Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia.

The biological and clinical behaviors of hematological malignancies can be influenced by the active crosstalk with an altered bone marrow (BM) microenvironment. In the present study, we provide a detailed picture of the BM vasculature in acute myeloid leukemia using intravital two-photon microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), such as vascular leakiness and increased hypoxia. Transcriptomic analysis in endothelial cells identified nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived biopsies. Moreover, induction chemotherapy failing to restore normal vasculature was associated with a poor prognosis. Inhibition of NO production reduced vascular permeability, preserved normal hematopoietic stem cell function, and improved treatment response in PDX.

Frequency and Dynamics of Leukemia-Initiating Cells during Short-term Ex Vivo Culture Informs Outcomes in Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.

Different Motile Behaviors of Human Hematopoietic Stem versus Progenitor Cells at the Osteoblastic Niche.

Despite advances in our understanding of interactions between mouse hematopoietic stem cells (HSCs) and their niche, little is known about communication between human HSCs and the microenvironment. Using a xenotransplantation model and intravital imaging, we demonstrate that human HSCs display distinct motile behaviors to their hematopoietic progenitor cell (HPC) counterparts, and the same pattern can be found between mouse HSCs and HPCs. HSCs become significantly less motile after transplantation, while progenitor cells remain motile. We show that human HSCs take longer to find their niche than previously expected and suggest that the niche be defined as the position where HSCs stop moving. Intravital imaging is the only technique to determine where in the bone marrow stem cells stop moving, and future analyses should focus on the environment surrounding the HSC at this point.

Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

RAS interaction with PI3K p110α is required for tumor-induced angiogenesis.

Direct interaction of RAS with the PI3K p110α subunit mediates RAS-driven tumor development: however, it is not clear how p110α/RAS-dependant signaling mediates interactions between tumors and host tissues. Here, using a murine tumor cell transfer model, we demonstrated that disruption of the interaction between RAS and p110α within host tissue reduced tumor growth and tumor-induced angiogenesis, leading to improved survival of tumor-bearing mice, even when this interaction was intact in the transferred tumor. Furthermore, functional interaction of RAS with p110α in host tissue was required for efficient establishment and growth of metastatic tumors. Inhibition of RAS and p110α interaction prevented proper VEGF-A and FGF-2 signaling, which are required for efficient angiogenesis. Additionally, disruption of the RAS and p110α interaction altered the nature of tumor-associated macrophages, inducing expression of markers typical for macrophage populations with reduced tumor-promoting capacity. Together, these results indicate that a functional RAS interaction with PI3K p110α in host tissue is required for the establishment of a growth-permissive environment for the tumor, particularly for tumor-induced angiogenesis. Targeting the interaction of RAS with PI3K has the potential to impair tumor formation by altering the tumor-host relationship, in addition to previously described tumor cell-autonomous effects.

A niche-like culture system allowing the maintenance of primary human acute myeloid leukemia-initiating cells: a new tool to decipher their chemoresistance and self-renewal mechanisms.

Acute myeloid leukemia-initiating cells (LICs) are responsible for the emergence of leukemia and relapse after chemotherapy. Despite their identification more than 15 years ago, our understanding of the mechanisms responsible for their self-renewal activity and their chemoresistance remains poor. The slow progress in this area is partly due to the difficulty of studying these cells ex vivo. Indeed, current studies are reliant on xenotransplantation assays in immunodeficient mice. In this paper, we report that by modeling key elements of the bone marrow niche using different stromal feeder layers and hypoxic culture conditions, we can maintain LICs over at least 3 weeks and support their self-renewal properties demonstrated through primary and secondary successful xenograft. We provide a proof of principle that this niche-like culture system can be used to study LIC chemoresistance following in vitro cytarabine treatment similarly to the xenograft chemotherapy model. We found that although LICs are believed to be more chemoresistant than non-LICs, functionally defined LICs are not enriched after cytarabine treatment, and heterogeneity in their resistance to treatment can be seen between patients and even within the same patient. We present a culture system that can be used as an in vitro surrogate for xenotransplantation and that has the potential to dramatically increase the throughput of the investigation of LICs. This would further provide the means by which to identify and target the functionality of the different signaling pathways involved in the maintenance and resistance of LICs to improve acute myeloid leukemia treatments.

