Targeting the MYC and PI3K pathways eliminates leukemia-initiating cells in T-cell acute lymphoblastic leukemia

Abstract

Disease relapse remains the major clinical challenge in treating T-cell acute lymphoblastic leukemia (T-ALL), particularly those with PTEN loss. We hypothesized that leukemia-initiating cells (LIC) are responsible for T-ALL development and treatment relapse. In this study, we used a genetically engineered mouse model of Pten(-/-) T-ALL with defined blast and LIC-enriched cell populations to demonstrate that LICs are responsible for therapeutic resistance. Unlike acute and chronic myelogenous leukemia, LICs in T-ALL were actively cycling, were distinct biologically, and responded differently to targeted therapies in comparison with their differentiated blast cell progeny. Notably, we found that T-ALL LICs could be eliminated by cotargeting the deregulated pathways driven by PI3K and Myc, which are altered commonly in human T-ALL and are associated with LIC formation. Our findings define critical events that may be targeted to eliminate LICs in T-ALL as a new strategy to treat the most aggressive relapsed forms of this disease.

Figure 1

Leukemia-initiating cells are responsible for therapeutic resistance in Pten null T-ALL. A, schematic diagram of strategy to investigate mechanisms underlying resistance to rapamycin. Pten null mice with T-ALL were treated for 7 days with rapamycin, and then bone marrow was harvested and transplanted into secondary and tertiary recipients in the absence of rapamycin. These recipients rapidly developed T-ALL and were re-treated for 2 days with rapamycin. After re-treatment, hematopoietic cells were harvested from Pten null T-ALL animals and analyzed for levels of phospho-S6, mutations in Notch1 and Fbxw7, and leukemia-initiating cell activity in blast and LIC compartments. B, quantitation of phospho-S6⁺ cells in Pten null TALL blast cells in bone marrow harvested from mice after 2 days of rapamycin treatment in vivo (4 mg/kg daily) using intracellular flow cytometric analysis. C, summary of mutational analysis of Notch1 exons 26, 27 and 34 and Fbxw7 exons 8, 9, and 10 using genomic DNA from splenocytes harvested from T-ALL mice after single or double rapamycin treatment. Ratios indicate number of T-ALL mice with mutations (Mut) over total number T-ALL mice screened. D, summary of transplantation assay with sorted fractions. Blast and LIC sorted populations were transplanted into NSG recipients at indicated doses and leukemia development was evaluated in recipients. Data in B are represented as mean ± SEM.

Figure 2

Rapamycin and JQ1 combination treatment reduces Pten null T-ALL disease burden and LICs. A, measurement of splenic mass and quantitation of blast and LIC populations in bone marrow of Pten null T-ALL mice by flow cytometric analysis after 7 day treatment in vivo. B, Kaplan-Meier survival curve representing morbidity of NSG recipients transplanted with 10⁴ and 10³ cell doses of treated bone marrow (BM) cells. C, fraction of secondary recipients that developed leukemia when transplanted with limiting dilutions of rapamycin or rapamycin and JQ1 combination treated donor cells, and logarithmic plot and limiting dilution analysis to calculate LIC frequency after rapamycin and JQ1 combination treatment. LIC frequency after 7 day rapamycin and JQ1 combination treatment is ~1/200,000. Data in A are represented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 3

The effects of rapamycin and JQ1 on T-ALL correlate with their associated genetic alterations. A, viability of T-ALL lines after rapamycin or JQ1 treatment for 48 hours measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Table below indicates mutational status of T-ALL lines. B, quantitation of cell cycle stage using propidium iodide and flow cytometric analysis after 24 hour treatment with 100 nM rapamycin in Pten null T-ALL. C, cell size and surface expression of CD71 and CD98 as measured by flow cytometry in Pten null T-ALL and Jurkat cells in response to 100 nM rapamycin at 24 hours. D, immunoblot for c-Myc, PARP, and cleaved caspase 3 (c-caspase 3) in Pten null T-ALL and Jurkat cells after 24 hour JQ1 treatment at indicated doses. Data in A through C are represented as mean ± SEM.

