Myeloid Differentiation Marker Research Articles

Nuclear factor I-C overexpression promotes monocytic development and cell survival in acute myeloid leukemia

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

Src-Family Protein Kinase Inhibitors Suppress MYB Activity in a p300-Dependent Manner.

Recent studies have disclosed transcription factor MYB as a potential drug target for malignancies that are dependent on deregulated MYB function, including acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often regarded as undruggable, successful targeting of MYB by low-molecular-weight compounds has recently been demonstrated. In an attempt to repurpose known drugs as novel MYB-inhibitory agents, we have screened libraries of approved drugs and drug-like compounds for molecules with MYB-inhibitory potential. Here, we present initial evidence for the MYB-inhibitory activity of the protein kinase inhibitors bosutinib, PD180970 and PD161570, that we identified in a recent screen. We show that these compounds interfere with the activity of the MYB transactivation domain, apparently by disturbing the ability of MYB to cooperate with the coactivator p300. We show that treatment of the AML cell line HL60 with these compounds triggers the up-regulation of the myeloid differentiation marker CD11b and induces cell death. Importantly, we show that these effects are significantly dampened by forced expression of an activated version of MYB, confirming that the ability to suppress MYB function is a relevant activity of these compounds. Overall, our work identifies several protein kinase inhibitors as novel MYB-inhibitory agents and suggests that the inhibition of MYB function may play a role in their pharmacological impact on leukemic cells.

Bcr-TMP, a Novel Nanomolar-Active Compound That Exhibits Both MYB- and Microtubule-Inhibitory Activity.

Simple SummaryRecent work has identified the transcription regulator MYB as an interesting therapeutic target for the treatment of certain leukemias and other cancers that are dependent on deregulated MYB activity, such as acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here we report the identification and characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a novel highly active MYB inhibitory compound. We show that nanomolar concentrations of Bcr-TMP are sufficient to down-regulate the expression of MYB target genes and induce both cell-death and differentiation in AML cell lines. Importantly, Bcr-TMP also and exerts stronger anti-proliferative effects on MYB-addicted primary AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Preliminary work shows that Bcr-TMP acts through p300, a protein interacting with MYB and stimulating its activity. Interestingly, Bcr-TMP has an additional activity as an anti-microtubule agent. Overall, Bcr-TMP is an interesting compound that warrants further research to understand its mechanism of action and its therapeutic potential for MYB-dependent malignancies.Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here, we present the initial characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a nanomolar-active MYB-inhibitory compound identified in a screen for novel MYB inhibitors. Bcr-TMP affects MYB function in a dual manner by inducing its degradation and suppressing its transactivation potential by disrupting its cooperation with co-activator p300. Bcr-TMP also interferes with the p300-dependent stimulation of C/EBPβ, a transcription factor co-operating with MYB in myeloid cells, indicating that Bcr-TMP is a p300-inhibitor. Bcr-TMP reduces the viability of AML cell lines at nanomolar concentrations and induces cell-death and expression of myeloid differentiation markers. It also down-regulates the expression of MYB target genes and exerts stronger anti-proliferative effects on MYB-addicted primary murine AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Surprisingly, we observed that Bcr-TMP also has microtubule-disrupting activity, pointing to a possible link between MYB-activity and microtubule stability. Overall, Bcr-TMP is a highly potent multifunctional MYB-inhibitory agent that warrants further investigation of its therapeutic potential and mechanism(s) of action.

Characterization of the MYB-inhibitory potential of the Pan-HDAC inhibitor LAQ824

A large body of work has shown that MYB acts as a master transcription regulator in hematopoietic cells and has pinpointed MYB as a potential drug target for acute myeloid leukemia (AML). Here, we have examined the MYB-inhibitory potential of the HDAC inhibitor LAQ824, which was identified in a screen for novel MYB inhibitors. We show that nanomolar concentrations of LAQ824 and the related HDAC inhibitors vorinostat and panobinostat interfere with MYB function in two ways, by inducing its degradation and inhibiting its activity. Reporter assays show that the inhibition of MYB activity by LAQ824 involves the MYB transactivation domain and the cooperation of MYB with co-activator p300, a key MYB interaction partner and driver of MYB activity. In AML cells, LAQ824-induced degradation of MYB is accompanied by expression of myeloid differentiation markers and apoptotic and necrotic cell death. The ability of LAQ824 to inhibit MYB activity is supported by the observation that down-regulation of direct MYB target genes MYC and GFI1 occurs without apparent decrease of MYB expression already after 2 h of treatment with LAQ824. Furthermore, ectopic expression of an activated version of MYB In HL60 cells counteracts the induction of myeloid differentiation by LAQ824. Overall, our data identify LAQ824 and related HDAC inhibitors as potent MYB-inhibitory agents that exert dual effects on MYB expression and activity in AML cells.

