Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Identification of a Musashi2 translocation as a novel oncogene in myeloid leukemia.

Myeloid leukemias, diseases marked by aggressiveness and poor outcomes, are frequently triggered by oncogenic translocations. In the case of chronic myelogenous leukemia (CML) the BCR-ABL fusion initiates chronic phase disease with second hits allowing progression to blast crisis. Although Gleevec has been transformative for CML, blast crisis CML remains relatively drug resistant. Here we show that MSI2-HOXA9, a translocation with an unknown role in cancer, can serve as a second hit in driving bcCML. Compared to BCR-ABL, BCR-ABL/MSI2-HOXA9 led to a more aggressive disease in vivo with decreased latency, increased lethality and a differentiation blockade that is a hallmark of blast crisis. Domain mapping revealed that the MSI2 RNA binding domain RRM1 had a preferential impact on growth and lethality of bcCML relative to RRM2 or the HOXA9 domain. Mechanistically, MSI2-HOXA9 triggered global downstream changes with a preferential upregulation of mitochondrial components. Consistent with this, BCR-ABL/MSI2-HOXA9 cells exhibited a significant increase in mitochondrial respiration. These data suggest that MSI2-HOXA9 acts, at least in part, by increasing expression of the mitochondrial polymerase Polrmt and augmenting mitochondrial function and basal respiration in blast crisis. Collectively, our findings demonstrate for the first time that translocations involving the stem and developmental signal MSI2 can be oncogenic, and suggest that MSI, which we found to be a frequent partner for an array of translocations, could also be a driver mutation across solid cancers.

AMD1 is required for the maintenance of leukemic stem cells and promotes chronic myeloid leukemic growth.

Polyamines are critical elements in mammals, but it remains unknown whether adenosyl methionine decarboxylase (AMD1), a rate-limiting enzyme in polyamine synthesis, is required for myeloid leukemia. Here, we found that leukemic stem cells (LSCs) were highly differentiated, and leukemia progression was severely impaired in the absence of AMD1 in vivo. AMD1 was highly upregulated as chronic myeloid leukemia (CML) progressed from the chronic phase to the blast crisis phase, and was associated with the poor prognosis of CML patients. In addition, the pharmacological inhibition of AMD1 by AO476 treatment resulted in a robust reduction of the progression of leukemic cells both in vitro and in vivo. Mechanistically, AMD1 depletion induced loss of mitochondrial membrane potential and accumulation of reactive oxygen species (ROS), resulting in the differentiation of LSCs via oxidative stress and aberrant activation of unfolded protein response (UPR) pathway, which was partially rescued by the addition of polyamine. These results indicate that AMD1 is an essential element in the progression of myeloid leukemia and could be an attractive target for the treatment of the disease.

Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning.

Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. In this study we show that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, we demonstrate that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. We show that d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly our work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, our results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning.

A stem cell reporter based platform to identify and target drug resistant stem cells in myeloid leukemia.

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus, identifying the cells responsible for residual disease and leukemia re-growth is critical to better understanding how they are regulated. Here, we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of leukemia stem cells in aggressive myeloid malignancies, and provides a strategy for defining their core dependencies. Specifically, we carry out a high throughput screen using Msi2-reporter blast crisis chronic myeloid leukemia (bcCML) and identify several adhesion molecules that are preferentially expressed in therapy resistant bcCML cells and play a key role in bcCML. In particular, we focus on syndecan-1, whose deletion triggers defects in bcCML growth and propagation and markedly improves survival of transplanted mice. Further, live imaging reveals that the spatiotemporal dynamics of leukemia cells are critically dependent on syndecan signaling, as loss of this signal impairs their localization, migration and dissemination to distant sites. Finally, at a molecular level, syndecan loss directly impairs integrin ß7 function, suggesting that syndecan exerts its influence, at least in part, by coordinating integrin activity in bcCML. These data present a platform for delineating the biological underpinnings of leukemia stem cell function, and highlight the Sdc1-Itgß7 signaling axis as a key regulatory control point for bcCML growth and dissemination.

MARCH Proteins Mediate Responses to Antitumor Antibodies.

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

An in vivo genome-wide CRISPR screen identifies the RNA-binding protein Staufen2 as a key regulator of myeloid leukemia.

