We reported that an acquired miR-142 deficit transforms chronic phase (CP) chronic myeloid leukemia (CML) leukemic stem cells (LSCs) into blast crisis (BC) LSCs. Given the role of miR-142 in the development and activity of the immune system, we postulated that this deficit also promotes LSC immune escape. Herein, we report on IL-6-driven miR-142 deficit occurring in T cells during BC transformation. In CML murine models, miR-142 deficit impairs thymic differentiation of lymphoid-primed multipotent progenitors (LMPP) into T cells and prevents T cells' metabolic reprogramming, thereby leading to loss of T cells and leukemia immune escape. Correcting miR-142 deficit with a miR-142 mimic compound (M-miR-142), alone or in combination with immune checkpoint antibodies, restores T cell number and immune activity, leading to LSC elimination and prolonged survival of BC CML murine and patient-derived xenograft models. These observations may open new therapeutic opportunities for BC CML and other myeloid malignancies.

Chronic inflammation is epidemiologically linked to the pathogenesis of gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer (CRC). However, our understanding of the molecular mechanisms controlling gut inflammation remains insufficient, hindering the development of targeted therapies for IBD and CRC. In this study, we uncovered C15ORF48/miR-147 as a negative regulator of gut inflammation, operating through the modulation of epithelial cell metabolism. C15ORF48/miR-147 encodes two molecular products, C15ORF48 protein and miR-147-3p microRNA, which are predominantly expressed in the intestinal epithelium. C15ORF48/miR-147 ablation leads to gut dysbiosis and exacerbates chemically induced colitis in mice. C15ORF48 and miR-147-3p work together to suppress colonocyte metabolism and inflammation by silencing NDUFA4 , a subunit of mitochondrial complex IV (CIV). Interestingly, the C15ORF48 protein, a structural paralog of NDUFA4, contains a unique C-terminal α-helical domain crucial for displacing NDUFA4 from CIV and its subsequent degradation. NDUFA4 silencing hinders NF-κB signaling activation and consequently attenuates inflammatory responses. Collectively, our findings have established the C15ORF48/miR-147 - NDUFA4 molecular axis as an indispensable regulator of gut homeostasis, bridging mitochondrial metabolism and inflammation.

Loss of function of T cells has been associated with AML growth and poor outcomes. MiR-142 reportedly regulates normal hematopoiesis. Loss of the mir142 gene in the mouse results in decreased hematopoietic output and reduced T and NK cells. Mutated MIR142 was found in AML patients (pts). Recently, while we observed that miR-142-3p levels were lower in blasts and associated with shorter survival (p=0.0288) in AML pts vs. healthy donors, we found that in these pts, T cells were also fewer, with reduced miR-142 levels, increased levels of PD-1 (a marker for activation/exhaustion) and reduced cytokine production, supporting impaired T cell immune activity. Similarly, in MllPTD/WTFlt3ITDITD mice, a murine AML model, T cells were also decreased, and had reduced miR-142 levels, increased spontaneous apoptosis and PD-1 expression, and reduced proliferation rate and cytokine production, compared to T cells from normal wild-type (wt) mice. Since we have shown that miR-142 deficit impairs T cell antileukemic activity in chronic myeloid leukemia (CML; ASH 2023, Abstract #3152), we hypothesize that during AML growth, T cells acquire a miR-142 deficit that contributes to immune escape and disease progression. To test this hypothesis, first we co-transplanted MllPTD/WTFlt3ITDITD AML leukemic stem cells (LSC, i.e., Lin-Sca-1+c-Kit+, LSKs) with Mir142+/+ T or Mir142−/− T cells into immunodeficient NSG recipients, which lack T cells. We observed significantly decreased T cell engraftment, increased leukemic blasts, and shorter survival in LSK+Mir142−/− T recipients compared with LSK+Mir142+/+ T recipients (median: 51 vs 59 days, p=0.0013). Second, we transplanted AML LSKs into Mir142−/− or Mir142+/+ recipients and observed significantly higher leukemic burden and shorter survival in Mir142−/− than in Mir142+/+ recipients (median: 32 vs 44 days, p<0.0001). Third, we