scholarly journals Higher Stability of Mutant IDH1/2 mRNA As Compared to Wild-Type mRNA in Patients with Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2730-2730
Author(s):  
Maher Albitar ◽  
Marina Y Konopleva ◽  
Ivan De Dios ◽  
Jeffrey Justin Estella ◽  
Spiraggelos Antzoulatos ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Research Funding ◽  
Variant Allele ◽  
Wild Type ◽  
Sequencing Data ◽  
Variant Allele Frequency ◽  
Advisory Committees ◽  
Dna And Rna ◽  
Equity Ownership ◽  
Mutant Idh1

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are homodimeric enzymes that play an important role in cellular metabolism, epigenetic regulation, and DNA repair. Early studies suggested that mutations in IDH1/2 were loss of function mutations associated with a tumor suppressor function. However, biallelic mutations are extremely rare, and studies demonstrate that mutant IDH1/2 enzymes are responsible for NADPH-dependent reduction of αKG to the oncometabolite d-2-hydroxyglutarate (D2HG), suggesting an oncoprotein. Cellular RNA levels are tightly regulated by very complex cellular processes, and the regulation of mutant mRNA in cancer cells is rarely studied. We explored the effects of IDH1/2 mutations on mRNA levels in patients with Acute myeloid leukemia (AML). Using next generation sequencing (NGS) and variant allele frequency (VAF) of mutant RNA, we compared relative mutant mRNA or variant allele frequency (RNA-VAF) with variant allele frequency of mutant DNA (DNA-VAF) in the same samples from patients with AML. Methods: RNA and DNA were extracted from 48 bone marrow and peripheral blood samples from patients with confirmed AML, including 12 patients with IDH1 mutations, 2 with IDH2 mutation and 34 samples from AML without IDH1/2 mutations. Samples were collected pretherapy as well as while on therapy. We sequenced the DNA using 177 gene panel and the RNA using 1408 gene panel. The DNA sequencing is based on Single Primer Extension (SPE) library preparation with unique molecular identifier (UMI) (Qiagen, Germantown, MD). Average coverage of DNA sequencing was >1000X. The RNA sequencing is based on hybrid capture and the number of reads ranged from 5 to 10 million. Sequencing data of DNA is analyzed using the DRAGEN Platform. Sequence duplicates were removed before calculating VAF. The RNA sequencing data is analyzed using Illumina basespace. RNA VAF is calculated also after removing duplicates using Isaac variant caller. Only mutations detected by both DNA and RNA variant callers are compared. Results: A total of 176 mutations were detected using the DNA panel and 122 mutations using the RNA panel. Some mutations were called by RNA variant caller, but not by DNA variant caller and vice versa. All mutations detected in IDH1 and IDH2 were detected in both DNA and RNA. When the IDH1/2 mutations are considered (#14), the VAF in RNA (median: 41%, range: 13%-74%) was significant higher (P=0.006, Wilcoxon matched pairs test ) as compared with DNA (median:28%, range: 13%-74%). The VAF of the other 31 mutations that were detected in both DNA and RNA varied dependent on the gene. ASXL1, DNMT3A, RUNX1, PTPN11, SRSF2, STAG2 and U2AF1 mutations showed no significant difference between DNA and RNA in VAF (P=0.71). Although the number is small, mutations in NRAS and NPM1 showed significantly higher VAF in RNA as compared with with that of DNA (P=0.008). Conclusion: This data suggests that, in general, stability of mutant RNA varies between genes and between the mutations in the same gene. Mutant IDH1/2 RNA is significantly more stable in myeloid leukemic cells a compared with the wild-type mRNA. Most likely this reflects increased levels of mutant IDH1/2 as compared with wild-type IDH1/2, confirming that IDH1/2 is oncoprotein and may explain the efficacy of therapeutic inhibition of IDH1/2 in treating cancers. Furthermore this suggests that mRNA testing might be more sensitive in monitoring minimal residual disease in patients with IDH1/2 mutations. Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Konopleva:Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Loghavi:GLG Consultants: Consultancy; AlphaSights: Consultancy; MDACC: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy. Kantarjian:Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Cyclacel: Research Funding; Novartis: Research Funding; Astex: Research Funding; Takeda: Honoraria; Agios: Honoraria, Research Funding; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding. DiNardo:medimmune: Honoraria; agios: Consultancy, Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; jazz: Honoraria; abbvie: Consultancy, Honoraria; celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; syros: Honoraria.

Higher Stability of Mutant mRNA As Compared to Wild-Type mRNA in Diffuse Large B-Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1499-1499
Author(s):  
Maher Albitar ◽  
Zijun Yidan Xu-Monette ◽  
Wanlong Ma ◽  
Yingjun Wang ◽  
Deng Manman ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Rna Sequencing ◽  
Allele Frequency ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Variant Allele ◽  
Mrna Levels ◽  
Variant Allele Frequency ◽  
Advisory Committees ◽  
Dna And Rna

