Normal Myeloid Differentiation Research Articles

Differential prognostic values of the three AKT isoforms in acute myeloid leukemia

The PI3K-AKT-mTOR pathway lies at the confluence of signaling pathways in which various components are subjected to activating genetic alterations in acute myeloid leukemia (AML), thus contributing to oncogenesis. Three AKT isoforms exist in humans. However, whether one isoform predominates in AML remains unknown. This study reveals that AKT3 behaves very distinctly than AKT1 or AKT2 in both normal myeloid differentiation and AML. During normal differentiation, AKT3 is preferentially expressed in hematopoietic stem cells whilst AKT1 becomes preferentially expressed as cells differentiate into granulocytes or monocytes. AKT2 expression remains unchanged. In AML, AKT3 expression varies widely among patient samples and is counterintuitively high in mature/monocytic leukemia. Furthermore, a low level of AKT3 expression is strongly correlated to genetic alterations associated with a better outcome (NPM1 mutations and RUNX1-RUNX1T1 translocation), while a high level is correlated to alterations associated to a bad outcome (RUNX1 mutations; and SRSF2, U2AF1, SF3B1, ASXL1 and BCOR mutations occurring frequently in MDS and MPN). Consistently, a high AKT3 expression level appears as a very strong predictor of poor survival. Curiously, although modestly varying among AML samples, a high AKT1 expression shows in contrast as a strong predictor of a better patient outcome. These data suggest that AKT3 and AKT1 expressions have strong, yet opposite, prognostic values.

Autophagy Inhibition-induced Cytosolic DNA Sensing Combined with Differentiation Therapy Induces Irreversible Myeloid Differentiation in Leukemia Cells.

Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling, thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1)

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5′ and 3′UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

IKZF2 Specifically Participates in AML1-ETO Mediated Blockage of Myeloid Differentiation

Objectives:The chromosomal translocation (8;21) is one of the most recurrent cytogenetic aberrations in acute myeloid leukemia (AML). Although most patients achieve complete remission, one third still relapse due to the persistent leukemia stem cells (LSCs). The lack of significant improvements in outcomes, especially in elderly patients ineligible for standard chemotherapy, has spurred further investigation in t(8;21) leukemia. In this study, we investigate the mechanism of AML1-ETO (AE) fusion gene induced differentiation blockage from earlier stages of HSPCs, aiming to identify potential therapeutic targets that can effectively overcome treatment resistance and minimize long-term side effects. Methods:The AML1-ETO conditional knock-in mouse (Aml1 Eto/+; Mx1-Cre) was previously established, hereafter called AE KI. The Cas9-expressing AE KI model, generated by crossing AE KI and Cas9 mice strains, was employed to define the regulatory program of AE fusion gene. For knockout of AE and IKZF2, Kasumi-1 cells were transduced with the single-vector lentiviral system containing sgRNAs for the respective genes, or a nontargeting control. Colony formation and serial bone marrow transplantation assay were performed to evaluate the function of HSPCs. ATAC sequencing, transcriptional profiling (RNA-seq) and Cut&Tag for H3K4me3, H3K27me3, H3K27ac were performed and analyzed comprehensively to characterize the regulatory profile of AE fusion gene. Results:Our previous research demonstrated that induction expression of AE fusion gene in vivo resulted in excessive accumulation of Lin -Sca-1 +c-Kit + (LSK) cells, leading to differentiation blockage from HSCs to HPCs. IKZF2 showed significantly higher expression in LSK cells following PIPC induction of AE expression compared to that of the control group. IKZF2 was highly increased in AML patients harboring AE fusion gene. And the level of IKZF2 was significantly higher in human t(8;21) cell lines Kasumi-1 and SKNO-1 when compared to that of non-AE controls. Depletion of the AE fusion gene led to a dramatic decrease in IKZF2 expression, while constitutive expression of the AE fusion protein in U-937 cells resulted in elevated levels of IKZF2. Loss of IKZF2 resulted in apoptosis in AE-expressing leukemia cells, and led to an increase in the myeloid differentiation markers CD11b and CD14. These findings suggested a functional relevance of IKZF2 in AE-driven AML. In our Cas9-expressing AE KI model, a significant improvement in the peripheral chimerism rate was observed following IKZF2 knockout at various time points. The population of HSCs, mainly the ST-HSCs, was reduced, while the more differentiated MPP and Lin -Sca-1 -c-Kit + (LK) pools, especially the GMP and CMP groups, were expanded. These findings indicated that the differentiation block triggered by AE was partially relieved by IKZF2 knockout. Furthermore, the deletion of IKZF2 had minimal impact on normal myeloid differentiation, highlighting its specific role in AE settings. We further investigated the underlying mechanisms through multi-omics analysis. The induction of AE led to increased accessibility of the myeloid differentiation inhibitory elements, including IKZF2. CUT&Tag data confirmed a significant decrease in H3K27me3 signals at the promoter regions of IKZF2 compared to that of the control group. Regarding the role of IKZF2 in chromatin remodeling, it is likely to interact with AE to form an epigenetic-transcriptional control module. This may expand the signal for differentiation blockage and drive critical leukemogenic gene expression profiles. Conclusions: Our results demonstrate that IKZF2 is a target gene of AML1-ETO crucial for AML1-ETO-induced differentiation blockage from HSCs to HPCs. The epigenetic regulation mechanism may contribute to the AML1-ETO-IKZF2 regulatory axis. And targeting IKZF2 may become a potential therapeutic strategy in human t(8;21) AML especially for the eradication of AML1-ETO-associated LSCs.

