Together with other luminal Ca2+ buffer proteins, SAR plays a critical role in modulation of Ca2+ uptake and Ca2+ release during excitation–contraction coupling in muscle fibers. SAR appears to be important in a wide range of other physiological functions, such as Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) stabilization, Store-Operated-Calcium-Entry (SOCE) mechanisms, muscle fatigue resistance and muscle development. The function and structural features of SAR are very similar to those of calsequestrin (CSQ), the most abundant and well-characterized Ca2+ buffer protein of junctional SR. Despite the structural and functional similarity, very few targeted studies are available in the literature. The present review provides an overview of the role of SAR in skeletal muscle physiology, as well as of its possible involvement and dysfunction in muscle wasting disorders, in order to summarize the current knowledge on SAR and drive attention to this important but still underinvestigated/neglected protein.Sarcalumenin (SAR) is a luminal Ca In muscle physiology, the main resources of Ca2+ for proper functioning of the intracellular Ca2+ signals derive from two Ca2+ pools: Ca2+ sequestered in stores (endoplasmic/sarcoplasmic reticulum (ER/SR)) and extracellular Ca2+. The movements of contractile proteins require the correct intracellular level of Ca2+ ions to be released from the SR, a specialized membrane system and a component of the cellular reticular network. The presence of these pools guarantees the correct amount of calcium essential for the muscle to perform its pivotal function, i.e., the well-understood excitation–contraction (EC) coupling. EC coupling is a process mediated by mechanical coupling between the dihydropyridine receptor (DHPR) located on the transverse tubule membrane (invaginations of the plasma membrane) and ryanodine receptor type 1 (RyR1) on the SR membrane. Following conduction of impulses through motor neurons (neuromuscular transmission) and the activation of the nicotinic acetylcholine receptor at neuromuscular junctions, muscle membrane depolarization  induces a conformational change in the DHPR required for the RyR1 interaction. The latter opens to release Ca2+ ions from the Ca2+ SR. Subsequently, Ca2+ uptake in the SR via Ca2+-Mg2+ ATPase (SERCA) leads back to a resting level with the replenishment of the SR calcium content [2+ homeostasis in muscle cells and the correct Ca2+ sequestration in the SR are essential requirements for proper muscle contraction. During muscle contraction, resting intracellular calcium (nM range) increases up to the μM range [2+ released from the SR, this Ca2+ increase is obtained through processes involving different molecules and Ca2+ transport channels. Among these, it is worth mentioning the entry of extracellular calcium into the cell through store-operated calcium entry (SOCE) via Orai1 and/or the transient receptor potential canonical (TRPC) channels [2+ entry (ECCE). Calcium entry into the cell via SOCE is important for Ca2+ replenishment of the SR in order to maintain SR Ca2+ content for maximal muscle performance [2+ entry during muscle contraction [2+ buffer proteins residing in the cytoplasm  or in the lumen of the longitudinal and junctional SR of skeletal muscle are believed to regulate EC coupling, SOCE and ECCE processes by binding Ca2+ ions  or calmodulin-regulated  [2+ buffer protein expressed primarily in fast skeletal muscle fibers. It binds Mg2+ when the muscle is in a resting state and dissociates from it and binds Ca2+ ions after SR Ca2+ release, thereby contributing to muscle relaxation [Regucalcin  is a 34 kDa cytosolic multifunctional Ca and PKC . Calmodusticity) . Insteadlaxation .2+-buffer proteins residing in the SR, the most abundantly expressed and well-studied Ca2+ buffer located in the junctional SR is calsequestrin (CSQ), followed by the calsequestrin-like proteins CLP-150, CLP-170 and CLP-220, as well as HRC, junctate and calreticulin. The most abundant Ca2+ buffer protein located in the longitudinal SR is SAR [In regard to the CaR is SAR . To the The sarcoplasmic reticulum (SR) is a specialized membrane system and a component of the cellular reticular network of striated muscle cells that surrounds each myofibril. Two well-defined structural and functional SR regions can be distinguished , namely 2+ ATPases (SERCAs), which pump Ca2+ from the cytosol to the lumen of the SR [2+ movements in skeletal muscle. Therefore, in the triad, upon membrane depolarization, the DHPR channels undergo a conformational change necessary for RyR1 interaction and activation for Ca2+ release into the cytosol and for myofilament contraction. In contrast, junctional SR represents the region of the terminal membrane cisternae that faces the t-tubule/SR membrane [2+ released from the SR, such as Stim1, inositol-trisphosphate receptor (InsP3R) and mitsugumin 53 (MG53), are located. Furthermore, during muscle differentiation, the formation of triad junctions can be favored by mitsugumin 29 (MG29), a structural protein exclusively expressed in skeletal muscle and localized both on t-tubules and SR terminal cisternae [2+ transients [The longitudinal SR represents the largest part of the SR in which is located the SR Caf the SR ,22,23. If the SR . JPHs arf the SR  and DHPRf the SR , and regmembrane , where Risternae ,28. It h2+ is bound to high-capacity Ca2+ binding proteins within the terminal cisternae and longitudinal tubules [2+ buffer protein of junctional SR is CSQ, which belongs to the class of high-capacity and medium-affinity Ca2+ buffer proteins [2+ binding, CSQ monomers polymerize to form large polymers, increasing Ca2+ binding ability [2+, both calsequestrin-1 and 2 are important for regulation of Ca2+ release during muscle contraction, interacting with ryanodine receptors (RyRs) and contributing to the regulation of Ca2+ homeostasis in muscle cells [2+ stores [Luminal Ca tubules . The mosproteins ,32,33. Tproteins . Followi ability . In addile cells ,37,38. F+ stores ,40.2+ buffer protein is HRC, which has structural similarities to CSQ and, like CSQ, binds Ca2+ with high capacity and low affinity. It is located within the SR lumen as a multimer [2+ levels, it dissociates from pentamers to trimers and dimers and is less closely folded and more sensitive to trypsin digestion [2+ directly and could interact with triadin, mediating RyRs activity [Another minor SR Camultimer ,42, and igestion . HRC binactivity ,44. Alteactivity  and in tactivity ,46,47; oactivity .2+ buffer proteins, it is worth mentioning junctate protein, an integral SR membrane protein of 33 kDa. Due to the presence of a negative luminal C-terminal Ca2+ binding domain, junctate is able to bind approximately 21 mol Ca2+/mol protein [2+ capacity but low Ca2+ affinity. Instead, unlike CSQ and HRC, it has been shown that in HEK cells, junctate is associated with IP3 receptors and TRPC channels, contributing to the SOCE mechanism with its N-terminal domain [With respect to Ca protein . Thus, ll domain .2+ binding protein located in the lumen of the SR [2+ buffering, with an amino acid sequence very similar to that of CSQ [2+ concentrations; normally, it is globular, while Ca2+ binding causes a change to an α-helical mode [2+ buffer protein due to its high binding capacity (20–30 mol Ca2+/ mol protein) and as a molecular chaperone, participating in different folding processes of the protein sequence in association with calnexin. The overexpression of calreticulin leads to calcium accumulation in cellular deposits and influences the SOCE mechanism [Lastly, calreticulin is an ubiquitous 46 kDa Caf the SR . The strt of CSQ . Its concal mode ,53. Calrechanism ,55,56.2+-binding glycoprotein composed of 473 acidic amino acids with a molecular weight of 160 KDa (long isoform) first isolated from skeletal muscle by Leberer et al. in 1989  and moderate affinity (Kd 0.6 mM) [2+ buffering by acting in the release and uptake of Ca2+ and favoring the excitation–contraction–relaxation cycle [2+ ATPase located on the SR [2+-mobilizing machinery during EC coupling [2+ regulatory proteins that impact the SOCE mechanism. Thus, in addition to the Ca2+ buffer function, SAR can have different and context-dependent functions. In consideration of the limited findings reported on this topic to date, new investigations are needed to confirm and uncover molecular mechanisms underlying SAR function.Similarly to CSQ, SAR has a high capacity to bind calcium (35 mol Ca 0.6 mM) ,59. It ron cycle ,21,60. In the SR , suggestn the SR . The samn the SR ,62. It hcoupling . Lastly,2+ regulation in muscle cells works with precision. High levels of cytosolic Ca2+ are essential for muscle contraction onset, and the reuptake of Ca2+ ions is pivotal to trigger muscle relaxation. Defects in the proteins that make up this system and modifications of Ca2+ cycling often represent the main cause of neuromuscular pathologies. Importantly, it is necessary to stress that Ca2+ SR binding proteins are not simply ion traps that facilitate Ca2+ reuptake and increase luminal Ca2+ storage capacity but are also multifunctional SR proteins that act as endogenous regulators of Ca2+ SR channels and as luminal chaperones [2+ binding proteins may play an important role in altering the cyclic Ca2+ system in skeletal muscle diseases. Here, we discuss current knowledge of the involvement of altered SAR expression and activity in neuromuscular diseases such as Duchenne muscular dystrophy, sarcopenia and malignant hyperthermia.The elaborate mechanism of Caaperones . Thus, e2+ due to augmented extracellular Ca2+ entry could be an important initiating event in dystrophic muscle and could play a central role in the pathophysiological mechanisms leading to muscle weakness [2+ levels may contribute to a cycle of activated protease and Ca2+-leak channel activation [2+ concentration, the Ca2+ buffering capacity of the dystrophic SR is also significantly altered [2+ homeostasis and impaired luminal Ca2+ buffering are considered the major downstream effects of sarcolemmal rupture, eventually leading to muscle weakness and accelerating the protein degradation process in dystrophic muscles. All these results corroborate the fact that not only CSQ but also SAR can be considered an important luminal calcium buffer that may play a role in the dystrophic phenotype. Therefore, comprehension of the mechanisms underlying SAR alteration in dystrophic settings could potentially lead to the identification of novel therapeutic targets for DMD.Duchenne muscular dystrophy (DMD) is an X-linked lethal disease of childhood that affects approximately 1 in 5000 live births and represents