The Leukemic Isocitrate Dehydrogenase (IDH) 1/2 Mutations Impair Myeloid and Erythroid Cell Differentiation of Primary Human Hematopoietic Stem and Progenitor Cells (HSPCs)

Simple Summary Acute myeloid leukemia (AML) arises from a stepwise acquisition of multiple genetic alterations in the hematopoietic stem and progenitor cell and is characterized by an accumulation of immature leukemic cells, called blasts, in the bone marrow and tissues. How the single gene alterations mediate the underlying processes is unclear in the majority of cases. IDH1/2 mutations are among the most frequent mutations in AML, accounting for about 15–20%, and are targets of specific small molecule inhibitors. Experimental evidence suggests that they occur early in leukemogenesis. Mostly, murine models of IDH1/2 mutations have been studied. Our study is the first to highlight that—in the human system—IDH1/2 mutants drive a complete block of hematopoietic cell differentiation in vitro, which is completely unleashed by the specific inhibitor, providing a model for early leukemogenesis studies in this setting. Abstract How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.


Introduction
Acute myeloid leukemia (AML) arises from genetic abnormalities in hematopoietic stem or progenitor cells (HSPCs), and it is responsible for uncontrolled growth and accumulation of neoplastic blasts in bone marrow (BM).AML is an aggressive, clinically and biologically heterogeneous disease, in which a large number of recurrently mutated genes have been identified [1].A stepwise acquisition of more than one alteration is required for leukemia development, although it is not always clear how single mutations contribute to leukemogenesis [2,3].
Isocitrate dehydrogenases are homodimeric enzymes that play an important role in cellular metabolism and the regulation of the response to hypoxia [15].Physiologically, the isoform IDH1, localized in the cytoplasm and peroxisomes, is involved in lipid and carbohydrate metabolism, providing NADPH to cells and protecting them from reactive oxygen species.IDH2, on the other hand, is localized in the mitochondrial matrix and participates in the tricarboxylic acid cycle to protect cells from oxidative damage.Both isoforms catalyze the oxidative decarboxylation of isocitrate, generating α-ketoglutarate (αKG) and carbon dioxide (CO 2 ), and producing reduced NADPH from NADP+, a process that is essential for preserving cellular homeostasis [16].
The bona fide correlation between mutations and leukemogenic mechanisms has prompted intense drug discovery, targeting the mutant IDH.In particular, two first-inclass small molecule inhibitors have been approved by the FDA: enasidenib (AG-221) and ivosidenib (AG-120) [26,27].Enasidenib is a selective allosteric inhibitor of the IDH2 mutation: its function involves the binding and stabilization of the open conformation of the IDH-mutated enzyme, and, primarily, the inhibition of the conversion of αKG to 2-HG2 [19,28].Ivosidenib is a reversible, allosteric competitive inhibitor of the mutated IDH1.Ivosidenib competes for binding with the magnesium ion, an essential cofactor for the IDH1 mutant, thereby preventing the formation of a catalytically active site [19,28].
IDH1/2 mutations and the other genetic lesions in genes encoding for epigenetic modifiers (DNMT3A, ASXL1, and TET2) are early events in the development of AML, like those observed in deep sequencing and single-cell studies determining clonal evolution in myeloid malignancies [11,29].These mutations are typically found in the founding clone of AML and are rarely found alone [11], suggesting that they are not sufficient to drive leukemia.Effectively, numerous studies in mouse models have demonstrated that IDH mutations alone cannot cause overt leukemia in vivo [30,31] but cooperate with additional genetic lesions to initiate cancer [32][33][34][35][36][37][38].The direct effects of single IDH AML-associated mutations on human HSPCs are poorly studied, so it remains unclear how they affect cell fate decisions, promote leukemogenesis, and contribute to the maintenance and progression of the malignant phenotype.Although some mouse model studies have elucidated how IDH mutations affect mouse HSPCs [30][31][32][33][34], specific studies in humans are needed to address the species-specific differences in basic biology and hematology.
Here, in order to investigate the direct effects of these mutations on the HSPC, we express IDH1-R132H or IDH2-R140Q mutants into human CD34+ cells from healthy donors via lentiviral transduction; we analyze changes in their hematopoietic fitness using the colony-forming unit (CFU) assay, which allows measuring the proliferation and differentiation abilities of individual hematopoietic stem cells and evaluates the effects of specific inhibitors, ivosidenib and enasidenib, respectively.Moreover, we studied the fitness dynamics of CD34+ cells isolated directly from a patient with IDH2-mutated AML undergoing treatment with the IDH2-inhibitor enasidenib.

