Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory, or High-Risk Leukemias: A Report from the LEAP Consortium

Abstract

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. SIGNIFICANCE: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations.See related commentary by Bornhauser and Bourquin, p. 1322.This article is highlighted in the In This Issue feature, p. 1307.

Figure 1:

LEAP Consortium clinical trial schema. The diagram outlines the clinical genomics data that was obtained during this trial and informed matched targeted therapy recommendations.

Figure 2:

Landscape of targetable genomic alterations for the LEAP Consortium Clinical Trial. Co-mutation plot showing targetable genomic alterations for the 153 patients enrolled on the LEAP trial. Samples are sorted according to the recommendation tier (across top) and grouped by drug target along left axis. For patients with more than one targetable alteration, the highest tier recommendation is used for diagram order.

Figure 3:. RAS pathway mutations are a therapeutic target in pediatric acute leukemia.

A) VAF for NRAS, KRAS, and PTPN11 mutations in patients with B-ALL on the LEAP trial compared to patients with newly-diagnosed B-ALL treated at Dana-Farber Cancer Institute. Leukemia samples were profiled using the same assay. **P<0.005 using Mann-Whitney test. B) VAF for NRAS, KRAS, and PTPN11 mutations in patients with AML on the LEAP trial. Primary leukemia samples from patients enrolled on the LEAP Consortium trial were tested ex vivo in response to MEK inhibitors. Samples with Ras pathway mutations (NRAS, KRAS, and PTPN11) were more sensitive to selumetinib (C) and trametinib (D), compared to WT samples. Shown is average area under the curve (AUC) for individual samples, with average +/− SD. *P<0.05 and **P<0.005 using unpaired t-test. Genome-scale CRISPR-Cas9 screen of 739 cell lines showed RAS mutant leukemia and other cell lines to be differentially dependent on NRAS (E) or KRAS (F) compared to RAS WT leukemia cell lines. Shown is the average gene effect +/− SEM. ****P<0.0001 using Mann-Whitney test. G) Expression of NRAS p.G12D in murine MLL-AF9 cells increased their sensitivity to MEK inhibitors, PD-0325901 and trametinib. Shown is average dose response +/− SEM, with 4 replicates. H) Patient-derived xenograft (PDX) model characterized by TCF3-HLF NRAS p.G12D was treated with venetoclax, selumetinib or venetoclax plus selumetinib. The combination of venetoclax with selumetinib decreased leukemia burden compared to mice treated with each drug individually. Data are flow cytometric quantification of total human CD45+ CD19+ ALL cells in harvested murine spleens (N=5). * P<0.01 and ****P<0.0001 using one-way ANOVA with Tukey’s post-test for multiple comparisons. I) Combination of selumetinib with venetoclax is synergistic across Ras pathway mutant acute leukemia cell lines. Synergy was assessed by Chou-Talalay combination index (CI) across the indicated cell lines. Normalized isobolograms depict CI scores over a range of concentrations. The coordinates of the CI scores are d1/Dx1 and d2/Dx2, where Dx1 is the concentration of drug 1 (selumetinib) that alone produces the fractional inhibition effect x, and Dx2 is the concentration of drug 2 (venetoclax) that alone produces the fractional inhibition effect x. The red line displayed is the line of additivity. Points below the line are synergistic and above the line are antagonistic. Cell viability was measured at 3 days of combination treatment using an ATP-based assay.

Figure 4:. FLT3 activating mutations increased ex vivo sensitivity to FLT3 inhibitors.

Primary leukemia samples from patients enrolled on the LEAP Consortium trial were tested ex vivo with FLT3 inhibitors. Samples with FLT3 mutations were more sensitive to crenolanib (A), gilteritinib (B), midostaurin (C), and quizartinib (D), compared to FLT3 wildtype samples. Shown is area under the curve (AUC) for individual samples, mean +/− SD. *P<0.05 and **P<0.005, using unpaired t test. The first trial patient had AML with a FLT3 p.A680V mutation, which has been described but had unknown functional significance. (E) Crystal structure of auto-inhibited FLT3 kinase domain (grey) and JM domain (pink). Area surrounding residue 680 is highlighted. (F) Zoom in on residues A680 (blue) and (G) A680V (yellow) from active-like models of FLT3 shown relative to the Y599/E604 H-bond observed in the auto-inhibited crystal structure. (H) Prevalence of H-bonds between: Y599/E604 (left) and Y599/water molecules (right) in molecular dynamics simulations of inactive-like models. (I) Proposed mechanism of activation of the A680V mutation. J) Ba/F3 cells expressing empty vector, FLT3 WT or FLT3 p.A680V were grown without IL-3, and cells counted using trypan blue exclusion. Shown is the average number of cells ± SD of 3 replicates. K) Ba/F3 cells expressing FLT3-ITD or FLT3 p.A680V were tested with a range of gilteritinib concentrations and viability evaluated at day 2 by an ATP-based assay as a percentage of cells relative to DMSO control. Shown are the mean ± SD of 4 replicates. L) Western immunoblotting showing inhibition of FLT3 phosphorylation in response to gilteritinib treatment.