transplanted AML LSKs into Mir142flox(f)/fLck-cre+ (i.e., miR-142 KO in T cells only) or cre- mice and monitored these mice for survival (ongoing). Fourth, we co-transplanted human AML blasts with miR-142 KD or wt T cells into NSGS mice. The AML+KD-T recipients have a higher leukemia burden and shorter survival than the AML+wt-T recipients (median: 32 vs 41 days, p=0.0017). Collectively, these results suggest that miR-142 deficit in T cells mediates T cell dysfunction and promotes LSC growth and disease progression. To determine how T cells acquire miR-142 deficit during AML growth, we co-cultured murine T cells with BM cells from MllPTD/WTFlt3ITDITD AML or normal wt mice in a transwell and observed lower miR-142 levels and increased PD-1 in T cells co-cultured with AML blasts (p<0.05 for both). Next, we transplanted BM cells from AML or normal wt mice into congenic wt recipients and observed lower miR-142 and increased PD-1 levels in the host T cells from the recipients of AML blasts (p<0.05 for both). This data suggested a possible role of blast-secreted cytokines in the induction of T-cell miR-142 deficit. To this end, we discovered that higher levels of IL-6 in AML were associated with lower T cell levels of miR-142. To determine if increased PD-1 on host T cells contributes to miR-142 deficit-induced T cell exhaustion, we also transplanted AML blasts into PD-1−/− or wt recipients. We observed significantly longer survival in PD-1−/− vs. wt recipients (median: 47 vs 36.5 days, p=0.0027). Next, to test if restoring miR-142 improves T cell control of leukemia growth, we treated a cohort of MllPTD/WTFlt3ITDITD AML mice with M-miR-142 (a synthetic miR-142) or scrambled control (SCR) for 3 weeks. M-miR-142-treated mice had lower disease burden and longer survival (median: 43 vs 38 days, p=0.0006). Recipients of BM from M-miR-142-treated donors also live longer (median: 35 vs 28 days, p<0.0001), supporting reduced LSC burden. Next, AML patient-derived xenograft (PDX) mice were given 106 autologous human T cells and treated with M-miR-142 or SCR for 3 weeks. T+M-miR-142-treated mice had reduced leukemic burden and prolonged survival (median: 60 vs 51 days, p=0.0002) compared with T+SCR-treated controls. Recipients of M-miR-142-treated donor BM also lived significantly longer than recipients of SCR-treated donor BM (median: 92 vs 21 days, p<0.0001). We concluded that T-miR-142 deficit is acquired during AML progression, causing decreased antileukemic surveillance and contributing to disease growth, which can be rescued by M-miR-142.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

CML may evolve from a chronic phase (CP) into blast crisis (BC), but the underlying mechanisms of the transformation remain to be fully elucidated. We reported that microRNA (miR)-142 is downregulated in BC compared with CP patients; and in a murine model of CP CML (i.e., BCR-ABL mouse), miR-142 knock-out (KO) induced a BC-like phenotype, substantiating a mechanistic role of miR-142 deficit in BC transformation. Mechanistically, miR-142 KO induced mitochondrial fusion and increased oxidative phosphorylation (OxPhos) in leukemic stem cells (LSCs), causing a shift of leukemic phenotype from CP to BC ( Nat Commun, in press). Herein, we report that miR-142 deficit also occurs in T lymphocytes of BC patients due to inflammatory cytokines that are aberrantly produced by the proliferating leukemic cells. Using the Mir142 −/−BCR-ABL mouse, we observed that miR-142 deficit resulted in loss of T cell number and activity, suppressed the antileukemic immune surveillance, and contributed to BC transformation. In fact, miR-142 KO hampered thymic lymphoid-primed multipotent progenitor (LMPP) differentiation into T cells and rendered mature T cells dysfunctional and exhausted, with increase of PD-1 levels, and decrease of the apoptotic threshold, cell cycling and cytokine production, via blockade of OxPhos/glycolysis switch that regulates the metabolism of otherwise activated T cells. These changes translated into a decrease of T-cell antileukemic surveillance as demonstrated by increased numbers of BC murine Lin -Sca-1 +c-Kit + (LSKs) or human CD34+ blasts cocultured with Mir142 −/−T-cells vs those