Our understanding of the molecular and cellular response to ionizing radiation (IR) has progressed considerably. This is notably the case for the repair and signaling of DNA double-strand breaks (DSB) that, if unrepaired, can result in cell lethality, or if misrepaired, can cause cancer. However, through the different protocols, techniques, and cellular models used during the last four decades, the DSB repair kinetics and the relationship between cellular radiosensitivity and unrepaired DSB has varied drastically, moving from all-or-none phenomena to very complex mechanistic models. To date, personalized medicine has required a reliable evaluation of the IR-induced risks that have become a medical, scientific, and societal issue. However, the molecular bases of the individual response to IR are still unclear: there is a gap between the moderate radiosensitivity frequently observed in clinic but poorly investigated in the publications and the hyper-radiosensitivity of rare but well-characterized genetic diseases frequently cited in the mechanistic models. This paper makes a comprehensive review of semantic issues, correlations between cellular radiosensitivity and unrepaired DSB, shapes of DSB repair curves, and DSB repair biomarkers in order to propose a new vision of the individual response to IR that would be more coherent with clinical reality. The statement that radiation-induced damage inside the cell nucleus are the key-elements of the individual response to ionizing radiation (IR) is much anterior to the discovery of the DNA structure itself. Indeed, at the beginning of the 20th century, from pioneer observations on irradiated cells, Claudius Regaud showed that “the most radiosensitive component of the cell is the nucleus” ,2. ThereThe first DSB repair pathway that was discovered, namely the recombination pathway, was pointed out with micro-organisms, whose major characteristics are a high rate of proliferation and a small genome length ,5,6. In In the 1980s, a new DSB repair pathway, the DNA end-joining pathway, was discovered. The end-joining pathway is active in G0/G1 phase but also in G2/M phase. However, again, since mammalian cells are mostly quiescent while micro-organisms are generally proliferating cells, the end-joining pathway is predominant in mammalian cells and less frequent in micro-organisms ,8. BecauHowever, some cells elicit a significant radiosensitivity while showing both normal homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways, thereby suggesting the existence of other DSB repair pathways ,9. HenceIn the 1980s, Fertil and Malaise showed that in vitro clonogenic cell survival is quantitatively correlated to the in vivo local tumour control . To bettWhile the other CHO-xrs cell lines were neglected, further investigations pointed out that the hyper-radiosensitive xrs-5 and xrs-6 cell lines were both mutated for the Ku80 protein, an essential component of the DNA-PK kinase, a key-actor of NHEJ ,18. ThisATM gene similar to those observed with the AT syndrome: it was later discovered that this young patient was suffering from a mutation of ligase IV (LIG4), which is essential for the NHEJ pathway [LIG4-mutated human cells [All the works described above were based on rodent cell lines. While the hyper-radiosensitivity of fibroblasts derived from ataxia telangiectasia (AT) patients was pointed out in 1975 , the fir pathway ,28,29. Tan cells . Hence, an cells   techniques , were baMore recently, such biphasic description was at the basis of the hypothesis of the backup/alternative NHEJ pathways ,38. IndeSome years later, from mutagenesis or genetic investigations, and independently of the biphasic description of the DSB repair curves, other models of NHEJ variants have been hypothesized and were described as “alternative” NHEJ pathway (A-NHEJ) or as a “classical” or “canonical” NHEJ pathway (C-NHEJ). However, this “A- and C-NHEJ” model is still not consensual and a number of different A-NHEJ flourished. This is notably the case of an ATM- and NBS1-dependent switch recombination pathway , a PARP1In parallel to these numerous molecular models, a deeper mathematical kinetic analysis of the DSB repair curves showed that their shape is more multiphasic than biphasic ,51. IndeLIG4-mutated fibroblast 180BR cell line [ATM-mutated cells show very few γH2AX foci, if any, whatever the post-irradiation time. Conversely, the hyper-radiosensitive DNA-PK or LIG4-mutated cells show normal yields of γH2AX foci some minutes after irradiation and significant residual DSB 24 h after irradiation, suggesting that ATM but not DNA-PK or LIG4 is required for an early radiation-induced phosphorylation of H2AX [In the 2000s, the immunofluorescence technique appeared that permits us to assess the subcellular localization of a given protein after irradiation via specific antibodies ,53,54,55ell line . For exa of H2AX ,57,58. M of H2AX ,59  the foci appearance phase during which the number of foci increases and reaches its maximum. This phase can be considered as a DNA damage recognition phase; 2) the foci disappearance phase during which the number of foci decreases. Such a phase can be considered as the result of the repairing of DNA damage . At thisThe diversity of immunofluorescence biomarkers that formed nuclear foci has not facilitated the elucidation of consensual mechanisms of DSB repair: to date, nearly all the foci-making proteins were found to co-localize. This is notably the case for γH2AX and MRE11 , γH2AX aATM mutations during their anti-leukemia or lymphoma treatment [LIG4- and ATM-mutated cells and moderate radiosensitivity caused by impairment of alternative signaling and DSB repair pathways remains.As discussed above, the great majority of the radiobiological investigations have been done with very radioresistant and hyper-radiosensitive cells. However, the hyper-radiosensitive and hyper-radioresistant cellular models are not representative of the clinical reality. For example, there is no known human genetic syndromes caused by mutations of Ku or DNA-PKcs proteins, the major NHEJ actors. The only human case of gross defect in NHEJ function to non-viability was caused by mutations of DNA ligase IV (LIG4) ,62. The reatment ,64,65,66reatment , Huntingreatment , and tubreatment . It mustTo date, the ATM protein has been considered as an early actor of the DSB recognition via its phosphorylation of H2AX and the individual radiation response ,70. SurpThree groups of radiosensitivity have been defined ,74,75,76Group I (about 75% to 85% whole population) represents the normosensitive (radioresistant) patients with a rapid RIANS after 2 Gy, a low risk of post-radiotherapy tissue reaction and of cancerGroup II (about 5–20% of whole population) represents the patients who elicit a moderate radiosensitivity with a delay in the RIANS due to the sequestration of ATM by mutated and over-expressed substrates. These patients are moderately radiosensitive and may have a high risk of cancer or neurodegeneration.ATM-mutated patients with no functional ATM kinase or those who show strong DSB repair defects (like the LIG4-mutated patient described above), hyper-radiosensitivity, and either high cancer proneness or severe accelerated aging [Group III (<1% whole population) represents the ed aging ,73.While it provides relevant explanation of the radiosensitivity of syndromes caused by mutations of cytoplasmic proteins, the RIANS model appears to be compatible with all the alternative models, as ATM is upstream of all the cascades of radiation-induced phosphorylations involved in the individual response to IR . ObviousAt the beginning of the 20th century, radiobiologists believed that individual response to IR was dependent on the DNA damage induced in the nucleus alone. To date, radiosensitivity also appeared to be caused by mutated cytoplasmic proteins. At the end of the 1980s, the “NHEJ or else paradigm” supported the theory that only two pathways were required to repair DSB. To date, by focusing on clinical reality, semantic issues, and technical artefacts linked to DSB assays or cellular models, evidence can be found suggesting that the individual response to IR is complex and that deciphering the DSB repair mechanisms needs more rigorous methodology and a constant link towards clinicians. To better quantify the radiation-induced risks, a general survey of human radiosensitivity from clinical and not molecular data is required to identify the proteins involved in DSB repair in realistic conditions of medical exposure to IR.


BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients’ outcome.In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients’ bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of  Nevertheless, crosstalk between leukemic stem cells and the associated bone marrow (BM) stroma appears to be essential for leukemic progression and response to therapy3. More globally, understanding interactions between tumor stem cells (SCs) and their microenvironment is a challenge to develop strategies to avoid relapses after therapy. Among the main elements implicated in the crosstalk between the microenvironment and both normal and tumor SCs, we have investigated the role of bone morphogenetic proteins (BMPs), because they govern SC regulation including hematopoietic5, neural and epithelial systems6 by directly and indirectly affecting their niche9. Alterations of the BMP signaling pathway have been observed in numerous cancers, in some cases closely associated with cancer stem cells (CSC) properties10. According to the context, BMPs could participate in initial tumor suppression or favor CSC maintenance and metastasis8. Within the BMP family, BMP2 and BMP4 have emerged as key regulators of normal and cancer SCs13. We have previously demonstrated that alterations in the BMP pathway at intrinsic (BMP receptors and downstream partners) and extrinsic (BMP extracellular ligands) levels constitute major events in transformation, expansion and persistence of immature cells in chronic phase chronic myeloid leukemia (CML) and breast cancer, by diverting their normal functions15.The current paradigm on the initiation of leukemogenesis implies a multistep process involving different types of genetic alterations, with no obvious hierarchy and understanding of the sequential clonal selection16, is a heterogeneous disease, in which the accumulation of genetic aberrations results in the uncontrolled growth of malignant undifferentiated cells. Relapse in the first years following complete remission is prevalent and may reflect the survival of resistant immature-like tumor cells able to regenerate the entire tumor17. The BMP pathway has been implicated in adult AML. For example, the overexpression of the transcription factor MIXL1, which is sufficient to initiate AML, can be induced by BMP4. Consistent with this induction, AML cells that express MIXL1 are sensitive to type BMP type 1 receptors (BMPR1) inhibitors18. In addition, in acute megakaryoblastic leukemia, the appearance of a specific fusion protein CBFA2T3-GLIS2 leads to the overexpression of BMP2 and BMP4 by leukemic cells and is associated with colony-forming capacities, a property ascribed to immature cells19.Acute myeloid leukemia (AML), the first tumor where CSCs were describedHere we have identified alterations of the BMP pathway and revealed their importance in immature properties exhibited by AML cells. Initially focusing on the analysis of AML patient samples collected at diagnosis and subsequently experimentally deregulating the BMP pathway, we have identified alterations in BMP ligands, receptors and target genes. Our data highlight a new signaling cascade likely involved in the cell survival and features of immature AML cells in their microenvironment.Bone marrow plasma obtained from allogeneic BM healthy donors and AML patients was harvested and cleared. BMPs concentration was determined using the human BMP2-ELISA or BMP4-ELISA kits (RayBiotech) following the manufacturer’s instructions.20. Normal goat IgG control (AB-108-C) and anti-hBMPR1A (AF346) (R&D Systems) were used at 4 µg/mL.Patient samples were obtained after informed consent in accordance with the Declaration of Helsinki in the hematology departments involved in this study. Mononuclear cells (MNCs) from 54 blood and BM samples were obtained from AML, excluding acute promyelocytic leukemia, patients at diagnosis. AML characteristics are presented in Table 20. LTC-IC number was expressed as W5-CFC/10,000 initial cells.Colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays were performed as described-ΔNp7321, pMXS-NANOG or empty vector (control) (Addgene) using a Neon (Thermofisher Scientific) electroporation device according to the manufacturer’s instructions. For luciferase assays, the pNANOG-Luc reporter vector was cotransfected with empty pCDNA3 or pCDNA3-ΔNp73 in KG1A cells. Luciferase assays were performed using Dual-Glo® Luciferase Assay System (Promega) following the manufacturer’s instructions. Relative activation as compared with cells transfected with the empty vector22.KG1A cells were transfected with pBabe20. MNCs were isolated by a Ficoll gradient and total RNA was purified using TRI REAGENT™ (Sigma) and the mini-RNA extraction kit . For RT-qPCR, cDNA was produced using Superscript II (Invitrogen) and amplified using Sybr-green  and the Real-Time PCR system (Roche). TBP (TATA-binding protein) and HPRT (hypoxanthine-guanine phosphoribosyl transferase) genes were used for normalization. Arbitrary unit (AU) corresponds to the ratio of expression between samples and a single normal sample used as a reference in each PCR. Primer sequences are presented in the Table Quantitative RT-PCR was performed using standard protocolsCells were incubated with antibodies specifically recognizing CD34, CD38 (Becton Dickinson) and/or BMPR1A (R&D System), or an irrelevant isotype-matched control antibody. MNCs separated by Ficoll gradient were subjected to sorting using BMPR1A antibody and an FACSARIA III cell sorter (Becton Dickinson). ALDH  activity was determined using an Aldefluor kit according to the manufacturer’s recommendations (StemCell Technologies).MNCs from AML samples were treated with BMP4 (20 ng/ml) during 24 h, then proteins were extracted. Per lane, 40 µg of proteins were loaded on SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad). Membranes were incubated with monoclonal antibodies anti-ΔNp73  or anti-βactin  from mouse, or anti-NANOG  from rabbit. Then membranes were incubated with their relevant HRP-coupled secondary antibodies (Jackson ImmunoResearch).Normalized Reads Per Kilobase Million (RPKM) Illumina GA-IIX RNA-seq profiles were downloaded from the TCGA AML data portal. One hundred and sixty-one patients in the TCGA AML cohort had both RNA-seq and clinical data. RPKM values were log-transformed to the base two after adding a value of 1. Expression levels were extracted from the log-transformed data. A representative probe set with the highest average intensity was selected for each gene in each dataset. The median of expression of each gene was used for patients’ stratification (high vs. low score).U test. Except for overall survival, the statistical analyses and graphs were performed with Graphpad prism (version 6). Significant P values are given in the text or symbolized by asterisks . Overall survival (OS) curves from the date of sampling onwards are illustrated using the Kaplan−Meier method. Log-rank tests were conducted to evaluate the effect of markers on survival. Best cut-off values for expression of each gene of interest were determined with the Youden index computed on Cumulative/Dynamic time-dependent ROC curve using Inverse Probability of Censoring Weighting (IPCW) estimation between 12 and 36 months of follow-up. The best cut-off value for NANOG expression that discriminates the most death events during time was 13 at 24 months of follow-up . The level of significance was set at 5%. OS analysis and graph were performed with the R program (version 3.2.3) using the “survival” and “ggplot2” packages.Unless otherwise specified, statistical analysis was performed using the Mann−Whitney P = 0.0100) and 3.6-fold (P < 0.0001), respectively , while BMP4 expression was the same. Interestingly, when we analyzed BMPs transcripts in BM samples, both BMP2 and BMP4 mRNA levels were reduced in AML samples  higher levels of BMPR1A transcripts, but not of BMPR1B and BMPR2, as compared to MNCs from healthy donors  in leukemic cells  in AML than in normal samples   Fig.  and that17) Fig. a, we obslls Fig. b. Furthe23, we evaluated its involvement in immature features observed in leukemic cells. We first analyzed the effect of BMP4 exposure of AML cells on the number of long-term culture-initiating cells (LTC-IC) as a read-out of SCs properties maintenance through time (8 weeks assay). As previously reported for normal hematopoietic cells24, BMP4 treatment of AML MNCs induced a reproducible increase in the number of LTC-IC  but as many more mature progenitors identified as CFCs as BMPR1Alow AML samples , recognized as an SC marker in several cancer types including AML  overexpression of ΔNp73    Fig. . This cound Fig. a. FurtheNp73 has been described as a regulator of NANOG33, we evaluated the potential link between ΔNp73 and genes known to drive SC features in AML samples. For this, we analyzed the expression of the three self-renewal regulators NANOG, SOX2, and OCT435. Although no difference was observed as compared with normal MNCs for OCT4 (P = 0.5500), a significantly higher expression of NANOG (P = 0.0025) and SOX2 (P = 0.0359) was measured in AML MNCs , showed again the association between high expression level of NANOG and shorter survival . Altogether, it suggests that NANOG could be considered as an independent risk factor and of high interest for patient within the intermediate risk group.Finally, we analyzed the significance of us Table . Conversars Fig. . Combiniars Fig. . In ordeval Fig. . We perfBMPR1A, ΔNp73 and even more so of NANOG, as they could contribute to predicting at diagnosis the risk of relapse of AML patients.These results demonstrate the importance of measuring levels of 37 and contribute to cancer SC emergence14, maintenance and expansion15. Here, we found an abnormally elevated concentration of BMP4, and to a lesser extent of BMP2 in the BM plasma of AML patients. These cytokines are likely to be produced by the BM microenvironment of leukemic cells, since we did not observe any increase in BMP2 and BMP4 transcripts levels in primitive leukemic cells themselves as compared to normal hematopoietic cells. This appears to be a general mechanism as we have found similar profiles in CML at the time of diagnosis12 and also in luminal breast cancer11. Conversely, we observed an increased expression of intrinsic actors of the BMP pathway in MNCs of AML patients. In particular, we identified a highly significant increase in the expression of BMPR1A both at the mRNA and protein levels, in agreement with the important role of BMPR1 in the early steps of the AML transforming process, as previously described18. Interestingly, unlike the chronic phase of CML, in which we identified BMP2 and BMPR1B as driving the deregulation of the BMP pathway and SC/progenitor maintenance and expansion12, in AML BM cells, BMP4 and BMPR1A alterations were already detected at diagnosis. Interestingly, with CML progression towards more advanced phases and the acquisition of resistance to treatment, the BMP4 signal becomes predominant38. This suggests that the BMP4 signal is related to more aggressive or advanced disease as identified in cancers of other origins such as liver (HCC)39, thyroid (PTC)40 and bladder41. In malignant glioma, while BMP4 appears to induce differentiation of glioma cancer stem-like cells43, an epigenetic repression of the BMPR1B receptors contributes to the CSC phenotype in these tumors10.Resistance to treatment is often associated with the persistence of cancer SCs within their microenvironment. In AML, owing to the poor clinical efficacy