The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or ...modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele ...frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation.
In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platform. Viable cells were sorted from 90 samples from 78 patients with CH, AML, and MPN/post-MPN AML followed by single cell amplification/sequencing. Mutation calls were filtered based on read depth, quality, and alleles genotyped per cell. We reconstructed a random distribution of clones by permuting genotype calls across cells and generated empirical p values for each clone. To identify dominant clones, we used a Poisson test to determine clones were significantly enriched compared to the mean clone size. Clones with significant p-values (p <0.05) were used to generate plots of clonal architecture of each sample (Figure 1A).
Despite significant clonal complexity, the majority of MM patients (80%;72/90) present with one (51/90; 56.7%) or two (21/90; 23.3%) dominant clones. These data show there are specific genotypic combinations which lead to clonal dominance with increased fitness relative to other clones and/or suppression of minor clones by dominant clone(s). We next investigated whether specific molecularly defined AML subtypes had increased clonal complexity. FLT3-ITD mutant AML samples had a significantly greater number of clones (p < 0.002) compared to AML samples with multiple epigenetic modifier mutations. Similar findings were not observed when comparing AML samples with epigenetic mutations to RAS pathway mutant samples.
We next investigated whether specific mutations were likely to co-occur/be mutually exclusive at a single cell level. We observed evidence of oligoclonality in CH, including parallel acquisition of DNMT3A mutations and clones with multiple mutations in the absence of progression to MM. By contrast, in MM the dominant clone(s) almost always harbored multiple epigenetic modifier mutations, suggesting cooperative epigenetic remodeling in myeloid transformation. Mutations in signaling effectors (FLT3-ITD/TKD; RAS/RAS) were mutually exclusive. We observed distinct FLT3-mutant clones in FLT3-mutant AML patients and parallel acquisition of different RAS pathway mutations. We used this data to develop clonal architecture trees in each patient, giving us a definitive picture of mutational acquisition and transformation at a single cell level. We calculated a Shannon diversity score and observed an increase in clonal complexity with disease evolution; CH samples had the lowest clonal diversity and FLT3-ITD AML patients the highest clonal diversity (Figure 1B).
We extended our findings by combining cell surface marker assessment and single cell mutational analysis. Patient samples were stained with an antibody cocktail of 6 oligo-conjugated antibodies with barcode tags prior to single cell sequencing, which allowed simultaneous acquisition of single cell immunophenotypic and genotypic data. This allows us to identify distinct populations of stem/progenitor cells with distinct clonal/mutational repertoires (Figure 1C). Additional data will be presented with this novel approach, which allows us to combine an assessment of stem/progenitor cell frequency with genetic data. This includes studies of CD34+ and CD34- AML, which show striking differences in mutational representation in different stem/progenitor compartments.
In summary, our studies of clonal architecture at a single cell level provide us novel insights into the pathogenesis of myeloid transformation and give us new insights into how clonal complexity contributes to disease progression.
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Ooi:Mission Bio: Employment, Equity Ownership. Mendez:Mission Bio: Employment, Equity Ownership. Carroll:Janssen Pharmaceuticals: Consultancy; Incyte: Research Funding; Astellas Pharmaceuticals: Research Funding. Papaemmanuil:Celgene: Research Funding. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Roche: Consultancy, Research Funding; Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Gilead: Consultancy; Celgene: Consultancy, Research Funding.
The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies ...to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C.sup.pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C.sup.pro, we measured cleavage kinetics of predicted SVV-001 3C.sup.pro substrates. An efficient substrate, L/VP4 (k.sub.cat /K.sub.M = 1932 #177; 183 M.sup.-1 s.sup.-1 ), was further optimized by a P2' Nright arrowP substitution yielding L/VP4.1 (k.sub.cat /K.sub.M = 17446 #177; 2203 M.sup.-1 s.sup.-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C.sup.pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C.sup.pro, with direct implications for anti-cancer therapeutic development.
Background: Over 300 somatic molecular variants in hematologic diseases are either specified as diagnostic criteria in the World Health Organization (WHO) Classification of Tumors of Hematopoietic ...and Lymphoid Tissues, recognized as potentially actionable biomarkers in the National Comprehensive Cancer Network (NCCN) compendia, or supported by published well-powered clinical studies. Moreover, new molecular alterations with potential clinical implications in hematologic disease are continuously emerging in the scientific literature. These have critical use for a wide spectrum of clinicians, including hematopathologists who diagnose patient-specific hematologic malignancies, heme-oncologists who direct patient care, and clinical trial nurses who assist patients in finding appropriate clinical trials. Importantly, the utility of this information critically depends on the clinician's ability to interpret the significance of variants in a point-of-care setting. Therefore, there is an urgent and unmet need for a clinical decision support system that 1) distills the clinical implications associated with molecular alterations into a standardized and easily interpretable format and 2) democratizes access of this information to all members of the heme-oncology community.
