AXL Inhibition in Macrophages Stimulates Host-versus-Leukemia Immunity and Eradicates Naïve and Treatment-Resistant Leukemia.

Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell- and T cell-dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1-refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells. SIGNIFICANCE: Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy.This article is highlighted in the In This Issue feature, p. 2659.

Microenvironmental cues for T-cell acute lymphoblastic leukemia development.

Intensive chemotherapy regimens have led to a substantial improvement in the cure rate of patients suffering from T-cell acute lymphoblastic leukemia (T-ALL). Despite this progress, about 15% and 50% of pediatric and adult cases, respectively, show resistance to treatment or relapse with dismal prognosis, calling for further therapeutic investigations. T-ALL is an heterogeneous disease, which presents intrinsic alterations leading to aberrant expression of transcription factors normally involved in hematopoietic stem/progenitor cell development and mutations in genes implicated in the regulation of cell cycle progression, apoptosis, and T-cell development. Gene expression profiling allowed the classification of T-ALL into defined molecular subgroups that mostly reflects the stage of their differentiation arrest. So far this knowledge has not translated into novel, targeted therapy. Recent evidence points to the importance of extrinsic signaling cues in controlling the ability of T-ALL to home, survive, and proliferate, thus offering the perspective of new therapeutic options. This review summarizes the present understanding of the interactions between hematopoietic cells and bone marrow/thymic niches during normal hematopoiesis, describes the main signaling pathways implicated in this dialog, and finally highlights how malignant T cells rely on specific niches to maintain their ability to sustain and propagate leukemia.

ISG15 modulates development of the erythroid lineage.

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.

Erythroblast transformation by FLI-1 depends upon its specific DNA binding and transcriptional activation properties.

FLI-1 is a transcriptional regulator of the ETS family of proteins. Insertional activation at the FLI-1 locus is an early event in F-murine leukemia virus-induced erythroleukemia. Consistent with its essential role in erythroid transformation, enforced expression of FLI-1 in primary erythroblasts strongly impairs the response of these cells to erythropoietin (Epo), a cytokine essential to erythropoiesis. We show here that point mutations in the ETS domain that abolished FLI-1 binding to specific DNA elements (ETS-binding sites) suppressed the ability of FLI-1 to transform erythroblasts. The exchange of the entire ETS domain (DNA binding domain) of FLI-1 for that of PU.1 changed the DNA binding specificity of FLI-1 for that of PU.1 and impaired FLI-1 transforming properties. In contrast, ETS domain swapping mutants that maintained the DNA binding specificity of FLI-1 did not affect the ability of FLI-1 to transform erythroblasts. Deletion and swapping mutants that failed to inhibit the DNA binding activity of FLI-1 but impaired its transcriptional activation properties were also transformation-defective. Taken together, these results show that both the ability of FLI-1 to inhibit Epo-induced differentiation of erythroblasts and to confer enhanced cell survival in the absence of Epo critically depend upon FLI-1 ETS-binding site-dependent transcriptional activation properties.

Up-regulation of SLAP in FLI-1-transformed erythroblasts interferes with EpoR signaling.

Rearrangement of the FLI-1 locus and ensuing overexpression of FLI-1 protein is an early event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. When overexpressed in primary erythroblasts, FLI-1 converts erythropoietin (Epo)-induced terminal differentiation into a proliferative response. We found that SLAP, a gene encoding a recently described negative regulator of T-cell antigen receptor function during thymocyte development, is up-regulated both at the RNA and protein levels in FLI-1-transformed erythroblasts. Src-like adaptor protein (SLAP) was found in a specific complex with erythropoietin receptor (EpoR), a cytokine receptor essential to erythroid differentiation. Constitutive expression of SLAP severely impairs hemoglobinization and late survival during Epo-induced terminal differentiation of erythroblasts. This impairment is associated with the specific inhibition of several critical Epo-dependent signaling events, including signal transducer and activator of transcription 5 (STAT5) activation and up-regulation of the expression of the antiapoptotic BCL-X gene. Our data support a model by which FLI-1 inhibits normal erythroid differentiation through the deregulation of genes encoding adaptors/effectors that modify the signaling output of cytokine receptors normally required for terminal differentiation.

Direct regulation of BCL-2 by FLI-1 is involved in the survival of FLI-1-transformed erythroblasts.

Rearrangement of the FLI-1 locus with ensuing overexpression of FLI-1 is an early event in Friend murine leukemia virus-induced disease. When overexpressed in primary erythroblasts, FLI-1 blocks erythropoeitin (Epo)-induced terminal differentiation and inhibits apoptosis normally induced in response to Epo withdrawal. We show here that the survival-inducing property of FLI-1 is associated with increased transcription of BCL-2. We further show that FLI-1 binds BCL-2 promoter sequences in transformed erythroblasts, and in vitro studies identify specific FLI-1-binding sites essential for the transactivation of the BCL-2 promoter by FLI-1. Analysis of FLI-1 mutants showed a correlation between the ability of FLI-1 to transactivate BCL-2 promoter sequences and their ability to inhibit apoptosis in the absence of Epo. Moreover, inhibitor studies confirmed the essential role of BCL-2 for FLI-1-transformed erythroblast survival. Finally, enforced expression of BCL-2 was sufficient to promote survival and terminal differentiation of erythroblasts in the absence of Epo. These results show that BCL-2 is an in vivo target of FLI-1 in FLI-1-transformed erythroblasts and that its deregulated expression is instrumental in the survival of these cells.