of current treatments, deciphering mechanisms through which the microenvironment can sustain and promote the survival of immature-like cancer cells remains an important issue for patient outcome. Among the mechanisms through which the microenvironment can affect leukemic SCs’ survival, cytokine production by stromal cells is of major importance. We focused on the BMP pathway as BMP2 and BMP4 regulate SC fate, maintenance and differentiation processes2. Here, the elevated concentrations of both BMP2 and BMP4 in BM of AML patients at diagnosis also support their involvement in leukemic cells survival, as described in CML12. These results, obtained in the context of AML, highlight the importance under pathological conditions of this pathway already found to be clearly involved in SC regulation in neural44, epithelial14 and hematopoietic systems24 under physiological conditions.We identify here that the compartment of cells expressing surface BMPR1A is enriched in very immature AML cells as demonstrated by functional and ALDH assays. In addition, an increase in LTC-IC after exposure to BMP4 confirmed the involvement of this cytokine provided by the tumor niche and of its receptor in promoting AML stem-like cells. The BM-niche was previously reported to promote survival of leukemic cells in AML by activating different cytokine-related pathways, such as SDF1/CXCR4, Wnt/β-catenin, integrins30. Crosstalk between the p53 family and the BMP pathway has already been reported during breast45 and epidermis46 development, as well as in tumorigenesis47, though no evidence for a link between BMPs and the ΔNp73 isoform has so far been reported. Here, we clearly demonstrate a functional link between BMP4 and ΔNp73. First, we identified a specific ΔNp73 upregulation already present at the time of diagnosis in MNCs of AML patients, while no such effect is seen for TAp73. Subsequently, we showed that this expression was higher within the BMPR1A+ subpopulation and after exposure to BMP4. Finally, we established the link between both the BMP pathway and ΔNp73 with immature cell features. Indeed, AML MNCs expressing high levels of BMPR1A and ΔNp73 were mainly ALDH positive, contained more LTC-IC and, after BMP4 treatment, exhibited an increased capacity to form colonies in an in vitro human functional SC assay.We investigated the interplay between BMP4/BMPR1A signaling and the ΔNp73 isoform, as this member of the p53 family has been shown to be overexpressed in AML and to regulate CSCsNp73 expression, contribute to the increase in the number of immature cells in AML BM. The molecular mechanisms underlying such a phenomenon were evaluated focusing on the expression of some genes associated with immaturity, in particular NANOG, a known ΔNp73-target gene33. We observed a significant correlation between the expression of ΔNp73 and NANOG and also of BMPR1A and NANOG in AML patients. These results highlight for the first time a cascade of events in AML, initiated by the binding of BMP4 to its type 1A receptor that leads to ΔNp73 expression, which in turn induces NANOG by promoter transactivation. This activation is p53-independent, since it was confirmed using the KG1A AML cell line, which does not express functional p5331.Our results suggest that extrinsic (BMP4) and intrinsic (BMPR1A) alterations of the BMP pathway, associated with the induction of ΔNp7332NANOG and SOX248, in AML patients and the induction of ΔNp73 and NANOG upon BMP4 treatment supports the hypothesis that BMP4 could promote the reprogramming of cells towards immature leukemic cells. This is consistent with a recent study that identified BMP4 and its signaling pathway as a driving element of adult cell reprogramming, including fibroblasts towards functional hematopoietic SCs49.The increased expression of three genes involved in self-renewal and in cell reprogramming, Δ50 and ovarian cancer51. BMP type 1 receptors have also been associated with resistance in CML38 and AML18, as has ΔNp7352. These latter, as well as NANOG, are also associated with resistance and progression in other cancers55.Resistance to treatment and relapse are often associated with the persistence of cancer SCs within their microenvironment. Indeed, BMP4 involvement in resistance to treatment has been demonstrated in several tumor types, including HCCBMPR1A, ΔNp73, and NANOG is likely to increase the ability to predict patient outcome at diagnosis. Since involvement of CSC in clinical outcome may be a common feature of relapsing cancers, it will be worthwhile to investigate the potential involvement of this novel cascade in other cancer types.In conclusion, our study deciphers a new signaling cascade through which alterations of BMPs secretion in the microenvironment can sustain and promote resistant immature-like leukemic cells Fig. . ActivatSupplementary Information


The significance of measurable residual disease (MRD) in hematopoietic stem cell transplantation (HSCT) is well recognized in different hematological malignancies, but the evidence indicate that pre-transplant MRD status is of particular importance in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In ALL, inadequate response at the level of MRD is a commonly accepted risk factor for relapse and thus an indication for allogeneic HSCT. Similarly, growing evidence from the literature strongly suggest that MRD detected by multiparameter flow cytometry or molecular techniques should be also used for risk stratification in AML at the time of HSCT. Despite the well-defined association of MRD and outcomes of HSCT in acute leukemias, there are still many open issues such as the role of additional pre-transplant consolidation for MRD eradication, the ability of HSCT to overcome negative influence of MRD positivity on survival, the impact of conditioning regimen intensity on MRD clearance post HSCT, and transplantation outcomes or the selection of optimal donor with regards to MRD status. In addition, the role of MRD assessment in guiding post-transplant maintenance treatment should also be addressed in prospective trials. These open issues mostly awaiting further clinical studies will be discussed in our current review. In acute leukemias, measurable residual disease (MRD) is defined as the presence of residual leukemic cells in bone marrow (BM) or peripheral blood (PB) in patients who achieved morphologic complete remission (mCR). Notably, the new definition of LeukemiaNet for CR includes MRD negativity  indicatiThe significance of MRD in hematopoietic stem cell transplantation is well recognized in different hematological malignancies, but the evidence indicate that pre-transplant MRD status is of particular importance in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as it correlates with the risk of relapse after hematopoietic stem cell transplantation (HSCT). The significance of MRD in acute leukemias has recently been highlighted in recommendations for diagnosis and management in ALL and AML published by different international groups of experts ,2,3,4.In ALL, inadequate response at the level of MRD is a commonly accepted risk factor for relapse and thus an indication for allo-hematopoietic stem cell transplantation  as was recently emphasized in the consensus paper by panel of international experts on behalf of the European Working Group for Adult Acute Lymphoblastic Leukemia (EWALL) and the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation .Similarly, growing evidence from the literature strongly suggests that MRD detected by MFC or molecular techniques should be also used for risk stratification in AML at the time of HSCT. The clinical significance of MRD status in AML has been confirmed recently by a position paper by the European LeukemiaNet (ELN) . Indeed,Despite the well-defined association of MRD and outcomes of HSCT in acute leukemias, there are still many open issues such as the role of additional pre-transplant consolidation for MRD eradication, the ability of HSCT to overcome positive MRD by achieving MRD negativity and the potential price in term of toxicity and transplant related mortality, the impact of conditioning regimen intensity on MRD clearance post HSCT, and transplantation outcomes or the selection of optimal donor with regards to MRD status. In addition, the role of MRD assessment in guiding post-transplant prophylactic and pre-emptive treatment should also be addressed in prospective trials. These open issues mostly awaiting further clinical studies will be discussed in our current review.2 is the most important adverse prognostic factor for treatment outcome, independent from “the old” classical risk factors identified at diagnosis [In acute lymphoblastic leukemia, detection of residual disease persisting below the mCR threshold of 10iagnosis ,6,7,8. Miagnosis ,6,7,8,9,iagnosis ,12,13.−4 to 10−5 [−5 to 10−6 [Sensitivities of clone-specific detection of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements using polymerase chain reaction (Ig/TCR PCR) range from 10 to 10−5 . However to 10−5 ,16,17. A to 10−5 . Yet, an to 10−5 ; therefo to 10−5 ,18. The  to 10−5 . With th to 10−5 ,21. Addi to 10−6 . Some au to 10−6 ,21,23. H to 10−6 . The fiv to 10−6 . The sumTCR and Ig gene rearrangements, it was demonstrated that the probability of disease-free survival (DFS) after 5 years was significantly higher for patients with persistent MRD > 104 who underwent HSCT in first CR than for those patients that did not undergo HSCT in first CR  [−3  assessed by RQ-PCR. In contrast, no benefit of HSCT on RFS was demonstrated in good MRD responders [MRD positivity in post-induction therapy is the universal strongest risk factor for relapse, regardless of the heterogeneity in MRD definitions applied, techniques used, and the time point being assessed in the different studies. Its negative impact on leukemia-free survival (LFS) may be at least partially overcome by HSCT, as strongly suggested by the results of all three cooperative trials conducted by the GMALL, PETHEMA, and GRALL research groups ,7,9. In Although outcomes of patients with persistent MRD who undergo HSCT is better compared with those who are treated with chemotherapy, the relapse rate after HSCT is significantly higher in MRD positive patients in comparison to those with undetectable MRD before transplant. Consequently, one can assume that eradication of MRD before HSCT may significantly improve the outcomes of transplant. A proof of principle is ALL. The efficacy of blinatumomab, a bispecific T cell–engager antibody in MRD eradication was evaluated in a single-arm study in adult patients with ALL in CR who exhibited MRD positivity after chemotherapy . A complThe other issue is the role of pre- and post-transplant MRD monitoring in guiding maintenance therapy after HSCT. This approach is intensively investigated in Ph-positive ALL. The use of tyrosine kinase inhibitors (TKIs) in post-transplant maintenance treatment results in reduced relapse incidence and improved long-term outcomes of HSCT, as was demonstrated by several prospective and retrospective studies ,29,30,31Furthermore, the development of highly sensitive techniques for MRD assessment may allow for revisiting the role of autologous transplantation in ALL. On behalf of the Acute Leukemia Working Party of EBMT, Giebel et al. retrospectively compared autologous versus allogeneic transplantation with myeloablative conditioning in patients with Ph+ ALL in first molecular remission and found no differences in outcomes . The autIn acute myeloid leukemia, genetic risk