Differential effects of itacitinib, fedratinib, and ruxolitinib in mouse models of hemophagocytic lymphohistiocytosis.

ABSTRACT: Hemophagocytic lymphohistiocytosis (HLH) comprises a severe hyperinflammatory phenotype driven by the overproduction of cytokines, many of which signal via the JAK/STAT pathway. Indeed, the JAK1/2 inhibitor ruxolitinib has demonstrated efficacy in preclinical studies and early-phase clinical trials in HLH. Nevertheless, concerns remain for ruxolitinib-induced cytopenias, which are postulated to result from the blockade of JAK2-dependent hematopoietic growth factors. To explore the therapeutic effects of selective JAK inhibition in mouse models of HLH, we carried out studies incorporating the JAK1 inhibitor itacitinib, JAK2 inhibitor fedratinib, and JAK1/2 inhibitor ruxolitinib. All 3 drugs were well-tolerated and at the doses tested, they suppressed interferon-gamma (IFN-γ)-induced STAT1 phosphorylation in vitro and in vivo. Itacitinib, but not fedratinib, significantly improved survival and clinical scores in CpG-induced secondary HLH. Conversely, in primary HLH, in which perforin-deficient (Prf1-/-) mice are infected with lymphocytic choriomeningitis virus (LCMV), itacitinib, and fedratinib performed suboptimally. Ruxolitinib demonstrated excellent clinical efficacy in both HLH models. RNA-sequencing of splenocytes from LCMV-infected Prf1-/- mice revealed that itacitinib targeted inflammatory and metabolic pathway genes in CD8 T cells, whereas fedratinib targeted genes regulating cell proliferation and metabolism. In monocytes, neither drug conferred major transcriptional impacts. Consistent with its superior clinical effects, ruxolitinib exerted the greatest transcriptional changes in CD8 T cells and monocytes, targeting more genes across several biologic pathways, most notably JAK-dependent proinflammatory signaling. We conclude that JAK1 inhibition is sufficient to curtail CpG-induced disease, but combined inhibition of JAK1 and JAK2 is needed to best control LCMV-induced immunopathology.

Childhood B-Cell Preleukemia Mouse Modeling.

Leukemia is the most usual childhood cancer, and B-cell acute lymphoblastic leukemia (B-ALL) is its most common presentation. It has been proposed that pediatric leukemogenesis occurs through a "multi-step" or "multi-hit" mechanism that includes both in utero and postnatal steps. Many childhood leukemia-initiating events, such as chromosomal translocations, originate in utero, and studies so far suggest that these "first-hits" occur at a far higher frequency than the incidence of childhood leukemia itself. The reason why only a small percentage of the children born with such preleukemic "hits" will develop full-blown leukemia is still a mystery. In order to better understand childhood leukemia, mouse modeling is essential, but only if the multistage process of leukemia can be recapitulated in the model. Therefore, mouse models naturally reproducing the "multi-step" process of childhood B-ALL will be essential to identify environmental or other factors that are directly linked to increased risk of disease.

Lessons from mouse models in the impact of risk factors on the genesis of childhood B-cell leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) stands as the primary contributor to childhood cancer-related mortality on a global scale. The development of the most conventional forms of this disease has been proposed to be conducted by two different steps influenced by different types of risk factors. The first step is led by a genetic insult that is presumably acquired before birth that transforms a healthy cell into a preleukemic one, which is maintained untransformed until the second step takes place. This necessary next step to leukemia development will be triggered by different risk factors to which children are exposed after birth. Murine models that recap the stepwise progression of B-ALL have been instrumental in identifying environmental and genetic factors that contribute to disease risk. Recent evidence from these models has demonstrated that specific environmental risk factors, such as common infections or gut microbiome dysbiosis, induce immune stress, driving the transformation of preleukemic cells, and harboring genetic alterations, into fully transformed leukemic cells. Such models serve as valuable tools for investigating the mechanisms underlying preleukemic events and can aid in the development of preventive approaches for leukemia in child. Here, we discuss the existing knowledge, learned from mouse models, of the impact of genetic and environmental risk factors on childhood B-ALL evolution and how B-ALL prevention could be reached by interfering with preleukemic cells.

Immune stress suppresses innate immune signaling in preleukemic precursor B-cells to provoke leukemia in predisposed mice.

The initial steps of B-cell acute lymphoblastic leukemia (B-ALL) development usually pass unnoticed in children. Several preclinical studies have shown that exposure to immune stressors triggers the transformation of preleukemic B cells to full-blown B-ALL, but how this takes place is still a longstanding and unsolved challenge. Here we show that dysregulation of innate immunity plays a driving role in the clonal evolution of pre-malignant Pax5+/- B-cell precursors toward leukemia. Transcriptional profiling reveals that Myd88 is downregulated in immune-stressed pre-malignant B-cell precursors and in leukemic cells. Genetic reduction of Myd88 expression leads to a significant increase in leukemia incidence in Pax5+/-Myd88+/- mice through an inflammation-dependent mechanism. Early induction of Myd88-independent Toll-like receptor 3 signaling results in a significant delay of leukemia development in Pax5+/- mice. Altogether, these findings identify a role for innate immunity dysregulation in leukemia, with important implications for understanding and therapeutic targeting of the preleukemic state in children.

Transient Inhibition of the JAK/STAT Pathway Prevents B-ALL Development in Genetically Predisposed Mice.

Preventing development of childhood B-cell acute lymphoblastic leukemia (B-ALL), a disease with devastating effects, is a longstanding and unsolved challenge. Heterozygous germline alterations in the PAX5 gene can lead to B-ALL upon accumulation of secondary mutations affecting the JAK/STAT signaling pathway. Preclinical studies have shown that this malignant transformation occurs only under immune stress such as exposure to infectious pathogens. Here we show in Pax5+/- mice that transient, early-life administration of clinically relevant doses of ruxolitinib, a JAK1/2 inhibitor, significantly mitigates the risk of B-ALL following exposure to infection; 1 of 29 animals treated with ruxolitinib developed B-ALL versus 8 of 34 untreated mice. Ruxolitinib treatment preferentially targeted Pax5+/- versus wild-type B-cell progenitors and exerted unique effects on the Pax5+/- B-cell progenitor transcriptional program. These findings provide the first in vivo evidence for a potential strategy to prevent B-ALL development. SIGNIFICANCE: JAK/STAT inhibition suppresses tumorigenesis in a B-ALL-susceptible mouse model, presenting a novel approach to prevent B-ALL onset.

The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia.

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies.