Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer, accounting for ~25% of all pediatric tumors. Nearly 20% of patients do not respond to current treatments, making cALL one of the leading cause of disease-related mortality amongst children. Moreover, sizable portion of childhood leukemia survivors develop significant long-term or late effects of treatment. This rate of refractory/relapse cases and the increased risk of chronic morbidity and early mortality among survivors highlight the pressing need for new treatment strategies in cALL. These can be developed through in-depth investigation of the genetic architecture underlying cALL. For this purpose, we performed whole exome sequencing (WES) of matched tumor-normal pair genomic DNA from 200 cALL patients to identify putative somatic driving mutations. A repertoire of 132 candidate drivers were further validated by performing a targeted knockdown functional screen in 3 pre-B ALL cell lines (NALM6, REH and 697). Through this screen, we identified WHSC1 as putative driver gene. WHSC1 is a methyltransferase that dimethylates lysine 36 on histone H3 (H3K36me2). Eight of the 200 (4%) cALL patients harboured a mutation in WHSC1 (7 pre-B and 1 pre-T ALL). In all 8 cases, the mutation led to a change at amino acid 1099 from a glutamic acid to lysine (E1099K). It has been shown that this activating mutation is associated with global increase in H3K36me2, which is accompanied by substantial changes in gene expression. Transcriptome analysis, using RNA-seq technique, of 6 cALL patients (2 with the mutation and 4 without) revealed distinctive differential expression pattern. We observed a specific enrichment in Wnt pathway genes in presence of the WHSC1 E1099K mutation (6.52 fold, P= 0.05), suggesting a stem cell signature. MCTP39 has been identified as an inhibitor of WHSC1, but has never been tested in ALL. Thus, we treated 4 pre-B ALL cell lines, 3 wild-type for WHSC1 (697, REH and Nalm6) and 1 carrying the mutation E1099K (RS411), as well as the non-leukemic human B cell precursor GM12878 cell line with variable concentrations of MCTP39 and calculated IC50. The IC50 in the leukemic cell lines 697, REH, Nalm6 and RS411 were 2.5µM, 1.7µM, 0.88µM and 0.89µM respectively, whereas it reached 25µM in the non-leukemic GM12878, suggesting a specificity for transformed cells. Although Nalm6 and RS411 had almost the same IC50 at 48h, cell growth curves showed differences in late effect (beyond 3 days), with RS411 being more sensitive to MCTP39. The latter differential effect has been confirmed by colony assay with reduction in plating efficiency of 69% and 45% in RS411 and Nalm6, respectively, when treated 48h with MCTP39 at 0.25µM. This result suggests that MCTP39 targets specifically stem/progenitor cells, particularly those that harbor the mutation WHSC1 E1099K. To confirm that the cellular effect of MCTP39 is due to WHSC1 inhibition, we assessed the impact of MCTP39 on H3K36me2 expression, using fluorometric assay and western blot. In both Nalm6 and RS411cell lines, MCTP39 led to significant decrease in H3K36me2. Then, we analyzed whether MCTP39 synergizes with doxorubicin, an anthracycline that is commonly used in cALL treatment. In both Nalm6 and RS411 cell lines, MCTP39 showed a clear synergistic effect at all concentration tested, permitting 40% reduction in doxorubicin concentration to obtain equal effect. Altogether, this study indicates that inhibition of WHSC1 is a promising treatment strategy in cALL patients, particularly in those carrying WHSC1 mutation. Further investigations are needed to recapitulate these results in vivo and to elucidate the pathways affected as result of the inhibition of H3K36me2 by MCTP39.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.