EVI1 in Acute Myeloid Leukemia Triggers Metabolic Reprograming Associated with Leukemogenesis and Increases Sensitivity to L-Aspalaginase

Abstract Leukemia cells survive and proliferate under conditions of metabolic stress by acquiring mutations that increase energy metabolism. Here, we aimed to identify a specific metabolic inhibitor and examine transcription factor-enhanced changes in energy metabolism by refractory leukemia cells. Overexpression of Ecotropic Virus Integration site 1 protein homolog (EVI1) in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. We focused on metabolic reprograming of MLL leukemia cells expressing EVI1, since the metabolic relationship between MLL and EVI1 is unclear. We used an extracellular flux analyze to examine metabolic changes during leukemia development in a mouse model of MLL-r AML expressing high levels of EVI1 (EVI1+). To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used transgenic mice expressing EVI1 (TG) in LSK and GMP cells model in which AML is driven by the MLL-AF9 oncogene. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a flux analyzer. TG MLL-AF9 mice showed a significantly higher basal and capacity of OCR than WT MLL-AF9 mice ex vivo. EVI1+ cells showed accelerated oxidative phosphorylation (OXPHOS) prior to activation of glycolysis, and higher dependency on glutamine as an energy source. To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-fight mass spectrometry-based metabolome profiling of WT and TG MLL-AF9 leukemia cells. We found significant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. Fructose 1,6-bisphosphate and lactate were up-regulated in TG MLL-AF9 cells, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in TG MLL-AF9 cells. EVI1 played a role in glycolysis as well as driving expression of genes engaged in the tricarboxylic acid cycle. Next, we tested whether pharmacological inhibition of glycolysis and glutaminolysis suppresses MLL-AF9. L-asparaginase (ASP) [which catalyzes hydrolysis of asparagine (Asn) and glutamine (Gln) to asparatic acid or glutamic acid, respectively] markedly suppressed proliferation of TG MLL-AF9 cells, EVI1highAML cell lines. To examine the therapeutic potential of ASP in vivo, we treated secondary recipients of TG MLL-AF9 AML cells with ASP or control (vehicle), beginning 5 days post-transplantation. Mice then received intraperitoneal injections (five times per week) of distilled water or ASP (1000 U/kg). ASP led to a significant reduction in the number of GFP+ AML cells in the peripheral blood and increased the survival of recipient mice. Next, we examined an AML xenograft model. Two groups of NOG mice were injected subcutaneously with UCSD/AML1 cells and then treated with ASP or control. ASP -treated mice showed a significant reduction in the growth of AML tumors. Overall, these findings indicate that ASP -mediated inhibition of OXPHOS is a potential treatment for AML. We clarified that increased glutamine dependency by MLL-r AML cells showing high EVI1 expression makes them sensitive to ASP. We found that the energy advantage of AML cells is acquired via transcription factor-mediated activation of mitochondrial metabolism, leading to a poor prognosis. Furthermore, we show that new therapeutic options can be identified by examining the energy-based metabolic characteristics of leukemia cells. Disclosures No relevant conflicts of interest to declare.

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Abstract 282A: The Nrf2 transcription factor is a positive regulator of differentiation of acute myeloid leukemia cells induced by vitamin D derivatives and plant polyphenols

10.1158/1538-7445.am2011-282a ◽

2011 ◽

Author(s):

Victoria Novik ◽

Irene Bobilev ◽

Andrzej Kutner ◽

Itai Levi ◽

Ofer Shpilberg ◽

...

