Combinatorial efficacy of entospletinib and chemotherapy in patient-derived xenograft models of infant acute lymphoblastic leukemia

Abstract

Survival of infants with KMT2A-rearranged (R) acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2A-R (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-R [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-R ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS/ or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (p.

Trial registration: ClinicalTrials.gov NCT00557193.

Figures

Figure 1.

Constitutive SYK signaling occurs in infant acute lymphoblastic leukemia (ALL). Simple Western analysis of splenic lysates from human ALL patient-derived xenograft (PDX) models demonstrated high basal phosphorylated SYK (pSYK) levels in the majority of infant non-KMT2A-rearranged (R) (light blue) and KMT2A-R (dark blue) ALL specimens. pSYK levels were lower in most childhood non-KMT2A-R ALL specimens (red) and absent in splenic tissue from non-leukemia-injected NSG mice (gray). Total SYK levels were similar across all models. ALL PDX model names are specified above corresponding Simple Western data.

Figure 2.

Activity and dose optimization of entospletinib monotherapy in KMT2A-R acute lymphoblastic leukemia (ALL). (A) Viably cryopreserved harvested human KMT2A-R ALL cells from murine PDX spleens (model ALL3103 with KMT2A-MLLT3 fusion) demonstrated dose-dependent inhibition of colony formation in vitro in methylcellulose colony assays after ENTO treatment for 14 days. Samples were plated in triplicate in methylcellulose-based medium and grown in 10% leukocyteconditioned medium with 25% FBS and 2% BSA. Data are displayed as mean ± SEM. (B) ALL3103 PDX mice were treated with vehicle (control) or ENTO chow at the specified concentrations for 4 weeks. Human CD45+ CD19+ ALL flow cytometric analysis of murine blood at weekly time points and (C) spleens at study endpoint demonstrated significant inhibition of ALL proliferation with ENTO treatment (mean ± SEM). No difference in ALL burden was observed in 0.03% versus 0.07% ENTOtreated animals. (D) Terminal blood was collected from animals after 4 weeks of continuous ENTO chow consumption and evaluated for entospletinib levels. Data from individual animals are plotted as median interquartile range. ns: not significant by t-test. (E) Terminal spleens from individual mice were harvested, viably cryopreserved, lysed, and evaluated for levels of pSYK, SYK, cMYC, pERK and β-actin by Simple Western. *P<0.05, **P<0.01, ****P<0.0001 as compared to control chow-fed mice by ANOVA with Tukey’s post-test.

Figure 3.

In vitro activity of entospletinib in KMT2A-R acute lymphoblastic leukemia (ALL). Viably cryopreserved KMT2A-R ALL PDX cells were exposed in vitro to 0.1% DMSO (vehicle control) or increasing concentrations of entospletinib (200 nM, 500 nM, 1 uM) for 2 hours, then lysed and analyzed by Simple Western. Additional untreated (baseline) cells were lysed immediately following sample thaw. (A) Dose-dependent inhibition of the specified phosphoproteins was observed with ENTO in the ALL135MR PDX model (KMT2A-MLLT1, RAS wild-type), while (B) no treatment effect was seen in the 142MR PDX model (KMT2A-AFF1, NRAS-mutant).

Figure 4.

HOXA9 and MEIS1 expression signatures of KMT2A-R and non-KMT2A-R acute lymphoblastic leukemia (ALL) patient-derived xenograft (PDX) specimens. (A) Splenic PDX samples were analyzed for expression of mRNA for HOXA9 and MEIS1 by NanoString, with human bone marrow mononuclear cells (BMMC) and KG- 1 cell line as negative and positive controls, respectively. Increased MEIS1 and/or HOXA9 expression was seen in KMT2A-R ALL PDX models versus non-KMT2A-R (WT) models and generally clustered by genetic subtype. (B) Total and phosphorylated signal transduction proteins from murine splenic lysates were evaluated using Simple Western. Basal kinase signaling activation differed among KMT2A-R and non-KMT2A-R ALL samples and stratified by genetic subgroup (KMT2A-AFF1, KMT2A-MLLT1, KMT2A-MLLT3, and non-KMT2A-R). β-actin was used as a protein loading control.

Figure 5.

Entospletinib potently inhibits in vivo acute lymphoblastic leukemia (ALL) proliferation with enhanced efficacy in combination with chemotherapy. Animals engrafted with KMT2A-R (ALL3103, ALL135MR, ALL142MR, ALL150MD, ALL3113) or non-KMT2A-R (ALL132GD, ALL185GD, ALL83GD) ALL were treated with control chow, 0.05% ENTO chow, 0.1 mg/kg vincristine (VCR) IP weekly, or both ENTO and VCR. Human CD45+CD19+ ALL cells were quantified by flow cytometry in end-of-study murine spleens and peripheral blood. (A) Combined ENTO+VCR significantly inhibited leukemia proliferation with enhanced activity compared to ENTO and/or VCR monotherapies in KMT2A-R PDX models without RAS mutations. (B) Conversely, potent VCR effects were observed in KMT2A-R ALL PDX models with NRAS or KRAS mutations without additional activity of combination treatment. (C) A KMT2A-R RAS wild-type ALL PDX model was sensitive to ENTO and not to VCR. (D) No treatment effects of ENTO or VCR were observed in a non-KMT2A-R KRAS-mutant ALL PDX model, while single-agent activity of VCR and/or ENTO and enhanced effects of combination treatment were detected in (E) non-KMT2A-R RAS wild-type PDX control models with other ALL-associated translocations. Data were analyzed by one-way ANOVA with Tukey’s post-test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6.

Superior preclinical activity of entospletinib and selumetinib in KMT2A-R acute lymphoblastic leukemia (ALL) patient-derived xenograft (PDX) models. (A) PDX models 142MR (KMT2A-AFF1, NRAS-mutant) and (B) ALL3113 (KMT2A-AFF1, RAS wild-type) were treated with vehicle control chow, 0.05% ENTO chow, 100 mg/kg selumetinib (SEL) via oral gavage twice daily 5 days/week, or both ENTO and SEL for 2 or 4 weeks. Human CD45+ CD19+ ALL cells were quantified by flow cytometry in peripheral blood and end-study murine spleens. Enhanced anti-leukemia efficacy was observed in both models with combined ENTO and SEL treatment versus ENTO or SEL alone, as measured by one-way ANOVA with post Tukey’s post-test for multiple comparisons. *P<0.05, **P<0.01, ****P<0.0001. (C) Ex vivo phosphoflow cytometry analysis of gated human CD19+ CD45+ ALL cells in end-study murine spleens after 2 weeks (ALL142MR) or 4 weeks (ALL3113) of ENTO and/or SEL treatment demonstrate inhibition of pSYK, pERK, and/or pS6 versus vehicle control (gray). ns: not significant, *P<0.05, **P<0.01 by one-way ANOVA with post Tukey’s post-test for multiple comparisons.