RUNX1::RUNX1T1 Impairs the Differentiation Potential of Primary AML Cells

Acute myeloid leukemias (AMLs) are often driven by genomic translocations, of which t(8;21) is the most frequent. The resulting gene fusion RUNX1::RUNX1T has been shown in cell line and transgenic expression models to impede cell differentiation and promote leukemic self-renewal. However, it has remained unclear if and how RUNX1::RUNX1T1 influences leukemic propagation in patient-derived AML cells. To address the role of RUNX1::RUNX1T1 in the differentiation status and heterogeneity of patient-derived leukemic blasts, we developed an siRNA lipid nanoparticle (LNP) delivery system for the fusion transcript knockdown (Issa et al, Leukemia 2023). For efficient delivery to primary cells, the LNPs were functionalized with a Leu-Asp-Val (LDV) tripeptide binding the Very Late antigen-4 receptor on leukocytes. Primary AML cells were cultured and expanded for several weeks using a serum-free co-culture system with human bone marrow-derived mesenchymal stromal cells (MSCs) (Swart et al, Pharmaceutics 2023). Treatment of t(8;21) patient-derived xenograft (PDX) and primary bone marrow aspirates depleted RUNX1::RUNX1T1 transcript and protein, as determined by RT-qPCR and western blotting. We unraveled the transcriptional repercussions of RUNX1::RUNX1T1 knockdown using bulk RNAseq and ATACseq in a PDX and identified 1068 up- and 1521 downregulated genes upon RUNX1::RUNX1T1 knockdown, as compared to mismatched siRNA-treated cells (padj < 0.001, log2FC > 2). Gene set enrichment analysis revealed loss of self-renewal potential and enhanced myeloid differentiation. Similarly, ATACseq revealed an increased GMP/monocyte-like chromatin accessibility and a decreased HCS/MPP-like one upon RUNX1::RUNX1T1 knockdown (Figure 1). Surprisingly and in contrast to previous cell line results, RUNX1::RUNX1T1 depletion correlated with increased cell cycle and DNA repair signatures. These combined data demonstrate that RUNX1::RUNX1T1 perturbance promotes differentiation of leukemic stem cells towards granulocytic and monocytic lineages. Furthermore, they suggest that the initial stages of differentiation are linked to increased proliferation in line with the increased proliferation capacity of GMPs compared to more immature progenitor states. To investigate the impact of RUNX1::RUNX1T1 on leukemic blast populations at greater granularity, we knocked RUNX1::RUNX1T1 down in 2 primary bone marrow aspirates and 1 PDX and performed single-cell (sc) RNAseq. Notably, principal component analysis revealed that loss of RUNX1::RUNX1T1 shifted all leukemic populations, while hardly affecting normal hematopoietic cells or MSCs (Figure 2). In all samples, pseudotime analysis showed a more differentiated state of cells upon RUNX1::RUNX1T1 depletion. For cell type annotation, we projected our datasets onto a reference normal bone marrow dataset (van Galen et al, Cell 2019). This projection confirmed differentiation toward granulocyte-monocyte progenitors (GMPs), granulocytes and monocytes. Strikingly, the confidence of cell type prediction according to the reference normal bone marrow was significantly and substantially higher upon RUNX1::RUNX1T1 depletion than in cells with unimpaired fusion transcript level, suggesting restoration of normal myeloid differentiation. In addition to that, primary RUNX1::RUNX1T1 AML samples also contained a new, distinct cell subpopulation that emerged upon RUNX1::RUNX1T1 depletion. That subpopulation could not be annotated as resembling any normal bone marrow cell type. However, it expressed eosinophil marker genes such as RNASE3 and EPX. We, therefore, labeled this cell subpopulation as aberrant eosinophilic. This reveals RUNX1::RUNX1T1's role in disrupting differentiation towards multiple lineages. Our work illustrates a strong dependency of the differentiation potential of t(8;21) AML cells on their driver gene RUNX1::RUNX1T1 in an ex vivo setting with PDX or primary cells. We demonstrate that RUNX1::RUNX1T1 hijacks more than one differentiation trajectory of the AML blasts. Our findings also highlight siRNA-loaded LNPs as an innovative therapeutic approach for fusion gene-driven AML by directly targeting the fusion gene as the leukemic driver.

Abstract 3729: Targeting the writers of enhancers as a new therapeutic strategy in pediatric AML

Abstract Introduction: Children with acute myeloid leukemia (AML) urgently need new targeted therapeutics to improve their outcomes. Super-enhancers (SEs) are extensive chromatin regions denoted by abundant histone 3 lysine 27 acetylation (H3K27ac), which are associated with high-level expression of cell-identity genes, including oncogenes. SE-regulated genes have unique sensitivity to transcriptional inhibitors, allowing for cancer type-specific approaches. Thus, targeting CBP/EP300 (the highly homologous enzymes responsible for catalyzing H3K27 acetylation) may be a specific strategy to target lineage-specifying oncogenes in AML. However, to date, the high degree of homology between EP300 and CBP has limited chemical strategies to tissue-specific disruption. Gene editing studies have shown that CBP, and not EP300, is essential for maintaining normal hematopoiesis, while EP300 regulates AML growth and myeloid differentiation. We hypothesized that selectively targeting EP300 would suppress AML growth while sparing effects on normal myeloid differentiation. Methods: We studied publicly available expression and CRISPR screening databases for EP300 and CBP data in AML. We treated pediatric AML (pAML) cell lines and patient samples with the EP300/CBP HAT inhibitor A485 and the EP300-selective PROTAC [PROteolysis TArgeting Chimera]) JQAD1, and measured effects on cell growth by CellTiter-Glo, and apoptosis, differentiation, and cell cycle changes by flow cytometry. We treated pAML cells with compounds for 2 hours and performed mass spectrometry on HPLC-purified H3 proteins. Results: pAML patient samples have higher EP300 expression than normal bone marrow samples in the TARGET dataset, and AML cell lines have enhanced dependency on EP300 over CBP by CRISPR knockout analysis. Treatment of pAML cell lines with A485 slowed proliferation and induced apoptosis, cell cycle arrest, and differentiation. In contrast, equimolar concentrations of JQAD1 had more marked antileukemic effects in both pAML cell lines and primary samples. Immunoblotting analysis demonstrated reduced H3K27ac in pAML cells treated with both JQAD1 and A485, but EP300 degradation only with treatment with JQAD1, consistent with findings in other cancer models. Studies on the effects of EP300 degradation versus combined EP300/CBP inhibition on global and SE-regulated gene expression are ongoing. Middle-down mass spectrometry recapitulates decreased H3K27 acetylation with both agents. JQAD1 combined with the BCL2 inhibitor Venetoclax has a synergistic effect on cell line and patient sample growth, with coordinate induction of apoptosis and differentiation. A four-arm study of this combination in a pAML patient-derived xenograft model is ongoing. Conclusion: Selective degradation of EP300 using the PROTAC JQAD1 is a new strategy that demonstrates preclinical efficacy in pAML as a single agent and in combination with BCL2 inhibitors. Citation Format: Joanna S. Yi, Kevin Duong, Abiola Obawemimo, Tingjian Wang, Faith Joseph, Nicolas L. Young, Stephen Mack, Jun Qi, Adam Durbin. Targeting the writers of enhancers as a new therapeutic strategy in pediatric AML. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3729.