Sequences and Expression Vector
Wild-type coding sequences used for lentiviral transduction are as follows: -Homo sapiens isocitrate dehydrogenase (NADP(+)) All sequences were synthesized and provided by GenScript ® (Piscataway, NJ, USA) into the pLVX-EF1α-IRES-ZsGreen1 (Takara Bio, Shiga, Japan) vector, in which a bicistronic RNA is transcribed by the same promoter and the internal ribosome entry (IRES) site allows for internal/cap-independent translation initiation, obtaining the coordinated expression of genes of interest (GOIs) and the reporter gene (ZsGreen1).

hCD34+ Lentiviral Infection
Lentiviral infections of CD34+ primary cells were conducted according to the RetroNectin ® (Takara Bio Inc., Kusatsu, Japan) protocol guidelines, which were optimized for our needs.For each well of a 6-well plate, 2 mL of growth HSPC-retention medium was added along with lentiviral 100× concentrate supernatant of IDH1 wild-type/R132H, or IDH2 wild-type/R140Q, or an empty vector at a multiplicity of infection = 30.The experimental conditions are illustrated in Supplementary Table S1.The plate was centrifuged at 2000× g for 2 h at 32 • C for virus pre-loading.The target cells CD34+ were counted and resuspended in HSPC-retention medium at 2 × 10 6 /mL and 1 mL per well was added.The plate was then centrifuged at 1800 RPM for 45 min at 32 • C and then incubated at 37 • C in a 5% CO 2 atmosphere for 72 h.

hCD34+ Cell-Staining and Sorting Strategies
After incubation, cells were harvested, washed with PBS, and stained for 20 min in the dark at RT with APC/Cyanine7 anti-human CD34 antibody.Cells were re-suspended in PBS, HSA (0.5%), and EDTA (2 mm) before sorting.An unstained control was made to set up forward and side scatter parameters on the cell sorter and to set positive gates.The cells were sorted by FACS using a BD FACSAria™ III cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA).Transduced bright ZsGreen1+/CD34+ double-positive cells expressing either the empty vector as the control, IDH1/2 WT, or IDH1-R132H/IDH2-R140Q were used for CFU assays in methylcellulose medium.In our 13 human CD34+ cell samples transduced with GOIs, we achieved ZsGreen1 reporter gene cell positivity.The sorting strategy is depicted in Supplementary Figure S1.

CD34+ Cells from a IDH2-Mutated AML Patient
Primary cells from a patient with IDH2-mutated R140Q AML were obtained, upon written informed consent, at the Hematology Institute of Perugia (Italy).The study was conducted according to the Declaration of Helsinki and following a protocol approved by the local Ethics Committee.Samples were obtained from BM aspirates performed at diagnosis and under therapy with enasidenib (AG-221) at 100 mg/die (for 28 days = 1 cycle), administered as third-line chemotherapy, on day 0 (before starting the treatment), and after the I, II, and IV therapy cycles.Mononuclear cells were separated by Lymphoprep™ (Serumwerk Bernburg AG for Alere Technologies AS, Oslo, Norway) according to the manufacturer's protocol.APC/Cyanine7 anti-human CD34 antibody staining was performed and CD34+ cells were isolated by the cell sorter.Sorted CD34+ AML cells were plated in MethoCult™-enriched medium for CFU assays, and incubated for 14 days at 37 °C, in a 5% CO2 atmosphere, with images captured by STEMvision™ as described above.

