Therapy-related acute myeloid leukemia with minimal myeloid differentiation (AML-MO) associated with a t(11;19)(q23;p13.3) translocation

The success of all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL) pioneered the concept of differentiation therapy. However, comparable approaches to overcome differentiation blockage for non-APL acute myeloid leukemia (AML) are hampered by lack of an effective drug discovery platform. Recently, we analyzed gene signatures of compounds (ATRA, arsenic trioxide, zalcitabine, and sodium butyrate) that trigger myeloid differentiation in the NCI-60 collection datasets and identified CD38 as the top gene upregulated by differentiation induction. We next initiated an in silico screen in the DTP database of >20,000 compounds to identify compounds that increase CD38 levels. Among those retrieved from "CellMiner" with CD38 as input, we assessed the top 193 available from NCI (r>0.6, p=0) for effects on differentiation utilizing a conditional murine myeloid differentiation-arrest model overexpressing estrogen receptor-HoxA9 (ER-HoxA9) fusion proteins (Cell, 2016). We identified NSC755985 (Nelarabine, NEL) in that screen. NEL is an orphan drug approved to treat relapsed or refractory T-cell acute lymphoblastic leukemia (ALL). NEL at clinically achievable doses (Cmax: 6.73 μM~ 26.91 μM, at a proposed adult dosing schedule of 1,500 mg/m2/day) markedly induced primary AML cell differentiation and death while sparing normal hematopoietic cells (AML vs normal, IC50: 14.7 ± 4.3 μM, n=7, vs 45.3 ± 1.3 μM, n=3), suggesting a therapeutic window in AML. Ex vivo NEL treatment compromised BM engraftment of CD34+ cells from one primary AML specimen in immunodeficient NSG mice at 12 weeks post-transplant (human CD45+ cells in BM: NEL 0.73% vs vehicle 33.42%, n=6/group, p<0.01). NEL administration in vivo (130 mg/kg/day, i.v. for 5 consecutive days) reduced leukemic burden of NSG mice xenografted with luciferase-expressing U937 cells (Radiance: NEL 2.83×107 vs vehicle 1.65×108 photons/s/cm2, n=9/group,p<0.01) and extended mouse survival. Transcriptome analyses in U397 cells and primary AML specimens revealed that NEL treatment upregulated RAS-related pathways. NEL-elicited RAS activation was confirmed by pull-down assay using a GST-Raf1-RBD affinity probe, followed by blotting with a pan-RAS antibody. We performed functional analysis by infecting ER-HoxA9 cells with lentiviral vector expressing oncogenic RAS and observed enhanced myeloid differentiation, as evidenced by increased CD11b/GFP levels relative to MOCK-infected controls. Given that NEL's active metabolite Ara-GTP perturbs guanine nucleotide metabolism, we asked if NEL-evoked RAS activation was associated with accrual of intracellular GTP. HPLC/MS analyses of U937 cells showed that NEL treatment resulted in a marked increase in GTP (approximately 5-fold higher than baseline at sub-millimolar levels) which was secondary to Ara-GTP. Importantly, either electroporation of GTP into U937 cells or indirect introduction of GTP by addition of guanine utilizing purine salvage pathways activated RAS and recapitulated differentiation induction phenotypes. To determine whether AML cells with higher RAS activity exhibited greater NEL sensitivity, we pretreated U937 cells with a RAS agonist KRA-553 or ectopically expressed RAS mutants and observed enhanced NEL inhibitory effects in both cases. We also observed enhanced vulnerability to NEL treatment in MLL-AF9 transformed murine hematopoietic cells from KrasLox-Stop-Lox (LSL) G12D/+/Vav-Cre mice (Blood, 2009) versus Cre+ counterparts. Relevant to AML line THP-1 which is poorly responsive to NEL (IC50>100 µM), we observed extremely low levels of Ara-GTP, no GTP increase or RAS hyperactivation after NEL treatment; Ara-GTP is inactivated by SAM domain and HD domain-containing protein 1 (SAMHD1), a dNTP hydrolase, whose high expression reportedly underlies NEL resistance in T-ALL. Indeed, SAMHD1 deletion remarkably increased RAS activity in THP-1 cells treated with NEL, thereby fully reversing NEL resistance. Our study provides a preclinical basis for testing NEL efficacy in a large cohort of AML patients, given that RAS activity is generally high in AML, or even against other malignancies harboring RAS mutations, which are considered "undruggable". Additionally, further study to test whether SAMHD1 inhibition enhances NEL efficacy against RAS active cancers is warranted. Disclosures Marcucci: Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Novartis: Speakers Bureau; Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Merck: Other: Research Support (Investigation Initiated Clinical Trial); Abbvie: Speakers Bureau. Sykes:Clear Creek Bio: Current equity holder in private company, Other: co-founder.

