Proteomics: a new era in pediatric acute myeloid leukemia research

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Clinical relevance of proteomic profiling in de novo pediatric acute myeloid leukemia: a Children’s Oncology Group study

Pediatric acute myeloid leukemia (AML) remains a fatal disease for at least 30% of patients, stressing the need for improved therapies and better risk stratification. As proteins are the unifying feature of (epi)genetic and environmental alterations, and are often targeted by novel chemotherapeutic agents, we studied the proteomic landscape of pediatric AML. Protein expression and activation levels were measured in 500 bulk leukemic patient samples and 30 control CD34+ samples, using the reverse phase protein arrays with 296 strictly validated antibodies. The multi-step “MetaGalaxy” analysis methodology was applied and identified nine protein expression signatures (PrSIG), based on strong recurrent protein expression patterns. PrSIGs were associated with cytogenetics and mutational state, and with both favorable or unfavorable prognosis. Analysis based on treatment (i.e., ADE vs. ADE plus bortezomib (ADEB)) identified three PrSIGs that did better with ADEB vs. ADE. When PrSIGs were studied in the context of genetic subgroups, PrSIGs were independently prognostic after multivariate analysis, suggesting a potential value for proteomics in combination with current classification systems. Proteins with universally increased (n=7) or decreased (n=17) expression were observed across PrSIGs. Expression of certain proteins significantly differentially expressed from normal could be identified, forming a hypothetical platform for personalized medicine.

Regulatory Networks of Prognostic mRNAs in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) in children refers to a malignant tumor caused by the abnormal proliferation of immature myeloid cells in the bone marrow and peripheral blood. The prognosis of patients with pediatric acute myeloid leukemia (AML) remains poor, highlighting the need for improved targeted therapy. The expression data of lncRNAs, mRNAs, and miRNAs and survival information of pediatric AML patients were collected from The Cancer Genome Atlas (TCGA) database. Cox regression analysis was used to screen the lncRNAs, mRNAs, and miRNAs that significantly affect the overall survival (OS) of patients as OS-related genes (included lncRNAs, mRNAs, and miRNAs). Enrichment analysis and protein-protein interaction (PPI) network construction were performed for the OS-related mRNAs. We further established a ceRNAs regulatory network. In addition, the potential prognostic role of genes was further evaluated by risk score. We have identified 5275 lncRNAs, 176 miRNAs, and 6221 mRNAs that significantly affect the prognosis of pediatric AML patients. It is worth noting that OS-related mRNAs are mainly involved in ribosome, RNA transport, and spliceosome. We identified the top 10 most connected mRNAs in the PPI network as important mRNAs and constructed a ceRNAs regulatory network (including NCBP2, RPLP0, UBC, RPS2, and RPS9). The risk score and nomogram results suggest that NCBP2 may be a risk factor for pediatric AML, while RPLP0, UBC, RPS2, and RPS9 may be protective factors. Our results construct 5 gene signals as new prognostic indicators for predicting the survival of pediatric AML patients. Our research has demonstrated the ceRNAs regulatory network may become a new target for pediatric AML treatment.

Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

Single Cell Sequencing of Pediatric Acute Myeloid Leukemia Reveals Clonal Evolution to Relapse on Combination Chemotherapy with Sorafenib

Introduction: Relapse of pediatric acute myeloid leukemia (AML) remains a leading cause of childhood cancer mortality, and leukemias with activation of the Fms-like tyrosine kinase 3 (FLT3) are particularly susceptible to relapsed disease. Risk-directed therapy to prevent relapse is based both on genetic changes known to drive drug resistance, and measurable residual disease (MRD) at the end of induction therapy (EOI). In adult AML, resistance to type II FLT3-inhibitors, like sorafenib, is primarily driven by on-target FLT3 kinase domain (KD) mutations. However, the resistance mechanisms for pediatric leukemias, which are treated on combination therapies, have not been fully elucidated. MRD is considered the among the most predictive markers of future relapsed disease. It has been assumed that the major clone at the time of MRD assessment will predict the majority clone at relapse. However, this assumption has not been proven. The definition of the most specific genetic and MRD markers of relapse are essential to prognosticate and personalize therapy to prevent relapsed disease. Methods: We performed single cell sequencing (SCS) with a high-throughput DNA sequencing platform, Mission Bio Tapestri, on bone marrow or peripheral blood samples from 24 samples from 8 pediatric patients treated on COG AAML1031 with serial samples from diagnosis, EOI, and relapse. Results: We analyzed a total of 94,833 cells from 8 pediatric patients (median cells per patient 12,428) all treated on AAML1031. SCS revealed a sensitive and specific description of clonal evolution on the combination of sorafenib with cytotoxic chemotherapy. The FLT3 internal tandem duplication (ITD) was controlled by the therapy in only half of the patients. In five of the patients, the FLT3-ITD was present in multiple clones. The FLT3-ITD co-mutated with additional mutations (NRAS, SH2B3, WT1, TET2, or NPM1) in half of the patients. However, the presence of a co-mutation did not necessarily correlate with whether or not the ITD-containing clone persisted at the time of relapse. Of the leukemias whose relapse was not driven by FLT3, the most likely mutational driver of resistance was NRAS. Notably, however, despite the fact that FLT3 KD mutations make up the bulk of mutational resistance to type II FLT3i such as sorafenib in adult patients, there were no on-target FLT3 mutations found in any of these pediatric patients. Further, SCS allows for an unprecedented depth of analysis of the genetic complexity of pediatric AML. Phylogenic analysis revealed that the same mutations may arise independently in different cells (NPM1 W288fs, NRAS G60E). Additionally, the same gene may be mutated twice within the same cell (WT1, TET2). These data, consistent with our prior work, suggest that some leukemias may have a predilection to mutations within specific loci. Finally, although there is a standing assumption that the dominant MRD population will proliferate into relapsed disease, in 3/8 patients, the dominant MRD clone did not predict the dominant relapse clone. Conclusions: SCS allows for direct measurement of clonal hierarchy and evolution, phylogeny, co-mutational status, and zygosity, which can only be inferred through traditional bulk NGS. The mutational mechanisms of resistance seen in adult leukemias treated with sorafenib monotherapy are not necessarily relevant to the pediatric population; rather than on-target FLT3 mutations, off target mutations including NRAS are found. This corroborates prior findings that off-target RAS pathway mutations may drive resistance to FLT3i. Non-RAS off-target mutations found in this cohort do not necessarily predict sorafenib resistance, so may be passenger mutations. The lack of consistent resistance mutations suggests that other mechanisms of resistance such as epigenetic modifications may also drive resistance to combination chemotherapy with FLT3i in pediatric leukemia. Further, SCS exposes more genetic complexity in pediatric AML than has previously been appreciated: the same mutation may independently arise in more than one cell or the same cell may have multiple mutations within the same gene. Finally, the sensitivity of SCS reveals that the major clone at the time of MRD assessment is not necessarily the major clone at relapse. This suggests a benefit of more frequent MRD monitoring to track clonal evolution in real time. Disclosures Smith: Daiichi Sankyo: Consultancy; Revolutions Medicine: Research Funding; AbbVie: Research Funding; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.