Extended Data Fig. 10: Pharmacological reactivation of suppressed divergent pathways as a therapeutic strategy in B-ALL.

a, h, Patient-derived B-ALL cells (STAT5-driven) LAX7 (a) and JFK125R (h) were treated overnight with vehicle control (DMSO), BCI-215 (1 μM), ruxolitinib (500 nM), or a combination of both. Western blotting was performed to measure levels of STAT5-pY694, STAT5, ERK1/2-pT202/pY204 and ERK1/2 (n = 3 independent experiments). b, Patient-derived B-ALL cells (ERK-driven, LAX7R) were treated overnight with vehicle control, DPH (1 μM), trametinib (500 nM), or a combination of both. Levels of STAT5-pY694, STAT5, ERK1/2-pT202/Y204 and ERK1/2 were assessed (n = 3 independent experiment). c, i, Patient-derived B-ALL cells (LAX7 and LAX7R (c) and JFK125R (i)) were treated with increasing concentrations of BCI-215, ruxolitinib or both, or DPH, trametinib, or both for three days. Percentage growth inhibition at each concentration is shown as heatmaps (n = 3 independent experiments). Combination indices (Cis) were calculated to determine synergy for treatment combinations. DPH and trametinib concentrations used for LAX7, LAX7R and JFK125R (in μM): DPH, 0, 0.16, 0.31, 0.63,1.3, 2.5, 5; trametinib, 0, 0.032, 0.063, 0.13, 0.25, 0.5, 1. Ruxolitinib and BCI-215 concentrations used for LAX7 (in μM): ruxolitinib, 0, 0.125, 0.25, 0.5, 1, 2; BCI-215, 0, 0.04, 0.08, 0.16, 0.31, 0.63. Ruxolitinib and BCI-215 concentrations used for LAX7R and JFK125R (in μM): ruxolitinib, 0, 0.063, 0.125, 0.25, 0.5, 1, 2; BCI-215, 0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63. d, j, Single-cell phosphoprotein analyses for STAT5-pY694 and ERK-pT202/Y204 were performed for patient-derived B-ALL cells LAX7 and LAX7R (d) and JFK125R (j) before in vivo treatment (n = 3 independent experiments). e, k, Patient-derived LAX7R (e) or JFK125R (k) B-ALL cells were injected into sublethally irradiated (2 Gy) NSG mice. Recipient mice injected with LAX7R were treated six times a week for four weeks with 2 mg kg⁻¹ DPH, 0.5 mg kg⁻¹ trametinib or both (e). Recipient mice injected with JFK125R were treated five times a week for four weeks with 2 mg kg⁻¹ BCI-215, 30 mg kg⁻¹ ruxolitinib or both (k). Mice were killed when they showed signs of overt leukaemia (hunched back, weight loss and inability to move). The y-axes indicate overall survival (%).Survival curves are shown (n = 6 per group). To assess additive versus synergistic activity of single versus combination treatments in vivo, we adapted the Bliss independence model for survival analysis. With this approach, treatments are Bliss ‘independent’ if the fraction of cells surviving combination therapy equals the product of fractions that survive the individual treatments. A Weibull distribution, {exp[−(t/β)α]}, is fitted to survival data for each condition, and distributions of survival benefits (treated survival time minus untreated survival time) are computed for each treatment. Survival benefits of drugs 1 and 2 are summed and added to the untreated survival distribution to compose a ‘sum of benefits’ survival distribution (see Methods). f, g, l, m, Although combination treatments prolonged overall survival, transplant-recipient mice ultimately developed overt leukaemia. B-ALLs that developed in mice bearing LAX7R (after treatment with DPH plus trametinib; e) and JFK125R (after treatment with BCI-215 plus ruxolitinib; k) were isolated from bone marrow, suspended in cell culture medium and treated with increasing concentrations of BCI-215, ruxolitinib or both, or DPH, trametinib, or both. f, l, Percentage growth inhibition at each concentration is shown as heatmaps (n = 3 independent experiments). Combination indices were calculated to determine synergy of treatment combinations. g, m, To elucidate the clonal composition of LAX7R B-ALL post-treatment (e) and that of JFK125R post-treatment (k), we performed single-cell phosphoprotein analyses for STAT5-pY694 and ERK-pT202/Y204 (n = 3 independent experiments). For gel source data, see Supplementary Fig. 1.

Source data