Page 299 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
Figure 3. Efficacy of DYRK1A inhibition in novel human Down syndrome acute lymphoblastic leukemia cell lines and patient-de- rived 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 assess- ing the constitutive phosphorylation of JAK2, STAT5 and ERK1/2 in DS-PER962 and DS-PER961 cell lines following a 6-hour star- vation; 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.
tion (IC50) values than its disomic counterpart WT-KRASG12D (Figure 2D, E; Online Supplementary Figure S2A).
In order to expand our observations, we next established two human DS-ALL cell lines: DS-PER961 and DS-PER962 from our previously reported DS06 (KRASG12S-positive) and DS02 (CRLF2-rearranged/JAK2I682F-positive) PDX.6 These unique models represent the first human cell lines for DS- ALL, with comprehensive characterization at the genomic, transcriptomic and phenotypic levels, confirming their re- semblance to the PDX models from which they originated (Figure 3A; Online Supplementary Figure S3A-E). Western blot analyses confirmed phosphorylation of ERK1/2 and STAT5 downstream of KRASG12S and JAK2I682F mutants in DS-PER961 and DS-PER962 respectively (Figure 3B). Using NOD-SCID-γc-/- (NSG) mice, we also showed that both DS- ALL cell lines engrafted into immunocompromised recipi- ents with DS-PER962 cells being more aggressive than their PDX counterparts (Figure 3C). Using these human cells, we confirmed that Leucettinib-21, AM30 and AM45 were potent inhibitors in human DS-ALL cells and in MHH-CALL4 (a non- DS CRLF2-rearranged and JAK2 mutant ALL cell line known to be sensitive to DYRK1A inhibition) (Figure 3D, E; Online Supplementary Figure S2B-D).8 No significant effect was seen for AM28 nor for the inactive isomer of Leucettinib-21 (Figure 3D; Online Supplementary Figure S2B, C). Importantly, efficacy of these DYRK1A inhibitors was demonstrated in DS- ALL blasts freshly harvested from four DS-ALL PDX models (described in 6), validating the suitability of our DS-ALL cells to assess efficacy of new therapies (Figure 3E). Compared to EHT1610 and AM30, Leucettinib-21 was the most potent compound in inhibiting phosphorylation of the known DYR- K1A target cyclin D3 in a dose-dependent manner shown in DS-PER962 (Figure 3F), DS-PER961 and in murine cells (Online Supplementary Figure S2E), but had limited effect on FOXO1-phosphorylation (Online Supplementary Figure S2F-I). Next, we evaluated in vitro drug combinations of Leucettinib-21 with targeted and conventional therapies and identified synergy with vincristine and an additive effect with dexamethasone and L-asparaginase (Figure 3G; Online Supplementary Figure S2J). An additive effect between Leu-
cettinib-21 and the targeted therapies ruxolitinib (a JAK1/2 inhibitor) and trametinib (a MEK1/2 inhibitor) was also seen in DS-PER962 (CRLF2-positive/JAK2I682F mutant) and in the DS-PER961 (KRASG12S) cell lines, respectively (Figure 3G). Finally, we assessed the efficacy of Leucettinib-21 in vivo in the DS06 and DS02 PDX models and observed that in vivo treatment with Leucettinib-21 decreased leukemia burden but did not fully eradicate leukemia (Figure 3H). Together, this data demonstrates the suitability and clinical relevance of the novel murine and human models we have established and emphasizes the key role of DYRK1A in DS-ALL. Compared to other children, higher sensitivity to treat- ment-related toxicity in children with DS-ALL remains a major clinical challenge. This has significantly limited the development of novel targeted therapies for this patient population, ultimately delaying translation into clinical tri- als. Recently, integration of immunotherapeutic approaches has offered promise in reducing toxicity. Indeed, Laetsch et al. reported comparable outcomes between DS- and non- DS children with relapsed/refractory B-ALL who received CD19-directed chimeric antigen receptor T-cell therapy, including similar rates of CD19-negative relapse,15 and an ongoing clinical trial is currently assessing whether blinatum- omab can replace two blocks of consolidation chemotherapy for treatment of de novo DS-ALL (clinicaltrial gov. Identifier: NCT03911128). As an alternative approach, identifying key vulnerabilities in DS-ALL blasts could provide the molecular basis for development of novel targeted therapies. While resources such as the recent in-depth characterization of the genetic landscape of DS-ALL can provide such insight,10 we have developed novel models of DS-ALL and shown that inhibiting dosage-sensitive mechanisms altered by trisomy 21 may also represent a new avenue to integrate agents with low toxicity and ultimately improve outcomes and quality of care for children with DS-ALL. We demonstrated that a reduction in DYRK1A expression is sufficient to decrease the growth of DS-ALL cell lines, confirmed the sensitivity of human and murine cells to DYRK1A inhibition and showed that the leading candidate, Leucettinib-21, potentiates the cytotoxic effect of other chemotherapeutic and targeted
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