Loss of glucocorticoid receptor expression mediates in vivo dexamethasone resistance in T-cell acute lymphoblastic leukemia

Abstract

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

Figure 1.. RIM-induced T-ALLs respond to dexamethasone alone or in combination with GDC-0941 and exhibit intrinsic glucocorticoid resistance upon re-treatment.

(A) Primary T-ALLs were first expanded in a single recipient. After euthanasia, 2×10⁶ bone marrow cells were harvested and transplanted into 14 recipient mice, which were then randomly assigned to one of the three indicated treatment groups. (B) Kaplan-Meier survival analysis of recipient mice transplanted with 10 independent primary T-ALLs (five Kras^WT and five Kras^G12D) and treated with vehicle (n=40), DEX (n=50), or DEX/GDC-0941 (n=50) indicated that DEX significantly extended survival (p<0.0001, Log-rank test), which was modestly but not significantly enhanced by GDC-0941 (p=0.0805, Log-rank test). (C) Southern blot analysis with a probe for the MOL4070 virus of DNA extracted from leukemia cells harvested from individual moribund recipients of T-ALL 20M that were treated with vehicle (V1-V3), DEX (D1-D4), or the DEX/GDC-0941 combination (C1-C5). Novel restriction fragments indicative of retroviral integrations that are not present in the dominant parental clone are visible in relapsed leukemias D3, D4, C1, and C4. (D) Kaplan-Meier survival analysis of resistant T-ALLs (dotted lines) treated with vehicle (n=3) or DEX (n=4) compared to the corresponding drug-sensitive parental T-ALLs (solid lines) treated with vehicle (n=4) or DEX (n=5). Resistant T-ALLs demonstrated significantly shorter survival after DEX treatment versus the corresponding parental leukemias; 20M.D1 (p=0.0039, Log-rank test), 73M.D4 (p=0.0027, Log-rank test), and JW-81.D3 (p=0.0025, Log-rank test).

Figure 2.. Relapsed mouse and human T-ALLs show reduced glucocorticoid receptor protein expression which confers dexamethasone resistance in vitro.

(A) Western blot analysis of bone marrow lysates from Kras^WT and Kras^G12D T-ALLs in parental (P), DEX treated (D) and DEX/GDC-0941 combination treated (C) mice at relapse. (B) Human bone marrow from T-ALL patients was subjected to immunohistochemistry analysis of GR protein expression and scored in a blinded fashion independently by two hematopathologists to classify samples as strongly positive (IV), positive (III), weakly positive (II) or negative (I). Scale bar represents 30 microns. Graphical representation of GR expression levels assessed by Western blot in primary murine T-ALLs (C) and by immunohistochemistry in human T-ALL patient samples (D). GR expression in murine T-ALLs was classified as low (examples in panel A include 2M.D1, 2M.C2, 20M.D4, 20M.C5), medium (examples include JW-81.C2, 5C.C2, 2M.C4, 20M.C4), or high (examples include 5C.D1, 2M.C1, 2M.C3, 20M.D3). Asterisk in panel C denotes a significant difference between high and medium/low classifications in parental versus relapsed murine T-ALLs (p-value=0.0109, Fisher’s test). Asterisk in panel D denotes a significant difference between strongly positive/positive and weakly positive/negative classifications in diagnostic versus relapsed human T-ALLs (p-value=0.0013, Fisher’s test). (E) Gene expression analysis using a TaqMan assay to measure Nr3c1 transcript levels in three independent parental T-ALLs (P), and corresponding DEX (D) or DEX/GDC-0941 combination (C) treated relapsed T-ALLs. Error bars represent standard error of the mean for technical replicates. (F) Western blot analysis of GR expression in lysates from the CCRF-CEM human T-ALL cell line edited using nucleofection with Cas9 ribonuleoproteins containing two independent sgRNAs targeting the Nr3c1 gene. (G) Analysis of viability in the CCRF-CEM cells CRISPR edited at the GR locus and exposed to increasing doses of DEX. Error bars represent standard deviation of technical replicates.

Figure 3.. Nr3c1 mutations in relapsed T-ALLs drive leukemia outgrowth in vivo and confer dexamethasone resistance in vitro.

(A) Mutant allele frequencies for mutations identified in the parental (P, x-axis) and the corresponding DEX-treated (D) or DEX/GDC-0941 combination-treated (C) relapsed T-ALLs (y-axis) highlighting the specific Nr3c1 mutations or indels. (B) Box and whiskers plot showing allele frequency of the Nr3c1 indel in each relapsed sample as compared to parental T-ALL 78A as determined from Sanger sequencing data (top), and corresponding Western blot data showing GR protein expression (bottom). Whiskers represent 10–90 percentile. (C) Sanger sequencing traces showing the site of the Nr3c1 point mutation (arrow) in parental and relapsed 20M T-ALLs. Note that representation of the mutant allele is stable after transplantation in the absence of treatment, but increases markedly in secondary recipients that received DEX. (D) Western blot showing GR protein expression in the parental (P), relapsed (D) and re-treated 20M T-ALLs shown in panel C. (E) Viability of independent CRISPR-edited CCRF-CEM cell clones nucleofected with a scramble guide (S1, S2, S3) or with a guide targeting the region of exon 4 in which the mutation in T-ALL 20M.D was identified (GR1, GR2, GR3) in response to increasing doses of DEX. Error bars represent standard deviation of technical replicates. (F) Western blot analysis showing GR expression in lysates from the CCRF-CEM cell clones edited with scramble or Nr3c1-targeting guides.

Figure 4.. Relapsed T-ALLs with pre-existing or acquired Nr3c1 mutations exhibit different evolutionary trajectories in response to dexamethasone treatment.

(A) Graphical representation of the mutant allele frequencies of missense mutations and indels shared between the parental T-ALL 78A (x-axis) and four independent relapsed leukemias (y-axis; grey), present only in the parental leukemia (green), shared only with other relapsed leukemias (blue), significantly enriched in the relapsed leukemia (purple), or specific to one of the relapsed leukemias (red). (B) Analysis of the evolutionary relationships among genetic alterations identified by WES in T-ALL 78A indicated a pattern of linear clonal evolution. (C) Graphical representation of the mutant allele frequencies shared between the parental T-ALL 20M (x-axis) and six independent relapsed leukemias (y-axis; grey), present in the parental and a subset of relapsed leukemias (yellow), shared only with other relapsed leukemias (blue), or specific to one of the relapsed leukemias (red). (D) Evolutionary analysis of the genetic alterations identified by WES in T-ALL 20M indicates a branching pattern of clonal dynamics involving multiple ancestral populations. Legends indicate the type of mutation represented by each dot shown in panels A and B (left), and each dot and branch in panels C and D. The same color scheme is used in the evolutionary analysis of panels A through D.

Figure 5.. Blunted transcriptional responses of relapsed T-ALLs to dexamethasone treatment in vivo.

(A) Heatmap showing fold increased (p-value<0.001, FDR=0.002) or decreased (p-value<0.001, FDR= 0.050) expression of selected GR-responsive genes after short-term in vivo DEX treatment measured by RNA-sequencing. (B) Gene expression analysis using TaqMan assays for each indicated gene in parental (P, grey bars) and relapsed (D) 20M T-ALLs with low GR protein expression with (blue bars) or without (red bars) an Nr3c1 mutation showing transcript levels of GC response genes as compared to vehicle-treated controls. Error bars represent standard error of the mean for technical replicates.