Exposure to Inflammatory Immune Responses As Driver of Clonal Evolution in Childhood Acute Lymphoblastic Leukemia
Authors: Klemm Lars , Swaminathan Srividya , Papaemmanuil Elli , Ford Anthony M , Greaves Mel , Casellas Rafael , Schatz David , Lieber Michael R. , Muschen Markus 
Published: October 2019

Abstract
Background: Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1 gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear.
Rationale: Activation Induced Cytidine Deaminase (AID) and Recombination Activation Genes 1 and 2 (RAG1-RAG2) are genetic modifiers of the immunoglobulin (Ig) genes that are expressed during normal B cell development. Although AID and RAG1/RAG2 are thought to be segregated to early (RAG1/RAG2) and late (AID) stages of B cell development, respectively, we found that the two enzymes can be concurrently expressed during early B-lymphopoiesis in the context of repeated inflammatory stimuli.
Results: Our experiments identified transitional pre-B cells as the subset that is particularly vulnerable to concomitant expression of AID and RAG1-RAG2 with earlier B cells being protected by IL7 signaling.Human B cells from children lacking a functional IL-7 receptor (IL-7R) displayed both increased expression and activity of AID concurrently with RAG1-RAG2. These results demonstrated that AID activation in both mouse and human early B cell compartments increases genetic instability. Although concurrent activation of AID and RAG1-RAG2 in patient samples implicated a correlation between the two enzymes in the pathogenesis of leukemia, this as such did not prove that the enzymes causally induce overt leukemogenesis. Therefore, we next evaluated the requirement of AID and RAG1-RAG2 in leukemogenic transformation, and identified a condition that leads to massive activation of these enzymes in a pre-leukemic B cell. Importantly, AID and RAG1-RAG2 expression increased dramatically during inflammatory immune responses (e.g. infection), where both these enzymes diversify the antibody repertoire and improve its affinity to antigens from infectious pathogens. We therefore tested whether the pre-B cell subset that concurrently expresses AID and RAG1-RAG2 can respond to an inflammatory stimulus, such as LPS. We observed that pre-B cells require protection from IL7, which prevents aberrant activation of AID. In the absence of protective IL-7, these pre-B cells acquired responsiveness to LPS and strongly activated AID concurrently with RAG1-RAG2 enzymes. We developed IL7-dependent pre-B cell cultures as a disease model for ETV6-RUNX1 pre-leukemia and tested the role of AID and RAG1 in the progression of pre-leukemic clones. To this end, we expanded ETV6-RUNX1 pre-B cells from wildtype (AID and RAG1 expressing) mice, or from mice lacking AID (Aid-/-Rag1+/+) or RAG1 (Aid+/+Rag1-/-). We then challenged pre-B cell cultures by withdrawal of IL7 (loss of protection) and inflammatory stimuli (LPS) and transplanted pre-B cells that had undergone five cycles of -IL7/LPS challenge. Upon transplanting -IL7/LPS-treated Aid+/+Rag1+/+ or Aid-/-Rag1+/+ or Aid+/+Rag1-/- pre-B cells containing ETV6-RUNX1 into NOD-SCID recipient mice, we observed that loss of either Aid or Rag1 dramatically prolonged the latency and reduced the penetrance of leukemia in transplant recipients. This proved that AID and RAG1-RAG2 causally accelerate clonal evolution of a pre-leukemic B cell towards leukemia. Our findings provide a mechanism by which pre-leukemic clones carrying a prenatal genetic lesion such as ETV6-RUNX1 can evolve through infectious and inflammatory stimuli ultimately leading to full blown leukemia.
Conclusion: The impact of inflammatory stimuli on leukemogenesis has been previously implicated in multiple epidemiological studies. For instance, day-care attendance primed the immune system during early childhood and is thought to protect against exacerbation of B cell responses and to prevent collateral damage driving clonal evolution towards leukemia. Although inflammation (LPS stimulation) seems to play a role in accelerating pre-B leukemogenesis in our model, further experiments testing actual infectious pathogens are needed to corroborate this concept. Moreover, it is crucial to test whether leukemogenesis is accelerated in individuals infected with restricted classes of pathogens, not all of which may activate AID in pre-B cells.

Disclosures
No relevant conflicts of interest to declare.