KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring  Although treatment has progressed, in some AML patients, particularly the elderly4, outcome is dismal due to the remarkable genetic complexity6, epigenetic alterations9, and the dynamics of the disease11. Treatment-related morbidity/mortality and resistance to chemotherapy are major causes of treatment failure12. However, longer life expectancy and possibly a cure is achievable if complete remission (CR) is attained14. Therefore, there is substantial research to improve and personalize therapies, and lessen treatment toxicity18.Acute myeloid leukemia (AML) is one of the most common hematologic malignancies, characterized by clonal, proliferative, and abnormally or poorly differentiated myeloid cells infiltrating the bone marrow, blood or extramedullary tissues19, gilteritinib20), IDH1/2 inhibitors , BCL-2 inhibitor (venetoclax25), and cyclin-dependent kinase (CDK) inhibitors , which cause cell cycle arrest and induce apoptosis.Currently, several novel agents acting through distinct molecular targets, identified in the pathophysiology of AML, are being investigated alone or combined with conventional chemotherapy, e.g., FLT3 inhibitors  transcriptional activity consequently inducing apoptosis33. Furthermore, CDK9 inhibition downregulates the anti-apoptotic protein myeloid cell leukemia 1 (MCL-1)34. Fadraciclib has already entered clinical trials in advanced solid tumors (NCT02552953), chronic lymphocytic leukemia (CLL) (NCT03739554) and AML (NCT04017546).The second generation CDKi fadraciclib is an orally available 2,6,9-trisubstituted purine analog designed to selectively inhibit CDK2 and CDK9In this study, fadraciclib was assessed for efficacy against AML cell lines and primary AML samples in vitro either as a single agent or in combination with venetoclax (VEN), cytarabine (AraC), or azacitidine (AZA).IC50 of fadraciclib was established by resazurin reduction assay at 24, 48, and 72 h. The IC50 of fadraciclib was lower at 72 h, compared to 24 and 48 h, showing that longer exposure increased drug potency Fig. . At 72 hTaken together, fadraciclib potently induces apoptosis, increases the subG0 population, and reduces proliferation as evidenced by reduced metabolic activity in AML cell lines.p < 0.0001) and greater than 99% at 24 h, with a corresponding reduction in the phosphorylated form (p-Rb). In MV4–11, only a modest reduction in the levels of Rb and p-Rb at 4 h, with Rb level returning to baseline and a modest increase in p-Rb observed at 24 h  and MV4–11 (p < 0.001), and at 24 h in MOLM-13, but not in OCI-AML3  are sensitive to fadraciclib29. Five primary AML samples harboring KMT2A-partial tandem duplication (PTD) and five primary AML samples with KMT2A wild-type (WT) were tested with fadraciclib as a single agent  and 1 µM , and in KMT2A WT patient samples following treatment with fadraciclib at 0.5 µM  and 1 µM  compared with NDC  and 1 µM , and in KMT2A WT patient samples at 0.5 µM  and 1 µM  following fadraciclib treatment compared with NDC  and %active caspase-3 at 6 h (p = 0.0351) as compared with NDC  and unfavorable mutations  and KMT2A-PTD), in order to assess the efficacy of fadraciclib and its synergistic activity when combined with VEN, AraC, or AZA, towards these mutations.For combination studies, six primary AML samples were selected based on the presence of the most common favorable  and 0.5 µM (FAD2) in combination with VEN, AraC or AZA were assessed for efficacy against primary AML cells. The concentrations selected were based on previous studies: 0.025 µM (VEN1) and 0.5 µM43 (VEN2); 0.01 µM (AraC1) and 0.1 µM44 (AraC2); 0.5 µM45 (AZA1) and 2 µM46 (AZA2), aiming to optimize results for both sensitive and more therapy-resistant patient samples. All drug concentrations were within clinically achievable concentrations . Apoptosis, active caspase-3, cell cycle analyses, and proliferation assays were performed at 72 h.Drug combination studies exploit opportunities for reduced drug resistance, decreased toxicity, and efficacy improvementp = 0.1059; and 64.6 ± 21.4% in VEN2 alone, p = 0.5166)      , as well as NDC , MOLM-13 (carries FLT3-ITD and KMT2A-MLLT3), and MV4–11 (carries FLT3-ITD and KMT2A-AFF1), aiming to encompass stratification of major risk profiles according to the European LeukemiaNet recommendations50. Results indicate MV4–11, harboring adverse prognosis molecular abnormalities, was less sensitive with the highest IC50 62. This highlights the important of assessing the efficacy of fadraciclib on specific AML subtypes based on its mechanism of action of inhibiting CDK9. As part of the Super Elongation Complex (SEC), CDK9 is important for transcriptional elongation, in particular in KMT2A-r AML63, it is therefore rational to evaluate the efficacy of fadraciclib in this AML subtype. Preliminary results indicate KMT2A-r AML cell lines are more sensitive to fadraciclib29. Patient samples, however, display a high diversity of genetic mutations  and unfavorable mutations . Initially, we hypothesized that the treatment response would be relevant to the risk profiles of the individual genetic lesions in primary AML samples. However, we found that complexity of the molecular genetic lesions and karyotypes have more influence. The more complex the molecular landscape, the less efficacious the combination therapy , exome sequencing and genome wide assays65, are being developed to replace single gene assays. This will facilitate better prognostication and more personalized therapy in the future66. A short delay in commencing therapy in newly diagnosed AML had no effect on outcome after accounting for other prognostic covariates67, providing a potential window to perform NGS.Considering the combination studies, primary AML samples were selected based on the most common favorable  (NCT04017546); and relapsed/refractory CLL (NCT03739554).As a global effect, an increase in cell death in primary human AML cells was shown for apoptosis, active caspase-3 assays, and cell cycle analysis when fadraciclib was combined with VEN, AraC, or AZA. In primary AML cells, combining the MCL-1 inhibitor S63845 with VEN results in greater efficacy than either inhibitor alone, with a more potent activity against leukemic rather than normal hematopoietic progenitors28. However, no increase in overall or event-free survival rates were observed. Potentially, this may be due to the higher toxicity of the combination regimen based on our preliminary data showing the impact of the fadraciclib + AraC on normal hematopoietic cells.When considering combining CDK9 inhibitors with AraC, a recent phase 2 study (NCT01349972), demonstrated a significantly higher efficacy of FLAM  as compared with the standard “7 + 3” regimen in terms of a CR rate72. In the same manner seen in fadraciclib + AZA combination presented here, alvocidib + AZA also increased %active caspase-3 as compared with single treatments. In a MOLM-13 xenograft model, the combination showed greater tumor growth inhibition compared to single agent treatments. Our results, demonstrated a potential therapeutic window with the FAD2 + AZA2 combination, as measured by %Undivided %undivided of AML cells in cell proliferation assay  with 20% or 10% fetal bovine serum (Gibco), supplemented with 1 mM glutamine and 1% penicillin-streptomycin (Invitrogen).OCI-AML3, MOLM-13, and MV4–11 (DSMZ) were cultured at 37 °C in a 5% COHealthy donor and diagnostic primary AML samples were taken in accordance with the Declaration of Helsinki, and Ethics Committee approval 15/WS/0077). Demographic data, cytogenetic abnormalities and genetic lesions of AML patients are shown in Table. 5/WS/0077Ten micromolar stock solutions of fadraciclib (Cyclacel), AZA (Stratech), and VEN (Stratech) were prepared in DMSO. 10 mM AraC (Sigma-Aldrich) was prepared in water. All stocks were stored at −20 °C and dilutions freshly prepared in cell culture media.Half-maximal inhibitory concentration (IC50) of AML cell lines treated with fadraciclib for up to 72 h was established using 7-Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt resazurin assay (Sigma-Aldrich) according to manufacturer’s instructions. IC50 were calculated using GraphPad Prism 8 software (GraphPad Software).