Next-Generation Targeted-DNA Sequencing (NGS)
Determination of the IDH2-mutant allele frequency was carried out by extracting DNA from the primary IDH2-R140Q AML BM sample (bulk, myeloid, erythroid, and CD34+ cells) at the fourth cycle of enasidenib therapy.The DNA sample collected for diagnostic procedures was used for DNA sequencing analysis to obtain a detailed overview of DNA mutations in our AML patient.The DNA concentration was assessed using a fluorometric quantitation via the Qubit 3.0 fluorometer (Thermo Fisher Scientific cat.no.Q33216, Waltham, MA, USA), while the DNA integrity numbers (DINs) were evaluated by microfluidic electrophoresis on an Agilent TapeStation using Agilent High Sensitivity D1000 ScreenTape System (Agilent, Santa Clara, CA, USA).The Qiagen Myeloid Neoplasm sequencing panel (Qiagen, Germantown, MD, USA, cat.nr.333502) was used to prepare libraries using 40 ng of DNA from each sample according to the manufacturerʹs instructions.The sequencing process was carried out on the MiSeq Illumina platform, generating 150 base-paired end reads.Variant calling was performed using the GeneGlobe online data analysis platform (Qiagen, USA).

Statistical Analysis
For the transduced hCD34+ CFU assay, technical replicates of 4 wells for each condition have been foreseen and subsequent statistical analysis was performed on the mean of a minimum of 3 wells per sample.A graphical representation of data was performed using GraphPad Prism [v7].Statistical analyses were performed by the one-way ANOVA test (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

CD34+ Cells from a IDH2-Mutated AML Patient
Primary cells from a patient with IDH2-mutated R140Q AML were obtained, upon written informed consent, at the Hematology Institute of Perugia (Italy).The study was conducted according to the Declaration of Helsinki and following a protocol approved by the local Ethics Committee.Samples were obtained from BM aspirates performed at diagnosis and under therapy with enasidenib (AG-221) at 100 mg/die (for 28 days = 1 cycle), administered as third-line chemotherapy, on day 0 (before starting the treatment), and after the I, II, and IV therapy cycles.Mononuclear cells were separated by Lympho-prep™ (Serumwerk Bernburg AG for Alere Technologies AS, Oslo, Norway) according to the manufacturer's protocol.APC/Cyanine7 anti-human CD34 antibody staining was performed and CD34+ cells were isolated by the cell sorter.Sorted CD34+ AML cells were plated in MethoCult™-enriched medium for CFU assays, and incubated for 14 days at 37 • C, in a 5% CO 2 atmosphere, with images captured by STEMvision™ as described above.

Next-Generation Targeted-DNA Sequencing (NGS)
Determination of the IDH2-mutant allele frequency was carried out by extracting DNA from the primary IDH2-R140Q AML BM sample (bulk, myeloid, erythroid, and CD34+ cells) at the fourth cycle of enasidenib therapy.The DNA sample collected for diagnostic procedures was used for DNA sequencing analysis to obtain a detailed overview of DNA mutations in our AML patient.The DNA concentration was assessed using a fluorometric quantitation via the Qubit 3.0 fluorometer (Thermo Fisher Scientific cat.no.Q33216, Waltham, MA, USA), while the DNA integrity numbers (DINs) were evaluated by microfluidic electrophoresis on an Agilent TapeStation using Agilent High Sensitivity D1000 ScreenTape System (Agilent, Santa Clara, CA, USA).The Qiagen Myeloid Neoplasm sequencing panel (Qiagen, Germantown, MD, USA, cat.nr.333502) was used to prepare libraries using 40 ng of DNA from each sample according to the manufacturer's instructions.The sequencing process was carried out on the MiSeq Illumina platform, generating 150 base-paired end reads.Variant calling was performed using the GeneGlobe online data analysis platform (Qiagen, USA).

Statistical Analysis
For the transduced hCD34+ CFU assay, technical replicates of 4 wells for each condition have been foreseen and subsequent statistical analysis was performed on the mean of a minimum of 3 wells per sample.A graphical representation of data was performed using GraphPad Prism [v7].Statistical analyses were performed by the one-way ANOVA test (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Either the IDH1-R132H or IDH2-R140Q Mutation Blocks CFU Ability of Human CD34+ HSPC
To investigate the direct effects of recurring AML IDH1/2 mutations, hCD34+ cells from HD were transduced by a lentiviral expression vector, containing either IDH1 wildtype or IDH1-R132H, along with a ZsGreen1 reporter gene (n = 7 donors), or containing either IDH2 wild-type or IDH2-R140Q, also with a ZsGreen1 reporter gene (n = 6 donors) (Supplementary Table S1), and sorted as described in the Materials and Methods section.
ZsGreen1-positive sorted cells were used for CFU assays, either in the presence or absence of the selective inhibitor AG-120 (n = 3 out of 7 donors for IDH1 experimental set) or AG-221 (n = 4 out of 6 donors in IDH2 set) (Figure 2).
To investigate the direct effects of recurring AML IDH1/2 mutations, hCD34+ cells from HD were transduced by a lentiviral expression vector, containing either IDH1 wildtype or IDH1-R132H, along with a ZsGreen1 reporter gene (n = 7 donors), or containing either IDH2 wild-type or IDH2-R140Q, also with a ZsGreen1 reporter gene (n = 6 donors) (Supplementary Table S1), and sorted as described in the Materials and Methods section.
ZsGreen1-positive sorted cells were used for CFU assays, either in the presence or absence of the selective inhibitor AG-120 (n = 3 out of 7 donors for IDH1 experimental set) or AG-221 (n = 4 out of 6 donors in IDH2 set) (Figure 2).
Colonies were assessed at 14 days.In our control groups, cloning efficiency from hCD34+ expressing the empty vector was comparable to hCD34+ bearing IDH1 wild-type or IDH2 wild-type and was not significantly influenced by treatment with ivosidenib or enasidenib.Instead, hCD34+ with the IDH1-R132H mutation showed an evident block of differentiation compared to hCD34+ transduced with the empty vector (p = 0.0002 ***) or wild-type gene (p = 0.0025 **) counterparts.This block was even more pronounced with the IDH2-R140Q mutation when compared with an empty vector (p < 0.0001 ****) or with the wild-type (p = 0.0004 ***) exogenous expression.In line with being a specific effect of the IDH1 or IDH2 mutations, the block was released, in both cases, by AG-120 (p = 0.0257 *) or AG-221 (p = 0.0028 **), respectively (Figure 2).Interestingly, no differences in the proportions of erythroid (BFU/CFU-E), myeloid (CFU-G/M/GM), and mixed (CFU-GEMM) colonies were observed in the different Colonies were assessed at 14 days.In our control groups, cloning efficiency from hCD34+ expressing the empty vector was comparable to hCD34+ bearing IDH1 wild-type or IDH2 wild-type and was not significantly influenced by treatment with ivosidenib or enasidenib.Instead, hCD34+ with the IDH1-R132H mutation showed an evident block of differentiation compared to hCD34+ transduced with the empty vector (p = 0.0002 ***) or wild-type gene (p = 0.0025 **) counterparts.This block was even more pronounced with the IDH2-R140Q mutation when compared with an empty vector (p < 0.0001 ****) or with the wild-type (p = 0.0004 ***) exogenous expression.In line with being a specific effect of the IDH1 or IDH2 mutations, the block was released, in both cases, by AG-120 (p = 0.0257 *) or AG-221 (p = 0.0028 **), respectively (Figure 2).
Interestingly, no differences in the proportions of erythroid (BFU/CFU-E), myeloid (CFU-G/M/GM), and mixed (CFU-GEMM) colonies were observed in the different con-ditions for both IDH-analyzed mutations, suggesting that the block of differentiation and release induced by the specific inhibitors affect all three lineages (Figure 3).conditions for both IDH-analyzed mutations, suggesting that the block of differentiation and release induced by the specific inhibitors affect all three lineages (Figure 3).