Molecular and epigenetic alterations in normal and malignant myelopoiesis in human leukemia 60 (HL60) promyelocytic cell line model.

In vitro cell line model systems are essential in supporting the research community due to their low cost, uniform culturing conditions, homogeneous biological resources, and easy experimental design to study the cause and effect of a gene or a molecule. Human leukemia 60 (HL60) is an in-vitro hematopoietic model system that has been used for decades to study normal myeloid differentiation and leukemia biology. Here, we show that IMDM supplemented with 20% FBS is an optimal culturing condition and induces effective myeloid differentiation compared with RPMI supplemented with 10% FBS when HL60 is induced with 1α,25-dihydroxyvitamin D3 (Vit D3) and all-trans retinoic acid (ATRA). The chromatin organization is compacted, and the repressive epigenetic mark H3K27me3 is enhanced upon HL60-mediated terminal differentiation. Differential gene expression analysis obtained from RNA sequencing in HL60 cells during myeloid differentiation showed the induction of pathways involved in epigenetic regulation, myeloid differentiation, and immune regulation. Using high-throughput transcriptomic data (GSE74246), we show the similarities (genes that did not satisfy |log2FC|>1 and FDR<0.05) and differences (FDR <0.05 and |log2FC|>1) between granulocyte-monocyte progenitor vs HL60 cells, Vit D3 induced monocytes (vMono) in HL60 cells vs primary monocytes (pMono), and HL60 cells vs leukemic blasts at the transcriptomic level. We found striking similarities in biological pathways between these comparisons, suggesting that the HL60 model system can be effectively used for studying myeloid differentiation and leukemic aberrations. The differences obtained could be attributed to the fact that the cellular programs of the leukemic cell line and primary cells are different. We validated several gene expression patterns for different comparisons with CD34+ cells derived from cord blood for myeloid differentiation and AML patients. In addition to the current knowledge, our study further reveals the significance of using HL60 cells as in vitro model system under optimal conditions to understand its potential as normal myeloid differentiation model as well as leukemic model at the molecular level.

Cooperative Inhibitory Effect of IDH1 Mutation and ASXL1 Mutation on Normal Myeloid Differentiation

Somatic mutations in the additional sex comb-like 1, ASXL1, have been described in various types of myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). In addition, ASXL1 mutations are frequently found in healthy population, which predispose them to not only myeloid malignancies, but also cardiovascular diseases. This condition is known as clonal hematopoiesis with intermediate potential (CHIP), although the full etiology of this preleukemic condition is not elucidated. As for AML, patients with ASXL1 mutations are known to have very poor prognosis compared to non-mutated patients. Although previous studies demonstrated that mutant ASXL1 promoted myeloid leukemogenesis via both loss-of-function and gain-of-function manners, mechanisms how ASXL1 mutations induce myeloid transformation is not fully elucidated. We analyzed publicly available datasets of AML patients, and found that isocitrate dehydrogenase 1 (IDH1) is co-mutated in 13% of AML with ASXL1. As the mechanism by which ASXL1 and IDH1 mutations cooperate to promote AML is not understood, we generated a model of AML with ASXL1 and IDH1 mutations using 32D-cl3 cells and discovered that mutant IDH1 could block myeloid differentiation only when mutant ASXL1 was present. At first, in our experiments, we retrovirally overexpressed mutant (G646WfsX12) or normal form of ASXL1 in 32D-cl3, which is a commonly used IL-3 dependent cell-line of murine myeloid precursor cell. We found that Gr.1 expression levels were decreased in these cells compared to 32D-cl3 with mutant ASXL1 (11.1% vs 26.5%, respectively; p=0.014), and that the proportion of cells in which Gr1 expression was induced by treatment with G-CSF was significantly lower (29.1% vs 54.8%, respectively; p=0.003). Next, we generated a tetracycline-inducible mutant (R132H) form of IDH1 lentiviral vector, and transfected them to these 32D-cl3 cells. We found that tetracycline-inducible mutant IDH1 inhibited G-CSF induced Gr.1 expression in the presence of mutant ASXL1 (7.1% with doxycycline vs 29.9% without doxycycline, respectively; p<0.01), while in the absence of mutant ASXL1, forced expression of mutant IDH1 could not block G-CSF induced Gr.1 expression (59.6% with doxycycline vs 59.2% without doxycycline, respectively; p=0.035). In accordance to these analyses of surface markers, we found that reduction of expression levels of CEBPβ induced by tetracycline-inducible ectopic expression of mutant IDH1 was only observed in 32D-cl3 cells with mutant ASXL1. Furthermore, we found that growth rate of 32D-cl3 cells with mutant ASXL1 and mutant IDH1 was significantly higher than those with mutant ASXL1 alone. These findings suggested that mutant IDH1 can exert its inhibitory effect of normal myeloid differentiation only when mutant ASXL1 is present. Herein, we hypothesized that mutant ASXL1 and mutant IDH1 had unknown cooperative mechanisms to suppress normal differentiation and promote leukemogenesis, and performed transcriptome analysis of the 32D-cl3 cells treated with G-CSF. Tendency toward decreased expression of genes associated with myeloid differentiation in the presence of IDH1 was only observed in cells with mutant ASXL1. Further, it was revealed that specifically dysregulated genes such as HDAC6 or KMT2A in leukemic stem cells were enriched in 32D-cl3 cells with mutant ASXL1, which suggested that mutant IDH1 could induce leukemogenesis only in the presence of ASXL1. In our study, we found that the presence of IDH1 R132H mutation in 32D-cl3 cells had an inhibitory effect on normal myeloid differentiation only in the presence of ASXL1 mutation. Transcriptome analysis was performed to explore genes dysregulated by cooperation of mutant ASXL1 and mutant IDH1. We demonstrated that there might be cooperative mechanisms between ASXL1 and IDH1 to induce differentiation and promote leukemogenesis. Our study would not only provide a new role for IDH1 mutation in ASXL1 mutated AML, but also identify a new therapeutic target for ASXL1-mutatated AML.