defined according to European LeukemiaNet (ELN) criteria is the most relevant factor in prognostication and decision-making processes ,34. HoweThe genetic and immunophenotypic diversity of AML is a major challenge for introducing an MRD assessment into daily clinical practice. In view of these difficulties, the ELN MRD Working Party has published guidelines for the use of MRD in clinical practice . The two−3 and 10−5 [−5 sensitivity can be reached mostly in experienced flow cytometry laboratories. MRD monitoring with MFC relies on defining leukemia-associated immunophenotypes (LAIPs) at diagnosis which is required in order to be able to detect MRD in the post-remission phase of the treatment. Alternatively, MRD assessment may be based on an analysis that screens for established aberrant differentiation/maturation immunophenotypic profiles at follow-up that is termed “different-from-normal” (DfN) approach [The most widely employed technique, applicable for more than 90% of the AML patients, remains the MFC with a sensitivity between 10and 10−5 ,38,39. Tapproach . The thiapproach ,41,42,43NPM1 mutation, RUNX1-RUNX1T1 and CBF-MYH11 in CBF-AML, and PML-RARA in acute promyelocytic leukemia [−4 to 10−6. However, these mutations accounts for no more than 50% of AML patients; in other words, RT-qPCR based MRD monitoring is not applicable for at least half of the AML patients [FLT3-ITD , FLT3-TKD , NRAS, KRAS, IDH1, IDH2, and MLL-PTD may be lost between diagnosis and relapse as a result of clonal evolution of leukaemia, thus being unstable throughout treatment. Therefore, ELN guidelines advise against using them as single markers, but they may be used to detect MRD in combination with other mutations [WT1) expression analysis in MRD assessment with the use of standardized and validated WT1 assay developed by ELN researches, who established reference ranges for WT1 expression in normal blood and bone marrow [WT1 encodes a transcription factor overexpressed in approximately 90% of AML patients, but the assessment of its expression for MRD monitoring has remained controversial due to limited data from large, controlled studies, as well as undetermined precise cut-off values and time-points for analysis. However, very recently researches from South Korea published a study on the optimal time-points and thresholds for the bone marrow WT1 expression assessment by real-time PCR with the use of the ELN normalized method in a large cohort of AML patients undergoing allo-HSCT [WT1 MRD assay useful for identifying patients with high risk of AML relapse.The main technical limitations of MRD monitoring by MFC result from (1) the hemodilution of the BM samples; (2) difficulties in proper gating of both pure WBC and blast cells, and (3) biological heterogeneity of the leukemic population. The 2017 ELN guidelines recommend RQ-PCR or NGS as alternative methods for MRD assessment. PCR-based methods  depend on identification of pretreatment-AML-associated mutation at diagnosis . RQ-PCR,leukemia ,36,45, fleukemia  with a spatients ,37. The e marrow . WT1 encllo-HSCT . The aut−3, down to 10−5. It is applicable for monitoring a variety of NPM1 mutations [DNMT3A and IDH mutations [Digital droplet PCR (ddPCR) is the new high-throughput technology that enables an absolute quantification of clonally amplified target genes without a reference standard amplification curve that is needed in conventional RQ-PCR. The ddPCR technique is characterized by high sensitivity exceeding 10utations , as wellutations  in AML autations ,51.NPM1 gene) and after applying error-correcting statistical models for raw sequencing data acquisition [DNMT3A, TET2, and ASXL1 (abbreviated as DTA) are known to be most frequent in CHIP, and thus may occur in healthy individuals with an increased incidence with age [NGS offers several advantages over PCR-based methods, such as the ability to study the entire leukemic genome, which can potentially be used in almost all AML patients. This molecular tool requires highly specialized bioinformatic approaches in order to identify all emerging clinically relevant variants or alleles. NGS used to detect MRD measures the variant allele frequency (VAF) of mutations. The sequencing approaches include Whole Genome Sequencing (WGS), Whole-Exome Sequencing (WES) and targeted-gene sequencing technology . The senuisition . Jongen-uisition . Howeveruisition ,55. Mutawith age ,56. Conswith age ,57. The NPM1-mutated AML treated with intensive chemotherapy may provide prognostic information useful in routine practice [NPM1-mutated transcripts after the second chemotherapy cycle in peripheral blood (PB) of patients in CR was the only significant prognostic factor. Notably as for the question of the timing of MRD assessment and the issue of bone marrow (BM) versus PB, assessment of MRD after the second chemotherapy cycle was more discriminatory than RT-qPCR positivity in PB at any other time point or assessing it in bone marrow samples. Moreover, the prognostic significance of the MRD status in the peripheral blood after the second chemotherapy cycle was also confirmed in the validation cohort. In fact, the presence of MRD compared to the absence of NPM1-mutated transcripts was associated with an increased 2-year cumulative incidence of relapse  and a lower OS rate . Additionally, MRD positive status after second cycle of chemotherapy in a subgroup of patients not harboring the FLT3-ITD mutation was also predictive for a very poor outcome with 2-year CI of relapse of 76% compared to 33% for MRD negative patients. Since the number of MRD positive patients who underwent HSCT in the first CR was small, the authors were not able to confirm a significant benefit of transplantation for those patients. This important question is currently addressed in an ongoing randomized phase III trial being conducted by the UK NCRI AML Working Group. The above UK NCRI AML findings are in line with the results of the Acute Leukemia French Association study, in which NPM1-mutated transcripts were assessed in PB after induction chemotherapy [NMP1 (<4-log) after chemotherapy was associated with poor outcome. Importantly, the study results indicated that HSCT improved OS and DFS only in patients with low reduction of NPM1. This finding is further supported by the results published by Freeman et al. demonstrating that in the subgroup of standard-risk patients without NPM1 mutations, only those with MRD+ benefit from HSCT in first CR [MRD assessment in the post-induction phase of the treatment of an AML patient is emerging as a new tool that can be used to predict poor outcome in standard-risk AML patients who may benefit from HSCT in the first CR. In this regard, the UK National Cancer Research Institute (UK NCRI) AML Working Group has evaluated prospectively the predictive value of MFC-MRD after first and second course of intensive chemotherapy in patients with AM . The patpractice . The resotherapy . Similarfirst CR .Regardless of the beneficial effect of allogeneic transplantation improving the outcomes of MRD-positive patients with low genetic risk, the results of HSCT in patients with intermediate and high-risk AML and positive MRD, for whom HSCT is the standard treatment approach, seem to be dismal, much worser than in patients with CR-MRD(−). Indeed, there is growing evidence confirming that also in AML, pre-transplant MRD assessment by either MFC or molecular techniques can be used for risk stratification at the time of HSCT following either myeloablative or reduced intensity conditioning ,62,63,64The molecular profiling and MRD assessment introduced into the prognostic stratification of patients with AML is a beginning of a new era of MRD-directed therapy, which will most probably allow patient-tailored and precision-type medicine avoiding under- or over-treatment of AML patients in the near future. In this regard, an important and critique question is the role of post-remission consolidation therapy in patients in CR with positive and negative MRD.The retrospective analyses of both the EBMT and CIBMTR registries strongly suggest no advantage of post-remission consolidation chemotherapy pre-transplantation with either MAC or RIC preparative regimens. Supporting the no role for post-remission consolidation chemotherapy are data published by Cahn et al. on behalf of the EBMT registry for AML patients undergoing HSCT from a matched related donors with MAC , and morAdditional important question is whether the kinetic of MRD clearance matter. Buccisano et al. analyzed the kinetics of reduction of MRD measured by MFC in bone marrow of AML patients treated with intensive chemotherapy according to the EORTC/GIMEMEA protocols . The autIn subsequent publication, Buccisano et al. addressed the question whether the patients in CR with positive MRD may benefit from allogeneic transplantation . In theiFLT3 internal tandem duplication (FLT3 ITD) inhibitors of FLT3, sorafenib, midostaurin, quizartinib, gilteritinib, and others, are also intensively assessed in active phase II–III trials [Because MRD emerges as a strong and independent risk factor for AML recurrence after allogeneic HSCT, a question arises regarding the prevention of post-transplant leukemia relapse in patients transplanted in CR-MRD(+) or in those with re-appearance of MRD after transplant. The increasing knowledge of both the molecular landscape of AML on one hand and better understanding of the biology of the GvL effect on the other opens the new perspectives on post-transplant MRD-targeted therapies, what has been very recently elegantly presented in expert review from the ALWP of the EBMT . PotentiI trials .In the context of suboptimal outcomes of HSCT in AML patients in complete remission but with positive MRD, the issues of whether selection of the type of graft or donor will improve results and whether the donor should differ in MRD positive as opposed to the MRD negative AML patient have been investigated. In other words, what is the optimal donor for MRD positive AML patients and whether alternative donors or mismatched donors with broader HLA disparities and thus theoretically stronger GVL are of advantage in eradicating MRD have been studied. Indeed, the impact of donor type in AML patients with pre-transplant MRD positivity was the subject of few retrospective studies also addressing the prognostic significance of MRD status for those type of transplantation. Specifically, the group from Seattle compared retrospectively the results of a first HSCT from an unrelated cord-blood (CB) donor versus transplantation from matched unrelated donor (MUD) or from an HLA-mismatched unrelated donor (mMUD) following myeloablative condition in a large group of patients with AML and myelodysplastic syndrome (MDS) who had MRD determined by MFC before transplantation . InteresThe molecular profiling and MRD assessment introduced into the prognostic stratification of patients with AML seem to begin a new era of MRD-directed therapy, which may allow avoiding under- or over-treatment of AML patients in the near future.WT1, mutated NPM1, t, inv(16), and t [WT1 analysis alone was with limited predictive value as well. Intriguingly, opposite results regarding the prognostic significance of testing the MRD status of the autograft by WT1 as the only tumor mutation were published by the group from Milan [WT1 expression level measured by RQ-PCR in autologous peripheral blood stem cells (PBSC) from leukapheresis discriminates the risk of relapse and is associated with DFS and OS after autologous HSCT.The