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Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.3513.3513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3513-3513

Author(s):

David B. Sykes ◽

Mark K Haynes ◽

Nicola Tolliday ◽

Anna Waller ◽

Julien M Cobert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Small Molecules ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Myeloid Differentiation ◽

Prostacyclin Receptor ◽

Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

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C/EBPα and MiR-182 Generate a Negative Feedback Loop Which Is Dysregulated in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.776.776 ◽

2014 ◽

Vol 124 (21) ◽

pp. 776-776

Author(s):

Alexander Arthur Wurm ◽

Dennis Gerloff ◽

Daniela Braeuer-Hartmann ◽

Christiane Katzerke ◽

Jens-Uwe Hartmann ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Feedback Loop ◽

Mononuclear Cells ◽

Myeloid Differentiation ◽

Negative Feedback Loop ◽

U937 Cells ◽

Leukemia Development ◽

Acute Myeloid

Abstract The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is silenced in approximately 50% of all acute myeloid leukemia (AML) cases. There are several mechanisms known how C/EBPα is inactivated in AML, including promoter hypermethylation, posttranslational modifications and mutations in the ORF of the CEBPA gene. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and leukemia development. We have already shown that microRNAs, such as miR-223, miR-34a and miR-30c, are essential elements in C/EBPα triggered granulocytic differentiation. But to our knowledge nothing is known about inactivation of C/EBPα by microRNAs in acute myeloid leukemia. In this study, we identified a novel network between C/EBPα and miR-182. In a next generation sequencing approach based on inducible K562-C/EBPα-ER cell line, we found miR-182 strongly downregulated by wildtype C/EBPα. We could further demonstrate an inverse correlation between C/EBPα protein amount and miR-182 expression level in several in vitro systems, including leukemic cell lines and G-CSF treated primary human CD34+progenitor cells. Additionally, C/EBPα and miR-182 showed reciprocal expression in sorted murine bone marrow subpopulations in vivo. To discover the mechanism how miR-182 is blocked by C/EBPα, we analyzed the minimal promoter region of miR-182 and performed chromatin immunoprecipitation (ChIP). Here, we could demonstrate a strong binding of C/EBPα to the miR-182 promoter, particularly to a conserved E2F binding site. Because E2F is a well known inhibitor of C/EBPα function, we tested whether E2F also effects miR-182 expression. An overexpression of E2F1 in U937 cells leads to an elevated miR-182 expression level. In addition, we measured the expression of miR-182 in bone marrow from AML patients regarding to their CEBPA mutation status. We could show that only patients with mutations in the C-terminal region of C/EBPα showed elevated miR-182 expression, while patients with N-terminal CEBPA mutations revealed no abnormal miR-182 expression compared to healthy donors or AML patients with no CEBPA mutation. The C-terminal domain of C/EBPα is necessary for E2F inhibition. These findings illustrate the importance of C/EBPα-E2F interaction during miR-182 regulation. Next, we found a highly conserved binding site of miR-182 in the 3’UTR of CEBPA itself, suggesting a possible negative feedback loop. To test this, we performed overexpression of miR-182 in U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) and primary blasts from AML patients. Here, we observed a strong reduction of C/EBPα protein after miR-182 overexpression in all cell types. Furthermore, we could demonstrate a direct binding of miR-182 to the 3’UTR of CEBPA via luciferase activity assay. Finally, we were interested in the functional impact of miR-182 in myeloid differentiation and leukemia development. We showed that enforced expression of miR-182 in U937 cells reduced the percentage of Mac-1 positive myeloid cells after treatment with all-trans retinoid acid (ATRA). Additionally, lentiviral overexpression of miR-182 induces a block of differentiation and hyperproliferation in G-CSF treated 32D cells and an enhanced replating capacity of primary mouse bone marrow mononuclear cells. Taken together, we identified miR-182 as novel oncogenic microRNA that directly blocks C/EBPα during myeloid differentiation and leukemia development. Thus, our data display a potential new strategy for therapeutics in C/EBPα dysregulated AML. Disclosures No relevant conflicts of interest to declare.

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