Enasidenib (AG-221) Treatment Induces a Progressive Improvement of CFU Ability of Primary CD34+ Cells in a Patient with IDH2-Mutated AML
In order to evaluate the dynamics of fitness of HSPCs in terms of their ability to form colonies, we isolated and studied CD34+ cells from the bone marrow of a patient with IDH2-R140Q-mutated AML, and we had the opportunity to follow under treatment with enasidenib.Specifically, we performed CFU assays with AML patient-derived CD34+ cells either at baseline or at the end of cycles I, II, and IV.Moreover, with cells obtained after the IV cycle, we explored the effect of adding pharmacological AG-221 inhibition in vitro during culture to assess whether maintaining the drug exposure in culture could enhance their CFU ability.Colonies were evaluated at 14 days as described above (Figure 4).Strikingly, we observed that in vivo treatment with enasidenib induced a progressive increase, over time, in the CFU capacity of the patient-derived CD34+ cells, whose fitness appeared progressively improved in terms of developing mature hematopoietic precursors across the different lineages, a phenomenon that was further strengthened when drug exposure was maintained in vitro in semi-solid culture (Figure 4).This occurred in parallel with the hematologically complete remission of the patient.
Remarkably, the NGS analysis performed after cycle IV, with respect to the diagnosis, showed the persistence of the IDH2-R140Q mutation, on the one hand, in the bulk, as well as in isolated myeloid and erythroid cells (variant allele frequency, VAF = 51% for both subpopulations), and on the other hand, in the CD34+ subpopulation (VAF = 52%) used for the CFU assay (Figure 4).

Enasidenib (AG-221) Treatment Induces a Progressive Improvement of CFU Ability of Primary CD34+ Cells in a Patient with IDH2-Mutated AML
In order to evaluate the dynamics of fitness of HSPCs in terms of their ability to form colonies, we isolated and studied CD34+ cells from the bone marrow of a patient with IDH2-R140Q-mutated AML, and we had the opportunity to follow under treatment with enasidenib.Specifically, we performed CFU assays with AML patient-derived CD34+ cells either at baseline or at the end of cycles I, II, and IV.Moreover, with cells obtained after the IV cycle, we explored the effect of adding pharmacological AG-221 inhibition in vitro during culture to assess whether maintaining the drug exposure in culture could enhance their CFU ability.Colonies were evaluated at 14 days as described above (Figure 4).Strikingly, we observed that in vivo treatment with enasidenib induced a progressive increase, over time, in the CFU capacity of the patient-derived CD34+ cells, whose fitness appeared progressively improved in terms of developing mature hematopoietic precursors across the different lineages, a phenomenon that was further strengthened when drug exposure was maintained in vitro in semi-solid culture (Figure 4).This occurred in parallel with the hematologically complete remission of the patient.
Remarkably, the NGS analysis performed after cycle IV, with respect to the diagnosis, showed the persistence of the IDH2-R140Q mutation, on the one hand, in the bulk, as well as in isolated myeloid and erythroid cells (variant allele frequency, VAF = 51% for both subpopulations), and on the other hand, in the CD34+ subpopulation (VAF = 52%) used for the CFU assay (Figure 4).
Despite being observed in a single patient, these findings are in line with clinical observations that complete remission in patients treated with the IDH2 inhibitor is achieved after more cycles of therapy (mean n = 5 cycles) without undergoing bone marrow aplasia; these results are also associated with the cell differentiation of bone marrow cells in the absence of clearance of the IDH2 mutation [26,41].