the most frequent neuromuscular disorder in humans . DMD is weakness ,82,83,84tivation ,86. In a altered . In part altered ,88,89. I altered ,87. A co altered . Therefo2+-activated proteases/proteasomes and dysfunction of satellite cells have been proposed to be involved [2+ homeostasis can play a key role in sarcopenia and age-related muscle weakness [2+ supply to the contractile apparatus and to a reduction in contractile force [2+ buffer proteins of the longitudinal SR, in particular SAR protein, occur in sarcopenic skeletal muscle [2+ release mechanisms, ultimately contributing to a significant decline in contractile force and related muscle function during normal aging [2+ uptake into the SR lumen and altered Ca2+ handling properties [2+ handling related to SAR reduction may contribute to the multifactorial etiology of sarcopenia and could be directly involved in contractile weakness. Unfortunately, very few studies focusing on the evaluation of the role of SAR in sarcopenia are currently available in the literature, and new evidence is needed to confirm preliminary results and to gain more insights.Sarcopenia is an age-related condition characterized by the presence of muscle atrophy and a progressive and generalized decline in muscle strength , leadinginvolved ,93. Furtweakness ,95,96,97le force ,98. Sevel muscle ,100,101.al aging . It has al aging . Therefooperties , with thoperties . Taking 2+ [2 production, hyperkalemia, muscle rigidity and rhabdomyolysis. Mutations in the RyR1 and CACNA1S genes encoding the RyR1 isoform and DHPR, respectively, have been associated with MH [2+ levels during muscle excitation. These defects favor the onset of the typical signs of MH, i.e., glycogenolysis, ATP depletion, mitochondrial oxidation, lactic acid production, electrolyte imbalance and muscle damage [2+ levels in the skeletal muscles of MH patients [2+ buffer proteins in MH pathology are available in the literature, and almost all are focused on the role of CSQ. To date, no differences in the expression of SR luminal Ca2+ binding proteins have been found for either CSQ or SAR levels [2+ release was shown, and considering that RyR1 is CSQ-interdependent, this event favors RyR1 opening in MH muscle [Malignant hyperthermia (MH) is a potentially fatal inherited severe myopathy characterized by a fulminant hypermetabolic state to inhalational anesthetics used during invasive procedures in predisposed individuals . Symptom2+  and incl with MH . Several with MH , which ie damage . In addipatients . Furtherpatients . Few stuR levels . HoweverH muscle . InteresH muscle  and CSQ H muscle  show sym2+ [2+ transport activity into the SR, even when the heart is subjected to stress. Specifically, biomechanical stress, such as pressure overload, was shown to promote progressive heart failure in this SAR-deficient mouse model, suggesting that cardiac SAR expression is essential for heart adaptation to this stress [Sarcalumenin is also expressed in the cardiac SR and exerts the same functions described for skeletal muscle, i.e., the regulation of intracellular Ca2+ . Importa2+ . Further2+ . Studiess stress . Likewiss stress . This ev2+ buffer protein located in the longitudinal part of SR muscle, dedicated studies have focused mainly on CSQ, likely due to its higher expression in SR. However, physiologically, SAR represents the major non-junctional SR protein responsible for Ca2+ buffering to support and regulate the muscle excitation–contraction cycle. In addition to this function, new roles of SAR have been recently revealed, with multiple context-dependent functions important for the stability of the SR membrane network. Importantly, even small changes in the expression levels of SAR may play an important role in altering the cyclic Ca2+ system in skeletal muscle diseases, corroborating the idea of the potential role of SAR as a pharmacological target. It is not clear whether and how the different Ca2+ buffer proteins interact with each other or whether protein–protein interactions can occur. Furthermore, the lack of SAR modulators represents a strong limitation. Our current understanding of the involvement of SAR and other Ca2+ buffer proteins in skeletal muscle diseases suggests that there may be a relationship between SAR and other SR Ca2+ buffer proteins through an adaptive or compensatory response of cells to the alteration of the expression of specific Ca2+ buffer proteins by enhancing or inhibiting other Ca2+ buffer proteins in a complementary manner. For this reason, it is important to characterize not only CSQ but also SAR, which continues to be underestimated. Once a complete map of the real role and involvement of the two main Ca2+ buffer proteins, SAR and CSQ (and others), in various pathophysiological conditions has been established, it may be possible to compose a three-dimensional map of each possible protein interaction and fully understand their complexity and involvement in skeletal muscle diseases. Furthermore, studying the interactions between Ca2+ buffer proteins and other proteins could pave the way for the identification of ligands and their relative binding sites.Although SAR was discovered 15 years ago as a CaThe number of patients with skeletal muscle diseases continues to increase, and improved understanding of their underlying mechanisms is fundamental for the development of new therapies. Investigation of the roles of SAR in skeletal muscle diseases could offer a great opportunity to meet currently unmet needs.


KMT2Ar (KMT2A/11q23 rearrangement) AML, normal karyotype AML with somatic mutations, unbalanced cytogenetic abnormalities AML, NUP98 11p15/NUP09 rearrangement AML, and acute promyelocytic leukemia (APL). Current risk group classification, the management algorithm in childhood AML, and novel treatment modalities such as targeted therapy, immune therapy, and chimeric antigen receptor (CAR) T-cell therapy are reviewed. Finally, the indications of hematopoietic stem cell transplantation (HSCT) in AML are discussed.Acute myeloid leukemia (AML) is the second most common hematologic malignancy in children. The incidence of childhood AML is much lower than acute lymphoblastic leukemia (ALL), which makes childhood AML a rare disease in children. The role of genetic abnormalities in AML classification, management, and prognosis prediction is much more important than before. Disease classifications and risk group classifications, such as the WHO classification, the international consensus classification (ICC), and the European LeukemiaNet (ELN) classification, were revised in 2022. The application of the new information in childhood AML will be upcoming in the next few years. The frequency of each genetic abnormality in adult and childhood AML is different; therefore, in this review, we emphasize well-known genetic subtypes in childhood AML, including core-binding factor AML (CBF AML),  Acute leukemia (AL) stands as the most common form of cancer observed among children. Among childhood leukemias, the majority (around 80%) are acute lymphoblastic leukemia (ALL), while a smaller proportion 15–20%) are diagnosed as acute myeloid leukemia (AML) 5–20% are, its proProgress in understanding AML at the genetic and molecular levels has led to a better grasp of patient risk and categorization. The WHO classification of myeloid neoplasms and the International Consensus Classification (ICC) of Myeloid Neoplasms and Acute Leukemias were both revised in 2022 ,6. Also,Despite the integration of findings from adult AML studies into the pediatric context, disparities between childhood and adult AML still persist. Variations in how treatment regimens are tolerated underscore the need for meticulous consideration and tailoring of treatment strategies for childhood AML ,11. In tRUNX1-RUNX1T1 and CBFβ-MYH11, respectively, which disrupt the CBF complex (consisting of RUNX1-3 + CBFβ), crucial for hematopoiesis. These fusion genes serve as indicators to assess minimal residual disease (MRD) [RAS-KIT and FLT3 are commonly observed molecular alterations in CBF AML [KIT mutations experienced bettered disease survival rates through the incorporation of dasatinib [The largest subgroup within pediatric AML is core-binding factor AML (CBF AML), comprising around 25% of cases, typically affecting individuals with a median age of 8–9 years . These gse (MRD) . Notablyse (MRD) . While tse (MRD) . Howeverse (MRD) . The sec CBF AML . The the CBF AML ,17. Over CBF AML . Additioasatinib . Combiniasatinib . Individasatinib .KMT2A, situated on chromosome 11q23, is a pivotal player in normal hematopoietic development. Mutations within KMT2A constitute 16–20% of pediatric AML cases. Applying the FAB classification, approximately 73% of KMT2ArAML falls under the categories of AML M4 or M5. Importantly, KMT2Ar AML is more common in infants, with a prevalence of 47–55% observed in children aged below 2 years [KMT2Ar mutations is generally unfavorable [KMT2Ar AML enrolled in the 2016 WHO classification is AML characterized by t KMT2A-MLLT3 [KMT2A-MLLT3”, because over 80 KMT2A fusion partners have been described, with MLLT3, AFDN, ELL, and MLLT10 being most common. The fusion partner identified could provide prognostic information, which may have an impact on monitoring disease [ELL (19p13.1) or MLLT1 (ENL) (19p13.3) partners, contributes to 8% and 6% of KMT2Ar cases, respectively, and carries intermediate prognosis [KMT2A-MLLT11(AF1Q), shows promise with a favorable prognosis, yet its rarity prevents conclusive assessment. On the other hand, gene fusions associated with unfavorable prognoses encompass cases with t/KMT2A-ABI1, t/KMT2A-AF10, and t/KMT2A-MLLT4, constituting 2%, 16%, and 6% of KMT2Ar instances, respectively [KMT2A fusions is not certain in childhood AML, but t/KMT2A-MLLT2, t/KMT2A-MLLT4, t/KMT2A-AF10, and t/KMT2A-ABI1 were identified with a poor prognosis in most of the literature [MLLT3, ELL, MLLT10 (10p12), and ABI1 (10p11.2) are often linked to infant AML instances. In contrast, MLLT4 (6q27) is more commonly found in older children, with a median age of 12 years [KMT2A-r AML [KMT2Ar AML [ 2 years . The proavorable . The fir disease . The t, mixed-lineage leukemia gene-partial tandem duplications (MLLPTD), or nucleophosmin (NPM1) mutations [FLT3-ITD mutations, present in 20–25% of childhood AML cases with an otherwise regular karyotype, stand as a crucial marker for poor prognosis [MLL-PTD is another mutation linked to poorer prognosis, leading to lowered overall survival (OS) and event-free survival (EFS) in pediatric AML [NPM1 mutations with FLT3-ITD is correlated with a positive prognosis, whereas WT1 mutations alongside FLT3-ITD exacerbate the