STAT5 Signaling Promotes Resistance to Ivosidenib in IDH1-Mutated AML

Mutations in isocitrate dehydrogenase 1 (IDH1) promote leukemic transformation through the production of an oncometabolite, 2-hydroxyglutarate (2-HG). Ivosidenib is an inhibitor of mutant IDH1 approved for the treatment of IDH1-mutated AML. Treatment with ivosidenib can induce terminal differentiation of leukemic blasts via suppression of 2-HG. However, ivosidenib as a single agent has limited efficacy, highlighting the need to better understand the mechanisms of drug resistance.The study of drug resistance mechanisms has been hindered by the lack of IDH1/2-mutated AML cell lines models. To address this issue, we derived an Idh1-mutated AML cell line from bone marrow cells of a murine AML model generated by crossing mice expressing mutant Idh1R132H with mice expressing mutant Npm1 (Npm1c). This cell line (henceforth termed “OCI-mIDH1/N”) undergoes partial myeloid differentiation in response to ivosidenib treatment in vitro.The establishment of OCI-mIDH1/N enabled us to perform a genome-wide CRISPR knockout screen to identify genes that upon inactivation, increased the differentiation response to ivosidenib. Through this screen, we identified C-type lectin member 5a (Clec5a) as one of the top hits. Clec5a encodes a cell surface receptor that signals through the intracellular spleen tyrosine kinase (SYK). To confirm this hit, we generated Clec5a knockout clones of OCI-mIDH1/N cells. Consistent with results of the screen, ivosidenib treatment induced higher levels of Gr-1, a myeloid differentiation marker, on Clec5a-/- cells than on parental Clec5a+/+ cells. Next, we investigated the role in SYK. Clec5a-/- cells had lower levels of pSYK compared with Clec5a+/+ cells, and overexpression of Syk in Clec5a-/- cells reversed their sensitization to ivosidenib. Furthermore, direct inhibition of SYK with fostamatinib was sufficient to sensitize Clec5a+/+ cells to ivosidenib. Our findings show that CLEC5A-SYK signaling promotes resistance to ivosidenib-induced differentiation.Mechanistically, we found that CLEC5A-SYK signaling drives ivosidenib resistance through STAT5 dependent expression of self-renewal genes in the HOX family. Clec5a-/- and Syk-/- OCI-mIDH1/N cells exhibited lower levels of STAT5 activation compared with wildtype cells. Furthermore, SYK inhibitor treatment downregulated pSTAT5 and decreased STAT5 occupancy at HOX gene clusters. Functionally, overexpression of constitutively active STAT5 in Clec5a-/- and Syk-/- cells reversed their heightened sensitivity to ivosidenib and increased the expression of key HOX self-renewal genes. To determine the clinical relevance of these findings, we analyzed two independent RNA-seq datasets from patients treated with IDH inhibitors (Quek et al., 2018; Wang et al., 2021). Gene set enrichment analysis revealed that poor responders expressed significantly higher levels of STAT5 target genes compared with responders in both datasets.Mutations in the receptor tyrosine kinase (RTK) pathway have previously been shown to be associated with resistance to IDH inhibitors. Given that these pathways signal through STAT5, we hypothesized that the mechanism by the RTK mutations confer ivosidenib resistance is through STAT5 activation. To test this hypothesis, we ectopically expressed KRASG12D or PTPN11E76G in OCI-mIDH1/N cells. Expression of the oncogenes increased pSTAT5 and conferred resistance to ivosidenib-induced differentiation. Importantly, knockdown of Stat5 completely restored their sensitivity to ivosidenib, in support for our hypothesis.Next, we tested if the STAT5 inhibitor pimozide synergizes with ivosidenib to treat patient-derived xenograft (PDX) models of human IDH1-mutated AML. In two PDX models where ivosidenib alone induced moderate differentiation, the addition of pimozide to ivosidenib significantly increased the expression of myeloid differentiation markers on AML cells. In two PDX models where ivosidenib alone induced no differentiation, pimozide alone was sufficient to induce profound differentiation of AML cells. Collectively, these results suggest that ivosidenib resistant cells shift their dependence from 2-HG to STAT5 to maintain their undifferentiated state.In summary, our findings demonstrate that STAT5 is a critical mediator of resistance to ivosidenib, and combination therapy with pimozide and ivosidenib is a promising therapeutic approach for IDH1-mutated AML. DisclosuresChan: BMS: Research Funding; AbbVie: Research Funding.

