The DMT1 isoform lacking the iron‐response element regulates normal and malignant hematopoiesis via NOTCH pathway activation

Natural resistance‐associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron‐response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non‐canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non‐canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T‐cell leukemia homeobox protein 1 (TLX1)‐defective leukemia. This work sets the stage for future investigation using a multiple‐hit T‐cell acute lymphoblastic leukemia (T‐ALL) model to further investigate the function of DMT1 nonIRE in T‐ALL disease development and progression.

Divalent Metal Transporter 1 (DMT1/NRAMP2/ SLC11A9) is responsible for the transport of divalent metals, including iron (Fe 2+ ) [1].Once absorbed and bound to transferrin, iron is internalized in cells by binding the transferrin receptor and endocytosis.Upon acidification of the endosome, iron is released from transferrin and transported across the endosomal membrane into the cytosol by DMT1 [2].The DMT1 gene encodes four isoforms: 1A nonIRE, 1A IRE, 1B nonIRE and 1B IRE, respectively [3].The IRE isoforms contain an iron-responsive element (IRE) in their 3 0 UTR, at which iron regulatory proteins (IRPs) together with Fe 2+ regulate its translation [4].The nonIRE isoforms contain an additional poly(A)tail, which improves mRNA stability.Due to its contribution in iron transport, Dmt1 loss-of-function mutations cause anemia [5,6].DMT1 mutant patients respond well to erythropoietin (EPO) treatment, by reducing erythrocyte apoptosis.Recently, we have shown that Dmt1 isoforms possess opposite functions in stem cell regulation in intestinal organoids through control of NOTCH1 signaling [7].
NOTCH signaling is a master regulator of hematopoiesis during development and in adult tissues and it exerts pleiotropic roles required for T-cell development differentiation, maturation, memory, and cytotoxic function [8].NOTCH receptors are transmembrane type 1 receptors activated by ligands from adjacent cells and activated by consecutive juxta-membrane and intramembrane cleavage events that result in the release of the NOTCH intracellular domain (NICD) that in conjunction with the DNA binding protein RBP-J controls the transcription of many hematopoietic lineage genes.Dysregulation and gain of function mutations in the NOTCH1 receptor is the most common alteration found in adult and pediatric leukemias [9].The c-MYC enhancer is a direct and rate-limiting transcriptional target major downstream target of NOTCH1 activation and acts as an oncogene in T-ALL, where it regulates cell growth and proliferation, but also the initiation of leukemic cells [10,11].
In this letter, we elucidate a novel role of the DMT1 nonIRE isoform in the regulation of normal hematopoiesis and leukemia, via activation of the NOTCH-MYC pathway.

Animal studies
Ethical permission for the performed experiments was obtained at the KU Leuven animal ethics committee (ECD; approval P145/2014).Lin À bone marrow cells from 6 to 8 weeks old male C57BL/6 mice (KU Leuven animalium in house breeding, filter-top cages, maximum 5 per cage) were transduced, puromycin selected for 24 h and 0.5 9 10 6 cells were intravenously injected into sub-lethally irradiated 6-8-week-old female C57BL/6 recipient mice.A blood drop was collected at day 10 by facial vein puncture and analyzed using a scil VET hemocytometer.Animals were sacrificed at day 21, when one animal presented physical illness; ruffled fur, curved posture, less active.Blood, serum, and bone marrow was collected and further analyzed by scil VET hemocytometer, flow cytometry (CD4 and CD19, Miltenyi, Leiden, Netherlands), and immunoblot.Iron levels in mouse serum were quantified using an IRON2 kit (Roche, cat.#03183696) analyzed on a Cobas 8000 (Roche Diagnostics, Basel, Switzerland).

Results and Discussion
Dmt1 nonIRE dictates hematopoietic stem/progenitor cell fate through regulation of NOTCH1 activity Bone marrow (BM) cells from three C57BL/6 mice were pooled and hematopoietic stem/progenitor cells were isolated by lineage depletion (Lin À ).The Lin À BM cells were transduced with either a scrambled control or our previously validated Dmt1 nonIRE targeting shRNA and puromycin selected before functionally analyzing these cells in long-term replating colony forming cell (CFC) assays (Fig. 1A).The shDmt1 non-IRE resulted in 60% Dmt1 nonIRE knockdown (KD) (Fig. S1A).In shDmt1 nonIRE cultures, we observe the presence of a quiescent hematopoietic stem/progenitor cell population that maintains its primitive phenotype over time, preserving a source of relatively slow-cycling cells that retain the long-term capacity to generate progeny compared to control (Fig. 1B).While the replating capacity dropped for control cells as they were completely exhausted in the 5th replate, shDmt1 nonIRE expressing cells showed an initial decrease followed by a relative and absolute increase in cell numbers in long-term replating capacity (3rd-5th replate, Fig. 1B and Fig. S1B).In CFC cultures of shDmt1 nonIRE BM, we observed an increased population of Sca1 hi and Kit1 hi expressing cells confirming the initial quiescent primitive phenotype of Dmt1 nonIRE KD cells (Fig. 1C).The reduced initial replating capacity of Dmt1 nonIRE KD cells was associated with loss of cleaved (activated) Notch1 and a decrease in expression of c-secretase complex members Nicastrin and Presenilin 2 (Fig. 1D).A reduced percentage of Notch1 expressing cells was confirmed by flow cytometry in shDmt1 nonIRE CFC assays (Fig. S1C).Taken together these data suggest that Dmt1 nonIRE isoforms interfere with Notch pathway activity; a master regulator of T cell development and among the most frequently mutated oncogenes in different B and T cell leukemias [13].

