Deconvoluting clonal and cellular architecture in IDH-mutant Acute Myeloid Leukemia

Isocitrate dehydrogenase 1/2 (IDH) mutations are early initiating events in acute myeloid leukemia (AML). The complex clonal architecture and cellular heterogeneity in IDH-mutant AML underlies the heterogeneous clinical presentation and outcomes. Integrating single-cell genotyping and transcriptomics, we demonstrate a stem-like and inflammatory phenotype of IDH-mutant AML and identify clone-specific programs associated with NPM1, NRAS, and SRSF2 co-mutations. Furthermore, these clones had distinct responses to treatment with combination IDH inhibitors and chemotherapy, including elimination, reconstitution of myeloid differentiation, or retention within progenitor populations. At relapse after IDH inhibitor monotherapy, we identify upregulated stemness, inflammation, mitochondrial metabolism, and anti-apoptotic factors, as well as downregulated major histocompatibility complex (MHC) class II antigen presentation. At the pre-leukemic stage, we observe upregulation of IDH2-associated pathways, including inflammation. We deliver a detailed phenotyping of IDH-mutant AML and a framework for dissecting contributions of recurrently mutated genes in AML at diagnosis and following therapy, with implications for precision medicine. To study the role of co-mutations in shaping disease presentation and treatment response in IDH1/2-mutant AML to either conventional cytotoxic chemotherapy and/or IDH-directed inhibitors +/- hypomethylating agents, we screened the OSU biobank for samples from patients receiving targeted IDH1/2 inhibitors and prioritized samples on the basis of availability of sequential samples, including matched diagnostic and post-treatment samples. In total, we obtained 30 cryopreserved bone marrow aspirate or peripheral blood samples from 9 patients at longitudinal timepoints beginning at diagnosis. We profiled 9 newly diagnosed patients (AML07-11) receiving IDH1/2 inhibitor monotherapy or in combination with Azacytidine (AML12-13) or chemotherapy alone (AML14-15). Serial samples were collected at remission (n= 9 patients, median time after diagnosis (41 days) and relapse timepoints (n=4 patients; median time 985 days). Frozen bone marrow samples were thawed and transferred into 50ml conical tubes containing 1x PBS +2% fetal bovine serum (FBS). Cell suspensions were centrifuged at 300 x g for 5 minutes at 4℃, and supernatant was removed. Samples were then enriched for >90% alive cells by using a dead cell removal kit, or stained with DAPI before sorted for alive cells using SY3200 Cell Sorter (SONY). For cell sorting, DAPI low cells are collected into 15ml conical tubes containing 3ml FBS on ice. Samples were then centrifuged at 300 x g for 5 mins at 4℃. For CITE-seq, enriched alive cells were first stained with cell-hashing oligo-tagged antibodies (Biolegend) according to manufacturer’s instructions. Then samples were counted and pooled together and stained with a CITE-seq antibody cocktail (Biolegend) according to the manufacturer’s instructions. After staining, samples were processed using 10X Genomics Chromium controller with the Single Cell Gene Expression 3’ Reagent Kits (v3 and v3.1). Libraries were sequenced on an Illumina NovaSeq 6000. The detailed information of antibody panels, antibody barcodes, hashtag assignment for each sequencing library is provided in the “CITEseq_panels.xlsx” file. To study the role of co-mutations in shaping disease presentation and treatment response in IDH-mutant AML to either conventional cytotoxic chemotherapy and/or IDH-directed inhibitors +/- hypomethylating agents, we screened the MSKCC and OSU biobank sfor samples from patients receiving targeted IDH inhibitors and chemotherapy and prioritized samples on the basis of availability of sequential samples, including matched diagnostic and post-treatment samples. We obtained cryopreserved bone marrow aspirate or peripheral blood samples from 20 patients. Two peripheral blood samples from IDH-mutant clonal hematopoiesis were also profiled. All samples were profiled by 10X RNA-seq. Please see manuscript methods for more details. We ascertained 20 IDH-mutated AML patients with representative co-mutations by clinical sequencing (Figure 1D, STAR Methods, and Table S1). Six had IDH1 mutations, 13 had IDH2 mutations, and 1 had both IDH1 and IDH2 (IDH1 25% VAF and IDH2 12% VAF). IDH mutations had clonal VAFs, indicating early acquisition. The co-occurring mutations in DNMT3A (n = 15 patients), NPM1 (n = 7), and SRSF2 (n = 7) reflect the genomic landscape of IDH-mutant AML (Figure 1A). Five patients had copy-number aberrations reported by clinical cytogenetics. In total, 57 longitudinal bone marrow or peripheral blood samples (median 3 samples per patient) were profiled under treatment with IDHi monotherapy (n = 6), IDHi in combination with intensive chemotherapy (NCT02632708, n = 6) or in combination with hypomethylating agent (n = 3), or chemotherapy alone (n = 4). Finally, to study how IDH2 mutations affect hematopoiesis at a pre-leukemic state, we profiled two individuals with IDH2-mutant CH (VAF 17%). The single-cell RNA sequencing (scRNA-seq) dataset comprises 386,794 cells from 20 AML patients (n = 317,317 cells), 2 CH individuals (n = 16,705), and 12 unaffected age-matched donors (n = 52,772 cells; n = 8 donors previously published24) (Figure 1E).