development of highly sensitive and specific MRD assessment methods seems to lead to the rejuvenation of the concept of autologous transplantation in AML. Autologous transplantation can potentially allow a selected group of patients, especially with low and potentially intermediate genetic risk, to avoid over-treatment with allogeneic transplantation, providing 50–60% probability of long-term LFS . The rect . In the om Milan . They foIn conclusion, there is growing evidence that results of autologous HSCT in patients with low and intermediate risk AML are very good, provided that MRD status is negative before transplant.NPM1 mutation who are MRD positive after the second course of chemotherapy should be considered for early allogeneic transplantation. As for the role of pre-transplant consolidation, there are still controversies about its necessity in patients that achieved remission but are MRD positive and are about to go allogeneic transplantation, since there is no evidence that consolidation can eradicate MRD in AML. On the other hand, there are strong data regarding the independent impact of pre-transplant MRD status on the outcomes of HSCT, with similar significance whether MAC and RIC were applied. The broad availability and predictive capabilities of MRD as summarized in the current review may open new perspectives for novel targeted therapies both for pre-transplant MRD eradication as well as for post-transplant maintenance therapy in both ALL and AML. Lastly, as for the question of optimal selection of donor depending on AML patients’ MRD status, the published data, although on limited number of patients, may suggest that the negative impact of MRD on transplant outcomes might be partially overcome by allotransplant from cord blood graft or haploidentical donors in comparison to transplantation from unrelated or sibling donors. However, due to small size of study group and retrospective nature of the analysis these somewhat provocative data should be confirmed in further studies.Measurable residual disease has been used to guide therapeutic decisions and therapies in pediatric acute lymphoblastic leukemia for more than two decades. The lessons learnt from pediatric ALL were gradually incorporated into adult ALL and AML therapeutic strategies. Data are currently emerging for both adult ALL and AML that, similar to pediatric ALL, MRD is a new, extremely important prognostic factor in both types of adult acute leukemia. MRD should be most probably incorporated in the treatment decision process for both adult ALL and AML, similar to being used in pediatric ALL, although there are still many open questions and some controversies discussed in our review. The widely available MFC and RQ-PCR methods for MRD measurement may allow the introduction of MRD into the risk stratification in AML in daily clinical practice in the nearest future. It may lead to treatment de-escalation in patients with intermediate genetic risk who achieve CR with MRD negativity during the post-remission therapy. The era of MRD-directed therapy in AML may even lead to revisiting and new perspectives for autologous transplantation for patients with low or even intermediate risk AML achieving MRD negative status, although this approach is controversial as GVL is of major importance in curing AML. On the other hand, there is strong evidence that patients with


In this review, we first introduce general concepts on neuronal Ca2+ signaling and then summarize the current knowledge on fundamental properties of substantia nigra pars compacta dopaminergic neurons, on the role of the interplay between Ca2+ and dopamine signaling in neuronal activity and susceptibility to cell death. We also discuss the possible involvement of a “neglected” player, the Neuronal Calcium Sensor-1 (NCS-1), which has been shown to participate to dopaminergic signaling by regulating dopamine dependent receptor desensitization in normal brain but, data supporting a direct role in PD pathogenesis are still missing. However, it is intriguing to speculate that the Ca2+-dependent modulation of NCS-1 activity could eventually counteract dopaminergic neurons degeneration.Parkinson’s disease (PD) is a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. The causes of PD in humans are still unknown, although metabolic characteristics of the neurons affected by the disease have been implicated in their selective susceptibility. Mitochondrial dysfunction and proteostatic stress are recognized to be important in the pathogenesis of both familial and sporadic PD, and they both culminate in bioenergetic deficits. Exposure to calcium overload has recently emerged as a key determinant, and pharmacological treatment that inhibits Ca Intracellular Ca2+ signaling in neurons is extremely fine-tuned, because it controls gene transcription, membrane excitability, neurotransmitters secretion and many other cellular processes, including synaptic plasticity  opening, cytochrome c release and can activate apoptotic cell death  to avoid that their excessive accumulation could trigger mitochondrial dysfunction , and Ca2+ binding proteins that act as Ca2+ buffer and/or transducer  is mainly responsible for translating the increases of the cytosolic Ca2+ concentration into a biochemical signal through conformational changes of its targets , calretinin (CR), and parvalbumin (PV) are also present in the nervous system. By buffering Ca2+ levels with different capacity, affinity and kinetics  was reduced in an age-related manner in humans, suggesting a role for CB-28K also in the selective neuronal vulnerability in AD  and generate metabolic stress and mitochondrial damage , which are also autonomous pacemakers, are significantly less vulnerable than SNc DA neurons from which they differ in respect with two main features: they have smaller Ca2+ currents  by measuring changes in Ca2+-bound buffer and dividing by the free Ca2+ increase. Interestingly, despite the intrinsic Ca2+ buffering in DA cells increases with postnatal age , it remains low at both age ranges. Other neuronal populations , that are not endowed with pacemaking activity, display similar or higher values and Purkinje cells have the highest KS values   .2+ binding proteins, also mitochondria play a role in buffering cytosolic Ca2+, a reduction of mitochondria amount or/and the presence of dysfunctional mitochondria could account for differences in Ca2+ buffering capacity among different neuronal midbrain populations. In line with these considerations, a study has found that the mitochondrial mass in SNc DA neurons is reduced in respect with that of other neurons from the midbrain  could contribute to SNc DA neurons vulnerability. In this respect, defects in intracellular Ca2+ stores handling and ER stress have been frequently documented in PD cellular models  are particularly abundant in neurons and photoreceptors and deserve special attention since their properties distinguish them from CaM or CB-28K, CR and PV and allow them to play non-reduntant roles. Differences in Ca2+ affinities, in cellular expression and distribution and in target proteins are at the basis of the specialization of NCS function (2+ Sensor-1 (NCS-1) is the most ancient member of the family  does not severely compromise pacemaking activity both in juvenile and adult SNc DA neurons, but rather altered its precision and regular occurrence . The app KO mice .2+ and DA are critical components in the disease and underline the complexity of their interplay in the modulation of dopaminergic response.All together these observations strongly support the idea that Ca2+ concentration) and high dendritic arborization distinguishes these neurons from the other neurons in the brain. Cav1.3 mediated Ca2+ influx is essential to sustain DA release, to guarantee high energy demands that are required for this function and to provide necessary amount of ATP at axonal and dendrites sites. But if continuous Ca2+ entry sustains DA secretion and mitochondrial metabolism, at the same time it exposes cells to “Ca2+ stress,” that may synergize with intrinsic low Ca2+ buffering capacity, aging, mutations or mitochondria damage and culminate in cell degeneration. In vitro and in vivo studies strongly implicated Cav1.3 activity in the high vulnerability of SNc DA neurons, however the complexity of DA metabolism that includes an autoregulatory nature of DA secretion underlines that selective vulnerability of SNc neurons is still an obscure issue. The characterization of the Cav1.3 Ca2+ channels physiology and of the alternative pathways that are engaged to compensate pharmacological inhibition of Cav.1.3 channels upon isradipine treatment certainly deserves more investigations. The outcome of isradipine phase III clinical trial will shed light on these aspects.The distinctive physiology of the DA midbrain neurons within the SNc has attracted attention as possible reason for their selective vulnerability. Slow rhythmic activity (accompanied by oscillations in intracellular CaAt this point we can conclude that the deciphering of the molecular mechanisms involved in dopaminergic signaling is the best we can do to develop therapeutic strategy, but we have to be aware that the complexity of the system is increased by interactive pathways that are engaged in compensatory mechanisms and this makes the investigations very challenging.All datasets generated for this study are included in the manuscript and/or the supplementary files.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca2+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca2+ mobilization induced by ROS.A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca These processes combine the action of a variety of channels, both in the plasma membrane and in the membrane of the intracellular stores, such as the endoplasmic or sarcoplasmic reticulum, including Ca2+ pumps, exchangers, and buffers. Many components of this large set of molecules have different isoforms with different characteristics and properties, which gives them the ability to make the system extremely versatile c is approximately 100 nM and in certain intracellular organelles, such as the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), the free luminal Ca2+ concentration ([Ca2+]L) is around 0.2–1 mM; therefore, there is a clear concentration gradient between compartments that is essential for the regulation of the cellular processes in which Ca2+ participates c is determined by the balance between the Ca2+ “on” mechanisms, including Ca2+ influx from the extracellular medium and the output from intracellular stores, and Ca2+ “off” mechanisms, involving Ca2+ extrusion across the plasma membrane and sequestration into the stores or mitochondria c reaches the micromolar range, both NCX and MCU can also transport Ca2+. NCX is a bidirectional transporter that combines the movement of three Na+ ions towards the cytosol with the transport of one Ca2+ in the opposite direction. NCX participates in the regulation of [Ca2+]c in a number of cell types acting either in forward mode, as explained above, or in reverse mode, introducing Ca2+ in the cell when the Na+ concentration at the inner face of the plasma membrane substantially increases. Mitochondrial membrane potential confers the driving force necessary for the activity of the MCU. The threshold [Ca2+]c for the activation of these four transporters is different as well as the transport rate. The lowest threshold is for the PMCA, which, by the way, has the lowest transport rate. This characteristic confers