BCR::ABL1 -Dependent Promotion of Hematopoietic Stem Cells (HSC) and Progenitors in an Inducible Embryonic Stem Cell (ESC) Model

Our current understanding of the effect of BCR::ABL1 oncogene in leukemia development largely relies on studies using in vitro and in vivo leukemogenesis models. Among them, in vivo models using retrovirus-mediated BCR-ABL gene transfer in murine embryonic stem cell (ESC)-derived hematopoietic cells suggested that the enforced expression of BCR::ABL1 was sufficient to confer them an in vivo long-term repopulation potential. However, in these models, the unique BCR::ABL1 dependence of the generation of this potential has not been shown. Our study aimed at getting a better understanding of the early events regulated by BCR::ABL1 during the course of ESC differentiation without retroviral-mediated gene transfer. The elucidation of these early events could also be of major interest in therapy as generation of bona fide HSCs from normal ESC or iPSC remains a major challenge. To this end, we engineered a unique Tet-ON inducible system in which BCR::ABL1(p210) expression level can be tightly controlled using doxycycline (Dox). As compared to previously reported ESC-derived models, our system contains a single copy of BCR::ABL1 inserted into the murine Col1a1 locus under the control of Tetracycline operator, is sensitive to Dox, leakproof and reversible. The first striking results was the timing of Dox-inducible BCR-ABL expression during the course of ESC differentiation, impacting both the frequency and emergence of hematopoietic progenitors. Indeed, the expression of BCR::ABL1 at day+5 favors significantly the emergence and maintenance of immature hematopoietic progenitors. Importantly, these cells can self-renew only in the presence of Dox. Several hematopoietic stem-like cell lines were established and two of them (HSCL-A and HSCL-C) have been explored in detail. A major BCR-ABL-dependent proliferative ability was documented in both (Figure 1D). Both expressed Lin-Sca-1+c-Kit+CD150+CD48- and eSLAM, corresponding to phenotypically identified adult mouse HSCs. Importantly, these cells exhibited a normal myeloid and erythroid differentiation capacity in CFC assays, giving rise also to CFU-GEMM. We then evaluated the ability of HSCL to self-renew using long-term culture-initiating cell (LTC-IC) assays. To this purpose, EPCR+CD45+/loCD48-CD150+ (ESLAM) cells were sorted at a single cell level and co-cultured on a supportive AFT024 feeder layers for 4 weeks. LTC-IC-derived progenitors were then assessed using CFC assays. The frequency of ESLAM LTC-ICs was 0.53 from adult bone marrow and 0.52 from HSCL lines demonstrating their self-renewal ability. To better characterize HSCL cell lines, we sorted LSK cell population from HSCL#A, HSCL#C, E14.5 fetal liver and murine adult bone marrow and analyzed their transcriptome signatures by RNA-Seq. Unsupervised principal component analysis with the 1333 genes exhibiting the highest variability revealed that HSCL cell lines clustered together and exhibited distinct molecular signature from fetal and adult LSK. This stratification was validated by clustering with the 50 best variable genes. Among them, we found 17 genes highly expressed in HSCL cell lines associated with HSC quiescence (such as NupR1). GSEA analyses confirmed an enrichment of several cell cycle genes as well as genes involved in unfolded protein response (UPR) and autophagy (p <0.001). Thus, we report here for the first time, a unique BCR-ABL inducible ESC-derived hematopoiesis model which demonstrates the ability of BCR-ABL expression to induce a proliferative signal for extended periods of time in highly primitive hematopoietic cells. This novel tool can be used to dissect CML ontogeny both in vitro and in vivo and perform drug screening at the most primitive stem cell level. It can also be of major interest to uncover molecular events and signaling pathways activated during normal ESC-derived hematopoiesis. Such knowledge will open up new avenues to differentiate human iPSC towards bona fide HSCs for potential clinical applications. Figure 1. Establishment of ES cell-derived hematopoietic cell lines expressing BCR::ABLp210.(A) Representation of the timeline of ES cell differentiation protocol and doxycycline induction. (B) HSCL#A cell morphology. (C) MGG staining of HSCL#A cells. (D) Proliferation curves of HSCL#A and HSCL#C cell lines over 40 days in culture. (E)Kinetics of the effect of doxycycline removal on HSCL#A cells cultured on OP9 cells for 4 consecutive days. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia

Homeobox genes encode transcription factors that control basic developmental decisions. Knowledge of their hematopoietic activities casts light on normal and malignant immune cell development. Recently, we constructed the so-called lymphoid TALE-code that codifies expression patterns of all active TALE class homeobox genes in early hematopoiesis and lymphopoiesis. Here, we present the corresponding myeloid TALE-code to extend this gene signature, covering the entire hematopoietic system. The collective data showed expression patterns for eleven TALE homeobox genes and highlighted the exclusive expression of IRX1 in megakaryocyte-erythroid progenitors (MEPs), implicating this TALE class member in a specific myeloid differentiation process. Analysis of public profiling data from acute myeloid leukemia (AML) patients revealed aberrant activity of IRX1 in addition to IRX3 and IRX5, indicating an oncogenic role for these TALE homeobox genes when deregulated. Screening of RNA-seq data from 100 leukemia/lymphoma cell lines showed overexpression of IRX1, IRX3, and IRX5 in megakaryoblastic and myelomonocytic AML cell lines, chosen as suitable models for studying the regulation and function of these homeo-oncogenes. Genomic copy number analysis of IRX-positive cell lines demonstrated chromosomal amplification of the neighboring IRX3 and IRX5 genes at position 16q12 in MEGAL, underlying their overexpression in this cell line model. Comparative gene expression analysis of these cell lines revealed candidate upstream factors and target genes, namely the co-expression of GATA1 and GATA2 together with IRX1, and of BMP2 and HOXA10 with IRX3/IRX5. Subsequent knockdown and stimulation experiments in AML cell lines confirmed their activating impact in the corresponding IRX gene expression. Furthermore, we demonstrated that IRX1 activated KLF1 and TAL1, while IRX3 inhibited GATA1, GATA2, and FST. Accordingly, we propose that these regulatory relationships may represent major physiological and oncogenic activities of IRX factors in normal and malignant myeloid differentiation, respectively. Finally, the established myeloid TALE-code is a useful tool for evaluating TALE homeobox gene activities in AML.

High MYC Expression Predicts Poor Survival Outcomes in IDH1/2 Mutant AML Patients

BackgroundIn AML, IDH1/2 somatic mutations can be frequent in patients at about 20%. Physiologically, IDH1/2 catalyze isocitrate conversion to alpha-ketoglutarate (α-KG). α-KG is an activating co-factor for ten-eleven translocation 2 (TET2), an important epigenetic regulator which converts 5mC to 5hmC in the genomic DNA ultimately turning on the myeloid differentiation. In contrast, mutant IDH1/2 proteins promote α-KG conversion to 2-hydroxyglutarate (2-HG), an onco-metabolite. In pre-clinical studies, 2-HG inhibits TET2 activity by suppressing 5mC-to-5hmC conversion and thus allowing AML development and maintenance. Clinical trials of IDH1/2 inhibitors demonstrated promising outcomes in IDH1/2 mut AML patients. However, a substantial fraction of patients fail to respond to IDH1/2 inhibitors in AML. Our recent preclinical studies have shown MYC prevents the normal myeloid differentiation and cell death by suppressing IDH1/2-TET2 signaling. We evaluated if high MYC expression is associated with poor survival outcomes in IDH1/2 mut AML patients treated with Ivosidenib or EnasidenibPatients and MethodsPatients diagnosed with AML at Moffitt Cancer Center between 2013-2020 were evaluated. IDH1/2 mutation status was assessed via next generation sequencing and those with documented IDH1/2 mut were included for the analysis. Clinical and demographic data were retrospectively extracted from the medical records. Pre-treatment MYC protein levels were assessed by immunohistochemistry staining using BM biopsy specimens. Statistical analyses were performed using GraphPad Prism (v.7.03) and SPSS (v.24.0). Baseline characteristics were compared by chi square (categorical variables) and t- test (continuous variables). Survival estimates were calculated using the Kaplan-Meier method from date of diagnosis and groups were compared using log-rank test. Multivariate survival analysis was performed by means of Cox proportional hazards regressionResultsA total of 28 (IDH1 mut, n=11; IDH2 mut, n=18; IDH1/2 mut, n=1) patients were included in the study. Median age at AML diagnosis was 66 years and 60% of patients were male. Twelve (42%) patients had secondary AML and 9 (32%), 11 (39%), and 6 (21%) patients had favorable, intermediate, and adverse risk, respectively, at AML diagnosis. A total of 17 (60.7%) and 6 (21.4%) patients received intensive chemotherapy and hypomethylating agents as their 1 st-line therapy. Among 28 patients, 22 (78.5%) and 6 (21.4%) patients had low and high MYC expression (≥5% by IHC staining), respectively, prior to IDH1/2 inhibitor treatment. Median number of treatments prior to IDH1/2 inhibitors was 3 (1-6) and the median duration of IDH1/2 inhibitor treatment was 3.2 (0.3-30) months (IDH1 mut, 3.3 [0.3-30] months; IDH2 mut, 2.5 [0.7-14.5] months). Treatment response was assessed in 27 patients and 11 (40.7%) had CR/CRi (high vs. low MYC, 20 vs. 45.5%, p=.6185). The median PFS was shorter in high MYC patients (1.6 vs. 4.4 months, HR=2.348, p=.0515). The median OS was also significantly shorter in high MYC patients (2.5 vs. 8.5 months, HR=5.971, p<.0001).ConclusionIn our study, we demonstrated that high MYC expression was associated with significantly shorter PFS and OS in IDH1/2 mut AML patients. In consistent with our recent studies that showed MYC represses IDH1/2 and TET2 expression in AML, this study suggests that MYC may play an important role in the resistance mechanism of IDH1/2 inhibitors. Additional studies with larger cohorts are warranted to further confirm and validate these findings. DisclosuresTalati: Jazz: Speakers Bureau; Astellas: Speakers Bureau; BMS: Honoraria; AbbVie: Honoraria; Pfizer: Honoraria. Sweet: Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuykendall: Pharmaessentia: Honoraria; Celgene/BMS: Honoraria; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding. Komrokji: Geron: Honoraria; Acceleron: Honoraria; Novartis: Honoraria; Agios: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau. Sallman: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intellia: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Takeda: Consultancy; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Padron: Kura: Research Funding; Incyte: Research Funding; BMS: Research Funding; Stemline: Honoraria; Blueprint: Honoraria; Taiho: Honoraria.

