Irinotecan Induces Complete Remission in MLL -Rearranged Acute Lymphoblastic Leukemia Xenografts

BACKGROUND: Acute lymphoblastic leukemia (ALL) in infants (diagnosis < 1 year) is characterized by a high incidence of rearrangements of the MLL ( KMT2A ) gene, occurring in ~80% of the cases. The resulting MLL fusions drive a leukemia biologically and clinically distinct from other subtypes. Therefore, while current ALL treatments achieve cure rates of 80-90%, MLL -rearranged ( MLL -R) infant ALL is still beset by poor outcome, with a 5-year event-free survival of ~40%. Further intensification of current treatment is not possible, highlighting an urgent need for new therapeutic strategies.

AIM: While novel drug classes have shown pre-clinical promise in MLL -R ALL, clinical implementation has become a major bottle-neck, as set-up of clinical trials with new drugs, as well as their FDA-approval, is a protracted process. Hence, we explored a drug repurposing approach, performing screens of >3000 FDA-approved drugs to identify compounds able to both target MLL -RALL cells and expeditiously transition into clinics.

METHODS: MLL -RALL cell lines (SEM, KOPN8 and RS411) were screened using commercially available drug libraries comprising in total 3685 compounds; drug sensitivity to the initial concentration of 1 μM was assessed using 4-day MTS-assays. The most efficacious hits were further screened at 100 nM and 10 nM, to refine selection of potent candidate compounds. Among the top hits were the topoisomerase I inhibitor camptothecin and its derivatives, 10-hydroxycamptothecin and 7-ethyl-10-hydroxycamptothecin (SN38). The most potent derivative, SN38, was further analyzed in vitro to assess SN38-induced molecular changes in MLL -RALL cells using immunoblotting and cell death assays.

To assess the anti-leukemic effects of SN38 in vivo , immunodeficient NOD.Cg- Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice were injected with the luciferase -expressing MLL -RALL reporter cell line SEM-SLIEW, as well as primary leukemic cells of two MLL -Rinfant ALL patients. Mice were treated 3 times per week with either the pro-dug irinotecan (40 mg/kg), which is metabolised into SN38, or vehicle, for up to 4 weeks. Treatment was initiated after engraftment was confirmed by either bioluminescence imaging (SEM-SLIEW) or presence of >1% human leukemic cells in the blood was detected by multi-color flow cytometry (patient-derived xenografts, PDX). Disease progression over time was measured accordingly. To investigate the curative potential of irinotecan, treatment with the drug was initiated in a subset of mice displaying advanced, full-blown leukemia.

At the end of treatment, mice were killed to assess the effect of irinotecan on human leukemic cell engraftment in bone marrow (BM), blood and spleen using flow cytometry and histochemistry. In addition, a subset of irinotecan-treated mice was kept alive for up to 42 days without treatment, to monitor for disease relapse.

RESULTS: Camptothecins target MLL -RALL cells in vitro ; in particular SN38 had nanomolar IC50-values in both cell lines and primary leukemicpatient cells. Exposure with 25 nM SN38 inhibited proliferation and induced cell death of the MLL -RALL cell line SEM; characteristic DNA damage markers, such as accumulation of γ-H2AX and CHK2 phosphorylation could also be detected already after 2h of SN38 exposure. The anti-leukemic effect of SN38 could be validated in vivo in MLL -RALL xenografts, where the SN38 pro-drug irinotecan completely blocked leukemia expansion. At the experimental end-point, flow cytometric analyses showed human leukemic cells to be completely eradicated from the BM, blood and spleen in irinotecan-treated cell line xenografts, and to be sporadically present at minimal residual disease levels in irinotecan-treated PDX mice. Histochemistry stainings confirmed lack of leukemic infiltration in extramedullary tissues. Moreover, irinotecan induced complete remission in mice with advanced leukemia, as shown by flow cytometry as well as loss of bioluminescence. While ablation of human leukemic cells persisted in cell line-derived xenografts after treatment stop, PDX mice relapsed after 4 weeks, possibly due to presence of leukemic cells at low levels; this could possibly be improved by extending treatment duration.

CONCLUSION: Irinotecan inhibits MLL -R ALL very potently, and as an FDA-approved drug already used in other pediatric cancers, it is an ideal candidate for repurposing and implementation in future MLL -R ALL treatment strategies.