CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

CEBPA-bZip mutations are associated with favorable prognosis in de novo AML: a report from the Children's Oncology Group.

Biallelic CEBPA mutations are associated with favorable outcomes in acute myeloid leukemia (AML). We evaluated the clinical and biologic implications of CEBPA-basic leucine zipper (CEBPA-bZip) mutations in children and young adults with newly diagnosed AML. CEBPA-bZip mutation status was determined in 2958 patients with AML enrolled on Children's Oncology Group trials (NCT00003790, NCT0007174, NCT00372593, NCT01379181). Next-generation sequencing (NGS) was performed in 1863 patients (107 with CEBPA mutations) to characterize the co-occurring mutations. CEBPA mutational status was correlated with disease characteristics and clinical outcomes. CEBPA-bZip mutations were identified in 160 (5.4%) of 2958 patients, with 132 (82.5%) harboring a second CEBPA mutation (CEBPA-double-mutated [CEBPA-dm]) and 28 (17.5%) had a single CEBPA-bZip only mutation. The clinical and laboratory features of the 2 CEBPA cohorts were very similar. Patients with CEBPA-dm and CEBPA-bZip experienced identical event-free survival (EFS) of 64% and similar overall survival (OS) of 81% and 89%, respectively (P = .259); this compared favorably to EFS of 46% and OS of 61% in patients with CEBPA-wild-type (CEBPA-WT) (both P < .001). Transcriptome analysis demonstrated similar expression profiles for patients with CEBPA-bZip and CEBPA-dm. Comprehensive NGS of patients with CEBPA mutations identified co-occurring CSF3R mutations in 13.1% of patients and GATA2 mutations in 21.5% of patients. Patients with dual CEBPA and CSF3R mutations had an EFS of 17% vs 63% for patients with CEBPA-mutant or CSF3R-WT (P < .001) with a corresponding relapse rate (RR) of 83% vs 22%, respectively (P < .001); GATA2 co-occurrence did not have an impact on outcome. CEBPA-bZip domain mutations are associated with favorable clinical outcomes, regardless of monoallelic or biallelic status. Co-occurring CSF3R and CEBPA mutations are associated with a high RR that nullifies the favorable prognostic impact of CEBPA mutations.

Functional consequence and therapeutic targeting of cryptic ALK fusions in monosomy 7 acute myeloid leukemia.

Acute myeloid leukemia (AML) patients have a wide array of cytogenetic and molecular aberrations, which can influence response to therapy. Monosomy 7 is a rare subset within pediatric AML (prevalence of <2%) that is highly associated with poor outcomes. Fusions involving the anaplastic tyrosine kinase (ALK) gene were exclusively identified in 14.3% of this high-risk cohort, while absent across all other AML. Given the dismal outcomes of monosomy 7, we evaluated the use of crizotinib, an FDA-approved tyrosine kinase inhibitor, used to treat patients with ALK fusions. Our findings suggest that crizotinib may serve as a novel therapy for these patients.

Novel CD33 antibodies unravel localization, biology and therapeutic implications of CD33 isoforms.

The aim of this study was to establish the therapeutic relevance of the CD33D2 isoform by developing novel antibodies targeting the IgC domain of CD33. Two novel IgC-targeting antibodies, HL2541 and 5C11-2, were developed, and CD33 isoforms were assessed using multiple assays in cells overexpressing either CD33FL or CD33D2 isoforms, unmodified acute myeloid leukemia (AML) cell lines and primary AML specimens representing different genotypes for the CD33 splicing single nucleotide polymorphism. CD33D2 was recognized on cells overexpressing CD33D2 and unmodified AML cell lines; however, minimal/no cell surface detection of CD33D2 was observed in primary AML specimens. Both isoforms were detected intracellularly using novel antibodies. Minimal cell surface expression of CD33D2 on primary AML/progenitor cells warrants further studies on anti-CD33D2 immunotherapeutics.

Long Noncoding RNA Expression Independently Predicts Outcome in Pediatric Acute Myeloid Leukemia.