Cellular RNA levels are tightly regulated by very complex nuclear and cytoplasmic processes. The regulation of mutant mRNA in cancer cells is rarely studied. We explored the effects of mutations on mRNA levels in patients with diffuse large B-cell lymphoma (DLBCL). Using next generation sequencing (NGS) and variant allele frequency (VAF) of mutant RNA, we compared relative mutant mRNA or variant allele frequency (RNA-VAF) with variant allele frequency of mutant DNA (DNA-VAF) in the same samples from patients with DLBCL. Methods: RNA and DNA were extracted from 427 FFPE samples from patients with DLBCL. We sequenced the DNA using 177 gene panel and the RNA using 1408 gene panel. The DNA sequencing is based on Single Primer Extension (SPE) library preparation with unique molecular identifier (UMI) (Qiagen, Germantown, MD). The RNA sequencing is based on hybrid capture. Sequencing data of DNA is analyzed using the DRAGEN Platform. Sequence duplicates were removed before calculating VAF. The RNA sequencing data is analyzed using Illumina basespace. RNA VAF is calculated also after removing duplicates using Isaac variant caller. Only mutations detected by both DNA and RNA variant callers are compared. Results: A total of 1770 mutations were detected using the DNA panel and 2207 mutations were detected using the larger RNA sequencing panel. We focused on the most commonly mutated genes that included in both DNA and RNA panels and compared the VAF of the same mutations between DNA and RNA. The selected genes are: KMT2D, NOTCH2, CARD11, MYC, MYD88, EZH2, TP53, CD79B, BCL2, and TET2. The overall VAF in the RNA was significantly higher (P<0.00001) (median:43.9%, minimum: 6%, maximum: 100%) as compared with that of the DNA (median: 28.8%, minimum: 3.5%, maximum: 95%). When each gene is considered individual, all genes showed significantly higher VAF in RNA as compared with DNA. As expected some mutations were detected in DNA, but not in in RNA and vice versa. However, the number of mutations detected in these 10 genes using DNA sequencing was significantly (P= 0.0001) higher (#658) as compared with mutations detected in RNA (#471). Most of the missed mutations by RNA were termination mutations. The most striking RNA-missed mutations were in NOTCH2. The DNA testing showed 81 mutations, while the RNA testing listed only 19 mutations. Almost all NOTCH2 mutations missed by RNA sequencing wer Pro6ArgfsTer27, which leads to early termination of mRNA (loss of function). When we looked at overall NOTCH2 mRNA levels, NOTCH2 mRNA was significantly higher (P=0.002, Kruskal-Wallis ANOVA) in samples with NOTCH2 mutation detected in both DNA and RNA as compared with mutations detected in DNA only. The NOTCH2 mRNA levels were also lower in samples with mutations detected in DNA only (P=0.046) as compared with wild-type NOTCH2. Conclusion: This data suggests that stability of mutant mRNA is significantly higher for most mutations and most genes. However, there are exceptions, especially when the mutations are termination at early amino acid. NOTCH2 pro6ArgfsTer27 mutation is an example of early termination of transcription, which leads to significant instability and reduction in NOTCH2 mRNA levels acting as a tumor suppressor, while other mutations in the gene lead to over expression and more oncogenenic function. This data suggests that molecular profiling of cancer should include evaluating RNA mutations and expression levels and not all mutations detected in a gene are the same. Furthermore, increased stability of most mutant mRNA may have some implication on methods used for detected minimal residual disease. Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Jazz: Consultancy; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2. Piris:Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees. Kantarjian:Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; Novartis: Research Funding; Jazz Pharma: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; Astex: Research Funding; Takeda: Honoraria; BMS: Research Funding; Cyclacel: Research Funding; Ariad: Research Funding.

scholarly journals Changes of the Mutational Landscape in Relapsed Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 599-599
Author(s):  
Franck Rapaport ◽  
Marc Robert de Massy ◽  
Adil al Hinai ◽  
Mathijs A. Sanders ◽  
Todd Hricik ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Allele Frequency ◽  
Combination Chemotherapy ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Variant Allele ◽  
Chemotherapy Treatment ◽  
Variant Allele Frequency ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Unfortunately, a significant proportion of patients relapse after responding to initial treatment reflecting our poor understanding of the mechanisms mediating therapy resistance and relapse. We hypothesized that understanding the evolution of the mutational landscape between diagnosis and relapse is essential in order to identify mutational markers associated with sensitivity or resistance to treatment. To address this hypothesis we assembled a cohort of 53 clinically annotated, paired AML patient samples (diagnosis, relapse and patient-matched germline samples; mean age = 52 years). All patients achieved clinical remission after treatment with combination chemotherapy (cytarabine arabinoside and an anthracycline) during induction phase followed by consolidation chemotherapy treatment with or without a stem cell transplantation in first remission. Serial samples were collected at the time of initial diagnosis and within three months of relapse (mean time to relapse 455 days). We performed whole-exome and targeted capture followed by high-throughput sequencing. We aligned samples with BWA, recalibrated them with The Genome Analysis Toolkit (GATK) and then compiled integrated calls from substitution and indel callers (Mutect, Scalpel, Strelka, Varscan and Somatic Sniper). We performed several layers of post-processing filtering on these calls, including removing non-oncogenic mutations and previously documented non-somatic variants, and correcting for the variant allele fraction of indel calls. We filtered out the variants that were found to occur in non-copy number neutral re-arrangements using the clinically determined cytogenetic data. Furthermore, we assessed for copy number events, including loss of heterozygosity events, and for the presence and the variant allele frequency of the FLT3-ITD in our samples. We observed a median of 4.5 and 5 mutations per patient at diagnosis and relapse, respectively, with 3.5 mutations being shared by paired diagnosis and relapse samples. When limiting our analysis to genes previously shown to contribute to leukemogenesis, we found a median of 1.5 and 2 mutations per patient at diagnosis and relapse, with 1 mutation being shared. FLT3, DNMT3A, IDH2, NRAS, RUNX1 and TET2 were among the most commonly mutated genes, with a detected presence rate of 28%, 25%, 19%, 19%, 11% and 11%, respectively, in the diagnosis samples and 39%, 23%, 19%, 4%, 13% and 11% in the relapse samples. We identified significant variation in the variant allele frequency (VAF) for several of the mutations related to these genes and others, denoting variations in the cellular prevalence of the related clones after adjustment for tumor content using the mutations with the highest VAF to delineate clonal architecture. Specifically, we observed that DNMT3A, IDH2, TET2 variants are most commonly present in the bulk AML clone, and persist after treatment. WT1, GATA2 and FLT3mutations are predicted to confer relative resistance to standard combination chemotherapy treatment based on their increased VAF at relapse, whereas KRAS and NRAS subclone(s) are more sensitive to chemotherapy since their VAFs decrease following multiagent chemotherapy. Fifteen patients presented new events in leukemogenesis-related genes at relapse. Overall, our results support a model of AML as a disease with a complex mutational hierarchy and clonal architecture and provide further insight into how these change in response to standard induction therapy. Our data suggests that future efforts to develop targeted therapies with maximal clinical benefit in combination with standard induction treatments should be placed on mutated genes identified to be more strongly associated with disease relapse. Authors contributed equally: F. Rapaport and M.R. De Massy Authors contributed equally: A. al Hinai and M.A. Sanders Disclosures Guzman: Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