to PMCA an important role in maintaining resting [Ca2+]c [2+ but higher transport rates than PMCA [2+]c for NCX and MCU is the highest but they also display much higher transport rates than the ATPases. Agonist-induced changes in [Cachondria . The Ca2the cell . There athe cell . These iame cell . PhosphoSR lumen . In addi [Ca2+]c . SERCA hhan PMCA . On the 2+ “on” mechanisms includes Ca2+ release from the intracellular pools and entry through plasma membrane channels. There are three major types of intracellular Ca2+ channels responsible for Ca2+ release from the ER and the SR: the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor (RyR) and the nicotinic acid-adenine dinucleotide phosphate (NAADP) receptor. Mammalian cells possess three different isoforms for IP3R and RyR. Occupation of phospholipase C (PLC)-coupled membrane receptors by agonists results in the activation of phosphoinositide-specific PLC. As a result, inositol 1,4,5-trisphosphate (IP3) is generated and activates IP3R leading to Ca2+ efflux from the ER c upon agonist stimulation and the development of regenerative Ca2+ waves. Finally, NAADP-mediated Ca2+ efflux from intracellular stores involves endolysosomal two-pore channels (TPC), especially TPC1 c c and subsequent cell dysfunction. PMCA can be reversibly inactivated by ROS by an unclear mechanism that is suggested to be a reversible cysteine modification c rises over the resting value. The interrelationship between ROS production and Cafication ,53,54. Cresidues . Furtherresidues . As a reROS can also alter NCX function although the effect of ROS on NCX activity remains controversial. Hydrogen peroxide formed by the xanthine/xanthine oxidase system and superoxide anion increase the activity of NCX in myocytes . Howeverin vitro by modifying sulphydryl groups c is higher in cells in patients with type 2 DM than in healthy donors c can trigger the synthesis of thromboxane A2 and hyperaggregability causing platelet hyperactivation.There is evidence linking the decrease in vascular NO production with increased production of ROS that alter platelet function c [Different antioxidants has been shown to reverse DM-associated platelet hyperactivity and hyperaggregability by reducing [Ca2+]c ,129,130.2+]c ,132,133.2+]c ,135. For2+]c . The enz2+]c ,138. The2+]c ,140.2+ is essential for neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways [2+]c directly by activating α-amino-3-hydroxy-5-methylisoxazole-4-propionate acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor channels and indirectly by activating voltage-dependent Ca2+ channel. Besides, the activation of glutamate receptors coupled to the GTP-binding protein Gq11 stimulates the release of IP3 leading to the opening of channels in the ER, following by a sustained entry of Ca2+ across the plasma membrane by store-operated or/and receptor-operated channels c is reduced, and this dysfunction can lead to neuronal death in three ways. First, direct or indirect cysteine proteases activation, such as calpain and caspases, which degrade a variety of substrates including cytoskeletal proteins, membrane receptors and metabolic enzymes. Calpain also has a role in the apoptotic cascade through its ability to activate caspases [2+ induces oxidative stress through the activation of oxygenases in arachidonic acid metabolism, disturbance of mitochondrial Ca2+ and energy metabolism. ROS generated in response to Ca2+ influx induced by glutamate includes superoxide, hydrogen peroxide, hydroxyl radical and peroxynitrite [2+ induces apoptosis through activation of pro-apoptotic proteins such Bax, Par-4 and p53, enhancing mitochondrial membrane permeability and release of cytochrome c [Under pathological conditions, the ability of neurons to control the increases in [Cacaspases ,146,147.ynitrite . Finallychrome c ,150.Oxidative stress plays a key role in the development of many neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. ROS induce blood-brain barrier disruption, mediate the transendothelial migration of monocytes and myelin phagocytosis, and finally induce cellular damage ,152,153.Alzheimer Disease (AD) is a disease that affects more than 35 million people worldwide. It is characterized by progressive loss of memory and other cognitive functions. Neurons progressively die and some areas of the brain show atrophy. The average life of AD patients is 8-10 years after its detection .2+ homeostasis in neurons contribute to the neurodegenerative process. It have been shown that in AD patients the proteolytic processing of β-amyloid precursor protein and presenilin 1 and 2 are altered, leading to increased production of neurotoxic Aβ [2+ homeostasis by generating ROS, a process catalyzed by Cu+ and Fe2+. The neurotoxic Aβ induces oxidative stress, which leads to lipid peroxidation and breaks the Ca2+ homeostasis through the production of 4-hydroxy-2,3-nonenal, resulting in damage of the Na+/K+ and Ca2+-ATPases, and glucose and glutamate transporters [2+ that sensitizes neurons to apoptosis [2+ homeostasis by oxidative stress, Aβ oligomers can form pores for Ca2+ in cell membranes [2+ homeostasis might be able to prevent or at least delay the onset of AD. However, despite the success of drugs such as L-type Ca2+ channel blockers [2+ fluxes may affect normal function of neurons, so the therapies should aim at stimulating the production of neurotrophic factors that protect neurons from cell death. Recent advances in AD treatment are focused on the use of the immune system. Current studies are mainly focused on the production of specific Aβ antibodies, on inhibitors of the enzymatic machinery involved in the production of Aβ from APP and also uncovered Aβ aggregation inhibitors [Alterations in Catoxic Aβ ,160,161.sporters . Finallypoptosis ,164. Besembranes . These fblockers , therapihibitors ,168,169.2+ homeostasis, there are treatments that attempt to reduce oxidative stress in the cells, for instance inhibition of NADPH oxidase, responsible for most of the ROS production in microglia [6 and B12 has been ineffective [Finally, because ROS can affect Caicroglia , which sicroglia ,172,173.ffective . Currentffective ,176.2+ mobilization [2+ uptake [Among other antioxidants, the use of flavonoids in therapeutic strategies has been extensively investigated. Flavonoids group a large and complex number of polyphenolic compounds that contain a three-ring structure with two aromatic centers and a central oxygenated heterocycle. On the basis of structural differences flavonoids are classified into flavonols, flavones, flavanones, catechins, anthocyanidins, isoflavones, dihydroflavonols, chalcones and proanthocyanidins . The antlization ,185,186.+ uptake . Plant f+ uptake  and supp+ uptake . Future + uptake .2+ homeostasis. The use of exogenous oxidant scavengers has been demonstrated to exert beneficial effects on a number of disorders, although further studies are necessary to design therapeutic strategies specific for the different diseases and scavengers. In summary, ROS have been reported to play an important functional role at physiological concentrations; however, when the concentration of oxidants exceeds the cellular scavenging mechanisms, ROS might be involved in the development of a number of cellular disorders, including abnormal Ca


Chemotherapy resistance represents a major obstacle for the treatment of patients with breast cancer (BC) and greatly restricts the therapeutic effect of the first-line chemotherapeutic agent doxorubicin (DOX). The present study aimed to investigate the feasibility of the recombinant dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor in reversing the DOX resistance of BC. Both DOX-resistant human breast carcinoma cell lines exhibited a multidrug resistance (MDR) phenotype. The ability of the dual-target MDM2/MDMX inhibitor in reversing doxorubicin resistance was subsequently verified,  respectively. We observed that the MDM2/MDMX inhibitor in combination with DOX could suppress proliferation, promote cell cycle arrest and induce apoptosis. In addition, it was capable of reducing rhodamine123 efflux in DOX-resistance BC cell lines and further played a key role in BC nude mice model. The groups that were treated with the combination of the drugs had decreased P-glycoprotein/multidrug resistance-associated protein/cdc 2/Bcl-2 expression and increased CyclinB1/Bax expression. These effects were caused due to activation of the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)/TAK1/p38 mitogen-activated protein kinase (MAPK) signaling pathway, as shown by small interfering RNA (siRNA) silencing and immumohistochemical staining of BC tissue sections. Furthermore, high MDM2/MDMX expression was positively associated with weak TAB1 expression in BC patients. Therefore, the recombinant dual-target MDM2/MDMX inhibitor could reverse doxorubicin resistance via the activation of the TAB1/TAK1/p38 MAPK pathway in wild-type p53 multidrug-resistant BC. Breast cancer (BC) remains the most common malignancy among women in western countriesIt is well established that the tumor suppressor gene p53 plays a pivotal role in coordinating the cellular responses to a wide variety of stress signalsin vitro and in vivo basic research, which requires further clinical evaluationSeveral studies have explored the developmental effects of the dual inhibitors of the p53-MDM2/X pathways, highlighting their prospects to confront cancer. However, the availability of such compounds is still limited and mostly focuses on We synthesized the cell-permeable dual-target MDM2/MDMX inhibitory protein that could simultaneously disrupt the interactions of MDM2 and MDMX with p53. The process included construction of an expression vector, followed by gene expression, protein purification and protein refolding, as described in detail in previous studies. Afterwards, co-immunoprecipitation-western blot analysis showed the protein was able to be immunoprecipitated by anti-MDM2 and anti-MDMX antibodies, indicating that this protein is functional. Enzyme-linked immunosorbent assay (ELISA) proved that the recombinant dual-target MDM2/MDMX inhibitor strongly inhibited interaction of MDM2/MDMX with p53, which was in a dose-dependent mannerThe human breast adenocarcinoma cell line MCF-7 and MCF-7/DOX cells (DOX-resistant MCF-7 cells) were purchased from KeyGEN BioTECH . The human breast infiltrating duct carcinoma cell line ZR-75-30 was obtained from the Translational Medical Center of the Medical College of Xi'an Jiaotong University. ZR-75-30/DOX cells (DOX-resistant ZR-75-30 cells) were established from the corresponding sensitive cell line ZR-75-30 with a gradual increase of DOX  concentration. The culture conditions were initially the same as those used for the ZR-75-30 cell line. Subsequently, DOX was added and the concentration was increased every two weeks with a medium exchange every two days. The DOX-resistant ZR-75-30 cell line was obtained following one year of culture. It is worth mentioning that both cell lines were wild-type p53.2. MCF‑7/ DOX and ZR-75-30/ DOX cells were cultured in media containing 1 μg/ml DOX to maintain the MDR phenotype, and prior to their use, the cells were maintained in drug-free media for at least two days.All cell lines were cultured in RPMI-1640 medium (KeyGEN BioTECH) containing 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin  at 37ºC in a humidified atmosphere of 5% COThe cells were lyzed with ice-cold lysis buffer including protease and phosphatase inhibitors . Following centrifugation at 4ºC for 20 min, the supernatants were collected and the protein concentration was determined using a bicinchoninic acid protein assay kit . Equivalent amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinyl difluoride membrane . The membranes were blocked in 5% (w/v) non-fat milk at room temperature for 2 h, and incubated with primary antibodies at 4ºC overnight, followed by incubation with the corresponding HRP-conjugated secondary antibodies  at room temperature for 1 h. Finally, the blots were visualized with the enhanced chemiluminescence kit  detection system according to the manufacturer's instructions. GAPDH  was used as a loading control. The following primary antibodies were used: MDM2 , MDMX ; p53 , P-gp , MRP , CyclinB1 , cdc2 , Bax , Bcl-2 , TAB1 , TAK1 , p38 MAPK .4 cells/well in a 96-well plate and overnight incubation to allow adherence, the cells were treated with at least eight concentration gradients of DOX, cis-Dichlorodiamineplatinum (DDP), cyclophosphamide (CTX) and tamoxifen (TAM), respectively. The treatments were performed in triplicate and the maximum concentration used was 80 μg/ml. Dimethyl sulfoxide  was used as a vehicle control and a blank group containing blank phosphate buffer saline (PBS) was also used. Cell monolayers were fixed with 50% (w/v) trichloroacetic acid and stained with 0.4% (w/v in acetic acid) SRB (Sigma-Aldrich) solution for 30 min at room temperature. The stained cells were destained with 1% acetic acid to remove the excess dye. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density (OD) determination at 540 nm using a microplate reader . The rate of cell inhibition was calculated using the following formula: inhibition rate = [1 - (OD test/OD negative control)] × 100%. After plotting the dose-response curve, the half maximal inhibitory concentration (IC50) values of different drugs were calculated using SPSS software  and the resistant fold (RF) value was determined by the following formula: IC50 (resistant cells)/IC50 (sensitive cells). Seeded cells were afterwards treated with different concentrations of MDM2/MDMX inhibitor  in a SRB assay in order to obtain the 10% inhibitory concentration (IC10) values as working concentrations. Moreover, the cells were exposed to various concentrations of DOX again after treatment with the MDM2/MDMX inhibitor to verify its reversal effect, and the reversal index (RI) value was determined by the following formula: IC50 (resistant cells before treatment with MDM2/MDMX inhibitor)/IC50 (resistant cells after treatment with MDM2/MDMX inhibitor).The SRB assay was performed for the toxicity screening of cells using a range of widely used anticancer drugs. Following seeding of the cells at a density of 1×102 cells/well) and then treated with DOX and/or MDM2/MDMX inhibitor for 24 h. The cells were cultured in drug-free media for approximately 2 weeks until they were evaluated macroscopically and subsequently washed twice with PBS. The cells were then fixed with methanol and stained with 0.1% (w/v in PBS) crystal violet for 20 min. The colonies (more than 50 cells) were photographed and manually counted. The relative clone formation ability was calculated as follows:  ×100%.The cells were seeded in 6-well plates (3 × 105 cells/well) that were separately treated with DOX and/or MDM2/MDMX inhibitor for 24 h. Adherent cells were collected, washed twice with cold PBS, and fixed with cold 70% ethanol at 4ºC overnight. Subsequently, the cells were stained with a solution that contained 25 μg/ml of propidium iodide with RNase A (50 μg/ml) for 30 min at 37ºC in the dark. The samples were analyzed with FCM . For measurements of cell apoptosis, the cells were double stained with Phycoerythrin (PE) Annexin V and 7-aminoactinomycin D (7-AAD) after harvesting and washing twice with PBS. Finally, stained cells were also detected by FCM according to the manufacturer's instructions .For cell cycle measurements, the cells were seeded in 6-well plates (3×105 cells/well), pre-treated with DOX and/or MDM2/MDMX inhibitor for 24 h and then exposed to 10 μM rhodamine123 (Sigma-Aldrich) for an additional 2 h in the dark. After harvesting and washing twice with PBS, the cells were detected with FCM with excitation and emission wavelengths of 485 and 535 nm, respectively and finally the cell-associated mean fluorescence intensity was determinedThe cells were seeded in 6-well plates (3×104 cells/well) with sterile glass coverslips and were subsequently fixed with 4% paraformaldehyde. Following blocking of the nonspecific binding sites with 5% (w/v) bovine serum albumin for 30 min, the slides were incubated with primary antibodies against P-gp  and MRP  at 4°C overnight. An additional reaction was carried out with Cy3-conjugated IgG  at room temperature for 1 h. Finally, the cells were sealed with a fluorescence quenching sealing tablet containing 4', 6-diamidino-2-phenylindole . The images were captured with a fluorescence microscope .Following relevant treatment of the cells with the MDM2/MDMX inhibitor for 24 h, the cells were seeded in 24-well plates (5×106 cells) were re-suspended in PBS, mixed with matrigel and were injected into the mammary fat pads of mice to establish mouse models of breast carcinoma in situ. When the average tumor size reached approximately 200 mm3, tumor-bearing mice were randomly divided into four groups (six in each group) and treated with the following regimens: (1) Normal saline (NS); (2) single treatment of DOX; (3) single treatment of MDM2/MDMX inhibitor; and (4) pre-treatment with MDM2/MDMX inhibitor followed by DOX. The epigenetic therapy used in the present study comprised i.v. administration of MDM2/MDMX inhibitor at 10 mg/kg, for the time period of d 1 to d 3. The DOX chemotherapy regimen was 2.5 mg/kg, iv, twice per week3) = a × b a was the largest diameter and b the smallest diameter). After several days, the mice were killed and tumor tissues were excised, weighed and fixed in 10% neutral buffered formalin for pathological analysis. The IRs were calculated according to the formula: inhibition rate (IR) =  ×100%.Four-week-old female athymic BALB/c nude mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and housed under specific-pathogen-free conditions in the Animal Center of the Medical College of Xi'an Jiaotong University. All animal experimental procedures were carried out according to the protocols approved by the Ethics Committee for Animal Experimentation of the Medical College of Xi'an Jiaotong University, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. MCF-7/DOX cells  and baked at 65ºC overnight. Following antigen retrieval in 10 mM of citrate buffer using a microwave, IHC staining was performed using a streptavidin-biotin peroxidase kit  according to the manufacturer's instructions. Subsequently, diaminobenzidine  was added to the sections, according to the manufacturer's instructions. Finally, the sections were counterstained with hematoxylin, dehydrated and mounted. Anti-P-gp polyclonal  or anti-MRP polyclonal antibodies  were applied and the slides were placed into a humid incubation chamber at 4ºC overnight. PBS was used as negative control instead of the primary antibody. Expression intensities were quantified as the sum of the integrated optical densities(IOD)/sum of the area of threshold pixels for all signals measured in each image using IPP 6.0 software (Media Cybernetics).5 cells/well) in medium without antibiotics. Following treatment for 24 h, the cells were transfected with double-stranded siRNA against TAB1 or nonspecific control siRNA using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. The efficiency of RNA interference was verified by western blot assays.The siRNA targeting TAB1 and the corresponding negative controls were designed and synthesized . To knock down endogenous TAB1, the following target sequences were constructed in a siRNA vector: siRNA#1-TAB1-Homo-219: sense: 5′- GGAGUGAGAACAACUGCUUTT -3′, antisense: 5′- AAGCAGUUGUUCUCACUCCTT -3′; siRNA#2-TAB1-Homo-685: sense: 5′- GGAUGAGCUCUUCCGUCUUTT -3′, antisense: 5′- AAGACGGAAGAGCUCAUCCTT -3′; siRNA#3-TAB1-Homo-970: sense: 5′- GGAGAUUGCUGCGAUGAUUTT -3′, antisense: 5′- AAUCAUCGCAGCAAUCUCCTT -3′. A scrambled siRNA sequence was used as a negative control: 5′- UUCUCCGAACGUGUCACGUTT -3′, antisense: 5′- ACGUGACACGUUCGGAGAATT -3′. The cells were seeded in a 6-well plate , containing 70 breast cancer tissue samples , were used to evaluate associations between MDM2/MDMX expression and TAB1 expression. The patients included 2 males and 68 females aged 30-89 years . None of the patients had received anti-cancer treatment prior to tumor excision. All patients were classified according to the p-TNM staging system of the American Joint Committee on CancerWe performed IHC staining of TMA slides with MDM2 , MDMX  and TAB1  antibodies by the procedure described in the IHC section. The Sinicrope scoring methodP<0.05) was considered to indicate a statistically significant difference.Statistical analysis was performed using the SPSS software. The data were expressed as mean ± SD. The differences between the two groups were analyzed using the student's t-test. The differences between three or more groups were analyzed using one-way ANOVA and the least-significant difference (LSD) test. The Chi-square test was used to analyze differences between clinicopathological variables. All statistical tests were two sided. A P value less than 0.05  for both cell types, confirming their DOX resistance. MDR has been previously reported as the underlying reason for drug resistanceWe confirmed the antitumor activity of the MDM2/MDMX inhibitor in a previous study50 values of DOX from 6.58 ± 1.50 µg/ml and 16.88 ± 5.64 µg/ml to 0.72 ± 0.08 µg/ml and 1.21 ± 0.17 µg/ml in MCF-7/DOX and ZR-75-30/DOX cells, respectively. The RIs of MCF-7/DOX and ZR-75-30/DOX cells were 9.15 and 13.92, respectively. This indicated that the MDM2/MDMX inhibitor significantly reversed the resistance of the cells to DOX. In addition, we used the IC10 values of DOX as the working concentrations in the following experiments. This corresponded to 0.08 ± 0.01 µg/ml in MCF-7/DOX cells and 0.20 ± 0.07 µg/ml in ZR-75-30/DOX cells.To evaluate the reversal effect of MDM2/MDMX inhibitor on the DOX resistance of MCF-7/DOX and ZR-75-30/DOX cells, the DOX concentration-cell viability curves were plotted. MCF-7/DOX and ZR-75-30/DOX cells exhibited apparent resistance to DOX compared to MCF-7 and ZR-75-30 cells Figure E and 1F.P<0.001) and the MDM2/MDMX inhibitor group . Furthermore, single treatments of DOX (P<0.001) and/or the MDM2/MDMX inhibitor (P<0.001) could also inhibit the growth of MCF-7/DOX cells significantly, whereas no significant difference was noted between them (P=0.055).A colony formation assay was performed to study the anti-proliferative effects of the combination of the MDM2/MDMX inhibitor and DOX in MCF-7/DOX cells. The combination of the MDM2/MDMX inhibitor and DOX inhibited the proliferation of MCF-7/DOX cells significantly, with a colony formation rate of 24.33 ± 5.03% detected for this group, which was significantly lower than the DOX group , the DOX (P<0.001) and the MDM2/MDMX inhibitor groups (P<0.001)  and ZR-75-30/DOX (P=0.017) cells to the G2/M phase, although single treatment with DOX did not exhibit this effect. Co-treatment with the MDM2/MDMX inhibitor and DOX increased the levels of the G2/M-phase dominant protein cyclin B1 and decreased the levels of cdc2, which provided further evidence that the MDM2/MDMX inhibitor in combination with