−ΔΔCT compared to no drug control (NDC) as previously described73. Internal sample control was ensured by subtracting the average of six housekeeping genes ATP5F1B, B2M, CYC1, RNF20, TYW1, and UBE2D2 from the Ct value of each gene of interest. Mean and standard deviation of fold change in expression were calculated. For Primer sequences see Supplementary Table RNA was extracted using RNA Easy Micro kits (Qiagen) and converted to cDNA using high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using Fluidigm Biomark technology and data were collected as per manufacturer’s instructions. Data was analyzed using the 26 cells were harvested and washed twice in ice-cold PBS and pelleted at 300 × g for 5 min. Then, the pellets were lyzed in ice-cold solubilization buffer containing phosphatase and protease inhibitors at a concentration of 1 × 106 cells per 10 μl solubilization buffer . This suspension was left on ice and vortexed every 15 min for 1 h, then centrifuged at 14,000 × g for 10 min to pellet all cell debris. The supernatant was transferred to a fresh eppendorf and stored at −80 o C until required.In all, 1–5 × 10Protein concentration was quantified using the Quick Start Bradford Dye reagent and compared to a standard curve prepared using Quick StartTM BSA protein. The colorimetric protein assay was measured by visual absorbance on the Spectramax M5 plate reader at 595 nm at using SoftMax Pro software.Cell lysates were prepared and 30 µg of protein was resolved. Western blotting was performed using the NuPAGE electrophoresis system (Invitrogen) as per manufacturer’s instructions, and protein detection via Odyssey Fc Imaging System. Densitometry was performed using Image Studio Lite version 5.2. Antibodies and experimental conditions are outlined in Supplementary Table 5 cells per condition. Propidium iodide (PI) staining buffer to assess cell cycle progression as per manufacturer’s protocol. Fixation/Permeabilization Solution Kit (BD Biosciences) was used prior to MCL-1 or active caspase-3 staining. For staining reagents used, see Supplementary Table For proliferation analysis, primary AML and normal CD34+ cells were stained with CellTrace Violet (Thermo Fisher Scientific) as per manufacturer’s protocol. For establishing a maximum point of fluorescence staining, cells were cultured with Colcemid  to determine non-dividing cells. All primary cells were treated with fadraciclib, VEN, AraC, AZA or fadraciclib in combination with VEN, AraC or AZA in physiological growth factor conditions. Annexin V/7-aminoactinomycin D (7-AAD) or DAPI  (BD Biosciences) staining to assess apoptosis by flow cytometry using 1 × 1074. The CI provides a quantitative definition for additive effect (CI = 1), synergism (CI < 1) or antagonism (CI > 1) in drug combinations.CompuSyn software  was used to investigate the synergism in the cell proliferation assays. The software was based on the Chou-Talalay method for drug combination based on the median-effect equation derived from the mass-action law principlet-test and one-way ANOVA .Average responses from at least three independent experiments are shown (mean ± SD). Statistical analysis used GraphPad Prism 8 for Student’s Supplementary Figure LegendsSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Fig. S1Supplementary Fig. S2Supplementary Fig. S3Supplementary Fig. S4Supplementary Fig. S5Supplementary Fig. S6Supplementary Fig. S7Supplementary Fig. S8Supplementary Fig. S9Supplementary Fig. S10


The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia.The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca They contain calcium carbonate (90–97% by mass), biological macromolecules including glycoproteins and proteoglycans and trace mineral additives, especially Sr2+ and Ba2+6. The ear stones have a higher density than the endolymph, and during movement of the head, they move relative to the sensory epithelium, which allows to sense linear acceleration. They also contribute to spatial orientation due to the sensing of the gravitational field2. The ear stones differ among vertebrates. In general, in fish, they form large continuous crystals called otoliths; in terrestrial vertebrates, they appear as tiny multiple otoliths, specifically named otoconia, which are embedded in a gelatinous membrane7. These biominerals contain calcium carbonate of different polymorphs. Most often, teleost fish otoliths contain aragonite or vaterite8. Otoconia can contain aragonite (amphibians), calcite , or a mixture of both polymorphs (reptiles)7. More detailed reviews of the evolution of otoliths and otoconia can be found elsewhere9. The mechanism of biomineralization of otoliths and otoconia, especially the mechanism of polymorph selection, is poorly understood11.Balance sensing in vertebrates depends on signals received in the inner ear, specifically in the semicircular canals and in the vestibule. The semicircular canals allow the detection of angular acceleration thanks to the inertial flow of the endolymph, a fluid that fills the inner ear, which deflects the cupula and thus stimulates mechanoreceptors in the sensory epithelium. The vestibule consists of an utricle, a saccule, and in all vertebrates besides mammals, of a lagena, which are collectively called vestibular organs. The vestibular organs contain biominerals called “ear stones” anchored to the sensory epithelium through a gelatinous membrane2+)11. For example, intrinsically disordered Starmaker protein (Stm) is necessary to form properly shaped aragonite otoconia in zebrafish Danio rerio12. The biomineralization activity of Stm and its analogues from other fish was thoroughly analysed in vitro16, but although these proteins strongly influenced the size and shape of calcium carbonate crystals grown in vitro, they were not sufficient to induce the formation of aragonite instead of calcite, the most stable polymorph of calcium carbonate. In the case of otolith matrix macromolecule-64 (OMM-64) from rainbow trout Oncorhynchus mykiss, an analogue of Stm, aggregated otolith matrix proteins were indeed shown to be required together with OMM-64 to form aragonite in vitro17. Recently, transcriptomic and proteomic studies have allowed to identify proteins that constitute complex protein mixtures of the endolymph and otolith matrix18. Remarkably, a major constituent of the aggregate is otolin-1—a short chain collagen-like protein, which is strongly conserved in evolution19. Proteolytic cleavage of otolin-1 has been suggested to be a key step in temporally regulated deposition of aragonite in otoliths18.Numerous studies have shown that biomineralization of otoliths and otoconia is controlled by proteins, many of which are acidic and bind calcium ions 25, insulin sensitivity (adiponectin)26, cell adhesion 28 and biomineralization 30. In addition to a collagen-like domain, otolin-1 contains a short N-terminal domain rich in cysteine residues and a globular C1q-like (gC1q) domain at the C-terminus. Function of the N-terminal domain is not known. In adiponectin, a serum protein hormone similar to otolin-1, the N-terminal fragment is responsible for the aggregation of adiponectin trimers to biologically active high molecular weight aggregates32. The gC1q domain is inherently able to form trimers that, together with the ability of the collagen-like domain to form a triple helix, enable it to form fibrils, which could be interconnected by N-terminal domains and gC1q domains. Such a fibrillary network could provide a scaffold for biomineralization of otoliths and otoconia33. Moreover, murine otolin-1 has been shown to interact with otoconin-90 (Oc90), a major otoconial matrix protein, through the collagen-like and gC1q domains34. This interaction enhances the biomineralization activity of otolin-1 and Oc9030, indicating that otolin-1 has two critical functions in the biomineralization of otoliths and otoconia, namely, structural function by providing a scaffold for biomineralization and regulatory function by interacting with other matrix proteins, which allows biomineralization to be controlled. The affinity for Ca2+ also suggests the direct involvement of otolin-1 in the localized precipitation of calcium carbonate through interactions with the mineral phase33.The term “otolin” was first used in the 1960s for a component of the otolith matrix, which maintained similar amino acid compositions among different species of fish2+ in their gC1q domains near the axes of the trimers36. In this work, we substituted two conserved acidic residues of the gC1q domain of human and zebrafish otolin-1 , which may bind Ca2+, with neutral alanine, and we analysed the influence of mutations on the effect of Ca2+ on the secondary structure, oligomerization propensity and stability of gC1q otolin-1. We also checked whether an abundant natural missense polymorphism in the human otolin-1 gene impacts the structure of the protein. Single mutations of the putative Ca2+-binding residues caused mixed effects. Surprisingly, when both residues were mutated, trimers of gC1q domains were highly stable even in the absence of Ca2+. Nevertheless, from all studied variants and conditions, native forms of the gC1q domain of otolin-1 saturated with Ca2+ were the most stable. We observed significant differences between hOtolC1q and dOtolC1q. A single mutation in the calcium binding site was enough to diminish the ability of dOtolC1q to bind Ca2+, which was not the case for hOtolC1q. Moreover, dOtolC1q and its mutants had lower thermal stability than all variants of hOtolC1q in all tested conditions. These observations allow us to propose a mechanism of oligomerization of otolin-1, in which the neutralization of the negative charge by Ca2+ is required at the axis of the gC1q trimer.The molecular mechanism of otolin-1 trimerization is not known. Similar proteins from the C1q superfamily such as collagen X, adiponectin or C1q contain Ca37. Following this definition, we found a single SNP in a region coding hOtolC1q, in which glutamate-470 is replaced by alanine (E470A) due to a substitution of adenine with cytosine (variant ID: rs3921595). The prevalence of this variant is close to 50%. Such a widespread genetic variant should not