Transcription factor MEF2D is required for the maintenance of MLL-rearranged acute myeloid leukemia

AbstractAcute myeloid leukemia (AML) with MLL-rearrangement (MLL-r) comprises ∼10% of all AML cases and portends poor outcomes. Much remains uncovered on how MLL-r AML drives leukemia development while preventing cells from normal myeloid differentiation. Here, we identified that transcription factor MEF2D is a super-enhancer-associated, highly expressed gene in MLL-r AML. Knockout of MEF2D profoundly impaired leukemia growth, induced myeloid differentiation, and delayed oncogenic progression in vivo. Mechanistically, MEF2D loss led to robust activation of a CEBPE-centered myeloid differentiation program in AML cells. Chromatin profiling revealed that MEF2D binds to and suppresses the chromatin accessibility of CEBPE cis-regulatory regions. In human acute leukemia samples, MEF2D expression showed a strong negative correlation with the expression of CEBPE. Depletion of CEBPE partially rescued the cell growth defect and myeloid cell differentiation induced by the loss of MEF2D. Lastly, we show that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced antileukemia effects. Collectively, our findings suggest that MEF2D plays a critical role in human MLL-r AML and uncover the MEF2D-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block.

IDH Mutated AML: Beyond Enasidenib and Ivosidenib Monotherapy

Introduction Mutations in the isocitrate dehydrogenase (IDH) genes, IDH1 or IDH2, occur in approximately 20% of patients with acute myeloid leukemia (AML) and promote leukemogenesis through a block in normal myeloid differentiation and through the production of the oncometabolite 2-hydroxyglutarate (2HG).1 Selective oral inhibitors of mutant IDH1 and IDH2, leading to restoration of normal myeloid differentiation and reduction of 2HG have been developed, and are now approved as single-agent therapy, based on clinical efficacy observed within the original first-in-human trials.

AML-170: High MYC Expression Predicts Poor Survival Outcomes in IDH1/2 Mutant AML Patients

<h3>Context:</h3> Clinical trials of IDH1/2 inhibitors demonstrated promising outcomes in <i>IDH1/2^mut</i> AML patients. However, a substantial fraction of patients fail to respond to IDH1/2 inhibitors in AML. Recent preclinical studies have shown MYC prevents the normal myeloid differentiation and cell death by suppressing IDH1/2-TET2-5hmC signaling. <h3>Objective:</h3> Evaluate if high MYC expression is associated with poor survival outcomes in <i>IDH1/2^mut</i> AML patients treated with ivosidenib or enasidenib. <h3>Design:</h3> We retrospectively identified AML patients who had <i>IDH1/2</i> somatic mutations based on NGS assessments. Clinical and demographic data were extracted from the medical records. Pre-treatment MYC protein levels were assessed by immunohistochemistry (IHC) staining using BM biopsy specimens. <h3>Results:</h3> A total of 28 (<i>IDH1^mut</i>, n=11; <i>IDH2^mut,</i> n=18; <i>IDH1/2^mut,</i> n=1) patients were included in the study. Median age at AML diagnosis was 66 years, and 60% of patients were male. Twelve (42%) patients had secondary AML and 9 (32%), 11 (39%), and 6 (21%) patients had favorable, intermediate, and adverse risk, respectively, at AML diagnosis. A total of 17 (60.7%) and 6 (21.4%) patients received intensive chemotherapy and hypomethylating agents as their first-line therapy. Among 28 patients, 22 (78.5%) and 6 (21.4%) patients had low and high MYC expression (≥5% by IHC staining), respectively, prior to IDH1/2 inhibitor treatment. Median number of treatments prior to IDH1/2 inhibitors was 3 (1–6) and the median duration of IDH1/2 inhibitor treatment was 3.2 (0.3–30) months (<i>IDH1^mut</i>, 3.3 [0.3–30] months; <i>IDH2^mut</i>, 2.5 [0.7–14.5] months). Treatment response was assessed in 27 patients, and 11 (40.7%) had CR/CRi (high <i>vs</i> low MYC, 20 <i>vs</i> 45.5%, p=0.6185). The median PFS was shorter in high MYC patients (1.6 <i>vs</i> 4.4 months, HR=2.348, p=0.0515). The median OS was also significantly shorter in high MYC patients (2.5 <i>vs</i> 8.5 months, HR=5.971, p<0.0001). <h3>Conclusions:</h3> High MYC expression was associated with significantly shorter PFS and OS in <i>IDH1/2^mut</i> AML patients. Consistent with our recent studies that showed MYC represses IDH1/2 and TET2 expression in AML, this study suggests that MYC may play an important role in the resistance mechanism of IDH1/2 inhibitors. Additional studies with larger cohorts are warranted to further confirm and validate these findings.