Keyword(s):

Vitamin D ◽

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Plant Polyphenols ◽

Positive Regulator ◽

Acute Myeloid Leukemia Cells ◽

Nrf2 Transcription Factor ◽

Acute Myeloid

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RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

Blood ◽

10.1182/blood.v116.21.2164.2164 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2164-2164

Author(s):

Benjamin J Schmiedel ◽

Constantin M Wende ◽

Tina Baessler ◽

Carolin Scheible ◽

Stefan Wirths ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Conflicts Of Interest ◽

Immune Surveillance ◽

Surface Protein ◽

Leukemia Cells ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

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Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1288.1288 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1288-1288

Author(s):

Rong Chen ◽

Bonnie Leung ◽

Yuling Chen ◽

William Plunkett

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Synthesis ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Bh3 Mimetics ◽

Abl Kinase ◽

Acute Myeloid

Abstract Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations. To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis. Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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High IDH1 Expression Is Associated with a Poor Prognosis in Cytogenetically Normal Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.5314.5314 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5314-5314 ◽

Cited By ~ 1

Author(s):

Qiu-Ling Ma ◽

Jing-Han Wang ◽

Yun-gui Wang ◽

Chao Hu ◽

Qi-Tian Mu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Prognostic Value ◽

Conflicts Of Interest ◽

Remission Rate ◽

Strong Association ◽

Gene Mutations ◽

Micro Rna ◽

Acute Myeloid

Abstract ABSTRACT The prognostic value of IDH1 mutations has been systematically evaluated in acute myeloid leukemia (AML) patients recently. However, the role of IDH1 expression in AML is still under exploration. To investigate the clinical significance, we analyzed the IDH1 expression in 320 patients with cytogenetically normal AML (CN-AML) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). High expression of IDH1 was predominant in patients with FLT3-ITD and DNMT3A mutations, and less prevalent in cases with CEBPA double allele mutations. Strong association was observed between high IDH1 expression and low expression of micro-RNA 181 family. Prognosis was adversely affected by high IDH1 expression with shorter overall survival (OS) and event free survival (EFS) in the context of clinical characteristics including age, WBC, and gene mutations of NPM1, FLT3-ITD, CEBPA, IDH1, IDH2, and DNMT3A in CN-AML. Moreover, the clinical outcome of IDH1 expression in terms of OS, EFS and complete remission rate still remained in multivariate models in CN-AML. Importantly, the prognostic value was validated using the published microarray data from 79 adult patients treated according to the German AMLCG-1999 protocol. Our results demonstrated that high IDH1expression is associated with a poor prognosis of CN-AML. Disclosures No relevant conflicts of interest to declare.

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Acute Myeloid Leukemia Cells Acquire Chemo-Resistance By Inducing Osteoblast Differentiation in Mesenchymal Stem Cells through up-Regulation of RUNX2

Blood ◽

10.1182/blood.v124.21.2929.2929 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2929-2929

Author(s):

Venkata Lokesh Battula ◽

Phuong M Le ◽

Jeffrey Sun ◽

Teresa McQueen ◽

Anitha Somanchi ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Alkaline Phosphatase ◽