have any adverse effect on human health. However, in the model of hOtolC1q obtained through ensemble optimization method (EOM) analysis of small angle X-ray scattering (SAXS) data33, E470 is predicted to be near the trimerization interface  of hOtolC1q and dOtolC1q for their abundance and possible effect on protein function and stability. To be qualified as an SNP, a variant must be found in at least 1% of the population2+ by the gC1q domains of collagen X and adiponectin are also strongly conserved in otolin-1  and glutamate (hOtolC1q E417) residues with alanine would affect hOtolC1q and dOtolC1q. Mutations of the putative Ca2+ binding sites of hOtolC1q and dOtolC1q are clearly predicted to be damaging to the protein by all tools used 38, native hOtolC1q and dOtolC1q precipitated completely. Dialysis against Ca2+-depleted buffer did not result in satisfactory removal of bound Ca2+. Upon concluding that the proteins were not obtainable in a Ca2+-free form without the addition of EDTA or EGTA, we decided to indirectly estimate the affinity of the proteins to Ca2+ by measuring the binding of terbium(III) ions (Tb3+), as Ca2+-binding proteins can also bind lanthanide ions due to similar effective ionic radii and positive charges of Ca2+ and lanthanide ions39. The binding of lanthanide ions by Ca2+-binding proteins is usually stronger than the binding of Ca2+, due mainly to a higher positive charge42. Therefore, protein-bound Ca2+ should be substituted by Tb3+. Specifically, the use of Tb3+ in such studies gives a significant advantage, as when bound to proteins, Tb3+ can be detected thanks to luminescence resonance energy transfer (LRET) from aromatic side chains  to the bound Tb3+. Upon excitation of the proteins at the aromatic absorption band (280 nm), sensitized emission from the bound Tb3+ can be observed with a maximum at 545 nm43. Both hOtolC1q and dOtolC1q contain numerous aromatic residues, which are a prerequisite for LRET to occur. The results of the titration experiments are shown in Fig. We attempted to measure Ca3+, fluorescence emission in the visible part of the spectrum with a maximum at 545 nm was observed upon excitation at 280 nm. The intensity of fluorescence emission increased when subsequent portions of TbCl3 were added  calculated using a model of a single binding site per protein monomer were 2.0 µM for hOtolC1q and 18.2 µM for dOtolC1q  seemed to bind Tb3+ stronger than E417A (Kd = 20.0 µM) or D425A (Kd = 24.6 µM) mutants. E470A substitution is in this case indistinguishable from native hOtolC1q. In the case of dOtolC1q, the D440A mutant bound Tb3+ weaker than dOtolC1q (Kd = 32.3 µM). Surprisingly, the D432A mutant (Kd = 2.4 µM) seemed to have a higher affinity for Tb3+ than native dOtolC1q (Kd = 18.2 µM). A double mutant dOtolC1q D432A D440A had the lowest affinity to Tb3+ of all tested forms of dOtolC1q (Kd = 85.9 µM).When the samples of hOtolC1q and dOtolC1q contained Tb2+-binding residues did not abolish the ability of OtolC1q to bind Tb3+. For all mutants except dOtolC1q D432A and hOtolC1q E470A, the affinity was decreased compared to the affinity of the native proteins, as indicated by an increase in Kd. The E470 residue of hOtolC1q did not seem to bind Tb3+, as substitution with alanine did not affect the affinity of hOtolC1q towards Tb3+. In the case of dOtolC1q D432A, we noted an increase in affinity (decrease in Kd). Notably, the emission intensity of dOtolC1q D432A was the highest of all tested forms of OtolC1q , caused visible precipitation of both hOtolC1q and dOtolC1q (not shown).Circular dichroism spectroscopy (CD) is a useful, sensitive technique that allows us to estimate the secondary structure of proteins and to detect changes in the structure induced by denaturation, stabilization or ligand binding2+ by 1 mM EDTA indicate that mutations involving the Ca2+ binding site led to changes in β-structure content  and Tb3+ (163.8 µM), precipitation became visible after prolonged incubation. The protein precipitated immediately at higher concentrations of dOtolC1q and Tb3+. For the mutants, the spectra collected in the presence of Tb3+ were similar to those collected in the presence of Ca2+. We conclude that, contrary to hOtolC1q, the binding of Ca2+ to dOtolC1q cannot be reliably simulated using Tb3+. Nevertheless, we can conclude that the double mutants (hOtolC1q E417A D425A and dOtolC1q D432A D440A) are completely unable to bind Ca2+, as their secondary structure is the same in the presence of 1 mM EDTA, 100 mM CaCl2 and an appropriate excess of TbCl3.In the case of dOtolC1q, all spectra collected in the presence of 1 mM EDTA, despite that for the D432A D440A mutant, are similar  to see how mutations of Ca2+ binding sites affected the oligomerization propensity of gC1q otolin-1. We hypothesized that as Ca2+ stabilizes trimers of hOtolC1q and dOtolC1q33, alanine mutations in the Ca2+ binding site could affect the formation of gC1q trimers. We also checked the influence of Ca2+ and Tb3+ on the structure of the mutants to see how the eventual changes correspond to the results of LRET and CD measurements. The results of SV AUC are summarized in Fig. The CD measurements showed significant secondary structure changes induced by mutations in the Ca33. Alanine mutations in the Ca2+ binding site induced stable oligomerization even in the absence of Ca2+, as evidenced by the apparent molecular weight values (MWapp) derived from SEDFIT analysis (Suppl. Table MW is 17.0 kDa for hOtolC1q and 17.1 kDa for dOtolC1q). This observation was unexpected, as a decrease in trimerization propensity was anticipated to occur due to the introduced mutations. hOtolC1q E417A also seems to form trimers in the absence of Ca2+, but a wider c(s) distribution than for D425A and E417A D425A mutants  distribution of dOtolC1q D432A compared to native dOtolC1q seems to be skewed towards heavier forms. Analysis of the concentration dependence of c(s) distributions  distributions, but it is not as strong as in the case of dOtolC1q. When the proteins were analysed in the presence of 10 mM CaCl2, it became apparent that the oligomers formed by the dOtolC1q D432A mutant were not as stable as the oligomers formed by native dOtolC1q  distributions reveal a small concentration dependence  distribution shows signs of aggregation at 0.25 mg/ml protein and 14-fold molar excess of TbCl3 . In the presence of Tb3+, dOtolC1q D432A finally formed stable trimers. c(s) distributions of dOtolC1q D440A and dOtolC1q D432A D440A did not change compared to the c(s) distributions calculated for proteins with EDTA or Ca2+  distribution of the natural variant hOtolC1q E470A in the absence of Ca2+ has a maximum at a lower sedimentation coefficient and a tail at a higher sedimentation coefficient  distributions. Under these conditions, both the hOtolC1q and E470A variants formed stable trimers without any trace of unbound subunits or heavier aggregates  with SYPRO Orange as a probe33. In this work, we used DSF to analyse how the mutations affected the thermal stability of hOtolC1q and dOtolC1q. We also analysed the effect of Ca2+ and Tb3+ on the stability of the mutants. Both hOtolC1q and dOtolC1q were strongly stabilized by Ca2+  increased from 41 to 44 °C in the absence of Ca2+ to 90 °C or more at 100 mM CaCl2 49, it simulates intracrystalline conditions of otoconia. Tb3+ ions stabilized hOtolC1q and all its mutants, but the effect was not the same as the effect for Ca2+.hOtolC1q and dOtolC1q were stabilized by Caa2+ Fig. , Table 2Cl2 Fig. , Table 2Tm was approximately 45 °C in the absence of Ca2+. The double mutant D432A D440A was very stable under these conditions, as its Tm reached 80 °C. The addition of Ca2+ stabilized dOtolC1q and, to a lesser extent, the D432A mutant. Native dOtolC1q and the D432A mutant were also stabilized by Tb3+, but the effect was different than for 100 mM CaCl2: weaker for dOtolC1q and stronger for D432A. Stabilization of dOtolC1q D432A by Tb3+ relative to Ca2+ is another indication that the mutation changed the structure of the protein, resulting in preferential binding of Tb3+. Strikingly, D440A and the double mutant D432A D440A were not stabilized by Ca2+. Similar to hOtolC1q E417A D425A, they were destabilized by 100 mM CaCl2. These mutants were also not stabilized by Tb3+. We can therefore conclude that hOtolC1q E417A D425A, dOtolC1q D440A and dOtolC1q D432A D440A are completely unable to bind Ca2+ or Tb3+ at the mutated binding site. The fluorescence signal observed in Tb3+ binding experiments for these mutants may come from nonspecific binding of Tb3+ at adventitious sites40, seemingly without an effect on their thermal stability. Mutation of a single residue, D440, seems to be sufficient to diminish the ability of dOtolC1q to bind Ca2+. An analogous mutant of hOtolC1q, D425A, seems to retain minor affinity to Ca2+.In the case of dOtolC1q, the D432A and D440A mutants were slightly more stable than dOtolC1q, as their 2+ binding properties of hOtolC1q and dOtolC1q. The results are summarized in Table 2+ binding site of hOtolC1q (E417A and D425A) strongly decreased its affinity to Ca2+, the mutants were still stabilized by Ca2+ and Tb3+, as evidenced by DSF. The double mutant (hOtolC1q