Clonotype-Immunophenotype Relationships in TET2 and IDH-Mutant Myeloid Transformation

Large scale molecular profiling studies in AML patients have suggested that stepwise acquisition of somatic mutations is crucial in driving leukemic development. High variant allele frequency (VAF) mutations in epigenetic modifier genes, such as TET2 and IDH1/2, are thought to occur early in AML pathogenesis while oncogenic mutations with typically lower VAF mutations, including FLT3 and NRAS, are suggested to occur late in disease evolution. While bulk DNA sequencing has catalogued co-mutations found in individual AMLs, it cannot unveil the heterogeneity and composition of clones that makes up the disease. Elucidating the architecture and clone-specific molecular profiles at the single cell resolution will be key to understanding how sequential and/or parallel mutation acquisition drives myeloid transformation.To assess the clonal architecture of AML, we previously performed single cell DNA sequencing (scDNA seq) in 146 patients with myeloid malignancies. We have further identified specific mutational combinations driving clonal expansion in TET2- or IDH1/2- mutant AML samples. These studies suggest TET2 and IDH1/2 can cooperate to promote clonal expansion with DNMT3A and NPM1 (Figure 1A). However, TET2 or IDH1/2 mutant clones that acquired KRAS mutations underwent minimal clonal expansion, suggesting mutant-pair specific fitness alterations (Figure 1B). To further identify how co-mutational pairing impacted clonal fitness and differentiation, we integrated the scDNA platform with immunophenotypic profiling of 45 cell surface markers and analyzed new TET2- and IDH1/2- mutant AML samples (Figure 1C). We identified clone-specific differences in lineage markers depending on co-mutational partners. NPM1 co-mutant clones were enriched for more primitive markers (CD33), whereas NRAS co-mutant clones possessed high expression of myeloid differentiation markers (CD14/CD11b), suggestive of clone-specific fitness landscapes across hematopoietic differentiation. We also identified divergent clonotype-immunophenotype patterns in TET2- and IDH2-mutant clones harboring NPM1/RAS mutations, suggesting that initiating mutations may prime mutant clones for very different evolutionary trajectories as they acquire similar mutations in leukemogenesis (Figure 1D).To deterministically delineate the relationship between clonal evolution and myeloid transformation, we generated Cre-inducible single (Tet2 -/-), double (Tet2 -/-/Nras G12D and Tet2 -/-/Npm1 cA/wt), and triple (Tet2 -/-/Npm1 cA/wt/Nras G12D) mutant mice and evaluated differences in chimerism, immunophenotype, and survival. We observed a shortened survival for double and triple mutant mice, compared to Tet2 -/- only mice (Figure 1E). As previously reported, Tet2 -/-/Nras G12D mice developed a CMML-like phenotype. Critically, the addition of Npm1 resulted in a more rapid disease onset and transformation to AML (Figure 1F). Moreover, triple mutant WBM transplanted to form a fully penetrant disease into secondary recipients, while double mutant Tet2 -/-/Nras G12D WBM failed to form disease within 3 months of transplant, suggesting a difference in the cell population responsible for disease propagation. Immunophenotypic alterations were evident with Tet2 -/-/ Nras G12D displaying an increase in Mac1 +Gr1 + cells compared to Tet2 -/-/Npm1 cA/wt/Nras G12D mice which possessed increased Mac1 +Gr1 - cells and expansion of lineage negative cells (Figure 1G). These findings align with the clonotype specific expression patterns observed in clinical specimen and suggest that myeloid transformation and maturation biases are influenced by specific mutational combinations. [Display omitted] DisclosuresMiles: Mission Bio: Honoraria, Speakers Bureau. Bowman: Mission Bio: Honoraria, Speakers Bureau. Carroll: Janssen Pharmaceutical: Consultancy; Incyte Pharmaceuticals: Research Funding. Levine: Astellas: Consultancy; Janssen: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Incyte: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Prelude: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Morphosys: Consultancy.

