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Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Navchannel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Despite significant efforts to improve therapies for acute myeloid leukemia (AML), clinical outcomes remain poor. Understanding the mechanisms that regulate the development and maintenance of leukemic stem cells (LSCs) is important to reveal new therapeutic opportunities. We have identified CD97, a member of the adhesion class of G protein–coupled receptors (GPCRs), as a frequently up-regulated antigen on AML blasts that is a critical regulator of blast function. High levels of CD97 correlate with poor prognosis, and silencing of CD97 reduces disease aggressiveness in vivo. These phenotypes are due to CD97’s ability to promote proliferation, survival, and the maintenance of the undifferentiated state in leukemic blasts. Collectively, our data credential CD97 as a promising therapeutic target on LSCs in AML.

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9–driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99’s role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.

Precision oncology relies on the accurate molecular characterization of individual patients with cancer at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection. Here, we performed genomic analyses of primary tumors, metastases, and plasma collected from individual patients to define the concordance of actionable genomic alterations and to identify drivers of metastatic disease progression. We observed a high degree of discordance of actionable genomic alterations, with 23% discordant between primary and metastatic disease sites. Among chromatin-modifying genes, ARID1Amutations, when discordant, were exclusive to the metastatic tumor samples. Our findings indicate that the high degree of lesion-to-lesion genomic heterogeneity may be a barrier to precision oncology approaches for bladder cancer and that circulating tumor DNA profiling may be preferred to tumor sequencing for a subset of patients.

Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types and to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1 bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, because miR-29b expression is unaltered in miR-29a/b-1-null HSCs, and only ectopic expression of miR-29a restores HSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1 exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29 target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1-null HSCs. The loss of negative regulation of Dnmt3a by miR-29a is a major contributor to the miR-29a/b-1-null HSPC phenotype, as both in vitro Dnmt3a short hairpin RNA knockdown assays and a genetic haploinsufficiency model of Dnmt3a restored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1-deficient mice. Overall, these data demonstrate that miR-29a is critical for maintaining HSC function through its negative regulation of Dnmt3a.

Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types and to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, because miR-29bexpression is unaltered in miR-29a/b-1-null HSCs, and only ectopic expression of miR-29arestores HSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1-null HSCs. The loss of negative regulation of Dnmt3aby miR-29ais a major contributor to the miR-29a/b-1-null HSPC phenotype, as both in vitro Dnmt3ashort hairpin RNA knockdown assays and a genetic haploinsufficiency model of Dnmt3arestored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1-deficient mice. Overall, these data demonstrate that miR-29ais critical for maintaining HSC function through its negative regulation of Dnmt3a.

Hematopoietic stem cells (HSCs) are heterogeneous with respect to their self-renewal, lineage, and reconstitution potentials. Although c-Kit is required for HSC function, gain and loss-of-function c-Kit mutants suggest that even small changes in c-Kit signaling profoundly affect HSC function. Herein, we demonstrate that even the most rigorously defined HSCs can be separated into functionally distinct subsets based on c-Kit activity. Functional and transcriptome studies show HSCs with low levels of surface c-Kit expression (c-Kitlo) and signaling exhibit enhanced self-renewal and long-term reconstitution potential compared with c-Kithi HSCs. Furthermore, c-Kitlo and c-Kithi HSCs are hierarchically organized, with c-Kithi HSCs arising from c-Kitlo HSCs. In addition, whereas c-Kithi HSCs give rise to long-term lymphomyeloid grafts, they exhibit an intrinsic megakaryocytic lineage bias. These functional differences between c-Kitlo and c-Kithi HSCs persist even under conditions of stress hematopoiesis induced by 5-fluorouracil. Finally, our studies show that the transition from c-Kitlo to c-Kithi HSC is negatively regulated by c-Cbl. Overall, these studies demonstrate that HSCs exhibiting enhanced self-renewal potential can be isolated based on c-Kit expression during both steady state and stress hematopoiesis. Moreover, they provide further evidence that the intrinsic functional heterogeneity previously described for HSCs extends to the megakaryocytic lineage.