cocultured with Mir142 +/+ T-cells. Furthermore, congenic B6 (lethally irradiated to eradicate host T cells) or immunodeficient NSG (no T cells) recipient mice transplanted with Mir142 −/−BCR-ABL LSK and Mir142 −/− T cells had reduced T cells (both: p<0.0001), increased blasts (B6: p=0.01; NSG: p<0.0001) and a shorter survival (median survival for B6: 58 days vs unreached, p=0.008; for NSG: 28 days vs unreached, p<0.0001) compared with the respective controls transplanted with Mir142 −/−BCR-ABL LSK and Mir142 +/+ T cells. Transplantation of Mir142 −/−BCR-ABL LSKs into Mir142 −/− or Mir142 flox(f)/fLck-cre+ (i.e., miR-142 KO only in T cells; Mir142T Δ/Δ) recipients also resulted in a shorter survival than Mir142 +/+ recipients (median survival for Mir142 −/− vs Mir142 +/+ recipients: 44 vs 53 days, p=0.001; for Mir142T Δ/Δ vs Mir142 +/+ recipients: 42 vs 53 days, p=0.0002). To correct miR-142 deficit, we produced a synthetic miR-142 mimic oligonucleotide (CpG-M-miR-142). We treated a cohort of NSG mice transplanted with Mir142 −/−BCR-ABL LSKs and Mir142 −/− T cells, with CpG-M-miR-142 (30mg/kg/day, IV) or CpG-scramble RNA (SCR) for 3 weeks. We observed increased T cells (p=0.04), decreased blasts (p=0.0029) and longer survival (median survival: unreached vs 37 days, p=0.0005) in CpG-M-miR-142-treated recipients vs SCR-treated controls. Similar results were observed in CpG-M-miR-142-treated Mir142 −/−BCR-ABL mice vs SCR-treated controls. To test CpG-M-miR-142 activity in human T cells, a cohort of BC CML patient-derived xenograft (PDX) mice were given patient's autologous T cells and CpG-M-miR-142. Mice receiving T+CpG-M-miR-142 had increased T cell expansion (p=0.04), reduced leukemic cells (p=0.0078), and longer survival (median survival: 53 vs 38 days, p=0.01) than controls receiving T+SCR. In secondary transplants, the recipients of BM from T+CpG-M-miR-142-treated donors survived longer (median survival: 73 vs 53 days, p<0.0001) than the recipients of BM from T+SCR-treated donors, suggesting a decrease in LSC burden. Finally, we tested the efficacy of tyrosine kinase inhibitors (TKIs), which represent the primary targeting approach for CML, in combination with CpG-M-miR-142 and/or PD-1 inhibitor in Mir142 −/−BCR-ABL mice. The mice treated with TKI+CpG-M-miR-142 (median survival: 107 days, p=0.001) or TKI+PD-1 inhibitor (104 days, p=0.002) survived longer than the controls treated with TKI alone (66 days); the triplet combination (TKI+CpG-M-miR-142+PD-1 inhibitor) is expected to improve survival further (ongoing experiment). In summary, CpG-M-miR-142 alone and in combination with TKI or PD-1 inhibitor rescued T-cell antileukemic activity and prolonged survival in BC CML murine and PDX models, thereby providing a potentially new therapeutic approach for BC CML.

The mechanisms underlying the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC) are not fully elucidated. Here, we show lower levels of miR-142 in CD34 + CD38 − blasts from BC CML patients than in those from CP CML patients, suggesting that miR-142 deficit is implicated in BC evolution. Thus, we create miR-142 knockout CML (i.e., miR-142 −/− BCR-ABL ) mice, which develop BC and die sooner than miR-142 wt CML (i.e., miR-142 +/+ BCR-ABL ) mice, which instead remain in CP CML. Leukemic stem cells (LSCs) from miR-142 −/− BCR-ABL mice recapitulate the BC phenotype in congenic recipients, supporting LSC transformation by miR-142 deficit. State-transition and mutual information analyses of “bulk” and single cell RNA-seq data, metabolomic profiling and functional metabolic assays identify enhanced fatty acid β-oxidation, oxidative phosphorylation and mitochondrial fusion in LSCs as key steps in miR-142-driven BC evolution. A synthetic CpG-miR-142 mimic oligodeoxynucleotide rescues the BC phenotype in miR-142 −/− BCR-ABL mice and patient-derived xenografts.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/- mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-κB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/- mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/- mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk between miR-146a and mir-146b in the regulation of LPS-induced neuroinflammation.