Discussion
Here, we report our experimental findings on how IDH1/2 mutations and their pharmacological inhibitions affect human hematopoietic stem cell fitness.Our model is the first to represent AML-associated IDH1/2 mutation modeling in the human system.Indeed, from the review of the models available so far, it emerges that the early effects of IDH1/2 mutations, particularly in human hematopoiesis, are mostly unexplored (Supplementary Table S2).
This work, as well as our previous preliminary reports [42,43], show for the first time that expressing the IDH1-R132H or IDH2-R140Q mutant is sufficient to drive a complete block of hematopoietic cell differentiation in primary human HSPCs in vitro; this block can be efficiently released by the specific inhibitor, restoring differentiation ability across all lineages.Despite the relatively low number of normal donors (n = 7, for IDH1-R132H; n = 6, for IDH2-R140Q) analyzed in our study, the results are very consistent among the different samples, supporting the need for further studies to more deeply explore our preliminary findings.
The block of differentiation represents a known early event in leukemogenesis, which is generally associated with increased self-renewal activity, forming the backbone for the development of leukemia upon the acquisition of other genetic alterations and blast proliferation [2].
In vitro cellular models of IDH mutations, either human or murine, highlight a significant increase in DNA methylation in IDH1/2 mutant-expressing cells [24], in keeping with the hypermethylation signature observed in AML patients [24], and is supported by experimental data showing impaired myeloid differentiation [24,35,36] (Supplementary Materials).The VAF values found in the different cell subpopulations suggest that the entire hemopoiesis, including the pool of CD34+ cells, were clonal (IDH2-R140Q positive), and that the improvement in the fitness of HSPC CD34+ relies on the protracted inhibitory action of enasidenib, which unleashes the block of differentiation mediated by the IDH2-R140Q mutation.
Despite being observed in a single patient, these findings are in line with clinical observations that complete remission in patients treated with the IDH2 inhibitor is achieved after more cycles of therapy (mean n = 5 cycles) without undergoing bone marrow aplasia; these results are also associated with the cell differentiation of bone marrow cells in the absence of clearance of the IDH2 mutation [26,41].