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and reconstitute all elements of the hematopoietic system. Acute myeloid leukemia (AML) is thought to arise from, and be maintained by, leukemic stem cells (LSCs), which exhibit similar features to HSCs, including the abilities to self-renew and differentiate into non-self-renewing cells. Acquisition of stem-cell-like characteristics by the LSCs is likely mediated in part by molecular mechanisms that normally regulate HSC function. Thus, understanding the shared and unique aspects of the molecular regulation of these cell populations will be important to understanding the relationship between normal hematopoiesis and leukemogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that act at the posttranscriptional level to regulate protein expression. Unfortunately, most investigations of the role of miRNAs in normal hematopoiesis have been restricted to studies of their effects on lineage commitment in progenitors and mature effector cell function, but not on HSCs. Recent studies have identified miRNAs that enhance HSC function, and an abundance of profiling studies using primary AML samples have identified dysregulated miRNAs that may target genes implicated in self-renewal (HOX genes, P53, and PTEN), thus providing a potential link between normal and malignant stem cells. While these studies as well as recent in vivo models of miRNA-induced leukemogenesis (e.g. miR-29a, miR-125b) suggest a role for miRNAs in the development of AML, future studies using serial transplantation of primary AML blasts, from both mouse models and primary human AML specimens, will be necessary to assess the roles of miRNAs in LSC biology.

No relevant conflicts of interest to declare.

Epigenetic regulators in normal and malignant hematopoiesis have been shown to be important in normal and malignant stem cell self-renewal function and myeloid leukemogenesis. While epigenetic dysregulation can occur through activating and/or loss-of-function mutations, these regulators can also be modulated by other regulators, such as microRNAs. Specifically, miR-29 has been previously identified as an “epi-mir” for contributing to epigenetic regulation by altering expression of DNMT3a and TET. We and others have previously shown that in the hematopoietic system, miR-29 is a positive regulator of hematopoietic stem cell (HSC) self-renewal, is upregulated in AML blasts, and when over-expressed in transplanted HSCs and immature progenitors, leads to a myeloproliferative-like disorder that progresses to acute myeloid leukemia (AML).

We recently reported that CD99 is highly expressed on disease initiating stem cells in myeloid malignancies. In acute myeloid leukemia (AML), high expression of CD99 identified human leukemia stem cells (LSCs) with enhanced leukemia-initiating capacity and gene signatures associated with hematopoietic stem cell (HSC) self-renewal. LSCs with high CD99 expression were also marked by significant depletion of ribosomal protein transcripts, and we have subsequently confirmed that they exhibit reduced levels of protein translation as measured by polysome profiling.

CD99 is a potential therapeutic target enriched on disease stem cells in myeloid malignancies.

No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is composed of functionally heterogeneous cells including leukemic stem cells (LSCs), which exhibit the ability to self-renew and propagate disease. It is thought that failure of common chemotherapy regimens is due to insufficient eradication of LSCs. However, the mechanisms that maintain stem cell function in the hematopoietic system are not well understood. MicroRNAs play an important role in the regulation of normal and malignant hematopoietic stem cells. Our studies showed that miR-99,a miRNA highly expressed in AML patient cell populations enriched for LSC activity, is among the most highly expressed miRNAs in hematopoietic stem cells (HSCs), suggesting that miR-99plays a role in regulating normal HSCs as well as LSCs.

No relevant conflicts of interest to declare.

