MHH-CALL-4

Synergistic Drug Interactions of the Givinostat (ITF2357) in CRLF2-Rearranged Pediatric BCP-ALL Identified by High-Throughput Drug Screening

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with CRLF2 rearrangements is a high-risk subtype, especially in children with Down Syndrome, who often suffer from treatment-related toxicity. Givinostat (ITF2357), a histone deacetylase inhibitor, has shown efficacy against CRLF2-rearranged BCP-ALL. Oikonomou et al. identify synergistic drug combinations with givinostat using high-throughput drug screening to improve treatment outcomes for these patients.

They previously identified givinostat as a promising anti-leukemic agent for CRLF2r BCP-ALL. To evaluate its synergistic potential with other compounds, they used a high-throughput drug screening library of 174 FDA/EMA-approved or preclinical drugs. Using the MHH-CALL-4 cell line, they constructed a per-drug DSS score to assess compound sensitivity in givinostat and DMSO conditions (Fig. 1A). Of the 174 compounds, 54 showed no activity, leaving 120 for further investigation (Fig. 1B). Unsupervised hierarchical clustering distinguished givinostat- and DMSO-treated cells (Fig. 1C), revealing 3 major clusters. One cluster contained drugs whose sensitivity was more modulated by givinostat. A t-test identified 19 compounds with significantly higher sensitivity in the presence of givinostat (Fig. 1D). These compounds belonged to 7 categories: BCL2, FLT3, HSP90, MAPK, PI3K/mTOR, topoisomerase, and p53-MDM2 inhibitors. Setting a DSS threshold of 25, we selected one drug from each category with the highest DSS score in givinostat condition and the largest difference between givinostat and DMSO conditions. The selected drugs were venetoclax, luminespib, trametinib, sapanisertib, etoposide, and idasanutlin (Fig. 1E).

Fig. 1. Givinostat combination high-throughput drug screening on MHH-CALL-4 (Oikonomou A, Watrin T, et al., 2024).

HTS Identifies Synergistic Drug Combinations with Ruxolitinib in Ph-Like ALL

Ph-like ALL is a high-risk leukemia subtype with frequent relapses and poor outcomes, often involving JAK-STAT pathway mutations. CRLF2-rearranged Ph-like ALL show resistance to single-agent JAK inhibitors. Bӧhm et al. identified effective combination treatments and elucidate the mechanisms of synergy for CRLF2-rearranged Ph-like ALL using high-throughput drug screening and molecular analysis.

To identify potential drug combinations with ruxolitinib for Ph-like ALL, they conducted high-throughput screening (HTS) of ruxolitinib with 5057 bioactive compounds against MHH-CALL-4 and MUTZ-5 cell lines, both overexpressing CRLF2 and harboring JAK2, IKZF1, and PAX5 mutations. Despite ruxolitinib inhibiting downstream signaling at 1 µM, both cell lines showed resistance to its cytotoxic effects (IC₅₀ >15 µM for MUTZ-5 and >30 µM for MHH-CALL-4). Thus, they used 1 µM ruxolitinib for HTS. In the primary screen, 460 compounds with <25% survival relative to control were selected for secondary screening. Compounds demonstrating >2-fold enhanced cell kill with ruxolitinib and high single-agent potency were tested in the tertiary screen to estimate IC₅₀ values (Fig. 2A–D). Overall, ~9% of compounds achieved >75% cell death, including kinase inhibitors, glucocorticoids, and anti-neoplastic drugs. Standard-of-care drugs like cyclophosphamide and methotrexate showed limited efficacy, while nucleotide analogs like cytarabine had high single-agent efficacy but only moderate synergy with ruxolitinib.

Fig. 2. HTS of the Ph-like ALL cell line MHH-CALL-4 to identify synergistic drug combinations with ruxolitinib (Bӧhm J W, Sia K C S, et al., 2021).