Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients

Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene

<div><p>Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of <i>HoxA9</i>, <i>HoxA10</i> and <i>Meis2</i>. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.</p></div

The N-terminal region of MN1 is required to block megakaryocyte/erythroid differentiation.

<p>(A–C) Percentage of transgene positive red blood cells engrafting in peripheral blood of transplanted mice at 4-week intervals. P values are calculated for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice and standard error is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112671#pone.0112671.s012" target="_blank">Table S3</a>. (D–F) Proportion of red blood cells (RBC) compared to white blood cells (WBC) expressing (D) strategy 1, (E) strategy 2, or (F) strategy 3 MN1 deletion constructs after transplantation. P values are calculated for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice and standard error is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112671#pone.0112671.s012" target="_blank">Table S3</a>. (G) Megakaryocyte colony-forming ability of mouse bone marrow cells transduced with MN1 deletion constructs (mean ± SD, n = 4). (H) Micrographs of representative CFC-Mk slides at the end of the first plating of bone marrow cells transduced with CTL vector, full-length MN1 or MN1Δ1. Images were visualised using a Nikon Eclipse 80i microscope (Nikon, Mississauga, ON, Canada) and a 20x/0.40 numerical aperture objective, or a 100x/1.25 numerical aperture objective and Nikon Immersion Oil (Nikon). A Nikon Coolpix 995 camera (Nikon) was used to capture images. * indicates P<0.05.</p

Functionally defined regions of MN1.

<p>Functionally defined regions of MN1.</p

Role of MN1 regions in leukemia cell fate regulation.

<p>Role of MN1 regions in leukemia cell fate regulation.</p

A 606 amino-acid C-terminal portion of MN1 prevents T-lymphoid differentiation.

<p>(A) Morphology of bone marrow cells from MN1Δ5-7 mice at death, showing a shift in leukemia from AML, as seen in MN1 leukemia, to an ALL leukemia upon loss of the C-terminal domains 5–7. (B) Representative immunophenotype of GFP⁺ MN1Δ5-7 bone marrow cells compared to wildtype MN1 bone marrow cells at death. (C) Representative immunophenotype of secondary transplants of GFP⁺ MN1Δ5-7 bone marrow cells at death. (D) Average cell surface marker expression for secondary transplants of GFP⁺ MN1Δ5-7 bone marrow cells at death (mean ± SEM, n = 3).</p

Hierarchical clustering of cells with N- and C-terminally deleted MN1.

<p>(A) Heat map of differentially regulated pathways for enhanced proliferation and blocked differentiation. (B) Comparison of top 60 enriched gene ontology gene sets for the comparison of MN1Δ1 and MN1Δ7 with wildtype MN1.</p

The N-terminal region of MN1 is required for its leukemogenic potential.

<p>(A) MN1 mutation constructs for structure-function analysis. In strategy 1 distinct stretches of approximately 200 amino acids were deleted throughout wildtype MN1. In strategy 2, stretches of approximately 200 amino acids were cumulatively deleted starting from the MN1 N-terminus. In strategy 3, stretches of approximately 200 amino acids were cumulatively deleted starting from the MN1 C-terminus. (B–D) Percentage of transgene-positive white blood cells engrafting in peripheral blood of transplanted mice at 4-week intervals. P values are given for the comparison of the indicated construct with CTL-transduced cells. The average engraftment is shown. Number of analysed mice and standard error can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112671#pone.0112671.s010" target="_blank">Table S1</a>. (E-G) Survival of mice receiving transplants of cells transduced with (E) strategy 1, (F) strategy 2, and (G) strategy 3 MN1 deletions. P values are given for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112671#pone.0112671.s010" target="_blank">Table S1</a>. (H) Morphology of bone marrow cells at death of diseased mice. The cells were Wright-Giemsa stained. Images were visualised using a Nikon Eclipse 80i microscope (Nikon, Mississauga, ON, Canada) and a 20x/0.40 numerical aperture objective, or a 100x/1.25 numerical aperture objective and Nikon Immersion Oil (Nikon). A Nikon Coolpix 995 camera (Nikon) was used to capture images. § engraftment in peripheral blood at the indicated time point or at death in cases where a mouse died before that time point. † all mice were dead at this time point due to disease. * indicates P<0.05, ** indicates P<0.001.</p

The C-terminal region of MN1 is required to block myeloid differentiation.

<p>(A–F) In vitro sensitivity to ATRA of ND13-immortalized cells that were transduced with MN1 deletion constructs. Dose-response curves are shown in the left panels (A, C, E) and IC50 values are shown in the right panels (B, D, F) for each deletion strategy (mean ± SD, n≥6).</p