LAUSR

The treatment of children diagnosed with acute lymphoblastic leukemia (ALL) has improved significantly over recent decades, with 5-year event-free survival (EFS) approaching 85% [1]. However, 5-year EFS for infants diagnosed at less than 1 year of age with MLL-rearranged ALL remains less than 40% [2]. MLL-rearranged infant ALL is both clinically and biologically distinct from other childhood ALL subtypes, which is reflected by a unique gene expression profile [3], a remarkably silent mutational landscape [4], and pronounced resistance to currently applied chemotherapeutics [5]. To date, one of the most effective treatments for infants with MLL-rearranged ALL is the international collaborative INTERFANT protocol [2, 6]. The relative success of this intensive treatment protocol can largely be ascribed to the implementation of the nucleoside analog drug cytarabine (Ara-C), throughout treatment. The addition of Ara-C to an intensified ALL treatment protocol was preceded by the observation that infant ALL cells were highly sensitive to Ara-C in vitro [5]. MLL-rearranged infant ALL cells have also shown sensitivity to other nucleoside analog drugs, including cladribine [7] and clofarabine [8]. From this perspective, we interrogated our drug library screens for drugs effective against MLL-rearranged ALL cells (Supplementary methods) and extracted data for all nucleoside and nucleobase analog drugs that are FDA approved or have been investigated in clinical trials. Cell viability was assessed for 20 different nucleoside and nucleobase analogs in MLL-rearranged ALL cell lines (n= 3) and primary MLL-rearranged infant ALL patient samples (n= 2) (Fig. 1a). Consistent with the known sensitivity of MLLrearranged ALL to several nucleoside analogs, top hits eliminating the vast majority of leukemic cells in both primary MLL-rearranged infant ALL patient samples and in all MLL-rearranged ALL cell lines, included cladribine, clofarabine, cytarabine, and fludarabine. We also identified two additional nucleoside analog drugs effective against MLL-rearranged ALL cells, namely gemcitabine (or dFdC/ 2′,2′-difluorodeoxycytidine) and floxuridine (or FUDR/5fluorodeoxyuridine). Both agents were highly effective in MLL-rearranged ALL cell lines, but had limited effect on the nonproliferating primary patient samples (Fig. 1a). Nucleoside analogs often exert their cytotoxic effects by interfering with DNA synthesis and therefore require proliferating cells [9]. This provides a plausible explanation as to why gemcitabine and floxuridine did not affect the primary patient samples, which fail to proliferate as suspension cultures in vitro. In turn, the observed cytotoxicity of fludarabine and cladribine against primary ALL cells can be These authors contributed equally: Rishi S. Kotecha, Ronald W. Stam