PURPOSE: Optimized strategies for risk classification are essential to tailor therapy for patients with biologically distinctive disease. Risk classification in pediatric acute myeloid leukemia (pAML) relies on detection of translocations and gene mutations. Long noncoding RNA (lncRNA) transcripts have been shown to associate with and mediate malignant phenotypes in acute myeloid leukemia (AML) but have not been comprehensively evaluated in pAML. METHODS: To identify lncRNA transcripts associated with outcomes, we evaluated the annotated lncRNA landscape by transcript sequencing of 1,298 pediatric and 96 adult AML specimens. Upregulated lncRNAs identified in the pAML training set were used to establish a regularized Cox regression model of event-free survival (EFS), yielding a 37 lncRNA signature (lncScore). Discretized lncScores were correlated with initial and postinduction treatment outcomes using Cox proportional hazards models in validation sets. Predictive model performance was compared with standard stratification methods by concordance analysis. RESULTS: Training set cases with positive lncScores had 5-year EFS and overall survival rates of 26.7% and 42.7%, respectively, compared with 56.9% and 76.3% with negative lncScores (hazard ratio, 2.48 and 3.16; P < .001). Pediatric validation cohorts and an adult AML group yielded comparable results in magnitude and significance. lncScore remained independently prognostic in multivariable models, including key factors used in preinduction and postinduction risk stratification. Subgroup analysis suggested that lncScores provide additional outcome information in heterogeneous subgroups currently classified as indeterminate risk. Concordance analysis showed that lncScore adds to overall classification accuracy with at least comparable predictive performance to current stratification methods that rely on multiple assays. CONCLUSION: Inclusion of the lncScore enhances predictive power of traditional cytogenetic and mutation-defined stratification in pAML with potential, as a single assay, to replace these complex stratification schemes with comparable predictive accuracy.

Inflammatory bone marrow signaling in pediatric acute myeloid leukemia distinguishes patients with poor outcomes.

High levels of the inflammatory cytokine IL-6 in the bone marrow are associated with poor outcomes in pediatric acute myeloid leukemia (pAML), but its etiology remains unknown. Using RNA-seq data from pre-treatment bone marrows of 1489 children with pAML, we show that > 20% of patients have concurrent IL-6, IL-1, IFNα/ß, and TNFα signaling activity and poorer outcomes. Targeted sequencing of pre-treatment bone marrow samples from affected patients (n = 181) revealed 5 highly recurrent patterns of somatic mutation. Using differential expression analyses of the most common genomic subtypes (~60% of total), we identify high expression of multiple potential drivers of inflammation-related treatment resistance. Regardless of genomic subtype, we show that JAK1/2 inhibition reduces receptor-mediated inflammatory signaling by leukemic cells in-vitro. The large number of high-risk pAML genomic subtypes presents an obstacle to the development of mutation-specific therapies. Our findings suggest that therapies targeting inflammatory signaling may be effective across multiple genomic subtypes of pAML.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

A B-cell developmental gene regulatory network is activated in infant AML.

Infant Acute Myeloid Leukemia (AML) is a poorly-addressed, heterogeneous malignancy distinguished by surprisingly few mutations per patient but accompanied by myriad age-specific translocations. These characteristics make treatment of infant AML challenging. While infant AML is a relatively rare disease, it has enormous impact on families, and in terms of life-years-lost and life limiting morbidities. To better understand the mechanisms that drive infant AML, we performed integrative analyses of genome-wide mRNA, miRNA, and DNA-methylation data in diagnosis-stage patient samples. Here, we report the activation of an onco-fetal B-cell developmental gene regulatory network in infant AML. AML in infants is genomically distinct from AML in older children/adults in that it has more structural genomic aberrations and fewer mutations. Differential expression analysis of ~1500 pediatric AML samples revealed a large number of infant-specific genes, many of which are associated with B cell development and function. 18 of these genes form a well-studied B-cell gene regulatory network that includes the epigenetic regulators BRD4 and POU2AF1, and their onco-fetal targets LIN28B and IGF2BP3. All four genes are hypo-methylated in infant AML. Moreover, micro-RNA Let7a-2 is expressed in a mutually exclusive manner with its target and regulator LIN28B. These findings suggest infant AML may respond to bromodomain inhibitors and immune therapies targeting CD19, CD20, CD22, and CD79A.