In Vivo crispr Screening Identifies GLUT1 As a Key Metabolic Regulator of MLL-AF9-Driven Acute Myeloid Leukemia

Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting.The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation.Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production.To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136).Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1135: Antitumor activity of novel reversible LSD1 inhibitors in preclinical models of AML and SCLC

Abstract Lysine-specific demethylase 1 (LSD1) has become a biologically important and validated epigenetic target for cancer therapy since its identification in 2004. LSD1 mediates many cellular signaling pathways and is involved in the initiation and development of cancers. Aberrant overexpression of LSD1 has been observed in different types of cancers, and such epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we introduce HM97346 and its derivatives as novel LSD1 inhibitors, and report a therapeutic potential for AML and SCLC patients based on several preclinical characterization and assessment. HM97346 and its derivatives were designed and synthesized as novel reversible inhibitors of LSD1. Standard cell proliferation assay, immunoblotting, biomarker expression, and apoptosis analysis were carried out to validate their potency in vitro studies using different AML and SCLC cell lines. The in vivo antitumor activity was evaluated in AML and SCLC cell xenograft mouse models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. HM97346 and its derivatives showed strong inhibition on LSD1 enzymatic activity in biochemical assay and promoted the methylations of histone H3 lysine 4 (H3K4) in both SCLC and AML cells. They reduced the expression of neuroendocrine factors ASCL1 in SCLC cells, effectively and dose-dependently. Moreover, in human AML cell lines, they stimulated cellular differentiation by disrupting LSD1-GFI1 complex and induced transcription factor PU.1 as well as myeloid differentiation markers CD86 and CD11b. As a result, they significantly inhibited cellular proliferation and induced apoptosis in studies using AML and SCLC cells. Oral administration of HM97346 and its derivatives inhibited tumor growth in human AML and SCLC cell xenograft mouse models studies. These antitumor effects dramatically increased in combination with standard chemotherapies. In conclusion, HM97346 and its derivatives, new synthesized reversible LSD1 inhibitors, showed potential anticancer activity in preclinical AML and SCLC models. Moreover, these compounds could provide a new insight and possibility in cancer therapy through epigenetic modulation as not only monotherapy but also combination therapy. Citation Format: Joo-Yun Byun, Won Jong Lee, Won jung Kim, Minjeong Kim, Inhwan Bae, Seokhyun Hong, Yu-Yon Kim, Hyunjin Park, Eunyoung Kim, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitors in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1135.

The Age of the Hematopoietic Microenvironment Specifies Lineage Commitment in MLL-Rearranged Leukemia