AML1/ETO and FLT3-ITD Cooperate to Induce Acute Myeloid Leukemia in Mice

Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, accounts for more than 40% of all leukemia deaths. The AML1-ETO (A) fusion gene resulting from t(8;21) is a recurring genetic alteration in human AML, typically associated with a favorable outcome. Internal tandem duplications (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (Flt3) result in constitutive ligand-independent signaling. Flt3ITD is one of the most frequently identified molecular abnormalities in AML, and when present, portends unfavorable clinical outcomes. AML1/ETO and FLT3ITD can co-exist in human AML, indicating that these lesions cooperate to promote disease development. Neither AML1/ETO nor Flt3ITD alone are sufficient to induce AML in mice. To determine whether coordinated expression of AML1/ETO and Flt3ITD would cause AML, Mx1-Cre x LSL AML1-ETO/+ x FLT3ITD/ITD (MAFF) mice were generated. In this system, an Mx1 promoter-driven Cre recombinase removes a lox-stop-lox (LSL) cassette upstream of a knock-in human AML1/ETO fusion gene in hematopoietic progenitor cells, while FLT3ITD is constitutively expressed. MAFF mice developed AML at 4-5 weeks of age with 100% penetrance. The AML was associated with severe leukocytosis, splenomegaly and dense bone marrow infiltration by immature-appearing blasts, and was universally lethal. Flow cytometry of bone marrow from MAFF mice revealed a striking expansion of cells harboring a hematopoietic stem cell (HSC) and multipotent progenitor (MP) cell phenotype, indicative of a block in normal myeloid differentiation. No lymphoid leukemias were observed. Interestingly, two copies of the FLT3ITD allele were required for AML to develop in this model. Transplantation of as few as 105 bone marrow cells from primary MAFF mice into lethally-irradiated secondary recipients resulted in 100% engraftment and lethality, with a leukemia identical to that which developed in primary MAFF mice, albeit with delayed latency. Mixed bone marrow chimeras were generated using MAFF marrow along with marrow from syngeneic, wild-type C57BL/6 or Rag2-/-γC-/- mice (lacking mature B, T and NK cells). Chimeras generated with MAFF and Rag2-/-γC-/- marrow succumbed more rapidly than those with MAFF and wild-type marrow, indicating that adaptive immunity and/or NK cells provided some level of active immune surveillance against MAFF AML. MAFF mice were crossed with Rosa26-LSLSIY/+ mice (referred to as MAFFS mice) in order to incorporate inducible expression of a tractable peptide antigen (SIYRYYGL; SIY) in leukemia cells. Through the use of mixed bone marrow chimeric approaches, we are now capable of monitoring SIY antigen-specific CD8+ T cell responses in MAFFS AML-bearing animals, including contexts in which the SIY antigen is shared (leukemia-associated antigen) or expressed only by leukemia cells (leukemia-specific antigen). In conclusion, we have generated the first autochthonous murine AML model in which the role of host immune surveillance can be interrogated. We expect that the MAFF AML model will also be useful for studies regarding leukemia biology and treatment. DisclosuresNo relevant conflicts of interest to declare.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.

Core Binding Factor Leukemias Utilize a Physiologic Sense/Antisense Promoter Switch Employed By T-Cells

Introduction: Alterations of core binding factors (CBF), Runx1 and CBFβ are frequent mutational targets in acute myeloid leukemia (AML). Chromosomal translocations t(8;21)(q22;q22) and inv(16)(p13q22), creating the fusion proteins RUNX1-ETO and CBFβ-MYH11 respectively, account for 15% and thus the largest sub-group of AML called CBF-AML. CBF oncogenes induce global changes in chromatin structure and gene regulation, which lead to differentiation blockade. A critical leukemic event could be the inactivation of PU.1 transcription factor. Normal myeloid differentiation needs PU.1 levels to increase, failure to do so leads to a stop of differentiation and AML development. In contrast, T-cell differentiation requires PU.1 to be completely switched off. The exact mechanism of PU.1 suppression, physiological for T-lymphopoiesis or pathological for leukemia, remains elusive. Results: We assessed the activation of the PU.1 locus throughout human hematopoietic differentiation stages using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Interestingly, we observed high accessibility of a previously identified antisense promoter (AsPr) in intron 3 and antisense transcript (asRNA) expression during early lymphopoiesis which preceded locus shutdown in T cells. The ratio of AsPr/PrPr accessibility and of antisense/sense transcription clearly indicated cellular fate during hematopoiesis (Figure 1A). T-lymphoid differentiation was related to the timely expression of RUNX transcription factors. RUNX1, RUNX3, and the CBF fusions RUNX1-ETO and CBFβ-MYH11 were capable to transactivate PU.1 AsPr. In CBF-AML patient samples we strikingly found elevated asRNA/mRNA ratios compared to normal karyotype AML or healthy CD34+ cells (Figure 1B) and increased AsPr/PrPr ratios unsing DNaseI-seq data in RUNX1-ETO AML patients. Functionally we found that PU.1 asRNA depletion in t(8;21) xenografted immune-deficient (NOD/SCID) mice restored a normal survival (Figure 1C) demonstrating that PU.1 antisense transcripts are required for CBF leukemia outgrow in vivo. To further dissect the mechanism of how CBFs could drive PU.1 antisense transcription we applied active RNA polymerase mapping (PRO-seq) and chromatin accessibility (ATAC-seq) and found a shift from PU.1 antisense to sense transcription after RUNX1-ETO depletion. Using chromosomal conformation capture sequencing (3C, Hi-C and CHiC) in T-lymphoid, myeloid and RUNX1-ETO cells combined with transcript quantification we observed that competitive interaction of an upstream enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development (Figure 2A). Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription (Figure 2B). Conclusion: The data suggest that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation that is induced by CBF fusions. Sense/antisense promoter competition represents a crucial functional switch for gene expression perturbation by oncogenes and provide a potential strategy for future precise therapeutic targeting of oncogene-induced chromatin remodeling. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Staber:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; msd: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria.