Dmt1 nonIRE knockdown is associated with superior non-canonical hematopoietic stem cell regulation in vivo
The changes in Notch controlled hematopoietic regulation of this Dmt1 isoform challenged us to perform a mouse bone marrow transplant experiment with Dmt1 nonIRE KD Lin À BM cells into sub-lethally irradiated recipient mice.At 10 days after BM transplantation, we already observed differences in blood composition.The Dmt1 nonIRE KD animals presented reduced numbers of red blood cells, increased numbers of eosinophils, and reduced hemoglobin and hematocrit levels (Fig. 2A and Fig. S1D).After exactly 3 weeks (d21), the Dmt1 nonIRE KD mice presented physical signs of severe sickness, for one animal we could not take all samples due to severity of disease.Animal autopsy did not reveal major differences between animal groups, besides presenting very thin blood consistency (Fig. S1E), reflecting severe bone marrow failure in these animals.Hemolysis was slightly increased in Dmt1 nonIRE KD animals, while lipemia was marginally decreased (Fig. S1F, both no significant).The initial increase in eosinophilic compartment dropped drastically at d21, which has been linked to reduced T-cell activation [14].In line with this, we observed slightly lower numbers of CD4 expressing cells, but most remarkably, we observed that especially the number of CD19 expressing B cells was reduced in the BM of Dmt1 nonIRE KD mice (Fig. 2A, both not significant).The essential expression of Dmt1 nonIRE for B cells was confirmed using Ba/F3 mouse B cells, in which we observed complete eradication already 48 h after transduction by induction of massive apoptosis (Fig. S1G).This B cell-specific essential expression of Dmt1 nonIRE isoform has not previously been described.Furthermore, hematological blood examination showed a small increase in white blood cell (WBC) counts in the Dmt1 nonIRE KD animals (Fig. 2A, d21).
Lymphocyte counts remained stable.Most striking was the ~2-fold increased presence of platelets (PLT) (Fig. 2A).Furthermore, low initial hemoglobin levels and at a later stage a slightly increased mean corpuscular volume in co-occurrence with reduced numbers of red blood cells (RBC) in Dmt1 nonIRE KD animals indicate defective RBC development causing anemia in these animals (Fig. S1H).The iron blood serum levels in the shDmt1 nonIRE mice were not changed (Fig. 2B).
Next, we tested whether Notch pathway members were involved in dictating this hematopoietic stem cell differentiation regulation.As was previously observed in our in vitro experimental set-up, bone marrow cells from shDmt1 nonIRE mice showed reduced Nicastrin levels, which due to its essential function in the gamma secretase complex, may cause failure of Notch pathway activation (Fig. 2C).In addition, an important downstream Notch1 target, namely Myc, was found simultaneously lost in the bone marrow of shDmt1 nonIRE mice.Iron linked transferrin receptor (Trfc) levels remained stable in the bone marrow cells.
Overall, from this bone marrow transplant experiment, we conclude that the Dmt1 nonIRE isoform has non-canonical functions controlling the hematopoietic stem cell pool by dictating differentiation of the myeloid and B cell lymphoid lineages, while suppressing the production of platelets via Notch/Myc pathway activation.These findings indicate that there must be a strict regulation and fine-tuning of expression levels of the DMT1 iron (in)dependent isoforms during hematopoietic development.