Leukemias are a heterogenous group of hematologic malignancies classified by their pathobiologic characteristics including differentiation lineage (myeloid, B-lymphoid, T-lymphoid), oncogenic mutations, karyotypic abnormalities, and clinical characteristics, such as presenting white blood cell count, in order to guide therapy. It is well known that many such characteristics of leukemia are associated with a typical age of disease onset. For example, in children, B-acute lymphoblastic leukemia (B-ALL) occurs most commonly, while acute myeloid leukemia (AML) is more frequent in older adults. Furthermore, B-ALL of infancy, typically diagnosed before 12 months of age, is a unique clinical entity with a poor prognosis compared to B-ALL of later childhood. Infant B-ALL often aberrantly expresses markers of myeloid differentiation, is commonly driven by rearrangements involving the MLL gene, and is treated with hybrid chemotherapy regimens incorporating drugs directed against both myeloid and lymphoid differentiation. The mechanistic underpinnings of the age specificity of leukemia remain unknown, but enhanced understanding could improve disease models and lead to new therapies.The hematopoietic system undergoes a highly conserved process of temporal maturation on a regimented schedule in concert with development of the host. Postnatally, during aging of the hematopoietic system, hematopoietic stem and progenitor cells (HSPCs) alter their self-renewal and lineage biases. Normal development and aging of the blood forming system are associated with changes in the hematopoietic microenvironment. We therefore hypothesized that the normal process of development and aging of blood regulates leukemia phenotypes due to intrinsic changes in HSPCs and/or the hematopoietic niche over time.To test this, we used heterochronic transplantation in a model of MLL translocation-driven leukemogenesis. We first isolated normal HSPCs from midgestation murine fetal liver and adult bone marrow, and transformed these cells with the MLL-AF9 oncogene. Upon transplantation into congenic adult recipients, both cell sources gave rise to pure AML with identical latency and leukemia initiating cell (LIC) content. Next, we transplanted MLL-AF9 -transformed bone marrow HSPCs into congenic adult or neonatal recipients. We found that leukemia developing in neonates contained a small (3.5 ± 1%) population of cells expressing the B-cell marker B220, which were not present in pure AML developing in adults. Serial transplantation of this neonatal leukemia through neonatal recipients decreased the latency of disease onset to as short as 20 days, coincident with expansion of the B220+ component (to 45 ± 4% in tertiary recipients) and infiltration of thymus, lymph nodes, and testes. This mixed B-lymphoid/myeloid leukemia developing in infant mice bore a similar immunophenotype and transcriptional profile by gene set enrichment analysis (GSEA) to human MLL-AF9 -driven infant ALL. These results extend previous observations that MLL -driven leukemia is sensitive to signals from the microenvironment by demonstrating the importance of the age of the niche in dictating lineage differentiation (Cancer Cell 13: 483 [2008] and Science 316:600 [2007]).The sustainment of mixed lineage leukemia through serial transplantation in neonates near limiting dilution suggested the existence of a multipotent progenitor-like LIC. By sorting neonatal leukemia cells using markers of lymphoid commitment, we identified Flk2+ cells within the L-GMP compartment (Lin- kit-lo CD16/32+ CD34+) primed to produce both B-lymphoid and myeloid cells in single cell clonogenicity assays, while Flk2- LGMPs were strongly myeloid-biased (P < 0.05). By screening for differentially expressed cytokines between adult and neonatal bone marrow stroma, we implicated the chemokine Ccl5 as a factor restraining B-lymphoid commitment in leukemia in pro-myeloid adult niches. Taken together, our data show that the neonatal hematopoietic niche supports the development infant-like mixed B/myeloid leukemia, while an adult niche promotes pure AML from identical cells of origin. We attribute this to inhibition of B-lymphoid output from multipotent progenitor-like LICs by Ccl5 from adult bone marrow stroma. These findings connect normal maturation and aging of the hematopoietic system and its microenvironment to the age specificity of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

SUMMARYGenome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.

Identification and characterization of a novel adiponectin receptor agonist adipo anti-inflammation agonist and its anti-inflammatory effects in vitro and in vivo.

Adiponectin (APN) is an adipokine secreted from adipocytes that binds to APN receptors AdipoR1 and AdipoR2 and exerts an anti-inflammatory response through mechanisms not fully understood. There is a need to develop small molecules that activate AdipoR1 and AdipoR2 and to be used to inhibit the inflammatory response in lipopolysaccharide (LPS)-induced endotoxemia and other inflammatory disorders. We designed 10 new structural analogues of an AdipoR agonist, AdipoRon (APR), and assessed their anti-inflammatory properties. Bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PEMs) were isolated from mice. Levels of pro-inflammatory cytokines were measured by reverse transcription and real-time quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and microarray in LPS-induced endotoxemia mice and diet-induced obesity (DIO) mice in which systemic inflammation prevails. Western blotting, immunohistochemistry (IHC), siRNA interference and immunoprecipitation were used to detect signalling pathways. A novel APN receptor agonist named adipo anti-inflammation agonist (AdipoAI) strongly suppresses inflammation in DIO and endotoxemia mice, as well as in cultured macrophages. We also found that AdipoAI attenuated the association of AdipoR1 and APPL1 via myeloid differentiation marker 88 (MyD88) signalling, thus inhibiting activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and c-Maf pathways and limiting the production of pro-inflammatory cytokines in LPS-induced macrophages. AdipoAI is a promising alternative therapeutic approach to APN and APR to suppress inflammation in LPS-induced endotoxemia and other inflammatory disorders via distinct signalling pathways.

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

Accessible Source of Stem Cells: Expansion of CD34+ Cells in Bulk Peripheral Blood Mononuclear Cells Using UM171

Introduction UM171, a novel compound with hematopoietic stem cells (HPSCs) self-renewal properties has shown great potential for expanding CD34+ HPSCs while preserving their stemness and increasing their engrafting abilities. The compound has been increasingly used for the purpose of CD34+ HPSCs expansion, both in pre-clinical research and in clinical applications. Its use is particularly important in the gene therapy (GT) and bone marrow transplantation (BMT) fields, especially when CD34+ expansion is needed to ensure the success of the therapy. Recent reports have demonstrated UM171efficacy to expand enriched CD34+ HPSCs from peripheral blood (PB) in osteopetrosis patients, and after mobilization in lymphoma patients. Here, we demonstrate the feasibility of exploiting the UM171 molecule to directly expand CD34+ HPSCs in bulk PB mononuclear cells (MNCs) without the need of either HPSCs mobilization or CD34+ enrichment. Material and Methods Our protocol entails culturing of bulk MNCs directly after Ficoll gradient separation in IMDM media supplemented with 10% FBS, and HPSCs stemness sparing cytokines, used in clinical suitable concentrations: SCF 300 ng/ml, TPO 100 ng/ml, Flt3-L 300 ng/ml and IL3 60 ng/ml +/- 38 nM UM171 for 20 days. Briefly, quantities of 7.5± 0.5 ml of blood from 2 healthy donors were gradient-separated by Ficoll and yielded a total of 6.6±0.2X10^6 MNCs. Cells were divided in two conditions/donor (Cytokines +/- UM171) and 3.3±0.14X10^6 cell were seeded in 6 well plates at concentration of 1.1X10^6/ml in a total volume of 3 ml. Half of medium was changed every 5 days. Cell count and phenotype of HPSCs were periodically evaluated to determine the optimal expansion window. Cells were manually counted, and phenotype was measured through Symphony A5 flow-cytometry with this antibody panel: Zombie viability dye, CD45, CD34, and in the last timepoint (day 20) CD11b as an exclusion marker for myeloid differentiation. Results UM171 treatment efficiently achieved direct expansion of CD34+ HPSCs in bulk PB MNCs, reaching a peak expansion at day 12 with CD34+ Cells representing 3.9±0.3% of cells (mean±SD of live cells) compared to 0.08±0.04% at baseline (48.75x increase). This increase in CD34+ cell concentration was also partially due to the decline of total PB MNCs cell count during culture time (declined up to 90%). Despite the overall cell count decrease, the CD34+ HPSCs population had an absolute number increase of 6.5±0.916 times with respect to the initial count, peaking at day 12 with an absolute number of 19600±1344 cells compared to 2985±1466 cells at baseline. If projected to a larger scale, this yield proves the feasibility of obtaining of ∼20X10^6 CD34+ HPSCs from a buffy coat of 350 ml blood donation. This amount would be adequate for a pediatric HSCT, and very handy for multiple experiments with CD34+ HPSCs. Conclusion In conclusion, we have provided a proof of concept on the feasibility to directly expand CD34+ HPSCs in bulk PB MNCs. Further characterization of the expanded CD34+ will be needed to ensure stemness, plasticity, and engrafting ability. The advantage of expanding HPSCs in bulk is to preserve the rare naïve CD34+ population that can be lost during the immunoselection process and to take advantage of the myeloid niche, to further enhance the expansion along with UM171. This expansion approach can be performed at larger scale on the buffy coats often dismissed in blood banks to provide considerable number of CD34+ cells for either banking, research or future clinical application. Disclosures No relevant conflicts of interest to declare.

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

PU.1-Dependent Enhancer Decommissioning Drives Transformation of Tet2 deficient Hematopoietic Stem and Progenitor Cells

