Knockdown of miR-128a induces Lin28a expression and reverts myeloid differentiation blockage in acute myeloid leukemia

Lin28A is a highly conserved RNA-binding protein that concurs to control the balance between stemness and differentiation in several tissue lineages. Here, we report the role of miR-128a/Lin28A axis in blocking cell differentiation in acute myeloid leukemia (AML), a genetically heterogeneous disease characterized by abnormally controlled proliferation of myeloid progenitor cells accompanied by partial or total inability to undergo terminal differentiation. First, we found Lin28A underexpressed in blast cells from AML patients and AML cell lines as compared with CD34+ normal precursors. In vitro transfection of Lin28A in NPM1-mutated OCI-AML3 cell line significantly triggered cell-cycle arrest and myeloid differentiation, with increased expression of macrophage associate genes (EGR2, ZFP36 and ANXA1). Furthermore, miR-128a, a negative regulator of Lin28A, was found overexpressed in AML cells compared with normal precursors, especially in acute promyelocytic leukemia (APL) and in ‘AML with maturation’ (according to 2016 WHO classification of myeloid neoplasms and acute leukemia). Its forced overexpression by lentiviral infection in OCI-AML3 downregulated Lin28A with ensuing repression of macrophage-oriented differentiation. Finally, knockdown of miR-128a in OCI-AML3 and in APL/AML leukemic cells (by transfection and lentiviral infection, respectively) induced myeloid cell differentiation and increased expression of Lin28A, EGR2, ZFP36 and ANXA1, reverting myeloid differentiation blockage. In conclusion, our findings revealed a new mechanism for AML differentiation blockage, suggesting new strategies for AML therapy based upon miR-128a inhibition.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic stem cell neoplasm, characterized by rapid growth and/or impaired differentiation of leukemic cells with abnormal accumulation. [1][2][3] Recurring chromosomal aberrations and gene mutations contribute to AML pathogenesis and are the most important tools for classification and prognosis assessment of AML. [2][3][4] Furthermore, there are some known deregulated pathways involved in the maintenance of leukemic stem cells such as hedgehog, 5,6 tyrosine kinase receptors (e.g. Flt3), 3,7 Wnt and Notch. [8][9][10][11] Notwithstanding, a successful target therapy is not yet available. Improving our current knowledge on the biology of AML-associated leukemic processes represents a valuable tool to identify novel potential drug targets.
Lin28 is a conserved RNA-binding protein having an important role in cancer stem cells. 12,13 This protein is expressed in embryonic stem cells 14,15 and is capable, with OCT4, SOX2 and NANOG, of converting fibroblasts in induced pluripotent stem cells. 16 Lin28, by physical interaction with several RNA transcripts, exerts various forms of regulation ranging from alternative splicing, turnover, localization and translation. [17][18][19] It has been demonstrated that altered functionality of RNA-binding proteins, due to deregulated gene expression or gene mutations, often results in genetic disease and cancer. 20 Several studies reported the existence of regulatory pathways between Lin28 and different miRNAs. 15,[21][22][23] In murine model, overexpression of miR-125b leads to the downregulation of Lin28A and the preleukemic state characterized by overproduction of myeloid cells eventually progressing to a myeloid leukemia. [24][25][26] Conversely, ectopic expression of Lin28B reprograms hematopoietic progenitor cells from adult bone marrow (BM), endowing them to mediate multilineage reconstitution. 27 Moreover, Li et al. 22 showed that miR-181 promotes megakaryocytic differentiation repressing Lin28 and upregulating let-7 expression. Thus, Lin28 seems to be a pivotal regulator of hematopoiesis. Interestingly, Lin28 is also regulated by miR-128, 28 a microRNA able to hold hematopoietic cells in an early progenitor stage, blocking their differentiation towards more mature cells. 29,30 Moreover, this microRNA was found associated with AML. [31][32][33] Therefore, it will be appealing to gain further insights into the role of miR-128a/Lin28A axis in induction and maintenance of an early differentiation status in AML.