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Cell Surface ◽

Myeloid Leukemia ◽

Osteoblast Differentiation ◽

Leukemia Cells ◽

Acute Myeloid

Abstract The leukemia bone marrow micro-environment (BME) is comprised of the endosteal and vascular niches, provides vital support for cellular growth and conveys drug resistance to leukemia cells. Several reports suggest that mesenchymal stem/stromal cells (MSCs) present in the bone marrow niche induce cell survival and anti-apoptotic proteins in acute myeloid leukemia (AML) cells and protect them from chemotherapy. The mechanisms underlying BME-mediated chemo-resistance however have not been fully elucidated. Here, we hypothesize that AML cells induce functional changes and prime MSCs to protect leukemia cells from chemotherapy. To test our hypothesis, we have compared age matched (between 40-60 years) bone marrow derived MSCs from AML patients (AML-MSC, n=10) and normal (N-MSC, n=10) individuals and analyzed their proliferation, cell surface phenotype, multi-lineage differentiation and chemo-protection potential. AML-MSCs are phenotypically different, with their polygonal morphology and larger cell size compared to N-MSCs which are elongated and spindle shaped appearance. The average cell doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs during their exponential growth phase (p<0.01). Cell surface phenotyping by flow cytometry revealed that most of the markers known to be expressed on N-MSCs including CD105, CD90, CD73, CD51, CD44, SUSD2, CD106, CD140b, CD140a, CD106 and CD271 were also expressed on AML-MSCs at similar levels. Interestingly, tissue non-specific alkaline phosphatase (TNAP, clone W8B2), a cell surface protein highly expressed in naïve-MSCs and osteoblast progenitors (Battula VL et al., Haematologica, 2009) was 10-14 fold higher in AML- as compared to N-MSCs. Since TNAP is also a osteoblast specific marker, we compared osteoblast differentiation potential of N- vs AML-MSCs. Surprisingly, a dramatic increase in alkaline phosphatase activity (by BCIP/NBT substrate) was observed in AML-MSCs even without induction of osteoblast differentiation. mRNA analysis by qRT-PCR revealed that osteoblast specific genes including osteopontin, TNAP, osteocalcin, and osterix were 5-10 fold up-regulated in AML-MSCs compared to N-MSCs before induction. In N-MSCs, the expression of these markers was induced only under osteoblast differentiation conditions. These data indicate that AML-MSCs are primed to differentiate into-osteoblasts. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a > 95% reduction (p<0.0001) in the number mature adipocytes in AML-MSCs compared to N-MSCs suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To understand the mechanism inducing osteogenic specific differentiation of AML-MSCs, we performed mRNA expression analysis of genes that regulate this process. We found RUNX2, a transcription factor that induces osteogenic but inhibits adipogenic differentiation, was 4-5 fold increased in AML-MSCs compared to N-MSCs. To validate these observations, we co-cultured N-MSCs in the presence or absence of OCI-AML3 cells for 3-5 days and FACS sorted the MSCs for gene expression analysis. We observed a 3-4 fold up-regulation of TNAP protein expression by flow cytometry and 4-6 fold up-regulation of osteoblast specific markers including osteopontin, alkaline phosphatase and osterix in MSCs co-cultured with OCI-AML3 cells. In addition, RUNX2 was up-regulated in MSCs when co-cultured with OCI-AML3 cells. These data suggest that AML cells induce osteogenic differentiation in BM-MSCs by up-regulation of RUNX2. To identify the clinical significance of these observations, we examined the ability of AML- and N-MSCs to protect AML cells from chemotherapy. Co-culture of OCI-AML3 cells with either AML- or N-MSCs and treatment with Cytarabine revealed a 15±4.5% increase in the number of live leukemia cells when co-cultured with AML-MSCs compared to N-MSCs. These data indicate that AML-MSCs protect leukemia cells better from chemotherapy than normal MSCs. In conclusion, AML cells induce osteogenic differentiation in MSCs through up-regulation of the RUNX2 transcription factor. Increased chemo-protection of AML cells by AML-MSCs suggests a prominent role of these cells in AML relapse. Targeting RUNX2 and thereby inhibition of osteoblast differentiation of MSCs may provide enhanced treatment options for AML therapy. Disclosures No relevant conflicts of interest to declare.

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Acute Myeloid Leukemia-Derived Exosomes Transform Bone Marrow Niche into Leukemic Niche.

Blood ◽

10.1182/blood.v124.21.352.352 ◽

2014 ◽

Vol 124 (21) ◽

pp. 352-352

Author(s):

Bijender Kumar ◽

Mayra Garcia ◽

Lihong Weng ◽

Xiaoman Lewis ◽

Jodi Murakami ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Leukemia Cells ◽

