Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib

Abstract

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

Conflict of interest statement

Competing interests:

PV has received research grant support from Celgene and is on its speaker bureau. LQ has received research grant support from Celgene

Figures

Figure 1. Enasidenib treatment induces differentiation of AML progenitor and precursor cell populations and restores progenitor function.

a) Top, immunophenotyping of hematopoietic stem/progenitor/precursor and mature cell populations in AML bone marrow (BM) samples pre-treatment showing expanded progenitor (n=11 biologically independent samples) or precursor (n=4 biologically independent samples) populations with normal BM (n=8 biologically independent samples). Below, detailed composition of stem/progenitor compartments in AML pre-treatment (n=15 biologically independent samples) and normal BM (n=12 biologically independent samples). Error bars in normal BM= 95% confidence interval. HSC: hematopoietic stem cell, MPP: multipotent progenitor, LMPP: lymphoid-primed multipotent progenitor, CMP: common myeloid progenitor, MEP; megakaryocyte-erythroid progenitor; GMP: granulocyte-macrophage progenitor. b) Top, schematic representation of flow cytometric approach and sequential gates used to analyse samples in (a). Lin-, lineage negative; BMMC, bone marrow mononuclear cells. Bottom, example of flow plots from a representative sample prior to enasidenib treatment (Pre-ENA) and at complete remission (CR) in patients with expanded progenitor-like populations (#201-023; left, experiment performed once) or expanded myeloid precursor-like population (#201-010; right, experiment performed once). Numbers shown within the gate indicate percentage of the corresponding cell population compared to all cells in the plot. c) Top, immunophenotyping of hematopoietic cell populations in normal BM (left, as in (a)) and in samples from 5 patients (#201-023, #201-011, #201-022, #201-010, #203-002) pre-ENA, at intermediate time points during treatment, and at CR. Bottom, sizes of stem and progenitor compartments. Abbreviations and error bars in normal BM are as in (a). C=cycle, D=day. d) Number of mixed erythroid-myeloid colonies (GEM), granulocyte-macrophage (GM), granulocyte (G), macrophage (M) and erythroid (E) colonies produced per 100 plated flow-sorted CD34+ cells from normal BM (n=4 biologically independent samples), enasidenib-treated patients in CR (n=5 biologically independent samples) and enasidenib-treated patients not in CR (n=3 biologically independent samples). Patient samples were plated with addition of Enasidenib (1μM) to semi-solid media. Error bars= standard error of the mean. P-values determined by 2-sided Student’s paired t-test.

Figure 2. Differentiation response arising from wild-type cells in patients treated with enasidenib.

a) Schematic representation of varying possible clonal responses to enasidenib. Four mutations (A, B, C and D) are present in four clones that are arranged in a branching structure. A differentiation response to enasidenib treatment could potentially occur from either wild type cells or from ancestral or terminal clones. b) Summary of the type of differentiation response (from either wild type cells, ancestral or terminal clones) in samples from 6 patients. c) Variant allele frequencies (VAF) of the indicated mutations in AML blasts of patient #201-022 prior to enasidenib treatment (Pre-ENA) and in peripheral blood mononuclear cells (PMNC) at CR, as assessed by targeted re-sequencing. d) Clonal contribution to colony output from the CR sample from patient #201-022, as a percentage of all individually picked colonies genotyped. Clones were identified as wild type (WT), carrying the FEZ2 P118S mutation (F), or carrying the FEZ2 P118S and DNMT3B N738S mutations (FD). Lineage affiliations of the colonies are as in Fig. 1d. Numbers next to the bars indicate the number of colonies analyzed. e) Bar graph showing the lineage affiliation of colonies from Lin-CD34+ normal cord blood (CB) cells (n=5 biologically independent samples) and CD34+ BM cells in the CR sample of patient #201-022. Numbers next to the bar indicate the number of colonies produced per 100 plated cells. The GM:E (granulocyte-macrophage:erythroid) ratio of colonies and the 95% confidence interval for the GM:E ratio in normal BM are shown.

Figure 3. Enasidenib induces differentiation from an ancestral IDH2 mutant clone.