Results
Lin28A expression was downregulated in myeloid leukemic cells. To evaluate Lin28A expression in AML, we performed quantitative real-time-PCR (qRT-PCR) in isolated blast cell samples from 38 AML patients at diagnosis, 7 AML cell lines (OCI-AML3, KG-1, Kasumi-1, NB4, CMK, ME-1 and MOLM-14) and CD34+ purified samples from 13 healthy donors. Lin28A (Po0.01) and cell lines (OCI-AML3 and KG-1 Po0.001, Kasumi-1, NB4, CMK and ME-1, Po0.01) showed a significantly lower expression in AML patients as compared with controls ( Figure 1a). To support our data, we also analyzed two independent publicly available gene expression profiling data sets, one containing 16 CD34+ isolated samples from healthy subjects (GSE 42519), and one with 251 AML patients with newly diagnosed AML (GSE 15434) confirming a significant downregulation of Lin28A in AML patients (230 BM and 21 PB) compared with healthy subjects (Supplementary Figure 1a). Stratifying AML according to the WHO classification, 4 Lin28A value was found underexpressed in all AML subtypes ( Figure 1b) compared with controls. Stratifying AML cases according to the principal genomic alterations detected in our cohort of patients and in GSE 15434 data set, we found lower expression of Lin28A in AML patients independent of their specific alterations (Figure 1c and Supplementary Figure 1b). Moreover, we evaluated Lin28A protein by cytometric analysis detecting a lower percentage of Lin28A+ cells in AML blast cells compared with normal hematopoietic myeloid precursors (Po0.01) (Figure 1d). When we analyzed distinct subsets of normal CD34+ cells, we observed a higher percentage of Lin28A+ cells in normal myeloid precursors (CD33+) compared with the erythroid (CD71+) (Po0.01) and lymphoid (CD19+) (Po0.001) ones, suggesting its main involvement in myeloid differentiation (Figure 1e).  [34][35][36] and ANXA1, a gene normally stored in inside macrophage cytosol (Figure 2l) 37 after Lin28A overexpression at 24-48 h.
Lin28A expression increased during PMA or ATRA differentiation. To corroborate the involvement of Lin28A in myeloid differentiation, we stimulate AML cell lines to differentiate. In particular, we induced macrophage-like differentiation treating ME-1/OCI-AML3 cell lines with phorbol 12-myristate 13-acetate (PMA) and MOLM-14 with all-transretinoic acid (ATRA), and granulocyte-like differentiation treating NB4 and KG-1 cell lines with ATRA. After treatment, the cytometric data revealed a significant percent increase, from 24 to 72 h, of CD11b+ cells and CD14+ cells in ME-1, OCI-AML3 (Figures 3a and b) and NB4 (Supplementary MiR-128a expression was upregulated in myeloid leukemic cells. To further clarify Lin28A downregulation in AML, we analyzed its regulator, miR-128a. 28 We evaluated miR-128a expression in the same cohort of AML patients and in the AML cell line panel previously examined for Lin28A, observing a significant overexpression of this microRNA compared with healthy subjects (Figure 4a). Stratifying AML cases for morphologic features, we found, at variance with Lin28A, elevated expression levels of miR-128a in AML with maturation and acute promyelocytic leukemia (APL) cases compared with controls ( Figure 4b). Furthermore, considering patients for their gene mutations, we found a significantly higher expression of miR-128a in patients with FLT3, PML/RARα and other genomic alterations (Figure 4c).
Our results show different expression pattern of miR-128a in MOLM-14 and AML samples, both carrying FLT3-ITD (Figures 4a and c). Matsuo et al. 38 demonstrated that MOLM-14, along with FLT3-ITD, carries a series of genotypic aberrancies, such as the insertion ins(11;9) with the fusion hybrid MLL-AF9. 38 This complex pattern could justify the partially divergent behavior of MOLM-14 as compared with fresh AML samples. Moreover, we also evaluated, by qRT-PCR, miR-128a expression during macrophage-and granulocytic-like differentiation detecting a significant  of infected cells, highlighting that miR-128a overexpression led to less mature macrophage-like cells (Figure 5f). Moreover, lentiviral infection of miR-128a inhibited colony-forming activity of colony-forming unit-macrophage (CFU-M) in colony size and number (Figures 5g and h).
Inhibition of miR-128a improved myeloid differentiation in AML BM HSPC. Since significantly increased miR-128a expression was mainly observed in AML with maturation, we investigated how miR-128a inhibition could influence myeloid differentiation/maturation blockage. Lenti-miRZip-128a stably expresses hairpins that have anti-miRNA activity. We used BM HSPCs derived from two AML patients with maturation (myeloblastic AML3 and myelomonocyte AML2, respectively), both FLT3 mutated, and one APL patient (AML1) (Supplementary Table S1). BM HSPCs were infected with Lenti-miRZip-128a or Lenti-GFP and exposed to macrophage-like induction culture. Flow cytometric analysis showed a significant increased of CD11b and CD14 percentage of positive cells in AML HSPCs infected with Lenti-miRZip-128a compared with the control (Figures 6a  and b). Lenti-miRZip-128a infection decreased the levels of mature miR-128a ( Figure 6c) and significantly enhanced the expression of Lin28A, EGR2, ZFP36 and ANXA1 (Figure 6d). These results demonstrated that miR-128a inhibition in AML induce myeloid differentiation.

