Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia

Abstract

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Keywords: HoxA9; acute myeloid leukemia; brequinar; differentiation; dihydroorotate dehydrogenase; high-throughput screen; leukemia-initiating cell; metabolic inhibitor; phenotypic screen.

Figure 1.. ER-HoxA9 Cells Undergo Conditional Myeloid Differentiation

(A) Primary murine bone marrow cells transduced with MSCVneo-ER-HoxA9 grow as lineage-negative cells in the presence of beta-estradiol, (+) E2. Removal of E2 and inactivation of ER-HoxA9 result in the synchronous upregulation of the myeloid differentiation markers CD11b and Gr-1, as demonstrated by flow cytometry. (B) Terminal differentiation of the ER-HoxA9 cells is accompanied by exit from the cell cycle. (C) Morphologic changes that accompany myeloid differentiation are confirmed by Wright-Giemsa staining of cells in the presence and absence of E2. (D) Terminally differentiated cells, but not undifferentiated cells, are capable of phagocytosis of fluorescently labeled E. coli. (E-G) In (E), Lys-GFP-ER-HoxA9 cells demonstrate expected changes in myeloid gene expression over a 5-day differentiation time course. Their stepwise gene expression parallels the patterns of unmanipulated murine bone marrow myeloid cells (F) as well as purified populations of primary human bone marrow cells (G). HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; GRAN, granulocytes. See also Figure S1.

Figure 2.. High-Throughput Screening and Medicinal Chemistry Identifies ML390 as an Inducer of Myeloid Differentiation

(A and B) In (A), upon differentiation (following the removal of estradiol), Lys-GFP-ER-HoxA9 GMPs upregulate GFP fluorescence and (B) the cell-surface markers CD11b and Gr-1. (C) A high-throughput flow cytometry phenotypic screen was established to identify compounds that could trigger differentiation of Lys-GFP-ER-HoxA9 cells as monitored by the upregulation of GFP and CD11b. (D) Twelve biologically active compounds were identified, including compound 3 (C03) and compound 7 (C07). (E) The most potent small molecule was a derivative of (R)-C07, designated ML390. (F) C03 and (R)-C07 triggered myeloid differentiation, while (S)-C07 lacked activity. (G) ML390 is capable of causing myeloid differentiation in murine (ER-HoxA9) and human (U937 and THP1) AML models. (H) Imaging flow cytometry demonstrates upregulation of GFP and CD11b expression as well as the downregulation of KIT expression in Lys-GFP-ER-HoxA9 cells during differentiation in the absence of estradiol (-E2) or as the result of treatment with ML390. See also Figure S2.

Figure 3.. Resistant Cell Lines Identify DHODH as the Target of ML390

(A) ER-HoxA9 and U937 cell lines resistant to C03 and (R)-C07 were generated by continuous culture in slowly increasing concentration of compound. (B) A comparison of gene expression between DMSO control and compound-resistant cells demonstrated that only eight overexpressed genes (more than 2-fold upregulated, p < 0.01) were shared across the four resistant cell lines. (C) The eight genes were gene syntenic gene neighbors on chromosome 16 (human) and chromosome 8 (mouse). (D) Analysis of the whole-exome sequencing data revealed an increased coverage over a narrow region of chromosome 16, consistent with chromosomal amplification as the mechanism underlying increased gene expression. (E and F) In (E), DHODH was confirmed as the target of C03, C07, and ML390 using an in vitro enzyme inhibition assay, as well as (F) by demonstrating that the differentiating effects of C03 and ML390 could be abrogated by supplementation of uridine in the cell-culture media. (G) Treatment of the Lys-GFP-ER-HoxA9 cells with ML390 demonstrated gene-expression changes consistent with myeloid differentiation by gene set enrichment analysis. See also Figure S3.

Figure 4.. BRQ Causes Differentiation and Shows Anti-tumor Activity in In Vivo Xenotransplant Models of AML

(A) THP1 cells were implanted subcutaneously in the flank of SCID mice; treatment with brequinar (BRQ) decreased tumor growth. QD, once every day. (B) Explanted THP1 tumors analyzed by flow cytometry demonstrated that BRQ causes the upregulation of the myeloid differentiation marker CD11b. (C) The geometric mean fluorescence intensity of CD11b-APC expression was compared for the explanted tumors from three mice per group. (D) Metabolites from explanted tumors were extracted into methanol, and levels of intracellular uridine were measured by mass spectroscopy. (E) HL60 cells were implanted subcutaneously in the flank of SCID mice, and the mice were treated with vehicle or BRQ. BRQ causes differentiation of HL60 cells in culture, as demonstrated by the upregulation of CD14 (inset graph). (F) MOLM13 cells were implanted subcutaneously in the flank of SCID mice, and the mice were treated with vehicle or BRQ. BRQ causes differentiation of MOLM13 cells in culture, as demonstrated by the upregulation of CD14 (inset graph). (G) BRQ prolongs survival in an intravenous HL60 leukemia model. (H) BRQ prolongs survival in an intravenous OCI/AML3 leukemia model. Data in (A), (C), (E), and (F) are represented as the mean ± SD. *p % 0.05; ***p % 0.001; ****p %0.0001; ns, not significant. See also Figure S4.