scholarly journals Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 543-543
Author(s):  
Theodore Braun ◽  
Cody Coblentz ◽  
Sarah A Carratt ◽  
Mariam Okhovat ◽  
Amy Foley ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Median Survival ◽  
Stock Options ◽  
Research Funding ◽  
Wild Type ◽  
Advisory Committees ◽  
Dana Farber Cancer Institute ◽  
Equity Ownership ◽  
Cebpa Mutations

Acute Myeloid Leukemia (AML) results from the stepwise accumulation of mutations from distinct functional classes, ultimately culminating in malignant transformation. Based on their oncogenic activity, mutations can be classified into three distinct groups. Class I mutations activate signaling pathways, produce uncontrolled proliferation, and in isolation produce a myeloproliferative phenotype. Class II mutations result from point mutations or chromosomal translocation events in lineage determining transcription factors, producing differentiation arrest and myelodysplasia in isolation. A classic example of oncogene synergy between distinct mutational classes can be found in the co-occurrence of mutations in the transcription factor CCAAT-enhancer binding protein alpha (CEBPA) with mutations in colony stimulating factor receptor 3 (CSF3R). Mutations in CEBPA occur in approximately 10% of AML where they block differentiation and convey favorable risk. In contrast, CSF3R mutations lead to constitutive receptor activation and uncontrolled neutrophil proliferation. In the absence of co-occurring Class II mutations, membrane proximal CSF3R mutations produce the myeloproliferative neoplasm chronic neutrophilic leukemia (CNL). Interestingly, patients with CEBPA mutant AML that also harbor an oncogenic CSF3R mutation have worse prognosis than those with wild type CSF3R. However, the mechanism underlying this oncogene synergy remains unknown. To model the co-occurrence of these mutations, we expressed CSF3RT618I (The most common membrane proximal CSF3R mutation) in fetal liver hematopoietic stem cells harboring compound heterozygous CEBPA mutations in the endogenous allele (CEBPAK/L). Mice transplanted with mutant CEBPA alone developed a long latency AML with a median survival of 60 weeks. In contrast, mice transplanted with mutant CSF3RT618I/CEBPAK/L cells developed a much more rapid AML with a median survival of 13 weeks. These results were corroborated in an orthogonal model in which mutant CSF3R and a C-terminal mutant CEBPA were retrovirally expressed prior to bone marrow transplant. To dissect the underlying mechanism, we performed a comprehensive transcriptomic and epigenetic analysis on cells expressing each mutation in isolation as well as the combination. This analysis revealed that mutant CSF3R activates a distinct set of enhancers that regulate genes associated with differentiation and drive neutrophil differentiation. Co-expression of mutant CEBPA blocks the activation differentiation-associated enhancers but is permissive to those associated with proliferation. Differentiation but not proliferation-associated enhancers are bound by wild type CEBPA. Thus, the dominant negative impact of mutant CEBPA at these enhancers explains its differential impact on differentiative and proliferative transcriptional programs. Enhancer activation precedes promoter activation and CEBPA mutations are thought to represent early events in AML initiation. The epigenetic mechanism underlying the observed oncogene synergy argues that CEBPA mutations must occur prior to CSF3R to impact differentiation. We therefore developed a retroviral vector system enabling temporal control of Cre-mediated oncogene expression. Using this system, we found that only when mutant CEBPA is expressed prior to mutant CEBPA is differentiation arrest observed. Furthermore, AML develops in vivo only when mutant CEBPA is expressed prior to mutant CSF3R. To develop novel therapeutic strategies for this subclass of AML with adverse prognosis, we performed medium throughput drug screening on CSF3R/CEBPA mutant AML cells and identified sensitivity to inhibitors of JAK/STAT signaling as well as Lysine Demethylase 1 (LSD1). In other subtypes of AML, LSD1 inhibitors activate enhancers associated with differentiation. We confirmed that LSD1 inhibition promotes neutrophilic differentiation in CSF3R/CEBPA and through epigenetic and transcription profiling establish that this occurs via the reactivation of differentiation-associated enhancers. We further found that the combination of ruxolitinib (JAK/STAT inhibitor) and GSK2879552 produce a complete hematologic response and double median survival in mice harboring CSF3R/CEBPA mutant AML. Thus, the combination of JAK/STAT and LSD1 inhibitors represents and exciting therapeutic strategy for CSF3R/CEBPA mutant AML. Disclosures Druker: Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Beat AML LLC: Other: Service on joint steering committee; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding.

