METTL3 regulates WTAP protein homeostasis

The 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

Retinoic acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), 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. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, 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. 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. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML

Retinoic acid sensitizes acute myeloid leukemia cells to ER stress

Acute 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. Contribution of AIRC (StG 4841) and FILAS-RU-2014-1020 to FF was greatly appreciated

Retinoic Acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), 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. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, 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. 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. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML

Development of a combination strategy based on ER and oxidative stress in Acute Myeloid Leukemia

AML is a heterogeneous disease caused by different genetic aberrations that often result in the expression of oncogenic fusion or mutant proteins. At present, the only effective molecular targeted therapy for AML is based on retinoic acid (RA) and/or arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), expressing the promyelocytic leukemia (PML)–retinoic acid receptor α (RARα) fusion protein. We previously showed that in the presence of RA APL cell lines and primary blasts are sensitive to amounts of ER stress not detrimental for the same cells in its absence. Furthermore, the same cells resulted highly sensitive to a combination of ER stress inducers with ATO that generates oxidative stress. Interestingly, we observed that ER stress caused increased amounts of disulphide-bound high molecular weight aggregates of PML-RARα, exacerbating the alteration of cellular proteostasis. Thus, also in other types of AML the presence of oncogenic proteins, that are easily prone to mis-folding because of their mutant structure, could render the cells particularly sensitive to low levels of ER and oxidative stress. We found that AML cell lines, bearing the MLL-AF6 fusion protein, or the fusion protein MLL-AF4 and FLT-3-ITD, are highly sensitive to the combination of drugs inducing ER and oxidative stress in the presence of RA, at doses showing no or low toxicity when used alone. In the cells undergoing ER and oxidative stress in combination, we found a strong activation of the unfolded protein response (UPR) and of the antioxidant response, as indicated by the expression of CHOP, BiP, sXBP1 and HMOX. Accordingly, the chemical chaperone 4-PBA and the antioxidant agent N-acetyl-cystenine strongly reduce the toxicity of the treatments. Furthermore, the use of inhibitors of the UPR PERK pathway suggests it could be an interesting 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, while not affecting normal hematopoietic progenitor cells. Altogether our data suggest that the combination of RA and low levels of ER and oxidative stress specifically leads to AML cell death

Retinoic acid synergizes with the unfolded protein response and oxidative stress to induce AML cell death

Objectives Acute Myeloid Leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) and oxidative stress that could otherwise be overcome. We aim at exploiting cellular stresses to hit AML cells. Methods We treated AML cell lines and primary blasts expressing mutant proteins with the differentiating agent retinoic acid (RA), the ER stress-inducing drug Tunicamycin (Tm) and arsenic trioxide (ATO), able to generate oxidative stress, at doses that result no or only slightly toxic when each drug was used alone. Results We show that the triple combination of RA, Tm and ATO, leads to death of AML cell lines and primary leukemic cells bearing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, primary healthy hematopoietic progenitor cells are not affected by this treatment. We demonstrate in cell lines, that combination of these drugs not only generates ER and oxidative stress, but also impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stress combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation

A positive feed-forward regulatory loop between METTL3 and WTAP sustains the oncogenic role of the m6A methylation complex in myeloid leukemia

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumours, 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. Our results indicate the existence of a positive feedforward regulatory loop, where METTL3 upregulates WTAP, which is relevant to increase WTAP expression concomitantly to the METTL3/METTL14 core m6A methylation complex and sustain the oncogenic role reported for the m6A modification complex in leukemia

Retinoic acid synergizes with the unfolded protein response and oxidative stress to induce AML cell death

Acute Myeloid Leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) and oxidative stress that could otherwise be overcome. We aim at exploiting cellular stresses to hit AML cells. Methods We treated AML cell lines and primary blasts expressing mutant proteins with the differentiating agent retinoic acid (RA), the ER stress-inducing drug Tunicamycin (Tm) and arsenic trioxide (ATO), able to generate oxidative stress, at doses that result no or only slightly toxic when each drug was used alone. Results We show that the triple combination of RA, Tm and ATO, leads to death of AML cell lines and primary leukemic cells bearing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, primary healthy hematopoietic progenitor cells are not affected by this treatment. We demonstrate in cell lines, that combination of these drugs not only generates ER and oxidative stress, but also impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stress combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation

Retinoic acid sensitizes acute myeloid leukemia cells to ER stress

Acute 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.

Promyelocytic 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