Figure 5.. Inhibition of DHODH Leads to an Accumulation of Upstream Metabolites and to a Depletion of Downstream Metabolites

(A) A model of the enzymatic steps involved in de novo pyrimidine synthesis as well as lipid biogenesis. DHODH is located in the mitochondrial inner membrane and passes electrons to ubiquinone. (B and C) In (B), treatment of ER-HoxA9 cells with ML390 results in an accumulation of dihydroorotate and (C) to a depletion of downstream metabolites including UMP, uridine, UDP, UDP-GlcNAc, and UDP-glucose. (D) Treatment of ER-HoxA9 cells with pyrazofurin, an inhibitor of OMP decarboxylase, results in differentiation that can be rescued by uridine supplementation. (E) Treatment of cells with ML390 or BRQ, followed by immunoblotting, demonstrates a global decrease in the degree of protein N-acetyl glycosylation (GlcNAc). Data in (B) and (C) are represented as the mean ± SD.

Figure 6.. BRQ Causes Differentiation, Shows Anti-leukemia Activity, and Leads to Depletion of Leukemia Initiating Cell Activity in an In Vivo Syngeneic Model of HoxA9 AML

(A) Experimental outline of a syngeneic model of HoxA9 and Meis1-driven acute myeloid leukemia. (B) Flow-cytometric analysis of bone marrow leukemic cells from mice treated with BRQ demonstrates an increase in the expression of differentiation markers CD11b and Gr-1. (C) Mice treated with 25 mg/kg BRQ given on days 1 and 4 of a 7-day schedule, for a total of six doses, show a decrease in leukemia burden and increase in differentiation markers. Data are box and whisker plots where the mean, the minimum, and the maximum are indicated. (D) Leukemia cells from mice that were treated with vehicle or BRQ were purified via FACS. Cytospin preparations stained with Wright-Giemsa showed signs of granulocytic maturation, including nuclear condensation and cytoplasmic clearing, in leukemic cells isolated from BRQ-treated mice. (E) Treatment with 12 doses of BRQ prolongs overall survival. (F) The same number of purified live leukemia cells from mice treated with vehicle or BRQ was introduced into recipient mice as a functional assay for leukemia-initiating cell activity. These secondary recipient mice were not treated. BRQ treatment leads to a decrease in the frequency of leukemia-initiating cells. (G) HoxA9+Meis1 leukemia was introduced into mice without pre-conditioning, and the mice were treated with BRQ given every 3 days.(H) The extended treatment of mice with BRQ leads to prolonged survival. **p % 0.01; ***p % 0.001; ****p % 0.0001; ns, not significant. See also Figure S5.

Figure 7.. BRQ Causes Differentiation, Shows Anti-leukemia Activity, and Leads to Depletion of Leukemia-Initiating Cell Activity in an In Vivo Syngeneic Model of MLL/AF9 AML

(A) Experimental outline of MLL/AF9 leukemia; mice were treated with standard-of-care chemotherapy (Ara-C and doxorubicin) or BRQ. (B-E) Treatment with BRQ decreased leukemia burden in the bone marrow (B), increased expression of MAC1 (C), increased expression of Gr-1 (D), and decreased expression of CKIT (E) cell-surface markers. (F) Treatment with BRQ or chemotherapy led to a decrease in the number of phenotypic leukemic stem cells. (G) Leukemic cells isolated from mice treated with BRQ or chemotherapy were placed into methylcellulose colony formation assays. Treatment of the mice with BRQ or chemotherapy led to a decrease in colony formation activity. (H) Leukemia cells isolated from mice treated with BRQ show morphologic evidence of differentiation. (I) Treatment with BRQ leads to prolonged survival in the primary recipient mice beyond the benefit seen with chemotherapy. (J) Treatment with BRQ leads to a decrease in the frequency of leukemia-initiating cells, as evidenced in secondary transplant assays. In this assay, treatment with chemotherapy does not lead to a decrease in the frequency of leukemia-initiating cells. (K) Treatment of patient-derived xenograft (PDX) leukemia samples with BRQ in culture shows an increase in myeloid differentiation markers in three out of four models. Data in (F) and (G) are represented as the mean ± SD. **p % 0.01; ****p % 0.0001; ns, not significant. See also Figure S7.