scholarly journals Conifer: Clonal Tree Inference for Tumor Heterogeneity With Single-cell and Bulk Sequencing Data

2021 ◽  
Author(s):  
Leila Baghaarabani ◽  
Sama Goliaei ◽  
Mohammad-Hadi Foroughmand-Araabi ◽  
Seyed Peyman Shariatpanahi ◽  
Bahram Goliaei
Keyword(s):  
Single Cell ◽  
Allele Frequency ◽  
Tumor Heterogeneity ◽  
Variant Allele ◽  
Evolutionary Relationships ◽  
Sequencing Data ◽  
Variant Allele Frequency ◽  
Similar Frequency ◽  
Single Cell Sequencing ◽  
Tree Inference

Abstract Background: An important and effective step in cancer treatment is understanding the clonal evolution of cancer tumors. Clones are cell populations with different genotypes, resulting from the differences in the somatic mutations that occur and accumulate during cancer development. An appropriate approach for better understanding a tumor population is determining the variant allele frequency with which the mutation occurs in the entire population. Bulk sequencing data can be used to provide that information, but the frequencies are not informative enough in identifying different clones and their evolutionary relationships. On the other hand, single-cell sequencing data provides valuable information about branching events in the evolution of a cancerous tumor. However, in the single-cell sequencing data, the total population of sequenced cells is naturally much smaller than bulk sequencing so it is not precise enough for calculating cell prevalence.Result: In this study, a new method called Conifer (ClONal tree Inference For hEterogeneity of tumoR) is proposed which combines aggregated variant allele frequency from bulk sequencing data with branch evolution information from single-cell sequencing data, in order to better understand clones and their evolutionary relationships. It is proven that the accuracy of clone identification is increased by using Conifer compared to other existing methods in both real and simulated data. Also, it is shown that the approach of Conifer in using single-cell sequencing data together with bulk sequencing data has reduced the possibility of cloning mutations with similar frequency but belonging to different clones.Conclusions: In this study, we provided an accurate and robust method to identify clones of tumor heterogeneity and their evolutionary history by combining single-cell and bulk sequencing data.

scholarly journals Comprehensive Single-Cell RNA-Sequencing Mapping of Primary Acute Myeloid Leukemias and Profiling of NPM1-Mutated Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 995-995
Author(s):  
Vincent-Philippe Lavallee ◽  
Elham Azizi ◽  
Vaidotas Kiseliovas ◽  
Ignas Masilionis ◽  
Linas Mazutis ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Research Funding ◽  
Single Cells ◽  
Cell Types ◽  
Wild Type ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Introduction: Acute myeloid leukemia (AML) evolution is a multistep process in which cells evolve from hematopoietic stem and progenitor cells (HSPCs) that acquire genetic anomalies, such as chromosomal rearrangements and mutations, which define distinct subgroups. Mutations in Nucleophosmin 1 (NPM1), which occur in ~30% patients, are the most frequent subgroup-defining mutations in AML and appear to be a late driver event in this disease. Bulk RNA-sequencing studies have identified differentially expressed genes between AML subgroups, but they are uninformative of the composition of cell types populating each sample. Large scale Single-cell RNA sequencing (scRNA-seq) technologies now enable a detailed characterization of intra tumoral heterogeneity, and could help to better understand the stepwise evolution from normal to malignant cells. Methods: Twelve primary human AML specimens from MSKCC and Quebec Leukemia Cell Bank, including 8 with NPM1 mutations, were included in this cohort. Cells were subjected to scRNA-seq using 10X Genomics Chromium Single Cell 3' protocols and libraries were sequenced on Illumina HiSeq or NovaSeq platforms. FASTQ files were processed using SEQC pipeline (Azizi E et al, Cell 2018), resulting in a carefully filtered count matrix of > 100,000 single cells (4877 to 11532 cells per sample). Results: Using euclidean distance metrics and t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization, we explored the phenotypic overlap between samples and showed that leukemia cells from different patients were mostly dissimilar, suggesting inter-sample heterogeneity. However, samples with similar morphology and similar NPM1 mutational status were phenotypically closer (Fig A), as anticipated from bulk RNA-sequencing data (TCGA, NEJM 2013). We partitioned cells into distinct clusters using Phenograph (Levine J et al, Cell 2015) (Fig B) and measured the diversity of samples per cluster using Shannon's entropy metric, revealing that mature cell types (B/plasma cells, T/NK and erythroid cells, Fig C), presumably excluded from the tumor bulk, are transcriptionally similar across samples. Most notably, the next most diverse cluster (C36), comprising 438 cells from 11/12 samples, contains cells with a HSPC-like phenotype, as suggested by i) highest correlation of the centroid of this cluster with HSC1 (lin-/CD133+/CD34dim) population from sorted bulk RNA-sequencing data (Novershtern N et al, Cell 2011), and ii) marked GSEA enrichment for stem cell signatures (top enrichment: Jaatinen_hematopoeitic_stem_cell_up, NES = 9.04, FDR q-val = 0). To study the extent to which NPM1 or other mutations drive heterogeneity in leukemia populations, we interrogated 3'-derived single-cell sequences for all recurrent mutations in AML and found that NPM1 gene has unique features (e.g. relatively high single-cell expression and 3' localization) that allow specific identification of mutations in 5 to 34% of cells per mutated sample. To control for the high frequency of false negatives caused by dropouts in scRNA-seq data, we normalized the abundance of mutated vs wild-type cells to provide an estimation of mutation frequency in different cell types (Fig D). As expected, NPM1 mutations were rare in B and T/NK lymphoid cells (also observed using RT-qPCR in sorted populations by Dvorakova D et al, Leuk Lymphoma 2013) and were found in the majority of leukemia and myeloid cells. Interestingly, these mutations were detected at various frequencies in erythroid cells, suggesting that NPM1 mutations are acquired in cells with different lineage commitment in different patients. Most notably, the HSPC-like cluster C36 also contained a subpopulation of cells that have acquired NPM1 mutations and are transcriptionally different from wild-type cells. Conclusion: This study presents a first comprehensive single-cell map of primary AML, and the first 3'-based interrogation of mutations in single cells. It led to the identification phenotypically distinct cells presenting a HSPC-like expression profile which were sub-clonally harboring NPM1 mutations, providing the means to identify deregulated genes in these important leukemia subpopulations. Figure Figure. Disclosures Levine: Epizyme: Patents & Royalties; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; C4 Therapeutics: Equity Ownership; Prelude: Research Funding; Gilead: Honoraria; Imago: Equity Ownership; Novartis: Consultancy; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Completely Non-Myeloablative/Non-Lymphoablative Conditioning for BMT/HSCT Using Anti-Ckit Immunotoxins