Acute myeloid leukemia (AML) is the most frequent leukemia in elderly individuals with a median age at diagnosis of 67 years (Juliusson et al., Blood 2009). It arises in a step-wise process and originates from hematopoietic stem cells (HSC) (Jan et al.,Sci Transl Med. 2012). Genetic and epigenetic alterations drive the formation of pre-leukemic HSC clones with altered function, which can gain dominance and eventually give rise to AML upon the acquisition of cooperating lesions (Jan et al.,Sci Transl Med. 2012). However, it is currently impossible to predict which healthy elderly individuals with clonal hematopoiesis will eventually develop myeloid malignancies, as the pathways to leukemia are unknown. Heterozygous inactivating mutations of the epigenetic regulator Ten-Eleven Translocation-2 (TET2) are commonly found in patients with AML, yet also in a remarkable fraction of healthy elderly individuals in whom it is associated with clonal hematopoiesis (Busque, et al Nat Genet. 2012). These observations and studies in Tet2-deficient mice strongly suggest that TET2 inactivation is an early event in the pathogenesis of myeloid malignancies, but is not sufficient to fully transform HSC (Moran-Crusio et al., Cancel Cell 2011). TET2 cooperates with several transcription factors to regulate hematopoiesis (Rasmussen et al., Genome Res 2019), one of which is PU.1 (de la Rica et al., Genome Biol. 2013), an essential transcription factor governing normal hematopoiesis (Iwasaki et al., Blood 2005). In humans, PU.1 activity or expression is only moderately impaired in the majority of AML patients, and remarkably, also in aged HSC (Will et al., Nat Med. 2015), underscoring the essentiality of PU.1. Importantly, PU.1 target genes are frequently found hypermethylated in AML (Sonnet et al., Genome Med. 2014, Kaasinen et al., Nat Commun. 2019), suggesting a profound epigenetic inactivation of the PU.1 network. We hypothesized that moderate impairment of PU.1 abundance, as found in AML, can cooperate with loss-of-function mutations of Tet2 to initiate malignancy. We developed a novel tissue-specific compound mutant mouse model carrying heterozygous deletion of an upstream regulatory element (URE) of Pu.1 along with Tet2 deletion (Vav-iCre+ PU.1URΕ∆/+Tet2+/flox; Vav-iCre+ PU.1URΕ∆/+Tet2flox/flox). While none of the single mutant mice developed AML, compound mutant mice developed aggressive myeloid leukemia whose penetrance and latency exhibited Tet2 dose dependency. The disease presented with leukocytosis, anemia and splenomegaly. By cell morphology analysis of the peripheral blood, bone marrow and spleen, the leukemic mice exhibited accumulation of differentiation-blocked myeloblasts, myelocytes and/or metamyelocytes, that was confirmed using detailed myeloid differentiation markers, distinguishing the disease in immature or mature AML. Furthermore, gold standard in vitro and in vivo assays, assessing both self-renewal and differentiation capacity of double mutant mice-derived cells, revealed that the expanded differentiation-blocked stem and progenitor cells bear aberrant self-renewal and disease-initiating capacities. Comprehensive molecular profiling by next generation sequencing of disease-initiating cells uncovered a substantial overlap with human AML, such as functional GF1b loss with concomitant overexpression of CD90/Thy1 (Thivakaran et al., Haematologica 2018). Importantly, our analyses also revealed transcriptional dysregulation, hypermethylation of PU.1 regulated enhancers with concomitant loss of enhancer activity and alterations in chromatin accessibility of particularly genes co-bound by PU.1 and TET2. Current efforts focus on identifying key effectors of the dysregulated PU.1/TET2 sub-network driving malignant transformation in clonal hematopoiesis. Our collected data provide proof of concept that moderate PU.1 dose impairment can functionally cooperate with the inactivation of Tet2 in the initiation of myeloid leukemia and uncovers a likely unifying AML pathomechansim. Disclosures Will: Novartis Pharmaceuticals: Other: Service on advisory boards, Research Funding.

PRT4165 nanocomposite promoting epigenetic retardation through proteasomal depletion of polycomb in acute myeloid leukemia

The polycomb repressive complex 1 (PRC1) protein, Bmi1 is overexpressed in acute myeloid leukemia and mediates histone H2A monoubiquitination through its ubiquitin (E3) ligase activity. The 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) inhibits PRC1-mediated H2A monoubiquitination but the poor aqueous solubility, and physiological instability limit its application. In current study, the encapsulation of PRT4165 in human serum albumin nanoparticles (HSANPs) improved its therapeutic efficacy as evident through the enhanced apoptosis, sub-G1 cell cycle arrest, depolarized mitochondrial membrane, reactive oxygen species generation and caspase 3 activation. The nanoformulation repressed Bmi1 through ubiquitin-proteasome pathway (UPP) and regulated H2AK119 ubiquitination in AML cells. Co-immunoprecipitation studies revealed the direct interaction of Bmi1 and C-Myb, a crucial regulator of AML pathogenesis. Moreover, PRT4165 encapsulated HSANPs showed improved in vivo biodistribution, better dispersibility and biocompatibility, and exhibited enhanced suppression of leukemia stem cell marker, CD45+ and activation of myeloid monocytes differentiation marker, CD11b+ in AML xenograft mouse model.