Bone Marrow Niche ◽

Stromal Compartment ◽

Bm Niche ◽

Acute Myeloid

Abstract Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) niche, where they hide from chemotherapy and continue to divide. As yet, how leukemia cells alter the BM niche to facilitate their growth and assist them in evading chemotherapy is unclear. In this study, we provide compelling evidences that acute myeloid leukemia (AML), through exosome secretion, transformed the BM niche to facilitate their own growth and suppress normal hematopoiesis. Using AML xenograft and MLL-AF9 knock-in mouse model, we show that leukemia cells as well as AML-derived exosomes stimulate the growth of BM stromal progenitors and blocked the osteolineage development in our stromal compartment analysis. Histological analysis and micro-CT examination confirmed loss or thinning of the bone in both leukemia and leukemic exosome-treated animals. Expression of cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt2 and VEGF) are upregulated, whereas genes important for HSC maintenance (CXCL12 and SCF), osteoblast (OCN, OSX, Notch3 and IGF1) and chondrocyte (ACAN, SOX9) development are suppressed. While we observed increases in phenotypic LT-HSC in AML-derived exosomes treated mice, these mice show reduced multilineage reconstitution ability, increased cell cycle entry and higher sensitivity to myeloablative stress suggesting that HSCs from exosome-treated mice have lower stem cell activity than their counterparts from normal mice.In addition, leukemia-modified stroma cells exhibit marked reduction in ability to support normal HSCs. Pre-treatment of AML-derived exosome “prime” the animal for leukemia cell invasion and accelerate leukemia progression. Conversely,disruption of exosome secretion by targeting Rab27a in AML cells significantly delays leukemia progression. These data strongly support the notion that leukemia-modified niches favor leukemic cell proliferation and suppress normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Granulocyte Colony-Stimulating Factor Inhibits CXCR4/SDF-1 Signaling and Overcomes Stromal-Mediated Drug Resistance in Acute Myeloid Leukemia Via Mir-146a

Blood ◽

10.1182/blood.v126.23.4763.4763 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4763-4763 ◽

Cited By ~ 1

Author(s):

Hua Zhong ◽

Lan Xu ◽

Xin Li ◽

Xian-fu Sheng ◽

Fang-yuan Chen ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Western Blotting ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemia Cells ◽

Hl60 Cells ◽

Expression Levels ◽

Qrt Pcr ◽

Smad4 Expression ◽

Acute Myeloid

Abstract CAG regimen was widely used in the clinical treatment of acute myeloid leukemia (AML). However, the mechanisms of G-CSF in the CAG regimen remain unknown. For the purpose of better elucidating the function of G-CSF, we evidenced that G-CSF could enhance HL60 and primary leukemia cells proliferation in vitro. Meanwhile, transwell migration experiments demonstrated that G-CSF, similar as the CXCR4 antagonist AMD3100, could remarkably inhibit the chemotaxis of HL60 cells induced by the chemokines released from marrow stromal cells (Fig 1). qRT-PCR and Western blotting results showed that the expression of miR-146a was up-regulated after G-CSF treatment, while the expression levels of CXCR4 and smad4 were down-regulated (Fig 2). CXCR4 and Smad4 expression levels in miR-146a over expression lentivirus infected HL60 cells were significantly decreased, which manifested the direct or indirect targeted relationship between miR-146a and CXCR4/Smad4. Our qRT-PCR and Western blotting results also showed an involvement of NF-KB in G-CSF induced up-regulation of miR-146a in AML cells. G-CSF activated NF-KB in the cells. Activated NF-KB induced the up-regulation of miR-146a expression. Sanguinarine, an inhibitor of NF-KB significantly inhibited miR-146a expression. We further demonstrated the involvement of NF-KB in the regulation of G-CSF in CXCR4 and Smad4 expression (Fig 3). Our study demonstrated that G-CSF not only could induce resting leukemia cells into proliferation cell cycle, but also could inhibit chemotaxis of leukemia cells. We elucidated the mechanism of G-CSF/NF-KB/miR-146a/CXCR4 signaling pathway in CAG treatment of AML. The expression levels of MiR-146a /CXCR4 /Smad4 may be selected as the clinical markers for CAG protocol choosing. Disclosures No relevant conflicts of interest to declare.

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The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.3930.3930 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3930-3930

Author(s):

Mark D McKenzie ◽

Margherita Ghisi ◽

Luisa Cimmino ◽

Michael Erlichster ◽

Ethan P Oxley ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Significant Loss ◽

Polymorphonuclear Neutrophil ◽

Myeloid Differentiation ◽

Myeloid Lineage ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy. Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation. Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. Disclosures No relevant conflicts of interest to declare.

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