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Abstract Intrachromosomal amplification of chromosome 21 (iAMP21) B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) in children is a high‐risk subtype for which targeted drugs are lacking. In this study, we determined the frequency of secondary lesions in 28 iAMP21 BCP‐ALL patient samples and investigated cellular sensitivity for candidate‐targeted drugs. iAMP21 was enriched in FLT3 aberrations (10.7% vs. 50.0%, p = 0.003) and SH2B3 inactivation (7.14% vs. 46.4%, p = 0.002), compared with 28 B‐other cases, and these alterations co‐occurred in 21.4%. The occurrence of lesions in CRLF2 and IL7R was similar between iAMP21 and B‐other cases (25% vs. 17.9%, p = 0.746 and 7.14% vs. 0%, p = 0.491 respectively) as were mutations in JAK1 and JAK2 (3.57% vs. 0% and 10.7% vs. 10.7%, p = 1 for both). Sensitivity to the FLT3 inhibitor gilteritinib did not differ between iAMP21 and B‐other cases irrespective of FLT3 status. However, iAMP21 samples harboring both FLT3‐ITD and SH2B3 lesions showed the highest sensitivity. CRLF2‐rearranged iAMP21 samples were slightly more sensitive to JAK inhibitor ruxolitinib than those without, although a lack of sensitivity was present in 50% of iAMP21 cases. Trametinib sensitivity varied among iAMP21 samples with over half of iAMP21 samples being sensitive irrespective of RAS‐pathway mutation status or other secondary lesions. In summary, iAMP21 leukemias were enriched in FLT3 and in SH2B3 lesions, which when co‐occurring affected sensitivity to FLT3 inhibition by gilteritinib but not JAK inhibition by ruxolitinib. Together, our results suggest that FLT3 and RAS signaling inhibitors are of interest for further (pre)clinical evaluation in iAMP21 BCP‐ALL.

Background: Chromosome 21 is affected in ∼60% of paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients and includes somatic and constitutional gains, intrachromosomal amplification of chromosome 21 (iAMP21), and the translocation t(12;21) resulting in the ETV6::RUNX1 gene fusion. Methods: Since these numeric and structural chromosome 21 alterations are not targetable, we studied the type and frequency of yet-proven targetable events co-occurring with chromosome 21 alterations. Results: Among 307 primary paediatric BCP-ALL cases, JAK/STAT pathway lesions were most frequent in patients with constitutional gain of chromosome 21 (Down syndrome ALL; 35/71, 49%) and iAMP21 (9/22, 41%). RAS pathway lesions were most frequent in high hyperdiploidy (62/108, 57%) and FLT3 lesions were most frequent in iAMP21 (7/22, 32%). Virtually all cases expressed CD19 and CD22 at the cell surface. Positivity for CD20 surface expression ranged from 67% in iAMP21 (8/12) to 20% in ETV6::RUNX1 (26/129). Conclusion: Activated JAK/STAT, RAS or FLT3 signalling, and CD marker surface expression may provide targetable treatment options for the majority of chromosome 21-altered BCP-ALL cases.

Summary: SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low μM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.

Background and purposeIntrachromosomal amplification of chromosome 21 (iAMP21) is a rare subtype of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It is unknown how iAMP21 contributes to leukaemia. The currently known commonly amplified region is 5.1 Mb.MethodsWe aimed to narrow down the common region of amplification by using high resolution techniques. Array comparative genomic hybridization (aCGH) was used to determine copy number aberrations, Affymetrix U133 Plus2 expression arrays were used to determine gene expression. Genome-wide expression correlations were evaluated using Globaltest.ResultsWe narrowed down the common region of amplification by combining copy number data from 12 iAMP21 cases with 52 cases from literature. The combined common region of amplification was 1.57 Mb, located from 36.07 to 37.64 Mb (GRCh38). This region is located telomeric from, but not including, RUNX1, which is the locus commonly used to diagnose iAMP21. This narrow region, which falls inside the Down Syndrome critical region, includes 13 genes of which the expression of eight genes was significantly upregulated compared with 143 non-iAMP21 B-other cases. Among these, transcriptional repressor RIPPLY3 (also known as DSCR6) was the highest overexpressed gene (fold change = 4.2, FDR < 0.001) and most strongly correlated (R = 0.58) with iAMP21-related genome-wide expression changes.DiscussionThe more precise definition of the common region of amplification could be beneficial in the diagnosis of iAMP21 based on copy number analysis from DNA sequencing or arrays as well as stimulate functional research into the role of the included genes in iAMP21 biology.