Discussion
AMLs are clonal diseases of hematopoietic progenitor cells, characterized by marked heterogeneity in terms of phenotypic, genotypic and clinical features. 1,2,4,39 In this study, we showed that Lin28A, an RNA-binding protein, 12 was significantly underexpressed in AML samples without any The miR-128a/Lin28A axis in acute myeloid leukemia L De Luca et al association with genotypic and phenotypic stratification. Moreover, we found a higher percentage of Lin28A+ cells in myeloid precursors compared with that in erythroid and lymphoid normal precursors, suggesting a preferential involvement of this protein in myeloid lineage differentiation.
Recently, Chaudhuri et al. 26 demonstrated that the knockdown of Lin28A in mouse hematopoietic system led to myeloid cell expansion and decrease of B-cell number, thus triggering an alteration of hematopoiesis. Furthermore, its overexpression in normal HSC produced a significant reduction of total white blood cells, causing mice dead at 5 weeks, probably because of the impaired hematopoietic development. 26 Our data, instead, showed that Lin28A overexpression in AML cells activated myeloid maturation. We observed, in fact, an increase of myeloid differentiation markers and a cell-cycle arrest with p21 expression augment. Literature data demonstrated that p21, a cyclin-dependent kinase inhibitor, induced cell-cycle arrest if overexpressed in progenitor cells favoring macrophage differentiation because of the accumulation of PU.1, a lineage-determining factor. 40 Of importance, we also detected a significant increase of macrophage-specific genes like early growth response 2 (EGR2), an EGR protein involved in macrophage growth and differentiation, 34,41 tristetraprolin (ZFP36), an anti-inflammatory and anticarcinogenic protein that is also involved in monocyte/macrophage differentiation processes and annexin A1 (ANXA1) an anti-inflammatory protein stored in the macrophage cytosol. 37,42 In addition, we demonstrated that Lin28A is a positive regulator of granulocytic-and macrophage-like differentiation. In fact, we observed its significant increase simultaneously augmented different myeloid-specific markers, stimulated by ATRA or PMA treatment, in five AML cell lines with different genotype and morphology.
Previous studies reported that Lin28A is a direct target of miR-128, 28 a microRNA involved in hematopoiesis. 29,30 Different studies have associated miR-128a with leukemia, showing that miR-128a belongs to a set of miRNAs with stringent specificity for AML or ALL. [31][32][33] Moreover, miR-128a expression was found to be associated with a subgroup of AML patients with high-risk molecular features, refractoriness, relapse and death. 31,33 In our study, we evaluated miR-128a expression in our cohort of AML patients. Of interest, miR-128a showed a significantly higher level in APL and AML with mature phenotypes harboring FLT3 and/or other alterations. Qian et al. 28 sustained that miR-128 directly target BMI1, CSF1, KLF4, LIN28A, NANOG and SNAIL. Some of these genes are involved in self-renewal (Bmi1 and Nanog) 43 and The miR-128a/Lin28A axis in acute myeloid leukemia L De Luca et al differentiation (CSF1 and KLF4). 44,45 Similar to Lin28A, they are deregulated in AML. [45][46][47] KLF4, for example, a lineagespecific transcriptor factor that promotes monocyte differentiation is downregulated in undifferentiated subtype M0 and in FLT3-ITD and NPM1-mutant AML. 45 BMI1, instead, a polycomb group protein involved in self-renewal is overexpressed in different AML subtypes. 46 Given that gene regulation is complex and depend on different factors, 45,48-50 the relative  29,52 MiR-125b, for example, is overexpressed in certain types of AML (C/EBPα, t(2;11)(p21;q23), GATA1) and inhibits myeloid differentiation. 50,53 Moreover, its overexpression causes a dose-dependent myeloproliferative disorder progressing to a lethal myeloid leukemia in mice. 50 MiR-181 family, instead, was found abnormally upregulated in AML patients, with t(8;21) and t(15;17) inhibiting granulocyticand macrophage-like differentiations. 54 Here, we demonstrated that miR-128a was downregulated during induced granulocyteand macrophage-like differentiation of AML cell lines. Moreover, we showed a reduction of Lin28A-and myeloid-specific marker expression following enforced miR-128a expression, in spite of PMA treatment in vitro. Conversely, miR-128a transient inhibition in two cell lines enhanced myeloid maturation and Lin28A overexpression. Given the higher expression of miR-128a in AML with mature phenotypes and with FLT3 or PML/RARα alterations, we decided to inhibit miR-128a maturation in leukemic cells of these subsets of patients to stimulate further propensity to cell differentiation. In fact, Lenti-miRZip-128a infection remarkably repressed miR-128a and improved granulocytic/macrophage-like differentiation in BM-derived AML blasts. Finally, we detected an augment of Lin28A in all infected AML blasts patients, while an increase of macrophage-specific genes occurred only in AML with FLT3 mutation and mature phenotypes.