Despite recent advances in our understanding of the genetic origins of acute myeloid leukemia (AML), clinical outcomes remain poor. While standard induction chemotherapy induces remission in most patients, the majority of patients eventually relapses and dies from progressive disease. A number of cell surface proteins have been shown to be expressed at high levels on AML stem cells compared to normal HSCs including CD47, CD44, CD96, TIM3, CD123, CD25 and IL1RAP. Despite the attention these antigens have received, data supporting their roles as cell-intrinsic regulators of LSCs are more limited. We have identified CD97, a member of the adhesion class of GPCR, as a frequently upregulated antigen expressed on AML blasts that is a critical regulator of LSC function.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

MicroRNAs play an important role in the regulation of normal and malignant stem cells of the hematopoietic system. Analysis of microRNA-Seq data from 168 acute myeloid leukemia (AML) patients in the TCGA database reveals widespread dysregulation of miRNA expression, including miR-99b. Interestingly, miR-99bexpression inversely correlates with differentiation status of the AMLs as determined by the French-American-British classification scheme, suggesting miR-99is a negative regulator of differentiation. We also have shown that miR-99a and miR-99b arehighly expressed in mouse hematopoietic stem cells (HSCs) compared to their more differentiated progeny. Thus, we hypothesized that miR-99is a regulator of differentiation in normal and leukemic stem cells.

The MLL--AF9 fusion oncogene is associated with acute leukemias in adults and children. Retroviral expression of MLL-AF9 in mouse hematopoietic stem/progenitor cells (HSPCs) rapidly induces acute myeloid leukemia (AML) and therefore serves as an excellent model to study MLL-AF9 induced leukemogenesis. We previously showed that miR-29is highly expressed in human AML and positively regulates hematopoietic stem cell (HSC) self-renewal. Consistent with a role in AML, higher expression of miR-29predicts poor survival in AML patients using the TCGA dataset. In addition, transduction of MLL-AF9 into microRNA-29 (miR-29a/b1) null Lin-c-Kit+Sca1+ (LSK) cells was sufficient to induce leukemic colony formation in vitrosimilar to WT LSK cells, but transplantation of MLL-AF9+ miR-29null cells into lethally irradiated recipients resulted in an increased disease latency (median - 56 vs 151 days, p<0.001). In addition, with a short time transduction (1 weeks) of MLL-AF9, mice of miR-29null recipients exhibited long-term donor-derived lymphomyeloid engraftment. MiR-29null and MLL-AF9 positive HSPCs were serially transplantable, and did not generate AML during a 2 year time period during with 5 times transplantation, suggesting no significant alteration in HSPC self-renewal.

No relevant conflicts of interest to declare.

MicroRNAs are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Expression profiling studies has identified miRNAs that regulate normal and malignant hematopoietic stem cell function. Our previous studies showed that ectopic expression of miR-29a in mouse bone marrow cells induced a myeloproliferative disorder that progressed to acute myeloid leukemia (AML). Over-expression of miR-29b in AML cell lines has been reported to induce apoptosis by negatively regulating Dnmt3a. We recently found that miR-29a positively regulates hematopoietic stem cell (HSC) self-renewal and proliferation using a knockout mouse model of miR-29ab. miR-29a null mice contained significantly lower HSC numbers and miR-29a null HSCs exhibited markedly decreased reconstitution ability in both competitive and non-competitive transplantation assays. To investigate the mechanism of miR-29a action, we performed transcriptomal profiling of miR-29a null HSCs and found that miR-29a null HSCs exhibit a gene expression pattern more similar to wild-type committed progenitors than wild-type HSCs. We identified Dnmt3a as one dysregulated miR-29a target as showing increased expression in miR-29a null HSCs, and haplodeficiency of Dnmt3a partly restores miR-29a deficient HSC function. In order to test the requirement for miR-29a in myeloid leukemogenesis, we transduced miR-29a deficient Lin-c-Kit+Sca-1+ (LSK) cells with the oncogenic MLL-AF9 fusion gene, and found that the development of AML from these cells was markedly delayed. We found that Meis1, Ccna2, Hoxa5 and Hoxa9 transcripts were significantly downregulated in miR-29a null LSK cells compared to WT LSK cells, but they were similarly induced in MLL-AF9 transformed c-Kit+Mac-1+ cells. To investigate whether the epigenetic dysregulation resulting from miR-29a deletion may underlie this transformation-resistant phenotype, we examined the distribution of the active epigenetic mark, H3K79me2, in c-Kit+Mac-1+ miR-29a null cells using a ChIP-Seq assay. After analyzing H3K39me2 peaks using model-based analysis of ChIP-Seq, we identified 4281 and 3649 genes associated with this active epigenetic mark using a duplicated ChIP-Seq analysis, with an overlap of 3164 genes (66.39%). Using public available ChIP-Seq data, we compared our results with the genes associated with the H3K79me2 mark in normal immature LSK cells (9282 genes), granulocyte-macrophage progenitors (GMPs, 8556 genes), and MLL-AF9 transformed GMP cells (L-GMP, 8578 genes), and found 4234, 4111, 4046, and 4766 genes were also identified have an active H3K79me2 mark in MLL-AF9 transformed miR-29a null cells. These data indicate that miR-29a loss inactivates a large group of genes activated by the MLL-AF9 oncogene. We also found that 379 genes were associated with H3K79me2 peaks in both normal LSK and MLL-AF9 transformed miR-29a null c-Kit+Mac-1+ cells, but were absent of this epigenetic marker in L-GMP, suggesting that these genes confer self-renewal and proliferation capacities to normal HSCs. In addition, suppression of these genes are important in leukemic transformation by MLL-AF9, and finally the reactivation of these genes in miR-29a null cells compromises the leukemogenesis ability of MLL-AF9. Interestingly, out of these 379 genes, we were able to identify 18 genes that were potential miR-29a targets including Akt3, Map4k4, Dnmt3a, et al. This suggests the direct and indirect effects from miR-29a in regulating its target gene networks at transcriptional and post-transcriptional levels. Our studies found miR-29a is essential in maintaining HSC function and loss of miR-29a abrogate the leukemogenesis capacity of MLL-AF9.