prognosis [Approximately 22–26% of childhood AML cases exhibit normal karyotypes. These patients are categorized into an intermediate-risk group, displaying varying prognoses. Notably, the proportion of AML cases with a normal cytogenetic profile in children 15–30%) is smaller compared to adults (40–47%) [% is smalutations ,32. Amonrognosis ,33. MLL-tric AML . Converstric AML . Notablyrognosis .NUP98-NSD1 and the infrequent t/RUNX1-USP42. This underscores the significance of comprehensive cytogenetic and molecular assessments for such cases [FLT3-ITD mutations [About 40% of childhood AML cases involve unbalanced cytogenetic abnormalities, with notable prognostic implications like monosomy 5, del(5q), and monosomy 7. However, despite their poor prognostic influence, these abnormalities are detected in just 5% of cases . Monosomch cases . Trisomyutations .Nucleoporin 11p15/98Kd (NUP98) rearrangements are infrequent, occurring in approximately 3–5% of childhood AML cases and occasionally in young adults. Several different partners for NUP98 have been identified, with the most prevalent being the nuclear receptor-binding SET domain protein 1 (NSD1) gene (5q35), present in around 75% of pediatric NUP98r patients. In terms of molecular aspects, CDK6 emerges as a prominently expressed target directly regulated by NUP98-fusion proteins. Intervening with CDK6 activity leads to cell cycle arrest, myeloid differentiation, and apoptosis both in vitro and in vivo. This prompts the exploration of CDK6 suppression as a potential tactic to address NUP98 fusion-associated AML . AdditioPML-RARA rearrangement [PML-RARA reciprocal translocation, there are instances of other translocations involving the RARA gene and other genes apart from PML. The presence of PML-RARA can be determined through techniques like RQ-PCR , FISH (fluorescence in situ hybridization), and chromosome analysis [Acute promyelocytic leukemia (APL) is an extensively studied variant of AML and is identified by the translocation t, which leads to the generation of the angement . APL is angement . The mutangement . While aanalysis .DNMT3A, TET2, IDH1/2 , and UP98, WT1, RUNX1, MLLT10, SPECC1, and KMT2A [MLLT3-KMT2A and t/RBM15-MKL1, was eliminated from the list  to achieve complete remission. However, this approach comes with significant drawbacks such as a heightened risk of infection and cardiac dysfunction. Furthermore, primary chemotherapy faces substantial resistance across various AML subtypes, including nd KMT2A . AML casnd KMT2A . Rubnitzthe list . NPM1 mu version . The 202te group .KMT2A rearrangements, FLT3-ITD abnormalities, and single-nucleotide polymorphisms in ABCB1 could benefit from GO treatment [Gemtuzumab ozogamicin (GO) is a CD33 targeting monoclonal antibody attached to a toxin. Studies indicate that using GO in induction therapy can reduce relapse risk, though upfront use was linked to increased treatment-related mortality . Althougreatment ,49.CD123 (the IL-3 receptor α-chain), present in many AML cases including leukemic stem cells, is a promising target for therapy. While still in early phases, CD123-targeted treatments offer a potential avenue for treatment .FLT3. Type I inhibitors, in addition to being active against tyrosine kinase domain (TKD) mutations, possess a higher binding affinity for FLT3 [FLT3 mutation status. Nonetheless, this combination also raised concerns regarding increased toxicity [FLT3/ITD+ AML cases [FLT3 mutation status [FLT3 mutations [FLT3 inhibitors are a class of molecules that attach to the ATP-binding site on FLT3, resulting in competitive inhibition of kinase activity . These ifor FLT3 . While afor FLT3 . Sorafentoxicity ,53. SoraML cases . MoreoveML cases . Midostan status . The extutations ,57.FLT3-mutated AML [Gilteritinib, a second-generation type I FLT3 inhibitor, exerts inhibition on AXL, a tyrosine kinase that augments FLT3 activation and contributes to FLT3 inhibitor resistance. Although research involving pediatric patients is currently limited, forthcoming trials are going to explore the application of gilteritinib combined with chemotherapy in FLT3-mutated AML, in both relapse and upfront settings . Notablyated AML .BCL-2 inhibitors are significant in AML treatment. One such inhibitor, venetoclax, targets the antiapoptotic protein BCL-2 abundant in AML cells. There exists evidence indicating that the amalgamation of venetoclax and azacytidine might offer a secure and encouraging treatment alternative for pediatric patients . In a phThe phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway assumes a central role in regulating cell proliferation, growth, and survival under normal physiological circumstances. This pathway, along with other significant signals like RAS-MAPK-ERK and JAK2-STAT5, serves as a downstream response to various tyrosine kinases, such as FLT3 and ABL1. Amplification of the PI3K-AKT-mTOR pathway is evident in a notable proportion, roughly around 60%, of individuals with AML . In the KMT2A rearrangements (KMT2Ar) exhibits heightened expressions of HOXA9 and MEIS1 genes. These elevated levels arise from the interaction between oncogenic KMT2A fusion proteins and complex-forming counterparts like LEDGF, DOT1L, and menin. Recent efforts have focused on strategies to disrupt this link between KMT2A protein and menin [KMT2A rearrangements or NPM1 mutations [AML characterized by nd menin . Notablyutations .CD47, a cell surface protein that is widely distributed, has a pivotal role in regulating phagocytosis via innate immune cells like macrophages and dendritic cells. This modulation takes place via the engagement of CD47 with SIRP-alpha, a receptor found on these immune cells. This interaction imparts a suppressive signal that hinders phagocytosis . As suchKMT2A-mutated infant AML [CAR-T cell therapy faces challenges in AML due to the absence of a unique cancer surface marker. However, two cell markers, CD33 and CD123, are especially common on AML cells, with CD33 detected in 90% of instances, and CD123 found in 75% of AML cases. Notably, only a minimal proportion (<5%) lacks both . While Cfant AML .Checkpoint inhibitors such as pembrolizumab and nivolumab, which target PD-1, along with ipilimumab targeting CTLA-4, emerge as promising therapy options. Clinical trials are investigating the synergism between the above mentioned PD-1 inhibitors and anti-PD-L1 antibodies including atezolizumab and durvalumab ,74.Epigenetic therapies encompass hypomethylating agents (HMAs) like decitabine and azacitidine, which inhibit DNA methyltransferases, inducing tumor suppressor expression and apoptosis ,76. St. Histone deacetylase (HDAC) inhibitors like vorinostat, panobinostat, tricostatin regulate chromatin by removing acetyl groups from histones, impacting gene transcription, and promoting apoptosis. Synergy with standard chemotherapy is evident . PanobinTP53-mutated cancer cells by restoring wild-type conformation and specific DNA binding of mutant p53 [The TP53 protein plays a crucial role as a tumor suppressor by regulating cell division and apoptosis. TP53 mutations exist in around 30–40% of secondary AML cases and less than 10% of normal AML cases . There atant p53 ,84. A cltant p53 . APR-548CPX-351 involves a liposomal encapsulation of cytarabine and daunorubicin, combined in a synergistic 5:1 drug ratio. In older patients aged 60 to 75 years with newly diagnosed secondary AML, CPX-351 treatment is associated with significantly longer survival compared to conventional 7 + 3 regimen . In pediAtovaquone, an anti-infective agent, is typically administered to prevent and to treat pneumonia, particularly pneumocystis jiroveci pneumonia (PJP), a common concern in AML affected children. Atovaquone also hampers oxidative phosphorylation and thereby helps combat AML in patient-derived xenograft mouse models ,89.ATRA  combined with ATO (arsenic trioxide) and induction chemotherapy represents the current treatment approach for APL, resulting in significantly improved outcomes compared to previous years . This coATRA and ATO additions to initial APL therapy have caused relapsed patients to become insensitive to such treatment. For relapse treatment, options include gemtuzumab ozogamicin (GO), a monoclonal antibody–drug conjugate targeting CD33-expressing cells, which has demonstrated effectiveness in achieving molecular remissions in both newly diagnosed and relapsed patients . AnotherHSCT guidelines emphasize recipient characteristics, donor selection, and preparatory regimen. Such transplantation aims to decrease relapse rates, consolidate graft-versus-leukemia (GVL) effects, and minimize graft-versus-host disease. If unfavorable cytogenetic properties are observed or response to induction therapy is limited, HSCT will be commonly pursued in the first complete remission (CR1) to intensify treatment due to previously suboptimal results with chemotherapy alone ,95.FLT3/ITD, it is recommended that patients proceed to HSCT in CR1, as studies suggest this approach improves outcomes. Co-existing favorable risk mutations, such as NPM1, have been found to mitigate the poor prognostic impact of FLT3/ITD [FLT3/ITD patients [In situations involving FLT3/ITD ,97. Thispatients .For cases involving high-risk KMT2Ar, t chromosomal rearrangement, monosomies of 7 and 5 (or del 5q), abnormalities of 3q and 12q, complex karyotype, NUP98-NSD1 fusion, t, and cryptic chromosome 16 inv(16)(p13.3q24.3), the panel advises HSCT in CR1 for patients with minimal residual disease (MRD) at the end of induction (EOI) stage. If primary induction failure (PIF) persists beyond the second EOI, HSCT should be considered after a third chemotherapy cycle, as achieving complete remission (CR) with further chemotherapy becomes unlikely and risks of toxicity increase. However, factors like emerging therapeutic strategies and disease progression should also guide HSCT timing decisions. Children with therapy-related AML (tAML) are prone to relapse and respond poorly to multiple cycles of chemotherapy due to prior exposure. Early HSCT consideration, generally following 1 or 2 chemotherapy cycles to elicit remission, is advised for these cases .The treatment of pediatric AML still poses many challenges. Our understanding of the genetics and molecular characterization of AML has led to a greater understanding of this disease with better patient risk and categorization. Novel treatment approaches with new therapeutics are also in clinical trials and show promising results. We hope that these new therapies can soon lead to less toxic, more effective and targeted treatment therapies that can help pediatric patients especially with resistant or refractory disease.