E417A D425A) was not stabilized by these ions. All tested forms of hOtolC1q had notably higher thermal stability than the respective forms of dOtolC1q under all tested conditions. Moreover, hOtolC1q was stabilized at a lower concentration of Ca2+ than dOtolC1q, clearly showing that during evolution, the gC1q domain of otolin-1 became optimized to function at lower concentrations of Ca2+. Endolymph Ca2+ concentration in teleosts varies, as it was reported to be 1.0 mM in turbot50, 1.0–1.6 mM in trout52 and as high as 2.9 mM in oyster toadfish53. In mammals, endolymph Ca2+ concentration was determined to be much lower, between 92 and 133 µM, depending on the compartment (guinea pig)49.In this work, a set of biophysical techniques was used to assess the mechanism of trimerization and the influence of mutations of chosen acidic residues on the structure and Ca2+ binding site of hOtolC1q, were ambiguous in terms of the effect on protein stability. Thermal stability in the presence of Ca2+, binding of Ca2+ or Tb3+ and solution structure of hOtolC1q were not significantly affected by this substitution. The only notable effects were decreased thermal stability of the E470A mutant below 1 mM Ca2+ and increased tendency to aggregate in the absence of Ca2+. Normally, this effect would have been negligible in vivo. Otolin-1, as a secreted protein, is after translation exposed to the ER lumen, which contains 100–800 µM Ca2+54. This concentration should be sufficient to stabilize the gC1q domain of the E470A variant. In the utricle and saccule, otolin-1 could be protected from low concentrations of Ca2+ by being bound within otoconia and cross-linked with other proteins in the otoconial membrane. However, a decrease in local Ca2+ caused by dysregulated calcium homeostasis or degeneration of otoconia might destabilize the protein, possibly accelerating the degradation of otoconial matrix and/or otoconial membrane. Therefore, it would be interesting to examine whether the presence of the E470A allele in the genome is associated with the prevalence of benign paroxysmal positional vertigo (BPPV). BPPV is a balance disorder caused by displacement of degraded otoconial membrane fragments to the semicircular canals55, where they disrupt the flow of endolymph or movement of cupula, which occur normally due to the movement of the head. This effect is felt by the affected person as sudden attacks of vertigo, which may lead to falls and bone fractures and, consequently, to disability, especially later in life57. The hypothesized lability at low Ca2+ is consistent with our observation that hOtolC1q E470A is particularly difficult to produce in Escherichia coli. The E470A variant is the sole form of hOtolC1q studied here that required the addition of a detergent to remain soluble after cell lysis, and still the purification yield was much lower than the purification yield for native hOtolC1q. The concentration of Ca2+ in the cytoplasm of E. coli, which was shown to be in the range of 0.3–1.6 µM58, is too low to stabilize hOtolC1q E470A. In the context of recombinant protein production, an increased tendency to form aggregates at low Ca2+ would be especially detrimental, as it would facilitate the precipitation of the protein in the form of inclusion bodies59. E470 of human otolin-1 is conserved in fish. If such a mutation would have a similar effect on piscine otolin-1, the continuous growth of the otoliths could be disrupted in the event of catastrophic loss of endolymph Ca2+. However, as native dOtolC1q was stabilized at higher Ca2+ than hOtolC1q, such an event would likely also affect the fish not having this mutation.In silico predictions for the prevalent natural polymorphism E470A, involving a residue that was modelled to be away from the Ca2+-binding residues of hOtolC1q and dOtolC1q were, however, consistently predicted to be deleterious and functionally damaging for the protein. Our results show that the predictions were not fully correct. All forms of hOtolC1q and dOtolC1q with any residue from the Ca2+ binding site mutated formed more stable oligomers in the absence of Ca2+ than the native proteins. Significant deleterious effects of the mutations became apparent when the proteins were studied with 10–100 mM CaCl2. Under these conditions, the D432A mutant of dOtolC1q was unable to form stable trimers, contrary to native dOtolC1q. Interestingly, this mutant seemed to be accidentally optimized for the binding of Tb3+ relative to Ca2+, as the mutation increased the apparent affinity of the protein towards Tb3+. This mutant also formed stable trimers only in the presence of Tb3+ and not in the presence of Ca2+. The most important observation, however, is that although the mutation of the Ca2+ binding sites of hOtolC1q or dOtolC1q seemed to have a stabilizing effect, native hOtolC1q and dOtolC1q were the most thermally stable forms in the conditions simulating the interiors of otoliths and otoconia. Additionally, hOtolC1q E417A, hOtolC1q E470A and all mutants of dOtolC1q were difficult to handle in the laboratory: the expression yields were smaller than for the native forms, and protein loss during purification was significantly higher. The mutants were also difficult to concentrate. The highest achievable concentration of hOtolC1q was 4 mg/mL, and of dOtolC1q was 1.5 mg/mL. In contrast, hOtolC1q E417A and all mutants of dOtolC1q began to precipitate at approximately 0.5 mg/mL.Mutations of the conserved Ca2+ is bound at the axis of a gC1q domain trimer48. Mutation of the Ca2+-binding residues of the gC1q domain of C1q-like protein 3 (C1QL3) significantly decreased its thermal stability48. It was plausible to conclude that mutation of a Ca2+ binding site in the gC1q domain of otolin-1 could disrupt the formation of trimers due to the lack of a stabilizing effect of Ca2+. However, mutations of the acidic residues forming the Ca2+ binding site to alanine actually facilitated the trimerization of hOtolC1q and dOtolC1q, explained by the neutralization of the repulsive negative charge at the trimerization interface by replacing acidic residues with uncharged residues. Interestingly, a gC1q domain of collagen VIII also forms trimers in the absence of Ca2+27. In this case, the Ca2+ binding sites are occupied by side chains of lysine residues from the same subunits, also leading to neutralization of the negative charge. We conclude that Ca2+-dependent neutralization of a negative charge at the Ca2+-binding site is crucial for the formation of the gC1q domain trimer.Crystallographic data show that Ca3+ occurs, the binding of Tb3+ is known not to be fully equivalent to the binding of Ca2+, although these ions have similar ionic radii due to the higher charge and higher observed coordination number of Tb3+ complexes compared to Ca2+ complexes with proteins61. Moreover, changes in hydrogen bonding around the metal binding site were observed when lanthanide ions were bound instead of Ca2+ to compensate for the charge difference40. In our case, this inequivalence apparently leads to the formation of stable dOtolC1q D432A trimers in the presence of Tb3+ but not in the presence of Ca2+, possibly also the reason for severe destabilization of dOtolC1q in the presence of Tb3+.Although our data do not explain why an increased preference of dOtolC1q D432A for Tb3+-binding experiments, which seem to bind strongly to and thermally stabilize hOtolC1q, dOtolC1q and their mutants, it is surprising that the proteins were actually destabilized if we consider their solubility. For dOtolC1q, strong immediate precipitation in the presence of Tb3+ was observed at 0.5 mg/mL protein. During prolonged incubation, some precipitation was also observed at 0.2 mg/mL. Human isoforms seemed to better tolerate the presence of Tb3+. In the LRET experiment, Tb3+ seemed to bind to mutants that were not responsive to Ca2+. In the case of dOtolC1q, this did not lead to thermal stabilization of its nonbinding mutants. Tb3+ ions may have been bound away from the Ca2+ binding sites. Such “adventitious” binding of lanthanide ions at sites that do not bind Ca2+ was observed in the crystal structures of proteins and was characterized by a lower coordination number. The remaining coordination sites of lanthanide ions were speculated to be occupied by water40. In the case of dOtolC1q, the available coordination sites of Tb3+ could possibly become occupied by functional groups from other trimers and lead to the formation of heavy aggregates of dOtolC1q. Weak binding of Tb3+ at the adventitious sites could result in aggregation occurring at a relatively high protein concentration, above 0.2 mg/mL.In light of the results of Tb2+ in the formation of gC1q domain trimers is the neutralization of the negative charge at the Ca2+ binding site. Despite the apparently stabilizing effect of substitutions of acidic Ca2+-binding residues with neutral alanine residues, hOtolC1q and dOtolC1q were ultimately the most stable in their native forms saturated with Ca2+. We also determined that hOtolC1q and its mutants were more stable than dOtolC1q and its mutants under all tested conditions, including a Ca2+-free environment. Apparently, the human protein became optimized to function at lower