The cell of origin for the chronic lymphoproliferative disorder hairy cell leukemia is a long-term hematopoietic stem cell, as shown through human genetic data and murine genetic models.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29ain regulating HSC self-renewal. In order to determine the role of miR-29ain HSC function, we have evaluated our recently described miR-29a/b1null mouse. Homozygous deletion of miR-29a/b1resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29asince miR-29bexpression was not significantly altered in HSCs and reconstitution of miR-29a/b1null HSPCs with miR-29a, but not miR-29b, rescued in vitrocolony formation defects. Self-renewal defects were observed in miR-29adeficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29adeficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29atarget genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a,in miR-29adeficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3amediates part of miR-29anull HSPCs functional defects. miR-29aloss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29apositively regulates HSC self-renewal and is required for myeloid leukemogenesis.

Recent studies strongly suggest that aging results in the decreased regenerative capacity of adult somatic stem cells, but this process is poorly understood. Hematopoietic stem cell (HSC) aging is associated with myeloid-biased differentiation, reduced capacity for self-renewal, and reduced production of lymphoid and red blood cells. These functional changes lead to alterations in peripheral blood cell counts and reduction in adaptive immunity, as well as an increased incidence of myeloid malignancies. Previous studies have demonstrated that cell-intrinsic changes underlie HSC aging and persist through transplantation. MicroRNAs (miRNAs) are small non-coding RNAs that regulate HSC self-renewal and lineage specificity by suppressing the expression of hundreds of genes. Since rapamycin treatment has previously been shown to inhibit age-associated phenotypes in old HSCs, we hypothesized that investigating the expression and function of miRNAs in HSCs purified from mouse models of longevity may yield significant insights into the molecular mechanisms that underlie the phenotypic changes observed in the aging hematopoietic system.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder recently found to be characterized by somatic BRAFV600E mutations. The malignant cell in HCL exhibits features consistent with a mature B-lymphocyte, including cell-surface expression of the pan-B-cell marker CD19 and monotypic surface immunoglobulins with clonal rearrangements of immunoglobulin heavy and light chains. Despite possessing these stereotypic features, the cell of origin of HCL has been long debated, and no cell type along the continuum of developing B-lymphocytes has been definitively identified as the normal counterpart of HCL cells.

No relevant conflicts of interest to declare.

Abstract 2325

No relevant conflicts of interest to declare.

Abstract 2556