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 493-493 ◽  
Author(s):  
Agnieszka Czechowicz ◽  
Rahul Palchaudhuri ◽  
Amelia Scheck ◽  
Jonathan Hoggatt ◽  
Borja Saez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Host Immunity ◽  
Solid Organ ◽  
Wild Type ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Conditioning Regimens ◽  
Equity Ownership

Abstract Bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) holds the remarkable ability to correct any blood or immune disease. Unfortunately, despite the tremendous potential of this procedure, BMT remains fairly limited in part due to the severe risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy that are currently employed to enable donor HSC engraftment. Although significant work has been done to dose reduce the amount of these preparative agents, patients still experience many side effects including neutropenia/infections, anemia, mucositis, infertility, organ damage and secondary malignancies. Complete elimination of these toxic conditioning regimens could dramatically improve the safety profile of BMT and expand the potential applications to include many more non-malignant hematologic disorders, a wide variety of autoimmune disorders including diabetes, as well as facilitate solid organ tolerance. We have previously shown that competition with host HSC limits donor HSC engraftment, and that in immunocompromised hosts antagonistic anti-ckit monoclonal antibodies deplete host HSC and are an effective and safe alternative conditioning approach (Czechowicz, Science 2007). However, this modality of conditioning is not effective in hosts with competent immune systems. To further understand efficacy of antagonistic anti-ckit conditioning, we tested its functionality in multiple strains of immunocompromised mice and show that inhibition of SCF signaling is not sufficient to deplete host HSC in mouse strains with competent B-cells or T-cells, and that the addition of these cells interferes with the ability of antagonistic anti-ckit antibodies to effectively condition. In an attempt to overcome this hurdle, wildtype mice were immune-depleted with a variety of regimens but none enabled antagonistic anti-ckit conditioning in the immunocompetent setting. To strengthen the potency of anti-ckit mAbs we linked them to protein synthesis toxins, which when internalized by host HSC led to their rapid decline in vitro and in vivo. Administration of anti-ckit-saporin to wild-type mice resulted in >99% depletion of host HSC (Ckit+Lin-Sca1+CD150+CD48-), and lack of residual host HSC activity in the bone marrow was confirmed by CFC assays and competitive transplantation into lethally irradiated recipients. Interestingly, although ckit is expressed by a majority of HSPC, LT-HSC were most significantly affected and no cellularity changes in the bone marrow were observed. Uniquely this regimen was entirely non-peripheral blood ablative unlike other more broadly targeted conditioning regimens such as CD45 immunotoxins (Palchaudhuri, Nat Biotech 2016), and treated animals did not experience any significant depletion of myeloid, lymphoid, or erythroid cells. Figure 1 Figure 1. Treatment with anti-ckit-saporin effectively conditioned wild-type animals and near complete donor granulocyte chimerism was rapidly achieved post transplantation of whole bone marrow cells (99.54 ± 0.35 % vs. 6.79 ± 0.57 %, p<0.001), a >25-fold increase compared to unconditioned controls. Similarly, anti-ckit-saporin conditioning enable efficient engraftment of FACS purified donor HSC (Ckit+Lin-Sca1+CD150+CD48-). In both settings, donor HSC chimerism matched donor granulocyte chimerism further confirming replacement of host HSC. Importantly, host immunity was entirely intact in these animals throughout, with slower recalibration of the longer-lived immune cells given the lack of their direct depletion. Figure 2 Figure 2. This work sets the stage for redefining the way BMT/HSCT is performed, as it opens up the possibility for entirely safe, quick and easy transplantation that potentially could be done in the outpatient setting with no perturbation to host immunity. Extrapolation of these methods to humans may enable efficient yet gentle conditioning regimens for transplantation, which is especially exciting in the gene-therapy settings where no immune suppression is required, allowing for simple, safe and curative treatment of a wide magnitude of grievous blood and immune diseases ranging from sickle cell to hemophilia to HIV. As multiple anti-ckit mAbs are currently in development and being tested in clinical trials, such an approach may be rapidly translatable to patients. Disclosures Czechowicz: Third Rock Ventures: Consultancy; Global Blood Therapeutics: Equity Ownership; Editas Medicines: Equity Ownership, Patents & Royalties; Decibel Therapeutics: Equity Ownership; Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Forty Seven Inc: Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy; GlaxoSmithKline: Research Funding; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Bone Therapeutics: Consultancy. Rossi:Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Clonal Cytopenias of Undetermined Significance Are Common in Cytopenic Adults Evaluated for MDS in the National MDS Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4271-4271
Author(s):  
Johannes B Goll ◽  
R. Coleman Lindsley ◽  
William F. Hooper ◽  
Rafael Bejar ◽  
Jason Walker ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Allele Frequency ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Overlap Syndrome ◽  
The Other ◽  
Advisory Committees ◽  
Pathogenic Variants ◽  
Equity Ownership ◽  
Undetermined Significance