Dmt1 nonIRE and its relation to NOTCH in T-ALL
Since the majority of T-ALL cases are characterized by NOTCH pathway mutations [9], and loss of Dmt1 non-IRE diminishes Notch pathway activity in mouse bone marrow progenitors/stem cells, we were interested in Fig. 2. Dmt1 nonIRE knockdown bone marrow transplant reveals its function in altering stem cell lineage differentiation.(A) Lineage negative (Lin À ) bone marrow (BM) cells were transduced, puromycin selected and transplanted by tail vein injection into sub-lethally irradiated mice.After 10 days (d10) and at sacrifice (d21) the blood was analyzed for its composition.At day 21, for one animal we could not take all samples due to severity of disease.At d10 RBC levels were decreased (P = 0.021, two-tailed t-test).Horizontal bars indicate median.At d21 the bone marrow was collected and analyzed for percentages of CD4 and CD19 expressing lymphocytes.A two-tailed t-test was performed for statistical analysis to compare the percentages of CD4 and CD19 cells in the BM of scr and shDmt1 nonIRE mice.(B) At d21 blood serum was analyzed for iron levels.PLT, platelets; RBC, red blood cells; WBC, white blood cells.Statistical analysis using one-way ANOVA with Tukey's multiple comparison test.Two-tailed Mann-Whitney U t-test was used to compare combined RBC counts and iron blood serum levels between scr and shDmt1 nonIRE mice.(C) Bone marrow pellets were collected at sacrifice and analyzed by immunoblot analysis for Notch pathway targets Nicastrin (Ncstn) and c-Myc (Myc) and transferrin receptor as unchanged control (Trfc).Scr, scrambled control.Actin (Act) and vinculin (Vinc) serve as loading controls.Scr, scrambled control; shDmt1 non IRE, short hairpin RNA against Dmt1 isoform lacking the iron-response element.Student's two-way t-test was used for statistical analysis.*P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.Error bars indicate standard deviations.
DMT1 nonIRE isoform expression across T-ALL subgroups (Fig. 3A).We analyzed publicly available gene expression data (R2 AMC platform) of DMT1 nonIRE specific probes using the 'Meijerink' pediatric T-ALL dataset containing normal bone marrow controls and T-ALL patient samples.We found that specifically the TLX1 subgroup presented a 2-fold upregulation of DMT1 nonIRE mRNA expression levels compared to normal bone marrow (Fig. 3B).Analysis of published RNA sequencing data of the thymus from either control or TLX1 leukemic mice confirmed that overall Dmt1 expression is simultaneously increased with Notch1 and Nicastrin, but not with Myc expression in TLX1 thymus samples (GSE102209, Fig. 3C), which may require its second hit, NUP214-ABL1, as was previously shown [15].Using ChIP-on-chip analysis, it was previously found that TLX1 occupied the DMT1 promoter region in TLX1 driven leukemia [16].
In summary, besides its supportive role in hematopoietic stem/progenitor cell differentiation, our data suggests a non-canonical function for the DMT1 nonIRE overexpression in further boosting NOTCH pathway activity in TLX1 defective leukemia.This work sets the stage for future investigation using a multiple-hit T-ALL model, to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

Fig. 1 .
Fig. 1.Dmt1 nonIRE knockdown affects hematopoietic stem/progenitor cell fate via Notch pathway regulation.(A) Experimental design and functional evaluation of Dmt1 nonIRE knockdown in mouse bone marrow (BM) lineage negative (Lin À ) hematopoietic stem cells (HSC) using colony forming cell (CFC) assays.Horizontal bars indicate the median.Data includes three technical repeats of BM from pooled mice.(B) Replating capacity of mouse HSC in CFC assays, gray: Puromycin scrambled control, blue: shDmt1-IRE.(C) Membrane marker expression analysis of CD25/IL2Ra was performed at the 2nd replate.ShDmt1 nonIRE cells present increased expression of stem/progenitor markers Sca1 and cKit at the 3rd replate.(D) Immunoblot analysis of cleaved Notch1 at Val1744 revealed a decrease in the Notch1 pathway activation cascade in shDmt1 nonIRE cells.NICD, Notch1 intracellular domain, cleaved Notch1; PSEN2, Presenilin 2. Actin serves as a loading control.Representative of three independent blots.Scr, scrambled control; shDmt1 non IRE, short hairpin RNA against Dmt1 isoform lacking the iron-response element.Student's two-tailed t-test was used for statistical analysis, *P-value < 0.05, **P-value < 0.01.Error bars indicate standard deviations.

Fig. 3 .
Fig. 3. TLX1 driven leukemias show specific DMT1 nonIRE expression and Notch pathway expression.(A) Representation of T-ALL subgroups based upon their differentiation status.(B) DMT1 nonIRE mRNA expression in normal bone marrow (NBM) and T-ALL subgroups using the DMT1 nonIRE specific probe 210047 (GSE26713).Statistical analysis using one-way ANOVA with Tukey's multiple comparison test.(C) RNA sequencing expression levels of Dmt1, Notch1 and Nicastrin (Ncstn) in the thymus of ctrl and TLX1 mice (mThymus), GSE102209.Student's two-tailed t-test was used for statistical analysis.*P-value < 0.05, **P-value < 0.01.Horizontal bars indicate the median.Error bars indicate standard deviations.