Aggressive myeloid leukemias such as blast crisis chronic myeloid leukemia and acute myeloid leukemia remain highly lethal. Here we report a genome-wide in vivo CRISPR screen to identify new dependencies in this disease. Among these, RNA-binding proteins (RBPs) in general, and the double-stranded RBP Staufen2 (Stau2) in particular, emerged as critical regulators of myeloid leukemia. In a newly developed knockout mouse, loss of Stau2 led to a profound decrease in leukemia growth and improved survival in mouse models of the disease. Further, Stau2 was required for growth of primary human blast crisis chronic myeloid leukemia and acute myeloid leukemia. Finally, integrated analysis of CRISPR, eCLIP and RNA-sequencing identified Stau2 as a regulator of chromatin-binding factors, driving global alterations in histone methylation. Collectively, these data show that in vivo CRISPR screening is an effective tool for defining new regulators of myeloid leukemia progression and identify the double-stranded RBP Stau2 as a critical dependency of myeloid malignancies.

Stem cells in cancer initiation and progression.

While standard therapies can lead to an initial remission of aggressive cancers, they are often only a transient solution. The resistance and relapse that follows is driven by tumor heterogeneity and therapy-resistant populations that can reinitiate growth and promote disease progression. There is thus a significant need to understand the cell types and signaling pathways that not only contribute to cancer initiation, but also those that confer resistance and drive recurrence. Here, we discuss work showing that stem cells and progenitors may preferentially serve as a cell of origin for cancers, and that cancer stem cells can be key in driving the continued growth and  functional heterogeneity of established cancers. We also describe emerging evidence for the role of developmental signals in cancer initiation, propagation, and therapy resistance and discuss how targeting these pathways may be of therapeutic value.

CD98-Mediated Adhesive Signaling Enables the Establishment and Propagation of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML) is an aggressive disease associated with drug resistance and relapse. To improve therapeutic strategies, it is critical to better understand the mechanisms that underlie AML progression. Here we show that the integrin binding glycoprotein CD98 plays a central role in AML. CD98 promotes AML propagation and lethality by driving engagement of leukemia cells with their microenvironment and maintaining leukemic stem cells. Further, delivery of a humanized anti-CD98 antibody blocks growth of patient-derived AML, highlighting the importance of this pathway in human disease. These findings indicate that microenvironmental interactions are key regulators of AML and that disrupting these signals with targeted inhibitors such as CD98 antibodies may be a valuable therapeutic approach for adults and children with this disease.

Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma.

Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in patient-derived xenografts. Specifically, we developed Msi reporter mice that allowed image-based tracking of stem cell signals within cancers, revealing that Msi expression rises as pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumour penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signalling as a central regulator of pancreatic cancer.

Tetraspanin 3 Is Required for the Development and Propagation of Acute Myelogenous Leukemia.

Acute Myelogenous Leukemia (AML) is an aggressive cancer that strikes both adults and children and is frequently resistant to therapy. Thus, identifying signals needed for AML propagation is a critical step toward developing new approaches for treating this disease. Here, we show that Tetraspanin 3 is a target of the RNA binding protein Musashi 2, which plays a key role in AML. We generated Tspan3 knockout mice that were born without overt defects. However, Tspan3 deletion impaired leukemia stem cell self-renewal and disease propagation and markedly improved survival in mouse models of AML. Additionally, Tspan3 inhibition blocked growth of AML patient samples, suggesting that Tspan3 is also important in human disease. As part of the mechanism, we show that Tspan3 deficiency disabled responses to CXCL12/SDF-1 and led to defects in AML localization within the niche. These identify Tspan3 as an important regulator of aggressive leukemias and highlight a role for Tspan3 in oncogenesis.

Fearful symmetry: subversion of asymmetric division in cancer development and progression.

Asymmetric division is an evolutionarily conserved process that generates daughter cells with different fates through the unequal partitioning of fate determinants. While asymmetric division is critically important in generating diversity during development, its dysregulation can also promote oncogenesis. In particular, signals that shift the normal balance of symmetric and asymmetric division can lead to a differentiation arrest and trigger cancer progression. Here, we discuss the studies that have provided increasing support for this idea. Beginning with original work carried out in Drosophila, we trace more recent work in mammalian systems that suggest that the subversion of asymmetric division can contribute significantly to the development and progression of both hematologic malignancies and solid cancers.

