Earlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5^+/− leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development. Significance: These results demonstrate that delayed infection exposure is a causal factor in pB-ALL. Therefore, these findings have critical implications for the understanding of the pathogenesis of leukemia and for the development of novel therapies for this disease. Cancer Discov; 5(12); 1328–43. ©2015 AACR. See related commentary by Greaves and Müschen, p. 1244. This article is highlighted in the In This Issue feature, p. 1225

The majority of childhood leukemias are precursor B-cell acute lymphoblastic leukemias (pB-ALLs) caused by a combination of prenatal genetic predispositions and oncogenic events occurring after birth. Although genetic predispositions are frequent in children (>1% to 5%), fewer than 1% of genetically predisposed carriers will develop pB-ALL. Although infectious stimuli are believed to play a major role in leukemogenesis, the critical determinants are not well defined. Here, by using murine models of pB-ALL, we show that microbiome disturbances incurred by antibiotic treatment early in life were sufficient to induce leukemia in genetically predisposed mice, even in the absence of infectious stimuli and independent of T cells. By using V4 and full-length 16S ribosomal RNA sequencing of a series of fecal samples, we found that genetic predisposition to pB-ALL (Pax5 heterozygosity or ETV6-RUNX1 fusion) shaped a distinct gut microbiome. Machine learning accurately (96.8%) predicted genetic predisposition using 40 of 3983 amplicon sequence variants as proxies for bacterial species. Transplantation of either wild-type (WT) or Pax5+/– hematopoietic bone marrow cells into WT recipient mice revealed that the microbiome is shaped and determined in a donor genotype–specific manner. Gas chromatography-mass spectrometry (GC-MS) analyses of sera from WT and Pax5+/– mice demonstrated the presence of a genotype-specific distinct metabolomic profile. Taken together, our data indicate that it is a lack of commensal microbiota rather than the presence of specific bacteria that promotes leukemia in genetically predisposed mice. Future large-scale longitudinal studies are required to determine whether targeted microbiome modification in children predisposed to pB-ALL could become a successful prevention strategy.

Inherited susceptibility to pre B-ALL caused by germline transmission of PAX5 c.547G>A

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365–77. ©2017 AACR.

Preleukemic clones carrying BCR-ABLp190 oncogenic lesions are found in neonatal cord blood, where the majority of preleukemic carriers do not convert into precursor B-cell acute lymphoblastic leukemia (pB-ALL). However, the critical question of how these preleukemic cells transform into pB-ALL remains undefined. Here, we model a BCR-ABLp190 preleukemic state and show that limiting BCR-ABLp190 expression to hematopoietic stem/progenitor cells (HS/PC) in mice (Sca1-BCR-ABLp190) causes pB-ALL at low penetrance, which resembles the human disease. pB-ALL blast cells were BCR-ABL–negative and transcriptionally similar to pro-B/pre-B cells, suggesting disease onset upon reduced Pax5 functionality. Consistent with this, double Sca1-BCR-ABLp190+Pax5+/− mice developed pB-ALL with shorter latencies, 90% incidence, and accumulation of genomic alterations in the remaining wild-type Pax5 allele. Mechanistically, the Pax5-deficient leukemic pro-B cells exhibited a metabolic switch toward increased glucose utilization and energy metabolism. Transcriptome analysis revealed that metabolic genes (IDH1, G6PC3, GAPDH, PGK1, MYC, ENO1, ACO1) were upregulated in Pax5-deficient leukemic cells, and a similar metabolic signature could be observed in human leukemia. Our studies unveil the first in vivo evidence that the combination between Sca1-BCR-ABLp190 and metabolic reprogramming imposed by reduced Pax5 expression is sufficient for pB-ALL development. These findings might help to prevent conversion of BCR-ABLp190 preleukemic cells. Significance: Loss of Pax5 drives metabolic reprogramming, which together with Sca1-restricted BCR-ABL expression enables leukemic transformation. Cancer Res; 78(10); 2669–79. ©2018 AACR.