Requirement for interaction of PI3-kinase p110α with RAS in lung tumor maintenance.

RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities.

Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells.

Intravital microscopy of the calvarium is the only noninvasive method for high-resolution imaging of the bone marrow (BM) and hematopoietic stem cell (HSC) niches. However, it is unclear if the calvarium is representative of all BM compartments. Using the combination of whole body optical imaging, intravital microscopy, and "in vivo fluorescence trapping," a thorough comparison of HSCs and putative HSC niches in the calvaria, epiphyses, and diaphyses, at steady state or after HSC transplantation, can be made. We report substantial heterogeneity between different BM compartments in terms of bone-remodeling activity (BRA), blood volume fraction (BVF), and hypoxia. Although BVF is high in all BM compartments, including areas adjacent to the endosteum, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted upon HSC transplantation. Unexpectedly, the macroanatomical distribution of HSCs at steady state is homogeneous across these 3 areas and independent of these 2 parameters and suggests the existence of "reconstituting niches," which are distinct from "homeostatic niches." Both types of niches were observed in the calvarium, indicating that endochondral ossification, the process needed for the formation of HSC niches during embryogenesis, is dispensable for the formation of HSC niches during adulthood.

The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.

Heterogeneous sensitivity of human acute myeloid leukemia to ß-catenin down-modulation.

Dysregulation of the Wnt/ß-catenin pathway has been observed in various malignancies, including acute myeloid leukemia (AML), where the overexpression of ß-catenin is an independent adverse prognostic factor. ß-catenin was found upregulated in the vast majority of AML samples and more frequently localized in the nucleus of leukemic stem cells compared with normal bone marrow CD34(+) cells. The knockdown of ß-catenin, using a short hairpin RNA (shRNA) lentiviral approach, accelerates all-trans retinoic acid-induced differentiation and impairs the proliferation of HL60 leukemic cell line. Using in vivo quantitative tracking of these cells, we observed a reduced engraftment potential after xenotransplantation when ß-catenin was silenced. However, when studying primary AML cells, despite effective downregulation of ß-catenin we did not observe any impairment of their in vitro long-term maintenance on MS-5 stroma nor of their engraftment potential in vivo. Altogether, these results show that despite a frequent ß-catenin upregulation in AML, leukemia-initiating cells might not be 'addicted' to this pathway and thus targeted therapy against ß-catenin might not be successful in all patients.

"Microenvironmental contaminations" induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking.

Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

[The consortium of Biological Resource Centres (BRC) and tumour cell and tissue banks in the Marseilles metropolitan area]. / Le réseau des Centres de Ressources Biologiques (CRB) et tumorothèques de l'agglomération marseillaise.

The goal of this presentation is to describe current and future aspects of the operations within the consortium of Biological Resource Centres (BRC) and Tumour cell and tissue banks of the Marseilles metropolitan area. The consortium was created in year 2001, through the association of several tissue and cell banks that were operating for many years in Marseilles. Existing collections are not exclusively collections of tumour cells or tissues; however, the two tumour cell and tissue banks located at the Regional Cancer Research Centre and at the University Hospital account for a very significant proportion of the collections. Our collective work leads to the recognition and funding of the consortium by Inserm, through the "Collections 2003" grant. The consortium objectives are to define a common scientific strategy, to share professional practices in the logistics and database management of the banks, to establish a quality management program, and to build a common catalogue that describes existing biological resources. Through these efforts, the ultimate goal is to adopt rules that define BRC, as defined by the Organization for Economic Co-operation and Development (OECD).