The interactions between Acute Myeloid Leukaemia (AML) cells and the surrounding bone marrow (BM) tissue contribute to blocking the efficacy of current drug treatments and to the relapse of patients. Relapsing AML tumours are refractory to current therapies and remain untreatable. Developing new therapies for AML requires the development of new drug-screening methods using in vitro models that closely mimic the interactions of AML cells with cytoprotective BM cells. We have developed a new fluorescence-based in vitro model and an analytical method that takes into consideration the reciprocal interactions between AML cells and the protective BM stroma during drug treatments. Using this new method and combining it with bioinformatics, we have identified new combinations of drugs that may overcome resistance to drug treatments and lead to improved therapies for AML.The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment. Although 70–80% of Acute Myeloid Leukaemia (AML) patients undergo complete remission after chemotherapy ,2 long-tCurrently, treatments for AML patients fit for intensive or low-intensive chemotherapy regimens are frequently based on the combination of daunorubicin and cytarabine ,5,6. TheGiven the emerging key role of the BM microenvironment in AML, the identification of new therapeutic interventions to overcome cell adhesion-mediated drug resistance (CAM-DR) requires the development of in vitro co-culture models that recreate the in vivo BM setting for studies of the biology of AML, as well as for drug screening . DespiteWe have previously devised a fluorescence-based experimental model for drug screening against multiple myeloma (MM) that is scalable to high throughput. This model provides a significant improvement on previous methodologies  by allowWe now report a new fluorescence-based in vitro co-culture system-based high throughput analysis for the assessment of drug efficacy against AML. This AML experimental model is based on the same principles as our MM method. For AML, we tested the interactions of eGFP–AML-expressing cells with the BM mesenchymal fibroblastic cell line mCherry-HS5 and primary osteoblasts, as both BM cell types have been shown to provide cytoprotection against drug treatments in AML ,22,23.®, nexpovio®), an inhibitor of the nuclear export receptor CRM1/XPO1. By combining in silico analysis using transcriptomics data of AML patients with in vitro testing using our new AML experimental model, we also identified the potentially efficacious combination of KPT-330 with the AURKA pharmacological inhibitor alisertib to prevent CAM-DR. Our data warrant further investigation of these identified drug combinations in order to improve the efficacy of AML therapy.Using our new platform, we found distinctive patterns of interaction between AML and BM mesenchymal/fibroblastic stromal cells that differ from the interactions of MM cells with the same BM niche. We also performed a drug screening and identified some pharmacological candidates that overcome BM-mediated resistance to daunorubicin, including KPT-330 . Primary Human osteoblasts were obtained from Lonza Ltd. and were grown using the OGM™ Osteoblast Growth Mediu BulletKit™.The Human AML cell lines eGFP-MOLM-14, eGFP-MV4-11, eGFP-Kasumi, and eGFP-THP-1 were generated using lentiviral vectors, as previously described . The BM 3 cells per well in 96 well plates and incubated overnight in DMEM supplemented with 10% FBS. The following day, the culture media was aspirated and eGFP–AML cells were layered on mCherry-HS5 cells at a density of 2 × 105 cells/mL in 200 μL per well of RPMI supplemented with 10% FBS. Three technical replicas were seeded per experimental condition. The library of compounds tested in our high throughput studies was Library I-384 from Merck. To determine the proliferation index, the fluorescence intensity (FI) per well was read at λex488 nm/λem528 nm and at λex584 nm/λem607 nm to estimate the numbers of eGFP-AML cells and mCherry-HS5 cells, respectively, using a FLx800 multidetection microplate reader . Measurements were taken at d0 and d3 and the proliferation index was calculated as the ratio of the fluorescence emission at d3/d0 after subtracting the background emission. Each experiment was repeated three times. Quest Graph™ EC50 Calculator, AAT Bioquest, Inc., https://www.aatbio.com/tools/ec50-calculator (accessed on 15 February 2023) was used to calculate the half maximal inhibitory concentration (IC50) and the half maximal efficacious concentration (EC50) values.mCherry-HS5 BM fibroblastic stromal cells were seeded at 10 × 10The percentage of viable and apoptotic cells was determined using flow cytometry. Cell cultures were harvested and Annexin-V-APC and propidium iodide staining in the eGFP positive population (AML cells) was measured by flow cytometry using a BD FACSCanto II flow cytometer  equipped with a High Throughput Sampler.The percentage of cells in the different phases of the cell cycle was determined according to the DNA content using the propidium iodide staining detected by FACS as a readout. Cells seeded in 96 well plates were fixed with cold 70% ethanol for 30 min. Cells were washed twice with PBS and then resuspended in 50 μL of PBS with propidium iodide (50 μg/mL) and RNase (100 μg/mL) and incubated in the dark for 30 min. The levels of propidium iodide staining were detected using a BD FACSCanto II flow cytometer .XPO1 (the gene encoding CRM1) with the 135 genes collected in the CGA TARGET (Tumour Alterations Relevant for GEnomics-driven Therapy) database of the Broad Institute (https://software.broadinstitute.org/cancer/cga/target (accessed on 11 January 2019)) (herein referred to as TARGET genes) was performed using two previously described bioinformatics tools. CANCERTOOL [XPO1/TARGET gene pair in each tumor type analyzed, and the mean R value across the five tumor types was calculated and used as a criterion to select XPO1-co-expressed genes. Significant co-expression correlation values (p < 0.05) higher than 0.2 or lower than −0.2 were considered to calculate an average correlation value for the five types of cancer for each gene from the CGA TARGET database.The potential co-expression of NCERTOOL  was usedNCERTOOL ,26 was ut-test was determined from p < 0.05. The interaction of pairs of pharmacological compounds under study was analyzed using Calcusyn software  based on the Chou–Talalay method [For statistical analysis using the adequate tests, GraphPad Prism 9 software was used. Statistically significant difference using ANOVA or Student y method . Data frMethods such as the MTT assay, commonly used to determine the numbers of cells in the culture, are unable to differentiate the presence of stromal and AML cells when in co-culture. Previous AML studies have used luciferase-expressing AML cells to distinguish the proliferative response of AML in co-culture with BM stromal cells . HoweverThe AML cell lines MOLM-14, MV4-11, Kasumi, and THP-1 were chosen for assessment in our model as they represent some of the most common genetic and cytogenetic abnormalities observed in AML patients, including those poor prognostic subtypes carrying FLT3 internal tandem duplication  and MLL-The presence of mCherry-HS5 cells in co-culture did not alter the levels of expression of eGFP in eGFP–AML-viable cells . This sh3 mCherry-HS5 cells/well) [4 cells/well or above  . Using tor above A,C,E.3 cells/well). However, when seeded at a density above 2 × 104 cells/well, the co-culture with increasing numbers of mCherry-HS5 cells correlated with an inhibition of the proliferation of eGFP-MV4-11 cells (4 cells/well) was their expansion reduced by the presence of mCherry-HS5 cells  AML cells versus cells cultured alone A. The trOverall, these results show that our fluorescence-based experimental model provides insight for a better understanding of the behaviour of AML cells and BM mesenchymal/fibroblastic cells and the concomitant impact of drugs on both cell populations.In MM, the presence of mCherry-HS5 cells provided a cytoprotective microenvironment against therapeutic drugs by promoting both the survival and proliferation of MM cells . Our resThe Library I-384 from the Merck library of compounds, which includes inhibitors of a variety of pathways involved in cell survival and cell adhesion, was selected for screening. We tested the potential of these compounds to revert the cytoprotection of AML cells mediated by BM stromal cells, as well as their ability to inhibit the proliferation of BM cells. A compound was selected as a positive hit when it fulfilled the following two criteria: (a) It reverted the mCherry-HS5 cells-mediated cytoprotection by increasing the percentage of apoptosis of eGFP–AML cells in co-culture with mCherry-HS5 cells by at least 25% with respect to this heterotypic culture treated with daunorubicin as a single agent; and (b) It inhibited the proliferation of mCherry-HS5 cells by at least 10% with respect to daunorubicin as a single agent . Our datTo identify possible common pathways involved in BM-mediated drug resistance to daunorubicin in AML cells independently of their genetic background, we extended the analysis of the selected hits to a wider panel of eGFP-expressing AML cell lines . As negative controls, we used compounds that were not selected as hits in our initial screening. Eleven compounds  showed enhanced killing of daunorubicin-treated AML cells in all the cell lines tested . These iWe then tested the efficacy of the selected hit compounds to overcome the possible cytoprotection of AML cells mediated by osteoblasts. Osteoblasts comprise a critical haematopoietic BM niche that supports the survival of normal haematopoietic stem cells and long-term haematopoietic progenitors, as well as pre-leukaemic stem cells . TherefoFinally, we investigated a possible synergistic pro-apoptotic effect of daunorubicin in combination with inhibitors targeting the identified pathways. In the study, we used the inhibitor compounds selected in the high throughput drug screening to overcome cytoprotection mediated by mCherry-HS5 cells and osteoblasts, as well as several clinical inhibitors of the same pathways currently used as therapies or under investigation in clinical trials against various types of cancers. In eGFP–MOLM-14 cells cultured alone, we found some synergistic effect with the combination of daunorubicin and cytarabine . HoweverTaken together, our data suggest that the inhibition of the pathways regulated by PKC kinase, CDK1 to 7, and CRM1/XPO1 are critical to overcome BM-mediated drug resistance of AML cells against daunorubicin in both the mesenchymal/fibroblastic and osteoblast niches.The efficacy of daunorubicin in combination with the CRM1 inhibitor KPT-330 detected in our drug screening has been further validated by recently published preclinical and clinical studies. KPT-330 has been shown to increase in vitro and in vivo the AML-killing efficacy of daunorubicin and other anthracyclins by restoring the nuclear localization of topoisomerase IIα and by downregulating the expression of DNA damage repair genes in AML cells . These rXPO1, the gene coding for CRM1 in various solid tumours and AML patients. Specifically, we analyzed the correlation between XPO1 expression and the expression of a set of 135 cancer-related genes included in the CGA TARGET (Tumour Alterations Relevant for GEnomics-driven Therapy) database of the Broad Institute (https://software.broadinstitute.org/cancer/cga/target (accessed on 19 March 2023)). This database contains genes (hereafter referred to as TARGET genes) with diagnostic, prognostic, or predictive utility whose alteration in cancer is directly linked to a clinical action. Correlation analyses were carried out using publicly available datasets from several cancer types, summarized in An overexpression of CRM1 in AML patients inversely correlates with overall survival, constituting a predictive factor for poor prognosis . CRM1 alXPO1 (p ≤ 0.05) with Pearson’s correlation indexes R > 0.2 or R < −0.2  whose expression positively correlated with XPO1 expression in all the tumour types analyzed  regulate the DNA damage response, while EZH2 encodes for the protein enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that plays an important role in global transcriptional regulation [For each type of analyzed tumour type, we selected TARGET genes showing a statistically significant correlation with R < −0.2 . We thenR < −0.2  and idenanalyzed  that wergulation ,70. The gulation ,72,73,74®, EPZ-6438, E-7438) has recently received FDA approval for specific subsets of sarcoma and lymphoma patients ; https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-tazemetostat-advanced-epithelioid-sarcoma (accessed on 2 December 2020); https://www.fda.gov/drugs/fda-granted-accelerated-approval-tazemetostat-follicular-lymphoma (accessed on 2 December 2020).At present, there are no reliable pharmacological inhibitors available to target MSH2, MSH6, BRCA1, BRCA2, or NPM1. On the other hand, there is mounting evidence of AURKA (Aurora Kinase A) and EZH2 proteins as possible targets for cancer therapy in a variety of cancers ,80,81,82Treatment of eGFP–MV4-11 cells with concentrations of alisertib achievable in patients  inhibiteIn contrast, when eGFP–MV4-11 were treated with doses of tazemetostat detectable in plasma from patients , cells cTaken together, our data show increased efficacy of the combination of KPT-330 and alisertib to overcome the cytoprotection by BM stromal cells in comparison to the single agents. However, our results indicate that tazemetostat does not increase the efficacy of KPT-330 and, in fact, the combination with tazemetostat may even exert a detrimental effect on the efficacy of KPT-330.Numerous studies validate the role of the BM microenvironment in oncogenesis ,86 and tThe BM-derived stromal cell line HS5 largely reproduces critical patterns of activation of BM mesenchymal/fibroblastic stromal cells that contribute to the expansion and survival of malignant clones in different types of haematological malignancies ,19,20,88We then proceeded to use our model for screening of possible compounds that may block the BM stromal-mediated drug resistance of AML cells against daunorubicin, which has been shown to be mimicked by HS5 cells . Our iniTo devise possible strategies to overcome the mechanisms of resistance to daunorubicin, a drug library was screened and compounds were selected by their capacity to block the pro-survival cues of BM stromal cells so that apoptosis was restored to the levels observed in AML cells grown in monoculture, as well as for their ability to reduce the proliferation of HS5 cells in co-culture with AML cells. We have previously shown that a similar strategy works to identify drug combinations to overcome BM-mediated drug resistance in MM . The ideOne such signaling node identified in our screening was the pathway regulated by the CRM1 protein, as its specific inhibitor, KPT-330, was a hit compound in our study. CRM1 is a specialized nuclear export receptor that mediates the translocation of hundreds of cargo proteins bearing nuclear export signals to the cytoplasm (reviewed in ). The acXPO1 (gene coding for CRM1) with 135 cancer-related genes included in the CGA TARGET database of the Broad Institute in various types of solid tumours and AML. We reasoned that the co-expression of cancer-related genes and XPO1 may imply a possible cooperation to promote cancer cell progression and possibly drug resistance. Two of the eight genes identified in this in silico analysis (AURKA and EZH2) were targets for specific pharmacological inhibitors: the AURKA inhibitor alisertib under testing in clinical trials [Importantly, there is recent evidence indicating that the AML therapies based on the combination of KPT-330 with daunorubicin may not be as effective in populations of elderly AML patients . This prl trials  and the l trials . FurtherIn summary, we have devised a new fluorescence-based experimental model and sequence of procedures to effectively identify new therapeutic approaches for AML. Our method mimics essential reciprocal interactions between AML and cytoprotective BM stromal cells involved in the final response to therapeutic agents. The data obtained suggest that the combination of the CRM1 inhibitor KPT-330 with the AURKA inhibitor alisertib may overcome BM-mediated drug resistance in AML. These data warrant further in vitro and in vivo studies to investigate the possible efficacy of this drug combination against AML.


Introduction: Mentoring programs minimize stress and anxiety in recent graduates and in newly recruited nurses, guiding their careers and enabling them to retain their skills and correctly care for patients. The objective of this scoping review is to explore and summarize the existing literature on mentoring models and programs in the clinical nursing context. Methods: The databases searched include PubMed, Embase, Cochrane Library, Epistemonikos, Cuiden, Scielo, MEDES, OpenGrey, Trove and MedNar. Published and unpublished studies worldwide that included nurse mentoring programs in a clinical context, in public and private systems and primary, secondary and tertiary healthcare settings, and articles published in English, French, Spanish and Portuguese, were included. Nurse students and training specialists were excluded. The papers were screened by two independent reviewers. In cases of discrepancy, a third reviewer made the decision. Results: Eleven studies were included. Most of them were conducted in the USA. A wide range of nurse mentoring programs were identified with highly variable characteristics. The duration of the programs and the evaluation systems were different, but the expected results matched. Conclusions: Mentoring programs need more in-depth and extensive study. In spite of their differences, they all lead to improvements for nurses, patients and organizations. A gender influence was found in our results, which could be studied in future research. Mentoring is defined as “Intentional mentoring relationships within the setting of a structured program that is bound by a time frame and defined objectives” . It refeThe first reference to mentoring in the literature was in Greece, but the first reference to specifically nurse mentoring was in England in 1960 . When diA mentor is a professional with important experience and knowledge who assumes the responsibility of guiding, advising, teaching and helping others to learn competency, improve their professional expertise and favor leadership. These attributes are not only about labor requirements, are about personal requirements too. Mentees’ growth requires mentors to improve their trust; motivate them; and use knowledge and reflection (as important tools of learning) to teach them important strategies to resolve problems, make decisions and develop good organization capacity, always in a continuous process of evaluation, while remaining unprejudiced and being a supportive facilitator and partner. The mentee is the person who receives the support to be able to develop their skills, as well as to achieve integration in a group or an institution. Assistance quality and security are actually important points in the context of sanitation. Mentoring programs are used in nursing practice as an essential part of the curriculum. This is completely normalized in the student context, and in certain countries in the context of sanitation, especially English ones. Actual clinical nursing settings are complex and changing, making them a very interesting candidate for improvement in a clinical context. Nurse mentoring programs provide minimal opportunities for professionals who join new health services, ranging from new graduates to professional nurses who come from other services. The form, duration, organization, and even the impacts of mentoring programs in the literature are very diverse . This reMentorship, mentoring, training programs, and induction programs have been shown to be fundamental to guaranteeing adaptation and training at the healthcare level of new nurses , promotiThe objective of this scoping review is to explore and summarize the existing literature on mentoring models and programs in the clinical nursing context, including their forms, duration, organization, and impacts on nursing professionals and healthcare institutions.What kinds of mentoring model or processes exist in the clinic nursing context?What is the impact of mentoring programs on nursing professionals?What is the impact of nurse mentoring programs on healthcare institutions?Review question(s):This review included studies conducted worldwide that focused on mentoring programs directed specifically at nurses, ranging from bachelor-level nurses to those with doctorate degrees, in a clinical context. The studies primarily focused on nurse students, and academic experts or individuals other than bachelor-level nurses were excluded, with the objective of determining the impact of mentoring programs on people who have at least a bachelor ‘s degree.This review considered studies in which the outcomes were related to mentoring programs for bachelor nursing in any clinical context in any country, as well as their impact on nursing professionals, and the impact of apply them in health institutions. Examples of regulatory activities included programs that explained the different methods used, the positive or negative impacts on nursing, as well as the benefits or drawbacks for institutions, like promoting quality care and integrating theoretical and practical nursing training and reducing the risk of mistakes and professional stress, having already been shown to reduce the risks of clinical variability and staff mobility, as well as improving satisfaction.This review encompassed mentoring programs in various clinical areas within public and private healthcare systems. Primary (primary care providers), secondary  and tertiary  settings were included. The countries selected for inclusion in this scoping review were limited to English-speaking, Spanish-speaking, French-speaking and Portuguese-speaking countries. English was selected because 95% of publications are written in this language. The rest of the languages were selected based on the knowledge of the authors.This scoping review considered all of the information included in any of the databases and the grey literature for unpublished documents, trying our best to capture reality in the most reliable way. The reviewers decided to keep the source of information “open” to allow for the inclusion of any and all types of studies. Otherwise, important information could be lost.http://osf.io/uh7q2?view_only=3c7eec1ccbed4798a7e4223472d551c2 (accessed on 1 March 2021)). The Joanna Briggs Institute (JBI) methodology for scoping reviews was followed [The objectives, inclusion criteria and methods for this scoping review were specified in advance and documented in a protocol, registered on the Open Science Framework platform  and peer reviewed by another librarian (CCA) according to best practice recommendations . The seaTo identify potentially relevant documents, a comprehensive literature search was conducted using the most impactful bibliographic databases: MEDLINE (PubMed), Embase (via Embase.com (accessed on May 2020)) and Cochrane Library. We also searched in additional databases, including Epistemonikos, Cuiden (Index Foundation), Scielo (Scientific Electronic Library Online) and MEDES (Medicine in Spanish), as well as in some grey literature databases for unpublished documents: OpenGrey, Trove and MedNar. Finally, we used the search engine Google Scholar. All searches were run in May 2020. The analysis time was arduous, which prolonged the investigation, ending in October 2022. No time limits were applied to the search strategy. The studies were limited to those published in English, French, Spanish and Portuguese.The final search results were exported into Mendeley, which was used to manage the references and remove duplicates. The Rayyan  citationThe data extraction process followed a predefined data extraction form based on the review protocol. Data were extracted from papers included in the scoping review by three independent reviewers. There was no need to contact the authors of the papers for missing or additional data. Any disagreements that arose between the reviewers were resolved through discussion and consensus.JBI Reviewer’s Manual [A data extraction table based on recommendations from the s Manual  and specDuring data extraction, it was found that the information related to the characteristics of the nurse mentoring programs was highly variable. For this reason, a second table was created to facilitate the description of the mentoring model/processes that exist in the clinic nursing context. The information added included the hospital’s name and features, program, duration, participants, objectives, development, outcomes and measurement tools.The database searches identified 1521 records. In total, 71 additional records were identified through other sources. A total of 52 duplicates were removed, leaving 1540 records. A further 1153 were not relevant, as determined by the initial screening of titles and abstracts. A total of 387 articles were retrieved in full text. Of these, 376 were excluded. A total of 11 records were included in this review. The results of the search are presented in a Preferred Reporting Items for Systematic Reviews and Metanalyses extension for Scoping Reviews (PRISMAScR) flow diagram  Figure .Eleven articles met the inclusion criteria and were included in this review. The articles included nine journals, eight of which were nursing journals ,16,17,18Descriptive studies were the most common research design identified ,16,17,18. A few programs were developed with external support from sources such as universities or nursing associations [A wide range of nurse mentoring programs were identified with highly variable characteristics. Most of them were developed in third-level hospitals, and only one of the studies reported data from long-term-care facilities and community organizations . Only thciations ,17.All mentoring programs were orientated toward new nurses, with few/no programs with different objectives, and ranged from improving job environments and increasing retention to improving the evidence based on practice or determining the satisfaction and organizational commitment of new nurses. One study used a mentoring support to facilitate evidence-based practice . RegardiThe results of the programs were varied, as were their objectives. Regarding the impact on nursing professionals, seven studies talked about the enhancement of nursing ,15,18,19The measurement tools were all different. Further details of the characteristics of the nurse mentoring programs are shown in Mentoring programs are frequently used to retain professionals in centers or in zones with difficult access or with problems with generational relief, as is the case in New Zeeland, where it is practically required to perform programs that allow for fast adaptation and immersion in the practice, to ensure that there are problems with having nurses in theses contexts.Generalist and specialist nurses benefit from these programs, because they allow them to grow quickly in all of the situations . In addiMentoring programs in large hospitals contribute to increased nurse allegiance and reduce mobility and turnover, making these hospitals more attractive to new professionals ,22. ThesMentoring programs have the biggest influence on clinical professionals, who see increases in their professional growth and more security in their own abilities , as wellThere are important heterogeneity in all of the studies’ aspects, including the program duration, number of participants and objectives, as well as the structures and evaluations tools. These results suggest that mentoring programs need more in-depth and extensive studies. In spite of their differences, they all lead to improvements for nurses, patients and/or organizations.All mentoring programs aim to support the transition of graduate or experienced nurses to new service areas. Mentoring programs improve practice and retention, enhance nurse performance and create a supportive and positive work environment.The impact on institutions’ organization is positive, decreasing mobility, leading to references and making hospitals attractive to new professionals, enabling new leaders to be found in the organizations and promoting professional development. All of these factors are creating a global and lasting change in organizational culture, which is especially complicated in large hospitals.The feminine approach improves clinical skills but does not require competition for development or promotion within the profession. This finding suggests that it would be interesting to study or contemplate, in future studies, a gender perspective and its impact in mentoring.


Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny. Chromosomal instability (CIN) increases the rate of numerical and structural chromosomal aberrations and is a hallmark of cancer . StudiesHs , EXO1 and DNA2  or Edinburgh minimal media (EMM) at 32°C with shaking, unless otherwise stated . See SupS. pombe haploid deletion library were screened for sensitivity to MMS (0.005%), bleomycin (0.001%) and/or CPT (10 μM) in a 96 well-array format on solid YE6S medium plates and subsequently replica-plated onto the respective drug plates in quadruple spots in duplicate. Sensitive mutants were identified by reduced or absent colony size on the drug plates after 48 hours of incubation at 32°C and confirmed by spot dilution assay.Deletion strains from the Bioneer 16. Ch16 encodes an ade6-m216 heteroallele, which when lost results in an Ade− cell, leading to pink sectored or striated colonies when grown on low adenine plates  were generated. Ch16-LMYAU carries an endogenous copy of the HO-endonuclease gene under an nmt1 (no message in thiamine) promoter that is induced without thiamine and generates a DSB uniquely at the MATa site. Following growth on selective media, containing 2 μM of thiamine (T), cells were plated onto sectoring plates with media containing EMM + LRHUA  +/- T to identify break-dependent, and independent Ch16 loss; or EMM + RHUA (low adenine) +/– T to identify break-dependent, and independent LOH. Plates were incubated at 32°C for 2–3 days, and subsequently kept at 4°C for 2 days prior to analysis. Sectoring strains were confirmed by repeating the assay a further 2 times.The sectoring assay is used to detect CIN through an increased rate of loss of a non-essential minichromosome, Che plates , thus vi16-DSB (Ch16-RMYAH), or Ch16-DSB-Tel (Ch16-UA-Ch1MGTASTEL) were performed as outlined previously (MATa site in Ch16 RMYAH the percentage of colonies undergoing NHEJ/ SCC (Arg+ HygR Ade+ His+), gene conversion (Arg+ HygS Ade++His), Ch16 loss (Arg− HygS Ade− His−) or LOH (Arg+ HygS Ade− His−) were calculated. To determine the levels of break-induced GC, Ch16 loss and LOH, background events at 48h-T were subtracted from break-induced events at 48 h-T in cells expressing HO endonuclease. Following DSB induction in Ch16-DSB-Tel the percentage of colonies undergoing NHEJ/SCC (Ura+ Ade+ G418R), isochromosome (ChI) formation (Ura+ Ade− G418S), dnTA (Ura+ Ade+ G418S) or Ch16 loss (Ura− Ade− G418S) were calculated as described above. More than 1000 colonies were scored for each time point. Mean ± SEM values were obtained from triplicate experiments. Differences were deemed significant if p-values obtained using Student's t test were ≤ 0.05.DSB assays using strains carrying the minichromosomes Cheviously . Followi16-DSB-Tel cells were inoculated in EMM + U + A + L + R medium (+T or –T). Samples were collected and washed twice in 0.05 M EDTA at indicated time points before PFGE analysis.The procedures used in this study for PFGE analysis have been described previously . For theThe procedures used in this study for pedigree analysis have been described previously .Proteins were extracted using trichloroacetic acid (TCA) precipitation and analysed by western blotting as described previously . TAP-tag16-DSB-Tel carrying cells were inoculated in EMM medium in the presence or absence of thiamine at 32°C. Samples were collected at indicated time points, fixed in methanol/acetone, rehydrated and stained with 4′,6-diamidino-2-phenylindole (DAPI) before examination using Zeiss Axioplan 2ie microscope, Hamamatsu Orca ER camera. Open-source micromanager software was used to analyse the images (Che images .Live cell imaging was performed on agarose pads as previously described . ImagingS. pombe DNA was extracted from cells grown to log phase at 32°C using MasterPureTM Yeast DNA purification kit (Lucigen). Genomic DNA from strains carrying Ch16-DSB-Tel, and loss of heterozygosity (LOH) strains LOH1, LOH5 and LOH9 was sent to Novogen (UK) Company Limited for whole genome sequencing (Illumina PE150). The resulting reads were aligned to the reference genome Schizosaccharomyces pombe ASM294v2 using bowtie2 v2.2.6. Only the locations with best alignment scores were kept if the reads aligned to multiple locations. The average coverage rate over all samples and all chromosomes is 64.7 reads. The number of reads over equally sized regions were counted by python package HTSeq. The sequence data are available using the link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA795979?reviewer=op9hm1s1oocds7lod9aga90ikqTo explore the relationship between DSB misrepair and CIN we performed an unbiased genome-wide screen to identify DSB repair mutants exhibiting elevated levels of break-induced CIN. As a first step we screened a library of haploid deletion mutants  to iden16), experimentally derived from the centromeric region of chromosome III (ChIII)   . Ch16 wa6-LMYAU) . Using tN Figure .16 is efficiently repaired by HR, the major DSB repair pathway in fission yeast, resulting in gene conversion (GC) of a marker adjacent to the DSB site, using the homologous ChIII as a repair template (16 loss) or to extensive loss of heterozygosity (LOH), resulting in differential loss of distant genetic markers integrated within Ch16 through either isochromosome formation (de novo telomere addition (16-RMYAH (termed here Ch16–DSB), and DSB repair outcomes analysed. The majority (66/81) of the CIN mutants exhibited a significantly altered DSB repair profile compared to a wild-type background, including significantly altered levels of sister chromatid conversion (SCC), interchromosomal gene conversion (GC), break-induced Ch16 loss, or LOH  and +fbh1; genes impacting on HR, including +, cdt2+ddb1; DDR factors, +rad3 (ATR), +hus1, +rad9, +rad17, +rad26,  was observed  or to extensive loss of heterozygosity (LOH), resulting in differential loss of distant genetic markers integrated within Ch16 through either isochromosome formation  in a rad51Δ background  and found isochromosome formation was observed much earlier (48h)  and Exo1 in rqh1Δ exo1Δ Ch16-DSB-Tel cells, consistent with isochromosome formation requiring extensive DNA end resection (rqh1Δ exo1Δ background resulted in significantly elevated levels of genetic marker loss consistent with de novo telomere addition (dnTA) at or near the DSB . Ne. Ne16. S) Figure , B. Thisesection ,21   analysis of time course samples following DSB induction. Sequence samples were compared to the parental strain carrying Ch) Figure . NGS anad Figure . While tS. pombe is 3.5 h,  . These observations strongly suggest that wild-type fission yeast cells divide in the presence of an unrepaired broken chromosome, as a result of checkpoint adaptation, thereby permitting the propagation of an unrepaired broken chromosome.A prediction of the above results is that propagation of an unrepaired broken chromosome over multiple generations requires DNA damage checkpoint adaptation. Checkpoint adaptation has been previously described in fission yeast , but is revisiae . To deterevisiae ,28,36,59l Figure . In cont (ATRHs)  did not  (ATRHs) . Consist16-DSB-Tel cells was associated with increased Chk1 phosphorylation levels at earlier time points when cell division was arrested, consistent with G2-M checkpoint activation , together with the human equilibrative nucleoside transporter (hENT1) (adh-Dm-dNK-adh-hENT1), we found that EdU was not incorporated in elongated cells following DSB induction. In contrast, EdU incorporation was observed in 10–20% of the population by 30 h following DSB induction but was largely restricted to binucleate cells (G1/S phase), and normal length daughter and granddaughter cells following checkpoint adaptation  and indels detected on isochromosome was similar to the wild-type Ch16-DSB-Tel minichromosome in a wild-type background exhibited a unique series of colony sectoring patterns of ura4 marker loss, indicative of CIN, when replica-plated from a non-selective YES plates to EMM-Ura plates  GC/ (NHEJ or SCC) (0.8%); GC/LOH (12.0%); GC/Ch16 loss (6.7%); LOH/LOH (13.6%); LOH/Ch16 loss (11.0%); or Ch16 loss/ Ch16 loss (2.8%) daughter colony pairs , LOH/dead (19%) Ch16 loss/LOH (0.5%), dnTA/LOH (6%) dnTA/dnTA (2%) dead/dnTA (1%) and dead/dead (17%) daughter colony pairs  shown by high resolution PFGE Figure . WGS anal Figure . In contDSB site ,54 (SuppDSB site .2 ratio of 0) within the centromeric inverted repeats  exhibiting parental disomic copy number at the junction between deleted and duplicated segments of LOH1 and LOH9 . These cycles give rise to a population of daughter cells containing unrepaired broken chromosomes. SERPent cycles continue over multiple generations until unstable broken chromosomal elements are individually stabilized or lost within each of the progeny cells (Figure Together, these observations lead us to propose a new model for the initiation and perpetuation of CIN, in which an unrepaired single-ended DSB arising from failed HR, or telomere loss, gives rise to an unrepaired broken chromosome. This single-ended unrepaired broken chromosome is subject to post-adaptive cycles of s Figure . This mo16 loss, is considerably elevated following DSB induction in HR mutants, or following telomere loss, both of which generate single-ended DSBs. Further, we find that isochromosome formation results from transgenerational resection of a single-ended DSB and is therefore a product of CIN. Such single-ended DSBs are predicted to arise from exposure to radiation and other clastogens, as well as through collapsed replication forks or telomere loss (S. pombe orthologues of HR CIN genes as CIN genes in S. cerevisiae (Our data support a single-ended DSB as a potent driver of CIN. Consistent with this we find that CIN, as determined by increased colony sectoring or increased levels of extensive LOH and Chere loss , suggestrevisiae  (Supplemrevisiae , stronglrevisiae ,71–74. WS. pombe. These findings are consistent with reports of checkpoint adaptation contributing to genome instability in S. cerevisiae (We show that checkpoint adaptation facilitates CIN in revisiae ,38,75. Wrevisiae , suggestS. cerevisiae (A key feature of such break-induced CIN is that following checkpoint adaptation, inherited broken chromosomes are faithfully replicated by daughter cells. This finding has considerable significance as this explains how unstable