamycin (Tm). Indeed, RA-induced
differentiation of human APL cell lines and primary blasts dramatically increases their sensitivity to Tm, at doses
that are not toxic in the absence of RA. Importantly the combination of RA and Tm results not toxic on human
bone marrow progenitors cells derived from healthy donors. We also show that the PERK pathway, triggered by ER
stress, plays a major protective role and, by using a specific PERK inhibitor, we potentiated the toxic effect of the
combination of RA and Tm. Moreover we found that small amounts of pharmacologically induced ER stress are also
able to strongly increase ATO toxicity even in the absence of RA: the combination of ATO with Tm efficiently induces
apoptosis in RA-sensitive and RA-resistant APL cell lines, at doses ineffective in the absence of ER stress. Eventually,
we demonstrate that insurgency of oxidative stress, tightly linked with the UPR, is at the basis of the toxicity induced
by Tm in combination with RA and/or ATO. In conclusion, our findings identify the ER stress-related pathways as
potential targets in the search for novel therapeutic strategies in AML
METTL3 regulates WTAP protein homeostasis
Full text linkThe Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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