Therapeutic Targeting of Mesothelin with Chimeric Antigen Receptor T Cells in Acute Myeloid Leukemia.

PURPOSE: We previously identified mesothelin (MSLN) as highly expressed in a significant fraction of acute myeloid leukemia (AML) but entirely silent in normal hematopoiesis, providing a promising antigen for immunotherapeutic targeting that avoids hematopoietic toxicity. Given that T cells genetically modified to express chimeric antigen receptors (CAR) are effective at eradicating relapsed/refractory acute lymphocytic leukemia, we developed MSLN-directed CAR T cells for preclinical evaluation in AML. EXPERIMENTAL DESIGN: The variable light (VL) and heavy (VH) sequences from the MSLN-targeting SS1P immunotoxin were used to construct the single-chain variable fragment of the standard CAR containing 41-BB costimulatory and CD3Zeta stimulatory domains. The preclinical efficacy of MSLN CAR T cells was evaluated against AML cell lines and patient samples expressing various levels of MSLN in vitro and in vivo. RESULTS: We demonstrate that MSLN is expressed on the cell surface of AML blasts and leukemic stem cell-enriched CD34+CD38- subset, but not on normal hematopoietic stem and progenitor cells (HSPC). We further establish that MSLN CAR T cells are highly effective in eliminating MSLN-positive AML cells in cell line- and patient-derived xenograft models. Importantly, MSLN CAR T cells can target and eradicate CD34+CD38- cells without impacting the viability of normal HSPCs. Finally, we show that CAR T-cell functionality can be improved by inhibition of the ADAM17 metalloprotease that promotes shedding of MSLN. CONCLUSIONS: These findings demonstrate that MSLN is a viable target for CAR T-cell therapy in AML and that inhibiting MSLN shedding is a promising approach to improve CAR T-cell efficacy.

Comprehensive Transcriptome Profiling of Cryptic CBFA2T3-GLIS2 Fusion-Positive AML Defines Novel Therapeutic Options: A COG and TARGET Pediatric AML Study.

PURPOSE: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. EXPERIMENTAL DESIGN: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N = 24) and without (N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. RESULTS: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFß, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. CONCLUSIONS: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.

Functional Properties of KIT Mutations Are Associated with Differential Clinical Outcomes and Response to Targeted Therapeutics in CBF Acute Myeloid Leukemia.

PURPOSE: KIT mutations (KIT +) are common in core binding factor (CBF) AML and have been associated with varying prognostic significance. We sought to define the functional and clinical significance of distinct KIT mutations in CBF pediatric AML. EXPERIMENTAL DESIGN: Following transfection of exon 17 (E17) and exon 8 (E8) mutations into HEK293 and Ba/F3 cells, KIT phosphorylation, cytokine-independent growth, and response to tyrosine kinase inhibitors (TKI) were evaluated. Clinical outcomes of patients treated on COG AAML0531 (NCT01407757), a phase III study of gemtuzumab ozogamicin (GO), were analyzed according to mutation status [KIT + vs. wild-type KIT (KIT -)] and mutation location (E8 vs. E17). RESULTS: KIT mutations were detected in 63 of 205 patients (31%); 22 (35%) involved only E8, 32 (51%) only E17, 6 (10%) both exons, and 3 (5%) alternative exons. Functional studies demonstrated that E17, but not E8, mutations result in aberrant KIT phosphorylation and growth. TKI exposure significantly affected growth of E17, but not E8, transfected cells. Patients with KIT + CBF AML had overall survival similar to those with KIT - (78% vs. 81%, P = 0.905) but higher relapse rates (RR = 43% vs. 21%; P = 0.005). E17 KIT + outcomes were inferior to KIT - patients [disease-free survival (DFS), 51% vs. 73%, P = 0.027; RR = 21% vs. 46%, P = 0.007)], although gemtuzumab ozogamicin abrogated this negative prognostic impact. E8 mutations lacked significant prognostic effect, and GO failed to significantly improve outcome. CONCLUSIONS: E17 mutations affect prognosis in CBF AML, as well as response to GO and TKIs; thus, clinical trials using both agents should be considered for KIT + patients.