chromosomes are propagated across the population of daughter cells, thereby driving widespread cell-to-cell heterogeneity, rather than being otherwise limited to a single cell lineage. Further, replication of unstable intermediates coupled with independent processing of sister chromatids provides a mechanistic basis for rapid genetic variation, prior to chromosomal stabilization or loss. Our findings suggest that replication of the broken chromosome is accurate and consistent with bulk DNA replication. Thus, the post-adaptive DNA replication machinery, once initiated, appears unable to distinguish between an intact and a broken chromosome. These findings build on observations in which a broken chromosome was shown to be propagated in revisiae , suggestde novo telomere addition (16-based assay despite the presence of a homologous ChIII template (Our findings indicate that post-adaptive segregation, replication and transgenerational extensive processing of a single-ended DSB leads to a broad spectrum of chromosomal rearrangements across multiple generations. In this regard, we calculate that a single yeast cell carrying an unrepaired broken chromosome will have undergone up to ten SERPent cycles in the time taken to resect 200 kb from the break site to the centromeric inverted repeats, generating up to 1000 cells carrying unstable chromosomes. Crucially, each inherited and replicated sister chromatid is independently processed by daughter cells, and can thus undergo a number of possible fates including stabilization through partial or complete chromosomal arm duplication leading to telomere acquisition; stabilization through addition ,54, or caddition . Surprisaddition , BIR wastemplate , or whentemplate . Instead16 centromere. Our findings suggest that annealing of these single-stranded inverted repeats promotes fold-back inversion, which following DNA replication, results in inverted chromosomal arm duplication, as previously described (Candida albicans (Our data support a model in which extensive DNA 5' end resection, initiated through failed HR repair of a distal DSB, continues over hundreds of kilobases, and over multiple generations, until it resects through inverted repeats within the Chescribed ,64,78,79escribed . Similaralbicans . Importaalbicans , and mayA feature of this study is that to harness the genetic potential of fission yeast to study CIN, we have focused on the fate of a single enzymatically broken experimentally derived nonessential minichromosome. We nevertheless anticipate that this model system may contribute to a broader understanding of CIN and how it is suppressed. It will be important to determine whether this mechanism is evolutionarily conserved and can lead to CIN in human cancers. In this respect, fold-back inversions are associated with genetic disease ,81,82 anAll materials and raw data are available upon request.gkad160_Supplemental_FilesClick here for additional data file.


N-acetylmuramicacid and cyclization of the product to form muramic-δ-lactam.This activity is unique among peptidoglycan deacetylases and is notablebecause it may involve the direct ligation of a carboxylic acid witha primary amine. Our reconstitution products are nearly identicalto the cortex peptidoglycan found in spores, and we expect that theywill be useful substrates for future studies of enzymes that act onthe spore cortex.Some bacteria survivein nutrient-poor environments and resistkilling by antimicrobials by forming spores. The cortex layer of thepeptidoglycan cell wall that surrounds mature spores contains a uniquemodification, muramic-δ-lactam, that is essential for sporegermination and outgrowth. Two proteins, the amidase CwlD and thedeacetylase PdaA, are required for muramic-δ-lactam synthesisin cells, but their combined ability to generate muramic-δ-lactamhas not been directly demonstrated. Here we report an in vitro reconstitutionof cortex peptidoglycan biosynthesis, and we show that CwlD and PdaAtogether are sufficient for muramic-δ-lactam formation. Ourmethod enables characterization of the individual reaction steps,and we show for the first time that PdaA has transamidase activity,catalyzing both the deacetylation of  Indeed, spore formation is the basis for transmission,chronic infection, and antibiotic evasion by human pathogens suchas Clostridioides difficile.3 Characterization of the molecular steps involved in sporulationand germination is therefore of interest because these pathways maybe promising targets for new spore-specific antimicrobials.In responseto nutrient starvation,many species of Gram-positive bacteria form spores that can germinate,reinitiating vegetative growth when permissive conditions are established.N-acetylglucosamine (GlcNAc) and N-acetylmuramicacid (MurNAc) residues  residue cyclized togive muramic-δ-lactam . To generate asuitable substrate for the enzymes, we extracted Lipid II from B. subtilis and polymerized it into linear peptidoglycanstrands using SgtB, a monofunctional glycosyltransferase from Staphylococcus aureus.17 We thenincubated the individual CwlD or PdaA proteins with the linear polymerand analyzed the reaction products using a liquid chromatography-massspectrometry (LC-MS) assay. In this analysis, polymeric products aredigested into smaller fragments with the muramidase mutanolysin andreduced with sodium borohydride (NaBH4) to enable LC-MSdetection. Mutanolysin digestion of unmodified peptidoglycan givesdisaccharide products , but mutanolysincleaves MurNAc-β(1→4)-GlcNAc linkages only when the MurNAcbears a pentapeptide. Stem peptide removal by an amidase thereforeproduces an altered cleavage pattern, giving tetrasaccharide or longerfragments . Reactionswith B. subtilis PdaA, BsPdaA, alonegave only starting material A , indicating that PdaA could deacetylateMurNAc only once the stem peptide had been removed. Product D containedmuramic-δ-lactam, but the peak was small. However, we also detectedanother product, E, with a mass consistent with reduction of muramic-δ-lactamby NaBH4 . While most amidesdo not react with NaBH4, the decalin-like lactam is sufficientlystrained to be reduced.8 Product E is therefore derived from D and is evidenceof muramic-δ-lactam formation. When we allowed the BsPdaA reaction to run for 48 h, we observed E as the major product,occurring in yields of ≤75% . Analogous experiments conducted with the CwlD and PdaA variantsfrom C. difficile, CdCwlD and CdPdaA1, respectively, produced similar results, althoughthe amidase activity of CdCwlD was weak . This is presumably because our reactionmixtures did not contain GerS, an activator of CdCwlD required for germination in this organism.19 Nevertheless, CdPdaA1 activity was robust, withall product B converted to C or lactam .Reconstitution of enzymes that act on peptidoglycan is challengingbecause many of these proteins will use only large peptidoglycan oligomersas substrates. Producing these substrates by chemical synthesis isnontrivial, but recent advances in obtaining the peptidoglycan precursorLipid II have made it possible to prepare structurally defined peptidoglycansubstrates in vitro.ogeneity S1. To geogeneity S1. To geogeneity S1. To geogeneity S1. To geogeneity S1. To geogeneity S1. To geogeneity S1. To geSupporting Methods) and with themodified residues occurring every other monomer. This distributionis similar to what is observed in the cortex layer of spores in which30–50% of residues are muramic-δ-lactam, depending onthe bacterium.9 We expect that the ability to generate defined cortexpeptidoglycan substrates will prove to be useful for studying otherenzymes that act on the cell wall during sporulation and germination.We note that in our initial reactions with PdaA, we found that thelactam synthesis was maximal when Ca2+ ions were presentin the reaction buffer . Theabsence of these ions could explain why only deacetylase activity,not lactam cyclization, was observed previously.15 Because Ca2+ would be abundant in the intermembranecompartment where PdaA is localized,20 itis plausible that these ions are required to stabilize a conformationthat permits lactam synthesis.Together, these results show that CwlD and PdaAare sufficientfor muramic-δ-lactam synthesis. They also confirm the prevailingmodel whereby the synthesis is ordered; CwlD acts first followed byPdaA. Importantly, the products produced with longer PdaA reactiontimes (24–48 h) are consistent with peptidoglycan where approximately40% of MurNAc residues have been converted to muramic-δ-lactam.21 Their reactionscan therefore be quenched by addition of excess chelator such as EDTA.23 To assessactivity over time, we treated linear peptidoglycan with BsCwlD to generate polymer enriched in peptide-cleaved product B andincubated the resulting material with BsPdaA or CdPdaA1. Reaction aliquots were quenched with EDTA and analyzedvia our LC-MS method .LC-MS analysis of the eluted peptidoglycan showed polymer enrichedin product C ; however,re-addition of BsPdaA gave good conversion of MurNto muramic-δ-lactam . From these results, we conclude that the lactamcyclization is catalyzed by PdaA. Our data are therefore consistentwith a model for muramic-δ-lactam synthesis shown in We next sought to determine if the lactam cyclizationwas catalyzedby CwlD or PdaA. We noted in the time course analysis that e points 3c. We enproteins S8.LC-MSproteins S8.LC-MSproteins S8.LC-MSproteins S8.LC-MS24 All other characterized members of this enzyme family act solelyas deacetylases or esterases. Therefore, the transamidase activityexhibited by PdaA is unique. Notably, lactam cyclization does notrequire concomitant MurNAc deacetylation. D73 of BsPdaA acts as a catalytic base in the deacetylation reaction.23 A D73N mutation abolished deacetylase activity , but the mutant protein was able to catalyze cyclizationof a previously deacetylated substrate  and classified as carbohydrateesterase 4 (CE4) in the CAZy database.activity S10, but activity S10, but activity S10, but activity S10, but