Methods: OncoKB is an established expert-guided precision oncology knowledge base that annotates the oncogenic effect and therapeutic implications of somatic molecular alterations (Chakravarty, D. et al., JCOPO, 2017). Previously, OncoKB was focused primarily on solid tumor mutation annotation. Recently, we expanded OncoKB to include alterations in hematologic malignancies. The heme-specific annotation efforts were guided by heme-oncology and hematopathology physician scientists at Memorial Sloan Kettering (MSK). Supplementing the previously published therapeutic levels of evidence (Fig. 1a), we further added level of evidence systems for diagnostic and prognostic implications (Fig. 1b, c). These three sets of evidence levels are consistent with the criteria set forth by the joint consensus of the ASCO/CAP/AMP guidelines (Li, MM. et al., J Mol Diagn, 2017). We assigned the newly curated heme-specific molecular alterations with diagnostic, prognostic or therapeutic levels of evidence, when applicable. Finally, we annotated and analyzed 1569 hematologic tumor samples from the AACR Project GENIE (release 6.1) with these levels of evidence.
Results: In addition to alterations with both solid and heme clinical implications already curated in OncoKB, we annotated 288 unique heme-specific mutations, fusions, and copy number alterations in 156 newly curated cancer-associated genes. Based on MSK-expert consensus, the WHO and NCCN guidelines, and the scientific literature, we identified a total of 192 alterations with unique diagnostic levels of evidence, 65 alterations with unique prognostic levels of evidence and 55 alterations with unique therapeutic levels of evidence across 13 major hematologic tumor types (Fig. 2).
To test the utility of OncoKB, we annotated all genomic events in 1569 heme cancer samples in 89 hematologic malignancies in the AACR GENIE cohort (V6.1) (Fig. 3a). Thirty-eight percent of samples harbored at least one potentially actionable alteration, and 8% were predictive of clinical benefit from an FDA-approved drug (Fig. 3b).
Conclusions: OncoKB heme data is publicly available both through the web resource http://oncokb.org and through incorporation into the cBioPortal for Cancer Genomics. Heme-specific molecular alterations are used to make an accurate diagnosis, inform prognosis, optimize the use of stem cell transplant, and to link patients with the optimal mechanism-based therapies in the clinical trial setting and in routine clinical practice. This is the first study to annotate and analyze actionability of heme samples. In this proof-of-principle study, we demonstrate the ability to annotate clinical samples with their diagnostic, prognostic and therapeutic implications in a point-of-care setting.
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Roshal:Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Ho:Invivoscribe, Inc.: Honoraria. Knorr:Fate Therapeutics: Patents & Royalties. LaFave:Epizyme: Patents & Royalties. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Berger:Roche: Consultancy. Solit:Pfizer: Consultancy; Lilly Oncology: Honoraria; Vivideon Therapeutics: Consultancy; Loxo Oncology: Consultancy, Equity Ownership; Illumina: Consultancy. Dogan:Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding.
Abstract
Introduction: Acute myeloid leukemia (AML) has a poor prognosis, despite aggressive therapies and a recent expansion in the array of treatments. Treatment is often determined by mutations, ...which risk-stratify a patient’s leukemia or can identify mutations that serve as therapeutic targets. Although common mutations in AML have been extensively studied, less research has formally characterized the order by which mutations tend to occur and how this order relates to properties of the disease.
Methods: We leveraged published, single-cell DNA sequencing data from three institutions to model the clonal evolution of AML. Sequencing had been performed using targeted sequencing panels covering 19 to 37 genes, and driver mutations were identified using conservative filters. Mutation trees were created using Single Cell Inference of Tumor Evolution (SCITE). Clonal evolution patterns, including across clinical timepoints, were identified and compared to patient characteristics, disease phenotype, and outcomes. The BeatAML dataset was leveraged to explore associations between mutation order and gene expression.
Results: Using 835 driver mutations from 276 samples and 209 patients, we identified 223 total unique mutation patterns. Branched evolution primarily involves FLT3 and RAS pathway mutations, whereas certain mutation pairs, such as NPM1 and FLT3, always occurred linearly in the same clone. Although some mutation pairs, such as those related to DNA methylation versus the RAS pathway, tended to occur in a specific order, several cases exhibited atypical orderings. Early signaling gene mutations were associated with younger patient age and increased signaling mutation homozygosity while NRAS-first cases were associated with increased monocyte counts. NRAS-first cases were also associated with distinct gene expression patterns in the BeatAML dataset. Paired diagnosis and relapse samples revealed novel associations between mutations gained at relapse and their clonal context, and these analyses supported a relative fitness advantage for signaling mutations in clones containing mutations in NPM1 or genes affecting DNA methylation.
Conclusions: Clonal evolution in AML can be reconstructed at scale using single-cell DNA sequencing data, and different mutation acquisition patterns are associated with distinct leukemia and patient characteristics, despite cases having similar co-mutation patterns.
Citation Format: Matthew Schwede, Katharina Jahn, Linde A Miles, Robert L Bowman, Jack Kuipers, Troy M Robinson, Asiri Ediriwickrema, Andrew J Gentles, Ross Levine, Niko Beerenwinkel, Koichi Takahashi, Ravindra Majeti. Characterizing the order of mutation acquisition in acute myeloid leukemia using large-scale single-cell sequencing abstract. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A49.
The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies ...to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 plus or minus 183 M-1s-1), was further optimized by a P2' N arrow right P substitution yielding L/VP4.1 (kcat/KM = 17446 plus or minus 2203 M-1s-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.
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