Regulatory T (T reg ) cells are critical in preventing aberrant immune responses. Posttranscriptional control of gene expression by microRNA (miRNA) has recently emerged as an essential genetic element for T reg cell function. Here, we report that mice with T reg cell–specific ablation of miR-142 (hereafter Foxp3 Cre miR-142 fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T-cell tolerance. Foxp3 Cre miR-142 fl/fl mice displayed a significant decrease in the abundance and suppressive capacity of T reg cells. Expression profiling of miR-142 –deficient T reg cells revealed an up-regulation of multiple genes in the interferon gamma (IFNγ) signaling network. We identified several of these IFNγ-associated genes as direct miR-142-3p targets and observed excessive IFNγ production and signaling in miR-142 –deficient T reg cells. Ifng ablation rescued the T reg cell homeostatic defect and alleviated development of autoimmunity in Foxp3 Cre miR-142 fl/fl mice. Thus, our findings implicate miR-142 as an indispensable regulator of T reg cell homeostasis that exerts its function by attenuating IFNγ responses.

Aberrant inflammation plays an important role in the pathogenesis of several gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer (CRC). Insufficient understanding of the molecular mechanisms that control gut inflammation is one of the critical barriers to effective IBD and CRC therapies. To this end, we have uncovered C15ORF48/miR-147 as a novel negative regulator of gut inflammation. The C15ORF48/miR-147 gene encodes a small protein (C15ORF48) and a microRNA (miR-147-3p). Both molecular products are expressed predominantly in the large intestine, and their genetic ablation significantly exacerbates dextran sodium sulfate (DSS)-induced colitis in mice. The fulminant DSS-induced colitis phenotype in C15ORF48/miR-147−/− mice is driven by a cell-autonomous defect in gut epithelial cells and is mediated by gut dysbiosis. miR-147-3p and C15ORF48 synergize to silence the expression of NDUFA4, an accessory subunit of complex IV (CIV) in the mitochondrial electron transport chain. Our findings suggest that NDUFA4 plays an essential role in the activation of NF-kB signaling and the downstream inflammatory responses. Furthermore, C15ORF48, being an NDUFA4 structural paralog, can replace NDUFA4 in CIV and thus attenuate CIV activity and mitochondrial respiration, which may subsequently impact gut microbiome homeostasis. Collectively, our findings have established the C15ORF48/miR-147-NDUFA4 molecular axis as a novel, indispensable regulator of gut inflammation and a critical mediator between gut epithelial cells and microbiota.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b, which are both known to suppress a variety of immune responses. Here in this study, we show that miR-146b is abundantly expressed in neuronal cells, while miR-146a is mainly expressed in microglia and astroglia of adult mice. Accordingly, miR-146b deficient (Mir146b-/-) mice exhibited anxiety-like behaviors and enhanced cognition. Characterization of cellular composition of Mir146b-/- mice using flow cytometry revealed an increased number of neurons and a decreased abundancy of astroglia in the hippocampus and frontal cortex, whereas microglia abundancy remained unchanged. Immunohistochemistry showed a higher density of neurons in the frontal cortex of Mir146b-/- mice, enhanced hippocampal neurogenesis as evidenced by an increased proliferation, and survival of newly generated cells with enhanced maturation into neuronal phenotype. No microglial activation or signs of neuroinflammation were observed in Mir146b-/- mice. Further analysis demonstrated that miR-146b deficiency is associated with elevated expression of glial cell line-derived neurotrophic factor (Gdnf) mRNA in the hippocampus, which might be at least in part responsible for the observed neuronal expansion and the behavioral phenotype. This hypothesis is partially supported by the positive correlation between performance of mice in the object recognition test and Gdnf mRNA expression in Mir146b-/- mice. Together, these results show the distinct function of miR-146b in controlling behaviors and provide new insights in understanding cell-specific function of miR-146b in the neuronal and astroglial organization of the mouse brain.