concentrations of Ca2+. We conclude that this predisposes otolin-1 to years-long function in biomineral organic matrices.In summary, we discovered that the basis of the engagement of CaOTOL1 gene Ensembl accession ID: ENSG00000182447.Human OTOL1A gene Ensembl accession ID: ENSDARG00000001771.Zebrafish Human otolin-1 UniProt accession ID: A6NHN0.Zebrafish otolin-1 UniProt accession ID: A5PN28.Human otolin-1 E470A SNP variant ID: rs3921595.E. coli and provided by GeneArt . Nucleotide primers were provided by Genomed . The pQE-80 L plasmid expression vector was purchased from Qiagen . E. coli Top10 cells, Phusion DNA polymerase, restriction enzymes, T4 DNA ligase and LB broth were obtained from Thermo Fisher Scientific. One-fusion DNA polymerase was obtained from GeneOn . Agar, agarose, tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), carbenicillin, isopropyl β-d-1-thiogalactopyranoside (IPTG), NaCl, glycerol, 2-mercaptoethanol, imidazole, glycine, sodium dodecyl sulfate (SDS) and CaCl2 were obtained from Carl Roth . E. coli BL21(DE3) cells, TB broth, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), lysozyme, phenylmethylsulfonyl fluoride (PMSF), DNase I, RNase A, terbium(III) chloride hexahydrate, xylenol orange disodium salt and SYPRO Orange were from Sigma . The NP40 substitute was from Amresco . Empty Tricorn, Superdex 200 Increase 10/300 GL and Superdex 75 16/60 Prep Grade columns were obtained from GE Healthcare Life Sciences . TALON® Metal Affinity resin and 6xHis monoclonal antibody  were purchased from Takara Bio . HRP horse anti-mouse IgG antibody was purchased from Vector Laboratories .Synthetic cDNAs encoding full-length otolin-1 from human and zebrafish were codon-optimized for www.ensembl.org/) was queried for known single nucleotide polymorphisms (SNPs) in otolin-1 genes. The gene identifiers were ENSG00000182447 (OTOL1) for human otolin-1 and ENSDARG00000001771 (OTOL1A) for zebrafish otolin-1. Boundaries of gC1q domains were retrieved from the UniProt database (A6NHN0 for human otolin-1 and A5PN28 for zebrafish otolin-1) as 338–477 for human and 351–489 for zebrafish otolin-1.The Ensembl genome browser (https://sift.bii.a-star.edu.sg/)62 and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/)63 tools were chosen for comparisons, as these tools were also useful to predict the effects of artificial mutations for both hOtolC1q and dOtolC1q. Additionally, for all investigated mutations, the SNP MuSiC (https://soft.dezyme.com/) tool was used to predict the effects on protein stability64. Models of gC1q trimers, which were used as a template, were based on already published EOM analysis conducted on the basis of SAXS data33. Default parameters were used in all predictions. The structure model was visualized using VMD software 65. Sequences of otolin-1 from selected fish, birds and mammals were aligned using Clustal X66 and visualized using Jalview67.Natural and designed mutants were assessed using online tools to predict their effects on protein structure and function. Data for the natural E470A SNP accompanied a respective entry in the Ensembl database, from which the results for SIFT (Esp3I (BsmBI) restriction enzyme, which cleaves DNA 5 bp away from the recognition site. Cohesive ends of the cleavage products contained codons, which introduced the mutations. The gene fragments were subcloned into the pQE-80 L vector using BamHI and HindIII restriction sites. In the case of hOtolC1q E470A, synthetic cDNA of hOtolC1q subcloned into the pQE-80 L plasmid expression vector33 was used as a template in modified QuickChange®, which was conducted as described68. One-fusion DNA polymerase was used in the mutagenic PCR. For the calculation of the annealing temperatures of the primers, the concentration of KCl in the reaction mixture was assumed to be 0.1 M, as in the assay buffer of the polymerase. Plasmids were propagated in E. coli TOP10 cells. The progress of the cloning was followed by agarose gel electrophoresis. All mutated genes were analysed by DNA sequencing (Genomed).Genes coding the mutants of hOtolC1q: E417A, D425A and E417A D425A and of dOtolC1q: D432A, D440A and D432A D440A were prepared by amplifying the synthetic cDNAs of hOtolC1q and dOtolC1q in two parts using Phusion DNA polymerase. PCR products were digested using the Escherichia coli BL21(DE3) cells were chemically transformed by heat shock and grown overnight on plates containing LB broth with 1.5% agar and 100 µg/mL carbenicillin at 37 °C. Single colonies were picked and used to inoculate starter cultures containing 100 ml of TB broth with carbenicillin, which were incubated overnight at 37 °C and 200 rpm. Portions of 500 ml TB with carbenicillin were inoculated with a 2% volume of starter culture and incubated at 29 °C and 200 rpm. After reaching the optical density at 600 nm of 0.5, cultures were cooled to 15 °C, and the expression of the protein of interest was induced by 0.5 mM IPTG. The culture was continued overnight (16–18 h) at 15 °C and 200 rpm. Cells were collected by centrifugation at 5000×g at 4 °C for 15 min and resuspended in H10Na500G5 buffer  with 1 mM 2-mercaptoethanol and 1 mg/ml lysozyme. Suspensions of cells expressing hOtolC1q E470A were additionally supplemented with 0.1% NP40 substitute . The cells were kept frozen at − 80 °C.g for 30 min at 4 °C and incubated with 1 mL bed volume of TALON® Metal Affinity resin for 1 h in a cold room (4–6 °C) in an orbital mixer set at 5 rpm. The resin was separated by centrifugation at 700×g for 5 min at 4 °C, washed with 20 bed volumes of H10Na500G5, centrifuged again and packed in a glass Tricorn column. The column was connected to ÄKTA Avant chromatography system  with a flow rate of 1 mL/min. Contaminants were washed away with 20 bed volumes of H10Na500G5 and subsequently with 20 bed volumes of the buffer with 30 mM imidazole. The protein of interest was eluted with buffer containing 200 mM imidazole. The eluate was concentrated in Amicon Ultra centrifuge filters with a 10 kDa cut-off (Merck) and subjected to gel filtration using a Superdex 200 Increase 10/300 GL column operated at 0.75 mL/min (hOtolC1q and its mutants) or Superdex 75 16/60 Prep Grade operated at 1.5 mL/min (dOtolC1q and its mutants) with H10Na500G5 as a mobile phase. Eluate before void volume of the column was collected for use as a reference in further studies. Fractions containing proteins were collected and analysed using SDS-PAGE with an acrylamide percentage of 4% in a stacking gel and 12% in a resolving gel in a Laemmli buffer system (Tris–glycine-SDS)69. The identity of the protein was confirmed by Western blotting with murine 6xHis monoclonal IgG as the primary antibody and HRP horse anti-mouse IgG antibody as the secondary antibody. Pure protein samples were stored at − 80 °C.Cell lysis was initiated by thawing in a room-temperature water bath. After thawing, 0.2 mg/mL PMSF, 20 µg/mL DNase I and 20 µg/mL RNase A were added. Lysis was facilitated by 10 cycles of ultrasonic treatment for 30 s with 1-min breaks in a Cole-Parmer CPX 500 ultrasonic processor with a microtip and amplitude set at 35% . The cell suspension was kept on ice to maintain the temperature below 10 °C. Lysates were clarified by centrifugation at 18,500×3+ ions to hOtolC1q, dOtolC1q and their mutants was assessed using steady-state fluorescence. Terbium(III) chloride was dissolved in Milli-Q water to a final concentration of approximately 0.5 M. The exact concentration of TbCl3 was determined by titration of diluted stock solution with EDTA in the presence of xylenol orange. Aliquots of diluted TbCl3 were added to 2 ml 3.7 µM protein solution in a 10 × 10 mm quartz cuvette and incubated for 15 min at room temperature. Subsequently, fluorescence emission at 520–580 nm was recorded using an excitation wavelength of 280 nm using a Fluorolog-SPEX fluorimeter  equipped with a Peltier temperature control accessory set at 20 °C. The bandwidth was set at 5 nm for both excitation and emission monochromators. The fluorescence intensities obtained were processed and fitted to a model based on work by Gonzalez et al.71. Fluorescence intensity was corrected for sample dilution and background fluorescence using the following equation:Icorr—corrected fluorescence, Imax—fluorescence at emission maximum (here: 545 nm), Imax–15—fluorescence at local emission minimum (here: 530 nm), v0—initial sample volume, vi—added volume of working solution of TbCl3.The binding of TbKd:I0—corrected fluorescence intensity at 0 µM Tb3+ (fitted), cp—concentration of the protein, ct—concentration of Tb3+, a—proportionality constant (fitted).Corrected fluorescence was fitted to a single binding site model to determine the apparent dissociation constant Fitting of the data to the model above was conducted using Origin Pro 9.0 software .2, 1 mM CaCl2, 10 mM CaCl2 or 100 mM CaCl2 was measured in 1 mm quartz SUPRASIL® cuvettes  using a Jasco J-815 spectropolarimeter  with a Peltier temperature control accessory set at 20 °C. The proteins were incubated with EDTA/CaCl2 at room temperature for at least 1 h before the measurements. The spectra were collected between 200 and 260 nm every 1 nm at a scanning speed of 50 nm/min with five accumulations. Data