HMGA1 Is Upregulated in Myelodysplastic Syndromes with Mutation in Pre-mRNA Splicing Genes and Inhibits Leukemia Cell Differentiation

In myelodysplastic syndrome (MDS), genes associated with pre-mRNA splicing such as SRSF2 (Serine and Arginine Rich Splicing Factor 2) and U2AF1 (U2 small nuclear RNA auxiliary factor 1) are frequently mutated. While several reports demonstrated that MDS patients with these mutations had worse prognosis, few treatments specific to these mutations are available, partly due to the dearth of material on functions of these mutated genes. To assess the role of pre-mRNA splicing gene mutations in the pathogenesis of MDS, we performed RNA-seq analysis using CD34-postitive fractions of bone marrow samples of MDS patients. Patients with mutations in either SRSF2 or U2AF1 tended to have shorter 1-year leukemia free survival compared to patients without these mutations (66.7% vs 73.7%, respectively; p=0.067). Gene set enrichment analysis revealed that MYC target genes were enriched in patients with mutations in either SRSF2 or U2AF1 compared to patients (n=4) without these mutations (n=3). Among them, we found that HMGA1 (High Mobility Group A1) was distinctly upregulated in these patients. Expression levels of HMGA1 are known to be higher in hematopoietic stem cells. HMGA1 is involved in various cellular processes such as transcriptional regulation, DNA repair, cell differentiation and regulated cell death. We confirmed that expression levels of HMGA1 in murine bone marrow cells were higher in Lineage marker-negative, Sca1-positive, and c-Kit-positive (LSK) fractions than other more differentiated fractions. To determine whether upregulation of HMGA1 contributed to pathogenesis of MDS, we retrovirally transduced HMGA1 to murine hematopoietic progenitor cell line 32D-cl3. We found that proportions of Gr-1 positive cells after treatment with G-CSF were lower in HMGA1-transduced 32D-cl3 cells. Further, expression levels of myeloid-associated genes were also suppressed in these 32D-cl3 cells. These results suggested that 32D-cl3 with extrinsic expression of HMGA1 were more resistant to G-CSF induced myeloid differentiation. Similarly, primary murine hematopoietic progenitor cells retrovirally transduced with HMGA1 showed increased colony-forming capacities. Considering that differentiation block plays a major role in the onset of MDS, these results supported our hypothesis that upregulation of HMGA1 would contribute to pathogenesis of MDS through blocking normal myeloid differentiation. To investigate whether inhibition of HMGA1 could affect the pathogenesis of myeloid malignancies, we silenced expression of HMGA1 via short-hairpin RNA (shRNA) in various cell lines of acute myeloid leukemia (AML). Human acute monocytic leukemia cell line THP-1 became more easily induced to differentiate upon phorbol12-myristate13-acetate treatment when HMGA1 was silenced. Similarly, we silenced expression of HMGA1 in HL60, which is a human acute promyelocytic leukemia (APL) cell line. We treated HL60 cells with all-trance retinoic acid (ATRA) to induce myeloid differentiation. As expected, we found that expression levels of CEBP-β and Gr-1 in ATRA-treated HL60 cells were significantly higher when HMGA1 was silenced, which meant that HL60 cells became more sensitive to ATRA-induced myeloid differentiation by inhibition of HMGA1. Considering that differentiation induction therapy has improved clinical outcome not only in APL but also in AML, these results suggested that HMGA1 would be a potential therapeutic target for myeloid malignancies. In summary, our experiments demonstrated that expression levels of HMGA1 were higher in patients with splicing mutations. Considering its inhibitory effects on differentiation demonstrated by our experiments, upregulation of HMGA1 could be involved in the pathogenesis of MDS. Further, therapeutic intervention for HMGA1 had a potential to improve clinical outcome of myeloid malignancies. Disclosures Kurokawa: Nippon Shinyaku: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Teijin: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; Chugai: Consultancy, Research Funding, Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Targeting IDH Mutations in AML: Wielding the Double-edged Sword of Differentiation.

The genomic characterization of acute myeloid leukemia (AML) by DNA sequencing has illuminated subclasses of the disease, with distinct driver mutations, that might be responsive to targeted therapies. Approximately 15-23% of AML genomes harbor mutations in one of two isoforms of isocitrate dehydrogenase (IDH1 or IDH2). These enzymes are constitutive mediators of basic cellular metabolism, but their mutated forms in cancer synthesize an abnormal metabolite, 2- hydroxyglutarate, that in turn acts as a competitive inhibitor of multiple gene regulatory enzymes. As a result, leukemic IDH mutations cause changes in genome structure and gene activity, culminating in an arrest of normal myeloid differentiation. These discoveries have motivated the development of a new class of selective small molecules with the ability to inhibit the mutant IDH enzymes while sparing normal cellular metabolism. These agents have shown promising anti-leukemic activity in animal models and early clinical trials, and are now entering Phase 3 study. This review will focus on the growing preclinical and clinical data evaluating IDH inhibitors for the treatment of IDH-mutated AML. These data suggest that inducing cellular differentiation is central to the mechanism of clinical efficacy for IDH inhibitors, while also mediating toxicity for patients who experience IDH Differentiation Syndrome. Ongoing trials are studying the efficacy of IDH inhibitors in combination with other AML therapies, both to evaluate potential synergistic combinations as well as to identify the appropriate place for IDH inhibitors within existing standard-of-care regimens.