Figure 4

Pten null LICs and leukemic blasts respond differently to targeted therapies. A, quantitation of phospho-S6⁺ cells in blast and LIC compartments by flow cytometric analysis of bone marrow harvested after 2 day rapamycin treatment in vivo. The percentage of phospho-S6⁺ in blasts and LICs is shown relative to untreated levels in respective compartments. B, quantitation of cell size and surface expression of CD71 and CD98 in blast and LIC compartments as measured by flow cytometry in Pten null T-ALL in response to 100 nM rapamycin at 24 hours. C, representative fluorescence-activated cell sorting (FACS) histogram plots showing the frequency of c-Myc⁺ cells in blast and LIC populations in Pten null T-ALL after 24 hour JQ1 treatment in vitro. D, quantitation of geometric mean and median fluorescence intensities (FI) of c-Myc using flow cytometric analysis after 24 hour JQ1 treatment in vitro. Data in A, B, and D are represented as mean ± SEM.

Figure 5

Leukemia-initiating cells in Pten null T-ALL are actively cycling. A, percentage BrdU incorporation using flow cytometric analysis in primary and transplanted Pten null T-ALL mice and representative histogram plots of blast and LIC cell fractions of bone marrow (BM) harvested from transplanted Pten null T-ALL mice after 1 hour labeling in vivo. B, quantitation of BrdU incorporation by flow cytometric analysis in BM, spleen (SP), and thymus (Th) harvested from primary Pten null leukemic mice (left) and BM harvested from transplanted Pten null T-ALL mice (right) after 1 hour labeling in vivo. C, representative FACS plot showing overlay of BrdU incorporation in population with high c-Myc levels (Myc^high) and population with low c-Myc levels (Myc^low) in Pten null T-ALL after 2 hour labeling in vitro. D, quantitation of Myc^high and Myc^low populations in Pten null T-ALL after 24 hour JQ1 treatment in vitro using flow cytometric analysis. Data in B and D are represented as mean ± SEM.

Figure 6

Potent effects of combination therapy using rapamycin and VX-680 to eliminate blast and LIC populations in Pten null T-ALL. A, immunoblot for c-Myc and Aurora B in Pten null TALL after 24 hour treatment with JQ1 at indicated doses. B, measurement of splenic mass and quantitation of blast and LIC populations in bone marrow of Pten null T-ALL by flow cytometric analysis after 7 day treatment in vivo. C, Kaplan-Meier survival curve representing morbidity of NSG recipients transplanted with 10⁴ and 10³ cell doses of 14 day treated bone marrow (BM) cells. D, fraction of secondary recipients that developed leukemia when transplanted with limiting dilutions of rapamycin, VX-680, or rapamycin and VX-680 combination treated donor cells, and logarithmic plot and limiting dilution analysis to calculate LIC frequency after rapamycin and VX-680 combination treatment (~1/60,000). Data in B are represented as mean ± SEM (*p<0.05; **p<0.01).

Figure 7

VX-680 induces mitotic block and causes polyploidy and apoptosis in Pten null TALL. A, viability of Pten null T-ALL measured using an MTT assay after 48 hour VX-680 treatment and immunoblot of phosphorylation of Histone H3 at Ser-10, a surrogate assay for the activity of Aurora kinase B, in Pten null T-ALL after 300 nM VX-680 treatment over 4 to 24 hour (h) time course. B, viability of T-ALL lines measured using an MTT assay after 48 hour VX-680 treatment. Table below indicates mutational status of T-ALL lines. C, representative histogram plots of cell cycle profiles of Pten null T-ALL and Jurkat cells after 24 hour treatment with 300nM VX-680. D, quantitation of cell cycle effects in Pten null T-ALL and Jurkat cells using flow cytometric analysis after 300 nM VX-680 treatment at indicated time points. E, immunoblot of PARP, cleaved caspase 3 (c-caspase 3), phosphorylation of H2AX, p53 and p21 in Pten null T ALL and Jurkat cells after 24 hour treatment with VX-680. F, model of combination drug therapy as an approach to target LICs and blasts and thereby eradicate T-ALL. Data in A and B are represented as mean ± SEM.