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SYK inhibition for infant ALL
Table 1. Molecular and cytogenetic characteristics of acute lymphoblastic leukemia (ALL) patient-derived xenograft (PDX) models.
ALL PDX model COG USI
ALL185GD PAVVRD
ALL83GD PAUFHC
ALL132GD PAUXSA ALL150MD PAVEDG
KMT2A status wild-type
wild-type
wild-type
KMT2A-AFF1 KMT2A-AFF1 KMT2A-AFF1
KMT2A-AFF1
KMT2A-MLLT3 KMT2A-MLLT1 KMT2A-MLLT1 KMT2A-MLLT1 KMT2A-MLLT1
Translocation
P2RY8-CRLF2, PAX5-AUTS2 P2RY8-CRLF2, PAX5-C20orf112 t(1;19) (q23;p13) with TCF3-PBX1 t(4;11) (q21;q23) t(4;11) (q21;q23) t(4;11) (q21;q23)
t(4;11) (q21;q23)
t(9;11) (p21;q23) t(11;19) (q23;p13.3) t(11;19) (q23;p13.3) t(11;19) (q23;p13.3) t(11;19) (q23;p13.3)
Disease status
De novo
De novo
De novo
De novo De novo Relapse
De novo
Relapse
De novo
Relapse
De novo
Relapse
Other genetic alterations
JAK2 mut, CDKN2A/B del JAK2 del, CDKN2A/B del, RTEL del
KRAS mut, WHSC1 mut, gain CCND3, MYB, ESR1 KRAS mut
NRAS mut
NRAS mut, IKZF1 del, cnLOH of chr22 JAK2 mut, TP53 17p del, IKZF1 7p del None identified None identified None identified None identified Partial 10q del, including PTEN
ALL142MD
ALL142MR
ALL3113MR
ALL3103MR
ALL135MD
ALL135MR
ALL26MD PASHFM ALL26MR PASHFM
PAVBRV
PAVBRV
n/a
n/a PAUYJT PAUYJT
COG USI: Children’s Oncology Group unique specific identifier; cnLOH: copy-neutral loss of heterozygosity; del: deletion; mut: mutation; n/a: not available.
etry signaling analyses of human B-ALL cell lines and PDX model cells treated with vehicle, kinase inhibitors, or chemotherapy (in vitro or in vivo) are detailed in the Online Supplementary Methods with data shown in Online Supplementary Figures S1-S6.
In vivo drug testing in patient-derived xenograft models
Animal studies were conducted under a CHOP Institutional Animal Use and Care Committee (IACUC)-approved protocol in accordance with the Panel on Euthanasia of the American Veterinary Medical Association’s guidelines. After flow cytometric (FC) confirmation of ≥1% CD45+ CD19+ human ALL (fluo- rochrome-conjugated antibodies from EBioscience) in murine peripheral blood, engrafted ALL PDX models were randomized to treatment with vehicle, ENTO chow orally ad libitum, vincristine 0.1 mg/kg intraperitoneally (IP) weekly, or both ENTO and vin- cristine for 72 hours (pharmacokinetic [PK] and pharmacodynam- ics [PD] studies) or up to 28 days (treatment efficacy studies) as described.37,38 Vincristine dosing was previously optimized in ALL cell line and PDX models (not shown). Additional studies in some ALL PDX models assessed selumetinib 100 mg/kg administered orally twice daily40 5 days/week as (ALL135MR and ALL3113) or dexamethasone 1 mg/kg PO once daily 5 days/week (ALL3113, ALL83GD) as monotherapy or in combination with ENTO. Further details about in vivo drug testing in ALL PDX models and conduction of all other experimental studies are included in the Online Supplementary Methods.
Results
Characterization of constitutive SYK pathway activation in infant KMT2A-R acute lymphoblastic leukemia patient-derived xenograft models
Constitutive SYK pathway activation was detected across a genetic spectrum of infant ALL and some non- infant Philadelphia chromosome-like (Ph-like) ALL control specimens using harvested murine spleens from well- engrafted PDX models (Table 1). Assessment of phospho-
rylated and total SYK levels revealed that expression of high basal phosphorylated SYK (pSYK) was seen in the majority of infant non-KMT2A-R and KMT2A-R ALL specimens (Figure 1, left). pSYK levels were also elevated in some Ph-like ALL specimens and absent in splenic tis- sue from non-leukemia-injected NSG mice (Figure 1, right). Total SYK expression was relatively consistent across all models. The observed constitutive basal pSYK levels, coupled with a previously suggested role of upreg- ulated SYK as a driver in AML models with high HOXA9 and MEIS1 expression,25 and early reports of clinical responses in adults with relapsed KMT2A-R leukemias treated with entospletinib42,43 led us to investigate the role of SYK signaling and therapeutic potential of ENTO specifically in infant KMT2A-R ALL PDX models.
Entospletinib decreases leukemic burden and inhibits kinase signaling in KMT2A-R acute lymphoblastic leukemia
SYK plays a pivotal role upstream of several key leukemia-associated signaling pathways,26,29 including RAS/MAPK, PI3K/AKT/mTOR, and JAK/STAT. SYK inhi- bition by ENTO has the potential to impact multiple sig- nal transduction pathways in ALL (Visual Abstract), leading to potential anti-leukemic efficacy. Given our initial demonstration of constitutive SYK and other signaling pathway activation in infant ALL specimens by Simple Western, we first assessed leukemia cell growth inhibitory effects of ENTO in vitro using methylcellulose colony assays. Viably cryopreserved harvested KMT2A-R PDX ALL cells (model ALL3103 with KMT2A-MLLT3 fusion) were grown under anchorage-independent (non-adherent) conditions in serum-free methylcellulose and treated with a clinically-relevant dose range of ENTO for 14 days (Figure 2A). ENTO maximally inhibited colony formation (89% inhibition; P<0.0001 by t-test), suggesting that SYK plays a central role upstream of signaling pathways essen- tial to proliferation and survival.
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