Bone Marrow Endosteal Mesenchymal Progenitors Depend on HIF Factors for Maintenance and Regulation of Hematopoiesis

Summary Maintenance and differentiation of hematopoietic stem cells (HSCs) is regulated through cell-autonomous and non-cell-autonomous mechanisms within specialized bone marrow microenvironments. Recent evidence demonstrates that signaling by HIF-1α contributes to cell-autonomous regulation of HSC maintenance. By investigating the role of HIF factors in bone marrow mesenchymal progenitors, we found that murine endosteal mesenchymal progenitors express high levels of HIF-1α and HIF-2α and proliferate preferentially in hypoxic conditions ex vivo. Inactivation of either HIF-1α or HIF-2α dramatically affects their phenotype, propagation, and differentiation. Also, downregulation of HIF factors provokes an increase in interferon-responsive genes and triggers expansion and differentiation of hematopoietic progenitors by a STAT1-mediated mechanism. Interestingly, in conditions of demand-driven hematopoiesis HIF factors are specifically downregulated in mesenchymal progenitors in vivo. In conclusion, our findings indicate that HIF factors also regulate hematopoiesis non-cell-autonomously by preventing activation of a latent program in mesenchymal progenitors that promotes hematopoiesis

HIF factors cooperate with PML-RARα to promote acute promyelocytic leukemia progression and relapse

Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t(15;17) and the resulting oncogenic fusion protein PML-RARα. Although acting primarily as a transcriptional repressor, PML-RARα can also exert functions of transcriptional co-activation. Here, we find that PML-RARα stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through in vitro and in vivo studies, we find that PML-RARα exploits a number of HIF-1α-regulated pro-leukemogenic functions that include cell migration, bone marrow (BM) neo-angiogenesis and self-renewal of APL blasts. Furthermore, HIF-1α levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1α in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs)

A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells

The regulatory factors governing adult mesenchymal stem cells (MSCs) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal form of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSCs transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF-DLK1-SOX9 pathway in the pathogenesis of undifferentiated sarcoma with important therapeutic implications

Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples

Synthetic CD4+ T cell-targeted antigen-presenting cells elicit protective antitumor responses

CD4(+) helper T cells are critical for protective immune responses and yet suboptimally primed in response to tumors. Cell-based vaccination strategies are under evaluation in clinical trials but limited by the need to derive antigen-presenting cells (APC) from patients or compatible healthy donors. To overcome these limitations, we developed CD4(+) T cell-targeted synthetic microbead-based artificial APC (aAPC) and used them to activate CD4(+) T lymphocytes specific for a tumor-associated model antigen (Ag) directly from the naive repertoire. In vitro, aAPC specifically primed Ag-specific CD4(+) T cells that were activated to express high levels of CD44, produced mainly interleukin 2, and could differentiate into Th1-like or Th2-like cells in combination with polarizing cytokines. I.v. administration of aAPC led to Ag-specific CD4(+) T-cell activation and proliferation in secondary lymphoid organs, conferred partial protection against subcutaneous tumors, and prevented the establishment of lung metastasis. Taken together, our data support the use of cell-free, synthetic aAPC as a specific and versatile alternative to expand peptide-specific CD4(+) T cells in adoptive and active immunotherapy

A non-cell-autonomous role for Pml in the maintenance of leukemia from the niche

Disease recurrence after therapy, due to the persistence of resistant leukemic cells, represents a fundamental problem in the treatment of leukemia. Elucidating the mechanisms responsible for the maintenance of leukemic cells, before and after treatment, is therefore critical to identify curative modalities. It has become increasingly clear that cell-autonomous mechanisms are not solely responsible for leukemia maintenance. Here, we report a role for Pml in mesenchymal stem cells (MSCs) in supporting leukemic cells of both CML and AML. Mechanistically, we show that Pml regulates pro-inflammatory cytokines within MSCs, and that this function is critical in sustaining CML-KLS and AML ckit+ leukemic cells non-cell autonomously

miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV