Enhanced Efficacy of the SYK Inhibitor Entospletinib and Chemotherapy in Infant Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A -rearranged (R) B-cell acute lymphoblastic leukemia (B-ALL), who have extremely poor event-free survival (EFS). Addition of kinase inhibitors to chemotherapy has markedly improved outcomes for patients with some genetic subsets of B-ALL, such as BCR-ABL1 -R (Ph+) ALL. Unfortunately, addition of the FMS-like tyrosine kinase 3 (FLT3) inhibitor lestaurtinib did not improve 3-year EFS for infants with KMT2A -R and FLT3-overexpressing B-ALL in the Children's Oncology Group trial AALL0631 (Brown et al, SIOP 2016), indicating continued need for alternative therapeutic strategies. We recently observed constitutive phosphorylation (p) of spleen tyrosine kinase (SYK) at tyrosine 323 in infant ALL specimens (Figure 1A) and hypothesized that activated signaling is targetable with the selective SYK inhibitor entospletinib (ENTO). We further hypothesized that combining ENTO with chemotherapy would have superior activity in comparison to single-agent treatments.

Methods: To test these hypotheses, we established patient-derived xenograft (PDX) models of KMT2A -R (n=7) and non- KMT2A -R (n=4) infant ALL in NSG mice using diagnostic and relapse bone marrow specimens from patients treated on or as per the AALL0631 trial protocol. Total and phosphorylated (p) SYK and other phosphoprotein levels were assessed by Simple Western^TM analysis of splenic lysates from ALL-engrafted PDX mice to identify leukemias with constitutive SYK signaling activation. Infant ALL PDX models were then treated with control mouse chow or chow containing 0.07% ENTO ad libitum, 0.25 mg/kg vincristine (VCR) dosed intraperitoneally once weekly, or a combination of ENTO and VCR for up to 28 days to determine treatment efficacy. Cohorts of additional infant ALL PDX mice were treated for 72 hours to assess plasma levels and pharmacodynamic effects of ENTO administration.

Results: Constitutive SYK signaling was observed in several models of de novo KMT2A -R and non- KMT2A -R B-ALL. ENTO treatment of KMT2A -R ALL PDX models ALL3103 with t(9;11) and ALL135 with t(11;19) inhibited leukemic cell burden in vivo versus control animals by flow cytometric and immunohistochemical (IHC) analyses of murine spleens (representative data in Figure 1B). Importantly, combined ENTO and VCR treatment resulted in a greater reduction in ALL cell number, which was confirmed by human CD19+ IHC analysis of murine spleens and bone marrow. ENTO concentrations were maintained throughout study duration with terminal free fraction plasma levels of 61 and 230 nM (ALL3103) and 89 and 214 nM (ALL135) for ENTO and ENTO/VCR treatment arms, respectively. Evaluation of SYK pathway modulation in vivo demonstrated inhibition of pSYK^Y323 in human ALL cells within murine spleens that correlated with the level of ENTO exposure of treated mice (ALL3103 PDX; Spearman correlation of -0.63, p<0.05). No alterations in total SYK protein were observed. RNA sequencing of ALL3103 PDX tissues showed that ENTO treatment was associated with downregulation of MYC and of cell cycle and DNA damage gene expression signatures.

Conclusions: Constitutive activation of SYK signaling occurs in infant KMT2A -R B-ALL. Entospletinib treatment of infant B-ALL PDX models potently inhibited the SYK pathway and decreased leukemic cell burden in vivo . Superior anti-ALL activity was observed with combined ENTO and VCR compared to ENTO alone. Additional studies are ongoing to determine the activity of ENTO in non- KMT2A -R and other ALL PDX models.

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