DOX could induce G2/M arrest of MCF-7/DOX and ZR-75-30/DOX cells , the DOX group (P<0.01) and the MDM2/MDMX inhibitor group (P<0.01)  Figure B. Furthe) Figure C-3E. TakP<0.01), the DOX groups (P<0.01) and the MDM2/MDMX inhibitor groups (P<0.01), suggesting that the MDM2/MDMX inhibitor was able to reduce the drug efflux function of P-gp with the combined action of DOX , DOX (P=0.004) and MDM2/MDMX inhibitor groups (P=0.011) since day 3 until mice executed  staining of tumor tissue sections from the MCF-7/DOX model showed decreased P-gp and MRP levels in the tumor cell membrane in co-treatment group tumor tissues  mediate a wide variety of cellular behaviors in response to extracellular stimuliHaving shown that the MDM2/MDMX inhibitor could reverse DOX resistance of BC We therefore speculated that the MDM2/MDMX inhibitor could act through the TAB1/TAK1/p38 MAPK signaling pathway. Subsequently, western blot analysis was applied to confirm that co-treatment with the MDM2/MDMX inhibitor and DOX could lead to TAB1, TAK1 and p38 MAPK overexpression in the MCF-7/DOX cells Figure A and 6B,To investigate the interaction between the MDM2/MDMX inhibitor and the TAB1/TAK1/p38 MAPK pathway, we used small interfering RNA (siRNA) to knockdown TAB1 in both DOX-resistant cells. Subsequent western blot analysis revealed that siRNA-mediated TAB1 knockdown attenuated the effect of the MDM2/MDMX inhibitor on the expression of certain key proteins Figure C and 6D.P=0.003)     and consistently, MDMX over-expression was also related to low expression of TAB1 in BC patients (P<0.001), which confirmed that the MDM2/MDMX inhibitor acted via the TAB1/TAK1/p38 MAPK pathway in the patient specimens examined.Finally, the correlations between MDM2/MDMX and TAB1 expression levels were explored. The results indicated that high MDM2 expression was positively associated with weak TAB1 expression in the cytoplasm  which is available and continued to explore whether TAB1 overexpression could enhance the effect of MDM2/MDMX inhibitor. In addition, we haven't realized how MDM2/MDMX inhibitor act on TAB1 in detail. Is it direct action or indirect effect? We need to carry out a co-immunoprecipitation-western blot analysis using a co-immunoprecipitation kit  to explore the inter-reaction of MDM2/MDMX and TAB1. Recent studies have shown that miR-134 affects the sensitivity of breast cancer cells to DOX by down-regulating the expression level of MRP proteinA clinical experiment was performed to investigate the aforementioned hypothesis. We demonstrated that MDM2 and TAB1 expression was associated with histological grading, and that MDM2/MDMX expression was associated with tumor size. Histological grading and tumor size are two variables that are closely related to prognosis. Thus we speculated that MDM2/MDMX/TAB1 expression might also correlate with patient outcomes. However, the lack of follow-up data was one of the limitations to our study. We only evaluated MDM2/MDMX and TAB1 expression by IHC and collected the clinicopathological characteristics of 70 breast cancer patients in order to explore the correlations between MDM2/MDMX and TAB1 expression levels. The follow-up studies of breast cancer patients that could be used to monitor their survival would help to elucidate the mechanisms and patient outcomes related to MDM2/MDMX inhibition. We searched The Cancer Genome Atlas (TCGA) but found no differences between MDM2/MDMX/TAB1 expression and breast cancer prognosis in their data, which may need to be tested in a larger sample.Immune checkpoint inhibitors have demonstrated desirable anticancer effects, and the response rates for single agent immune checkpoint inhibitors in solid malignancies range from 20% to 40%In conclusion, our results indicated that the MDM2/MDMX inhibitor reversed DOX resistance of human BC by activating the TAB1/TAK1/p38 MAPK pathway, suggesting that the MDM2/MDMX inhibitor in combination with chemotherapy may provide a novel therapeutic strategy for cancer treatment. However, a prospective randomized study of MDM2/MDMX inhibitor versus standard therapy would be required in order to lead to an unbiased conclusion of comparative progression rates between DOX resistance and MDM2/MDMX inhibition.


Several immune checkpoint molecules and immune targets in leukemic cells have been investigated. Recent studies have suggested the potential clinical benefits of immuno-oncology (IO) therapy against acute myeloid leukemia (AML), especially targeting CD33, CD123, and CLL-1, as well as immune checkpoint inhibitors -1 and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) antibodies) with or without conventional chemotherapy. Early-phase clinical trials of chimeric antigen receptor (CAR)-T or natural killer (NK) cells for relapsed/refractory AML showed complete remission (CR) or marked reduction of marrow blasts in a few enrolled patients. Bi-/tri-specific antibodies  and dual-affinity retargeting (DART)) exhibited 11–67% CR rates with 13–78% risk of cytokine-releasing syndrome (CRS). Conventional chemotherapy in combination with anti-PD-1/anti-CTLA4 antibody for relapsed/refractory AML showed 10–36% CR rates with 7–24 month-long median survival. The current advantages of IO therapy in the field of AML are summarized herein. However, although cancer vaccination should be included in the concept of IO therapy, it is not mentioned in this review because of the paucity of relevant evidence. Since the Food and Drug Administration (FDA) approved ipilimumab, the first-in-class anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody, for melanoma in March 2011 ,4. CurreIntensive chemotherapy  with or without hematopoietic stem cell transplantation (SCT) has been the mainstay of the curative treatment of acute myeloid leukemia (AML), presenting a dismal prognosis for relapsed/refractory or intolerable cases which mostly require palliative care. For CD33-positive AML, GO showed an overall response rate of 63% in relapsed/refractory cases , along wPDCD1 gene on chromosome 2q37.3. Along with its ligands PD-L1 and PD-L2, also known as CD274 and CD273, PD-1 plays an important role in maintaining self-tolerance [The inhibitory surface receptor programmed cell death 1 (PD-1) (UniProtKB-Q15116) on activated T cells is encoded by the olerance  and is oolerance ,13. An iolerance .An observational study at the MD Anderson Cancer Center analyzed bone marrow and peripheral blood specimens from 124 patients with myelodysplastic syndrome (MDS), chronic myelo-monocytic leukemia (CMML), and AML who received hypomethylating agents (HMAs) and reported that PD-1 and PD-L1 expression on CD34-positive cells were found in 7% and 20% of the patients, respectively . In 57% The cell surface receptor T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVcr-2), is encoded by the HAVCR2 gene on chromosome 5q33.3. TIM-3 is normally expressed on T-helper type 1 (Th1) lymphocytes, regulatory T cells (Treg), and natural killer (NK) cells. TIM-3 regulates macrophage activation , promoteTIM-3 was found in approximately 6% of the bone marrow T cells in newly diagnosed AML patients, and the proportion was larger (11.5–18.5%) in FLT3-ITD mutated cases . SimultaTIM-3 inhibitors are currently under early phase evaluation as monotherapies or in combination with PD-1/PD-L1 inhibitors for patients with advanced tumors. Preclinically, CAR-T therapy bispecific for CD13 and TIM-3 has been efficiently screened by a nanobody-based technology and shows eradication of leukemic cells in mouse models .The inhibitory receptor lymphocyte activation gene-3 protein (LAG-3), also known as CD223, is encoded by the LAG-3 gene on chromosome 12p13.31. LAG-3 is normally expressed on activated T cells, NK cells, and plasmacytoid dendritic cells (DCs) ,42. NotaCD47, encoded by the CD47 gene on chromosome 3q13.12, is a transmembrane glycoprotein and a ligand for signal-regulatory proteins (SIRPs). SIRPα is expressed on macrophages, DCs, myeloid cells, neurons, and astrocytes. In terms of macrophages and DCs, SIRPα-CD47 binding inhibits their phagocytic function through the cytoplasmic domain of SIRPα called immunoreceptor tyrosine-based inhibition motifs (ITIMs) by recruiting SH2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2 ,54. In oSimilar to the CD47 receptor SIRPα, the B- and T-lymphocyte attenuator  and CD200R  have cytoplasmic ITIMs which induce inhibitory signaling in immune effector cells ,62,63. CInterleukin-3 receptor subunit alpha (IL-3RA), also known as CD123, contains the alpha-subunit of the receptor for IL-3 and is encoded by the IL3RA gene on the X chromosome. Studies have indicated that IL-3RA is overexpressed in AML cells and other hematologic malignancies but is scant in normal hematopoietic stem cells ,71,72. ECD33 is a sialic-acid-binding immunoglobulin-like lectin expressed in monocytic/myeloid lineage cells and is encoded by the CD33 gene on chromosome 19q13.41. CD33 is expressed on myeloid cell lines from the progenitor to well-differentiated cells, including neutrophils, monocytes, and tissue-resident macrophages . CD33 shC-type lectin domain family 12 member A , also known as c-type lectin-like molecule-1 (CLL-1), is an ITIM-containing inhibitory transmembrane glycoprotein expressed on more than 80% of AML blasts as well as leukemic stem cells. The protein is encoded by the CLEC12A gene on chromosome 12p13.31. Although the function of CLL-1 is not fully understood, its involvement in homeostasis in certain inflammatory situations, such as monosodium urate-induced reaction and collagen antibody-induced arthritis has been suggested ,90. A prA proto-oncogene protein c-KIT, also known as CD117, is a type III receptor tyrosine kinase expressed in 80–90% of AML blasts and is related to adverse clinical outcome . A seconPublished trials of ICIs are listed in TIM-3 blockade can be another strategy for disarming the immune-escaping mechanisms of tumor cells. A phase Ib study testing anti-TIM-3 antibody (MBG453) in combination with decitabine for patients with high-risk MDS and AML reported that 9 of 31 (29%) patients with AML achieved partial response or better response and eight (25.8%) additional patients showed more than a 50% reduction in marrow blasts . Severe CAR-T cells are engineered peripheral T cells, often of autologous origin, which have an extracellular antigen-recognition domain, commonly single-chain variable fragments derived from monoclonal antibodies, conjugated with intracellular signal domains , and DARTs are made by cross-linking two Fvs . A CD33-CD33 has been recognized for decades as the most popular immune target of AML. An anti-CD33 ADC, GO, showed CR rates of 63% with 2-year overall survival of 41% in patients with relapsed/refractory CD33-positive AML . A meta-As knowledge of immune inhibitory molecules and leukemic antigens has accumulated, clinical use of IO therapy for AML has come closer to reality. While conventional chemotherapy, with or without SCT, is still pivotal in curative treatment and/or disease control for AML because of its high proliferation intensity, molecular targeted approaches  remain a practical alternative, especially for patients with relapsed/refractory or intolerable AML to intensive treatment. Clinical experience of ICIs in solid tumors has shown that IO therapy brings long-term disease control in at least 10–20% of the patients with metastatic cancers  even after discontinuation of chemotherapy. Furthermore, concomitant use of chemotherapy and/or radiation could enhance the efficacy of IO therapy ,137, alt