for which the photomultiplier voltage was below 600 V were analysed. CD spectra of the proteins were corrected for buffer background signal and normalized for protein composition and concentration using the equation below72:MRW—mean residual weight of a protein [g/mol], c—protein concentration [g/L] and l—optical pathlength of a cuvette [cm].Circular dichroism of 0.2 mg/mL proteins in H10Na500G5 with 1 mM EDTA, 0.1 mM CaCl2. Assembled cells with the samples were preincubated in an ultracentrifuge for 3 h at 20 °C and then centrifuged at 50,000 rpm  overnight. Scans of absorbance at 280 nm were collected continuously with 0.003 cm resolution. The scans representing the whole sedimentation process were time-corrected73 and analysed in SEDFIT (available at https://sedfitsedphat.nibib.nih.gov/) using a continuous c(s) distribution model74 with at least 20 points per 1 S. Partial specific volumes of the proteins, densities and dynamic viscosities of the solvents were calculated using SEDNTERP (available at http://www.jphilo.mailway.com/download.htm). Maximum entropy regularization with p = 0.95 was used. Simplex and Marquardt–Levenberg algorithms were used until the RMSD value converged. Among the results of the calculations were sedimentation coefficients (s), sedimentation coefficients corrected for water at 20 °C , weight-averaged sedimentation coefficients (MWapp) and frictional ratios (f/f0). c(s) distributions were visualized using GUSSI (available at https://www.utsouthwestern.edu/labs/mbr/software/)75 and Origin Pro 9.0 software.Sedimentation velocity analytical ultracentrifugation (SV AUC) was conducted in a Beckman Coulter ProteomeLab XLI analytical ultracentrifuge  with an An60Ti rotor and assembled cells with two-channel 12 mm charcoal filled Epon® centerpieces and quartz windows. The proteins were analysed at approximate concentrations of 0.1, 0.25 and 0.5 mg/ml in H10Na500G5 cells with 1 mM EDTA or 10 mM CaCl76. Five micromolar solutions of the proteins in H10Na500G5 supplemented with 10 × SYPRO Orange (500-fold dilution of delivered stock solution) were measured in the presence of 1 mM EDTA, 0.1 mM CaCl2, 1 mM CaCl2, 10 mM CaCl2 or 100 mM CaCl2. Additionally, hOtolC1q and its mutants were measured in the presence of a sevenfold molar excess of TbCl3, dOtolC1q and its mutants in the presence of a 14-fold excess of TbCl3. Final sample volume was 20 µl. The samples were aliquoted into a 96-well plate in triplicate, covered with optically clear foil and incubated at room temperature for at least 1 h before the measurements. Fluorescence of SYPRO Orange was measured using an Applied Biosystems ImageQuant5 qPCR thermal cycler (Thermo Fisher Scientific) with optical filters set as x1 - m3  between 20 and 99 °C during heating at 0.033 °C/s. The data were analysed using Protein Thermal Shift software (Thermo Fisher Scientific). Tm were determined from the derivative of fluorescence with increasing temperature (dF/dT).Differential scanning fluorimetry assays were conducted as describedSupplementary Information.


Despite intensive chemotherapy regimens, up to 60% of adults with acute myeloid leukaemia (AML) will relapse and eventually succumb to their disease. Recent studies suggest that leukaemic stem cells (LSCs) drive AML relapse by residing in the bone marrow niche and adapting their metabolic profile. Metabolic adaptation and LSC plasticity are novel hallmarks of leukemogenesis that provide important biological processes required for tumour initiation, progression and therapeutic responses. These findings highlight the importance of targeting metabolic pathways in leukaemia biology which might serve as the Achilles’ heel for the treatment of AML relapse. In this review, we highlight the metabolic differences between normal haematopoietic cells, bulk AML cells and LSCs. Specifically, we focus on four major metabolic pathways dysregulated in AML; (i) glycolysis; (ii) mitochondrial metabolism; (iii) amino acid metabolism; and (iv) lipid metabolism. We then outline established and emerging drug interventions that exploit metabolic dependencies of leukaemic cells in the treatment of AML. The metabolic signature of AML cells alters during different biological conditions such as chemotherapy and quiescence. Therefore, targeting the metabolic vulnerabilities of these cells might selectively eradicate them and improve the overall survival of patients with AML. Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults , 2. It iDespite intensive treatment regimens, the median survival rate remains disappointingly low in adults and the majority of patients will eventually succumb to complications of drug treatment or disease relapse. Treatment outcome is significantly better in younger patients with a complete remission rate of ≥80%, however, due to relapse and refractory disease, there is still a 5-year overall survival rate of ~40%  cycle. This provides cancer cells with a high rate of ATP production to fuel rapid cell division . BesidesPKM2 impaired progenitor cell function without perturbing HSCs, while LDHA deletion significantly blocked the function of both HSCs and progenitors during haematopoiesis, demonstrating a key metabolic difference among distinct haematopoietic cells , a common leukaemic mutation that confers a poor prognosis in AML patients, glycolysis is associated with the pentose phosphate pathway to sustain a high flux of glucose for cell survival  in the mitochondria  by glutamate dehydrogenase as an auxiliary reaction to replenish TCA intermediates . In lined malate . In contd malate .R140Q mutations in vivo drive aberrant self-renewal activity and block differentiation in normal HSCs. Furthermore, co-mutant in vivo models of IDH2R140Q and FLT3-ITD induced acute leukaemia and were essential for leukaemia maintenance . However, it has been shown that somatic mutations in the active site of IDH1/2 in AML cells lead to the overproduction of the oncometabolite 2-D-hydroxyglutarate, which blocks differentiation and contributes to AML progression . Moreoventenance . Mutationtenance . These mntenance . In addintenance . In a sintenance .It was originally assumed that mitochondrial respiration was impaired in cancer cells as they mainly rely on glycolysis for survival . HoweverNormal HSCs maintain their quiescent state by reducing their mitochondrial respiration rate and relying on glycolysis . In line+ CD38− LSC population of primary AML samples  . In addiin vivo study, normal HSCs from healthy donors were resistant to apoptosis induction following glutamine removal. In contrast, depletion of glutamine by shRNA knockdown of SLC1A5, induced apoptosis in eight primary AML samples  is a multi-enzyme protein that catalyses FA synthesis. It is required for AML cell proliferation and was shown to be low in normal HSCs derived from healthy donors and highly overexpressed in AML patients. Inhibition of FASN with RNAi or epigallocatechin-3-gallate accelerated granulocytic differentiation in acute promyelocytic leukaemia cells and re-sensitised them to ATRA treatment . This prFatty acid oxidation (FAO) is another deregulated pathway in AML, playing an important role in promoting leukaemic cell survival and quiescence . FAO gen+ LSC population. These CD36+CD34+ cells are significantly enriched in gonadal adipose tissues of the BM which protects them from destructive effects of venetoclax and allows them to utilise FAO required for their maintenance . The anti-leukaemic activity of IACS-010759 is mediated by the AMPK pathway in OXPHOS-dependent primary AML blasts and cell lines . In sensThe pyruvate dehydrogenase complex is responsible for catalysing the carboxylation of pyruvate to acetyl-CoA. The pyruvate dehydrogenase complex acts as a central hub between three different metabolic pathways including glycolysis, the TCA cycle, and OXPHOS . HamperiAnother mitochondrial-related treatment approach is the exposure of AML cells to BCL-2 inhibitors. Venetoclax is a potent BCL-2 inhibitor that has shown activity in haematologic malignancies including both as a single agent or in combination with chemotherapeutics agents , 141. Trin vivo . HPLM contains biologically relevant levels of uric acid which inhibits de novo pyrimidine synthesis. Uric acid levels are higher in human blood than in mice serum and therefore can inhibit uridine monophosphate synthase, a crucial enzyme for pyrimidine synthesis. This has been shown to reduce the sensitivity of AML cells to chemotherapeutic drugs such as 5-fluorouracil and highlights the importance of the tumour microenvironment on cell metabolism and the efficacy of drugs  [Grant #: RSP-00122-19/20] to PC and National Health and Medical Research Council of Australia [NHMRC Fellowships APP1059804 and APP1157871] to RBL. We acknowledge the Research Training Program Scholarship (RTP) awarded to YM for study towards a PhD in the Faculty of Medicine. Figures were created with The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lyso-7 reduced cell contraction and accelerated relaxation while lowering diastolic Ca2+ and reducing Ca2+ transient amplitude. Lyso-7 also promoted abnormal ectopic diastolic Ca2+ events, which isoproterenol dramatically enhanced. Incorporation of Lyso-7 in NC attenuated drug effects on cell contraction and prevented its impact on relaxation, diastolic Ca2+, Ca2+ transient amplitude, Ca2+ transient decay kinetics, and promotion of diastolic Ca2+ events. Acute effects of Lyso-7 on cardiomyocytes in vitro at high concentrations (450 nM) were globally similar to those observed after repeated administration in vivo. In conclusion, we show evidence for off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations and after repeated treatment in mice. Nano-encapsulation of Lyso-7 in polymeric NC attenuated the unwanted effects, particularly ectopic Ca2+ events known to support life-threatening arrhythmias favored by stress or exercise.Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,β/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5–450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo . In previous work, we characterized Lyso-7-NC. We administered intravenously Lyso-7, Lyso-7-NC, control, and blank-NC once a day for seven days in mice. We assessed cell contraction and intracellular Ca Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family and function as ligand inducible transcription factors to regulate genes involved in lipid and glucose metabolism ,2. Thiaz2+ levels in neutrophils -3-(4-chlorobenzyl)-thiazolidine-2,4-dione] (CAS Number 1505484-42-3) (CAS Registry Number 1505484-42-3), Mw 447.73 g/mol, was synthesized, purified and characterized by Prof Ivan da Rocha Pitta as reported . PLA polv/v. We used this solution without Lyso-7 as a control in the acute experiments performed in vitro with isolated cardiomyocytes. We prepared the free Lyso-7 solution for intravenous (iv) treatment of mice as follows: we dissolved Lyso-7 in 20 μL of DMSO, 20 μL of polysobate 80, 380 μL of DMA, and 580 μL of PEG 300. This solution was mixed for 10 min and then appropriately diluted in isotonic glucose to obtain 0.5 mg/mL of free Lyso-7 solution used in vivo. We used the same solution without Lyso-7 as a control in vivo.Lyso-7 was dissolved in absolute ethanol further diluted in buffers used in each experiment with ethanol concentration no higher than 2% wt/v of PLA, 0.75% v/v of Miglyol 810N), and 0.75% wt/v of soybean lecithin dissolved in 10 mL of acetone. We added this organic solution into 0.75% wt/v of Poloxamer 188 solution (20 mL) via a syringe. We maintained dispersion under magnetic stirring for 10 min, and then all organic solvents and part of the water were evaporated under reduced pressure  to render 5 mL of the aqueous suspension of NC.We used the nanoprecipitation method to make monodispersed nanocapsules loading Lyso-7 (Lyso-7-NC) or without Lyso-7 (blank-NC) as previously described . We prepThe mean hydrodynamic diameters were 296 ± 4.2 nm and 265 ± 1.7 nm, respectively, with dispersion indexes less than 0.3 indicating the monodispersed population of particles in size. NC encapsulated Lyso-7 with high efficiency (83%) at a 0.5 mg/mL Lyso-7 concentration, attributed to Lyso-7 high lipophilicity. We have already characterized these NC in detail . We usedad libitum access to water, and behavioral needs . For repeated-dose intravenous administration (via tail vein), mice were blindly randomized to the four different groups: vehicle (intravenous solution diluted in isotonic glucose), Lyso-7 (1.6 mg/kg/day), blank-NC , and Lyso-7-NC (1.6 mg/kg/day). We administered mice vehicle and blank-NC as control groups for Lyso-7 and Lyso-7-NC, respectively, for comparisons. Repeated doses of both Lyso-7 and Lyso-7-NC for seven days produced no death in mice. We isolated myocytes freshly from mice of all experimental groups after the seven days of repeated administrations, as shown in We used 7-week-old male C57BL/6J mice  bred and housed (4 mice/cage) under pathogen-free conditions  with standard rodent chow diet, We assessed the acute effects of Lyso-7 in vitro following the application of 4.5 nM, 45 nM, and 450 nM of Lyso-7 for 15 min on single cardiomyocytes isolated from untreated animals. The hearts were excised after euthanasia of mice by cervical dislocation to ensure rapid death without injecting any substance interfering with cellular physiology. Whole hearts were submitted to the liberase action using a Langendorf perfusion system to obtain individual LV myocytes as described ,22,23,242+ transients recorded simultaneously using an IonOptix system  with a Zeiss microscope   as described [2, 1.8 CaCl2, 4.4 NaHCO3, 1.5 KH2PO4, and 21 HEPES (pH 7.4); all chemicals bought from Sigma-Aldrich, Saint-Quentin-Fallavier, France. We loaded cells with the dual-emission ratiometric Ca2+ indicator Indo-1AM  to monitor cytosolic Ca2+ determined by the ratio of 405 nm/480 nm fluorescence (a.u.: arbitrary units) [2+ diastolic events and contractions. In some experiments, we mimicked stress conditions by exposing cells to the β-adrenergic agonist isoproterenol  for 5 min before the experiments. Data were analyzed using Ionwizard® Software .We field-stimulated myocytes with 1-ms current pulses delivered at 1 Hz to assess cell sarcomere length (SL) shortening, an index of contraction, and intracellular Caescribed ,22,23,24y units) ,22,23,242+ sparks in quiescent myocytes incubated with the Ca2+ indicator Fluo-4AM (4 μM) , visualized by confocal imaging with a Zeiss LSM510 microscope  equipped with 63×/1.2 N.A. water immersion objective, at 25 °C in line-scan mode  as described [2+ sparks were determined by averaging the intensity of each sequential scan line and plotting the mean intensity as a function of time. We used the SparkMaster plugin for ImageJ software  to detect and analyze Ca2+ sparks.We visualized Caescribed ,22,23. T® Software . We expressed all data as mean ± SEM. We used D’Agostino and Pearson omnibus and Shapiro–Wilk normality tests, then a One-way ANOVA test for multiple comparisons. We performed post hoc tests where F was significant (p < 0.05) and no variance inhomogeneity. We used a Tukey post-test to compare all pairs of columns. We used a t-test for Ca2+ waves and Ca2+ sparks. p < 0.05 indicated a significant difference. N indicates the number of mice, and n indicates the number of independent cardiomyocytes isolated from these mice.We performed statistical analyses using GraphPad Prism2+ transient evoked by electrical stimulation of LV myocytes at 1 Hz. No mice died under treatment with the formulations. First, it is worth noting that blank-NC did not affect any parameter relative to the control group (2+ (−5%), reduced the Ca2+ transient amplitude (−35%) and accelerated the Ca2+ transient decay kinetics (16%) (p < 0.05), and the impact of Lyso-7 on cell relaxation was prevented .We investigated the effects of the repeated administration of Lyso-7 (free form) on contraction and Caol group A–D. In pcs (16%) E–H. Howerevented D as well2+ transient amplitude and accelerated Ca2+ transient decay kinetics in line with enhanced reuptake of Ca2+ in the sarcoplasmic reticulum (SR) via the SERCA2a pump [2+ transient amplitude by 81%, and accelerated Ca2+ transient decay kinetics by 60%. Overall, these effects were consistent with those reported in the literature [2+ and accelerated the Ca2+ transient decay kinetics  by encapsulation in NC.We next evaluated the effects of Lyso-7 under the challenge of a maximally active concentration of the β-adrenergic receptor agonist isoproterenol  to mimic the effects of physical exercise and stress . We expoA2a pump . For exaterature . Howeverkinetics D,F, simikinetics F,H excep compare E. The di2+ events, not observed in the control and NC groups, during resting periods following a train of stimulations , as such, favored their occurrence (2+ events (80% vs. 10% in the absence of ISO), which was abolished in the Lyso-7-NC group  (2+ transient (−45%)  E,F and rt (−45%) E,G. In cransient H.2+ transient (+80%), the diastolic Ca2+ (+12%), and accelerated the Ca2+ transient decay (+40%)  and the Ca2+ transient amplitude  approximately twice, except at 450 nM (+25%) due to the intrinsic positive effect of Lyso-7 at this concentration, and enhanced relaxation (+30%) in control myocytes  leakage, generating microscopic Ca2+ events called Ca2+ sparks [2+ sparks . In the presence of ISO, all cells exhibited spontaneous diastolic Ca2+ events at 450 nM , suggesting different mechanisms or that cell physiology has been impacted during the repeated administration. Nevertheless, Lyso-7 had many similar effects and profoundly modified the Ca2+ homeostasis of the myocytes when administered acutely and directly on the cardiomyocytes or repeatedly to mice. Lyso-7 lowered the resting Ca2+, decreased the amplitude of the Ca2+ transient, and accelerated its decay kinetics. Finally, Lyso-7 promoted abnormal firing of Ca2+ events during diastolic periods in both experimental conditions.A significant result of our study was evidence for some off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations or after repeated treatment in vivo. Indeed, most of the adverse effects, namely on contraction, intracellular Ca2+ sparks reflect discrete, elementary Ca2+ signaling events arising from abnormally leaky RyR2 channels [2+ waves. From a mechanistic point of view, the processes involved here are unclear. RyR2 