Specific microRNAs with established oncogenic functions, such as miR-155, miR-125b, miR-181 and miR-128a, appear to be associated with particular AML subtypes. 31,50,55 Selected sets of microRNAs could be used as a target therapy tailored to specific biological and molecular features of AML. 50 In particular, we hypothesize that in AML subtypes with t(8;21) and inv16, differentiation block could be released by miR-128a knockdown in combination with differentiation agents. In this setting, we previously demonstrated that G-CSF treatment of a The miR-128a/Lin28A axis in acute myeloid leukemia L De Luca et al patient with t(8;21) AML led to complete remission. 56 Moreover, the combined inhibition of miR-128a and miR-155 could be evaluated as a therapeutic option in high -isk AML patients harboring FLT3 mutation.
In conclusion, we revealed a new regulatory axis miR-128a/ Lin28A that affects hematopoiesis, favoring AML development. Our experiments suggest that the inhibition of miR-128a could provide a new strategy for AML therapy.  RNA isolation and qRT-PCR for mRNA and miRNA quantification. Mononuclear cells were obtained by Ficoll-Paque gradient centrifugation. Total RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturer's instructions. Reverse transcription was performed using 1 μg of total RNA from each sample by High Capacity cDNA Reverse Transcription Kit (Applied Biosistem, Foster City, CA, USA). qRT-PCR was performed as described previously. 57 Simultaneous quantification of ABL1 mRNA was used as a reference for mRNA TaqMan assay data normalization. miR-128 expression was normalized on RNU44. The comparative cycle threshold (Ct) method for relative quantification of mRNA and miRNA expression (User Bulletin No. 2; Applied Biosystems) was used to determine transcript levels.
Western blotting. Cells were lysed as reported previously. 58 Total proteins were extracted from AML cell lines. Equal amount of protein extract (60 μg) was transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h with 5% milk (Sigma-Aldrich) at room temperature, and then incubated with primary antibodies directed toward Lin28A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (Merck Millipore, Billerica, MA, USA) and β-actin (Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Protein bands were visualized and quantified as described previously. 59 Lentivirus production and infection. MiR-128a expression vector were made by cloning~60 bp 5′ and 3′ of the pre-miRNA into the multiple cloning site for pLKO.1 (Addgene, Cambridge, MA, USA). Lenti_GFP control and Lenti-miRZip-128a were purchased by System Biosciences (Palo Alto, CA, USA). The virus packaging was performed according to the manufacturer's instructions. The virus particles (lenti_128a, lenti_GFP control and Lenti-miRZip-128a) were harvested and concentrated using PEG-it Virus Precipitation Solution (System Biosciences). Virus titer was determined in 293TN cells using the global Ultrarapid Lentiviral Titer Kit (System Biosciences). For transduction, AML primary cells and OCI-AML3 were seeded onto 6-well plates at 800 000 cells per ml. Cells were infected with lentiviral stocks at an MOI of 5 in the presence of polybrene. AML primary cells were sorted for the expression of GFP using cell sorter MoFlo Atrios (Beckman Coulter, Brea, CA, USA). OCI-AML3 cells were maintained with puromycin 0.5 μg/ml.
Colony-forming assay. OCI-AML3 cells infected with pLKO.1_scr or pLKO.1_miR-128a were cultured in 35mm dishes in MethoCult Classic (Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer's instruction. CFU-M were visualized, measured and counted after being cultured in incubator at 37°C for 14 days.