Discussion
Here, we report our experimental findings on how IDH1/2 mutations and their pharmacological inhibitions affect human hematopoietic stem cell fitness.Our model is the first to represent AML-associated IDH1/2 mutation modeling in the human system.Indeed, from the review of the models available so far, it emerges that the early effects of IDH1/2 mutations, particularly in human hematopoiesis, are mostly unexplored (Supplementary Table S2).
This work, as well as our previous preliminary reports [42,43], show for the first time that expressing the IDH1-R132H or IDH2-R140Q mutant is sufficient to drive a complete block of hematopoietic cell differentiation in primary human HSPCs in vitro; this block can be efficiently released by the specific inhibitor, restoring differentiation ability across all lineages.Despite the relatively low number of normal donors (n = 7, for IDH1-R132H; n = 6, for IDH2-R140Q) analyzed in our study, the results are very consistent among the different samples, supporting the need for further studies to more deeply explore our preliminary findings.
The block of differentiation represents a known early event in leukemogenesis, which is generally associated with increased self-renewal activity, forming the backbone for the development of leukemia upon the acquisition of other genetic alterations and blast proliferation [2].In vitro cellular models of IDH mutations, either human or murine, highlight a significant increase in DNA methylation in IDH1/2 mutant-expressing cells [24], in keeping with the hypermethylation signature observed in AML patients [24], and is supported by experimental data showing impaired myeloid differentiation [24,35,36] (Supplementary Materials).
DNA hypermethylation in hematopoietic progenitors has also been reported via in vivo studies in conditional knock-in mice with the IDH1-R132H mutation, associated with a significant increase in the pool of hematopoietic precursors and self-renewal capacity [30].Data emerging from our CD34+ HSPC model of the IDH1-R132H mutation are in contrast with this previous study of the murine model.Indeed, in human CD34+ HSPCs from healthy donors, the expression of the mutated IDH1 transgene induces a significant block of differentiation that was not observed in the Sasaki et al. mouse model [30].
The expansion of early hematopoietic progenitors and increased self-renewal were also detected in a model of transgenic mice expressing IDH2-R140Q in the hematopoietic system [31], where, in keeping with our results, a potent block of differentiation was also observed.However, the block primarily affected erythroid colonies, which were severely reduced, while myeloid colonies were not compromised.Instead, the marked block of differentiation that we show-following the expression of the IDH2 mutation in human CD34+ HSPCs-features trilinear myeloid involvement, consistent across the different donors (Figure 3), suggesting that this is a peculiar effect in the human system.Indeed, strikingly, our findings are in line with the recent report from Landberg et al. [44,45], obtained in human CD34+ HSPCs but edited by the CRISPR/Cas9 and AAV6mediated homology-directed repair to express IDH1-R132H or IDH2-R140Q mutations.A pronounced reduction in the colony formation and differentiation capacity triggered by both IDH mutations as well as the ability of ivosidenib to restore the block of differentiation driven by IDH1-R132H were shown.The report from Landberg et al. [44] and our findings highlight the direct involvement of the AML-associated IDH1-R132H and IDH2-R140Q mutations in the hematopoietic differentiation process.
In this sense, the revertant actions of the specific inhibitors described in our (and other studies) underline the dependence of the cellular differentiation of hematopoietic precursors on the specific genetic mutation.The drug-induced 'unblock' of differentiation is unveiled in our experimental conditions in vitro using the CFU assay, showing that the ability of hematopoietic precursors harboring the IDH1/2 mutation consistently recover their potential to give rise to heterogeneous colonies similar to the negative control (Figures 2 and 3).These findings may suggest that in patients affected by IDH-mutated AML, treatment with the specific inhibitor leads to a phase of differentiation of leukemic cells and clonal trilineage hemopoietic recovery with peripheral blood count improvement, which occurs in clinically responding patients, who also show progressive transfusion independence, as reported in seminal clinical studies [26,27,41].
Our study on the fitness dynamics of primary HSPCs from an IDH2-mutated AML patient undergoing enasidenib treatment, despite being limited to a single case, appears to be in line with these findings and clinical reports.

Conclusions
Our report highlights how IDH1/2 mutations frequently found in acute myeloid leukemia act at the level of hematopoietic precursors in the human system by inducing a block of differentiation.The block involves all the hematopoietic myeloid lineages and is specifically reversed by the targeted therapy.Furthermore, we provide evidence that the specific inhibitor acts progressively on the colony-forming ability of HSPCs in patients in vivo, and sustains trilinear hematopoiesis over time in vivo, leading to hematological recovery and complete hematological remission.Our model also serves as a suitable model for further mechanistic studies to explore the role of IDH1/2 mutations in human leukemogenesis.

Figure 4 .
Figure 4. Enasidenib treatment induces the progressive improvement of CFU ability in primary CD34+ cells carrying the IDH2-R140Q mutation.Experimental scheme (left) and stacked columns chart (right) of colonies that rose after 14 days from CD34+ cells isolated from the patient's BM before the start of treatment (Day 0) and at I, II, and IV cycles of enasidenib therapy (Days 28, 56, and 112, respectively).The table in the middle shows IDH2-R140Q VAF obtained by targeted NGS on DNA extracted from either bulk BM or isolated cell subpopulations at cycle IV/Day 112 of treatment.

Figure 4 .
Figure 4. Enasidenib treatment induces the progressive improvement of CFU ability in primary CD34+ cells carrying the IDH2-R140Q mutation.Experimental scheme (left) and stacked columns chart (right) of colonies that rose after 14 days from CD34+ cells isolated from the patient's BM before the start of treatment (Day 0) and at I, II, and IV cycles of enasidenib therapy (Days 28, 56, and 112, respectively).The table in the middle shows IDH2-R140Q VAF obtained by targeted NGS on DNA extracted from either bulk BM or isolated cell subpopulations at cycle IV/Day 112 of treatment.