Chromatin Profiles Are Prognostic of Clinical Response to Bortezomib-Containing Chemotherapy in Pediatric Acute Myeloid Leukemia: Results from the COG AAML1031 Trial.

The addition of the proteasome inhibitor bortezomib to standard chemotherapy did not improve survival in pediatric acute myeloid leukemia (AML) when all patients were analyzed as a group in the Children's Oncology Group phase 3 trial AAML1031 (NCT01371981). Proteasome inhibition influences the chromatin landscape and proteostasis, and we hypothesized that baseline proteomic analysis of histone- and chromatin-modifying enzymes (HMEs) would identify AML subgroups that benefitted from bortezomib addition. A proteomic profile of 483 patients treated with AAML1031 chemotherapy was generated using a reverse-phase protein array. A relatively high expression of 16 HME was associated with lower EFS and higher 3-year relapse risk after AML standard treatment compared to low expressions (52% vs. 29%, p = 0.005). The high-HME profile correlated with more transposase-accessible chromatin, as demonstrated via ATAC-sequencing, and the bortezomib addition improved the 3-year overall survival compared with standard therapy (62% vs. 75%, p = 0.033). These data suggest that there are pediatric AML populations that respond well to bortezomib-containing chemotherapy.

Valosin-containing protein (VCP/p97) is prognostically unfavorable in pediatric AML, and negatively correlates with unfolded protein response proteins IRE1 and GRP78: A report from the Children's Oncology Group.

PURPOSE: The endoplasmic reticulum (ER) is the major site of protein synthesis and folding in the cell. ER-associated degradation (ERAD) and unfolded protein response (UPR) are the main mechanisms of ER-mediated cell stress adaptation. Targeting the cell stress response is a promising therapeutic approach in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Protein expression levels of valosin-containing protein (VCP), a chief element of ERAD, were measured in peripheral blood samples from in 483 pediatric AML patients using reverse phase protein array methodology. Patients participated in the Children's Oncology Group AAML1031 phase 3 clinical trial that randomized patients to standard chemotherapy (cytarabine (Ara-C), daunorubicin, and etoposide [ADE]) versus ADE plus bortezomib (ADE+BTZ). RESULTS: Low-VCP expression was significantly associated with favorable 5-year overall survival (OS) rate compared to middle-high-VCP expression (81% versus 63%, p < 0.001), independent of additional bortezomib treatment. Multivariable Cox regression analysis identified VCP as independent predictor of clinical outcome. UPR proteins IRE1 and GRP78 had significant negative correlation with VCP. Five-year OS in patients characterized by low-VCP, moderately high-IRE1 and high-GRP78 improved after treatment with ADE+BTZ versus ADE (66% versus 88%, p = 0.026). CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest the potential of the protein VCP as biomarker in prognostication prediction in pediatric AML.

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Therapeutic targeting of PRAME with mTCRCAR T cells in acute myeloid leukemia.

Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A∗02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

Integrated stem cell signature and cytomolecular risk determination in pediatric acute myeloid leukemia.

Relapsed or refractory pediatric acute myeloid leukemia (AML) is associated with poor outcomes and relapse risk prediction approaches have not changed significantly in decades. To build a robust transcriptional risk prediction model for pediatric AML, we perform RNA-sequencing on 1503 primary diagnostic samples. While a 17 gene leukemia stem cell signature (LSC17) is predictive in our aggregated pediatric study population, LSC17 is no longer predictive within established cytogenetic and molecular (cytomolecular) risk groups. Therefore, we identify distinct LSC signatures on the basis of AML cytomolecular subtypes (LSC47) that were more predictive than LSC17. Based on these findings, we build a robust relapse prediction model within a training cohort and then validate it within independent cohorts. Here, we show that LSC47 increases the predictive power of conventional risk stratification and that applying biomarkers in a manner that is informed by cytomolecular profiling outperforms a uniform biomarker approach.

Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia.

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. SIGNIFICANCE: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.

Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia.

In an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN-DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Heat shock factor 1 (HSF1-pSer326) predicts response to bortezomib-containing chemotherapy in pediatric AML: a COG report.

Bortezomib (BTZ) was recently evaluated in a randomized phase 3 clinical trial by the Children's Oncology Group (COG) that compared standard chemotherapy (cytarabine, daunorubicin, and etoposide [ADE]) vs standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia (AML). Although the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefiting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. Total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) were measured in leukemic cells from 483 pediatric patients using reverse phase protein arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared with CD34+ nonmalignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs 67% for low-HSF1-pSer326 treated with ADEB (P = .019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and nonphosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs those with wild-type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients who benefit from BTZ-containing chemotherapy.

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.

Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia.

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.