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. 2024 Jul 1;109(7):2309-2315.
doi: 10.3324/haematol.2023.284271.

Efficacy of DYRK1A inhibitors in novel models of Down syndrome acute lymphoblastic leukemia

Shannon L Carey-Smith  1 , Maryam H Simad  2 , Kunjal Panchal  1 , Carlos Aya-Bonilla  2 , Hannah Smolders  2 , Sang Lin  2 , Jesse D Armitage  2 , Vivien T Nguyen  2 , Kathryn Bentley  2 , Jette Ford  2 , Sajla Singh  2 , Joyce Oommen  2 , Anouchka P Laurent  3 , Thomas Mercher  3 , John D Crispino  4 , Andrew P Montgomery  5 , Michael Kassiou  5 , Thierry Besson  6 , Emmanuel Deau  7 , Laurent Meijer  7 , Laurence C Cheung  8 , Rishi S Kotecha  9 , Sébastien Malinge  10
Affiliations

Efficacy of DYRK1A inhibitors in novel models of Down syndrome acute lymphoblastic leukemia

Shannon L Carey-Smith et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Establishment of novel Down syndrome acute lymphoblastic leukemia models for preclinical testing. (A) Constitutive phosphorylation of Stat5 and Erk1/2 in KRASG12D and BCR-ABL-expressing murine cells (starved for 6 hours). (B) Growth of murine wild-type (WT)-KRASG12D, Tc1-KRASG12D and Ts1/Cdkn2a-KRASG12D cells with or without cytokines (Il-7, Scf and Flt3-L, 10 ng/mL) over 6 days. (C) Kaplan-Meier analysis comparing survival of primary (1R) and secondary (2R) sub-lethally irradiated recipient mice engrafted with 1-2x106 Tc1-KRASG12D (1R N=4, 2R N=6), WT-KRASG12D (1R N=5, 2R N=6) and Ts1Rhr/Cdkn2a-KRASG12D (1R N=5, 2R N=6) cell lines; **P<0.01. (D) Phenotype of the murine cell lines assessing surface expression of BP1 and CD24 (phenotype of the WT-BCR-ABL and Tc1-BCR-ABL cell lines are in the Online Supplementary Figure 1F), and representative flow plots showing phenotype of mCherry-positive cell lines in primary and secondary recipients. w/: with; w/o: without. Cyt: cytokine.
Figure 2.
Figure 2.
Genetic and pharmacological inhibition of DYRK1A decreases growth of Down syndrome acute lymphoblastic leukemia cells. (A) Ratio of wild-type (WT)-KRASG12D and Tc1-KRASG12D transduced with GFP-expressing Banshee vectors encoding two shDyrk1a compared to empty Banshee-U6 counterparts over 9 days (N=4 replicates); ***P<0.001. (B) Validation of Dyrk1a knockdown at the protein level 48 hours after transduction (GFP-sorted). Dyrk1a band intensities were quantified and normalized as a ratio of shDyrk1a-transduced to control U6-transduced WT-KRASG12D cells. (C) Cytotoxic effect of increasing doses (in μM) of the DYRK1A inhibitors EHT1610, AM30, Leucettinib-21 (LCTB-21) and its inactive isomer iso-Leucettinib-21 (Iso LCTB-21) at 48 hours in murine WT-KRASG12D and Tc1-KRASG12D cells assessed by flow cytometry (Annexin V staining). (D) Dose-response curves assessing efficacy of EHT1610, LCTB-21 and Iso LCTB-21 at 72 hours by alamarBlue cell viability assay in murine WT-KRASG12D and Tc1-KRASG12D cells. (E) Heatmap integrating relative half-maximal inhibitory concentration (IC50) values obtained for the DYRK1A inhibitors tested in our murine cell lines.
Figure 3.
Figure 3.
Efficacy of DYRK1A inhibition in novel human Down syndrome acute lymphoblastic leukemia cell lines and patient-derived xenograft models. (A) Representative flow plots assessing CD38 and TSLPR expression of the human DS-PER962 and DS-PER961 Down syndrome acute lymphoblastic leukemia (DS-ALL) cell lines compared to their corresponding DS06 and DS02 patient-derived xenografts (PDX), and to NSG recipients engrafted with 1x106 DS-ALL cell lines (PDXcells). (B) Western blots assessing the constitutive phosphorylation of JAK2, STAT5 and ERK1/2 in DS-PER962 and DS-PER961 cell lines following a 6-hour starvation; the non-DS MHH-CALL4 (CRLF2-rearranged/JAK2 mutant) ALL cell line was used as control. (C) Kaplan-Meier curves comparing the survival of the DS-PER962 and DS-PER961 PDX (N=3-4) to their corresponding DS02 and DS06 PDX (N=9-12); ***P<0.001. (D) Efficacy of EHT1610, Leucettinib-21 (LCTB-21) and its inactive isomer iso-Leucettinib-21 (Iso LCTB-21) in the human DS-PER962 and DS-PER961 cell lines (72 hours). (E) Heatmap representing the relative half-maximal inhibitory concentration (IC50) values obtained in the non-DS-ALL, DS-ALL human cell lines and DS-ALL cells freshly harvested from PDX models. (F) Western blot comparing the effect of EHT1610, LCTB-21 and AM30 on phospho-cyclin D3 and total cyclin D3 stability after 6 hours of treatment in the DS-PER962 cell line. (G) ZIP scores obtained from combining Leucettinib-21 (10 doses from 0.01 to 10 μM) with trametinib (0.0001-20 μM), ruxolitinib (0.0001-20 μM), vincristine (0.0005-0.5 μM), dexamethasone (0.00025-20 μM) and L-asparaginase (0.1-20 μM) in the human DS-ALL cell lines. ZIP scores <-10 =antagonism; -10 to 10 =additive; >10 =synergy. (H) In vivo effect of Leucettinib-21 on leukemia burden in the peripheral blood (PB) of the DS02 and DS06 PDX models (oral gavage, 2 weeks and 4 weeks of treatment respectively); *P<0.05, **P<0.01. Veh: vehicle.
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