Abstract A55: Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities

Abstract Introduction: Nearly 35% of children with acute myeloid leukemia (AML) die, despite aggressive but toxic and nonspecific therapies. New approaches to find and drug pediatric AML targets are clearly needed. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the RARA agonist tamibarotene. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by RARA regulation. Methods: Four AML/APL cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes, with an over-representation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters. Some of the highly correlated, SE-associated genes in these clusters encode proteins involved in inflammation. Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes. Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples reduced cell viability and increased apoptosis (detected by annexin V+ and activated caspase 3/7), with no effect in RARA SE- pAML samples. In the RARA SE+ samples, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) and DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial). Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and have seen a high frequency of a RARA SE in pAML. Tamibarotene has antiproliferative, proapoptotic, and prodifferentiation effects in RARA SE+ pAML. We are confirming these results in a RARA SE+ patient-derived xenograft mouse model, evaluating combinations, and interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Citation Format: Monika Perez, Alfred Daramola, Oscar Sias-Garcia, Helen Wei, Nikitha Cherayil, Charles Y. Lin, Joanna S. Yi. Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A55.

Human CD34+ hematopoietic stem cells culture in humanized culture medium for cell therapy

IntroductionHematopoietic stem cell transplantation (HSCT) is a proper treatment for many cancers, including malignant and non-malignant hematologic and immune disorders. However, due to the small quantity of HSCTs, their number should be increased ex vivo. Cell proliferation increase by animal serum can transmit certain diseases and immune problems to the recipient; therefore, replacement of culture media with media of human origin seems to be crucial. Acellular Wharton's jelly (AWJ) can be considered as an alternative medium in this regard. This study aimed to investigate the effect of AWJ substitution for fetal bovine serum (FBS) on the expression of myeloid and lymphoid specific markers and CD34+ cells proliferation pattern following co-culture with mesenchymal stem cells. Materials and methodsIn this study, CD34+ umbilical cord blood HSCs were co-cultured with MSCs in the presence of 10% FBS or AWJ, stem cell factor (SCF), thrombopoietin (TPO), and FLT3-L and the proliferation pattern and expression of myeloid as well as lymphoid lineage markers were then evaluated. ResultsThe expression of myeloid and lymphoid specific markers was increased and decreased in AWJ and FBS containing culture medium, respectively. ConclusionThis study provided evidence for the differentiation of UCB-derived CD34+ cells toward myeloid lineage and lack of their differentiation toward lymphoid lineage in AWJ containing culture medium.

SRC family kinase inihibitors enhance all-trans-retinoic acid or arsenic trioxide induced differentiation of NB4 Leukemia Cells

Background & Aim Background: The Src family kinases (SFK) are non-receptor tyrosine kinases that have been implicated in a variety of process, including proliferation, survival, differentiation and migration. Recent studies showed that inhibition of SFKs resulted in enhancement of retinoic acid-induced myeloid differentiation. In this study, we investigated whether SFK inhibitor PP2 and FDA approved dual Src/ABL inhibitor (bosutinib or dasatinib) could enhance the differentiation of NB4 leukemia cells when combined with all-trans-retinoic acid (ATRA) or arsenic trioxide (ATO). Methods, Results & Conclusion Methods: We demonstrated the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATRA or ATO. Subsequently, we investigated whether bosutinib and dasatinib could enhance the differentiation of NB4 cells when combined with ATRA or ATO. The cells were analyzed for myeloid differentiation marker CD11b expression using flow cytometry. These results were confirmed in morphologic analysis by Wright stain and NBT reduction assay. In addition, we attempted to determine the difference in retinoic acid-induced gene expression. Results Our data showed that SFK inhibitor PP2 enhanced myeloid differentiation when combined with ATRA or ATO. The synergistic effect was significantly stronger when PP2 was combined with ATRA and ATO (triple treatment) than when PP2 was combined with ATRA or ATO (dual treatment). The similar results were obtained when bosutinib and dasatinib were applied instead of PP2. Co-treatment with bosutinib plus ATRA or ATO, and dasatinib plus ATRA or ATO resulted in significant enhancement of CD11b-positive cells when compared to the each treatment group in bosutinib, dasatinib, ATRA, or ATO. Also, the synergistic effects of bosutinib and dasatinib in combination with ATRA were greater than in combination with ATO. Conclusions SFK inhibitor PP2 and dual Src/ABL inhibitor (bosutinib or dasatinib) can enhances the differentiation of NB4 leukemia cells when combined ATRA and/or ATO. These findings suggest that ATRA or ATO combinated with dual Src/ABL inhibitors may have therapeutic beneficial for the treatment of APL patients.