All data shown refer to samples from patient #201-023. a) Heat map of targeted re-sequencing of mutations (rows) in single cells (columns, n=63 cells) from flow-sorted BM populations isolated pre-ENA and at CR which are shown together. Clonal identification of each cell is shown below the heat map and the key to mutations is denoted by letters on the right. Mutation detection key: red=detected; blue=not detected; white= sequencing failed. b) Clonal structure of the AML sample based on single cell genotyping (SCG). Number next to a clone indicates the number of cells identified in that clone (data from a). The most likely clonal structure is shown in solid arrows with alternatives in dotted arrows. (*) indicates genotype “A” or “SIAR”, which were each detected in only 1 cell. § indicates genotype “AS” with ADO of an IDH2 allele in 2/3 cells. Ø “SI” with ADO of the ASXL1 allele in 3/12 cells. See also Supplementary Fig. 12a. c) Clonal composition in different immunophenotypic compartments pre-ENA and at CR. Number of cells studied are indicated. d) Clonal contribution (vertical bars) to immunophenotypic stem, progenitor, myeloid precursor and terminal mature GM populations in patient samples pre-ENA and at CR (horizontal bars). Data is from SCG except for mature GM population at CR, where the flow-sorted cell population was genotyped (*). Normal BM is shown for comparison of immunophenotypic populations. e) Clonal contribution to colonies grown from CR sample (percentage of genotyped, individually picked colonies). Key to mutations detected are as in (b). Numbers next to the bars indicate the numbers of colonies analyzed. Lineage affiliations are as in Fig. 1d. f) Lineage affiliation of colonies from BM CD34+ cells purified from CR sample compared with normal CB (as in Fig. 2e).

Figure 4. Enasidenib induces differentiation from a terminal IDH2m clone.

All the data here are from patient #201-011. a) Heat map of targeted re-sequencing of mutations (rows) in single cells (columns, n=110 cells) from flow-sorted BM populations pre-treatment and CR which are shown together. The key is as in Fig. 3a. b) Clonal structure of the AML sample based on SCG. The key to the panel is as in Fig. 3b. * marks the genotype “DIA” where ADO was detected in 6/7 cells. Ø denotes genotype “DId”. No heterozygous germline SNPs were available in the ASXL1 gene. Estimated ADO frequency of the ASXL1 allele was 12.1%. See also Supplementary Fig. 12c. c) Clonal composition in different immunophenotypic compartments pre-ENA and at CR, as in Fig. 3c. d) Clonal contribution (vertical bars) to immunophenotypic BM haematopoietic populations in patient samples pre-ENA and at CR (horizontal bars). Data is from SCG except for mature GM population pre-ENA, where the flow-sorted cell population was genotyped (*). e) Clonal contribution to colonies grown from CR sample (percentage of genotyped, individually picked colonies) as in Fig. 3e. f) Lineage affiliation of colonies from BM CD34+ cells purified from CR sample compared with normal CB (as in Fig. 2e).

Figure 5. Mechanisms leading to relapse of enasidenib-treated patients.

a) Summary of mechanisms (rows) leading to relapse in 12 patients (columns). Selected mutations detected at relapse by WES (all patients except #104-021) or by Heme Panel bait capture sequencing (#104-021) are shown. b) Longitudinal analysis of the percentage suppression of serum 2-HG concentrations prior to enasidenib treatment (pre-ENA), at best response (CR or PR) and at relapse in 14 patients with an IDH2 R140 codon mutation. c-d) Serum 2-HG levels and bone marrow blast percentages prior to enasidenib treatment (pre-ENA), at CR or CRp (complete remission without platelet recovery) during the course of treatment (C=cycle, D=day of treatment) and at relapse in patients #201-014 (c) and #201-022 (d). e-f) Serial mutation analyses in flow-sorted blasts prior to enasidenib treatment (pre-ENA) and at relapse in patients #201-014 (e) and #201-022 (f).

Figure 6. Relapse post-enasidenib occurs through clonal evolution/selection.

a) Patient #104-021: Heat map of targeted re-sequencing of mutations (rows) in single cells (n=214 cells, columns) from flow-sorted BM populations pre-treatment and at relapse which are shown together. The key is as in Fig. 3a. b) Clonal structure of patient #104-021 based on SCG. The key to the panel is as in Fig. 3b. Boxes in dotted red lines highlight clones which are only detected at relapse. c) Clonal composition in different immunophenotypic compartments pre-ENA and at relapse, as in Fig. 3c. d) Clonal contribution (vertical bars) to immunophenotypic BM haematopoietic populations in patient samples pre-ENA and at relapse (horizontal bars). Data is from SCG. e) Patient #201-011: Heat map of targeted re-sequencing of mutations (rows) in single cells (n=87 cells, columns) from flow-sorted BM populations at relapse. The key is as in Fig. 3a. f) Clonal structure of patient #201-011 at relapse. * indicates 6 cells with genotype “DIA” where we detected ADO in 4/5 cells in the DNMT3A allele. Ø denotes “DId”. The estimated ADO frequency of the ASXL1 allele was 12.1%. § indicates 6/7 “DIXF” cells where there was ADO for the DHX15 R222G mutant allele. See also Supplementary Fig. 12c. Boxes in dotted red lines as in (b). g) Clonal composition in different immunophenotypic compartments at relapse, as in Fig. 3c. h) Clonal contribution (vertical bars) to immunophenotypic BM haematopoietic populations in patient samples at relapse (horizontal bars). Data is from SCG.