Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia
Authors: Külp Marius , Siemund Anna Lena , Meyer Claus , Larghero Patrizia , Dietz Alissa , Alten Julia , Cario Gunnar , Caye-Eude Aurélie , Cavé Hélène , Bardini Michela , De Lorenzo Paola , Cazzaniga Giovanni , Diehl Laura , Bonig Halvard , Marschalek Rolf 
Published: November 2021

Abstract
Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse.
Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target.
We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene.
The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse.
To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression.
To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed.
In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression.

Disclosures
Cario: Novartis: Other: Lecture Fee.