Abstract Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA‐146a and microRNA‐146b (miR‐146a/b) are anti‐inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF‐κB) pathway and inhibit pro‐inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR‐146a/b could regulate cellular responses to RVs in HBECs and airways during RV‐induced asthma exacerbation. We demonstrated that expression of miR‐146a/b and pro‐inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell‐penetrating peptide (CPP)‐miR‐146a nanocomplexes before infection with RV significantly reduced the expression of the pro‐inflammatory chemokines CCL5, IL‐8 and CXCL1, increased interferon‐λ production, and attenuated infection with the green fluorescent protein (GFP)‐expressing RV‐A16 in HBECs. Concordantly, compared to wild‐type (wt) mice, Mir146a/b−/− mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV‐A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)‐induced allergic airway inflammation and RV‐induced exacerbation models. Interestingly, intranasal administration of CPP‐miR‐146a nanocomplexes reduced HDM‐induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild‐type mice. In conclusion, the overexpression of miR‐146a has a strong anti‐inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR‐146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV‐induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP‐miR‐146a nanocomplexes has therapeutic potential for targeting airway inflammation.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA-146a (miR-146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a-deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a-deficient mice are protected from ovariectomy-induced bone loss. In humans, the levels of miR-146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age-related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-kappa B, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a(-/-) myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a(-/-) HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a(-/-) mice. miR-146a(-/-) HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immune responses despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function and terminal differentiation. To more precisely dissect the role of miR 142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDcre). To label activated B cells in miR-142fl/fl AIDcre mice, we bred them to mice carrying a loxP-flanked STOP cassette, allowing for the expression of fluorescent Zsgreen1 reporter protein in the presence of Cre recombinase. As expected, B cell development was largely unperturbed in miR-142fl/fl AIDcreZsgreen+/− mice. In contrast, FACS analysis of naive and antigen-challenged miR-142fl/fl AIDcre Zsgreen+/− mice revealed a significant decrease in germinal center (GC) B cells and plasma cells (PCs). RNA sequencing of GC B cells suggests dysregulation of transcription factor networks such as miR-142-3p targets BACH2 and IRF4 may contribute to the phenotypes observed in miR 142fl/fl AIDcre Zsgreen+/− mice. Future experiments will investigate molecular pathways governing terminal B cell differentiation and effector function in miR-142fl/fl AIDcre Zsgreen+/− mice, thereby elucidating the role of miR-142 in promoting a healthy response to antigen.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from the BCR-ABL1 fusion gene that encodes a constitutively activated tyrosine kinase (TK). Although TK inhibitors (TKIs) induce disease remission and prolonged survival in CML patients, a subset are resistant and progress from chronic phase (CP) to blast crisis (BC) with poor prognosis. Understanding the molecular mechanisms of transformation from CP to BC is necessary in the development of effective treatments. Here, we used the inducible SCLtTA/BCR-ABL transgenic CP CML model to study the molecular mechanism of disease evolution. Upon tetracycline withdrawal to induce BCR-ABL expression, both the SCLtTA/BCR-ABL homozygous (homo, i.e., SCLtTA+/+BCR-ABL+/+, hereafter called BCR-ABL) and heterozygous (het, i.e., SCLtTA+/-BCR-ABL+/-) mice developed and died of CP CML without developing BC CML, implying that BCR-ABL dosage is insufficient to induce transformation. MicroRNA (miR)-142 is highly expressed in hematopoietic cells with a critical role in normal hematopoiesis. In miR-142 knockout (KO)(miR-142−/−) mice, hematopoietic stem and progenitor cells expanded with a decrease of hematopoietic output. Loss of miR-142 function has been reported in lymphoma, acute lymphocytic leukemia and acute myeloid leukemia. Of note, we also observed lower levels of miR-142 in CD34+CD38- cells from patients with BC CML versus (vs) patients with CP CML. Thus, we hypothesized that miR-142 insufficiency may promote CML transformation from CP to BC. To test our hypothesis, we generated miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mice and observed increasing leukemic blasts over time after BCR-ABL induction in the blood and bone marrow (BM), but not in miR-142 wt (miR-142+/+)BCR-ABL controls even when the latter became moribund. MiR-142−/−BCR-ABL mice had larger spleens and significantly shorter survival [median: 26 vs 54 days (d); p<0.0001] than miR-142+/+BCR-ABL controls. Of note, while both homo (miR-142−/−) and het (miR-142+/−) miR-142 KO BCR-ABL mice eventually developed BC CML, the former had a significantly faster progression to BC and shorter survival (median: 26 vs 45 d; p=0.003) than the latter, suggesting miR-142 deficiency alone is sufficient to initiate BC transformation in the CP CML model in a dose-dependent manner. Importantly, all these features were recapitulated in congenic recipient mice transplanted with BM Lin-Sca-1+c-Kit+ cells (LSKs, 2000/mouse) from diseased miR-142−/−BCR-ABL mice, suggesting LSKs were enriched in leukemic stem cells and able to reproduce BC. Of note, in an RNA-seq analysis comparing LSKs from diseased miR-142−/−BCR-ABL (BC) and miR-142+/+ BCR-ABL(CP) mice, 504 genes were found differentially expressed. Gene set enrichment analysis (GSEA) showed only four pathways differentially expressed (upregulated); three [i.e., oxidative phosphorylation, glycolysis and adipogenesis] regulating cell metabolism and the fourth regulating protein secretion. Next, we developed a novel CpG-miR-142 mimic oligonucleotide, hereafter called CpG-M-miR-142, to restore miR-142 levels. Treatment with CpG-M-miR-142 (20mg/kg/day, iv, 4 weeks) on day 2 after BCR-ABL induction significantly prolonged survival of miR-142−/−BCR-ABL mice compared with CpG-scramble (SCR) (75% vs 33% survival rate at day 40 after BCR-ABL induction; median survival: not reached vs 25 d; p=0.03). Since we observed lower miR-142 levels in TKI-resistant vs TKI-sensitive CML patients (p=0.02), we selected LSKs from diseased miR-142−/−BCR-ABL and miR-142+/+BCR-ABL mice and exposed them to TKI nilotinib (NIL; 2µM) or vehicle for 72 hours to evaluate if downregulation of miR-142 was associated with TKI resistance. We observed lower apoptosis and higher cell growth in NIL-treated miR-142−/−BCR-ABL LSKs vs NIL-treated miR-142+/+BCR-ABL LSKs. The decreased sensitivity of miR-142−/−BCR-ABL LSKs to TKI was rescued by treatment with CpG-M-miR-142. CpG-M-miR-142 (2µM) plus NIL significantly increased apoptosis and reduced cell growth in miR-142−/−BCR-ABL LSKs compared with SCR+ NIL. We showed a key role of miR-142 deficiency in the transformation of CP CML to BC CML associated with deregulation of metabolic pathways. Restoring miR-142 expression in vivo with CpG-M-miR-142 significantly decreased the BC transformation rate, prolonged survival of miR-142−/−BCR-ABL mice and may increase sensitivity to TKIs. Disclosures Marcucci: Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial).

NF-kappa B is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-kappa B using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-kappa B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Tolllike receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-kappa B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-kappa B activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-kappa B-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-kappa B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146-amimics for the treatment of inflammatory and myeloproliferative disorders.

