Page 295 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
Efficacy of DYRK1A inhibitors in novel models of Down syndrome acute lymphoblastic leukemia
Despite significant advances, outcomes for children with Down syndrome (DS, trisomy 21) who develop acute lym- phoblastic leukemia (ALL) remain poor. Reports of large DS- ALL cohorts have shown that children with DS have inferior event-free survival (EFS) and overall survival (OS) compared to children without DS.1-3
Children with DS also exhibit increased treatment-related mortality due to infections and toxicities following che- motherapy, higher cumulative risk of relapse and inferior outcomes following relapse.4 This situation highlights the dire need for the development of more potent and target- ed therapies to improve the survival and quality of care for these vulnerable children, who often have additional co-mor- bidities linked to trisomy 21 that complicates their clinical management. Targeted approaches and immunotherapies have shown promising results for pediatric leukemia.5 Hence, the development of new models of DS-ALL are needed to rapidly advance drug discovery and refine existing treatment strategies. We recently established DS-ALL patient-derived xenografts (PDX) and demonstrated that targeting somatic alterations found in DS-ALL using MEK inhibitors combined with conventional treatment has the potential to improve outcomes for these children.6 Targeting the dosage-sensitive mechanisms resulting from the extra copy of chromosome 21 is also an area of intense investigation.7-9 As such, inhibition of the chromosome 21 kinase DYRK1A using EHT1610 or of its direct targets FOXO1 and STAT3, has shown promising cytotoxic effects both in vitro and in vivo.8
In this study, we developed novel clinically relevant models of DS-ALL to facilitate the assessment of new therapeu- tic agents. First, we modeled oncogenic cooperation seen in DS-ALL in vitro.10 To this end, we transduced wild-type (WT) and trisomic (Tc1) 8-10-week-old bone marrow cells with retroviruses encoding the frequently observed mutant KRASG12D or the more rarely seen BCR-ABL fusion11 (animal experiments were approved by institutional ethics committee and followed Australian guidelines for the care and use of animals). Of note, the bone marrow stroma and hematopoi- etic stem cell and progenitor compartment composition did not significantly differ between disomic and trisomic mice, except from an increased proportion of multipotent pro- genitors MPP2 at the expense of the less committed MPP1 (Online Supplementary Figure S1A-C). Trisomic Tc1 progen- itors exhibited increased capacity to form colony-forming unit (CFU) pre-B colonies in vitro compared to WT (Online Supplementary Figure S1D), as seen previously in the partially trisomic Ts1Rhr (Ts1) model.7 Ectopic expression of KRASG12D or BCR-ABL enhanced the number and replating capacity of both WT and Tc1 CFU pre-B colonies compared to the
empty vector MIC (MSCV-IRES-mCherry) (Online Supple- mentary Figure S1E, F). Next, we established two murine DS-ALL cell lines (Tc1-KRASG12D and Tc1-BCR-ABL), disomic controls (WT-KRASG12D and WT-BCR-ABL), as well as an in- dependent Ts1/Cdkn2a-KRASG12D cell line (established from triple transgenic Ts1Rhr, Mb1-Cre, Cdkn2afl/fl donor mice); attempts to develop CRLF2-rearranged/JAK2 mutant murine DS-ALL cell lines were unsuccessful. Interestingly, although KRASG12D led to a constitutive phosphorylation of Erk1/2, the highest levels of Erk1/2 phosphorylation and cytokine independence for cell proliferation were only observed in WT-KRASG12D cells (Figure 1A, B). All KRASG12D-expressing mu- rine cell lines engrafted in sub-lethally irradiated C57Bl/6J mice in primary and secondary recipients and ultimately succumbed to leukemia with complete penetrance, with recipients displaying mCherry-positive cells in the peripheral blood, bone marrow, and in the spleen (data not shown). Engrafted recipient mice exhibited splenomegaly associated with a leukemia-driven disorganized architecture (Online Supplementary Figure S1G, H). We also confirmed that the engrafted cell lines exhibit a phenotype similar to the cell lines cultured in vitro; although we noted clonal selection of a CD24+/BP1- pro-B population from the WT-KRASG12D cells in vivo, which was retained in secondary recipients (Figure 1C, D; and data not shown). Altogether, we developed nov- el murine models of DS-ALL, providing a unique platform suitable for testing targeted therapies.
In order to validate the clinical relevance of these murine cells, we focused on the therapeutically targetable chromo- some 21 kinase DYRK1A, as recent reports have emphasized its role in childhood leukemia, regardless of Down syndrome.8 First, we used short hairpin RNA (shRNA) interference to show that all five cell lines, disomic (N=2) or trisomic (N=3) and expressing either KRASG12D or BCR-ABL oncogenes, were sensitive to Dyrk1a knock-down (KD) (Figure 2A, B; Online Supplementary Figure S1I). We next assessed the efficacy of new potent DYRK1A inhibitors using a DYRK1A-focused library which included EHT1610 used as control, Leucettinib-21 and its inactive isomer (compounds inspired by Leucettines and Leucettamine B, a natural substance produced by the marine sponge Leucetta microraphis),12,13 and three additional DYRK1A inhibitors whose chemical structure is based on the 7-azain- dole scaffold, AM28, AM30 and AM45.14 In dose-response experiments, we showed that AM30 and Leucettinib-21 were cytotoxic in both WT-KRASG12D and Tc1-KRASG12D cell lines (Figure 2C). We also observed that Leucettinib-21, AM30 and AM45 were more potent than EHT1610 in decreasing cellular growth in all cell lines tested, and that Tc1-KRASG12D cells always exhibited lower half-maximal inhibitory concentra-
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