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.

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.

Prognostic impact of cooccurring mutations in FLT3-ITD pediatric acute myeloid leukemia.

ABSTRACT: We sought to define the cooccurring mutational profile of FLT3-ITD-positive (ITDpos) acute myeloid leukemia (AML) in pediatric and young adult patients and to define the prognostic impact of cooperating mutations. We identified 464 patients with FLT3-ITD mutations treated on Children's Oncology Group trials with available sequencing and outcome data. Overall survival, event-free survival (EFS), and relapse risk were determined according to the presence of cooccurring risk stratifying mutations. Among the cohort, 79% of patients had cooccurring alterations across 239 different genes that were altered through mutations or fusions. Evaluation of the prognostic impact of the cooccurring mutations demonstrated that patients with ITDpos AML experienced significantly different outcomes according to the cooccurring mutational profile. Patients with ITDpos AML harboring a cooccurring favorable-risk mutation of NPM1, CEBPA, t(8;21), or inv(16) experienced a 5-year EFS of 64%, which was significantly superior to of 22.2% for patients with ITDpos AML and poor-risk mutations of WT1, UBTF, or NUP98::NSD1 as well to 40.9% for those who lacked either favorable-risk or poor-risk mutation (ITDpos intermediate; P < .001 for both). Multivariable analysis demonstrated that cooccurring mutations had significant prognostic impact, whereas allelic ratio had no impact. Therapy intensification, specifically consolidation transplant in remission, resulted in significant improvements in survival for ITDpos AML. However, patients with ITDpos/NUP98::NSD1 continued to have poor outcomes with intensified therapy, including sorafenib. Cooccurring mutational profile in ITDpos AML has significant prognostic impacts and is critical to determining risk stratification and therapeutic allocation. These clinical trials were registered at www.clinicaltrials.gov as NCT00002798, NCT00070174, NCT00372593, and NCT01371981.

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.

Characteristics and prognostic impact of IDH mutations in AML: a COG, SWOG, and ECOG analysis.

Somatic mutations in isocitrate dehydrogenase (IDH) genes occur frequently in adult acute myeloid leukemia (AML) and less commonly in pediatric AML. The objective of this study was to describe the prevalence, mutational profile, and prognostic significance of IDH mutations in AML across age. Our cohort included 3141 patients aged between <1 month and 88 years treated on Children's Cancer Group/Children's Oncology Group (n = 1872), Southwest Oncology Group (n = 359), Eastern Cooperative Oncology Group (n = 397) trials, and in Beat AML (n = 333) and The Cancer Genome Atlas (n = 180) genomic characterization cohorts. We retrospectively analyzed patients in 4 age groups (age range, n): pediatric (0-17, 1744), adolescent/young adult (18-39, 444), intermediate-age (40-59, 640), older (≥60, 309). IDH mutations (IDHmut) were identified in 9.2% of the total cohort (n = 288; IDH1 [n = 123, 42.7%]; IDH2 [n = 165, 57.3%]) and were strongly correlated with increased age: 3.4% pediatric vs 21% older, P < .001. Outcomes were similar in IDHmut and IDH-wildtype (IDHWT) AML (event-free survival [EFS]: 35.6% vs 40.0%, P = .368; overall survival [OS]: 50.3% vs 55.4%, P = .196). IDH mutations frequently occurred with NPM1 (47.2%), DNMT3A (29.3%), and FLT3-internal tandem duplication (ITD) (22.4%) mutations. Patients with IDHmut AML with NPM1 mutation (IDHmut/NPM1mut) had significantly improved survival compared with the poor outcomes experienced by patients without (IDHmut/NPM1WT) (EFS: 55.1% vs 17.0%, P < .001; OS: 66.5% vs 35.2%, P < .001). DNTM3A or FLT3-ITD mutations in otherwise favorable IDHmut/NPM1mut AML led to inferior outcomes. Age group analysis demonstrated that IDH mutations did not abrogate the favorable prognostic impact of NPM1mut in patients aged <60 years; older patients had poor outcomes regardless of NPM1 status. These trials were registered at www.clinicaltrials.gov as #NCT00070174, #NCT00372593, #NCT01371981, #NCT00049517, and #NCT00085709.

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.

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).

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.

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.

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.

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.

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.

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.

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.