Introduction: Myelodysplastic syndromes (MDS) are a heterogenous group of blood disorders defined by peripheral cytopenia(s), bone marrow failure, morphological dysplasia, and risk of progression. To understand the genetic, epigenetic and biological factors associated with the initiation and progression of MDS, NHLBI created the National MDS Study (NCT02775383). This is a prospective cohort study conducted at 92 community hospitals and 29 academic centers enrolling patients undergoing diagnostic work up for suspected MDS or MDS/myeloproliferative neoplasm (MPN) overlap syndrome. Eligible patients have yet to receive any therapy directed at their cytopenias. Previously untreated cytopenic participants underwent centralized histopathology and data review at the time of enrollment for assignment into distinct subcategories: MDS, MDS/MPN overlap, AML, and Other. Targeted exon sequencing of 96 genes was performed using marrow specimens from the first 300 consecutive individuals in the study. Here we report the genetic mutations for this cohort. Methods: NovaSeq 6000 was used for deep sequencing at a mean coverage of 1,286X and mean breadth (bases covered at ≥100X) of 99.8%. Reads were aligned against build GRCh38 using BWA-MEM, and VarScan2 was used to detect SNVs and INDELS. Variants were filtered for those with an allele base quality of >25 in combination with rule-based and manual review criteria. Subjects in the Other category without an identified malignancy were considered clonal cytopenias of undetermined significance (CCUS) when a mutation or a clonal cytogenetic change was present. Fisher's exact and Wilcoxon rank sum tests in combination with Bonferroni correction were applied to compare groups. Results : A total of 350 putative nonsynonymous pathogenic variants in 36 genes with an allele frequency of >.05 were identified across 150 patients (50%). At least one variant was noted in the following proportion of individuals: 61/72 (85%) with MDS, 13/13 (100%) with MDS/MPN, 15/17 (88%) with AML, and 61/198 (31%) in the Other category, of which 48 were CCUS and 13 were other cancers. Two CCUS patients only had a cytogenetic abnormality. Table 1 shows the distribution of variants in each subcategory of patients for the most commonly mutated genes in our cohort of 300 subjects. Mutations in these genes were enriched in specific groups: SF3B1, STAG2,TP53, and ASXL1 in MDS; TET2 in MDS/MPN; and IDH2 and TP53 in AML (one-sided p<0.0012). None were enriched in the Other group. Within the CCUS subset, 21 genes were mutated, with 37 of 50 (74%) patients having a mutation in TET2, ASXL1, SRSF2, SF3B1, or DNMT3A. The heatmap presented in Figure 1 summarizes variants by subject and allele frequency. Pair-wise comparisons of baseline characteristics of subjects between MDS, MDS/MPN, AML, or CCUS groups revealed no significant differences for age or sex. The CCUS group had significantly higher hemoglobins than the MDS group with median hemoglobin levels of 11.35 and 9.40 g/dL, respectively (p<0.0084). AML had significantly lower platelets compared to all other groups (median of 53 vs. > 110x109/L , respectively, p<0.0084). All groups significantly differed in their median ANC with AML having the lowest (0.8x109/L), followed by MDS (1.5x109/L), CCUS (2.45x109/L), and MDS/MPN overlap (5.95x109/L) (p<0.0084). There was no difference in the median number of variants per patient between groups or correlation with age (rs=0.11, p=0.18). The maximum variant allele frequency (maxVAF) per patient was highest in the MDS/MPN group (median = 0.42, range = 0.38-0.91) and lowest in the CCUS group (median = 0.37, range =0.06-0.98) with the MDS/MPN group having a significantly higher maxVAF compared to the MDS and the CCUS groups (p<0.0084). The proportions of subjects with mutations was similar for those who had abnormal (92% [34/37]) and normal (91% [80/88]) cytogenetics. Conclusions: Incorporation of gene-panel sequencing in the comprehensive evaluation of 300 adult cytopenic patients identified half of the cohort with potentially pathogenic variants. Ultimately, a diagnosis of CCUS was possible in 48 of 183 subjects (26%) not diagnosed with MDS, MDS/MPN overlap syndrome, AML, other cancers or clonal cytogenetics. This study continues to serially bank samples from patients with CCUS, in addition to MDS, MDS/MPN, and ICUS, with the goal to better understand the natural history of these diseases and their progression. Disclosures Lindsley: Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Bejar:Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Al Baghdadi:Celgene: Consultancy, Honoraria; Heron: Consultancy, Honoraria; Tracon: Equity Ownership; Epizyme: Equity Ownership; Bristol Myer Squibb: Consultancy, Honoraria; Sunesis: Equity Ownership; Portola: Equity Ownership; Heron therapeutics: Equity Ownership; Cardinal health: Consultancy, Honoraria; Bristol Myer Squibb: Equity Ownership; Celgene: Equity Ownership; Spectrum pharmaceutical: Equity Ownership; Astrazeneca: Equity Ownership; Seattle genetics: Equity Ownership; Roche: Consultancy, Honoraria. DeZern:Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Foran:Agios: Honoraria, Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Komrokji:DSI: Consultancy; Agios: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; celgene: Consultancy; Incyte: Consultancy; JAZZ: Consultancy. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Phase 1 Study of Milademetan in Combination with Quizartinib in Patients with Newly Diagnosed (ND) or Relapsed/Refractory (R/R) FLT3-ITD Acute Myeloid Leukemia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1389-1389 ◽  
Author(s):  
Naval G. Daver ◽  
Weiguo Zhang ◽  
Richard Graydon ◽  
Vikas K Dawra ◽  
Jingdong Xie ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Wild Type ◽  
Phase 1 Study ◽  
Advisory Committees ◽  
Once Daily ◽  
Flt3 Inhibitor ◽  
Equity Ownership ◽  
Mdm2 Inhibitor