leakage may participate in depleting SR Ca2+ content, therefore explaining the decrease of Ca2+ transient amplitude. However, since this depletion should increase intracellular Ca2+, it was counterintuitive to find that Lyso-7 decreases diastolic Ca2+. The Ca2+ levels are controlled mainly by the balance between uptake of Ca2+ via SERCA2a and Ca2+ release of the RyR2 receptors. In case of imbalance, abnormal diastolic Ca2+ discharge activates the electrogenic Na+/Ca2+ exchanger (NCX), producing delayed afterdepolarization and potentially ectopic Ca2+ transients [+/Ca2+ exchanger (NCX) activity, essential for removing intracellular Ca2+ during relaxation [2+.Cachannels ,28. Theyansients ,27,30. Tlaxation , to acco2+ handling parameters, but it dramatically enhanced its impact on the firing of macroscopic Ca2+ events as well as Ca2+ sparks at rest. Low RyR2 leakage is insufficient per se to produce arrhythmogenic diastolic Ca2+ release, particularly with enhanced NCX-mediated Ca2+ extrusion, because of the depletion in the SR Ca2+ content [2+ content, consistent with Ca2+ transient decay acceleration found here, is known to enhance RyR2 open probability and the associated arrhythmia risks [2+ waves and Ca2+ sparks . The amount of Ca2+ released depends on the amount of Ca2+ stored in the SR [2+ transient amplitude, which was paradoxical. Since this latter parameter is a crucial determinant of contraction, another mechanism must be responsible for the positive inotropy. An increase in the sensitivity of the contractile proteins to Ca2+ is a likely candidate. We did not investigate the underlying mechanism because that was not the main objective of our study. ISO could not prevent, or compensate for, the impact of Lyso-7 on Ca2+ handling parameters such as the decrease in diastolic Ca2+ and Ca2+ transient amplitude. Overall, our data suggest that Lyso-7 has multiple molecular off-targets, direct targeting of contractile protein activity being a likely possibility in addition to Ca2+ handling. Moreover, the divergent effect on contraction, particularly the decrease resulting from repeated exposure of cells to Lyso-7 in vivo, may reflect a long-term impact of the molecule on this parameter. This possibility warrants further thorough investigation on the effect of more prolonged, repeated administration to investigate whether morphological and functional changes occur during chronic exposure at the whole organ level and whether NC ensures long-term cardioprotection.The effects of Lyso-7 were complex, probably involving several mechanisms. In contrast with its impact on Can the SR . The pos2+ handling, spontaneous Ca2+ events, and contraction of cardiomyocytes. The overall result was somewhat unexpected because the administration of Lyso-7 encapsulated in polymeric NC increases Lyso-7 body exposure by 14-fold and heart concentration by 3.5-fold, respectively, according to the pharmacokinetics and biodistribution studies [β/δ or PPAR-γ [The most important result of our study was the safety provided by the administration of Lyso-7 in the NC dosage form for seven days. Indeed, Lyso-7 in NC was devoid of cardiotoxicity for most parameters investigated at the cellular level. Lyso-7-NC had only little effect on Ca studies . The lip studies . From our PPAR-γ ,13.2+ events known to support life-threatening Ca2+-dependent ventricular arrhythmias potentially favored during stress or exercise. The interest of this concept can extend to all TZD to treat type 2 diabetes because of risks of increased HF and mortality incidence [The association of Lyso-7 with PLA-NC is promising to provide a potential therapeutic option devoid of significant adverse cardiac effects in line with previous findings from our group showing that NC can attenuate the toxicity of artemether and lychnopholide both at the heart and cardiomyocytes levels ,19,20. Tncidence  and all


Atypical fibroxanthoma is an infrequent, low-grade superficial cutaneous neoplasm, usually presenting as a nodule or plaque of red color. It is considered as a superficial variant of pleomorphic dermal sarcoma. Although atypical fibroxanthoma has similar histologic features to pleomorphic dermal sarcoma, it has less aggressive behavior. Atypical fibroxanthoma usually occurs on sun-exposed regions of the head and neck of elderly patients. Ultraviolet light, specific genetic mutations and administration of immunosuppressive agents to transplant recipients have been associated with the pathogenesis of the tumor. The prognosis is typically excellent when treated with complete excision of the primary lesion. This report describes the rare case of a 84-year-old man with hemoptysis due to metastatic cutaneous atypical fibroxanthoma.  The term atypical fibroxanthoma of skin was described for the first time in 1963 in a group of cases by Helwig et al . It is aAtypical fibroxanthoma represents a low-grade neoplasm of atypical spindle cells with intermediate malignant behavior. Differential diagnosis includes squamous cell carcinoma, melanoma, angiosarcoma, leiomyosarcoma and dermatofibrosarcoma protuberans . UltraviAtypical fibroxanthoma is included in the same spectrum with pleomorphic dermal sarcoma, as both tumors are characterized by similar etiologic, histologic and clinical factors. The terminology and diagnostic criteria for the two tumors continue to be controversial. Tumors sited more superficially, with minimal subcutis involvement, can be called atypical fibroxanthoma, while pleomorphic dermal sarcoma has features like several atypical mitoses, perineural, or perivascular invasion and is associated with a poor prognosis and higher rates of metastasis . FactorsAtypical fibroxanthoma is rarely associated with distant metastases . CompletHerein we describe a rare case of a 84-year-old man with pulmonary metastases from cutaneous atypical fibroxanthoma presenting with hemoptysis. A 84-year-old man presented to our Pulmonology Department with cough accompanied with bloody sputum over the last month and weight loss, approximately 15 kg, over the last year. The patient had a medical history of coronary artery disease with percutaneous coronary intervention (PCI) and surgically resected head cutaneous atypical fibroxanthoma one year ago without abnormal findings at excision region within 6 months follow-up. The clinical examination was without remarkable signs.Complete blood count revealed normochromic normocytic anemia and the coagulation tests were normal. The other blood biochemistry parameters were normal, with the exception of an elevated serum lactate dehydrogenase (LDH) 501 U/L . Chest X-ray revealed multiple pulmonary nodules and left pleural effusion . The patient underwent computed tomography (CT) of the chest and abdominal CT. Chest CT showed several nodules of various size in all lung fields, ground glass opacities and a small left pleural effusion , while fBronchoscopy with bronchoalveolar lavage (BAL) was performed without abnormal findings. CT-guided fine needle biopsy of a lesion in the left lower lobe was conducted. Histopathological and examination of biopsy specimen demonstrated partially necrotic malignant neoplasm of spindle cells with evidence of recent and old hemorrhage. Immunohistochemical staining revealed that this neoplasm was positive for CD10, S100 and CD99. CK8/18, p63, GATA3, HMB45, CD34, podoplanin, CD68, Hepaticyte D, Desmin and PSA were negative. Morphological and immunophenotypic findings supported the diagnosis of metastatic cutaneous atypical fibroxanthoma. The patient declined to receive any specific therapy. He died two months after the diagnosis.Pulmonary metastasis from cutaneous atypical fibroxanthoma was described for the first time in 2003 by Kargi et al. . Very feKargi et al. described a case of a 68-year-old man with was diagnosed with atypical fibroxanthoma in his right thigh and presented with respiratory symptoms within 6 months after surgical excision. Chest CT revealed pulmonary metastasis and histological examination of the lesion, that was obtained with CT-guided fine needle biopsy, demonstrated metastasis from atypical fibroxanthoma . ArmstroSheth et al. reported a case of a 41-year-old woman with atypical fibroxanthoma of her left ankle. Staging fluorodeoxyglucose positron emission tomography (FDG PET)/CT revealed local extension of the lesion to bone and metastases to lung parenchyma, lymph nodes, bone marrow and muscles . Satter Thirty-one months later, the patient presented with cough, fever and imaging revealed loculated pleural effusion that did not respond to antibiotics administration and a lung biopsy demonstrated metastatic atypical fibroxanthoma to the lung . Wang et al., in their review of 152 cases of atypical fibroxanthoma from their institution and from a consultative dermatopathology practice, reported 7 cases of atypical fibroxanthoma, with minimal or no subcutaneous involvement who presented with lung metastases . WollinaThis is a rare case of pulmonary metastases caused by metastatic cutaneous atypical fibroxanthoma in patient presenting with hemoptysis. Clinicians should be aware of this potentially aggressive tumor and should take into consideration the possibility of recurrence and subsequent metastases.