CD66+ cells in cervical precancers are partially differentiated progenitors with neoplastic traits.

Cervical cancers, a malignancy associated with oncogenic papilloma viruses, remain a major disease burden in the absence of effective implementation of preventive strategies. CD66(+) cells have previously been identified as a tumor-propagating subset in cervical cancers. We investigated the existence, differentiation state, and neoplastic potential of CD66(+) cells in a precancer cell line harboring HPV31b episomes. The gene expression profile of CD66(high) cells overlaps with differentiated keratinocytes, neoplastic mesenchymal transition, cells of the squamocolumnar junction, and cervical cancer cell line-derived spheroids. There is elevated expression of DNMT1, Notch1, and the viral gene product E1⁁E4 in CD66(high) cells. Thus, CD66(high) cells, in the absence of differentiating signals, express higher levels of key regulators of keratinocytes stemness, differentiation, and the viral life cycle, respectively. We also find a striking association of neoplastic traits, including migration, invasion, and colony formation, in soft agar with CD66(high) cells. These properties and a distinct G2-M-enriched cell-cycle profile are conserved in cells from cervical cancers. Principally, using a precancerous cell line, we propose that CD66(high) cells have an intermediate differentiation state, with a cellular milieu connected with both viral replication and neoplastic potential, and validate some key features in precancer lesions. Such pathophysiologically relevant systems for defining cellular changes in the early phases of the disease process provide both mechanistic insight and potential therapeutic strategies. Collectively, our data provide a rationale for exploring novel therapeutic targets in CD66(+) subsets during cancer progression.

Lis1 regulates asymmetric division in hematopoietic stem cells and in leukemia.

Cell fate can be controlled through asymmetric division and segregation of protein determinants, but the regulation of this process in the hematopoietic system is poorly understood. Here we show that the dynein-binding protein Lis1 is critically required for hematopoietic stem cell function and leukemogenesis. Conditional deletion of Lis1 (also known as Pafah1b1) in the hematopoietic system led to a severe bloodless phenotype, depletion of the stem cell pool and embryonic lethality. Further, real-time imaging revealed that loss of Lis1 caused defects in spindle positioning and inheritance of cell fate determinants, triggering accelerated differentiation. Finally, deletion of Lis1 blocked the propagation of myeloid leukemia and led to a marked improvement in survival, suggesting that Lis1 is also required for oncogenic growth. These data identify a key role for Lis1 in hematopoietic stem cells and mark its directed control of asymmetric division as a critical regulator of normal and malignant hematopoietic development.

Notch signaling in CD66+ cells drives the progression of human cervical cancers.

Human epithelial tumor progression and metastasis involve cellular invasion, dissemination in the vasculature, and regrowth at metastatic sites. Notch signaling has been implicated in metastatic progression but its roles have yet to be fully understood. Here we report the important role of Notch signaling in maintaining cells expressing the carcinoembryonic antigen cell adhesion molecule CEACAM (CD66), a known mediator of metastasis. CD66 and Notch1 were studied in clinical specimens and explants of human cervical cancer, including specimens grown in a pathophysiologically relevant murine model. Gene expression profiling of CD66(+) cells from primary tumors showed enhanced features of Notch signaling, metastasis, and stemness. Significant differences were also seen in invasion, colony formation, and tumor forming efficiency between CD66(+) and CD66(-) cancer cells. Notably, CD66(+) cells showed a marked sensitivity to a Notch small molecule inhibitor. In support of studies in established cell lines, we documented the emergence of a tumorigenic CD66(+) cell subset within a metastatic lesion-derived cervical-cancer cell line. Similar to primary cancers, CD66 expression in the cell line was blocked by chemical and genetic inhibitors of ligand-dependent nuclear Notch signaling. Collectively, our work on the oncogenic properties of CD66(+) cells in epithelial cancers provides insights into the nature of tumor progression and offers a mechanistic rationale to inhibit the Notch signaling pathway as a generalized therapeutic strategy to treat metastatic cancers.