Background: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations occur in ≈ 25% of patients with AML and are associated with poor prognosis. Quizartinib is a once-daily, oral, highly potent and selective FLT3 inhibitor. In the phase 3 QuANTUM-R trial (NCT02039726; Cortes et al. Lancet Oncol 2019), quizartinib prolonged overall survival compared with salvage chemotherapy in patients with R/R FLT3-ITD AML. Murine double minute 2 (MDM2), an E3 ubiquitin ligase, negatively regulates the p53 tumor suppressor and has been shown to be upregulated in patients with AML; TP53 mutations in AML are infrequent except within complex karyotypes. Milademetan, a novel and specific MDM2 inhibitor, showed activity in an ongoing phase 1 trial in patients with AML or myelodysplastic syndromes (MDS) [DiNardo et al. ASH 2016, abstract 593]. Preclinical studies have shown that quizartinib plus milademetan may act synergistically to target FLT3-ITD and restore p53 activity in FLT3-ITD/TP53 wild-type AML [Andreeff et al. ASH 2018, abstract 2720]. Targeting MDM2 may restore p53 activity in cell signaling pathways altered by FLT3-ITD in patients with wild-type TP53 AML. Methods: This open-label, 2-part, phase 1 study (NCT03552029) evaluates quizartinib in combination with milademetan in patients with FLT3-ITD AML. Key inclusion criteria comprise a diagnosis of FLT3-ITD AML (de novo or secondary to MDS) and adequate renal, hepatic, and clotting functions. Key exclusion criteria include acute promyelocytic leukemia, prior treatment with a MDM2 inhibitor, QTcF interval &gt; 450 ms, significant cardiovascular disease, and unresolved toxicities from prior therapies. Dose-escalation (part 1) comprises patients with R/R AML. In part 1, quizartinib will be administered once daily in 28-day cycles, at 3 proposed levels (30, 40, and 60 mg) with appropriate dose modifications based on QTcF monitoring and concomitant use of strong CYP3A inhibitors. Milademetan will be administered on days 1-14 of each 28-day cycle, at 3 proposed levels (90, 120, and 160 mg). Dose escalation will be guided by modified continual reassessment with overdose control. The primary objectives of part 1 are to evaluate the safety and tolerability, optimum dosing schedule, maximum tolerated dose (MTD), and recommended dosing for the expansion (RDE) cohort. Dose expansion (part 2) comprises a cohort of patients with R/R FLT3-ITD AML who have not received &gt; 1 salvage therapy and not received &gt; 1 prior FLT3 inhibitor, and a second cohort including ND patients with FLT3-ITD AML who are unfit for intensive chemotherapy. Patients in part 2 will be treated with quizartinib plus milademetan at the RDE doses identified in part 1. The objectives of part 2 are to confirm the safety and tolerability of quizartinib plus milademetan at RDE and identify the recommended phase 2 dose. Pharmacokinetics and preliminary assessment of efficacy are also being evaluated as secondary outcomes. Pharmacodynamic and biomarker assessments such as leukemic stem cell numbers, STAT5 downstream signaling, minimal residual disease measured by flow cytometry, and gene mutations will be evaluated as exploratory endpoints. Approximately 24 to 36 dose-limiting toxicity-evaluable patients are needed in part 1 to determine the MTDs and the RDE; approximately 40 patients per cohort will be treated at the RDE in part 2. This study is currently recruiting at multiple sites in the United States for part 1; recruitment for part 2 may be expanded to additional sites worldwide as necessary. Disclosures Daver: Jazz: Consultancy; Glycomimetics: Research Funding; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Servier: Research Funding; Karyopharm: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy, Research Funding; Agios: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; Otsuka: Consultancy; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Genentech: Consultancy, Research Funding; Astellas: Consultancy; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; NOHLA: Research Funding. Graydon:Daiichi Sankyo, Inc.: Employment. Dawra:Daiichi Sankyo, Inc.: Employment; Pfizer Inc: Employment. Xie:Daiichi Sankyo, Inc.: Employment. Kumar:Daiichi Sankyo, Inc.: Employment, Equity Ownership. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Synergistic Anti-Leukemic Activity with Combination of FLT3 Inhibitor Quizartinib and MDM2 Inhibitor Milademetan in FLT3-ITD Mutant/p53 Wild-Type Acute Myeloid Leukemia Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2720-2720 ◽  
Author(s):  
Michael Andreeff ◽  
Weiguo Zhang ◽  
Prasanna Kumar ◽  
Oleg Zernovak ◽  
Naval G. Daver ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Combination Treatment ◽  
Research Funding ◽  
Single Agent ◽  
Wild Type ◽  
Advisory Committees ◽  
Mouse Xenograft Model ◽  
Equity Ownership ◽  
Mdm2 Inhibitor

