Mikhail Binnewies

FOXO3A directs a protective autophagy program in haematopoietic stem cells.

Blood production is ensured by rare, self-renewing haematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that mouse HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy after ex vivo cytokine withdrawal and in vivo calorie restriction. We demonstrate that FOXO3A is critical to maintain a gene expression program that poises HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FOXO3A-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.

IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development

Using a mouse model recapitulating the main features of human chronic myelogenous leukemia (CML), we uncover the hierarchy of leukemic stem and progenitor cells contributing to disease pathogenesis. We refine the characterization of CML leukemic stem cells (LSCs) to the most immature long-term hematopoietic stem cells (LT-HSCs) and identify some important molecular deregulations underlying their aberrant behavior. We find that CML multipotent progenitors (MPPs) exhibit an aberrant B-lymphoid potential but are redirected toward the myeloid lineage by the action of the proinflammatory cytokine IL-6. We show that BCR/ABL activity controls Il-6 expression thereby establishing a paracrine feedback loop that sustains CML development. These results describe how proinflammatory tumor environment affects leukemic progenitor cell fate and contributes to CML pathogenesis.

Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons

Quiescence acts as a safeguard mechanism to ensure survival of the HSC pool during chronic IFN-1 exposure

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1β, G-CSF, TGFβ and CXCL4) and activation of an inducible Irf8 and β-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

Understanding the tumor immune microenvironment (TIME) for effective therapy

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient’s tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.The tumor immune microenvironment influences tumor progression and response to immunotherapy; its further characterization will improve therapeutic outcome.

Initially disadvantaged, TEL-AML1 cells expand and initiate leukemia in response to irradiation and cooperating mutations

Although TEL-AML1 is present in one-fourth of childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL), the expression of the ETV6-RUNX1 gene encoding this fusion protein has been a poor initiator of leukemia in multiple mouse models.1, 2, 3, 4, 5, 6, 7, 8 To gain insight into why this common lesion does not readily initiate disease, we took advantage of a novel mouse model, in which TEL-AML1 expression was linked to a fluorescence marker and could be induced in a subset of hematopoietic cells during embryonic development. The laboratory of Corrinne Lobe had generated a reporter mouse in which enhanced green fluorescent protein (EGFP) was expressed following Cre-mediated excision (designated herein as ZEG mice), and subsequently generated an analogous mouse, in which a human TEL-AML1 encoding complementary DNA and an internal ribosomal entry site were placed upstream of EGFP (designated herein as TA1 mice). We crossed the inducible ZEG and TA1 mice with mice expressing Cre under the control of the Tie2(Tek) promoter, which is expressed in endothelial cells as well as during early hematopoietic development. These crosses resulted in mice carrying either the Tie2-Cre and inducible ZEG transgenes (Cre/ZEG mice) or the Tie2-Cre and inducible TA1 transgenes (Cre/TA1 mice). Mice carrying the inducible TA1 transgene but lacking Cre were also generated (Cre-/TA1+ control mice).

A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 ‘checkpoint’ immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell–directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.Cross-talk between innate immune cells helps to enhance the antitumor T cell response during checkpoint blockade therapy.

Abstract IA32: Visualizing tumor immune interaction in real time

The nature of an immune response is rarely defined by a unanimous decisions by the participating cells; cells with seemingly opposing functions pervade many immune sites and tumors are no exception. Multiple DC and Macrophage subsets exert push/pull on T cell responses in tumors. Fundamentals of this are gleaned by live-imaging in connection with conventional methods such as flow-cytometry and immunofluorescence. Here, we broadly treat real-time imaging as a discovery tool to understand the tumor microenvironment (TME) and the diversity of cellular interactions that comprise host-tumor interactions. We have advanced this technology from sites of primary, spontaneous and aggressive tumors into sites of metastasis. The results promote the concept of seeking “allies” for tumor therapies within the TME and provide a basis for considering the balance of push/pull signals that determine the consensus immune response. Citation Format: Edward W. Roberts, Mark B. Headley, Miranda Broz, Bijan Boldjipour, Kaitlin Corbin, Mikhail Binnewies, Adriaan Bins, Audrey Audrey Gerard, Matthew Krummel. Visualizing tumor immune interaction in real time [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA32.

Abstract B65: Dissecting the tumor myeloid compartment reveals rare activating antigen presenting cells, critical for T cell immunity

Incoming tumor-reactive cytotoxic T lymphocytes (CTL) are modulated by innate immune cells of the tumor microenvironment and we have used intravital live-imaging to characterize the predominant T cell-APC interactions there. To further hone in on the identities of the APC populations, we have extensively examined myeloid subset diversity across multiple spontaneous (GEMM) and ectopic models. Importantly, as a functional counterpart to the abundant tumor infiltrating macrophages, we have identified a small population of rare tumoral Dendritic Cells capable of robust antigen processing and T cell stimulation. We have identified the specific lineage requirements of these cells, dissecting the essential cytokines driving each tumor APC population. In addition we have found that T-cell dependent immune clearance relies upon these rare tumoral Dendritic Cells. Thus, through the dissection of the tumor myeloid compartment we have revealed a rare, yet highly stimulatory and targetable DC subset that could be exploited therapeutically to enhance anti-tumor immune responses locally. This work is supported by the Genentech Foundation and the Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award. Citation Format: Miranda Broz, Mikhail Binnewies, Bijan Boldajipour, Amanda Nelson, Joshua Pollock, David Erle, Andrea Barczak, Michael Rosenblum, Adil Daud, Diane Barber, Sebastian Amigorena, Laura J. van 9t Veer, Anne Sperling, Denise Wolf, Matthew F. Krummel. Dissecting the tumor myeloid compartment reveals rare activating antigen presenting cells, critical for T cell immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B65.