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J.P. Loftus et al.
ferase 2A (KMT2A, formerly mixed lineage leukemia [MLL]; located at chromosome 11q23) are at higher risk of relapse and have inferior overall survival.6-8 KMT2A rearrange- ments occur in approximately 10% of childhood and adult B-ALL cases with highest frequency (75%) in infants diag- nosed with leukemia at <365 days old.8,9 Children with KMT2A-rearranged (KMT2A-R) ALL have a poor progno- sis with 5-year event-free survival (EFS) of 20-50% in infants9-12 and approximately 58% in older children.13 Age <6 months at diagnosis, hyperleukocytosis with white blood cell count >300x109/L, and poor response to pred- nisone prophase chemotherapy have been associated with worst clinical outcomes and dismal long-term survival amongst infants with KMT2A-R ALL.10,11 Adults with KMT2A-R ALL have similarly poor outcomes with <50% 5-year EFS.14
Wild-type KMT2A is required for normal hematopoiesis and post-natal hematopoietic cell maintenance.15 Disruption of KMT2A via chromosomal translocation in acute lymphoid and myeloid leukemias was first described nearly three decades ago.16,17 In ALL, these translocations result in fusion of KMT2A to one of >100 currently known translocation partner genes, leading to production of fusion proteins which disrupt normal regu- lation of gene expression by wild-type KMT2A.18-20 Recruitment of the super elongation complex (SEC) and the H3K79 histone methyltransferase DOT1L by the fusion proteins consequently leads to new fusion-depen- dent functions of KMT2A.21 While numerous partner genes have been reported, five translocations account for the majority of KMT2A rearrangements in ALL across the age spectrum. These include t(4;11)(q21;q23) with KMT2A-AFF1 fusion (60%), t(11;19)(q23;p13.3) with KMT2A-MLLT1 fusion (18%), t(9;11)(p21;q23) with KMT2A-MLLT3 fusion (12%), t(10;11)(p12;q23) with KMT2A-MLLT10 fusion (3%), and t(6;11)(q27;q23) with KMT2A-MLLT4 fusion (1%).8,22-24
Preclinical studies of murine models and primary patient specimens demonstrate that KMT2A-R ALL cells harbor gene expression signatures with distinct arrest in B-cell development at the pro-B and pre-B cell stages. Recent publications have reported a strong link between increased expression of the HOX cluster of transcription factor genes (particularly HOXA9) and its co-factor MEIS1 in accelerating KMT2A-R leukemia development via upregulation of spleen tyrosine kinase (SYK),21,25 as well as constitutive activation of SYK signaling in several B-ALL subtypes.2,26 There specific mechanisms by which KMT2A translocations contribute to SYK signaling in B-ALL and their role in leukemogenesis and maintenance have not been completely characterized.
SYK is expressed in hematopoietic cells and involved in multiple signal transduction pathways downstream of the B-cell receptor (BCR). SYK is autophosphorylated and activated when its two tandem Src homology 2 (SH2) domains bind to immunoreceptor tyrosine based activa- tion motifs (ITAM).27 This binding then initiates down- stream signal transduction via activation of effector mole- cules, including phospholipase C gamma (PLCγ), B-cell linker protein (BLNK), phosphatidylinositol 3 kinase (PI3K), and mitogen activated protein kinase (MAPK) that converge to activate multiple downstream signaling path- ways involved in B-cell malignancies. This makes SYK an attractive potential therapeutic target.28,29 In vitro and in vivo activity of SYK inhibition in preclinical B-ALL models has
been previously established26,30,31 and several SYK inhibitors (e.g., entospletinib, fostamatinib) are under evaluation in patients with relapsed/refractory solid tumors, hematologic malignancies, or autoimmune dis- eases.
Entospletinib (ENTO, formerly GS-9973)32 is a potent and highly selective SYK inhibitor under current clinical investigation in adults with relapsed acute leukemias (clin- icaltrials.gov identifiers: NCT02343939 and NCT02404220). Interim analysis of a phase Ib/II study of ENTO and chemotherapy showed complete responses in two patients with relapsed KMT2A-R acute myeloid leukemia (AML) treated with ENTO monotherapy for 14 days, sug- gesting potential for particular clinical activity in KMT2A-rearranged leukemias.33 Translating the efficacy of SYK inhibition with ENTO and depth of response in combination with standard-of-care chemotherapy agents warrants further investigation at a molecular level. In the current study, we assessed the therapeutic potential of ENTO monotherapy and in combination with chemother- apy or other kinase inhibitors in preclinical infant KMT2A-R and non-KMT2A-R ALL patient-derived xenograft (PDX) models to delineate the potential anti- leukemic utility of SYK inhibition in this high-risk child- hood leukemia subtype.
Methods
KMT2A-rearranged acute lymphoblastic leukemia patient specimens and xenotransplantation models
Viably cryopreserved leukemia cells from infants with de novo KMT2A-R (n=4; corresponding relapse, n=3) and non-KMT2A-R ALL (n=3) enrolled on the Children’s Oncology Group (COG) trial AALL0631 were obtained via informed consent as previously described.34 Additional specimens from an infant with relapsed KMT2A-R (n=1; ALL3103) and an adult with de novo KMT2A-R ALL (n=1; ALL3113) were obtained from the University of California, San Francisco and University of Pennsylvania leukemia biorepositories under approved institutional research protocols after informed consent in accordance with the Declaration of Helsinki (Table 1). PDX models were established in NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice via an Institutional Animal Use and Care Committee-approved protocol at the Children’s Hospital of Philadelphia as described with serial transplantation of human ALL cells into secondary or tertiary recipients for experi- mental studies.35-38 Additional established non-KMT2A-R ALL PDX models (primarily of the Philadelphia chromosome-like [Ph- like] subtype)15,37-39 (Online Supplementary Table S1) were used as negative controls.
Kinase inhibitors and chemotherapy
The selective SYK inhibitor entospletinib (ENTO)32 was provid- ed as a dispersible powder for in vitro studies and in rodent chow formulation in 0.03%, 0.05%, and 0.07% concentrations for in vivo animal studies by Gilead Sciences Inc. (Foster City, CA, USA). Rodent chow concentrations were selected and optimized based upon PK levels achieved in ENTO-treated adult patients with acute leukemia (clinicaltrials.gov identifiers: NCT02404220 and NCT02343939).33 Vincristine and dexamethasone were purchased from the Children’s Hospital of Philadelphia investigational phar- macy (Philadelphia, PA, USA). The MEK inhibitor selumetinib, SYK inhibitor fostamatinib, and multi-kinase inhibitor dasatinib were purchased from Selleckchem (Houston, TX, USA) or LC Labs (Woburn, MA, USA). Cell viability and phosphoflow cytom-
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haematologica | 2021; 106(4)