Abstract Background: MDM2, a negative regulator of the tumor suppressor p53, is overexpressed in several cancers including hematological malignancies. Disrupting the MDM2-p53 interaction represents an attractive approach to treat cancers expressing wild-type functional p53. Anticancer activity of small molecule MDM2 inhibitor milademetan (DS-3032b) has been demonstrated in preclinical studies and in a phase 1 trial in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome. Quizartinib is a highly selective and potent FLT3 inhibitor that has demonstrated single-agent activity and improvement in overall survival in a phase 3 clinical study in relapsed/refractory AML with FLT3-internal tandem duplication (FLT3-ITD) mutations. We present here the preclinical studies exploring the rationale and molecular basis for the combination of quizartinib and milademetan for the treatment of FLT3-ITD mutant/TP53 wild-type AML. Methods: We investigated the effect of quizartinib and milademetan combination on cell viability and apoptosis in established AML cell lines, including MV-4-11, MOLM-13 and MOLM-14, which harbor FLT3-ITD mutations and wild type TP53. Cells were treated with quizartinib and milademetan at specified concentrations; cell viability and caspase activation were determined by chemiluminescent assays, and annexin V positive fractions were determined by flow cytometry. We further investigated the effect of the combination of quizartinib and the murine specific MDM2 inhibitor DS-5272 in murine leukemia cell lines Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD+F691L and Ba/F3-FLT3-ITD+D835Y, which harbor FLT3-ITD, ITD plus F691L and ITD plus D835Y mutations, respectively. F691L or D835Y mutations are associated with resistance to FLT3-targeted AML therapy. In vivo efficacy of combination treatment was investigated in subcutaneous and intravenous xenograft models generated in male NOD/SCID mice inoculated with MOLM-13 and MV-4-11 human AML cells. Results: Combination treatment with milademetan (or DS-5272) and quizartinib demonstrated synergistic anti-leukemic activity compared to the respective single-agent treatments in FLT3 mutated and TP53 wild type cells. Combination indices (CIs) were 0.25 ± 0.06, 0.61 ± 0.03, 0.62 ± 0.06, 0.29 ± 0.004 and 0.50 ± 0.03, respectively, in MV-4-11, MOLM-13, MOLM-14, Ba/F3-FLT3-ITD+F691L and D835Y cell lines, all of which harbor FLT3-ITD or ITD plus TKD point mutations. The combination regimen triggered synergistic pro-apoptotic effect in a p53-dependent manner as shown by annexin-V staining and caspase 3/7 assays. Mechanistically, the combination treatment resulted in significant suppression of phospho-FLT3, phospho-ERK and phospho-AKT and anti-apoptotic Bcl2 family proteins (eg, Mcl-1), as well as up-regulation of p53, p21 and pro-apoptotic protein PUMA, compared to single agent treatments. Of note, the combination regimen also exerted a synergistic pro-apoptotic effect on venetoclax (BCL-2 inhibitor)-resistant MOLM-13 cells (CI: 0.39 ± 0.04) through profound suppression of Mcl-1. In an in vivo study using the MOLM-13 subcutaneous mouse xenograft model, quizartinib at 0.5 and 1 mg/kg and milademetan at 25 and 50 mg/kg demonstrated a significant tumor growth inhibition compared with vehicle treatment or respective single-agent treatments. In MV-4-11 intravenous mouse xenograft model, the combination of quizartinib plus milademetan showed a significantly prolonged survival, with no animal death in the combination group during the study period, compared to respective single agent treatments and untreated control (Figure). Conclusion: Synergistic anti-leukemic activity was observed for quizartinib plus milademetan combination treatment in preclinical AML models. A phase I clinical trial of quizartinib/milademetan combination therapy in patients with FLT3-ITD mutant AML is underway. Figure. Effects of quizartinib, milademetan and their combination on survival of mice intravenously inoculated with human MV-4-11 AML cells Disclosures Andreeff: Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Reata: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership. Kumar:Daiichi Sankyo: Employment, Equity Ownership. Zernovak:Daiichi Sankyo: Employment, Equity Ownership. Daver:Pfizer: Research Funding; ImmunoGen: Consultancy; Otsuka: Consultancy; Karyopharm: Research Funding; Alexion: Consultancy; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Kiromic: Research Funding; Sunesis: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Sunesis: Consultancy. Isoyama:Daiichi SANKYO CO., LTD.: Employment. Iwanaga:Daiichi Sankyo Co., Ltd.: Employment. Togashi:Daiichi SANKYO CO., LTD.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment.

Sign in / Sign up

Export Citation Format

Share Document