Because there are few evidence-based guidelines and an extremely low incidence rate, managing and treating patients who have transitioned from acute promyelocytic leukemia (APL), which was diagnosed during pregnancy, to acute myeloid leukemia (AML), can be difficult.In this case, a 34-year-old pregnant patient was diagnosed with APL in medium-risk group in June 2017. After the all-trans retinoic acid and arsenic trioxide-based full-course treatment, the patients achieved complete remission (CR) and were well-tolerated. After 5 years, the patient complained of fatigue for 3 months.Bone marrow examination revealed hypercellularity with approximately 50% immunophenotypic abnormal myeloblasts with MLL-AF9 fusion gene. Based on the AML diagnosis criteria of the World Health Organization, the patient was eventually diagnosed with a rare transformation from APL to AML.The patient was treated with two cycles of induction chemotherapy and an allogeneic hematopoietic stem cell transplantation .Until now, the patient is in continuous remission with no signs of APL and AML.Despite the rarity of APL to AML transformation, it is crucial to track the disease’s progress and administer treatment on time. It remains uncertain whether the risk stratification and clinical outcomes of secondary AML with MLL-AF9 are equivalent to those of de novo AML with MLL-AF9. The management and treatment of these patients should be personalized and require further observation. Despite these advancements, a small minority of APL patients who achieve CR can undergo transformation to other types of AML. The diagnosis and treatment of this transformation remain challenging, as there are no clear guidelines to follow. In this case report, we presented a unique case of APL transformation to AML, which was diagnosed during pregnancy and has not been reported previously. We also described the management and treatment process and conducted the literature review on the transformation of APL into AML.Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by unique cytogenetic changes, molecular pathogenesis, clinical symptoms and therapeutic regimens. Previously, it was a highly fatal disease due to severe coagulation disorders. The introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) into therapy has significantly improved the clinical efficacy and outcomes with complete remission (CR) rates exceeding 90% and long-term remission rates exceeding 80%.9/L; neutrophils count, 0.19 × 109/L; hemoglobin concentration, 90 g/L; platelet count, 19 × 109/L; prothrombin time, 10.8 seconds; activated partial thromboplastin time, 29.5 seconds; fibrinogen, 2.38 g/L; thrombin time, 15.1 seconds; D-Dimer, 2530 ng/mL; creatinine, 37 μmol/L; K+, 3.88 mmol/L; Na+, 137 mmol/L. On May 28, 2017, she underwent a cesarean delivery with red blood cell and platelet infusion, and received ceftriaxone for infection prevention. She delivered a healthy baby. After the cesarean delivery, a bone marrow (BM) aspiration and biopsy were performed on June 2, 2017. The peripheral blood film showed 2% abnormal promyelocytes, while the BM film revealed 85% abnormal promyelocytic blasts with excess of granule and Auer bodied. Additionally, there was dysfunction erythroid differentiation, and rare megakaryocytes and platelets were observed  with no history of hematologic or solid malignancies, and no exposure to toxins or radioactive substances, was diagnosed with APL during the 35th week of gestation on May 27, 2017. She experienced slight vaginal bleeding for one day before her fist presentation, but had no other significant clinical pictures or physical signs. The findings of initial analysis of peripheral blood were as follows: white blood cell count, 0.56 × 10 with no 2/d for 42 days, ATO at a dose of 0.16 mg/kg/d for 42 days, and THP at a dose of 30 mg on days 3 to 6. Following the induction therapy, the patient achieved complete remission (CR) and underwent 2 cycles of consolidation therapy using ATRA, ATO, and THP. Additionally, intrathecal chemotherapy was administered to prevent central nervous system leukemia. On the September 28, 2017, the patient was initiated maintenance treatment with ATO and ATRA for 5 cycles. Throughout the treatment, the patients was tolerant without remarkable complications, and her APL remained CR. Therefore, from 2019 to 2021, the BM examination and PML-RARα fusion gene analyses were performed annually, consistently showing CR of APL . The specific regimen included ATRA at a dose of 20 mg/m2, days 1–5; cytarabine, 10 mg/m2, every 12 hours, days 1–14; aclacinomycin, 20mg/d, days 1–4; G-CSF, 150 μg/m2, q12h, days 0–14) and one cycle of idarubicin and cytarabine . The patient achieved a rapid CR. However, during the consolidation treatment, the patient experienced a sudden recurrence of leukemia after one cycle of azacytidine and venetoclax  and one cycle of azacytidine . Subsequently, she underwent cladribine, cytarabine and G-CSF (CLAG)  as bridging therapy to prepare for myeloablative allogeneic hematopoietic cell transplantation . The conditioning regimens consisted of busulfan (0.8 mg/kg every 6 hours) from days -7 to -3 , etoposide (12 mg/kg) on days -3 and -2 , and cyclophosphamide (60 mg/kg) on days 3 and 4   positivity was observed. Moreover, erythroid cells hyperplasia was inhibited and only one megakaryocyte was observed in the whole film  is a term that refers to the development of AML following an antecedent hematologic disorder , such as myelodysplastic syndrome, or myeloproliferative neoplasm, or aplastic anemia, or prior exposure to cytotoxic chemotherapy drugs or radiotherapy or immunosuppressive therapy . Across multicenter studies, the incidence of sAML ranged from 10% to 30% of all AML cases. In total, sAML-AHD accounted for 60% to 80% of sAML cases.–9 In particular, in APL, the incidence of therapy-related myeloid neoplasms which included myelodysplastic syndrome, AML and acute lymphoblastic leukemia, reported in a few studies for patients in CR ranged from 0.97% to 6.5%. The 6-year cumulative incidence of therapy-related myeloid neoplasms was only 2.2%. Therefore, it is rare for APL to undergo transformation to AML during CR period and the reasons and underlying mechanisms for its occurrence in our case remain unclear.Owing to the advancements in management and treatment of APL, the majority of patients achieve long-term CR and disease-free survival. However, a minority of APL patients still experience refractory/recurrent disease or other complications. These findings aligned with data from a Danish national population-based cohort study, which showed that “sAML” patients were older, had lower CR rates, and poorer survival compared to patients with de novo AML. Additionally, patients with sAML-AHD were more likely to have intermediate cytogenetics than patients with t-AML. However, adverse cytogenetics were more frequently found in t-AML patients, which may be a result of the cytotoxic effect of the drugs on hematopoietic progenitors. The drugs associated with t-AML can be mainly divided into 2 classes: alkylating agents  and topoisomerase-targeted drugs . Certian drugs tended to be associated with specific cytogenetic abnormal changes . Individual susceptibility to cytotoxic agents also participated in the development of t-AML. These findings provided support for the hypothesis that the development of t-AML may be attributed to the resistance of advantaged clones with specific cytogenetic abnormalities (single hit) to certain cytotoxic drugs, as well as the suppression of cytotoxic treatment in disadvantaged clones (double hit). However, the current studies did not establish a clear relationship between the dose-intensity of cytotoxic treatment and the incidence of t-AML. In the case of sAML-AHD, the transformation may be influenced by the inherent lineage plasticity of leukemia stem cells. In our case, the patient received THP, a topoisomerase inhibitor, during the induction and consolidation therapy. Although the patient was diagnosed with sAML in the cytogenetic intermediate group five years after achieving remission from APL, the potential role of cytotoxic drugs in the development of sAML cannot be excluded.In a study that evaluated the disease characteristics, treatment response and outcomes of sAML, patients with “sAML” were found to be older, and had poorer WHO performance status and higher rates of adverse karyotypes compared to patients with de novo AML. Similarly, the achievement of CR and survival were significantly inferior in “sAML” patients. Among them, sAML-AHD patients had a higher age and rates of intermediate-risk karyotypes compared to t-AML patients. There were no significant difference between sAML-AHD and t-AML in terms of gender, CR achievement, refractory rates, relapse rates and early death. In MLL-rearrangement (MLL-r) AML, the most common fusion partners are AF9 and AF6, resulting in the translocations t   and t   respectively. MLL rearrangements are often observed in t-AML following treatment with topoisomerase II inhibitors and are associated with poor clinical outcomes. According to current guidelines, except t  , t  , and t   translocations, which are considered adverse-risk cytogenetic abnormalities, all other MLL rearrangements are classified as intermediate risk groups. Several studies have shown that MLL-r patients tend to achieve remission after induction chemotherapy but have high rates of relapse.,17 Based on these findings of the above-mentioned studies, the patient in our case received azacytidine and venetoclax as consolidation treatment during the second remission period to prevent disease recurrence. However, the patient experienced early recurrence just four months after remission and had to undergo allo-HSCT. It remains uncertain whether the risk stratification and clinical outcomes of sAML with MLL-AF9 are equivalent to those of de novo AML with MLL-AF9.The mixed lineage leukemia (MLL) gene, located on chromosome 11q23, plays a crucial role in maintaining chromosomal stability. MLL rearrangements can occur with approximately 80 different partner genes.In summary, our study confirms the poorer outcomes of sAML following APL compared to de novo AML. Despite the positive outcomes of allo-HSCT in our case, there are certain restrictions, such as insufficient therapeutic observation time. Future developments are anticipated in the areas of basic and clinical research, with a focus on the need for better early diagnosis techniques and treatment regimens for sAML.We are grateful to all the participants of the study.Conceptualization: Ziyuan Lu.Data curation: Na Han, Yu Jiang.Formal analysis: Yang Gao.Funding acquisition: Ziyuan Lu.Writing – original draft: Yang Gao, Na Han.Writing – review & editing: Ziyuan Lu.