Background: NF-κB is a key regulator of inflammation, myeloproliferation and cancer progression, with an important role in leukemogenesis. Despite therapeutic potential, targeting NF-κB proved challenging. However, in non-malignant myeloid cells NF-κB activity is tightly regulated through many molecular mechanisms, including miRNA. Methods: Here, we describe an original approach to NF-κB inhibition using miR146a, which targets upstream regulators of NF-κB signaling. We generated a myeloid cell-targeted NF-κB inhibitor by tethering a chemically-modified miR146a mimic oligonucleotide to a scavenger receptor (SR)/Toll-like receptor 9 (TLR9) ligand (C-miR146a). Results: Unlike an unconjugated miR-146a molecule, C-miR146a was rapidly internalized and delivered to cytoplasm of target myeloid cells such as macrophages or myeloid leukemia cells. C-miR146a reduced protein levels of classic miR-146a targets, IRAK1 and TRAF6, thereby efficiently blocking NF-κB activation in target cells. Intravenous injections of C-miR146a mimic to miR-146-deficient mice prevented excessive NF-κB activation in myeloid cells, thereby alleviating myeloproliferation and exaggerated inflammatory responses to bacterial challenge. The NF-κB-driven release of IL-1 and IL-6 from monocytes is known to be responsible for cytokine release syndrome (CRS), which can occur in response to bacterial infections, antibody-based therapies and relatively frequently as a serious adverse effect of chimeric antigen receptor (CAR) T-cell therapies. While low expression of miR146a has not yet been implicated in CRS, C-miR146a treatments did reduce pro-inflammatory activity of human monocytes, at the level of IL-1 and IL-6 production, induced by the CD19-specific but not by the naive CAR T cells in vitro. Repeated systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent CRS in xenotransplanted B-cell lymphoma model without impeding the on-target therapeutic effects of CAR T-cells against lymphoma cells. Conclusions: Our results demonstrate potential of using myeloid cell-targeted miR146a mimics for treatment of inflammatory diseases and prevention of potential side effects of immunotherapies. The SR/TLR9-targeted miR-146a mimic design provides an outline for the development of miRNA therapeutics for a variety of myeloid cell-related diseases.

Abstract microRNA-142 (miR-142) is expressed predominantly in cells of hematopoietic origin and plays a vital role in the regulation of innate and adaptive immunity. We have previously shown that miR-142 ablation in mice results in a marked expansion of the immature and mature B cell compartments. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142 null mice using Hardy fraction analysis. Our findings indicate that miR-142 deletion significantly upregulates the frequency of pro-B cells, large pre-B cells, and immature B cells (Fractions B, C, and E), while decreasing the relative number of the earliest B cell progenitors (pre-pro-B cells; Fraction A). Our data links accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow to an increase in their survival capacity. Moreover, we established miR-142 as a negative regulator of interleukein-7 receptor (IL7R), a cytokine receptor which plays a critical role in B cell maturation from pro-B to pre-B cell stage. In agreement with this notion, miR-142-deficient pro-B cells displayed elevated IL7R levels on their surface and proliferated more vigorously than WT cells in response to IL-7 stimulation in vitro. To test the effect of miR-142 dysregulation on malignant B cell transformation, we bred miR-142−/− mice with Eμ-myc transgenic mice, which spontaneously develop a mix of B cell lymphoblastic leukemia and lymphoma. Interestingly, miR-142 haploinsufficiency dramatically accelerated development of the disease in Eμ-myc transgenic mice. Collectively, our results establish miR-142 as an essential negative regulator of primary B cell development, whose dysregulation may contribute to malignant B cell transformation.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142(-/-) mice demonstrated increased expression of the miR-142-3p target alpha V integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-beta (TGF-beta) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

The Philadelphia (Ph) chromosome or t(9;22) results in the generation of a fusion gene, namely BCR/ABL1, which encodes a chimeric protein with aberrant tyrosine kinase activity that drives leukemia cell growth and survival. This molecular/cytogenetic aberration occurs in ~20%-30% of ALL cases and confers poor prognosis. Ph+ ALL patients (pts) are often referred for allogeneic hematopoietic stem cell transplantation (alloHCT), although more recently BCR-ABL-specific tyrosine-kinase inhibitors (TKIs) and immunotherapeutic approaches seemingly induced long-term remission in some patients. Nevertheless, it is still a challenge to determine which Ph+ ALL of the pts could be treated more conservatively without alloHCT. Thus identification of new prognostic biomarkers and/or therapeutic targets may be helpful. Regulation of short non-coding microRNAs(miRNAs) associated with initiation and progression of acute leukemia has been reported. miR-142(both miR-142-3p and miR-142-5p) is expressed at a relatively high level in hematopoietic tissue, and plays a role in myeloid lineage differentiation. In fact, low miR-142-3p expression was associated with myeloid differentiation failure, and miR-142 mutations was reported to promote acute myeloid leukemia (AML). More recently, Kramer et al demonstrated a role of miR-142 in lymphopoiesis by showing that miR-142 deficiency impaired B cell production in a miR-142 knock-out(ko) mouse model (Blood. 2015).